33

CHAPTER 3

LITERATURE SURVEY

31 Analytical methods for single drugs

311 Epalrestat

Madhusudan N Saraf et al (2007) [39] reported an HPTLC method and

validated for estimation of Epalrestat (EPL) in plasma HPTLC was performed on

silica gel 60F 254 plates with ethyl acetate-toluene-acetic acid 60+40+2 (vv) as

mobile phase Nitrofurantoin was used as IS Densitometric scanning was

performed in absorbance mode at 390 nm The RF values of the IS and drug were

04 and 06 respectively The response was a linear function of concentration over

the range 01ndash60 microgml (r2=09974) Mean extraction recovery was gt63 Intra-

day and inter-day precision ( CV) of the assay were in the range 162ndash468 and

accuracy was gt95 This method can be applied to pharmacokinetic and

bioequivalence studies

Yang Wan-Hua et al (2000) [40] developed a reversed phase liquid

chromatographic method for the determination of EPL in human plasma and

studied pharmacokinetics The plasma sample was injected directly for

determination after being deproteinized with methanol The linearity range was

204 to 12750 ngml the LOD was 51 ngml within day RSD lt 448 and

between day RSD lt 453 the recoveries of EPL was 10109

34

312 Glibenclamide

Christelle Gedeon et al (2008) [41] described a simple rapid and sensitive

HPLC method for the analysis of Glyburide (GLB) in human plasma and

perfusate Samples were extracted by liquidndashliquid extraction (LLE) with

chloroform at neutral pH GLB was detected at 254 nm with a total run time of 7

min per sample Standard curves of 50 to 400 ngml of GLB were linear

(r2=0998) Inter-day and intra-day sample CV were 8 and 4 respectively

Recoveries ranged from 71 to 75 in human plasma samples for the 20ndash400 ngml

concentration range

Iaonnis Niopas et al (2002) [42] reported a rapid sensitive precise accurate and

specific HPLC assay for the determination of GLB in human plasma After

addition of Flufenamic acid as IS the analytes were isolated from human plasma

by liquid-liquid extraction The method was linear in the 10-400 ngml

concentration range (r gt 0999) Recovery for GLB was greater than 915 and for

IS was 935 Within-day and between-day precision expressed as the RSD

ranged from 14 to 59 and 58 to 66 respectively Assay accuracy was better

than 934 The assay was used to estimate the pharmacokinetics of GLB after

oral administration of a 5 mg tablet to 18 healthy volunteers

313 Gliclazide

Gulshan Bansal et al (2007) [43] conducted forced degradation study on

Gliclazide (GLC) under hydrolysis oxidation dry heat and photolysis conditions

and a isocratic stability-indicating HPLC-UV method was developed and

validated All the seven degradation products (IndashVII) formed under different

conditions were optimally resolved on a C18 column using mobile phase mixture

with 40 of acetonitrile and 60 0025M ammonium acetate solution (pH 35) at

a flow rate of 025 mlmin using 235 nm as detection wavelength The method was

linear between 5ndash500 microgml concentrations The RSD of intra- and inter-day

precision studies was lt1 and lt2 respectively Excellent recoveries (9981ndash

35

10097) proved the method sufficiently accurate Each peak resolved with a

resolution of gt190

Guixia Ling et al (2006) [44] developed a rapid sensitive and specific LC-ESI-

MS method for the quantification of GLC in human plasma The analyte and

Tolbutamide (IS) were extracted from plasma samples with n-hexanendash

dichloromethane (11 vv) and analyzed on a C18 column The chromatographic

separation was achieved within 40 min using methanol and 05 formic acid

(8020 vv) as mobile phase and the flow rate was 10 mlmin Ion signals mz

3240 and 2710 for GLC and IS were measured in the positive mode respectively

The method was linear within the range of 25ndash2000 ngml The LLOQ was 25

ngml The intra- and inter-day precisions were lower than 28 in terms of

RSD The inter-day Relative Error (RE) as determined from QC samples ranged

from 2193 to 185

314 Glimepiride

Yannis Dotsikas et al (2005) [45] developed a semi-automated LC-MSMS

method for the determination of Glimepiride (GLM) in human plasma The plasma

samples were treated by liquid-liquid extraction (LLE) in 12 ml 96-well format

micro-tubes GLM and the IS-GLB were extracted from human plasma by LLE

using a mixture of ethyl acetatediethyl ether 5050 (vv) as the organic solvent

After vortexing centrifugation and freezing the supernatant organic solvent was

evaporated The analyte and IS were dissolved in a small volume of a

reconstitution solution an aliquot of which was analyzed by reversed-phase

LCMSMS with positive ion electrospray ionization using multiple reaction

monitoring The method proved to be sensitive and specific for both drugs and

statistical evaluation revealed excellent linearity for the range of concentrations 2-

500 ngml with very good accuracy and inter- and intra-day precisions The

proposed method enabled the rapid and reliable determination of GLM in

36

pharmacokinetic or bioequivalence studies after administration of a 3 or 4 mg

tablet of GLM

Sacide Altinoz et al (2001) [46] developed a method using second order

derivative UV spectrophotometry for the quantification of GLM in

Dimethylformamide (DMF) in the wavelength range of 245ndash290 nm at n=6 The

second order derivative spectra was calculated using peak to peak (lDMF-2633ndash

2682 nm) peak to zero (lDMF-2682 nm) and tangent (lDMF-2633ndash2718 nm)

method for calibration curves the linearity range of 1-500 microgml by using the

second order derivative UV spectrophotometric method The developed method

was applied to directly and easily to the analysis of the pharmaceutical tablet

preparations RSD was found to be 418 and 221 The method was

completely validated and proven to be rugged The LOQ and LOD were found as

100 and 04 microgml respectively

315 Glipizide

Ghoneim et al (2007) [47] studied the electrochemical behavior of Glipizide

(GLP) at the Hanging Mercury Drop Electrode (HMDE) BndashR universal buffers of

pH 17ndash11 The voltammograms exhibited a well-defined 4-electron irreversible

cathodic peak which attributed to reduction of the two C N double bonds of the

pyrazine ring of GLP molecule GLP was found to have an interfacial adsorptive

character onto the mercury electrode surface A monolayer surface coverage of

102times10minus10 molcm2 was estimated and hence each adsorbed GLP molecule

occupied an area of 163 nm2 onto the mercury electrode surface A simple and

precise square-wave adsorptive cathodic stripping (SWAdCS) voltammetric

procedure was described for quantification of bulk GLP with a LOD of

15 times 10minus10 M and a LOQ of 5 times 10minus10 M

Dhawan et al (2003) [48] described the validation of a sensitive accurate and

reproducible method for the determination of a release profile of GLP from

37

controlled-release dosage forms In this method an in vitro dissolution profile of

commercial controlled-release dosage forms is determined using a reversed-phase

C18 column mobile phase (acetonitrilendashbuffer 005M KH2PO4 adjusted to pH 35

with orthophosphoric acid) and UV detection at a wavelength of 275 nm The

method is validated for linearity accuracy precision and detection and

quantification limits The same method can be exploited to determine the plasma

concentration of GLP The peak area versus plasma concentration is linear over the

range of 125ndash1000 ngml and the detection limit was 5 ngml in plasma The

average accuracy was 9990 with a RSD of not more than 3 Repeatability

and reproducibility were found to be good with an RSD of less than 3

316 Metformin

Sakalgaonkar et al (2008) [49] reported a simple rapid and precise isocratic

reversed phase chromatographic method was developed for assay of Metformin

HCl (MET) in bulk drug The chromatographic separation was achieved on Intersil

ODS 3V (250x46 mm 5micro) column using 0025 M dibasic potassium phosphate

and 0005 M hexane sulphonic acid sodium salt in 1000 ml water adjust pH-30

with phosphoric acid and ratio of buffer to acetonitrile 93 7 as a mobile phase

Forced degradation studies were performed for MET bulk drug using (Conc

H2So4) base (NaOH) oxidation (30 H2O2) Heat (60 oC) and UV light (254 and

365 nm) Degradation was not observed for MET sample during stress conditions

like UV light heat acid base and oxidation Peak purity test confirms MET peak

was homogenous in all stress conditions The mass balance of MET was close to

be 100 in all stress conditions The recovery was observed 10008 by the

formula recovery = slope x100 The MET sample solution and mobile phase

were found to be stable at least 48 hours Robustness of the analytical method was

determined by deliberately changing the analytical condition of wavelength

detection pH of mobile phase flow rate of mobile phase and percentage buffer

content So assay of MET sample obtained under each changed condition was

compared with mean value of method precision the overall relative standard

38

deviation should be below 20 The developed method was validated with

respective linearity accuracy precision system suitability specificity and forced

degradation studies

Collin et al (2004) [50] studied the antioxidant properties of Metformin HO

radical induced oxidation of MET was studied in aqueous solution in both aerated

and deaerated conditions Gamma radiolysis of water was used to generate HO

free radicals capable of initiating one-electron oxidation of MET Oxidation end-

products were identified by direct infusion mass spectrometry (MS) and high-

performance liquid chromatographymass spectrometry (HPLCMSn) for every

product structure elucidation was based on its mass (simple mass spectra

confirmed by HPLCMS) In addition fragmentation spectra (MS2 MS3 and MS4)

and the determination of deuteriumndashhydrogen exchange sites provided valuable

information allowing the complete identification of some of the end-products At

low radiation dose four products were identified as primary ones since they result

from the direct attack of HO radicals on MET These primary oxidation end-

products were identified respectively as hydroperoxide of MET covalent dimer of

MET methylbiguanide and 2-amino-4-imino-5-methyl-135-triazine At high

radiation dose seven other products were identified as secondary ones resulting

from the HO induced oxidation of the primary end-products A reaction scheme

was postulated for the interpretation of the results

Ashour et al (2003) [51] developed a simple sensitive and accurate

spectrophotometric method for the determination of MET The method is based on

the formation of Charge Transfer complex (CT) with iodine in acetonitrile

medium Beerrsquos law is obeyed within the concentration range 1656ndash7286 microgml

of MET whereas the optimum concentration range as evaluated by Ringbomrsquos

method was found to be 662ndash662 microgml The formation constant of the formed

complex is evaluated and was found to be 38 x 104 Lmol The molar

absorptivity Sandell sensitivity RSD and recovery are evaluated The results

39

are treated statistically applying the F- and t-tests and found highly accurate

precise and reproducible The method is used to determine MET in pure form and

pharmaceutical preparations There is no interference from the excipients

Zarghi et al (2003) [52] reported a rapid simple and sensitive ion-pair HPLC

method for quantification of MET in plasma The assay enables the measurement

of MET for therapeutic drug monitoring with a LOD 20 ngml The method

involves simple one-step extraction procedure and analytical recovery was

complete The separation was performed on an analytical 150 x 46 mm id

microbondapak C18 column The wavelength was set at 235 nm The mobile phase was

40 acetonitrile 001 M sodium dodecyl sulphate 001 M sodium dihydrogen

phosphate and distilled water to 100 adjusted to pH 51 at a flow rate of 15

mlmin The calibration curve was linear over the concentration range 02-25

mgml The coefficients of variation for inter-day and intra-day assay were within

the range of clinical usefulness

Ching-Ling Cheng et al (2001) [53] developed a simple selective sensitive and

precise HPLC plasma assay for MET Acidified samples of plasma were de-

proteinated with acetonitrile washed with dichloromethane and the resulting

supernatant injected Chromatography was performed at 40 oC by pumping a

mobile phase of acetonitrile (250 ml) in pH 7 003 M diammonium hydrogen

phosphate buffer (750 ml) at a flow-rate of 1 mlmin through a silica column MET

and the IS (Atenolol) were detected at 240 nm and eluted at 78 and 68 min

respectively after injection No endogenous substances were found to interfere

Calibration curves were linear (rgt0999) from 10 to 2000 ngml The absolute

recovery of both MET and IS was greater than 76 The detection limit and limit

of quantification were 25 and 10 ngml respectively The intra- and inter-day

precision (CV) was 12 or less and the accuracy was within 62 of the

nominal concentration

40

Florentin Tache et al (2001) [54] optimized a method for determination of MET

at ppb level in human plasma Derivatization of MET with p-nitrobenzoyl chloride

was carried out in a biphasic system The derivative extracted in the organic layer

(dichloromethane) was concentrated and analyzed by HPLC with diode array

detector using an isocratic elution water methanol mixture 6535 monitored at

280 nm Good selectivity against co-extracted matrix components was observed A

detection limit of 10 ppb MET in human plasma was reached

Saad SM Hassan et al (1999) [55] described a simple and convenient

potentiometric spectrofluorimetric and spectrophotometric methods for the

determination of MET in pharmaceutical preparations The potentiometric

technique is based on preparation of PVC membrane sensors incorporating

metformin plusmn reineckate and metformin plusmn tungstosilicate ion-pairs as electroactive

species with dioctylphthalate and o-nitrophenyloctylether as plasticizers

respectively A membrane consisting of carboxylated PVC plasticized with

dibutylsebacate is also prepared and tested These sensors give rapid Nernstian

response for 10-1-10-5 M MET at pH range 5-11 The metformin plusmn tungstosilicate

based sensor is used in a flow-through sandwich cell for flow injection

potentiometric determination of MET Graphite sensors coated or doped with

metforminplusmntungstosilicate-PVC Cu-diethyldithio-carbamate and Ni-

diethyldithiocarbamate are also prepared and used for monitoring the titration of

the drug with tetraphenyl borate (TPB-) Cu2+ and Ni2+ ions respectively The

spectrofluorimetric method depends on the reaction of MET with chrysenequinone

in alkaline medium to give a Schiffs base which upon hydrolysis gives the free

base The latter in the presence of 1-naphthol gives a fluorescent product with

excitation and emission maxima at 450 and 520 nm respectively The fluorescence

plusmn concentration relationship is linear over the range 20plusmn200 mgml MET The

proposed spectrophotometric technique involves reaction of MET with Cu2+ in

basic medium to form a Cu plusmn metformin complex The complex is dissolved in

cyclohexylamine and its maximum absorption at 540 nm is measured Beers law is

41

obeyed over the range 05-2 mgml MET Various cations some nitrogenous

compounds or drug excipients cause no interferences

Kah Hay Yuen et al (1998) [56] developed a simple HPLC method using UV

detection for the determination of MET in human plasma The method entailed

direct injection of the plasma sample after deproteination using perchloric acid

The mobile phase comprised 001 M potassium dihydrogen orthophosphate (pH

35) and acetonitrile (6040 vv) Analyses were run at a flow-rate of 10 ml min

with the detector operating at a detection wavelength of 234 nm The method is

specific and sensitive with a LOQ approximately 60 ngml and a LOD 15 ngml at

a Sn ratio of 31 The mean absolute recovery value was about 97 while the

within-day and between-day CV and error values of the assay method were all

less than 8 The calibration curve was linear over a concentration range of 625ndash

4000 ngml

Ole Vesterqvist et al (1998) [57] reported a rapid HPLC method for the

determination of MET in plasma Sample preparation entailed a 30-min

centrifugation of plasma through a micron filter with direct injection of the

protein-free ultra-filtrate into an HPLC system consisting of a cation-exchange

extraction column (75346 mm) a column switching valve and a cation-exchange

analytical column (250346 mm) The eluent was monitored at 232 nm MET was

well resolved at a retention time of about 5 min There was less than 2 loss of

MET during ultra-filtration and good linearity was established from 010 to 40

mgL of MET The LLOQ was about 005 mgL at which concentration the Sn

was above 10 The intra- and inter-assay CV at plasma concentrations of MET

between 025 and 25 mgL were typically 08ndash14 and 35ndash64 respectively

317 Nateglinide

Kanthi Kiran et al (2008) [58] studied in rats for drugndashdrug interaction of

Nateglinide (NAT) with Cilostazol (CLZ) an antiplatelet agent commonly used in

42

diabetics and developed a liquid chromatography tandem mass spectrometry (LCndash

MSMS) based method that is capable of simultaneous monitoring plasma levels of

NAT CLZ and its active metabolite 34-dehydro-cilostazol (DCLZ) All analytes

including the IS Repaglinide were chromatographed on reverse phase C18 column

(50 mm times 46 mm id 5 microm) using acetonitrile 2 mM ammonium acetate buffer

pH 34 (9010 vv) as mobile phase at a flow rate 04 mlmin in an isocratic mode

The detection of analyte was performed on LCndashMSMS system in the multiple

reaction monitoring (MRM) mode The quantitations for analytes were based on

relative concentration The method was validated over the concentration range of

20ndash2000 ngml and the LLOQ was 20 ngml The recoveries from spiked control

samples were gt79 for all analytes and IS Intra- and inter-day accuracy and

precision of validated method were with in the acceptable limits of lt15 at all

concentration The quantitation method was successfully applied for simultaneous

estimation of NAT CLZ and DCLZ in a pharmacokinetic drugndashdrug interaction

study in Wistar rats

Danai Malli et al (2007) [59] developed and validated a sensitive and selective

HPLC method for the determination of NAT in human plasma NAT and the IS

undecylenic acid were extracted from plasma by liquidndashliquid extraction using a

mixture of ethyl acetatendashdiethyl ether 5050 (vv) Pre-column derivatization

reaction was performed using a coumarin-type fluorescent reagent

N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide The derivati-

zation proceeded in acetone in the presence of potassium carbonate and catalyzed

by 18-crown-6 ether The fluorescent derivatives were separated under isocratic

conditions on a Hypersil BDS-C8 analytical column with a mobile phase that

consisted of 65 acetonitrile in water and pumped at a flow rate of 05 mlmin

The excitation and emission wavelengths were set at 345 and 435 nm respectively

The assay was linear over a concentration range of 005ndash16 microgml for NAT with a

LOQ 005 microgml QC samples (005 450 and 1600 microgml) in five replicates from

five different runs of analysis demonstrated intra-assay precision ( CV lt68)

43

inter-assay precision ( CV lt16) and an overall accuracy ( relative error) lt

-34

Jolly M Sankalia et al (2007) [60] reported a rapid simple and sensitive HPLC

method with UV detection for the quantification of NAT from rabbit plasma The

retention behavior of NAT and GLZ (IS) as a function of mobile phase pH

composition and flow rate was investigated Separation was developed on a

reverse-phase C18 column (250 times 46 mm id 5 microm) using a mixture of

acetonitrile 10 mM phosphate buffer (pH 30) in the ratio of 7030 ( vv) at a

flow rate of 10 mlmin with UV detection at 203 nm within 8 min and quantified

based on drugIS peak area ratios The plasma samples were prepared by a simple

deproteinization with a mixture of methanol and acetonitrile yielding more than

9786 extraction efficiencies The calibration curve was linear (r2 = 09984) in

the concentration range of 10ndash2500 ngml The LOD and LOQ were found to be

291 and 970 ngml respectively Both the intra-day and inter-day precisions at

four tested concentrations were below 132 RSD

Junfa Yin et al (2005) [61] reported a rapid and efficient chiral separation

method of NAT and its L-enantiomer on a monolithic Molecularly Imprinted

Polymers (MIP) The enantiomers were rapidly separated on this novel monolithic

MIP based Chiral Stationary Phase (MIP-CSP) whereas the enantioseparation was

not obtained on the non-imprinted polymer (NIP) Chiral recognition was found to

be dependent on the stereo structures and the arrangement of functional groups of

the imprinted molecule and the cavities on MIP Thermodynamic data (∆∆H and

∆∆S) obtained by Vanrsquot Hoff plots revealed an enthalpy-controlled

enantioseparation The binding capacity was evaluated by frontal analysis

Monolithic NAT-MIP had an effective number of binding sites Bt = 4115 micromolg

with a dissociation constant of Kd = 740 mM The morphological characteristics of

the monolithic MIP were investigated by pore analysis and Scanning Electron

Microscope (SEM) Results showed that both mesopores and macropores were

formed in the monolith Over all this study presents a new and practical possibility

44

for providing high rates of mass transfer fast separations and high efficiencies

without the pressure constraints of the traditional bulk molecularly imprinted

polymers through the monolithic MIPs

Hongyuan Yan et al (2004) [62] developed a Micellar Electro Kinetic

Chromatography (MEKC) method for the determination of NAT in animal plasma

by on-line sweeping technique in which plasma samples were simply

deproteinized with acetonitrile and analyzed with 16 mmolL NaH2PO4 + 6

mmolL Na2B4O7 + 60 mmolL sodium dodecyl sulfate (SDS) (pH 714) as the

running buffer a fused-silica capillary as the separation tube 21 kV as the running

voltage and UV detection at 214 nm Under these conditions more than 100-fold

enrichment of NAT was obtained with the good linear relation in the range of

nateglinide plasma concentration 02ndash7mgL (r = 0998) The method could be

applied successfully to determine trace drugs in clinical analysis

Steffen Bauer et al (2003) [63] developed a rapid and simple method for the

analysis of NAT in human plasma and validated with LOQ- 01 microgml is low

enough to allow determination of pharmacokinetic parameters of the substance

The intra-assay coefficients of variation ranged from 16 to 129 at NAT

concentrations of 05-75 microgml The inter-assay variation for the same plasma

concentrations ranged from 38 to 84 The calibration was linear in the range of

01-20 microgml To quantify NAT only 50 microl of plasma were needed Following

protein precipitation in human plasma the samples were separated by isocratic

reversed phase HPLC and analyzed using UV detection at 210 nm Sample

preparation time and analysis time are both short and allow rapid analysis of large

sample sets

318 Pioglitazone

Wanjari et al (2005) [64] developed a stability indicating simple rapid and

precise reversed-phase HPLC method for the quantitation of Pioglitazone (PIO) in

45

tablet on a Hypersil C-8 (250x46 mm) column using a mobile phase consisting

acetonitrile 015 vv triethylamine (4060 vv) adjusted to pH 46 with

orthophosphoric acid at a flow rate of 15 mlmin and detection at 220 nm The

retention time of PIO have been found to be 76 min and recoveries were between

99-101 Validation of the proposed method also been done

Zhongping John Lin et al (2003) [65] reported a LC-MSMS method for the

simultaneous determination of PIO and its two metabolites M-III (keto-derivative)

and M-IV (hydroxy-derivative) in human plasma Human plasma samples of 02

ml were extracted by a single step LLE procedure and analyzed using a HPLC-

electrospray (ESI) tandem mass spectrometer system The compounds were eluted

isocratically on a C-18 column ionized using a positive ion atmospheric pressure

electrospray ionization source and analyzed using MRM mode The ion transitions

monitored were mz 3570134 for PIO mz 3710148 for M-III mz 3730150 for

M-IV and mz 4130178 for the IS The chromatographic run time was 25 min per

injection with retention times of 145 102 and 095 min for PIO M-III and M-IV

respectively The calibration curves of PIO M-III and M-IV were well fit over the

range of 05-2000 ngml (r2 gt 0998759) by using a weighted (1x2) quadratic

regression The inter-day precisions of the quality control samples were le105

(N=15) coefficient of variation and the inter-day accuracy ( nominal) ranged

from 846 to 1035 for PIO 944 to 1040 for M-III and 968 to 1010 for M-

IV All three analytes demonstrated acceptable short-term long-term and

freezethaw stability

Radha Krishna et al (2002) [66] developed an HPLC and MEKC methods have

been developed for the determination of PIO its unsaturated impurity were

separated by MEKC in less than 7 min using a 43 cm times 50 microm id uncoated fused-

silica capillary with extended light path for better sensitivity (25 kV at 30 degC) and

a Back Ground Electrolyte (BGE) consisting of 20 acetonitrile (vv) in 20 mM

sodium borate buffer pH 93 containing 50 mM Sodium Dodecyl Sulphate (SDS)

The influence of various parameters on the separation such as pH of the buffer

46

SDS concentration buffer concentration organic modifiers temperature and

voltage were investigated The MEKC method was compared with HPLC method

using 5 microm symmetry C18 column (250times46 mm id) eluted with a mobile phase

consisting of a mixture of 50 (vv) acetonitrile and 10 mM potassium dihydrogen

phosphate buffer adjusting the pH to 60 with 01 M KOH The HPLC method is

capable of detecting all process related compounds which may be present at trace

levels in finished products

Kenji Yamashita et al (1996) [67] developed a HPLC method for the

simultaneous determination of PIO and its metabolites (M-I to M-V) in human

serum and urine The method for serum involved the solid-phase (SPE) and LLE

extraction Urine with and without enzymatic hydrolysis using β-glucuronidase

was treated with LLE The compounds in the extract were analyzed using HPLC

with UV detection at 269 nm The detection limits of PIO M-I M-II M-Ill M-IV

and M-V in serum were 001-005 microgml those in urine were 01-05 microgml and

those in urine after enzymatic hydrolysis were 03-05 microgml respectively

Zhong et al (1996) [68] reported a HPLC method for the simultaneous

determination of PIO and its potential metabolites (M-I to M-6) in human serum

The method involved a SPE of PIO its metabolites and the IS from serum using

C18 SPE columns with an elution solvent of 05 ml of acetonitrile-water (3565

vv) Separation of the eight analytes was achieved within 20 min using a reversed-

phase Zorbax RX-Cs analytical column with a mobile phase of acetonitrile-water

(4060 vv) containing acetic acid 3 mlL (apparent pH 55) An UV detector

operated at 269 nm was used with a linear response observed from 002 to 2 microgml

for these analytes except for M-4 which was best fitted with a polynomial

regression LOQ was found to be 002 microgml for PIO M-3 M-5 and M-6 004

microgml for M-2 and M-4 and 05 microgml for M-I when using a 05 ml serum sample

for extraction Intra- and inter-assay precision was le 9 and accuracy ranged from

-82 to 134 for all analytes were obtained from the method validation

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

34

312 Glibenclamide

Christelle Gedeon et al (2008) [41] described a simple rapid and sensitive

HPLC method for the analysis of Glyburide (GLB) in human plasma and

perfusate Samples were extracted by liquidndashliquid extraction (LLE) with

chloroform at neutral pH GLB was detected at 254 nm with a total run time of 7

min per sample Standard curves of 50 to 400 ngml of GLB were linear

(r2=0998) Inter-day and intra-day sample CV were 8 and 4 respectively

Recoveries ranged from 71 to 75 in human plasma samples for the 20ndash400 ngml

concentration range

Iaonnis Niopas et al (2002) [42] reported a rapid sensitive precise accurate and

specific HPLC assay for the determination of GLB in human plasma After

addition of Flufenamic acid as IS the analytes were isolated from human plasma

by liquid-liquid extraction The method was linear in the 10-400 ngml

concentration range (r gt 0999) Recovery for GLB was greater than 915 and for

IS was 935 Within-day and between-day precision expressed as the RSD

ranged from 14 to 59 and 58 to 66 respectively Assay accuracy was better

than 934 The assay was used to estimate the pharmacokinetics of GLB after

oral administration of a 5 mg tablet to 18 healthy volunteers

313 Gliclazide

Gulshan Bansal et al (2007) [43] conducted forced degradation study on

Gliclazide (GLC) under hydrolysis oxidation dry heat and photolysis conditions

and a isocratic stability-indicating HPLC-UV method was developed and

validated All the seven degradation products (IndashVII) formed under different

conditions were optimally resolved on a C18 column using mobile phase mixture

with 40 of acetonitrile and 60 0025M ammonium acetate solution (pH 35) at

a flow rate of 025 mlmin using 235 nm as detection wavelength The method was

linear between 5ndash500 microgml concentrations The RSD of intra- and inter-day

precision studies was lt1 and lt2 respectively Excellent recoveries (9981ndash

35

10097) proved the method sufficiently accurate Each peak resolved with a

resolution of gt190

Guixia Ling et al (2006) [44] developed a rapid sensitive and specific LC-ESI-

MS method for the quantification of GLC in human plasma The analyte and

Tolbutamide (IS) were extracted from plasma samples with n-hexanendash

dichloromethane (11 vv) and analyzed on a C18 column The chromatographic

separation was achieved within 40 min using methanol and 05 formic acid

(8020 vv) as mobile phase and the flow rate was 10 mlmin Ion signals mz

3240 and 2710 for GLC and IS were measured in the positive mode respectively

The method was linear within the range of 25ndash2000 ngml The LLOQ was 25

ngml The intra- and inter-day precisions were lower than 28 in terms of

RSD The inter-day Relative Error (RE) as determined from QC samples ranged

from 2193 to 185

314 Glimepiride

Yannis Dotsikas et al (2005) [45] developed a semi-automated LC-MSMS

method for the determination of Glimepiride (GLM) in human plasma The plasma

samples were treated by liquid-liquid extraction (LLE) in 12 ml 96-well format

micro-tubes GLM and the IS-GLB were extracted from human plasma by LLE

using a mixture of ethyl acetatediethyl ether 5050 (vv) as the organic solvent

After vortexing centrifugation and freezing the supernatant organic solvent was

evaporated The analyte and IS were dissolved in a small volume of a

reconstitution solution an aliquot of which was analyzed by reversed-phase

LCMSMS with positive ion electrospray ionization using multiple reaction

monitoring The method proved to be sensitive and specific for both drugs and

statistical evaluation revealed excellent linearity for the range of concentrations 2-

