recent developments of achiral hplc methods in...

Recent Developments of Achiral HPLCMethods in Pharmaceuticals Using

Various Detection Modes

Theresa K. Natishan*

Merck & Co., Inc., Merck Research Laboratories,

Rahway, New Jersey, USA

ABSTRACT

High performance liquid chromatography (HPLC) is used extensively in

the pharmaceutical industry due to the availability of fully automated

systems, excellent quantitative precision, accuracy, broad linear dynamic

range, and availability of a wide variety of column stationary phases. The

technique is used in drug discovery, pre-clinical, and clinical develop-

ment and factory finished product analysis. HPLC has greatly developed

through the years in terms of convenience, speed, increased selection of

column stationary phases, high sensitivity, applicability to a broad variety

1237

DOI: 10.1081/JLC-120030603 1082-6076 (Print); 1520-572X (Online)

Copyright # 2004 by Marcel Dekker, Inc. www.dekker.com

*Correspondence: Theresa K. Natishan, Merck & Co., Inc., Merck Research Labora-

tories, RY818-C215, P.O. Box 2000, Rahway, NJ 07065, USA; E-mail:

[email protected].

JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIESw

Vol. 27, Nos. 7–9, pp. 1237–1316, 2004

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of sample matrices, and ability to hyphenate the chromatographic method

to spectroscopic detectors.

Key Words: Reverse phase HPLC; Electrochemical detectors; Active

pharmaceutical ingredient; Formulation; Impurity profile method;

Stability indicating method; Fast HPLC.

INTRODUCTION

Reverse phase high performance liquid chromatography (RP-HPLC) is by

far the most widely used mode of modern liquid chromatography in pharma-

ceuticals. Stationary phases with C8 and C18 functionalities are most com-

monly used in pharmaceutical analysis with ultraviolet (UV) detection.

Other alternate column chemistries used in the reverse phase mode include

phenyl, cyano, monolith, zirconia, porous graphitized carbon, and fluorinated

columns. Other HPLC detectors used for pharmaceuticals include fluor-

escence (FL), chemiluminescence (CL), electrochemical (ECD), evaporative

light scattering (ELSD), and refractive index (RI). Other modes of HPLC

used in pharmaceutical analysis are normal phase HPLC (NP-HPLC), ion-

exchange HPLC (IEX-HPLC), size-exclusion chromatography (SEC), micel-

lar (MLC), and hydrophilic interaction chromatography (HILIC). These

modes have been previously discussed in detail.[1] The use of molecular

imprinted polymers (MIP) has also been applied recently[2] for specific

recognition of target molecules. Hyphenation of HPLC with spectroscopic

detectors[3] such as mass spectrometry (MS), nuclear magnetic resonance

(NMR), inductively coupled plasma mass spectrometry (ICP/MS), and infra-

red spectroscopy (IR) have been used for structural elucidation of pharma-

ceutical compounds.

The analytical chemist in the pharmaceutical industry plays a major role

in developing analytical methods that ensure the safety, efficacy, purity, stab-

ility, and quality of active pharmaceutical ingredient (API) and formulated

drug products. Regulatory requirements are increasing and HPLC method-

ology plays a critical role in ensuring the ruggedness and accuracy of methods

used in the pharmaceutical industry. Rapid chromatographic methods in fast

HPLC have made significant progress in reduction of analysis times and man-

agement of high throughput analysis. Miniaturization of HPLC columns in

applications using micro-HPLC methods has allowed for coupling of HPLC

systems with spectroscopic detectors, small sample sizes, and reduced solvent

consumption for pharmaceutical analysis.

The functional types of HPLC methods, which are typically used in phar-

maceuticals are assay, impurity profile, stability, in-process, and cleaning

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methods. The pharmaceutical HPLC methods will be reviewed and recently

published applications described. The intent of the review is to focus on recent

developments of HPLC achiral methods in pharmaceuticals using different

detection modes. The literature is extensive in this field and the references

cited are only a fraction of the literature published in the past few years.

Additional references can be found in the publications cited.

HPLC WITH UV DETECTION

Assay Method

The assay test is used to determine the concentration of an active ingre-

dient present in a dosage form or the purity of an API. HPLC is typically

chosen as the method of choice for the assay test due to its specificity.

There are numerous examples in the literature of assay methods in pharmaceu-

ticals using RP-HPLC with UV detection.[4–49] Typical samples, which are

analyzed by an HPLC assay method in pharmaceuticals are formulations, bio-

logicals, and proteins.

Formulation

Most of the assay methods used for formulation analysis use either exter-

nal or internal standard analysis with isocratic method conditions for ease of

method transfer to quality control (QC) laboratories. Gradient elution is not

performed to reduce timecycles as column re-equilibration is not required

and baseline disturbances are minimized. Table 1 gives some recent appli-

cations of assay HPLC methods used in the analysis of formulations. Advan-

tages and different strategies to obtain method selectivity of selected methods

given in Table 1 are described.

Method selectivity is critical for an assay method. The method should

resolve the API from any other potential impurity. An assay method selective

for the API and its synthetic precursors, intermediates, and degradates was

developed by Qi et al.[4] A simple and accurate HPLC assay using external

standard quantification for determination of oxcarbazepine in a tablet formu-

lation was developed (method conditions shown in Table 1). The method was

selective for oxcarbazepine, its synthetic precursors, intermediates, and

degradates.

A different strategy to increase method selectivity was demonstrated by

Zawilla et al.[5] A sensitive and reproducible assay method (refer to Table 1

for method conditions) was developed for meloxicam in API and pharmaceu-

tical formulations. The HPLC method is selective and the concentrations of

Developments of Achiral HPLC Methods in Pharmaceuticals 1239

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Table

1.

HPLC-U

Vassaymethodsusedforthequantitativeanalysisofform

ulations.

Analyte,form

ulation

Method

Column

Mobilephase

Quantification

UV

detection

wavelength

Reference

Oxcarbazepinein

tablets

RP-H

PLC

isocratic,

15min

DiamonsilC18

(150mm

�5mm,

5mm)

Acetonitrile:potassium

phosphatemonobasic

(aq)pH

6.8

External

standard

255nm

[4]

Meloxicam

intabletsand

suppositories

RP-H

PLC

isocratic,

20min

Spherisorb

ODS

(200mm

�4.6mm,

5mm)

45:55methanol:acetate

buffer

pH

4.3

External

standard

365nm

[5]

Lidocainehydrochloride,

tolpersione

hydrochloridein

tablet,

injectable,cream,and

jellyform

ulations

MLCisocratic,

10min

Zorbax

SBC18column

(12.5mm

�4.6mm

i.d.,5mm)

92.5:7.5

(v/v)0.075M

SDS(aq):pentanol

External

standard

210nm

[6]

Sodium

chondroitin

SECisocratic,

30min

TSK

gel

HW-40F

(250mm

�9.4mm

i.d.)

2:98acetonitrile:10mM

phosphate(aq),pH

6.0

External

standard

210nm

[7]

Glutaminein

thalidomide

IndirectUV,

segmented

isocratic,

25min

Zorbax

SB-Phenyl

(150mm

�4.6mm,

i.d.,5mm)

0.15%

H3PO4,1mM

2-naphthalenesulfonate

sodium:m

ethanol

External

standard

254nm

[8]

Aspirin,isosorbide

5-m

ononitrate

in

combined

tablet

form

ulation

RP-H

PLC

isocratic,

15min

ThermoquestC18

(150mm

�4.6mm,

5mm)

60:40aqueousphosphoric

acid,pH

3.4:m

ethanol

Internal

standard

215nm

[9]

Montelukast,loratadinein

tablets

RP-H

PLC

isocratic,

15min

Symmetry

C18

(250mm

�4.6mm,

5mm)

20:80sodium

phosphate

(aq)pH

3.7:acetonitrile

Internal

standard

215nm

[10]

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Dextro-propoxyphene

salts:napsalate

hydrochloride,aspirin,

acetam

inophen

caffeine

RP-H

PLC

gradient,

23min

Zorbax

SBC8

(150mm

�4.6mm

i.d.,5mm)oreclipse

XDBC8

(150mm

�4.6mm

i.d.,5mm)

50mM

potassium

phosphate

monobasic(aq)pH

2.4:acetonitrile

External

standard

Propoxyphene

related

substances:

210nm,

acetam

inophen

combinations:

280nm

[11]

Naproxen

RP-H

PLC

isocratic,

10min

Hypercarb

PGC

(100mm

�4.6mm

i.d.,7mm)

80:20tetrahydrofuran:

methanol

External

standard

272nm

[12]

Dorzolamide

hydrochloride,timolol

maleate

ineyedrops

RP-H

PLC

isocratic,5min

RP-Y

MCpackODS

A-132C18

(150mm

�6.0mm

i.d.,5mm)

5:85:10acetonitrile:

phosphate(aq)pH

2.5:m

ethanol

External

standard

Dorzolamide

hydrochloride:

250nm,timolol

maleate:

300nm

[13]

EDTA

intopical

cream

IEX

isocratic,

10min

PRP-X

100column

(150mm

�4.6mm

i.d.,10mm)

70:30(v/v)3mM

sulfuric

acid

(aq):m

ethanol

External

standard

254nm

[14]