500 ngml with very good accuracy and inter- and intra-day precisions The

proposed method enabled the rapid and reliable determination of GLM in

36

pharmacokinetic or bioequivalence studies after administration of a 3 or 4 mg

tablet of GLM

Sacide Altinoz et al (2001) [46] developed a method using second order

derivative UV spectrophotometry for the quantification of GLM in

Dimethylformamide (DMF) in the wavelength range of 245ndash290 nm at n=6 The

second order derivative spectra was calculated using peak to peak (lDMF-2633ndash

2682 nm) peak to zero (lDMF-2682 nm) and tangent (lDMF-2633ndash2718 nm)

method for calibration curves the linearity range of 1-500 microgml by using the

second order derivative UV spectrophotometric method The developed method

was applied to directly and easily to the analysis of the pharmaceutical tablet

preparations RSD was found to be 418 and 221 The method was

completely validated and proven to be rugged The LOQ and LOD were found as

100 and 04 microgml respectively

315 Glipizide

Ghoneim et al (2007) [47] studied the electrochemical behavior of Glipizide

(GLP) at the Hanging Mercury Drop Electrode (HMDE) BndashR universal buffers of

pH 17ndash11 The voltammograms exhibited a well-defined 4-electron irreversible

cathodic peak which attributed to reduction of the two C N double bonds of the

pyrazine ring of GLP molecule GLP was found to have an interfacial adsorptive

character onto the mercury electrode surface A monolayer surface coverage of

102times10minus10 molcm2 was estimated and hence each adsorbed GLP molecule

occupied an area of 163 nm2 onto the mercury electrode surface A simple and

precise square-wave adsorptive cathodic stripping (SWAdCS) voltammetric

procedure was described for quantification of bulk GLP with a LOD of

15 times 10minus10 M and a LOQ of 5 times 10minus10 M

Dhawan et al (2003) [48] described the validation of a sensitive accurate and

reproducible method for the determination of a release profile of GLP from

37

controlled-release dosage forms In this method an in vitro dissolution profile of

commercial controlled-release dosage forms is determined using a reversed-phase

C18 column mobile phase (acetonitrilendashbuffer 005M KH2PO4 adjusted to pH 35

with orthophosphoric acid) and UV detection at a wavelength of 275 nm The

method is validated for linearity accuracy precision and detection and

quantification limits The same method can be exploited to determine the plasma

concentration of GLP The peak area versus plasma concentration is linear over the

range of 125ndash1000 ngml and the detection limit was 5 ngml in plasma The

average accuracy was 9990 with a RSD of not more than 3 Repeatability

and reproducibility were found to be good with an RSD of less than 3

316 Metformin

Sakalgaonkar et al (2008) [49] reported a simple rapid and precise isocratic

reversed phase chromatographic method was developed for assay of Metformin

HCl (MET) in bulk drug The chromatographic separation was achieved on Intersil

ODS 3V (250x46 mm 5micro) column using 0025 M dibasic potassium phosphate

and 0005 M hexane sulphonic acid sodium salt in 1000 ml water adjust pH-30

with phosphoric acid and ratio of buffer to acetonitrile 93 7 as a mobile phase

Forced degradation studies were performed for MET bulk drug using (Conc

H2So4) base (NaOH) oxidation (30 H2O2) Heat (60 oC) and UV light (254 and

365 nm) Degradation was not observed for MET sample during stress conditions

like UV light heat acid base and oxidation Peak purity test confirms MET peak

was homogenous in all stress conditions The mass balance of MET was close to

be 100 in all stress conditions The recovery was observed 10008 by the

formula recovery = slope x100 The MET sample solution and mobile phase

were found to be stable at least 48 hours Robustness of the analytical method was

determined by deliberately changing the analytical condition of wavelength

detection pH of mobile phase flow rate of mobile phase and percentage buffer

content So assay of MET sample obtained under each changed condition was

compared with mean value of method precision the overall relative standard

38

deviation should be below 20 The developed method was validated with

respective linearity accuracy precision system suitability specificity and forced

degradation studies

Collin et al (2004) [50] studied the antioxidant properties of Metformin HO

radical induced oxidation of MET was studied in aqueous solution in both aerated

and deaerated conditions Gamma radiolysis of water was used to generate HO

free radicals capable of initiating one-electron oxidation of MET Oxidation end-

products were identified by direct infusion mass spectrometry (MS) and high-

performance liquid chromatographymass spectrometry (HPLCMSn) for every

product structure elucidation was based on its mass (simple mass spectra

confirmed by HPLCMS) In addition fragmentation spectra (MS2 MS3 and MS4)

and the determination of deuteriumndashhydrogen exchange sites provided valuable

information allowing the complete identification of some of the end-products At

low radiation dose four products were identified as primary ones since they result

from the direct attack of HO radicals on MET These primary oxidation end-

products were identified respectively as hydroperoxide of MET covalent dimer of

MET methylbiguanide and 2-amino-4-imino-5-methyl-135-triazine At high

radiation dose seven other products were identified as secondary ones resulting

from the HO induced oxidation of the primary end-products A reaction scheme

was postulated for the interpretation of the results

Ashour et al (2003) [51] developed a simple sensitive and accurate

spectrophotometric method for the determination of MET The method is based on

the formation of Charge Transfer complex (CT) with iodine in acetonitrile

medium Beerrsquos law is obeyed within the concentration range 1656ndash7286 microgml

of MET whereas the optimum concentration range as evaluated by Ringbomrsquos

method was found to be 662ndash662 microgml The formation constant of the formed

complex is evaluated and was found to be 38 x 104 Lmol The molar

absorptivity Sandell sensitivity RSD and recovery are evaluated The results

39

are treated statistically applying the F- and t-tests and found highly accurate

precise and reproducible The method is used to determine MET in pure form and

pharmaceutical preparations There is no interference from the excipients

Zarghi et al (2003) [52] reported a rapid simple and sensitive ion-pair HPLC

method for quantification of MET in plasma The assay enables the measurement

of MET for therapeutic drug monitoring with a LOD 20 ngml The method

involves simple one-step extraction procedure and analytical recovery was

complete The separation was performed on an analytical 150 x 46 mm id

microbondapak C18 column The wavelength was set at 235 nm The mobile phase was

40 acetonitrile 001 M sodium dodecyl sulphate 001 M sodium dihydrogen

phosphate and distilled water to 100 adjusted to pH 51 at a flow rate of 15

mlmin The calibration curve was linear over the concentration range 02-25

mgml The coefficients of variation for inter-day and intra-day assay were within

the range of clinical usefulness

Ching-Ling Cheng et al (2001) [53] developed a simple selective sensitive and

precise HPLC plasma assay for MET Acidified samples of plasma were de-

proteinated with acetonitrile washed with dichloromethane and the resulting

supernatant injected Chromatography was performed at 40 oC by pumping a

mobile phase of acetonitrile (250 ml) in pH 7 003 M diammonium hydrogen

phosphate buffer (750 ml) at a flow-rate of 1 mlmin through a silica column MET

and the IS (Atenolol) were detected at 240 nm and eluted at 78 and 68 min

respectively after injection No endogenous substances were found to interfere

Calibration curves were linear (rgt0999) from 10 to 2000 ngml The absolute

recovery of both MET and IS was greater than 76 The detection limit and limit

of quantification were 25 and 10 ngml respectively The intra- and inter-day

precision (CV) was 12 or less and the accuracy was within 62 of the

nominal concentration

40

Florentin Tache et al (2001) [54] optimized a method for determination of MET

at ppb level in human plasma Derivatization of MET with p-nitrobenzoyl chloride

was carried out in a biphasic system The derivative extracted in the organic layer

(dichloromethane) was concentrated and analyzed by HPLC with diode array

detector using an isocratic elution water methanol mixture 6535 monitored at

280 nm Good selectivity against co-extracted matrix components was observed A

detection limit of 10 ppb MET in human plasma was reached

Saad SM Hassan et al (1999) [55] described a simple and convenient

potentiometric spectrofluorimetric and spectrophotometric methods for the

determination of MET in pharmaceutical preparations The potentiometric

technique is based on preparation of PVC membrane sensors incorporating

metformin plusmn reineckate and metformin plusmn tungstosilicate ion-pairs as electroactive

species with dioctylphthalate and o-nitrophenyloctylether as plasticizers

respectively A membrane consisting of carboxylated PVC plasticized with

dibutylsebacate is also prepared and tested These sensors give rapid Nernstian

response for 10-1-10-5 M MET at pH range 5-11 The metformin plusmn tungstosilicate

based sensor is used in a flow-through sandwich cell for flow injection

potentiometric determination of MET Graphite sensors coated or doped with

metforminplusmntungstosilicate-PVC Cu-diethyldithio-carbamate and Ni-

diethyldithiocarbamate are also prepared and used for monitoring the titration of

the drug with tetraphenyl borate (TPB-) Cu2+ and Ni2+ ions respectively The

spectrofluorimetric method depends on the reaction of MET with chrysenequinone

in alkaline medium to give a Schiffs base which upon hydrolysis gives the free

base The latter in the presence of 1-naphthol gives a fluorescent product with

excitation and emission maxima at 450 and 520 nm respectively The fluorescence

plusmn concentration relationship is linear over the range 20plusmn200 mgml MET The

proposed spectrophotometric technique involves reaction of MET with Cu2+ in

basic medium to form a Cu plusmn metformin complex The complex is dissolved in

cyclohexylamine and its maximum absorption at 540 nm is measured Beers law is

41

obeyed over the range 05-2 mgml MET Various cations some nitrogenous

compounds or drug excipients cause no interferences

Kah Hay Yuen et al (1998) [56] developed a simple HPLC method using UV

detection for the determination of MET in human plasma The method entailed

direct injection of the plasma sample after deproteination using perchloric acid

The mobile phase comprised 001 M potassium dihydrogen orthophosphate (pH

35) and acetonitrile (6040 vv) Analyses were run at a flow-rate of 10 ml min

with the detector operating at a detection wavelength of 234 nm The method is

specific and sensitive with a LOQ approximately 60 ngml and a LOD 15 ngml at

a Sn ratio of 31 The mean absolute recovery value was about 97 while the

within-day and between-day CV and error values of the assay method were all

less than 8 The calibration curve was linear over a concentration range of 625ndash

4000 ngml

Ole Vesterqvist et al (1998) [57] reported a rapid HPLC method for the

determination of MET in plasma Sample preparation entailed a 30-min

centrifugation of plasma through a micron filter with direct injection of the

protein-free ultra-filtrate into an HPLC system consisting of a cation-exchange

extraction column (75346 mm) a column switching valve and a cation-exchange

analytical column (250346 mm) The eluent was monitored at 232 nm MET was

well resolved at a retention time of about 5 min There was less than 2 loss of

MET during ultra-filtration and good linearity was established from 010 to 40

mgL of MET The LLOQ was about 005 mgL at which concentration the Sn

was above 10 The intra- and inter-assay CV at plasma concentrations of MET

between 025 and 25 mgL were typically 08ndash14 and 35ndash64 respectively

317 Nateglinide

Kanthi Kiran et al (2008) [58] studied in rats for drugndashdrug interaction of

Nateglinide (NAT) with Cilostazol (CLZ) an antiplatelet agent commonly used in

42

diabetics and developed a liquid chromatography tandem mass spectrometry (LCndash

MSMS) based method that is capable of simultaneous monitoring plasma levels of

NAT CLZ and its active metabolite 34-dehydro-cilostazol (DCLZ) All analytes

including the IS Repaglinide were chromatographed on reverse phase C18 column

(50 mm times 46 mm id 5 microm) using acetonitrile 2 mM ammonium acetate buffer

pH 34 (9010 vv) as mobile phase at a flow rate 04 mlmin in an isocratic mode

The detection of analyte was performed on LCndashMSMS system in the multiple

reaction monitoring (MRM) mode The quantitations for analytes were based on

relative concentration The method was validated over the concentration range of

20ndash2000 ngml and the LLOQ was 20 ngml The recoveries from spiked control

samples were gt79 for all analytes and IS Intra- and inter-day accuracy and

precision of validated method were with in the acceptable limits of lt15 at all

concentration The quantitation method was successfully applied for simultaneous

estimation of NAT CLZ and DCLZ in a pharmacokinetic drugndashdrug interaction

study in Wistar rats

Danai Malli et al (2007) [59] developed and validated a sensitive and selective

HPLC method for the determination of NAT in human plasma NAT and the IS

undecylenic acid were extracted from plasma by liquidndashliquid extraction using a

mixture of ethyl acetatendashdiethyl ether 5050 (vv) Pre-column derivatization

reaction was performed using a coumarin-type fluorescent reagent

N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide The derivati-

zation proceeded in acetone in the presence of potassium carbonate and catalyzed

by 18-crown-6 ether The fluorescent derivatives were separated under isocratic

conditions on a Hypersil BDS-C8 analytical column with a mobile phase that

consisted of 65 acetonitrile in water and pumped at a flow rate of 05 mlmin

The excitation and emission wavelengths were set at 345 and 435 nm respectively

The assay was linear over a concentration range of 005ndash16 microgml for NAT with a

LOQ 005 microgml QC samples (005 450 and 1600 microgml) in five replicates from

five different runs of analysis demonstrated intra-assay precision ( CV lt68)

43

inter-assay precision ( CV lt16) and an overall accuracy ( relative error) lt

-34

Jolly M Sankalia et al (2007) [60] reported a rapid simple and sensitive HPLC

method with UV detection for the quantification of NAT from rabbit plasma The

retention behavior of NAT and GLZ (IS) as a function of mobile phase pH

composition and flow rate was investigated Separation was developed on a

reverse-phase C18 column (250 times 46 mm id 5 microm) using a mixture of

acetonitrile 10 mM phosphate buffer (pH 30) in the ratio of 7030 ( vv) at a

flow rate of 10 mlmin with UV detection at 203 nm within 8 min and quantified

based on drugIS peak area ratios The plasma samples were prepared by a simple

deproteinization with a mixture of methanol and acetonitrile yielding more than

9786 extraction efficiencies The calibration curve was linear (r2 = 09984) in

the concentration range of 10ndash2500 ngml The LOD and LOQ were found to be

291 and 970 ngml respectively Both the intra-day and inter-day precisions at

four tested concentrations were below 132 RSD

Junfa Yin et al (2005) [61] reported a rapid and efficient chiral separation

method of NAT and its L-enantiomer on a monolithic Molecularly Imprinted

Polymers (MIP) The enantiomers were rapidly separated on this novel monolithic

MIP based Chiral Stationary Phase (MIP-CSP) whereas the enantioseparation was

not obtained on the non-imprinted polymer (NIP) Chiral recognition was found to

be dependent on the stereo structures and the arrangement of functional groups of

the imprinted molecule and the cavities on MIP Thermodynamic data (∆∆H and

∆∆S) obtained by Vanrsquot Hoff plots revealed an enthalpy-controlled

enantioseparation The binding capacity was evaluated by frontal analysis

Monolithic NAT-MIP had an effective number of binding sites Bt = 4115 micromolg

with a dissociation constant of Kd = 740 mM The morphological characteristics of

the monolithic MIP were investigated by pore analysis and Scanning Electron

Microscope (SEM) Results showed that both mesopores and macropores were

formed in the monolith Over all this study presents a new and practical possibility

44

for providing high rates of mass transfer fast separations and high efficiencies

without the pressure constraints of the traditional bulk molecularly imprinted

polymers through the monolithic MIPs

Hongyuan Yan et al (2004) [62] developed a Micellar Electro Kinetic

Chromatography (MEKC) method for the determination of NAT in animal plasma

by on-line sweeping technique in which plasma samples were simply

deproteinized with acetonitrile and analyzed with 16 mmolL NaH2PO4 + 6

mmolL Na2B4O7 + 60 mmolL sodium dodecyl sulfate (SDS) (pH 714) as the

running buffer a fused-silica capillary as the separation tube 21 kV as the running

voltage and UV detection at 214 nm Under these conditions more than 100-fold

enrichment of NAT was obtained with the good linear relation in the range of

nateglinide plasma concentration 02ndash7mgL (r = 0998) The method could be

applied successfully to determine trace drugs in clinical analysis

Steffen Bauer et al (2003) [63] developed a rapid and simple method for the

analysis of NAT in human plasma and validated with LOQ- 01 microgml is low

enough to allow determination of pharmacokinetic parameters of the substance

The intra-assay coefficients of variation ranged from 16 to 129 at NAT

concentrations of 05-75 microgml The inter-assay variation for the same plasma

concentrations ranged from 38 to 84 The calibration was linear in the range of

01-20 microgml To quantify NAT only 50 microl of plasma were needed Following

protein precipitation in human plasma the samples were separated by isocratic

reversed phase HPLC and analyzed using UV detection at 210 nm Sample

preparation time and analysis time are both short and allow rapid analysis of large

sample sets

318 Pioglitazone

Wanjari et al (2005) [64] developed a stability indicating simple rapid and

precise reversed-phase HPLC method for the quantitation of Pioglitazone (PIO) in

45

tablet on a Hypersil C-8 (250x46 mm) column using a mobile phase consisting

acetonitrile 015 vv triethylamine (4060 vv) adjusted to pH 46 with

orthophosphoric acid at a flow rate of 15 mlmin and detection at 220 nm The

retention time of PIO have been found to be 76 min and recoveries were between

99-101 Validation of the proposed method also been done

Zhongping John Lin et al (2003) [65] reported a LC-MSMS method for the

simultaneous determination of PIO and its two metabolites M-III (keto-derivative)

and M-IV (hydroxy-derivative) in human plasma Human plasma samples of 02

ml were extracted by a single step LLE procedure and analyzed using a HPLC-

electrospray (ESI) tandem mass spectrometer system The compounds were eluted

isocratically on a C-18 column ionized using a positive ion atmospheric pressure

electrospray ionization source and analyzed using MRM mode The ion transitions

monitored were mz 3570134 for PIO mz 3710148 for M-III mz 3730150 for

M-IV and mz 4130178 for the IS The chromatographic run time was 25 min per

injection with retention times of 145 102 and 095 min for PIO M-III and M-IV

respectively The calibration curves of PIO M-III and M-IV were well fit over the

range of 05-2000 ngml (r2 gt 0998759) by using a weighted (1x2) quadratic

regression The inter-day precisions of the quality control samples were le105

(N=15) coefficient of variation and the inter-day accuracy ( nominal) ranged

from 846 to 1035 for PIO 944 to 1040 for M-III and 968 to 1010 for M-

IV All three analytes demonstrated acceptable short-term long-term and

freezethaw stability

Radha Krishna et al (2002) [66] developed an HPLC and MEKC methods have

been developed for the determination of PIO its unsaturated impurity were

separated by MEKC in less than 7 min using a 43 cm times 50 microm id uncoated fused-

silica capillary with extended light path for better sensitivity (25 kV at 30 degC) and

a Back Ground Electrolyte (BGE) consisting of 20 acetonitrile (vv) in 20 mM

sodium borate buffer pH 93 containing 50 mM Sodium Dodecyl Sulphate (SDS)

The influence of various parameters on the separation such as pH of the buffer

46

SDS concentration buffer concentration organic modifiers temperature and

voltage were investigated The MEKC method was compared with HPLC method

using 5 microm symmetry C18 column (250times46 mm id) eluted with a mobile phase

consisting of a mixture of 50 (vv) acetonitrile and 10 mM potassium dihydrogen

phosphate buffer adjusting the pH to 60 with 01 M KOH The HPLC method is

capable of detecting all process related compounds which may be present at trace

levels in finished products

Kenji Yamashita et al (1996) [67] developed a HPLC method for the

simultaneous determination of PIO and its metabolites (M-I to M-V) in human

serum and urine The method for serum involved the solid-phase (SPE) and LLE

extraction Urine with and without enzymatic hydrolysis using β-glucuronidase

was treated with LLE The compounds in the extract were analyzed using HPLC

with UV detection at 269 nm The detection limits of PIO M-I M-II M-Ill M-IV

and M-V in serum were 001-005 microgml those in urine were 01-05 microgml and

those in urine after enzymatic hydrolysis were 03-05 microgml respectively

Zhong et al (1996) [68] reported a HPLC method for the simultaneous

determination of PIO and its potential metabolites (M-I to M-6) in human serum

The method involved a SPE of PIO its metabolites and the IS from serum using

C18 SPE columns with an elution solvent of 05 ml of acetonitrile-water (3565

vv) Separation of the eight analytes was achieved within 20 min using a reversed-

phase Zorbax RX-Cs analytical column with a mobile phase of acetonitrile-water

(4060 vv) containing acetic acid 3 mlL (apparent pH 55) An UV detector

operated at 269 nm was used with a linear response observed from 002 to 2 microgml

for these analytes except for M-4 which was best fitted with a polynomial

regression LOQ was found to be 002 microgml for PIO M-3 M-5 and M-6 004

microgml for M-2 and M-4 and 05 microgml for M-I when using a 05 ml serum sample

for extraction Intra- and inter-assay precision was le 9 and accuracy ranged from

-82 to 134 for all analytes were obtained from the method validation

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

35

10097) proved the method sufficiently accurate Each peak resolved with a

resolution of gt190

Guixia Ling et al (2006) [44] developed a rapid sensitive and specific LC-ESI-

MS method for the quantification of GLC in human plasma The analyte and

Tolbutamide (IS) were extracted from plasma samples with n-hexanendash

dichloromethane (11 vv) and analyzed on a C18 column The chromatographic

separation was achieved within 40 min using methanol and 05 formic acid

(8020 vv) as mobile phase and the flow rate was 10 mlmin Ion signals mz

3240 and 2710 for GLC and IS were measured in the positive mode respectively

The method was linear within the range of 25ndash2000 ngml The LLOQ was 25

ngml The intra- and inter-day precisions were lower than 28 in terms of

RSD The inter-day Relative Error (RE) as determined from QC samples ranged

from 2193 to 185

314 Glimepiride

Yannis Dotsikas et al (2005) [45] developed a semi-automated LC-MSMS

method for the determination of Glimepiride (GLM) in human plasma The plasma

samples were treated by liquid-liquid extraction (LLE) in 12 ml 96-well format

micro-tubes GLM and the IS-GLB were extracted from human plasma by LLE

using a mixture of ethyl acetatediethyl ether 5050 (vv) as the organic solvent

After vortexing centrifugation and freezing the supernatant organic solvent was

evaporated The analyte and IS were dissolved in a small volume of a

reconstitution solution an aliquot of which was analyzed by reversed-phase

LCMSMS with positive ion electrospray ionization using multiple reaction

monitoring The method proved to be sensitive and specific for both drugs and

statistical evaluation revealed excellent linearity for the range of concentrations 2-

500 ngml with very good accuracy and inter- and intra-day precisions The

proposed method enabled the rapid and reliable determination of GLM in

36

pharmacokinetic or bioequivalence studies after administration of a 3 or 4 mg

tablet of GLM

Sacide Altinoz et al (2001) [46] developed a method using second order

derivative UV spectrophotometry for the quantification of GLM in

Dimethylformamide (DMF) in the wavelength range of 245ndash290 nm at n=6 The

second order derivative spectra was calculated using peak to peak (lDMF-2633ndash

2682 nm) peak to zero (lDMF-2682 nm) and tangent (lDMF-2633ndash2718 nm)

method for calibration curves the linearity range of 1-500 microgml by using the

second order derivative UV spectrophotometric method The developed method

was applied to directly and easily to the analysis of the pharmaceutical tablet

preparations RSD was found to be 418 and 221 The method was

completely validated and proven to be rugged The LOQ and LOD were found as

100 and 04 microgml respectively

315 Glipizide

Ghoneim et al (2007) [47] studied the electrochemical behavior of Glipizide

(GLP) at the Hanging Mercury Drop Electrode (HMDE) BndashR universal buffers of

pH 17ndash11 The voltammograms exhibited a well-defined 4-electron irreversible

cathodic peak which attributed to reduction of the two C N double bonds of the

pyrazine ring of GLP molecule GLP was found to have an interfacial adsorptive

character onto the mercury electrode surface A monolayer surface coverage of

102times10minus10 molcm2 was estimated and hence each adsorbed GLP molecule

occupied an area of 163 nm2 onto the mercury electrode surface A simple and

precise square-wave adsorptive cathodic stripping (SWAdCS) voltammetric

procedure was described for quantification of bulk GLP with a LOD of

15 times 10minus10 M and a LOQ of 5 times 10minus10 M

Dhawan et al (2003) [48] described the validation of a sensitive accurate and

reproducible method for the determination of a release profile of GLP from

37

controlled-release dosage forms In this method an in vitro dissolution profile of

commercial controlled-release dosage forms is determined using a reversed-phase

C18 column mobile phase (acetonitrilendashbuffer 005M KH2PO4 adjusted to pH 35

with orthophosphoric acid) and UV detection at a wavelength of 275 nm The

method is validated for linearity accuracy precision and detection and

quantification limits The same method can be exploited to determine the plasma

concentration of GLP The peak area versus plasma concentration is linear over the

range of 125ndash1000 ngml and the detection limit was 5 ngml in plasma The

average accuracy was 9990 with a RSD of not more than 3 Repeatability

and reproducibility were found to be good with an RSD of less than 3

316 Metformin

Sakalgaonkar et al (2008) [49] reported a simple rapid and precise isocratic

reversed phase chromatographic method was developed for assay of Metformin

HCl (MET) in bulk drug The chromatographic separation was achieved on Intersil

ODS 3V (250x46 mm 5micro) column using 0025 M dibasic potassium phosphate

and 0005 M hexane sulphonic acid sodium salt in 1000 ml water adjust pH-30

with phosphoric acid and ratio of buffer to acetonitrile 93 7 as a mobile phase

Forced degradation studies were performed for MET bulk drug using (Conc

H2So4) base (NaOH) oxidation (30 H2O2) Heat (60 oC) and UV light (254 and

365 nm) Degradation was not observed for MET sample during stress conditions

like UV light heat acid base and oxidation Peak purity test confirms MET peak

was homogenous in all stress conditions The mass balance of MET was close to

be 100 in all stress conditions The recovery was observed 10008 by the

formula recovery = slope x100 The MET sample solution and mobile phase

were found to be stable at least 48 hours Robustness of the analytical method was

determined by deliberately changing the analytical condition of wavelength

detection pH of mobile phase flow rate of mobile phase and percentage buffer

content So assay of MET sample obtained under each changed condition was

compared with mean value of method precision the overall relative standard

38

deviation should be below 20 The developed method was validated with

respective linearity accuracy precision system suitability specificity and forced

degradation studies

Collin et al (2004) [50] studied the antioxidant properties of Metformin HO

radical induced oxidation of MET was studied in aqueous solution in both aerated

and deaerated conditions Gamma radiolysis of water was used to generate HO

free radicals capable of initiating one-electron oxidation of MET Oxidation end-

products were identified by direct infusion mass spectrometry (MS) and high-

performance liquid chromatographymass spectrometry (HPLCMSn) for every

product structure elucidation was based on its mass (simple mass spectra

confirmed by HPLCMS) In addition fragmentation spectra (MS2 MS3 and MS4)

and the determination of deuteriumndashhydrogen exchange sites provided valuable

information allowing the complete identification of some of the end-products At

low radiation dose four products were identified as primary ones since they result

from the direct attack of HO radicals on MET These primary oxidation end-

products were identified respectively as hydroperoxide of MET covalent dimer of

MET methylbiguanide and 2-amino-4-imino-5-methyl-135-triazine At high

radiation dose seven other products were identified as secondary ones resulting

from the HO induced oxidation of the primary end-products A reaction scheme

was postulated for the interpretation of the results

Ashour et al (2003) [51] developed a simple sensitive and accurate

spectrophotometric method for the determination of MET The method is based on

the formation of Charge Transfer complex (CT) with iodine in acetonitrile

medium Beerrsquos law is obeyed within the concentration range 1656ndash7286 microgml

of MET whereas the optimum concentration range as evaluated by Ringbomrsquos

method was found to be 662ndash662 microgml The formation constant of the formed

complex is evaluated and was found to be 38 x 104 Lmol The molar

absorptivity Sandell sensitivity RSD and recovery are evaluated The results

39

are treated statistically applying the F- and t-tests and found highly accurate

precise and reproducible The method is used to determine MET in pure form and

pharmaceutical preparations There is no interference from the excipients

Zarghi et al (2003) [52] reported a rapid simple and sensitive ion-pair HPLC

method for quantification of MET in plasma The assay enables the measurement

of MET for therapeutic drug monitoring with a LOD 20 ngml The method

involves simple one-step extraction procedure and analytical recovery was

complete The separation was performed on an analytical 150 x 46 mm id

microbondapak C18 column The wavelength was set at 235 nm The mobile phase was

40 acetonitrile 001 M sodium dodecyl sulphate 001 M sodium dihydrogen

phosphate and distilled water to 100 adjusted to pH 51 at a flow rate of 15

mlmin The calibration curve was linear over the concentration range 02-25

mgml The coefficients of variation for inter-day and intra-day assay were within

the range of clinical usefulness

Ching-Ling Cheng et al (2001) [53] developed a simple selective sensitive and

precise HPLC plasma assay for MET Acidified samples of plasma were de-

proteinated with acetonitrile washed with dichloromethane and the resulting

supernatant injected Chromatography was performed at 40 oC by pumping a

mobile phase of acetonitrile (250 ml) in pH 7 003 M diammonium hydrogen

phosphate buffer (750 ml) at a flow-rate of 1 mlmin through a silica column MET

and the IS (Atenolol) were detected at 240 nm and eluted at 78 and 68 min

respectively after injection No endogenous substances were found to interfere

Calibration curves were linear (rgt0999) from 10 to 2000 ngml The absolute

recovery of both MET and IS was greater than 76 The detection limit and limit

of quantification were 25 and 10 ngml respectively The intra- and inter-day

precision (CV) was 12 or less and the accuracy was within 62 of the

nominal concentration

40

Florentin Tache et al (2001) [54] optimized a method for determination of MET

at ppb level in human plasma Derivatization of MET with p-nitrobenzoyl chloride

was carried out in a biphasic system The derivative extracted in the organic layer

(dichloromethane) was concentrated and analyzed by HPLC with diode array

detector using an isocratic elution water methanol mixture 6535 monitored at

280 nm Good selectivity against co-extracted matrix components was observed A

detection limit of 10 ppb MET in human plasma was reached

Saad SM Hassan et al (1999) [55] described a simple and convenient

potentiometric spectrofluorimetric and spectrophotometric methods for the

determination of MET in pharmaceutical preparations The potentiometric

technique is based on preparation of PVC membrane sensors incorporating

metformin plusmn reineckate and metformin plusmn tungstosilicate ion-pairs as electroactive

species with dioctylphthalate and o-nitrophenyloctylether as plasticizers

respectively A membrane consisting of carboxylated PVC plasticized with

dibutylsebacate is also prepared and tested These sensors give rapid Nernstian

response for 10-1-10-5 M MET at pH range 5-11 The metformin plusmn tungstosilicate

based sensor is used in a flow-through sandwich cell for flow injection

potentiometric determination of MET Graphite sensors coated or doped with

metforminplusmntungstosilicate-PVC Cu-diethyldithio-carbamate and Ni-

diethyldithiocarbamate are also prepared and used for monitoring the titration of

the drug with tetraphenyl borate (TPB-) Cu2+ and Ni2+ ions respectively The

spectrofluorimetric method depends on the reaction of MET with chrysenequinone

in alkaline medium to give a Schiffs base which upon hydrolysis gives the free

base The latter in the presence of 1-naphthol gives a fluorescent product with

excitation and emission maxima at 450 and 520 nm respectively The fluorescence

plusmn concentration relationship is linear over the range 20plusmn200 mgml MET The

proposed spectrophotometric technique involves reaction of MET with Cu2+ in

basic medium to form a Cu plusmn metformin complex The complex is dissolved in

cyclohexylamine and its maximum absorption at 540 nm is measured Beers law is

41

obeyed over the range 05-2 mgml MET Various cations some nitrogenous

compounds or drug excipients cause no interferences

Kah Hay Yuen et al (1998) [56] developed a simple HPLC method using UV

detection for the determination of MET in human plasma The method entailed

direct injection of the plasma sample after deproteination using perchloric acid

The mobile phase comprised 001 M potassium dihydrogen orthophosphate (pH

35) and acetonitrile (6040 vv) Analyses were run at a flow-rate of 10 ml min

with the detector operating at a detection wavelength of 234 nm The method is

specific and sensitive with a LOQ approximately 60 ngml and a LOD 15 ngml at

a Sn ratio of 31 The mean absolute recovery value was about 97 while the

within-day and between-day CV and error values of the assay method were all

less than 8 The calibration curve was linear over a concentration range of 625ndash

4000 ngml

Ole Vesterqvist et al (1998) [57] reported a rapid HPLC method for the

determination of MET in plasma Sample preparation entailed a 30-min

centrifugation of plasma through a micron filter with direct injection of the

protein-free ultra-filtrate into an HPLC system consisting of a cation-exchange

extraction column (75346 mm) a column switching valve and a cation-exchange

analytical column (250346 mm) The eluent was monitored at 232 nm MET was

well resolved at a retention time of about 5 min There was less than 2 loss of

MET during ultra-filtration and good linearity was established from 010 to 40

mgL of MET The LLOQ was about 005 mgL at which concentration the Sn

was above 10 The intra- and inter-assay CV at plasma concentrations of MET

between 025 and 25 mgL were typically 08ndash14 and 35ndash64 respectively

317 Nateglinide

Kanthi Kiran et al (2008) [58] studied in rats for drugndashdrug interaction of

Nateglinide (NAT) with Cilostazol (CLZ) an antiplatelet agent commonly used in

42

diabetics and developed a liquid chromatography tandem mass spectrometry (LCndash

MSMS) based method that is capable of simultaneous monitoring plasma levels of

NAT CLZ and its active metabolite 34-dehydro-cilostazol (DCLZ) All analytes

including the IS Repaglinide were chromatographed on reverse phase C18 column

(50 mm times 46 mm id 5 microm) using acetonitrile 2 mM ammonium acetate buffer

pH 34 (9010 vv) as mobile phase at a flow rate 04 mlmin in an isocratic mode

The detection of analyte was performed on LCndashMSMS system in the multiple

reaction monitoring (MRM) mode The quantitations for analytes were based on

relative concentration The method was validated over the concentration range of

20ndash2000 ngml and the LLOQ was 20 ngml The recoveries from spiked control

samples were gt79 for all analytes and IS Intra- and inter-day accuracy and

precision of validated method were with in the acceptable limits of lt15 at all

concentration The quantitation method was successfully applied for simultaneous

estimation of NAT CLZ and DCLZ in a pharmacokinetic drugndashdrug interaction

study in Wistar rats

Danai Malli et al (2007) [59] developed and validated a sensitive and selective

HPLC method for the determination of NAT in human plasma NAT and the IS

undecylenic acid were extracted from plasma by liquidndashliquid extraction using a

mixture of ethyl acetatendashdiethyl ether 5050 (vv) Pre-column derivatization

reaction was performed using a coumarin-type fluorescent reagent

N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide The derivati-

zation proceeded in acetone in the presence of potassium carbonate and catalyzed

by 18-crown-6 ether The fluorescent derivatives were separated under isocratic

conditions on a Hypersil BDS-C8 analytical column with a mobile phase that

consisted of 65 acetonitrile in water and pumped at a flow rate of 05 mlmin

The excitation and emission wavelengths were set at 345 and 435 nm respectively

The assay was linear over a concentration range of 005ndash16 microgml for NAT with a

LOQ 005 microgml QC samples (005 450 and 1600 microgml) in five replicates from

five different runs of analysis demonstrated intra-assay precision ( CV lt68)

43

inter-assay precision ( CV lt16) and an overall accuracy ( relative error) lt

-34

Jolly M Sankalia et al (2007) [60] reported a rapid simple and sensitive HPLC

method with UV detection for the quantification of NAT from rabbit plasma The

retention behavior of NAT and GLZ (IS) as a function of mobile phase pH

composition and flow rate was investigated Separation was developed on a

reverse-phase C18 column (250 times 46 mm id 5 microm) using a mixture of

acetonitrile 10 mM phosphate buffer (pH 30) in the ratio of 7030 ( vv) at a

flow rate of 10 mlmin with UV detection at 203 nm within 8 min and quantified

based on drugIS peak area ratios The plasma samples were prepared by a simple

deproteinization with a mixture of methanol and acetonitrile yielding more than

9786 extraction efficiencies The calibration curve was linear (r2 = 09984) in

the concentration range of 10ndash2500 ngml The LOD and LOQ were found to be

291 and 970 ngml respectively Both the intra-day and inter-day precisions at

four tested concentrations were below 132 RSD

Junfa Yin et al (2005) [61] reported a rapid and efficient chiral separation

method of NAT and its L-enantiomer on a monolithic Molecularly Imprinted

Polymers (MIP) The enantiomers were rapidly separated on this novel monolithic

MIP based Chiral Stationary Phase (MIP-CSP) whereas the enantioseparation was

not obtained on the non-imprinted polymer (NIP) Chiral recognition was found to

be dependent on the stereo structures and the arrangement of functional groups of

the imprinted molecule and the cavities on MIP Thermodynamic data (∆∆H and

∆∆S) obtained by Vanrsquot Hoff plots revealed an enthalpy-controlled

enantioseparation The binding capacity was evaluated by frontal analysis

Monolithic NAT-MIP had an effective number of binding sites Bt = 4115 micromolg

with a dissociation constant of Kd = 740 mM The morphological characteristics of

the monolithic MIP were investigated by pore analysis and Scanning Electron

Microscope (SEM) Results showed that both mesopores and macropores were

formed in the monolith Over all this study presents a new and practical possibility

44

for providing high rates of mass transfer fast separations and high efficiencies

without the pressure constraints of the traditional bulk molecularly imprinted

polymers through the monolithic MIPs

Hongyuan Yan et al (2004) [62] developed a Micellar Electro Kinetic

Chromatography (MEKC) method for the determination of NAT in animal plasma

by on-line sweeping technique in which plasma samples were simply

deproteinized with acetonitrile and analyzed with 16 mmolL NaH2PO4 + 6

mmolL Na2B4O7 + 60 mmolL sodium dodecyl sulfate (SDS) (pH 714) as the

running buffer a fused-silica capillary as the separation tube 21 kV as the running

voltage and UV detection at 214 nm Under these conditions more than 100-fold

enrichment of NAT was obtained with the good linear relation in the range of

nateglinide plasma concentration 02ndash7mgL (r = 0998) The method could be

applied successfully to determine trace drugs in clinical analysis

Steffen Bauer et al (2003) [63] developed a rapid and simple method for the

analysis of NAT in human plasma and validated with LOQ- 01 microgml is low

enough to allow determination of pharmacokinetic parameters of the substance

The intra-assay coefficients of variation ranged from 16 to 129 at NAT

concentrations of 05-75 microgml The inter-assay variation for the same plasma

concentrations ranged from 38 to 84 The calibration was linear in the range of

01-20 microgml To quantify NAT only 50 microl of plasma were needed Following

protein precipitation in human plasma the samples were separated by isocratic

reversed phase HPLC and analyzed using UV detection at 210 nm Sample

preparation time and analysis time are both short and allow rapid analysis of large

sample sets

318 Pioglitazone

Wanjari et al (2005) [64] developed a stability indicating simple rapid and

precise reversed-phase HPLC method for the quantitation of Pioglitazone (PIO) in

45

tablet on a Hypersil C-8 (250x46 mm) column using a mobile phase consisting

acetonitrile 015 vv triethylamine (4060 vv) adjusted to pH 46 with

orthophosphoric acid at a flow rate of 15 mlmin and detection at 220 nm The

retention time of PIO have been found to be 76 min and recoveries were between

99-101 Validation of the proposed method also been done

Zhongping John Lin et al (2003) [65] reported a LC-MSMS method for the

simultaneous determination of PIO and its two metabolites M-III (keto-derivative)

and M-IV (hydroxy-derivative) in human plasma Human plasma samples of 02

ml were extracted by a single step LLE procedure and analyzed using a HPLC-

electrospray (ESI) tandem mass spectrometer system The compounds were eluted

isocratically on a C-18 column ionized using a positive ion atmospheric pressure

electrospray ionization source and analyzed using MRM mode The ion transitions

monitored were mz 3570134 for PIO mz 3710148 for M-III mz 3730150 for

M-IV and mz 4130178 for the IS The chromatographic run time was 25 min per

injection with retention times of 145 102 and 095 min for PIO M-III and M-IV

respectively The calibration curves of PIO M-III and M-IV were well fit over the

range of 05-2000 ngml (r2 gt 0998759) by using a weighted (1x2) quadratic

regression The inter-day precisions of the quality control samples were le105

(N=15) coefficient of variation and the inter-day accuracy ( nominal) ranged

from 846 to 1035 for PIO 944 to 1040 for M-III and 968 to 1010 for M-

IV All three analytes demonstrated acceptable short-term long-term and

freezethaw stability

Radha Krishna et al (2002) [66] developed an HPLC and MEKC methods have

been developed for the determination of PIO its unsaturated impurity were

separated by MEKC in less than 7 min using a 43 cm times 50 microm id uncoated fused-

silica capillary with extended light path for better sensitivity (25 kV at 30 degC) and

a Back Ground Electrolyte (BGE) consisting of 20 acetonitrile (vv) in 20 mM

sodium borate buffer pH 93 containing 50 mM Sodium Dodecyl Sulphate (SDS)

The influence of various parameters on the separation such as pH of the buffer

46

SDS concentration buffer concentration organic modifiers temperature and

voltage were investigated The MEKC method was compared with HPLC method

using 5 microm symmetry C18 column (250times46 mm id) eluted with a mobile phase

consisting of a mixture of 50 (vv) acetonitrile and 10 mM potassium dihydrogen

phosphate buffer adjusting the pH to 60 with 01 M KOH The HPLC method is

capable of detecting all process related compounds which may be present at trace

levels in finished products

Kenji Yamashita et al (1996) [67] developed a HPLC method for the

simultaneous determination of PIO and its metabolites (M-I to M-V) in human

serum and urine The method for serum involved the solid-phase (SPE) and LLE

extraction Urine with and without enzymatic hydrolysis using β-glucuronidase

was treated with LLE The compounds in the extract were analyzed using HPLC

with UV detection at 269 nm The detection limits of PIO M-I M-II M-Ill M-IV

and M-V in serum were 001-005 microgml those in urine were 01-05 microgml and

those in urine after enzymatic hydrolysis were 03-05 microgml respectively

Zhong et al (1996) [68] reported a HPLC method for the simultaneous

determination of PIO and its potential metabolites (M-I to M-6) in human serum

The method involved a SPE of PIO its metabolites and the IS from serum using

C18 SPE columns with an elution solvent of 05 ml of acetonitrile-water (3565

vv) Separation of the eight analytes was achieved within 20 min using a reversed-

phase Zorbax RX-Cs analytical column with a mobile phase of acetonitrile-water

(4060 vv) containing acetic acid 3 mlL (apparent pH 55) An UV detector

operated at 269 nm was used with a linear response observed from 002 to 2 microgml

for these analytes except for M-4 which was best fitted with a polynomial

regression LOQ was found to be 002 microgml for PIO M-3 M-5 and M-6 004

microgml for M-2 and M-4 and 05 microgml for M-I when using a 05 ml serum sample

for extraction Intra- and inter-assay precision was le 9 and accuracy ranged from

-82 to 134 for all analytes were obtained from the method validation

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

36

pharmacokinetic or bioequivalence studies after administration of a 3 or 4 mg

tablet of GLM

Sacide Altinoz et al (2001) [46] developed a method using second order

derivative UV spectrophotometry for the quantification of GLM in

Dimethylformamide (DMF) in the wavelength range of 245ndash290 nm at n=6 The

second order derivative spectra was calculated using peak to peak (lDMF-2633ndash

2682 nm) peak to zero (lDMF-2682 nm) and tangent (lDMF-2633ndash2718 nm)

method for calibration curves the linearity range of 1-500 microgml by using the

second order derivative UV spectrophotometric method The developed method

was applied to directly and easily to the analysis of the pharmaceutical tablet

preparations RSD was found to be 418 and 221 The method was

completely validated and proven to be rugged The LOQ and LOD were found as

100 and 04 microgml respectively

315 Glipizide

Ghoneim et al (2007) [47] studied the electrochemical behavior of Glipizide

(GLP) at the Hanging Mercury Drop Electrode (HMDE) BndashR universal buffers of

pH 17ndash11 The voltammograms exhibited a well-defined 4-electron irreversible

cathodic peak which attributed to reduction of the two C N double bonds of the

pyrazine ring of GLP molecule GLP was found to have an interfacial adsorptive

character onto the mercury electrode surface A monolayer surface coverage of

102times10minus10 molcm2 was estimated and hence each adsorbed GLP molecule

occupied an area of 163 nm2 onto the mercury electrode surface A simple and

precise square-wave adsorptive cathodic stripping (SWAdCS) voltammetric

procedure was described for quantification of bulk GLP with a LOD of

15 times 10minus10 M and a LOQ of 5 times 10minus10 M

Dhawan et al (2003) [48] described the validation of a sensitive accurate and

reproducible method for the determination of a release profile of GLP from

37

controlled-release dosage forms In this method an in vitro dissolution profile of

commercial controlled-release dosage forms is determined using a reversed-phase

C18 column mobile phase (acetonitrilendashbuffer 005M KH2PO4 adjusted to pH 35

with orthophosphoric acid) and UV detection at a wavelength of 275 nm The

method is validated for linearity accuracy precision and detection and

quantification limits The same method can be exploited to determine the plasma

concentration of GLP The peak area versus plasma concentration is linear over the

range of 125ndash1000 ngml and the detection limit was 5 ngml in plasma The

average accuracy was 9990 with a RSD of not more than 3 Repeatability

and reproducibility were found to be good with an RSD of less than 3

316 Metformin

Sakalgaonkar et al (2008) [49] reported a simple rapid and precise isocratic

reversed phase chromatographic method was developed for assay of Metformin

HCl (MET) in bulk drug The chromatographic separation was achieved on Intersil

ODS 3V (250x46 mm 5micro) column using 0025 M dibasic potassium phosphate

and 0005 M hexane sulphonic acid sodium salt in 1000 ml water adjust pH-30

with phosphoric acid and ratio of buffer to acetonitrile 93 7 as a mobile phase

Forced degradation studies were performed for MET bulk drug using (Conc

H2So4) base (NaOH) oxidation (30 H2O2) Heat (60 oC) and UV light (254 and

365 nm) Degradation was not observed for MET sample during stress conditions

like UV light heat acid base and oxidation Peak purity test confirms MET peak

was homogenous in all stress conditions The mass balance of MET was close to

be 100 in all stress conditions The recovery was observed 10008 by the

formula recovery = slope x100 The MET sample solution and mobile phase

were found to be stable at least 48 hours Robustness of the analytical method was

determined by deliberately changing the analytical condition of wavelength

detection pH of mobile phase flow rate of mobile phase and percentage buffer

content So assay of MET sample obtained under each changed condition was

compared with mean value of method precision the overall relative standard

38

deviation should be below 20 The developed method was validated with

respective linearity accuracy precision system suitability specificity and forced

degradation studies

Collin et al (2004) [50] studied the antioxidant properties of Metformin HO

radical induced oxidation of MET was studied in aqueous solution in both aerated

and deaerated conditions Gamma radiolysis of water was used to generate HO

free radicals capable of initiating one-electron oxidation of MET Oxidation end-

products were identified by direct infusion mass spectrometry (MS) and high-

performance liquid chromatographymass spectrometry (HPLCMSn) for every

product structure elucidation was based on its mass (simple mass spectra

confirmed by HPLCMS) In addition fragmentation spectra (MS2 MS3 and MS4)

and the determination of deuteriumndashhydrogen exchange sites provided valuable

information allowing the complete identification of some of the end-products At

low radiation dose four products were identified as primary ones since they result

from the direct attack of HO radicals on MET These primary oxidation end-

products were identified respectively as hydroperoxide of MET covalent dimer of

MET methylbiguanide and 2-amino-4-imino-5-methyl-135-triazine At high

radiation dose seven other products were identified as secondary ones resulting

from the HO induced oxidation of the primary end-products A reaction scheme

was postulated for the interpretation of the results

Ashour et al (2003) [51] developed a simple sensitive and accurate

spectrophotometric method for the determination of MET The method is based on

the formation of Charge Transfer complex (CT) with iodine in acetonitrile

medium Beerrsquos law is obeyed within the concentration range 1656ndash7286 microgml

of MET whereas the optimum concentration range as evaluated by Ringbomrsquos

method was found to be 662ndash662 microgml The formation constant of the formed

complex is evaluated and was found to be 38 x 104 Lmol The molar

absorptivity Sandell sensitivity RSD and recovery are evaluated The results

39

are treated statistically applying the F- and t-tests and found highly accurate

precise and reproducible The method is used to determine MET in pure form and

pharmaceutical preparations There is no interference from the excipients

Zarghi et al (2003) [52] reported a rapid simple and sensitive ion-pair HPLC

method for quantification of MET in plasma The assay enables the measurement

of MET for therapeutic drug monitoring with a LOD 20 ngml The method

involves simple one-step extraction procedure and analytical recovery was

complete The separation was performed on an analytical 150 x 46 mm id

microbondapak C18 column The wavelength was set at 235 nm The mobile phase was

40 acetonitrile 001 M sodium dodecyl sulphate 001 M sodium dihydrogen

phosphate and distilled water to 100 adjusted to pH 51 at a flow rate of 15

mlmin The calibration curve was linear over the concentration range 02-25

mgml The coefficients of variation for inter-day and intra-day assay were within

the range of clinical usefulness

Ching-Ling Cheng et al (2001) [53] developed a simple selective sensitive and

precise HPLC plasma assay for MET Acidified samples of plasma were de-

proteinated with acetonitrile washed with dichloromethane and the resulting

supernatant injected Chromatography was performed at 40 oC by pumping a

mobile phase of acetonitrile (250 ml) in pH 7 003 M diammonium hydrogen

phosphate buffer (750 ml) at a flow-rate of 1 mlmin through a silica column MET

and the IS (Atenolol) were detected at 240 nm and eluted at 78 and 68 min

respectively after injection No endogenous substances were found to interfere

Calibration curves were linear (rgt0999) from 10 to 2000 ngml The absolute

recovery of both MET and IS was greater than 76 The detection limit and limit

of quantification were 25 and 10 ngml respectively The intra- and inter-day

precision (CV) was 12 or less and the accuracy was within 62 of the

nominal concentration

40

Florentin Tache et al (2001) [54] optimized a method for determination of MET

at ppb level in human plasma Derivatization of MET with p-nitrobenzoyl chloride

was carried out in a biphasic system The derivative extracted in the organic layer

(dichloromethane) was concentrated and analyzed by HPLC with diode array

detector using an isocratic elution water methanol mixture 6535 monitored at

280 nm Good selectivity against co-extracted matrix components was observed A

detection limit of 10 ppb MET in human plasma was reached

Saad SM Hassan et al (1999) [55] described a simple and convenient

potentiometric spectrofluorimetric and spectrophotometric methods for the

determination of MET in pharmaceutical preparations The potentiometric

technique is based on preparation of PVC membrane sensors incorporating

metformin plusmn reineckate and metformin plusmn tungstosilicate ion-pairs as electroactive

species with dioctylphthalate and o-nitrophenyloctylether as plasticizers

respectively A membrane consisting of carboxylated PVC plasticized with

dibutylsebacate is also prepared and tested These sensors give rapid Nernstian

response for 10-1-10-5 M MET at pH range 5-11 The metformin plusmn tungstosilicate

based sensor is used in a flow-through sandwich cell for flow injection

potentiometric determination of MET Graphite sensors coated or doped with

metforminplusmntungstosilicate-PVC Cu-diethyldithio-carbamate and Ni-

diethyldithiocarbamate are also prepared and used for monitoring the titration of

the drug with tetraphenyl borate (TPB-) Cu2+ and Ni2+ ions respectively The

spectrofluorimetric method depends on the reaction of MET with chrysenequinone

in alkaline medium to give a Schiffs base which upon hydrolysis gives the free

base The latter in the presence of 1-naphthol gives a fluorescent product with

excitation and emission maxima at 450 and 520 nm respectively The fluorescence

plusmn concentration relationship is linear over the range 20plusmn200 mgml MET The

proposed spectrophotometric technique involves reaction of MET with Cu2+ in

basic medium to form a Cu plusmn metformin complex The complex is dissolved in

cyclohexylamine and its maximum absorption at 540 nm is measured Beers law is

41

obeyed over the range 05-2 mgml MET Various cations some nitrogenous

compounds or drug excipients cause no interferences

Kah Hay Yuen et al (1998) [56] developed a simple HPLC method using UV

detection for the determination of MET in human plasma The method entailed

direct injection of the plasma sample after deproteination using perchloric acid

The mobile phase comprised 001 M potassium dihydrogen orthophosphate (pH

35) and acetonitrile (6040 vv) Analyses were run at a flow-rate of 10 ml min

with the detector operating at a detection wavelength of 234 nm The method is

specific and sensitive with a LOQ approximately 60 ngml and a LOD 15 ngml at

a Sn ratio of 31 The mean absolute recovery value was about 97 while the

within-day and between-day CV and error values of the assay method were all

less than 8 The calibration curve was linear over a concentration range of 625ndash

4000 ngml

Ole Vesterqvist et al (1998) [57] reported a rapid HPLC method for the

determination of MET in plasma Sample preparation entailed a 30-min

centrifugation of plasma through a micron filter with direct injection of the

protein-free ultra-filtrate into an HPLC system consisting of a cation-exchange

extraction column (75346 mm) a column switching valve and a cation-exchange

analytical column (250346 mm) The eluent was monitored at 232 nm MET was

well resolved at a retention time of about 5 min There was less than 2 loss of

MET during ultra-filtration and good linearity was established from 010 to 40

mgL of MET The LLOQ was about 005 mgL at which concentration the Sn

was above 10 The intra- and inter-assay CV at plasma concentrations of MET

between 025 and 25 mgL were typically 08ndash14 and 35ndash64 respectively

317 Nateglinide

Kanthi Kiran et al (2008) [58] studied in rats for drugndashdrug interaction of

Nateglinide (NAT) with Cilostazol (CLZ) an antiplatelet agent commonly used in

42

diabetics and developed a liquid chromatography tandem mass spectrometry (LCndash

MSMS) based method that is capable of simultaneous monitoring plasma levels of

NAT CLZ and its active metabolite 34-dehydro-cilostazol (DCLZ) All analytes

including the IS Repaglinide were chromatographed on reverse phase C18 column

(50 mm times 46 mm id 5 microm) using acetonitrile 2 mM ammonium acetate buffer

pH 34 (9010 vv) as mobile phase at a flow rate 04 mlmin in an isocratic mode

The detection of analyte was performed on LCndashMSMS system in the multiple

reaction monitoring (MRM) mode The quantitations for analytes were based on

relative concentration The method was validated over the concentration range of

20ndash2000 ngml and the LLOQ was 20 ngml The recoveries from spiked control

samples were gt79 for all analytes and IS Intra- and inter-day accuracy and

precision of validated method were with in the acceptable limits of lt15 at all

concentration The quantitation method was successfully applied for simultaneous

estimation of NAT CLZ and DCLZ in a pharmacokinetic drugndashdrug interaction

study in Wistar rats

Danai Malli et al (2007) [59] developed and validated a sensitive and selective

HPLC method for the determination of NAT in human plasma NAT and the IS

undecylenic acid were extracted from plasma by liquidndashliquid extraction using a

mixture of ethyl acetatendashdiethyl ether 5050 (vv) Pre-column derivatization

reaction was performed using a coumarin-type fluorescent reagent

N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide The derivati-

zation proceeded in acetone in the presence of potassium carbonate and catalyzed

by 18-crown-6 ether The fluorescent derivatives were separated under isocratic

conditions on a Hypersil BDS-C8 analytical column with a mobile phase that

consisted of 65 acetonitrile in water and pumped at a flow rate of 05 mlmin

The excitation and emission wavelengths were set at 345 and 435 nm respectively

The assay was linear over a concentration range of 005ndash16 microgml for NAT with a

LOQ 005 microgml QC samples (005 450 and 1600 microgml) in five replicates from

five different runs of analysis demonstrated intra-assay precision ( CV lt68)

43

inter-assay precision ( CV lt16) and an overall accuracy ( relative error) lt

-34

Jolly M Sankalia et al (2007) [60] reported a rapid simple and sensitive HPLC

method with UV detection for the quantification of NAT from rabbit plasma The

retention behavior of NAT and GLZ (IS) as a function of mobile phase pH

composition and flow rate was investigated Separation was developed on a

reverse-phase C18 column (250 times 46 mm id 5 microm) using a mixture of

acetonitrile 10 mM phosphate buffer (pH 30) in the ratio of 7030 ( vv) at a

flow rate of 10 mlmin with UV detection at 203 nm within 8 min and quantified

based on drugIS peak area ratios The plasma samples were prepared by a simple

deproteinization with a mixture of methanol and acetonitrile yielding more than

9786 extraction efficiencies The calibration curve was linear (r2 = 09984) in

the concentration range of 10ndash2500 ngml The LOD and LOQ were found to be

291 and 970 ngml respectively Both the intra-day and inter-day precisions at

four tested concentrations were below 132 RSD

Junfa Yin et al (2005) [61] reported a rapid and efficient chiral separation

method of NAT and its L-enantiomer on a monolithic Molecularly Imprinted

Polymers (MIP) The enantiomers were rapidly separated on this novel monolithic

MIP based Chiral Stationary Phase (MIP-CSP) whereas the enantioseparation was

not obtained on the non-imprinted polymer (NIP) Chiral recognition was found to

be dependent on the stereo structures and the arrangement of functional groups of

the imprinted molecule and the cavities on MIP Thermodynamic data (∆∆H and

∆∆S) obtained by Vanrsquot Hoff plots revealed an enthalpy-controlled

enantioseparation The binding capacity was evaluated by frontal analysis

Monolithic NAT-MIP had an effective number of binding sites Bt = 4115 micromolg

with a dissociation constant of Kd = 740 mM The morphological characteristics of

the monolithic MIP were investigated by pore analysis and Scanning Electron

Microscope (SEM) Results showed that both mesopores and macropores were

formed in the monolith Over all this study presents a new and practical possibility

44

for providing high rates of mass transfer fast separations and high efficiencies

without the pressure constraints of the traditional bulk molecularly imprinted

polymers through the monolithic MIPs

Hongyuan Yan et al (2004) [62] developed a Micellar Electro Kinetic

Chromatography (MEKC) method for the determination of NAT in animal plasma

by on-line sweeping technique in which plasma samples were simply

deproteinized with acetonitrile and analyzed with 16 mmolL NaH2PO4 + 6

mmolL Na2B4O7 + 60 mmolL sodium dodecyl sulfate (SDS) (pH 714) as the

running buffer a fused-silica capillary as the separation tube 21 kV as the running

voltage and UV detection at 214 nm Under these conditions more than 100-fold

enrichment of NAT was obtained with the good linear relation in the range of

nateglinide plasma concentration 02ndash7mgL (r = 0998) The method could be

applied successfully to determine trace drugs in clinical analysis

Steffen Bauer et al (2003) [63] developed a rapid and simple method for the

analysis of NAT in human plasma and validated with LOQ- 01 microgml is low

enough to allow determination of pharmacokinetic parameters of the substance

The intra-assay coefficients of variation ranged from 16 to 129 at NAT

concentrations of 05-75 microgml The inter-assay variation for the same plasma

concentrations ranged from 38 to 84 The calibration was linear in the range of

01-20 microgml To quantify NAT only 50 microl of plasma were needed Following

protein precipitation in human plasma the samples were separated by isocratic

reversed phase HPLC and analyzed using UV detection at 210 nm Sample

preparation time and analysis time are both short and allow rapid analysis of large

sample sets

318 Pioglitazone

Wanjari et al (2005) [64] developed a stability indicating simple rapid and

precise reversed-phase HPLC method for the quantitation of Pioglitazone (PIO) in

45

tablet on a Hypersil C-8 (250x46 mm) column using a mobile phase consisting

acetonitrile 015 vv triethylamine (4060 vv) adjusted to pH 46 with

orthophosphoric acid at a flow rate of 15 mlmin and detection at 220 nm The

retention time of PIO have been found to be 76 min and recoveries were between

99-101 Validation of the proposed method also been done

Zhongping John Lin et al (2003) [65] reported a LC-MSMS method for the

simultaneous determination of PIO and its two metabolites M-III (keto-derivative)

and M-IV (hydroxy-derivative) in human plasma Human plasma samples of 02

ml were extracted by a single step LLE procedure and analyzed using a HPLC-

electrospray (ESI) tandem mass spectrometer system The compounds were eluted

isocratically on a C-18 column ionized using a positive ion atmospheric pressure

electrospray ionization source and analyzed using MRM mode The ion transitions

monitored were mz 3570134 for PIO mz 3710148 for M-III mz 3730150 for

M-IV and mz 4130178 for the IS The chromatographic run time was 25 min per

injection with retention times of 145 102 and 095 min for PIO M-III and M-IV

respectively The calibration curves of PIO M-III and M-IV were well fit over the

range of 05-2000 ngml (r2 gt 0998759) by using a weighted (1x2) quadratic

regression The inter-day precisions of the quality control samples were le105

(N=15) coefficient of variation and the inter-day accuracy ( nominal) ranged

from 846 to 1035 for PIO 944 to 1040 for M-III and 968 to 1010 for M-

IV All three analytes demonstrated acceptable short-term long-term and

freezethaw stability

Radha Krishna et al (2002) [66] developed an HPLC and MEKC methods have

been developed for the determination of PIO its unsaturated impurity were

separated by MEKC in less than 7 min using a 43 cm times 50 microm id uncoated fused-

silica capillary with extended light path for better sensitivity (25 kV at 30 degC) and

a Back Ground Electrolyte (BGE) consisting of 20 acetonitrile (vv) in 20 mM

sodium borate buffer pH 93 containing 50 mM Sodium Dodecyl Sulphate (SDS)

The influence of various parameters on the separation such as pH of the buffer

46

SDS concentration buffer concentration organic modifiers temperature and

voltage were investigated The MEKC method was compared with HPLC method

using 5 microm symmetry C18 column (250times46 mm id) eluted with a mobile phase

consisting of a mixture of 50 (vv) acetonitrile and 10 mM potassium dihydrogen

phosphate buffer adjusting the pH to 60 with 01 M KOH The HPLC method is

capable of detecting all process related compounds which may be present at trace

levels in finished products

Kenji Yamashita et al (1996) [67] developed a HPLC method for the

simultaneous determination of PIO and its metabolites (M-I to M-V) in human

serum and urine The method for serum involved the solid-phase (SPE) and LLE

extraction Urine with and without enzymatic hydrolysis using β-glucuronidase

was treated with LLE The compounds in the extract were analyzed using HPLC

with UV detection at 269 nm The detection limits of PIO M-I M-II M-Ill M-IV

and M-V in serum were 001-005 microgml those in urine were 01-05 microgml and

those in urine after enzymatic hydrolysis were 03-05 microgml respectively

Zhong et al (1996) [68] reported a HPLC method for the simultaneous

determination of PIO and its potential metabolites (M-I to M-6) in human serum

The method involved a SPE of PIO its metabolites and the IS from serum using

C18 SPE columns with an elution solvent of 05 ml of acetonitrile-water (3565

vv) Separation of the eight analytes was achieved within 20 min using a reversed-

phase Zorbax RX-Cs analytical column with a mobile phase of acetonitrile-water

(4060 vv) containing acetic acid 3 mlL (apparent pH 55) An UV detector

operated at 269 nm was used with a linear response observed from 002 to 2 microgml

for these analytes except for M-4 which was best fitted with a polynomial

regression LOQ was found to be 002 microgml for PIO M-3 M-5 and M-6 004

microgml for M-2 and M-4 and 05 microgml for M-I when using a 05 ml serum sample

for extraction Intra- and inter-assay precision was le 9 and accuracy ranged from

-82 to 134 for all analytes were obtained from the method validation

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

37

controlled-release dosage forms In this method an in vitro dissolution profile of

commercial controlled-release dosage forms is determined using a reversed-phase

C18 column mobile phase (acetonitrilendashbuffer 005M KH2PO4 adjusted to pH 35

with orthophosphoric acid) and UV detection at a wavelength of 275 nm The

method is validated for linearity accuracy precision and detection and

quantification limits The same method can be exploited to determine the plasma

concentration of GLP The peak area versus plasma concentration is linear over the

range of 125ndash1000 ngml and the detection limit was 5 ngml in plasma The

average accuracy was 9990 with a RSD of not more than 3 Repeatability

and reproducibility were found to be good with an RSD of less than 3

316 Metformin

Sakalgaonkar et al (2008) [49] reported a simple rapid and precise isocratic

reversed phase chromatographic method was developed for assay of Metformin

HCl (MET) in bulk drug The chromatographic separation was achieved on Intersil

ODS 3V (250x46 mm 5micro) column using 0025 M dibasic potassium phosphate

and 0005 M hexane sulphonic acid sodium salt in 1000 ml water adjust pH-30

with phosphoric acid and ratio of buffer to acetonitrile 93 7 as a mobile phase

Forced degradation studies were performed for MET bulk drug using (Conc

H2So4) base (NaOH) oxidation (30 H2O2) Heat (60 oC) and UV light (254 and

365 nm) Degradation was not observed for MET sample during stress conditions

like UV light heat acid base and oxidation Peak purity test confirms MET peak

was homogenous in all stress conditions The mass balance of MET was close to

be 100 in all stress conditions The recovery was observed 10008 by the

formula recovery = slope x100 The MET sample solution and mobile phase

were found to be stable at least 48 hours Robustness of the analytical method was

determined by deliberately changing the analytical condition of wavelength

detection pH of mobile phase flow rate of mobile phase and percentage buffer

content So assay of MET sample obtained under each changed condition was

compared with mean value of method precision the overall relative standard

38

deviation should be below 20 The developed method was validated with

respective linearity accuracy precision system suitability specificity and forced

degradation studies

Collin et al (2004) [50] studied the antioxidant properties of Metformin HO

radical induced oxidation of MET was studied in aqueous solution in both aerated

and deaerated conditions Gamma radiolysis of water was used to generate HO

free radicals capable of initiating one-electron oxidation of MET Oxidation end-

products were identified by direct infusion mass spectrometry (MS) and high-

performance liquid chromatographymass spectrometry (HPLCMSn) for every

product structure elucidation was based on its mass (simple mass spectra

confirmed by HPLCMS) In addition fragmentation spectra (MS2 MS3 and MS4)

and the determination of deuteriumndashhydrogen exchange sites provided valuable

information allowing the complete identification of some of the end-products At

low radiation dose four products were identified as primary ones since they result

from the direct attack of HO radicals on MET These primary oxidation end-

products were identified respectively as hydroperoxide of MET covalent dimer of

MET methylbiguanide and 2-amino-4-imino-5-methyl-135-triazine At high

radiation dose seven other products were identified as secondary ones resulting

from the HO induced oxidation of the primary end-products A reaction scheme

was postulated for the interpretation of the results

Ashour et al (2003) [51] developed a simple sensitive and accurate

spectrophotometric method for the determination of MET The method is based on

the formation of Charge Transfer complex (CT) with iodine in acetonitrile

medium Beerrsquos law is obeyed within the concentration range 1656ndash7286 microgml

of MET whereas the optimum concentration range as evaluated by Ringbomrsquos

method was found to be 662ndash662 microgml The formation constant of the formed

complex is evaluated and was found to be 38 x 104 Lmol The molar

absorptivity Sandell sensitivity RSD and recovery are evaluated The results

39

are treated statistically applying the F- and t-tests and found highly accurate

precise and reproducible The method is used to determine MET in pure form and

pharmaceutical preparations There is no interference from the excipients

Zarghi et al (2003) [52] reported a rapid simple and sensitive ion-pair HPLC

method for quantification of MET in plasma The assay enables the measurement

of MET for therapeutic drug monitoring with a LOD 20 ngml The method

involves simple one-step extraction procedure and analytical recovery was

complete The separation was performed on an analytical 150 x 46 mm id

microbondapak C18 column The wavelength was set at 235 nm The mobile phase was

40 acetonitrile 001 M sodium dodecyl sulphate 001 M sodium dihydrogen

phosphate and distilled water to 100 adjusted to pH 51 at a flow rate of 15

mlmin The calibration curve was linear over the concentration range 02-25

mgml The coefficients of variation for inter-day and intra-day assay were within

the range of clinical usefulness

Ching-Ling Cheng et al (2001) [53] developed a simple selective sensitive and

precise HPLC plasma assay for MET Acidified samples of plasma were de-

proteinated with acetonitrile washed with dichloromethane and the resulting

supernatant injected Chromatography was performed at 40 oC by pumping a

mobile phase of acetonitrile (250 ml) in pH 7 003 M diammonium hydrogen

phosphate buffer (750 ml) at a flow-rate of 1 mlmin through a silica column MET

and the IS (Atenolol) were detected at 240 nm and eluted at 78 and 68 min

respectively after injection No endogenous substances were found to interfere

Calibration curves were linear (rgt0999) from 10 to 2000 ngml The absolute

recovery of both MET and IS was greater than 76 The detection limit and limit

of quantification were 25 and 10 ngml respectively The intra- and inter-day

precision (CV) was 12 or less and the accuracy was within 62 of the

nominal concentration

40

Florentin Tache et al (2001) [54] optimized a method for determination of MET

at ppb level in human plasma Derivatization of MET with p-nitrobenzoyl chloride

was carried out in a biphasic system The derivative extracted in the organic layer

(dichloromethane) was concentrated and analyzed by HPLC with diode array

detector using an isocratic elution water methanol mixture 6535 monitored at

280 nm Good selectivity against co-extracted matrix components was observed A

detection limit of 10 ppb MET in human plasma was reached

Saad SM Hassan et al (1999) [55] described a simple and convenient

potentiometric spectrofluorimetric and spectrophotometric methods for the

determination of MET in pharmaceutical preparations The potentiometric

technique is based on preparation of PVC membrane sensors incorporating

metformin plusmn reineckate and metformin plusmn tungstosilicate ion-pairs as electroactive

species with dioctylphthalate and o-nitrophenyloctylether as plasticizers

respectively A membrane consisting of carboxylated PVC plasticized with

dibutylsebacate is also prepared and tested These sensors give rapid Nernstian

response for 10-1-10-5 M MET at pH range 5-11 The metformin plusmn tungstosilicate

based sensor is used in a flow-through sandwich cell for flow injection

potentiometric determination of MET Graphite sensors coated or doped with

metforminplusmntungstosilicate-PVC Cu-diethyldithio-carbamate and Ni-

diethyldithiocarbamate are also prepared and used for monitoring the titration of

the drug with tetraphenyl borate (TPB-) Cu2+ and Ni2+ ions respectively The

spectrofluorimetric method depends on the reaction of MET with chrysenequinone

in alkaline medium to give a Schiffs base which upon hydrolysis gives the free

base The latter in the presence of 1-naphthol gives a fluorescent product with

excitation and emission maxima at 450 and 520 nm respectively The fluorescence

plusmn concentration relationship is linear over the range 20plusmn200 mgml MET The

proposed spectrophotometric technique involves reaction of MET with Cu2+ in

basic medium to form a Cu plusmn metformin complex The complex is dissolved in

cyclohexylamine and its maximum absorption at 540 nm is measured Beers law is

41

obeyed over the range 05-2 mgml MET Various cations some nitrogenous

compounds or drug excipients cause no interferences

Kah Hay Yuen et al (1998) [56] developed a simple HPLC method using UV

detection for the determination of MET in human plasma The method entailed

direct injection of the plasma sample after deproteination using perchloric acid

The mobile phase comprised 001 M potassium dihydrogen orthophosphate (pH

35) and acetonitrile (6040 vv) Analyses were run at a flow-rate of 10 ml min

with the detector operating at a detection wavelength of 234 nm The method is

specific and sensitive with a LOQ approximately 60 ngml and a LOD 15 ngml at

a Sn ratio of 31 The mean absolute recovery value was about 97 while the

within-day and between-day CV and error values of the assay method were all

less than 8 The calibration curve was linear over a concentration range of 625ndash

4000 ngml

Ole Vesterqvist et al (1998) [57] reported a rapid HPLC method for the

determination of MET in plasma Sample preparation entailed a 30-min

centrifugation of plasma through a micron filter with direct injection of the

protein-free ultra-filtrate into an HPLC system consisting of a cation-exchange

extraction column (75346 mm) a column switching valve and a cation-exchange

analytical column (250346 mm) The eluent was monitored at 232 nm MET was

well resolved at a retention time of about 5 min There was less than 2 loss of

MET during ultra-filtration and good linearity was established from 010 to 40

mgL of MET The LLOQ was about 005 mgL at which concentration the Sn

was above 10 The intra- and inter-assay CV at plasma concentrations of MET

between 025 and 25 mgL were typically 08ndash14 and 35ndash64 respectively

317 Nateglinide

Kanthi Kiran et al (2008) [58] studied in rats for drugndashdrug interaction of

Nateglinide (NAT) with Cilostazol (CLZ) an antiplatelet agent commonly used in

42

diabetics and developed a liquid chromatography tandem mass spectrometry (LCndash

MSMS) based method that is capable of simultaneous monitoring plasma levels of

NAT CLZ and its active metabolite 34-dehydro-cilostazol (DCLZ) All analytes

including the IS Repaglinide were chromatographed on reverse phase C18 column

(50 mm times 46 mm id 5 microm) using acetonitrile 2 mM ammonium acetate buffer

pH 34 (9010 vv) as mobile phase at a flow rate 04 mlmin in an isocratic mode

The detection of analyte was performed on LCndashMSMS system in the multiple

reaction monitoring (MRM) mode The quantitations for analytes were based on

relative concentration The method was validated over the concentration range of

20ndash2000 ngml and the LLOQ was 20 ngml The recoveries from spiked control

samples were gt79 for all analytes and IS Intra- and inter-day accuracy and

precision of validated method were with in the acceptable limits of lt15 at all

concentration The quantitation method was successfully applied for simultaneous

estimation of NAT CLZ and DCLZ in a pharmacokinetic drugndashdrug interaction

study in Wistar rats

Danai Malli et al (2007) [59] developed and validated a sensitive and selective

HPLC method for the determination of NAT in human plasma NAT and the IS

undecylenic acid were extracted from plasma by liquidndashliquid extraction using a

mixture of ethyl acetatendashdiethyl ether 5050 (vv) Pre-column derivatization

reaction was performed using a coumarin-type fluorescent reagent

N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide The derivati-

zation proceeded in acetone in the presence of potassium carbonate and catalyzed

by 18-crown-6 ether The fluorescent derivatives were separated under isocratic

conditions on a Hypersil BDS-C8 analytical column with a mobile phase that

consisted of 65 acetonitrile in water and pumped at a flow rate of 05 mlmin

The excitation and emission wavelengths were set at 345 and 435 nm respectively

The assay was linear over a concentration range of 005ndash16 microgml for NAT with a

LOQ 005 microgml QC samples (005 450 and 1600 microgml) in five replicates from

five different runs of analysis demonstrated intra-assay precision ( CV lt68)

43

inter-assay precision ( CV lt16) and an overall accuracy ( relative error) lt

-34

Jolly M Sankalia et al (2007) [60] reported a rapid simple and sensitive HPLC

method with UV detection for the quantification of NAT from rabbit plasma The

retention behavior of NAT and GLZ (IS) as a function of mobile phase pH

composition and flow rate was investigated Separation was developed on a

reverse-phase C18 column (250 times 46 mm id 5 microm) using a mixture of

acetonitrile 10 mM phosphate buffer (pH 30) in the ratio of 7030 ( vv) at a

flow rate of 10 mlmin with UV detection at 203 nm within 8 min and quantified

based on drugIS peak area ratios The plasma samples were prepared by a simple

deproteinization with a mixture of methanol and acetonitrile yielding more than

9786 extraction efficiencies The calibration curve was linear (r2 = 09984) in

the concentration range of 10ndash2500 ngml The LOD and LOQ were found to be

291 and 970 ngml respectively Both the intra-day and inter-day precisions at

four tested concentrations were below 132 RSD

Junfa Yin et al (2005) [61] reported a rapid and efficient chiral separation

method of NAT and its L-enantiomer on a monolithic Molecularly Imprinted

Polymers (MIP) The enantiomers were rapidly separated on this novel monolithic

MIP based Chiral Stationary Phase (MIP-CSP) whereas the enantioseparation was

not obtained on the non-imprinted polymer (NIP) Chiral recognition was found to

be dependent on the stereo structures and the arrangement of functional groups of

the imprinted molecule and the cavities on MIP Thermodynamic data (∆∆H and

∆∆S) obtained by Vanrsquot Hoff plots revealed an enthalpy-controlled

enantioseparation The binding capacity was evaluated by frontal analysis

Monolithic NAT-MIP had an effective number of binding sites Bt = 4115 micromolg

with a dissociation constant of Kd = 740 mM The morphological characteristics of

the monolithic MIP were investigated by pore analysis and Scanning Electron

Microscope (SEM) Results showed that both mesopores and macropores were

formed in the monolith Over all this study presents a new and practical possibility

44

for providing high rates of mass transfer fast separations and high efficiencies

without the pressure constraints of the traditional bulk molecularly imprinted

polymers through the monolithic MIPs

Hongyuan Yan et al (2004) [62] developed a Micellar Electro Kinetic

Chromatography (MEKC) method for the determination of NAT in animal plasma

by on-line sweeping technique in which plasma samples were simply

deproteinized with acetonitrile and analyzed with 16 mmolL NaH2PO4 + 6

mmolL Na2B4O7 + 60 mmolL sodium dodecyl sulfate (SDS) (pH 714) as the

running buffer a fused-silica capillary as the separation tube 21 kV as the running

voltage and UV detection at 214 nm Under these conditions more than 100-fold

enrichment of NAT was obtained with the good linear relation in the range of

nateglinide plasma concentration 02ndash7mgL (r = 0998) The method could be

applied successfully to determine trace drugs in clinical analysis

Steffen Bauer et al (2003) [63] developed a rapid and simple method for the

analysis of NAT in human plasma and validated with LOQ- 01 microgml is low

enough to allow determination of pharmacokinetic parameters of the substance

The intra-assay coefficients of variation ranged from 16 to 129 at NAT

concentrations of 05-75 microgml The inter-assay variation for the same plasma

concentrations ranged from 38 to 84 The calibration was linear in the range of

01-20 microgml To quantify NAT only 50 microl of plasma were needed Following

protein precipitation in human plasma the samples were separated by isocratic

reversed phase HPLC and analyzed using UV detection at 210 nm Sample

preparation time and analysis time are both short and allow rapid analysis of large

sample sets

318 Pioglitazone

Wanjari et al (2005) [64] developed a stability indicating simple rapid and

precise reversed-phase HPLC method for the quantitation of Pioglitazone (PIO) in

45

tablet on a Hypersil C-8 (250x46 mm) column using a mobile phase consisting

acetonitrile 015 vv triethylamine (4060 vv) adjusted to pH 46 with

orthophosphoric acid at a flow rate of 15 mlmin and detection at 220 nm The

retention time of PIO have been found to be 76 min and recoveries were between

99-101 Validation of the proposed method also been done

Zhongping John Lin et al (2003) [65] reported a LC-MSMS method for the

simultaneous determination of PIO and its two metabolites M-III (keto-derivative)

and M-IV (hydroxy-derivative) in human plasma Human plasma samples of 02

ml were extracted by a single step LLE procedure and analyzed using a HPLC-

electrospray (ESI) tandem mass spectrometer system The compounds were eluted

isocratically on a C-18 column ionized using a positive ion atmospheric pressure

electrospray ionization source and analyzed using MRM mode The ion transitions

monitored were mz 3570134 for PIO mz 3710148 for M-III mz 3730150 for

M-IV and mz 4130178 for the IS The chromatographic run time was 25 min per

injection with retention times of 145 102 and 095 min for PIO M-III and M-IV

respectively The calibration curves of PIO M-III and M-IV were well fit over the

range of 05-2000 ngml (r2 gt 0998759) by using a weighted (1x2) quadratic

regression The inter-day precisions of the quality control samples were le105

(N=15) coefficient of variation and the inter-day accuracy ( nominal) ranged

from 846 to 1035 for PIO 944 to 1040 for M-III and 968 to 1010 for M-

IV All three analytes demonstrated acceptable short-term long-term and

freezethaw stability

Radha Krishna et al (2002) [66] developed an HPLC and MEKC methods have

been developed for the determination of PIO its unsaturated impurity were

separated by MEKC in less than 7 min using a 43 cm times 50 microm id uncoated fused-

silica capillary with extended light path for better sensitivity (25 kV at 30 degC) and

a Back Ground Electrolyte (BGE) consisting of 20 acetonitrile (vv) in 20 mM

sodium borate buffer pH 93 containing 50 mM Sodium Dodecyl Sulphate (SDS)

The influence of various parameters on the separation such as pH of the buffer

46

SDS concentration buffer concentration organic modifiers temperature and

voltage were investigated The MEKC method was compared with HPLC method

using 5 microm symmetry C18 column (250times46 mm id) eluted with a mobile phase

consisting of a mixture of 50 (vv) acetonitrile and 10 mM potassium dihydrogen

phosphate buffer adjusting the pH to 60 with 01 M KOH The HPLC method is

capable of detecting all process related compounds which may be present at trace

levels in finished products

Kenji Yamashita et al (1996) [67] developed a HPLC method for the

simultaneous determination of PIO and its metabolites (M-I to M-V) in human

serum and urine The method for serum involved the solid-phase (SPE) and LLE

extraction Urine with and without enzymatic hydrolysis using β-glucuronidase

was treated with LLE The compounds in the extract were analyzed using HPLC

with UV detection at 269 nm The detection limits of PIO M-I M-II M-Ill M-IV

and M-V in serum were 001-005 microgml those in urine were 01-05 microgml and

those in urine after enzymatic hydrolysis were 03-05 microgml respectively

Zhong et al (1996) [68] reported a HPLC method for the simultaneous

determination of PIO and its potential metabolites (M-I to M-6) in human serum

The method involved a SPE of PIO its metabolites and the IS from serum using

C18 SPE columns with an elution solvent of 05 ml of acetonitrile-water (3565

vv) Separation of the eight analytes was achieved within 20 min using a reversed-

phase Zorbax RX-Cs analytical column with a mobile phase of acetonitrile-water

(4060 vv) containing acetic acid 3 mlL (apparent pH 55) An UV detector

operated at 269 nm was used with a linear response observed from 002 to 2 microgml

for these analytes except for M-4 which was best fitted with a polynomial

regression LOQ was found to be 002 microgml for PIO M-3 M-5 and M-6 004

microgml for M-2 and M-4 and 05 microgml for M-I when using a 05 ml serum sample

for extraction Intra- and inter-assay precision was le 9 and accuracy ranged from

-82 to 134 for all analytes were obtained from the method validation

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

38

deviation should be below 20 The developed method was validated with

respective linearity accuracy precision system suitability specificity and forced

degradation studies

Collin et al (2004) [50] studied the antioxidant properties of Metformin HO

radical induced oxidation of MET was studied in aqueous solution in both aerated

and deaerated conditions Gamma radiolysis of water was used to generate HO

free radicals capable of initiating one-electron oxidation of MET Oxidation end-

products were identified by direct infusion mass spectrometry (MS) and high-

performance liquid chromatographymass spectrometry (HPLCMSn) for every

product structure elucidation was based on its mass (simple mass spectra

confirmed by HPLCMS) In addition fragmentation spectra (MS2 MS3 and MS4)

and the determination of deuteriumndashhydrogen exchange sites provided valuable

information allowing the complete identification of some of the end-products At

low radiation dose four products were identified as primary ones since they result

from the direct attack of HO radicals on MET These primary oxidation end-

products were identified respectively as hydroperoxide of MET covalent dimer of

MET methylbiguanide and 2-amino-4-imino-5-methyl-135-triazine At high

radiation dose seven other products were identified as secondary ones resulting

from the HO induced oxidation of the primary end-products A reaction scheme

was postulated for the interpretation of the results

Ashour et al (2003) [51] developed a simple sensitive and accurate

spectrophotometric method for the determination of MET The method is based on

the formation of Charge Transfer complex (CT) with iodine in acetonitrile

medium Beerrsquos law is obeyed within the concentration range 1656ndash7286 microgml

of MET whereas the optimum concentration range as evaluated by Ringbomrsquos

method was found to be 662ndash662 microgml The formation constant of the formed

complex is evaluated and was found to be 38 x 104 Lmol The molar

absorptivity Sandell sensitivity RSD and recovery are evaluated The results

39

are treated statistically applying the F- and t-tests and found highly accurate

precise and reproducible The method is used to determine MET in pure form and

pharmaceutical preparations There is no interference from the excipients

Zarghi et al (2003) [52] reported a rapid simple and sensitive ion-pair HPLC

method for quantification of MET in plasma The assay enables the measurement

of MET for therapeutic drug monitoring with a LOD 20 ngml The method

involves simple one-step extraction procedure and analytical recovery was

complete The separation was performed on an analytical 150 x 46 mm id

microbondapak C18 column The wavelength was set at 235 nm The mobile phase was

40 acetonitrile 001 M sodium dodecyl sulphate 001 M sodium dihydrogen

phosphate and distilled water to 100 adjusted to pH 51 at a flow rate of 15

mlmin The calibration curve was linear over the concentration range 02-25

mgml The coefficients of variation for inter-day and intra-day assay were within

the range of clinical usefulness

Ching-Ling Cheng et al (2001) [53] developed a simple selective sensitive and

precise HPLC plasma assay for MET Acidified samples of plasma were de-

proteinated with acetonitrile washed with dichloromethane and the resulting

supernatant injected Chromatography was performed at 40 oC by pumping a

mobile phase of acetonitrile (250 ml) in pH 7 003 M diammonium hydrogen

phosphate buffer (750 ml) at a flow-rate of 1 mlmin through a silica column MET

and the IS (Atenolol) were detected at 240 nm and eluted at 78 and 68 min

respectively after injection No endogenous substances were found to interfere

Calibration curves were linear (rgt0999) from 10 to 2000 ngml The absolute

recovery of both MET and IS was greater than 76 The detection limit and limit

of quantification were 25 and 10 ngml respectively The intra- and inter-day

precision (CV) was 12 or less and the accuracy was within 62 of the

nominal concentration

40

Florentin Tache et al (2001) [54] optimized a method for determination of MET

at ppb level in human plasma Derivatization of MET with p-nitrobenzoyl chloride

was carried out in a biphasic system The derivative extracted in the organic layer

(dichloromethane) was concentrated and analyzed by HPLC with diode array

detector using an isocratic elution water methanol mixture 6535 monitored at

280 nm Good selectivity against co-extracted matrix components was observed A

detection limit of 10 ppb MET in human plasma was reached

Saad SM Hassan et al (1999) [55] described a simple and convenient

potentiometric spectrofluorimetric and spectrophotometric methods for the

determination of MET in pharmaceutical preparations The potentiometric

technique is based on preparation of PVC membrane sensors incorporating

metformin plusmn reineckate and metformin plusmn tungstosilicate ion-pairs as electroactive

species with dioctylphthalate and o-nitrophenyloctylether as plasticizers

respectively A membrane consisting of carboxylated PVC plasticized with

dibutylsebacate is also prepared and tested These sensors give rapid Nernstian

response for 10-1-10-5 M MET at pH range 5-11 The metformin plusmn tungstosilicate

based sensor is used in a flow-through sandwich cell for flow injection

potentiometric determination of MET Graphite sensors coated or doped with

metforminplusmntungstosilicate-PVC Cu-diethyldithio-carbamate and Ni-

diethyldithiocarbamate are also prepared and used for monitoring the titration of

the drug with tetraphenyl borate (TPB-) Cu2+ and Ni2+ ions respectively The

spectrofluorimetric method depends on the reaction of MET with chrysenequinone

in alkaline medium to give a Schiffs base which upon hydrolysis gives the free

base The latter in the presence of 1-naphthol gives a fluorescent product with

excitation and emission maxima at 450 and 520 nm respectively The fluorescence

plusmn concentration relationship is linear over the range 20plusmn200 mgml MET The

proposed spectrophotometric technique involves reaction of MET with Cu2+ in

basic medium to form a Cu plusmn metformin complex The complex is dissolved in

cyclohexylamine and its maximum absorption at 540 nm is measured Beers law is

41

obeyed over the range 05-2 mgml MET Various cations some nitrogenous

compounds or drug excipients cause no interferences

Kah Hay Yuen et al (1998) [56] developed a simple HPLC method using UV

detection for the determination of MET in human plasma The method entailed

direct injection of the plasma sample after deproteination using perchloric acid

The mobile phase comprised 001 M potassium dihydrogen orthophosphate (pH

35) and acetonitrile (6040 vv) Analyses were run at a flow-rate of 10 ml min

with the detector operating at a detection wavelength of 234 nm The method is

specific and sensitive with a LOQ approximately 60 ngml and a LOD 15 ngml at

a Sn ratio of 31 The mean absolute recovery value was about 97 while the

within-day and between-day CV and error values of the assay method were all

less than 8 The calibration curve was linear over a concentration range of 625ndash

4000 ngml

Ole Vesterqvist et al (1998) [57] reported a rapid HPLC method for the

determination of MET in plasma Sample preparation entailed a 30-min

centrifugation of plasma through a micron filter with direct injection of the

protein-free ultra-filtrate into an HPLC system consisting of a cation-exchange

extraction column (75346 mm) a column switching valve and a cation-exchange

analytical column (250346 mm) The eluent was monitored at 232 nm MET was

well resolved at a retention time of about 5 min There was less than 2 loss of

MET during ultra-filtration and good linearity was established from 010 to 40

mgL of MET The LLOQ was about 005 mgL at which concentration the Sn

was above 10 The intra- and inter-assay CV at plasma concentrations of MET

between 025 and 25 mgL were typically 08ndash14 and 35ndash64 respectively

317 Nateglinide

Kanthi Kiran et al (2008) [58] studied in rats for drugndashdrug interaction of

Nateglinide (NAT) with Cilostazol (CLZ) an antiplatelet agent commonly used in

42

diabetics and developed a liquid chromatography tandem mass spectrometry (LCndash

MSMS) based method that is capable of simultaneous monitoring plasma levels of

NAT CLZ and its active metabolite 34-dehydro-cilostazol (DCLZ) All analytes

including the IS Repaglinide were chromatographed on reverse phase C18 column

(50 mm times 46 mm id 5 microm) using acetonitrile 2 mM ammonium acetate buffer

pH 34 (9010 vv) as mobile phase at a flow rate 04 mlmin in an isocratic mode

The detection of analyte was performed on LCndashMSMS system in the multiple

reaction monitoring (MRM) mode The quantitations for analytes were based on

relative concentration The method was validated over the concentration range of

20ndash2000 ngml and the LLOQ was 20 ngml The recoveries from spiked control

samples were gt79 for all analytes and IS Intra- and inter-day accuracy and

precision of validated method were with in the acceptable limits of lt15 at all

concentration The quantitation method was successfully applied for simultaneous

estimation of NAT CLZ and DCLZ in a pharmacokinetic drugndashdrug interaction

study in Wistar rats

Danai Malli et al (2007) [59] developed and validated a sensitive and selective

HPLC method for the determination of NAT in human plasma NAT and the IS

undecylenic acid were extracted from plasma by liquidndashliquid extraction using a

mixture of ethyl acetatendashdiethyl ether 5050 (vv) Pre-column derivatization

reaction was performed using a coumarin-type fluorescent reagent

N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide The derivati-

zation proceeded in acetone in the presence of potassium carbonate and catalyzed

by 18-crown-6 ether The fluorescent derivatives were separated under isocratic

conditions on a Hypersil BDS-C8 analytical column with a mobile phase that

consisted of 65 acetonitrile in water and pumped at a flow rate of 05 mlmin

The excitation and emission wavelengths were set at 345 and 435 nm respectively

The assay was linear over a concentration range of 005ndash16 microgml for NAT with a

LOQ 005 microgml QC samples (005 450 and 1600 microgml) in five replicates from

five different runs of analysis demonstrated intra-assay precision ( CV lt68)

43

inter-assay precision ( CV lt16) and an overall accuracy ( relative error) lt

-34

Jolly M Sankalia et al (2007) [60] reported a rapid simple and sensitive HPLC

method with UV detection for the quantification of NAT from rabbit plasma The

retention behavior of NAT and GLZ (IS) as a function of mobile phase pH

composition and flow rate was investigated Separation was developed on a

reverse-phase C18 column (250 times 46 mm id 5 microm) using a mixture of

acetonitrile 10 mM phosphate buffer (pH 30) in the ratio of 7030 ( vv) at a

flow rate of 10 mlmin with UV detection at 203 nm within 8 min and quantified

based on drugIS peak area ratios The plasma samples were prepared by a simple

deproteinization with a mixture of methanol and acetonitrile yielding more than

9786 extraction efficiencies The calibration curve was linear (r2 = 09984) in

the concentration range of 10ndash2500 ngml The LOD and LOQ were found to be

291 and 970 ngml respectively Both the intra-day and inter-day precisions at

four tested concentrations were below 132 RSD

Junfa Yin et al (2005) [61] reported a rapid and efficient chiral separation

method of NAT and its L-enantiomer on a monolithic Molecularly Imprinted

Polymers (MIP) The enantiomers were rapidly separated on this novel monolithic

MIP based Chiral Stationary Phase (MIP-CSP) whereas the enantioseparation was

not obtained on the non-imprinted polymer (NIP) Chiral recognition was found to

be dependent on the stereo structures and the arrangement of functional groups of

the imprinted molecule and the cavities on MIP Thermodynamic data (∆∆H and

∆∆S) obtained by Vanrsquot Hoff plots revealed an enthalpy-controlled

enantioseparation The binding capacity was evaluated by frontal analysis

Monolithic NAT-MIP had an effective number of binding sites Bt = 4115 micromolg

with a dissociation constant of Kd = 740 mM The morphological characteristics of

the monolithic MIP were investigated by pore analysis and Scanning Electron

Microscope (SEM) Results showed that both mesopores and macropores were

formed in the monolith Over all this study presents a new and practical possibility

44

for providing high rates of mass transfer fast separations and high efficiencies

without the pressure constraints of the traditional bulk molecularly imprinted

polymers through the monolithic MIPs

Hongyuan Yan et al (2004) [62] developed a Micellar Electro Kinetic

Chromatography (MEKC) method for the determination of NAT in animal plasma

by on-line sweeping technique in which plasma samples were simply

deproteinized with acetonitrile and analyzed with 16 mmolL NaH2PO4 + 6

mmolL Na2B4O7 + 60 mmolL sodium dodecyl sulfate (SDS) (pH 714) as the

running buffer a fused-silica capillary as the separation tube 21 kV as the running

voltage and UV detection at 214 nm Under these conditions more than 100-fold

enrichment of NAT was obtained with the good linear relation in the range of

nateglinide plasma concentration 02ndash7mgL (r = 0998) The method could be

applied successfully to determine trace drugs in clinical analysis

Steffen Bauer et al (2003) [63] developed a rapid and simple method for the

analysis of NAT in human plasma and validated with LOQ- 01 microgml is low

enough to allow determination of pharmacokinetic parameters of the substance

The intra-assay coefficients of variation ranged from 16 to 129 at NAT

concentrations of 05-75 microgml The inter-assay variation for the same plasma

concentrations ranged from 38 to 84 The calibration was linear in the range of

01-20 microgml To quantify NAT only 50 microl of plasma were needed Following

protein precipitation in human plasma the samples were separated by isocratic

reversed phase HPLC and analyzed using UV detection at 210 nm Sample

preparation time and analysis time are both short and allow rapid analysis of large

sample sets

318 Pioglitazone

Wanjari et al (2005) [64] developed a stability indicating simple rapid and

precise reversed-phase HPLC method for the quantitation of Pioglitazone (PIO) in

45

tablet on a Hypersil C-8 (250x46 mm) column using a mobile phase consisting

acetonitrile 015 vv triethylamine (4060 vv) adjusted to pH 46 with

orthophosphoric acid at a flow rate of 15 mlmin and detection at 220 nm The

retention time of PIO have been found to be 76 min and recoveries were between

99-101 Validation of the proposed method also been done

Zhongping John Lin et al (2003) [65] reported a LC-MSMS method for the

simultaneous determination of PIO and its two metabolites M-III (keto-derivative)

and M-IV (hydroxy-derivative) in human plasma Human plasma samples of 02

ml were extracted by a single step LLE procedure and analyzed using a HPLC-

electrospray (ESI) tandem mass spectrometer system The compounds were eluted

isocratically on a C-18 column ionized using a positive ion atmospheric pressure

electrospray ionization source and analyzed using MRM mode The ion transitions

monitored were mz 3570134 for PIO mz 3710148 for M-III mz 3730150 for

M-IV and mz 4130178 for the IS The chromatographic run time was 25 min per

injection with retention times of 145 102 and 095 min for PIO M-III and M-IV

respectively The calibration curves of PIO M-III and M-IV were well fit over the

range of 05-2000 ngml (r2 gt 0998759) by using a weighted (1x2) quadratic

regression The inter-day precisions of the quality control samples were le105

(N=15) coefficient of variation and the inter-day accuracy ( nominal) ranged

from 846 to 1035 for PIO 944 to 1040 for M-III and 968 to 1010 for M-

IV All three analytes demonstrated acceptable short-term long-term and

freezethaw stability

Radha Krishna et al (2002) [66] developed an HPLC and MEKC methods have

been developed for the determination of PIO its unsaturated impurity were

separated by MEKC in less than 7 min using a 43 cm times 50 microm id uncoated fused-

silica capillary with extended light path for better sensitivity (25 kV at 30 degC) and

a Back Ground Electrolyte (BGE) consisting of 20 acetonitrile (vv) in 20 mM

sodium borate buffer pH 93 containing 50 mM Sodium Dodecyl Sulphate (SDS)

The influence of various parameters on the separation such as pH of the buffer

46

SDS concentration buffer concentration organic modifiers temperature and

voltage were investigated The MEKC method was compared with HPLC method

using 5 microm symmetry C18 column (250times46 mm id) eluted with a mobile phase

consisting of a mixture of 50 (vv) acetonitrile and 10 mM potassium dihydrogen

phosphate buffer adjusting the pH to 60 with 01 M KOH The HPLC method is

capable of detecting all process related compounds which may be present at trace

levels in finished products

Kenji Yamashita et al (1996) [67] developed a HPLC method for the

simultaneous determination of PIO and its metabolites (M-I to M-V) in human

serum and urine The method for serum involved the solid-phase (SPE) and LLE

extraction Urine with and without enzymatic hydrolysis using β-glucuronidase

was treated with LLE The compounds in the extract were analyzed using HPLC

with UV detection at 269 nm The detection limits of PIO M-I M-II M-Ill M-IV

and M-V in serum were 001-005 microgml those in urine were 01-05 microgml and

those in urine after enzymatic hydrolysis were 03-05 microgml respectively

Zhong et al (1996) [68] reported a HPLC method for the simultaneous

determination of PIO and its potential metabolites (M-I to M-6) in human serum

The method involved a SPE of PIO its metabolites and the IS from serum using

C18 SPE columns with an elution solvent of 05 ml of acetonitrile-water (3565

vv) Separation of the eight analytes was achieved within 20 min using a reversed-

phase Zorbax RX-Cs analytical column with a mobile phase of acetonitrile-water

(4060 vv) containing acetic acid 3 mlL (apparent pH 55) An UV detector

operated at 269 nm was used with a linear response observed from 002 to 2 microgml

for these analytes except for M-4 which was best fitted with a polynomial

regression LOQ was found to be 002 microgml for PIO M-3 M-5 and M-6 004

microgml for M-2 and M-4 and 05 microgml for M-I when using a 05 ml serum sample

for extraction Intra- and inter-assay precision was le 9 and accuracy ranged from

-82 to 134 for all analytes were obtained from the method validation

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

39

are treated statistically applying the F- and t-tests and found highly accurate

precise and reproducible The method is used to determine MET in pure form and

pharmaceutical preparations There is no interference from the excipients

Zarghi et al (2003) [52] reported a rapid simple and sensitive ion-pair HPLC

method for quantification of MET in plasma The assay enables the measurement

of MET for therapeutic drug monitoring with a LOD 20 ngml The method

involves simple one-step extraction procedure and analytical recovery was

complete The separation was performed on an analytical 150 x 46 mm id

microbondapak C18 column The wavelength was set at 235 nm The mobile phase was

40 acetonitrile 001 M sodium dodecyl sulphate 001 M sodium dihydrogen

phosphate and distilled water to 100 adjusted to pH 51 at a flow rate of 15

mlmin The calibration curve was linear over the concentration range 02-25

mgml The coefficients of variation for inter-day and intra-day assay were within

the range of clinical usefulness

Ching-Ling Cheng et al (2001) [53] developed a simple selective sensitive and

precise HPLC plasma assay for MET Acidified samples of plasma were de-

proteinated with acetonitrile washed with dichloromethane and the resulting

supernatant injected Chromatography was performed at 40 oC by pumping a

mobile phase of acetonitrile (250 ml) in pH 7 003 M diammonium hydrogen

phosphate buffer (750 ml) at a flow-rate of 1 mlmin through a silica column MET

and the IS (Atenolol) were detected at 240 nm and eluted at 78 and 68 min

respectively after injection No endogenous substances were found to interfere

Calibration curves were linear (rgt0999) from 10 to 2000 ngml The absolute

recovery of both MET and IS was greater than 76 The detection limit and limit

of quantification were 25 and 10 ngml respectively The intra- and inter-day

precision (CV) was 12 or less and the accuracy was within 62 of the

nominal concentration

40

Florentin Tache et al (2001) [54] optimized a method for determination of MET

at ppb level in human plasma Derivatization of MET with p-nitrobenzoyl chloride

was carried out in a biphasic system The derivative extracted in the organic layer

(dichloromethane) was concentrated and analyzed by HPLC with diode array

detector using an isocratic elution water methanol mixture 6535 monitored at

280 nm Good selectivity against co-extracted matrix components was observed A

detection limit of 10 ppb MET in human plasma was reached

Saad SM Hassan et al (1999) [55] described a simple and convenient

potentiometric spectrofluorimetric and spectrophotometric methods for the

determination of MET in pharmaceutical preparations The potentiometric

technique is based on preparation of PVC membrane sensors incorporating

metformin plusmn reineckate and metformin plusmn tungstosilicate ion-pairs as electroactive

species with dioctylphthalate and o-nitrophenyloctylether as plasticizers

respectively A membrane consisting of carboxylated PVC plasticized with

dibutylsebacate is also prepared and tested These sensors give rapid Nernstian

response for 10-1-10-5 M MET at pH range 5-11 The metformin plusmn tungstosilicate

based sensor is used in a flow-through sandwich cell for flow injection

potentiometric determination of MET Graphite sensors coated or doped with

metforminplusmntungstosilicate-PVC Cu-diethyldithio-carbamate and Ni-

diethyldithiocarbamate are also prepared and used for monitoring the titration of

the drug with tetraphenyl borate (TPB-) Cu2+ and Ni2+ ions respectively The

spectrofluorimetric method depends on the reaction of MET with chrysenequinone

in alkaline medium to give a Schiffs base which upon hydrolysis gives the free

base The latter in the presence of 1-naphthol gives a fluorescent product with

excitation and emission maxima at 450 and 520 nm respectively The fluorescence

plusmn concentration relationship is linear over the range 20plusmn200 mgml MET The

proposed spectrophotometric technique involves reaction of MET with Cu2+ in

basic medium to form a Cu plusmn metformin complex The complex is dissolved in

cyclohexylamine and its maximum absorption at 540 nm is measured Beers law is

41

obeyed over the range 05-2 mgml MET Various cations some nitrogenous

compounds or drug excipients cause no interferences

Kah Hay Yuen et al (1998) [56] developed a simple HPLC method using UV

detection for the determination of MET in human plasma The method entailed

direct injection of the plasma sample after deproteination using perchloric acid

The mobile phase comprised 001 M potassium dihydrogen orthophosphate (pH

35) and acetonitrile (6040 vv) Analyses were run at a flow-rate of 10 ml min

with the detector operating at a detection wavelength of 234 nm The method is

specific and sensitive with a LOQ approximately 60 ngml and a LOD 15 ngml at

a Sn ratio of 31 The mean absolute recovery value was about 97 while the

within-day and between-day CV and error values of the assay method were all

less than 8 The calibration curve was linear over a concentration range of 625ndash

4000 ngml

Ole Vesterqvist et al (1998) [57] reported a rapid HPLC method for the

determination of MET in plasma Sample preparation entailed a 30-min

centrifugation of plasma through a micron filter with direct injection of the

protein-free ultra-filtrate into an HPLC system consisting of a cation-exchange

extraction column (75346 mm) a column switching valve and a cation-exchange

analytical column (250346 mm) The eluent was monitored at 232 nm MET was

well resolved at a retention time of about 5 min There was less than 2 loss of

MET during ultra-filtration and good linearity was established from 010 to 40

mgL of MET The LLOQ was about 005 mgL at which concentration the Sn

was above 10 The intra- and inter-assay CV at plasma concentrations of MET

between 025 and 25 mgL were typically 08ndash14 and 35ndash64 respectively

317 Nateglinide

Kanthi Kiran et al (2008) [58] studied in rats for drugndashdrug interaction of

Nateglinide (NAT) with Cilostazol (CLZ) an antiplatelet agent commonly used in

42

diabetics and developed a liquid chromatography tandem mass spectrometry (LCndash

MSMS) based method that is capable of simultaneous monitoring plasma levels of

NAT CLZ and its active metabolite 34-dehydro-cilostazol (DCLZ) All analytes

including the IS Repaglinide were chromatographed on reverse phase C18 column

(50 mm times 46 mm id 5 microm) using acetonitrile 2 mM ammonium acetate buffer

pH 34 (9010 vv) as mobile phase at a flow rate 04 mlmin in an isocratic mode

The detection of analyte was performed on LCndashMSMS system in the multiple

reaction monitoring (MRM) mode The quantitations for analytes were based on

relative concentration The method was validated over the concentration range of

20ndash2000 ngml and the LLOQ was 20 ngml The recoveries from spiked control

samples were gt79 for all analytes and IS Intra- and inter-day accuracy and

precision of validated method were with in the acceptable limits of lt15 at all

concentration The quantitation method was successfully applied for simultaneous

estimation of NAT CLZ and DCLZ in a pharmacokinetic drugndashdrug interaction

study in Wistar rats

Danai Malli et al (2007) [59] developed and validated a sensitive and selective

HPLC method for the determination of NAT in human plasma NAT and the IS

undecylenic acid were extracted from plasma by liquidndashliquid extraction using a

mixture of ethyl acetatendashdiethyl ether 5050 (vv) Pre-column derivatization

reaction was performed using a coumarin-type fluorescent reagent

N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide The derivati-

zation proceeded in acetone in the presence of potassium carbonate and catalyzed

by 18-crown-6 ether The fluorescent derivatives were separated under isocratic

conditions on a Hypersil BDS-C8 analytical column with a mobile phase that

consisted of 65 acetonitrile in water and pumped at a flow rate of 05 mlmin

The excitation and emission wavelengths were set at 345 and 435 nm respectively

The assay was linear over a concentration range of 005ndash16 microgml for NAT with a

LOQ 005 microgml QC samples (005 450 and 1600 microgml) in five replicates from

five different runs of analysis demonstrated intra-assay precision ( CV lt68)

43

inter-assay precision ( CV lt16) and an overall accuracy ( relative error) lt

-34

Jolly M Sankalia et al (2007) [60] reported a rapid simple and sensitive HPLC

method with UV detection for the quantification of NAT from rabbit plasma The

retention behavior of NAT and GLZ (IS) as a function of mobile phase pH

composition and flow rate was investigated Separation was developed on a

reverse-phase C18 column (250 times 46 mm id 5 microm) using a mixture of

acetonitrile 10 mM phosphate buffer (pH 30) in the ratio of 7030 ( vv) at a

flow rate of 10 mlmin with UV detection at 203 nm within 8 min and quantified

based on drugIS peak area ratios The plasma samples were prepared by a simple

deproteinization with a mixture of methanol and acetonitrile yielding more than

9786 extraction efficiencies The calibration curve was linear (r2 = 09984) in

the concentration range of 10ndash2500 ngml The LOD and LOQ were found to be

291 and 970 ngml respectively Both the intra-day and inter-day precisions at

four tested concentrations were below 132 RSD

Junfa Yin et al (2005) [61] reported a rapid and efficient chiral separation

method of NAT and its L-enantiomer on a monolithic Molecularly Imprinted

Polymers (MIP) The enantiomers were rapidly separated on this novel monolithic

MIP based Chiral Stationary Phase (MIP-CSP) whereas the enantioseparation was

not obtained on the non-imprinted polymer (NIP) Chiral recognition was found to

be dependent on the stereo structures and the arrangement of functional groups of

the imprinted molecule and the cavities on MIP Thermodynamic data (∆∆H and

∆∆S) obtained by Vanrsquot Hoff plots revealed an enthalpy-controlled

enantioseparation The binding capacity was evaluated by frontal analysis

Monolithic NAT-MIP had an effective number of binding sites Bt = 4115 micromolg

with a dissociation constant of Kd = 740 mM The morphological characteristics of

the monolithic MIP were investigated by pore analysis and Scanning Electron

Microscope (SEM) Results showed that both mesopores and macropores were

formed in the monolith Over all this study presents a new and practical possibility

44

for providing high rates of mass transfer fast separations and high efficiencies

without the pressure constraints of the traditional bulk molecularly imprinted

polymers through the monolithic MIPs

Hongyuan Yan et al (2004) [62] developed a Micellar Electro Kinetic

Chromatography (MEKC) method for the determination of NAT in animal plasma

by on-line sweeping technique in which plasma samples were simply

deproteinized with acetonitrile and analyzed with 16 mmolL NaH2PO4 + 6

mmolL Na2B4O7 + 60 mmolL sodium dodecyl sulfate (SDS) (pH 714) as the

running buffer a fused-silica capillary as the separation tube 21 kV as the running

voltage and UV detection at 214 nm Under these conditions more than 100-fold

enrichment of NAT was obtained with the good linear relation in the range of

nateglinide plasma concentration 02ndash7mgL (r = 0998) The method could be

applied successfully to determine trace drugs in clinical analysis

Steffen Bauer et al (2003) [63] developed a rapid and simple method for the

analysis of NAT in human plasma and validated with LOQ- 01 microgml is low

enough to allow determination of pharmacokinetic parameters of the substance

The intra-assay coefficients of variation ranged from 16 to 129 at NAT

concentrations of 05-75 microgml The inter-assay variation for the same plasma

concentrations ranged from 38 to 84 The calibration was linear in the range of

01-20 microgml To quantify NAT only 50 microl of plasma were needed Following

protein precipitation in human plasma the samples were separated by isocratic

reversed phase HPLC and analyzed using UV detection at 210 nm Sample

preparation time and analysis time are both short and allow rapid analysis of large

sample sets

318 Pioglitazone

Wanjari et al (2005) [64] developed a stability indicating simple rapid and

precise reversed-phase HPLC method for the quantitation of Pioglitazone (PIO) in

45

tablet on a Hypersil C-8 (250x46 mm) column using a mobile phase consisting

acetonitrile 015 vv triethylamine (4060 vv) adjusted to pH 46 with

orthophosphoric acid at a flow rate of 15 mlmin and detection at 220 nm The

retention time of PIO have been found to be 76 min and recoveries were between

99-101 Validation of the proposed method also been done

Zhongping John Lin et al (2003) [65] reported a LC-MSMS method for the

simultaneous determination of PIO and its two metabolites M-III (keto-derivative)

and M-IV (hydroxy-derivative) in human plasma Human plasma samples of 02

ml were extracted by a single step LLE procedure and analyzed using a HPLC-

electrospray (ESI) tandem mass spectrometer system The compounds were eluted

isocratically on a C-18 column ionized using a positive ion atmospheric pressure

electrospray ionization source and analyzed using MRM mode The ion transitions

monitored were mz 3570134 for PIO mz 3710148 for M-III mz 3730150 for

M-IV and mz 4130178 for the IS The chromatographic run time was 25 min per

injection with retention times of 145 102 and 095 min for PIO M-III and M-IV

respectively The calibration curves of PIO M-III and M-IV were well fit over the

range of 05-2000 ngml (r2 gt 0998759) by using a weighted (1x2) quadratic

regression The inter-day precisions of the quality control samples were le105

(N=15) coefficient of variation and the inter-day accuracy ( nominal) ranged

from 846 to 1035 for PIO 944 to 1040 for M-III and 968 to 1010 for M-

IV All three analytes demonstrated acceptable short-term long-term and

freezethaw stability

Radha Krishna et al (2002) [66] developed an HPLC and MEKC methods have

been developed for the determination of PIO its unsaturated impurity were

separated by MEKC in less than 7 min using a 43 cm times 50 microm id uncoated fused-

silica capillary with extended light path for better sensitivity (25 kV at 30 degC) and

a Back Ground Electrolyte (BGE) consisting of 20 acetonitrile (vv) in 20 mM

sodium borate buffer pH 93 containing 50 mM Sodium Dodecyl Sulphate (SDS)

The influence of various parameters on the separation such as pH of the buffer

46

SDS concentration buffer concentration organic modifiers temperature and

voltage were investigated The MEKC method was compared with HPLC method

using 5 microm symmetry C18 column (250times46 mm id) eluted with a mobile phase

consisting of a mixture of 50 (vv) acetonitrile and 10 mM potassium dihydrogen

phosphate buffer adjusting the pH to 60 with 01 M KOH The HPLC method is

capable of detecting all process related compounds which may be present at trace

levels in finished products

Kenji Yamashita et al (1996) [67] developed a HPLC method for the

simultaneous determination of PIO and its metabolites (M-I to M-V) in human

serum and urine The method for serum involved the solid-phase (SPE) and LLE

extraction Urine with and without enzymatic hydrolysis using β-glucuronidase

was treated with LLE The compounds in the extract were analyzed using HPLC

with UV detection at 269 nm The detection limits of PIO M-I M-II M-Ill M-IV

and M-V in serum were 001-005 microgml those in urine were 01-05 microgml and

those in urine after enzymatic hydrolysis were 03-05 microgml respectively

Zhong et al (1996) [68] reported a HPLC method for the simultaneous

determination of PIO and its potential metabolites (M-I to M-6) in human serum

The method involved a SPE of PIO its metabolites and the IS from serum using

C18 SPE columns with an elution solvent of 05 ml of acetonitrile-water (3565

vv) Separation of the eight analytes was achieved within 20 min using a reversed-

phase Zorbax RX-Cs analytical column with a mobile phase of acetonitrile-water

(4060 vv) containing acetic acid 3 mlL (apparent pH 55) An UV detector

operated at 269 nm was used with a linear response observed from 002 to 2 microgml

for these analytes except for M-4 which was best fitted with a polynomial

regression LOQ was found to be 002 microgml for PIO M-3 M-5 and M-6 004

microgml for M-2 and M-4 and 05 microgml for M-I when using a 05 ml serum sample

for extraction Intra- and inter-assay precision was le 9 and accuracy ranged from

-82 to 134 for all analytes were obtained from the method validation

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

40

Florentin Tache et al (2001) [54] optimized a method for determination of MET

at ppb level in human plasma Derivatization of MET with p-nitrobenzoyl chloride

was carried out in a biphasic system The derivative extracted in the organic layer

(dichloromethane) was concentrated and analyzed by HPLC with diode array

detector using an isocratic elution water methanol mixture 6535 monitored at

280 nm Good selectivity against co-extracted matrix components was observed A

detection limit of 10 ppb MET in human plasma was reached

Saad SM Hassan et al (1999) [55] described a simple and convenient

potentiometric spectrofluorimetric and spectrophotometric methods for the

determination of MET in pharmaceutical preparations The potentiometric

technique is based on preparation of PVC membrane sensors incorporating

metformin plusmn reineckate and metformin plusmn tungstosilicate ion-pairs as electroactive

species with dioctylphthalate and o-nitrophenyloctylether as plasticizers

respectively A membrane consisting of carboxylated PVC plasticized with

dibutylsebacate is also prepared and tested These sensors give rapid Nernstian

response for 10-1-10-5 M MET at pH range 5-11 The metformin plusmn tungstosilicate

based sensor is used in a flow-through sandwich cell for flow injection

potentiometric determination of MET Graphite sensors coated or doped with

metforminplusmntungstosilicate-PVC Cu-diethyldithio-carbamate and Ni-

diethyldithiocarbamate are also prepared and used for monitoring the titration of

the drug with tetraphenyl borate (TPB-) Cu2+ and Ni2+ ions respectively The

spectrofluorimetric method depends on the reaction of MET with chrysenequinone

in alkaline medium to give a Schiffs base which upon hydrolysis gives the free

base The latter in the presence of 1-naphthol gives a fluorescent product with

excitation and emission maxima at 450 and 520 nm respectively The fluorescence

plusmn concentration relationship is linear over the range 20plusmn200 mgml MET The

proposed spectrophotometric technique involves reaction of MET with Cu2+ in

basic medium to form a Cu plusmn metformin complex The complex is dissolved in

cyclohexylamine and its maximum absorption at 540 nm is measured Beers law is

41

obeyed over the range 05-2 mgml MET Various cations some nitrogenous

compounds or drug excipients cause no interferences

Kah Hay Yuen et al (1998) [56] developed a simple HPLC method using UV

detection for the determination of MET in human plasma The method entailed

direct injection of the plasma sample after deproteination using perchloric acid

The mobile phase comprised 001 M potassium dihydrogen orthophosphate (pH

35) and acetonitrile (6040 vv) Analyses were run at a flow-rate of 10 ml min

with the detector operating at a detection wavelength of 234 nm The method is

specific and sensitive with a LOQ approximately 60 ngml and a LOD 15 ngml at

a Sn ratio of 31 The mean absolute recovery value was about 97 while the

within-day and between-day CV and error values of the assay method were all

less than 8 The calibration curve was linear over a concentration range of 625ndash

4000 ngml

Ole Vesterqvist et al (1998) [57] reported a rapid HPLC method for the

determination of MET in plasma Sample preparation entailed a 30-min

centrifugation of plasma through a micron filter with direct injection of the

protein-free ultra-filtrate into an HPLC system consisting of a cation-exchange

extraction column (75346 mm) a column switching valve and a cation-exchange

analytical column (250346 mm) The eluent was monitored at 232 nm MET was

well resolved at a retention time of about 5 min There was less than 2 loss of

MET during ultra-filtration and good linearity was established from 010 to 40

mgL of MET The LLOQ was about 005 mgL at which concentration the Sn

was above 10 The intra- and inter-assay CV at plasma concentrations of MET

between 025 and 25 mgL were typically 08ndash14 and 35ndash64 respectively

317 Nateglinide

Kanthi Kiran et al (2008) [58] studied in rats for drugndashdrug interaction of

Nateglinide (NAT) with Cilostazol (CLZ) an antiplatelet agent commonly used in

42

diabetics and developed a liquid chromatography tandem mass spectrometry (LCndash

MSMS) based method that is capable of simultaneous monitoring plasma levels of

NAT CLZ and its active metabolite 34-dehydro-cilostazol (DCLZ) All analytes

including the IS Repaglinide were chromatographed on reverse phase C18 column

(50 mm times 46 mm id 5 microm) using acetonitrile 2 mM ammonium acetate buffer

pH 34 (9010 vv) as mobile phase at a flow rate 04 mlmin in an isocratic mode

The detection of analyte was performed on LCndashMSMS system in the multiple

reaction monitoring (MRM) mode The quantitations for analytes were based on

relative concentration The method was validated over the concentration range of

20ndash2000 ngml and the LLOQ was 20 ngml The recoveries from spiked control

samples were gt79 for all analytes and IS Intra- and inter-day accuracy and

precision of validated method were with in the acceptable limits of lt15 at all

concentration The quantitation method was successfully applied for simultaneous

estimation of NAT CLZ and DCLZ in a pharmacokinetic drugndashdrug interaction

study in Wistar rats

Danai Malli et al (2007) [59] developed and validated a sensitive and selective

HPLC method for the determination of NAT in human plasma NAT and the IS

undecylenic acid were extracted from plasma by liquidndashliquid extraction using a

mixture of ethyl acetatendashdiethyl ether 5050 (vv) Pre-column derivatization

reaction was performed using a coumarin-type fluorescent reagent

N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide The derivati-

zation proceeded in acetone in the presence of potassium carbonate and catalyzed

by 18-crown-6 ether The fluorescent derivatives were separated under isocratic

conditions on a Hypersil BDS-C8 analytical column with a mobile phase that

consisted of 65 acetonitrile in water and pumped at a flow rate of 05 mlmin

The excitation and emission wavelengths were set at 345 and 435 nm respectively

The assay was linear over a concentration range of 005ndash16 microgml for NAT with a

LOQ 005 microgml QC samples (005 450 and 1600 microgml) in five replicates from

five different runs of analysis demonstrated intra-assay precision ( CV lt68)

43

inter-assay precision ( CV lt16) and an overall accuracy ( relative error) lt

-34

Jolly M Sankalia et al (2007) [60] reported a rapid simple and sensitive HPLC

method with UV detection for the quantification of NAT from rabbit plasma The

retention behavior of NAT and GLZ (IS) as a function of mobile phase pH

composition and flow rate was investigated Separation was developed on a

reverse-phase C18 column (250 times 46 mm id 5 microm) using a mixture of

acetonitrile 10 mM phosphate buffer (pH 30) in the ratio of 7030 ( vv) at a

flow rate of 10 mlmin with UV detection at 203 nm within 8 min and quantified

based on drugIS peak area ratios The plasma samples were prepared by a simple

deproteinization with a mixture of methanol and acetonitrile yielding more than

9786 extraction efficiencies The calibration curve was linear (r2 = 09984) in

the concentration range of 10ndash2500 ngml The LOD and LOQ were found to be

291 and 970 ngml respectively Both the intra-day and inter-day precisions at

four tested concentrations were below 132 RSD

Junfa Yin et al (2005) [61] reported a rapid and efficient chiral separation

method of NAT and its L-enantiomer on a monolithic Molecularly Imprinted

Polymers (MIP) The enantiomers were rapidly separated on this novel monolithic

MIP based Chiral Stationary Phase (MIP-CSP) whereas the enantioseparation was

not obtained on the non-imprinted polymer (NIP) Chiral recognition was found to

be dependent on the stereo structures and the arrangement of functional groups of

the imprinted molecule and the cavities on MIP Thermodynamic data (∆∆H and

∆∆S) obtained by Vanrsquot Hoff plots revealed an enthalpy-controlled

enantioseparation The binding capacity was evaluated by frontal analysis

Monolithic NAT-MIP had an effective number of binding sites Bt = 4115 micromolg

with a dissociation constant of Kd = 740 mM The morphological characteristics of

the monolithic MIP were investigated by pore analysis and Scanning Electron

Microscope (SEM) Results showed that both mesopores and macropores were

formed in the monolith Over all this study presents a new and practical possibility

44

for providing high rates of mass transfer fast separations and high efficiencies

without the pressure constraints of the traditional bulk molecularly imprinted

polymers through the monolithic MIPs

Hongyuan Yan et al (2004) [62] developed a Micellar Electro Kinetic

Chromatography (MEKC) method for the determination of NAT in animal plasma

by on-line sweeping technique in which plasma samples were simply

deproteinized with acetonitrile and analyzed with 16 mmolL NaH2PO4 + 6

mmolL Na2B4O7 + 60 mmolL sodium dodecyl sulfate (SDS) (pH 714) as the

running buffer a fused-silica capillary as the separation tube 21 kV as the running

voltage and UV detection at 214 nm Under these conditions more than 100-fold

enrichment of NAT was obtained with the good linear relation in the range of

nateglinide plasma concentration 02ndash7mgL (r = 0998) The method could be

applied successfully to determine trace drugs in clinical analysis

Steffen Bauer et al (2003) [63] developed a rapid and simple method for the

analysis of NAT in human plasma and validated with LOQ- 01 microgml is low

enough to allow determination of pharmacokinetic parameters of the substance

The intra-assay coefficients of variation ranged from 16 to 129 at NAT

concentrations of 05-75 microgml The inter-assay variation for the same plasma

concentrations ranged from 38 to 84 The calibration was linear in the range of

01-20 microgml To quantify NAT only 50 microl of plasma were needed Following

protein precipitation in human plasma the samples were separated by isocratic

reversed phase HPLC and analyzed using UV detection at 210 nm Sample

preparation time and analysis time are both short and allow rapid analysis of large

sample sets

318 Pioglitazone

Wanjari et al (2005) [64] developed a stability indicating simple rapid and

precise reversed-phase HPLC method for the quantitation of Pioglitazone (PIO) in

45

tablet on a Hypersil C-8 (250x46 mm) column using a mobile phase consisting

acetonitrile 015 vv triethylamine (4060 vv) adjusted to pH 46 with

orthophosphoric acid at a flow rate of 15 mlmin and detection at 220 nm The

retention time of PIO have been found to be 76 min and recoveries were between

99-101 Validation of the proposed method also been done

Zhongping John Lin et al (2003) [65] reported a LC-MSMS method for the

simultaneous determination of PIO and its two metabolites M-III (keto-derivative)

and M-IV (hydroxy-derivative) in human plasma Human plasma samples of 02

ml were extracted by a single step LLE procedure and analyzed using a HPLC-

electrospray (ESI) tandem mass spectrometer system The compounds were eluted

isocratically on a C-18 column ionized using a positive ion atmospheric pressure

electrospray ionization source and analyzed using MRM mode The ion transitions

monitored were mz 3570134 for PIO mz 3710148 for M-III mz 3730150 for

M-IV and mz 4130178 for the IS The chromatographic run time was 25 min per

injection with retention times of 145 102 and 095 min for PIO M-III and M-IV

respectively The calibration curves of PIO M-III and M-IV were well fit over the

range of 05-2000 ngml (r2 gt 0998759) by using a weighted (1x2) quadratic

regression The inter-day precisions of the quality control samples were le105

(N=15) coefficient of variation and the inter-day accuracy ( nominal) ranged

from 846 to 1035 for PIO 944 to 1040 for M-III and 968 to 1010 for M-

IV All three analytes demonstrated acceptable short-term long-term and

freezethaw stability

Radha Krishna et al (2002) [66] developed an HPLC and MEKC methods have

been developed for the determination of PIO its unsaturated impurity were

separated by MEKC in less than 7 min using a 43 cm times 50 microm id uncoated fused-

silica capillary with extended light path for better sensitivity (25 kV at 30 degC) and

a Back Ground Electrolyte (BGE) consisting of 20 acetonitrile (vv) in 20 mM

sodium borate buffer pH 93 containing 50 mM Sodium Dodecyl Sulphate (SDS)

The influence of various parameters on the separation such as pH of the buffer

46

SDS concentration buffer concentration organic modifiers temperature and

voltage were investigated The MEKC method was compared with HPLC method

using 5 microm symmetry C18 column (250times46 mm id) eluted with a mobile phase

consisting of a mixture of 50 (vv) acetonitrile and 10 mM potassium dihydrogen

phosphate buffer adjusting the pH to 60 with 01 M KOH The HPLC method is

capable of detecting all process related compounds which may be present at trace

levels in finished products

Kenji Yamashita et al (1996) [67] developed a HPLC method for the

simultaneous determination of PIO and its metabolites (M-I to M-V) in human

serum and urine The method for serum involved the solid-phase (SPE) and LLE

extraction Urine with and without enzymatic hydrolysis using β-glucuronidase

was treated with LLE The compounds in the extract were analyzed using HPLC

with UV detection at 269 nm The detection limits of PIO M-I M-II M-Ill M-IV

and M-V in serum were 001-005 microgml those in urine were 01-05 microgml and

those in urine after enzymatic hydrolysis were 03-05 microgml respectively

Zhong et al (1996) [68] reported a HPLC method for the simultaneous

determination of PIO and its potential metabolites (M-I to M-6) in human serum

The method involved a SPE of PIO its metabolites and the IS from serum using

C18 SPE columns with an elution solvent of 05 ml of acetonitrile-water (3565

vv) Separation of the eight analytes was achieved within 20 min using a reversed-

phase Zorbax RX-Cs analytical column with a mobile phase of acetonitrile-water

(4060 vv) containing acetic acid 3 mlL (apparent pH 55) An UV detector

operated at 269 nm was used with a linear response observed from 002 to 2 microgml

for these analytes except for M-4 which was best fitted with a polynomial

regression LOQ was found to be 002 microgml for PIO M-3 M-5 and M-6 004

microgml for M-2 and M-4 and 05 microgml for M-I when using a 05 ml serum sample

for extraction Intra- and inter-assay precision was le 9 and accuracy ranged from

-82 to 134 for all analytes were obtained from the method validation

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

41

obeyed over the range 05-2 mgml MET Various cations some nitrogenous

compounds or drug excipients cause no interferences

Kah Hay Yuen et al (1998) [56] developed a simple HPLC method using UV

detection for the determination of MET in human plasma The method entailed

direct injection of the plasma sample after deproteination using perchloric acid

The mobile phase comprised 001 M potassium dihydrogen orthophosphate (pH

35) and acetonitrile (6040 vv) Analyses were run at a flow-rate of 10 ml min

with the detector operating at a detection wavelength of 234 nm The method is

specific and sensitive with a LOQ approximately 60 ngml and a LOD 15 ngml at

a Sn ratio of 31 The mean absolute recovery value was about 97 while the

within-day and between-day CV and error values of the assay method were all

less than 8 The calibration curve was linear over a concentration range of 625ndash

4000 ngml

Ole Vesterqvist et al (1998) [57] reported a rapid HPLC method for the

determination of MET in plasma Sample preparation entailed a 30-min

centrifugation of plasma through a micron filter with direct injection of the

protein-free ultra-filtrate into an HPLC system consisting of a cation-exchange

extraction column (75346 mm) a column switching valve and a cation-exchange

analytical column (250346 mm) The eluent was monitored at 232 nm MET was

well resolved at a retention time of about 5 min There was less than 2 loss of

MET during ultra-filtration and good linearity was established from 010 to 40

mgL of MET The LLOQ was about 005 mgL at which concentration the Sn

was above 10 The intra- and inter-assay CV at plasma concentrations of MET

between 025 and 25 mgL were typically 08ndash14 and 35ndash64 respectively

317 Nateglinide

Kanthi Kiran et al (2008) [58] studied in rats for drugndashdrug interaction of

Nateglinide (NAT) with Cilostazol (CLZ) an antiplatelet agent commonly used in

42

diabetics and developed a liquid chromatography tandem mass spectrometry (LCndash

MSMS) based method that is capable of simultaneous monitoring plasma levels of

NAT CLZ and its active metabolite 34-dehydro-cilostazol (DCLZ) All analytes

including the IS Repaglinide were chromatographed on reverse phase C18 column

(50 mm times 46 mm id 5 microm) using acetonitrile 2 mM ammonium acetate buffer

pH 34 (9010 vv) as mobile phase at a flow rate 04 mlmin in an isocratic mode

The detection of analyte was performed on LCndashMSMS system in the multiple

reaction monitoring (MRM) mode The quantitations for analytes were based on

relative concentration The method was validated over the concentration range of

20ndash2000 ngml and the LLOQ was 20 ngml The recoveries from spiked control

samples were gt79 for all analytes and IS Intra- and inter-day accuracy and

precision of validated method were with in the acceptable limits of lt15 at all

concentration The quantitation method was successfully applied for simultaneous

estimation of NAT CLZ and DCLZ in a pharmacokinetic drugndashdrug interaction

study in Wistar rats

Danai Malli et al (2007) [59] developed and validated a sensitive and selective

HPLC method for the determination of NAT in human plasma NAT and the IS

undecylenic acid were extracted from plasma by liquidndashliquid extraction using a

mixture of ethyl acetatendashdiethyl ether 5050 (vv) Pre-column derivatization

reaction was performed using a coumarin-type fluorescent reagent

N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide The derivati-

zation proceeded in acetone in the presence of potassium carbonate and catalyzed

by 18-crown-6 ether The fluorescent derivatives were separated under isocratic

conditions on a Hypersil BDS-C8 analytical column with a mobile phase that

consisted of 65 acetonitrile in water and pumped at a flow rate of 05 mlmin

The excitation and emission wavelengths were set at 345 and 435 nm respectively

The assay was linear over a concentration range of 005ndash16 microgml for NAT with a

LOQ 005 microgml QC samples (005 450 and 1600 microgml) in five replicates from

five different runs of analysis demonstrated intra-assay precision ( CV lt68)

43

inter-assay precision ( CV lt16) and an overall accuracy ( relative error) lt

-34

Jolly M Sankalia et al (2007) [60] reported a rapid simple and sensitive HPLC

method with UV detection for the quantification of NAT from rabbit plasma The

retention behavior of NAT and GLZ (IS) as a function of mobile phase pH

composition and flow rate was investigated Separation was developed on a

reverse-phase C18 column (250 times 46 mm id 5 microm) using a mixture of

acetonitrile 10 mM phosphate buffer (pH 30) in the ratio of 7030 ( vv) at a

flow rate of 10 mlmin with UV detection at 203 nm within 8 min and quantified

based on drugIS peak area ratios The plasma samples were prepared by a simple

deproteinization with a mixture of methanol and acetonitrile yielding more than

9786 extraction efficiencies The calibration curve was linear (r2 = 09984) in

the concentration range of 10ndash2500 ngml The LOD and LOQ were found to be

291 and 970 ngml respectively Both the intra-day and inter-day precisions at

four tested concentrations were below 132 RSD

Junfa Yin et al (2005) [61] reported a rapid and efficient chiral separation

method of NAT and its L-enantiomer on a monolithic Molecularly Imprinted

Polymers (MIP) The enantiomers were rapidly separated on this novel monolithic

MIP based Chiral Stationary Phase (MIP-CSP) whereas the enantioseparation was

not obtained on the non-imprinted polymer (NIP) Chiral recognition was found to

be dependent on the stereo structures and the arrangement of functional groups of

the imprinted molecule and the cavities on MIP Thermodynamic data (∆∆H and

∆∆S) obtained by Vanrsquot Hoff plots revealed an enthalpy-controlled

enantioseparation The binding capacity was evaluated by frontal analysis

Monolithic NAT-MIP had an effective number of binding sites Bt = 4115 micromolg

with a dissociation constant of Kd = 740 mM The morphological characteristics of

the monolithic MIP were investigated by pore analysis and Scanning Electron

Microscope (SEM) Results showed that both mesopores and macropores were

formed in the monolith Over all this study presents a new and practical possibility

44

for providing high rates of mass transfer fast separations and high efficiencies

without the pressure constraints of the traditional bulk molecularly imprinted

polymers through the monolithic MIPs

Hongyuan Yan et al (2004) [62] developed a Micellar Electro Kinetic

Chromatography (MEKC) method for the determination of NAT in animal plasma

by on-line sweeping technique in which plasma samples were simply

deproteinized with acetonitrile and analyzed with 16 mmolL NaH2PO4 + 6

mmolL Na2B4O7 + 60 mmolL sodium dodecyl sulfate (SDS) (pH 714) as the

running buffer a fused-silica capillary as the separation tube 21 kV as the running

voltage and UV detection at 214 nm Under these conditions more than 100-fold

enrichment of NAT was obtained with the good linear relation in the range of

nateglinide plasma concentration 02ndash7mgL (r = 0998) The method could be

applied successfully to determine trace drugs in clinical analysis

Steffen Bauer et al (2003) [63] developed a rapid and simple method for the

analysis of NAT in human plasma and validated with LOQ- 01 microgml is low

enough to allow determination of pharmacokinetic parameters of the substance

The intra-assay coefficients of variation ranged from 16 to 129 at NAT

concentrations of 05-75 microgml The inter-assay variation for the same plasma

concentrations ranged from 38 to 84 The calibration was linear in the range of

01-20 microgml To quantify NAT only 50 microl of plasma were needed Following

protein precipitation in human plasma the samples were separated by isocratic

reversed phase HPLC and analyzed using UV detection at 210 nm Sample

preparation time and analysis time are both short and allow rapid analysis of large

sample sets

318 Pioglitazone

Wanjari et al (2005) [64] developed a stability indicating simple rapid and

precise reversed-phase HPLC method for the quantitation of Pioglitazone (PIO) in

45

tablet on a Hypersil C-8 (250x46 mm) column using a mobile phase consisting

acetonitrile 015 vv triethylamine (4060 vv) adjusted to pH 46 with

orthophosphoric acid at a flow rate of 15 mlmin and detection at 220 nm The

retention time of PIO have been found to be 76 min and recoveries were between

99-101 Validation of the proposed method also been done

Zhongping John Lin et al (2003) [65] reported a LC-MSMS method for the

simultaneous determination of PIO and its two metabolites M-III (keto-derivative)

and M-IV (hydroxy-derivative) in human plasma Human plasma samples of 02

ml were extracted by a single step LLE procedure and analyzed using a HPLC-

electrospray (ESI) tandem mass spectrometer system The compounds were eluted

isocratically on a C-18 column ionized using a positive ion atmospheric pressure

electrospray ionization source and analyzed using MRM mode The ion transitions

monitored were mz 3570134 for PIO mz 3710148 for M-III mz 3730150 for

M-IV and mz 4130178 for the IS The chromatographic run time was 25 min per

injection with retention times of 145 102 and 095 min for PIO M-III and M-IV

respectively The calibration curves of PIO M-III and M-IV were well fit over the

range of 05-2000 ngml (r2 gt 0998759) by using a weighted (1x2) quadratic

regression The inter-day precisions of the quality control samples were le105

(N=15) coefficient of variation and the inter-day accuracy ( nominal) ranged

from 846 to 1035 for PIO 944 to 1040 for M-III and 968 to 1010 for M-

IV All three analytes demonstrated acceptable short-term long-term and

freezethaw stability

Radha Krishna et al (2002) [66] developed an HPLC and MEKC methods have

been developed for the determination of PIO its unsaturated impurity were

separated by MEKC in less than 7 min using a 43 cm times 50 microm id uncoated fused-

silica capillary with extended light path for better sensitivity (25 kV at 30 degC) and

a Back Ground Electrolyte (BGE) consisting of 20 acetonitrile (vv) in 20 mM

sodium borate buffer pH 93 containing 50 mM Sodium Dodecyl Sulphate (SDS)

The influence of various parameters on the separation such as pH of the buffer

46

SDS concentration buffer concentration organic modifiers temperature and

voltage were investigated The MEKC method was compared with HPLC method

using 5 microm symmetry C18 column (250times46 mm id) eluted with a mobile phase

consisting of a mixture of 50 (vv) acetonitrile and 10 mM potassium dihydrogen

phosphate buffer adjusting the pH to 60 with 01 M KOH The HPLC method is

capable of detecting all process related compounds which may be present at trace

levels in finished products

Kenji Yamashita et al (1996) [67] developed a HPLC method for the

simultaneous determination of PIO and its metabolites (M-I to M-V) in human

serum and urine The method for serum involved the solid-phase (SPE) and LLE

extraction Urine with and without enzymatic hydrolysis using β-glucuronidase

was treated with LLE The compounds in the extract were analyzed using HPLC

with UV detection at 269 nm The detection limits of PIO M-I M-II M-Ill M-IV

and M-V in serum were 001-005 microgml those in urine were 01-05 microgml and

those in urine after enzymatic hydrolysis were 03-05 microgml respectively

Zhong et al (1996) [68] reported a HPLC method for the simultaneous

determination of PIO and its potential metabolites (M-I to M-6) in human serum

The method involved a SPE of PIO its metabolites and the IS from serum using

C18 SPE columns with an elution solvent of 05 ml of acetonitrile-water (3565

vv) Separation of the eight analytes was achieved within 20 min using a reversed-

phase Zorbax RX-Cs analytical column with a mobile phase of acetonitrile-water

(4060 vv) containing acetic acid 3 mlL (apparent pH 55) An UV detector

operated at 269 nm was used with a linear response observed from 002 to 2 microgml

for these analytes except for M-4 which was best fitted with a polynomial

regression LOQ was found to be 002 microgml for PIO M-3 M-5 and M-6 004

microgml for M-2 and M-4 and 05 microgml for M-I when using a 05 ml serum sample

for extraction Intra- and inter-assay precision was le 9 and accuracy ranged from

-82 to 134 for all analytes were obtained from the method validation

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

42

diabetics and developed a liquid chromatography tandem mass spectrometry (LCndash

MSMS) based method that is capable of simultaneous monitoring plasma levels of

NAT CLZ and its active metabolite 34-dehydro-cilostazol (DCLZ) All analytes

including the IS Repaglinide were chromatographed on reverse phase C18 column

(50 mm times 46 mm id 5 microm) using acetonitrile 2 mM ammonium acetate buffer

pH 34 (9010 vv) as mobile phase at a flow rate 04 mlmin in an isocratic mode

The detection of analyte was performed on LCndashMSMS system in the multiple

reaction monitoring (MRM) mode The quantitations for analytes were based on

relative concentration The method was validated over the concentration range of

20ndash2000 ngml and the LLOQ was 20 ngml The recoveries from spiked control

samples were gt79 for all analytes and IS Intra- and inter-day accuracy and

precision of validated method were with in the acceptable limits of lt15 at all

concentration The quantitation method was successfully applied for simultaneous

estimation of NAT CLZ and DCLZ in a pharmacokinetic drugndashdrug interaction

study in Wistar rats

Danai Malli et al (2007) [59] developed and validated a sensitive and selective

HPLC method for the determination of NAT in human plasma NAT and the IS

undecylenic acid were extracted from plasma by liquidndashliquid extraction using a

mixture of ethyl acetatendashdiethyl ether 5050 (vv) Pre-column derivatization

reaction was performed using a coumarin-type fluorescent reagent

N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide The derivati-

zation proceeded in acetone in the presence of potassium carbonate and catalyzed

by 18-crown-6 ether The fluorescent derivatives were separated under isocratic

conditions on a Hypersil BDS-C8 analytical column with a mobile phase that

consisted of 65 acetonitrile in water and pumped at a flow rate of 05 mlmin

The excitation and emission wavelengths were set at 345 and 435 nm respectively

The assay was linear over a concentration range of 005ndash16 microgml for NAT with a

LOQ 005 microgml QC samples (005 450 and 1600 microgml) in five replicates from

five different runs of analysis demonstrated intra-assay precision ( CV lt68)

43

inter-assay precision ( CV lt16) and an overall accuracy ( relative error) lt

-34

Jolly M Sankalia et al (2007) [60] reported a rapid simple and sensitive HPLC

method with UV detection for the quantification of NAT from rabbit plasma The

retention behavior of NAT and GLZ (IS) as a function of mobile phase pH

composition and flow rate was investigated Separation was developed on a

reverse-phase C18 column (250 times 46 mm id 5 microm) using a mixture of

acetonitrile 10 mM phosphate buffer (pH 30) in the ratio of 7030 ( vv) at a

flow rate of 10 mlmin with UV detection at 203 nm within 8 min and quantified

based on drugIS peak area ratios The plasma samples were prepared by a simple

deproteinization with a mixture of methanol and acetonitrile yielding more than

9786 extraction efficiencies The calibration curve was linear (r2 = 09984) in

the concentration range of 10ndash2500 ngml The LOD and LOQ were found to be

291 and 970 ngml respectively Both the intra-day and inter-day precisions at

four tested concentrations were below 132 RSD

Junfa Yin et al (2005) [61] reported a rapid and efficient chiral separation

method of NAT and its L-enantiomer on a monolithic Molecularly Imprinted

Polymers (MIP) The enantiomers were rapidly separated on this novel monolithic

MIP based Chiral Stationary Phase (MIP-CSP) whereas the enantioseparation was

not obtained on the non-imprinted polymer (NIP) Chiral recognition was found to

be dependent on the stereo structures and the arrangement of functional groups of

the imprinted molecule and the cavities on MIP Thermodynamic data (∆∆H and

∆∆S) obtained by Vanrsquot Hoff plots revealed an enthalpy-controlled

enantioseparation The binding capacity was evaluated by frontal analysis

Monolithic NAT-MIP had an effective number of binding sites Bt = 4115 micromolg

with a dissociation constant of Kd = 740 mM The morphological characteristics of

the monolithic MIP were investigated by pore analysis and Scanning Electron

Microscope (SEM) Results showed that both mesopores and macropores were

formed in the monolith Over all this study presents a new and practical possibility

44

for providing high rates of mass transfer fast separations and high efficiencies

without the pressure constraints of the traditional bulk molecularly imprinted

polymers through the monolithic MIPs

Hongyuan Yan et al (2004) [62] developed a Micellar Electro Kinetic

Chromatography (MEKC) method for the determination of NAT in animal plasma

by on-line sweeping technique in which plasma samples were simply

deproteinized with acetonitrile and analyzed with 16 mmolL NaH2PO4 + 6

mmolL Na2B4O7 + 60 mmolL sodium dodecyl sulfate (SDS) (pH 714) as the

running buffer a fused-silica capillary as the separation tube 21 kV as the running

voltage and UV detection at 214 nm Under these conditions more than 100-fold

enrichment of NAT was obtained with the good linear relation in the range of

nateglinide plasma concentration 02ndash7mgL (r = 0998) The method could be

applied successfully to determine trace drugs in clinical analysis

Steffen Bauer et al (2003) [63] developed a rapid and simple method for the

analysis of NAT in human plasma and validated with LOQ- 01 microgml is low

enough to allow determination of pharmacokinetic parameters of the substance

The intra-assay coefficients of variation ranged from 16 to 129 at NAT

concentrations of 05-75 microgml The inter-assay variation for the same plasma

concentrations ranged from 38 to 84 The calibration was linear in the range of

01-20 microgml To quantify NAT only 50 microl of plasma were needed Following

protein precipitation in human plasma the samples were separated by isocratic

reversed phase HPLC and analyzed using UV detection at 210 nm Sample

preparation time and analysis time are both short and allow rapid analysis of large

sample sets

318 Pioglitazone

Wanjari et al (2005) [64] developed a stability indicating simple rapid and

precise reversed-phase HPLC method for the quantitation of Pioglitazone (PIO) in

45

tablet on a Hypersil C-8 (250x46 mm) column using a mobile phase consisting

acetonitrile 015 vv triethylamine (4060 vv) adjusted to pH 46 with

orthophosphoric acid at a flow rate of 15 mlmin and detection at 220 nm The

retention time of PIO have been found to be 76 min and recoveries were between

99-101 Validation of the proposed method also been done

Zhongping John Lin et al (2003) [65] reported a LC-MSMS method for the

simultaneous determination of PIO and its two metabolites M-III (keto-derivative)

and M-IV (hydroxy-derivative) in human plasma Human plasma samples of 02

ml were extracted by a single step LLE procedure and analyzed using a HPLC-

electrospray (ESI) tandem mass spectrometer system The compounds were eluted

isocratically on a C-18 column ionized using a positive ion atmospheric pressure

electrospray ionization source and analyzed using MRM mode The ion transitions

monitored were mz 3570134 for PIO mz 3710148 for M-III mz 3730150 for

M-IV and mz 4130178 for the IS The chromatographic run time was 25 min per

injection with retention times of 145 102 and 095 min for PIO M-III and M-IV

respectively The calibration curves of PIO M-III and M-IV were well fit over the

range of 05-2000 ngml (r2 gt 0998759) by using a weighted (1x2) quadratic

regression The inter-day precisions of the quality control samples were le105

(N=15) coefficient of variation and the inter-day accuracy ( nominal) ranged

from 846 to 1035 for PIO 944 to 1040 for M-III and 968 to 1010 for M-

IV All three analytes demonstrated acceptable short-term long-term and

freezethaw stability

Radha Krishna et al (2002) [66] developed an HPLC and MEKC methods have

been developed for the determination of PIO its unsaturated impurity were

separated by MEKC in less than 7 min using a 43 cm times 50 microm id uncoated fused-

silica capillary with extended light path for better sensitivity (25 kV at 30 degC) and

a Back Ground Electrolyte (BGE) consisting of 20 acetonitrile (vv) in 20 mM

sodium borate buffer pH 93 containing 50 mM Sodium Dodecyl Sulphate (SDS)

The influence of various parameters on the separation such as pH of the buffer

46

SDS concentration buffer concentration organic modifiers temperature and

voltage were investigated The MEKC method was compared with HPLC method

using 5 microm symmetry C18 column (250times46 mm id) eluted with a mobile phase

consisting of a mixture of 50 (vv) acetonitrile and 10 mM potassium dihydrogen

phosphate buffer adjusting the pH to 60 with 01 M KOH The HPLC method is

capable of detecting all process related compounds which may be present at trace

levels in finished products

Kenji Yamashita et al (1996) [67] developed a HPLC method for the

simultaneous determination of PIO and its metabolites (M-I to M-V) in human

serum and urine The method for serum involved the solid-phase (SPE) and LLE

extraction Urine with and without enzymatic hydrolysis using β-glucuronidase

was treated with LLE The compounds in the extract were analyzed using HPLC

with UV detection at 269 nm The detection limits of PIO M-I M-II M-Ill M-IV

and M-V in serum were 001-005 microgml those in urine were 01-05 microgml and

those in urine after enzymatic hydrolysis were 03-05 microgml respectively

Zhong et al (1996) [68] reported a HPLC method for the simultaneous

determination of PIO and its potential metabolites (M-I to M-6) in human serum

The method involved a SPE of PIO its metabolites and the IS from serum using

C18 SPE columns with an elution solvent of 05 ml of acetonitrile-water (3565

vv) Separation of the eight analytes was achieved within 20 min using a reversed-

phase Zorbax RX-Cs analytical column with a mobile phase of acetonitrile-water

(4060 vv) containing acetic acid 3 mlL (apparent pH 55) An UV detector

operated at 269 nm was used with a linear response observed from 002 to 2 microgml

for these analytes except for M-4 which was best fitted with a polynomial

regression LOQ was found to be 002 microgml for PIO M-3 M-5 and M-6 004

microgml for M-2 and M-4 and 05 microgml for M-I when using a 05 ml serum sample

for extraction Intra- and inter-assay precision was le 9 and accuracy ranged from

-82 to 134 for all analytes were obtained from the method validation

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

43

inter-assay precision ( CV lt16) and an overall accuracy ( relative error) lt

-34

Jolly M Sankalia et al (2007) [60] reported a rapid simple and sensitive HPLC

method with UV detection for the quantification of NAT from rabbit plasma The

retention behavior of NAT and GLZ (IS) as a function of mobile phase pH

composition and flow rate was investigated Separation was developed on a

reverse-phase C18 column (250 times 46 mm id 5 microm) using a mixture of

acetonitrile 10 mM phosphate buffer (pH 30) in the ratio of 7030 ( vv) at a

flow rate of 10 mlmin with UV detection at 203 nm within 8 min and quantified

based on drugIS peak area ratios The plasma samples were prepared by a simple

deproteinization with a mixture of methanol and acetonitrile yielding more than

9786 extraction efficiencies The calibration curve was linear (r2 = 09984) in

the concentration range of 10ndash2500 ngml The LOD and LOQ were found to be

291 and 970 ngml respectively Both the intra-day and inter-day precisions at

four tested concentrations were below 132 RSD

Junfa Yin et al (2005) [61] reported a rapid and efficient chiral separation

method of NAT and its L-enantiomer on a monolithic Molecularly Imprinted

Polymers (MIP) The enantiomers were rapidly separated on this novel monolithic

MIP based Chiral Stationary Phase (MIP-CSP) whereas the enantioseparation was

not obtained on the non-imprinted polymer (NIP) Chiral recognition was found to

be dependent on the stereo structures and the arrangement of functional groups of

the imprinted molecule and the cavities on MIP Thermodynamic data (∆∆H and

∆∆S) obtained by Vanrsquot Hoff plots revealed an enthalpy-controlled

enantioseparation The binding capacity was evaluated by frontal analysis

Monolithic NAT-MIP had an effective number of binding sites Bt = 4115 micromolg

with a dissociation constant of Kd = 740 mM The morphological characteristics of

the monolithic MIP were investigated by pore analysis and Scanning Electron

Microscope (SEM) Results showed that both mesopores and macropores were

formed in the monolith Over all this study presents a new and practical possibility

44

for providing high rates of mass transfer fast separations and high efficiencies

without the pressure constraints of the traditional bulk molecularly imprinted

polymers through the monolithic MIPs

Hongyuan Yan et al (2004) [62] developed a Micellar Electro Kinetic

Chromatography (MEKC) method for the determination of NAT in animal plasma

by on-line sweeping technique in which plasma samples were simply

deproteinized with acetonitrile and analyzed with 16 mmolL NaH2PO4 + 6

mmolL Na2B4O7 + 60 mmolL sodium dodecyl sulfate (SDS) (pH 714) as the

running buffer a fused-silica capillary as the separation tube 21 kV as the running

voltage and UV detection at 214 nm Under these conditions more than 100-fold

enrichment of NAT was obtained with the good linear relation in the range of

nateglinide plasma concentration 02ndash7mgL (r = 0998) The method could be

applied successfully to determine trace drugs in clinical analysis

Steffen Bauer et al (2003) [63] developed a rapid and simple method for the

analysis of NAT in human plasma and validated with LOQ- 01 microgml is low

enough to allow determination of pharmacokinetic parameters of the substance

The intra-assay coefficients of variation ranged from 16 to 129 at NAT

concentrations of 05-75 microgml The inter-assay variation for the same plasma

concentrations ranged from 38 to 84 The calibration was linear in the range of

01-20 microgml To quantify NAT only 50 microl of plasma were needed Following

protein precipitation in human plasma the samples were separated by isocratic

reversed phase HPLC and analyzed using UV detection at 210 nm Sample

preparation time and analysis time are both short and allow rapid analysis of large

sample sets

318 Pioglitazone

Wanjari et al (2005) [64] developed a stability indicating simple rapid and

precise reversed-phase HPLC method for the quantitation of Pioglitazone (PIO) in

45

tablet on a Hypersil C-8 (250x46 mm) column using a mobile phase consisting

acetonitrile 015 vv triethylamine (4060 vv) adjusted to pH 46 with

orthophosphoric acid at a flow rate of 15 mlmin and detection at 220 nm The

retention time of PIO have been found to be 76 min and recoveries were between

99-101 Validation of the proposed method also been done

Zhongping John Lin et al (2003) [65] reported a LC-MSMS method for the

simultaneous determination of PIO and its two metabolites M-III (keto-derivative)

and M-IV (hydroxy-derivative) in human plasma Human plasma samples of 02

ml were extracted by a single step LLE procedure and analyzed using a HPLC-

electrospray (ESI) tandem mass spectrometer system The compounds were eluted

isocratically on a C-18 column ionized using a positive ion atmospheric pressure

electrospray ionization source and analyzed using MRM mode The ion transitions

monitored were mz 3570134 for PIO mz 3710148 for M-III mz 3730150 for

M-IV and mz 4130178 for the IS The chromatographic run time was 25 min per

injection with retention times of 145 102 and 095 min for PIO M-III and M-IV

respectively The calibration curves of PIO M-III and M-IV were well fit over the

range of 05-2000 ngml (r2 gt 0998759) by using a weighted (1x2) quadratic

regression The inter-day precisions of the quality control samples were le105

(N=15) coefficient of variation and the inter-day accuracy ( nominal) ranged

from 846 to 1035 for PIO 944 to 1040 for M-III and 968 to 1010 for M-

IV All three analytes demonstrated acceptable short-term long-term and

freezethaw stability

Radha Krishna et al (2002) [66] developed an HPLC and MEKC methods have

been developed for the determination of PIO its unsaturated impurity were

separated by MEKC in less than 7 min using a 43 cm times 50 microm id uncoated fused-

silica capillary with extended light path for better sensitivity (25 kV at 30 degC) and

a Back Ground Electrolyte (BGE) consisting of 20 acetonitrile (vv) in 20 mM

sodium borate buffer pH 93 containing 50 mM Sodium Dodecyl Sulphate (SDS)

The influence of various parameters on the separation such as pH of the buffer

46

SDS concentration buffer concentration organic modifiers temperature and

voltage were investigated The MEKC method was compared with HPLC method

using 5 microm symmetry C18 column (250times46 mm id) eluted with a mobile phase

consisting of a mixture of 50 (vv) acetonitrile and 10 mM potassium dihydrogen

phosphate buffer adjusting the pH to 60 with 01 M KOH The HPLC method is

capable of detecting all process related compounds which may be present at trace

levels in finished products

Kenji Yamashita et al (1996) [67] developed a HPLC method for the

simultaneous determination of PIO and its metabolites (M-I to M-V) in human

serum and urine The method for serum involved the solid-phase (SPE) and LLE

extraction Urine with and without enzymatic hydrolysis using β-glucuronidase

was treated with LLE The compounds in the extract were analyzed using HPLC

with UV detection at 269 nm The detection limits of PIO M-I M-II M-Ill M-IV

and M-V in serum were 001-005 microgml those in urine were 01-05 microgml and

those in urine after enzymatic hydrolysis were 03-05 microgml respectively

Zhong et al (1996) [68] reported a HPLC method for the simultaneous

determination of PIO and its potential metabolites (M-I to M-6) in human serum

The method involved a SPE of PIO its metabolites and the IS from serum using

C18 SPE columns with an elution solvent of 05 ml of acetonitrile-water (3565

vv) Separation of the eight analytes was achieved within 20 min using a reversed-

phase Zorbax RX-Cs analytical column with a mobile phase of acetonitrile-water

(4060 vv) containing acetic acid 3 mlL (apparent pH 55) An UV detector

operated at 269 nm was used with a linear response observed from 002 to 2 microgml

for these analytes except for M-4 which was best fitted with a polynomial

regression LOQ was found to be 002 microgml for PIO M-3 M-5 and M-6 004

microgml for M-2 and M-4 and 05 microgml for M-I when using a 05 ml serum sample

for extraction Intra- and inter-assay precision was le 9 and accuracy ranged from

-82 to 134 for all analytes were obtained from the method validation

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

44

for providing high rates of mass transfer fast separations and high efficiencies

without the pressure constraints of the traditional bulk molecularly imprinted

polymers through the monolithic MIPs

Hongyuan Yan et al (2004) [62] developed a Micellar Electro Kinetic

Chromatography (MEKC) method for the determination of NAT in animal plasma

by on-line sweeping technique in which plasma samples were simply

deproteinized with acetonitrile and analyzed with 16 mmolL NaH2PO4 + 6

mmolL Na2B4O7 + 60 mmolL sodium dodecyl sulfate (SDS) (pH 714) as the

running buffer a fused-silica capillary as the separation tube 21 kV as the running

voltage and UV detection at 214 nm Under these conditions more than 100-fold

enrichment of NAT was obtained with the good linear relation in the range of

nateglinide plasma concentration 02ndash7mgL (r = 0998) The method could be

applied successfully to determine trace drugs in clinical analysis

Steffen Bauer et al (2003) [63] developed a rapid and simple method for the

analysis of NAT in human plasma and validated with LOQ- 01 microgml is low

enough to allow determination of pharmacokinetic parameters of the substance

The intra-assay coefficients of variation ranged from 16 to 129 at NAT

concentrations of 05-75 microgml The inter-assay variation for the same plasma

concentrations ranged from 38 to 84 The calibration was linear in the range of

01-20 microgml To quantify NAT only 50 microl of plasma were needed Following

protein precipitation in human plasma the samples were separated by isocratic

reversed phase HPLC and analyzed using UV detection at 210 nm Sample

preparation time and analysis time are both short and allow rapid analysis of large

sample sets

318 Pioglitazone

Wanjari et al (2005) [64] developed a stability indicating simple rapid and

precise reversed-phase HPLC method for the quantitation of Pioglitazone (PIO) in

45

tablet on a Hypersil C-8 (250x46 mm) column using a mobile phase consisting

acetonitrile 015 vv triethylamine (4060 vv) adjusted to pH 46 with

orthophosphoric acid at a flow rate of 15 mlmin and detection at 220 nm The

retention time of PIO have been found to be 76 min and recoveries were between

99-101 Validation of the proposed method also been done

Zhongping John Lin et al (2003) [65] reported a LC-MSMS method for the

simultaneous determination of PIO and its two metabolites M-III (keto-derivative)

and M-IV (hydroxy-derivative) in human plasma Human plasma samples of 02

ml were extracted by a single step LLE procedure and analyzed using a HPLC-

electrospray (ESI) tandem mass spectrometer system The compounds were eluted

isocratically on a C-18 column ionized using a positive ion atmospheric pressure

electrospray ionization source and analyzed using MRM mode The ion transitions

monitored were mz 3570134 for PIO mz 3710148 for M-III mz 3730150 for

M-IV and mz 4130178 for the IS The chromatographic run time was 25 min per

injection with retention times of 145 102 and 095 min for PIO M-III and M-IV

respectively The calibration curves of PIO M-III and M-IV were well fit over the

range of 05-2000 ngml (r2 gt 0998759) by using a weighted (1x2) quadratic

regression The inter-day precisions of the quality control samples were le105

(N=15) coefficient of variation and the inter-day accuracy ( nominal) ranged

from 846 to 1035 for PIO 944 to 1040 for M-III and 968 to 1010 for M-

IV All three analytes demonstrated acceptable short-term long-term and

freezethaw stability

Radha Krishna et al (2002) [66] developed an HPLC and MEKC methods have

been developed for the determination of PIO its unsaturated impurity were

separated by MEKC in less than 7 min using a 43 cm times 50 microm id uncoated fused-

silica capillary with extended light path for better sensitivity (25 kV at 30 degC) and

a Back Ground Electrolyte (BGE) consisting of 20 acetonitrile (vv) in 20 mM

sodium borate buffer pH 93 containing 50 mM Sodium Dodecyl Sulphate (SDS)

The influence of various parameters on the separation such as pH of the buffer

46

SDS concentration buffer concentration organic modifiers temperature and

voltage were investigated The MEKC method was compared with HPLC method

using 5 microm symmetry C18 column (250times46 mm id) eluted with a mobile phase

consisting of a mixture of 50 (vv) acetonitrile and 10 mM potassium dihydrogen

phosphate buffer adjusting the pH to 60 with 01 M KOH The HPLC method is

capable of detecting all process related compounds which may be present at trace

levels in finished products

Kenji Yamashita et al (1996) [67] developed a HPLC method for the

simultaneous determination of PIO and its metabolites (M-I to M-V) in human

serum and urine The method for serum involved the solid-phase (SPE) and LLE

extraction Urine with and without enzymatic hydrolysis using β-glucuronidase

was treated with LLE The compounds in the extract were analyzed using HPLC

with UV detection at 269 nm The detection limits of PIO M-I M-II M-Ill M-IV

and M-V in serum were 001-005 microgml those in urine were 01-05 microgml and

those in urine after enzymatic hydrolysis were 03-05 microgml respectively

Zhong et al (1996) [68] reported a HPLC method for the simultaneous

determination of PIO and its potential metabolites (M-I to M-6) in human serum

The method involved a SPE of PIO its metabolites and the IS from serum using

C18 SPE columns with an elution solvent of 05 ml of acetonitrile-water (3565

vv) Separation of the eight analytes was achieved within 20 min using a reversed-

phase Zorbax RX-Cs analytical column with a mobile phase of acetonitrile-water

(4060 vv) containing acetic acid 3 mlL (apparent pH 55) An UV detector

operated at 269 nm was used with a linear response observed from 002 to 2 microgml

for these analytes except for M-4 which was best fitted with a polynomial

regression LOQ was found to be 002 microgml for PIO M-3 M-5 and M-6 004

microgml for M-2 and M-4 and 05 microgml for M-I when using a 05 ml serum sample

for extraction Intra- and inter-assay precision was le 9 and accuracy ranged from

-82 to 134 for all analytes were obtained from the method validation

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

45

tablet on a Hypersil C-8 (250x46 mm) column using a mobile phase consisting

acetonitrile 015 vv triethylamine (4060 vv) adjusted to pH 46 with

orthophosphoric acid at a flow rate of 15 mlmin and detection at 220 nm The

retention time of PIO have been found to be 76 min and recoveries were between

99-101 Validation of the proposed method also been done

Zhongping John Lin et al (2003) [65] reported a LC-MSMS method for the

simultaneous determination of PIO and its two metabolites M-III (keto-derivative)

and M-IV (hydroxy-derivative) in human plasma Human plasma samples of 02

ml were extracted by a single step LLE procedure and analyzed using a HPLC-

electrospray (ESI) tandem mass spectrometer system The compounds were eluted

isocratically on a C-18 column ionized using a positive ion atmospheric pressure

electrospray ionization source and analyzed using MRM mode The ion transitions

monitored were mz 3570134 for PIO mz 3710148 for M-III mz 3730150 for

M-IV and mz 4130178 for the IS The chromatographic run time was 25 min per

injection with retention times of 145 102 and 095 min for PIO M-III and M-IV

respectively The calibration curves of PIO M-III and M-IV were well fit over the

range of 05-2000 ngml (r2 gt 0998759) by using a weighted (1x2) quadratic

regression The inter-day precisions of the quality control samples were le105

(N=15) coefficient of variation and the inter-day accuracy ( nominal) ranged

from 846 to 1035 for PIO 944 to 1040 for M-III and 968 to 1010 for M-

IV All three analytes demonstrated acceptable short-term long-term and

freezethaw stability

Radha Krishna et al (2002) [66] developed an HPLC and MEKC methods have

been developed for the determination of PIO its unsaturated impurity were

separated by MEKC in less than 7 min using a 43 cm times 50 microm id uncoated fused-

silica capillary with extended light path for better sensitivity (25 kV at 30 degC) and

a Back Ground Electrolyte (BGE) consisting of 20 acetonitrile (vv) in 20 mM

sodium borate buffer pH 93 containing 50 mM Sodium Dodecyl Sulphate (SDS)

The influence of various parameters on the separation such as pH of the buffer

46

SDS concentration buffer concentration organic modifiers temperature and

voltage were investigated The MEKC method was compared with HPLC method

using 5 microm symmetry C18 column (250times46 mm id) eluted with a mobile phase

consisting of a mixture of 50 (vv) acetonitrile and 10 mM potassium dihydrogen

phosphate buffer adjusting the pH to 60 with 01 M KOH The HPLC method is

capable of detecting all process related compounds which may be present at trace

levels in finished products

Kenji Yamashita et al (1996) [67] developed a HPLC method for the

simultaneous determination of PIO and its metabolites (M-I to M-V) in human

serum and urine The method for serum involved the solid-phase (SPE) and LLE

extraction Urine with and without enzymatic hydrolysis using β-glucuronidase

was treated with LLE The compounds in the extract were analyzed using HPLC

with UV detection at 269 nm The detection limits of PIO M-I M-II M-Ill M-IV

and M-V in serum were 001-005 microgml those in urine were 01-05 microgml and

those in urine after enzymatic hydrolysis were 03-05 microgml respectively

Zhong et al (1996) [68] reported a HPLC method for the simultaneous

determination of PIO and its potential metabolites (M-I to M-6) in human serum

The method involved a SPE of PIO its metabolites and the IS from serum using

C18 SPE columns with an elution solvent of 05 ml of acetonitrile-water (3565

vv) Separation of the eight analytes was achieved within 20 min using a reversed-

phase Zorbax RX-Cs analytical column with a mobile phase of acetonitrile-water

(4060 vv) containing acetic acid 3 mlL (apparent pH 55) An UV detector

operated at 269 nm was used with a linear response observed from 002 to 2 microgml

for these analytes except for M-4 which was best fitted with a polynomial

regression LOQ was found to be 002 microgml for PIO M-3 M-5 and M-6 004

microgml for M-2 and M-4 and 05 microgml for M-I when using a 05 ml serum sample

for extraction Intra- and inter-assay precision was le 9 and accuracy ranged from

-82 to 134 for all analytes were obtained from the method validation

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

46

SDS concentration buffer concentration organic modifiers temperature and

voltage were investigated The MEKC method was compared with HPLC method

using 5 microm symmetry C18 column (250times46 mm id) eluted with a mobile phase

consisting of a mixture of 50 (vv) acetonitrile and 10 mM potassium dihydrogen

phosphate buffer adjusting the pH to 60 with 01 M KOH The HPLC method is

capable of detecting all process related compounds which may be present at trace

levels in finished products

Kenji Yamashita et al (1996) [67] developed a HPLC method for the

simultaneous determination of PIO and its metabolites (M-I to M-V) in human

serum and urine The method for serum involved the solid-phase (SPE) and LLE

extraction Urine with and without enzymatic hydrolysis using β-glucuronidase

was treated with LLE The compounds in the extract were analyzed using HPLC

with UV detection at 269 nm The detection limits of PIO M-I M-II M-Ill M-IV

and M-V in serum were 001-005 microgml those in urine were 01-05 microgml and

those in urine after enzymatic hydrolysis were 03-05 microgml respectively

Zhong et al (1996) [68] reported a HPLC method for the simultaneous

determination of PIO and its potential metabolites (M-I to M-6) in human serum

The method involved a SPE of PIO its metabolites and the IS from serum using

C18 SPE columns with an elution solvent of 05 ml of acetonitrile-water (3565

vv) Separation of the eight analytes was achieved within 20 min using a reversed-

phase Zorbax RX-Cs analytical column with a mobile phase of acetonitrile-water

(4060 vv) containing acetic acid 3 mlL (apparent pH 55) An UV detector

operated at 269 nm was used with a linear response observed from 002 to 2 microgml

for these analytes except for M-4 which was best fitted with a polynomial

regression LOQ was found to be 002 microgml for PIO M-3 M-5 and M-6 004

microgml for M-2 and M-4 and 05 microgml for M-I when using a 05 ml serum sample

for extraction Intra- and inter-assay precision was le 9 and accuracy ranged from

-82 to 134 for all analytes were obtained from the method validation

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

47

319 Repaglinide

Abu Bakar Ruzilawati et al (2007) [69] developed and validated an HPLC

assay for determination of Repaglinide (REP) in human plasma for

pharmacokinetic studies Plasma samples containing REP and an IS Indomethacin

were extracted with ethylacetate at pH 74 The recovery of REP was 92 plusmn 5531

Chromatographic separations were performed on Purospherreg STAR C-18

analytical column (48times150 mm 5 microm) The mobile phase composed of

acetonitrilendashammonium formate (pH 27 001 M) (6040 vv) The flow rate was

1 mlmin The retention time for REP and IS were approximately 62 and 53 min

respectively Calibration curves of REP were linear in the concentration range of

20ndash200 ngml in plasma The LOQ and LOQ were 10 ngml and 20 ngml

respectively The inter-day precision was from 521 to 1184 and the intra-day

precision ranged from 390 to 667 The inter-day accuracy ranged 8995 to

10575 and intra-day accuracy ranged from 9237 to 10466

Vipul P Rane et al (2007) [70] developed and validated a simple isocratic

normal phase chiral HPLC method for the enantiomeric separation of REP

(S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonyl-

methyl]benzoic acid in bulk drug substance The enantiomers of REP were

resolved on a ChiralPak AD-H (amylose based stationary phase) column using a

mobile phase consisting of n-hexane 2-propanol trifluoroacetic acid (95502

vvv) at a flow rate of 10 mlmin The resolution between the enantiomers was

found to be not gt35 in optimized method The presence of trifluoroacetic acid in

the mobile phase played an important role in enhancing chromatographic

efficiency and resolution between the enantiomers The developed method was

extensively validated and proved to be robust The calibration curve for (R)-

enantiomer showed excellent linearity over the concentration range of 900 ngml

(LOQ) to 6000 ngml The LOD and LOQ for (R)-enantiomer were 300 and 900

ngml respectively The recovery of the (R)-enantiomer ranged between 983

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

48

and 10105 in bulk drug samples of REP Sample solution and mobile phase

were found to be stable up to 48 h

3110 Rosiglitazone

He et al (2007) [71] reported a sensitive and selective HPLCndashESIndashMSndashMS

method for determination of Rosiglitazone (ROS) in human plasma After the

addition of the IS plasma samples were precipitated by acetonitrile The

compounds were separated on a proC18 column using a mixture of ammonium

acetate buffer (002M pH 65) and acetonitrile (in the ratio of 4753 vv) as

mobile phase Linearity was established for the range of concentrations 1ndash1000

ngml with a coefficient of determination (r2) of 0999 The intra-day accuracy for

ROS ranged from 1100 to 992 at low medium and high levels The inter-day

accuracy was less than 15 The LLOQ was identified reproducible at 10 ngml

with a precision of 57 After validation the method was used to study the

pharmacokinetic profile of ROS in five healthy volunteers administered with a

single oral dose (40 mg) The proposed method enabled the unambiguous

evaluation and quantification of ROS for pharmacokinetic bioavailability or drugndash

drug interaction studies A possible chromatography peak (mz 121 its parent ion

mz 344) of N-demethyl- ROS was observed at 349 min during determining ROS

This may be also a potential method for simultaneous determination of ROS and

its metabolite N-demethyl- ROS concentrations in plasma

Chi-Chi Chou et al (2005) [72] evaluated solid-phase extraction combined with

LCndashMSMS to determine the trace amount of ROS in human urine The analytical

performance of four modes of LCndashMS and tandem MS operation (APCI ESI)

positive and negative ionization) was compared for two mass spectrometers a

triple-quadrupole and a quadrupole ion trap instrument ROS was extracted from

urine using a SPE cartridge of 50 mg C8 sorbent and acetonitrile used as the

eluting solvent Samples were then separated on a RP-18 column interfaced with a

tandem mass spectrometer The recovery of ROS was gt 912 The urine assay

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

49

combining SPE and LCndashAPCI-MSMS of triple-quadrupole was proved a very

selective and sensitive method for determination of trace ROS The assay was

linear over a wide range with a LLOQ of 01 ngml using 1ml of urine The intra-

and inter-day precisions were lt98 and lt79 respectively and the accuracies

were in the range 910ndash1036 The ROS concentration profile in human urine

was also determined The results of this study reveal the adequacy of SPEndashLCndash

APCI-MSMS method for analyzing ROS from diabetic patientsrsquo urines The

concentrations of ROS were detected to range from 760 to164 pgml

Patracuteıcia Gomes et al (2004) [73] reported a validated MEKC and HPLC

methods for the determination of ROS in coated tablet The electrophoretic

separation was performed in a fused-silica capillary of total length 480 cm

(effective length 395 cm 75 microm id) using 10 mM sodium tetraborate buffer (pH

90) containing 30 mM sodium dodecyl sulfate (SDS) as the Background

Electrolyte (BGE) The separating voltage used was of 20 kV at 25 oC and the

diode array detector was set at 247 nm The MEKC method was compared with

HPLC method using a RP-18 column (125 times 40mm id) eluted with a mobile

phase consisting of mixture of 25 mM KH2PO4 buffer and acetonitrile (5545 vv)

adjusting the pH to 62 with dilute KOH Statistical analysis by Studentrsquos t-test

showed no significant differences between the results obtained by two methods

The results indicated that MEKC can be used an alternative method to HPLC for

the determination of ROS in pharmaceutical dosage form

3111 Sitagliptin

Ramakrishna Nirogi et al (2008) [74] reported a sensitive HPLCndashpositive ion

ESIMSMS method for the quantification of Sitagliptin (SGP) Following LLE

the analytes were separated using an isocratic mobile phase on a reverse-phase

column and analyzed by MSMS in the MRM mode using the respective [M+H]+

ions mz 408ndash235 for SGP and mz 310ndash148 for the IS The assay exhibited a

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

50

linear dynamic range of 01ndash250 ngml for SGP in human plasma The LLOQ was

01 ngml with a RSD of lt 6

Wei Zeng et al (2008) [75] developed a High Turbulence Liquid

Chromatography (HTLC or Turbulent Flow online extraction) and tandem mass

spectrometry (MSMS) methods for the determination of SGP in human urine and

hemodialysate to support clinical studies A narrow bore large particle size

reversed-phase column (Cyclone 50 mm times 10 mm 60 microm) and a BDS Hypersil

C18 column (30 mm times 21 mm 3 microm) were used as extraction and analytical

columns respectively The LLOQ was 01 microgml for the urine assay the linear

calibration range was 01 to 50 microgml the inter-day precision ( RSD n = 5) was

23-65 and the accuracy was 969-106 of the nominal value For the urine QC

samples the intra-day precision ( RSD n = 5) and accuracy were 18ndash26 and

962ndash106 of the nominal value respectively The inter-day precision ( RSD)

for 56 sets of urine QC samples over a 6-month period varied from 38 to 55

and the accuracy from 102 to 105 of the nominal value The LLOQ was 001

ngml for the hemodialysate assay the linear dynamic range was 001-50 ngml

the inter-day precision was 16ndash41 and the accuracy was 898ndash104 of the

nominal value and the intra-day precision and accuracy varied from 23 to 89

and from 998 to 111 of the nominal value respectively

3112 Voglibose

Jong Soo Woo et al (2006) [76] developed and validated a highly sensitive liquid

chromatographic procedure with post-column derivatization using fluorescence

detection (LCndashFD) for the determination of Voglibose (VGB) in pharmaceutical

tablets Sample pre- treatment included a simple extraction and centrifugation

without pre-column derivatization Taurine and sodium periodate dissolved in

water was used as a post-column reagent Detection was performed at an excitation

wavelength of 350 nm and an emission wavelength of 430 nm LC separation was

carried out in less than 25 min In addition to the LC procedure with post-column

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

51

derivatization an LCndashMS assay procedure was also investigated for the analysis of

VGB without derivatization VGB was detected in an ESI mode with single ion

recording (SIR mz 2681) After selection of the optimum conditions both assay

methods were validated providing good performances with respect to precision

linearity and accuracy Linearity of both methods were obtained with an average

r2gt 0999 The LLOD were 94 and 18 ngml for LCndashFD and LCndashMS

respectively

32 Analytical methods for Anti-diabetic drugs in combinations

Deepti Jain et al (2008) [77] developed a simple precise rapid and

reproducible reverse-phase HPLC method for the simultaneous estimation of

MET PIO and GLM present in multi-component dosage forms Chromatography

is carried out isocratically at 25plusmn05 degC on an Inertsil-ODS-3 (C-18) Column (250

times 460 mm 5 microm) with a mobile phase composed of methanolndashphosphate buffer

(pH 43) in the ratio of 7525 vv at a flow rate of 1 mlmin Detection is carried

out using a UV-PDA detector at 258 nm Parameters such as linearity precision

accuracy recovery specificity and ruggedness are studied as reported in the

International Conference on Harmonization (ICH) guidelines The retention times

for MET PIO and GLM are 266 plusmn 05 min 712 plusmn 05 min and 1017 plusmn 05 min

respectively The linearity range and percentage recoveries for MET PIO and

GLM are 10ndash5000 10ndash150 and 1ndash10 microgml and 1004 10006 and 1002

respectively The correlation coefficients for all components are close to 1 The

RSD for three replicate measurements in three concentrations of samples in tablets

are always less than 2

Karthik et al (2008) [78] developed a simple fast and precise reverse phase

isocratic HPLC method for the separation and quantification of PIO and GLM in

bulk drug and pharmaceutical dosage form The quantification was carried out

using Inertsil ODS (250 times 46 mm 5micro) column and mobile phase comprised of

acetonitrile and ammonium acetate (pH 45 20 mM) in proportion of 6040 (vv)

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

52

The flow rate was 10 mlmin and the effluent was monitored at 230 nm The

retention time of PIO and GLM were 70plusmn01 and 102plusmn01 min respectively The

method was validated in terms of linearity precision accuracy and specificity

LOD and LOQ Linearity of PIO and GLM were in the range of 2-200 microgml and

05-50 microgml respectively The recoveries of both the drugs were 9985 and

10206 for PIO and GLM respectively from the tablet formulation

Bytul M Rahman et al (2007) [79] reported a simple rapid and precise method

for the simultaneous determination of MET and ROS in a combined

pharmaceutical dosage form The method is based on a reversed-phase column

shimpack CLC ODS (C18) 46times 250 5 microm using a mobile phase of phosphate

buffer and acetonitrile (6040 vv) adjusted the pH 45 plusmn 01 with 1 M NaOH or

diluted orthophosphoric acid The buffer used in the mobile phase contains

KH2PO4 and hexane-1 sulfonic acid sodium salt in double-distilled water The

instrumental settings are flow rate 1 mlmin column temperature at 40 oC and

detector wavelength of 268 nm Both the drugs were well resolved on the

stationary phase and the retention times were around 2 min for MET and 4 min for

ROS The correlation coefficients for MET and ROS are 099997 and 099946

respectively The RSD for six replicate measurements in two sets of each drug in

the tablets is always lt 2 and mean error of active recovery not more than plusmn

15 The method was validated for precision and accuracy

Ceren Yardımcı et al (2007) [80] reported a novel fast and simple liquid

chromatographic method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The separation was achieved on a phenyl column

(250x46 mm id 5microm) using a mobile phase composed of acetonitrile 10 mM

phosphate buffer pH 55 (7030 vv) The flow rate was 1 mlmin UV detection

was performed at 245 nm and Verapamil was used as internal standard The

developed method was validated in terms of stability specificity sensitivity

linearity accuracy precision and robustness The LOQ was 002 microgml for both

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

53

drugs The method developed was successfully applied to the simultaneous

determination of ROS and MET in pharmaceutical preparations

Hiren N Mistri et al (2007) [81] developed a simple and rapid LCndashMSMS

method for the simultaneous quantification of MET and GLB in human plasma

using GLM as IS After acidic acetonitrile-induced protein precipitation of the

plasma samples MET GLB and IS were chromatographed on reverse phase C18

(50times46 mm id 5 microm) analytical column Quantification was performed on a

triple quadrupole mass spectrometer employing ESI technique and operating in

MRM and positive ion mode The total chromatographic run time was 35 min and

calibration curves were linear over the concentration range of 20ndash2500 ngml for

MET and 5ndash500 ngml for GLB The method was validated for selectivity

sensitivity recovery linearity accuracy and precision dilution integrity and

stability studies The recoveries obtained for the analytes and IS (ge69) were

consistent and reproducible Inter-batch and intra-batch CV across four validation

runs (LLOQ LQC MQC and HQC) was less than 8 The accuracy determined at

these levels was within plusmn8 in terms of RE The method was applied to a

bioequivalence study of 500 mg MET and 5 mg of GLB tablet after oral

administration to 28 healthy human subjects under condition of fasting

Cun-Gang Ding et al (2007) [82] developed a method for the simultaneous

quantification of MET (I) and GLP (II) in human plasma It is based on HPLC

with ESIMSMS detection in positive ionization mode Phenformin (III) and GLC

(IV) were used as IS for MET and GLP respectively The MSMS detection was

performed in MRM mode The precursor-product ion combinations of mz

130rarr71 446rarr321 206rarr60 and 324rarr127 were used to quantify I II III and IV

respectively This method was validated in the concentration ranges of 002ndash4

microgml for I and 0004ndash08 microgml for II It was utilized to support a clinical

pharmacokinetic study after single dose oral administration of a combination of I

and II

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

54

Jing Yao et al (2007) [83] reported an isocratic reverse phase HPLC method for

screening counterfeit medicines and adulterated dietary supplement products The

developed method could be employed to separate and determine simultaneously

six anti-diabetic drugs (Glipizide Gliclazide Glibenclamide Glimepiride

Gliquidone Repaglinide) on an isocratic solvent system using an Alltima C18

column (5 microm 150times46 mm) with an isocratic mobile phase of methanolndash

phosphate buffer (pH 30 001 molL) (7030 vv) at a flow rate of 10 mlmin

and at a wavelength of 230 nm

Lu Zhang et al (2007) [84] developed a selective and sensitive electrospray LC-

MSMS method for simultaneous determination of MET and ROS in human

plasma using Phenformin as IS Plasma samples were precipitated by acetonitrile

and the analytes were separated on a prepacked Phenomenex Luna 5u CN 100A

(150times20 mm) column using a mobile phase comprised of methanol 30 mM

ammonium acetate pH 50 (8020 vv) delivered at 02 mlmin Detection was

performed on a Finnigan TSQ triple-quadrupole tandem mass spectrometer in

positive ion Selected Reaction Monitoring (SRM) mode using ESI technique The

ion transitions monitored were mz 13027 rarr 7111 for MET mz

35814 rarr 13507 for ROS and mz 20620 rarr 10519 for the IS The standard

curves were linear (r2 gt 099) over the concentration range of 5ndash3000 ngml for

MET and 15ndash500 ngml for ROS with acceptable accuracy and precision

respectively The within- and between-batch precisions were lt 15 RSD The

LOD of both MET and ROS was 1 ngml The method described is precise and

sensitive and has been successfully applied to the study of pharmacokinetics of

compound MET and ROS capsules in 12 healthy Chinese volunteers

Ghassempour et al (2006) [85] developed a method on HPTLC for the

simultaneous determination of MET and GLB in pharmaceutical formulations

Normal phase TLC plate (silica gel 60 F254) was used as stationary phase and

watermethanolammonium sulfate (2105 wv) as mobile phase to determine two

pharmaceutically active ingredients in three different formulations of Glucovance

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

55

This system gave a good resolution for MET (Rf value of 043 plusmn 001) and GLB (Rf

value of 064 plusmn 002) Determination was by densitometry in the absorbance mode

at 237 nm The linear regression data for the calibration plot showed a good

relationship with r=099581 and 099982 for MET and GLB respectively The

method was validated for precision and recovery The LOD and LOQ were 2524

and 8412 ngspot for MET and 1226 and 4086 ngspot for GLB respectively

Stability study has been carried out for samples and standard solutions

Venkatesh et al (2006) [86] described a convenient method for the separation

and simultaneous determination of six anti-diabetic drugs GLB GLC GLP PIO

REP and ROS in pharmaceutical formulations Also the assay has been shown

applied to support quantification of the six anti-diabetic drugs in human plasma

The analytes were either injected directly onto the column after suitable dilution

(pharmaceutical formulation analysis) or a simple extraction procedure using

acetonitrile from human plasma spiked with anti-diabetic drugs and Celecoxib

(IS) Ternary gradient elution at a flow rate of 1 mlmin was employed on an

Intersil ODS 3V column (46times250 mm 5 microm) at ambient temperature The mobile

phase consisted of 005 M formic acid (pH 30) acetonitrile Milli Q water and

methanol The six anti-diabetic drugs were monitored at a wavelength of 260 nm

The assay developed for formulation analysis was found to be accurate and

precise The calibration curves ranged from 01 to 100 microgml for all analytes with

the exception of GLB where the range was 03ndash100 microgml The plasma assay was

validated for parameters such as specificity accuracy and extraction recovery The

proposed method is simple selective and can be extended for routine analysis of

anti-diabetics in pharmaceutical preparations and in biological matrices

Aburuz et al (2005) [87] described the development of SPE and HPLC methods

for the simultaneous determination of MET and GLP GLC GLB or GLM in

plasma Several extraction and HPLC methods have been described previously for

the determination of each of these analytes in plasma separately The simultaneous

determination of these analytes is important for the routine monitoring of diabetic

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

56

patients who take combination medications and for studying the pharmacokinetics

of the combined dosage forms In addition this developed method can serve as a

standard method for the plasma determination of these analytes therefore saving

time effort and money The recoveries of the developed methods were found to be

between 763 and 1019 The LOQrsquos were between 5 and 225 ngml The

intra-day and inter-day precision ( CV) was always lt 9 The accuracy ( RE)

was always lt 12 Stability analysis showed that all analytes are stable for at least

3 months when stored at -70 oC

Anna Gumieniczek et al (2005) [88] investigated the TLC behavior of PIO

ROS and REP Chemically bonded cyanopropyl plates with mobile phases

comprising 14-dioxane with phosphate buffers were used for reversed phase

chromatography The influence of the pH on the separation of the drugs was also

examined Then a simple rapid and stability-indicating HPTLC method has been

developed and validated for the quantitative determination of PIO in tablets

Analysis was performed with 14-dioxane-phosphate buffer of pH 44 (55) as the

mobile phase Detection and quantification were performed by classical

densitometry at the wavelength of maximum absorption of PIO 266 nm A

calibration plot was constructed in the range of 04-24 microg10 microl and was linear

with a good correlation coefficient (r = 09957) Precision was validated by

replicate analyses of standard solutions and accuracy by analysis of fortified

samples The precision of the proposed chromatographic method expressed as

mean RSD was 499 and 257 respectively for the lowest and the highest

calibration levels Recovery from the fortified samples ranged from 9809 to

10328 The mean (plusmnSD) recovery from tablets was 9979 plusmn 157

Ceren Yardımcı et al (2005) [89] reported a novel and simple capillary zone

electrophoresis method for the simultaneous determination of ROS and MET in

pharmaceutical preparations The optimum separation for these analytes was

achieved in lt9 min at 25 oC with a fused-silica capillary column (805 cm times 75 microm

id effective length 720 cm) and a running buffer containing 25 mM acetate

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

57

buffer at pH 40 The samples were injected hydrodynamically for 3 sec at 50 mbar

and the applied voltage was +250 kV Detection wavelength was set at 203 nm

Verapamil was used as IS The method was suitably validated with respect to

specificity linearity LOD and LOQ accuracy precision and robustness The

LOD and LOQ were 05 and 10 microgml for ROS and MET respectively

Guo-ping Zhong et al (2005) [90] reported a rapid and sensitive LCMSMS

method to simultaneously determine GLC and MET in human plasma using

huperzine A (IS) After acetonitrile-induced protein precipitation of the plasma

samples GLC MET and the IS were subjected to LCMSMS analysis using

electro-spray ionization (ESI) Chromatographic separation was performed on a

Hypersil BDS C18 column (50times21 mm id 3 microm) The method had a

chromatographic running time of 20 min and linear calibration curves over the

concentration ranges of 10ndash10000 ngml for GLC and 78ndash46789 ngml for MET

The recoveries of the method were found to be 71ndash104 The LLOQ of the

method were 100 and 78 ngml for GLC and MET respectively The intra- and

inter day precision was less than 15 for all quality control samples at

concentrations of 100 500 and 2000 ngml The validated LCMSMS method has

been used to study bioequivalence in healthy volunteers These results indicate that

the method was efficient with a very short running time (20 min) for MET and

GLC The method was used in clinical bioequivalence study

Kolte et al (2005) [91] developed a simple rapid and precise reversed-phase LC

method for the simultaneous determination of MET in combination with GLM

Good separation of the analytes was achieved in short analysis time under the

developed conditions Several parameters affecting the separation of the analytes

were studied including pH and the concentration of SDS The method is validated

and shown to be linear in the range of 25-150 microgml for MET and 01-06 microgml

for GLM The method is applied for the analysis of these analytes in commercially

available tablets

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

58

Shankar Madhira et al (2005) [92] developed two simple and accurate methods

of analysis for the determination of PIO and MET in combined dosage forms using

second-derivative spectrophotometry and reverse-phase LC Tablets were

quantified using the second-derivative responses at 22755 nm for PIO and 25725

nm for MET in spectra of their solutions in a mixture of methanol and acetonitrile

(3070) The calibration curves were linear [(r) = 09984 for PIO and 09986 for

MET] in the concentration range of 8-40 microgml for PIO and 4-12 microgml for MET

In the LC method analysis was performed on a Hypersil ODS-C18 column with

5microm particle size using the mobile phase acetonitrile-water-acetic acid (752503)

adjusted to pH 55 with ammonia at a flow rate of 05 mlmin Measurement was

made at a wavelength of 230 nm Both the drugs were well resolved on the

stationary phase and the retention times were 85 min for PIO and 160 min for

MET The calibration curves were linear (r= 09933 for PIO and 09958 for MET)

in the concentration range of 4-20 microgml for PIO and MET Both methods were

validated and the results were compared statistically

Emmie NM Ho et al (2004) [93] described a convenient method for the

separation and simultaneous detection of 10 anti-diabetic drugs (namely GLP

GLB GLM GLC Tolazamide (TLZ) Tolbutamide (TLB) NAT REP ROS and

PIO) in equine plasma and urine by LCndashMS-MS The anti-diabetics were isolated

from equine plasma and urine by LLE with 12-dichloroethane at acidic pH and

analysed by LCndashMS-MS in the positive electro-spray ionization mode Separation

of 10 anti-diabetic drugs was achieved with a reversed phase C8 column using a

mixture of aqueous ammonium formate (pH 30 10mM) and methanol as the

mobile phase GLB and its metabolites were also quantified using the same

method The method is sensitive to detect 10 ngml concentration of each drug

The inter-day precision of the peak area ratios were about 20ndash30 and those of the

relative retention times were lt 031 for all targeted drugs except for ROS which

gave RSD of 33 for the peak area ratios and 077 for the relative retention

time

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET

59

Sane et al (2004) [94] reported a rapid and accurate HPLC method for the

simultaneous determination of PIO and GLM Chromatographic separation of the

two pharmaceuticals was performed on a Cosmosil C18 column (150times46 mm 5

microm) with a 453520 (vv) mixture of 001 mM triammonium citrate (pH adjusted

to 695 with orthophosphoric acid) acetonitrile and methanol as mobile phase at a

flow rate of 1 mlmin and detection at 228 nm Separation was complete in less

than 10 min The method was validated for linearity accuracy precision LOQ

and robustness Linearity accuracy and precision were found to be acceptable

over the ranges 250ndash3000 microgml for PIO and 010ndash1000 microgml for GLM

Kolte et al (2003) [95] developed HPLC procedure for the simultaneous

determination of MET in combination with GLP and GLC in pharmaceutical

preparations A Zorbax XDB C18 15 cm analytical column and UV detection at

wavelength 226 nm were used In this study various mobile phase variables were

studied to determine the effects that each had on MET GLP and GLC The

reproducibilities in combination I for MET and GLP were 139 130 and in

combination II for MET and GLC were 128 175 respectively The

determinations of combination I and combination II gave recoveries with respect

to the values declared by the manufacturers are in the range 961ndash1034 950ndash

1000 using peak areas and 964ndash1036 973ndash1025 using peak heights

respectively for GLP and MET The recoveries for the combination II were in the

range 947ndash1008 962ndash1012 using peak areas and 943ndash1015 978ndash

1025 using peak heights respectively for GLC and MET