Hydrocortisoneacetate,

methylparaben,

propylparaben

intopical

cream

RP-H

PLC

isocratic,

13min

Supelco

discoveryC18

(125mm

�4.0mm

i.d.,5mm)

58:27:15

water:acetonitrile:

methanol

External

standard

238nm

[15]

Sulfonam

ides

intablets,

capsules,suspensions,

anddrops

MLCisocratic,

20min

Spherisorb

ODS-2

C18

column

(125mm

�4.6mm

i.d.,5mm)

96:4

(v/v)0.1M

sodium

dodecylsulfate(SDS)

(aq),pH

3:pentanol

External

standard

490and550nm

[16]

Methyltesteronein

sugar-

coated

pills

MLCisocratic,

15min

HypersilC18column

(150mm

�3.0mm

i.d.,5mm)

90:10(v/v)40mM

SDS

(aq):propanolmobile

phases

Internal

standard

245nm

[17]


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meloxicam are quantified in the presence of its degradation products by moni-

toring at a UV wavelength where the degradates are not detected. This strategy

is useful for elimination of UV interferences from the HPLC method.

A different mode of HPLC can be used to improve selectivity. A single

chromatographic method suitable for control of several drugs is very useful

for the pharmaceutical laboratory to streamline the number of methods and

HPLCs used for analysis. MLC was used by Youngvises et al.[6] for simul-

taneous separation and determination of lidocaine hydrochloride and tolper-

sione hydrochloride in tablet, injectable, cream, and jelly formulations. The

MLC method procedure (refer to Table 1) was more streamlined than the

RP-HPLC method used previously since there was no sample pretreatment

other than dilution, less organic solvent used and the method run time shorter.

Similarly, Choi et al.[7] used a size exclusion HPLC method to determine

sodium chondroitin in pharmaceutical dosage forms. The method conditions

are shown in Table 1. Size exclusion chromatography was chosen for the sep-

aration to remove excipient interferences from the chromatography observed

with UV detection. The method was found to be applicable to pharmaceutical

tablet and capsule formulations.

Indirect UV HPLC methods have been useful for assay determination in

formulations containing non-UV absorbing compounds. The technique is

adapted easily to existing laboratory HPLC instrumentation since UV detec-

tors are readily available. Li et al.[8] developed an HPLC method (refer to

Table 1 for method conditions) with indirect UV detection for detecting glu-

tamine in thalidomide. The quantitative and automated HPLC glutamine

method was a significant improvement compared to the previously used

TLC method.

Biologicals

Typical biological samples analyzed in the pharmaceutical laboratory are

blood, plasma, tissues, and urine. The results of the biological sample assay

methods are used for drug monitoring and pharmacokinetic studies. The

assay of pharmaceuticals in biological fluids and tissues presents analytical

challenges. The drug substance is typically present at low concentrations,

bound to proteinaceous material present in the samples and endogenous com-

pounds typically present can interfere with the analysis. For these reasons, the

HPLC analysis of pharmaceuticals in biological material usually requires a

sample pretreatment procedure to isolate the analyte from the complex bio-

logical matrix. The sample pretreatment techniques typically used are classi-

cal liquid–liquid extraction (LLE) or solid phase extraction (SPE) to remove

the proteinaceous compounds and other endogenous compounds prior to

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analysis. Another alternate HPLC mode for biological samples, which does

not require sample pretreatment, is MLC. Direct injection of the biological

sample can be performed in MLC since the protein components are solubilized

and the stationary phase is coated with surfactant monomers to inhibit irre-

versible adsorption of the protein components in the sample matrix to the

stationary phase. There have been numerous biological assay HPLC methods

recently published.[30–48] Several recently published biological sample assay

HPLC methods, which used different approaches for sample pretreatment, are

described.

Liquid–Liquid Extraction

A rapid, simple, and sensitive ion-pair RP-HPLC method was developed

for quantification of metformin in human plasma,[39] which enabled the

measurement of metformin for therapeutic drug monitoring. The method

involved a one-step LLE extraction procedure in which aliquots of plasma,

internal standard, and acetonitrile solvent were centrifuged and the superna-

tent analyzed. The 6min isocratic separation was performed on a m-Bondapak

C18 (150mm � 4.6mm i.d., 4mm) analytical column with 60 : 40 (v/v)10mM sodium dodecyl sulfate/10mM sodium dihydrogen phosphate (aq)

pH 5.1 : acetonitrile mobile phases and UV detection at 235 nm. Metformin

and the internal standard were well-resolved and the endogeneous plasma

components did not give any interfering peaks. The average recovery of met-

formin was 100.1% and limit of detection 20 ng/mL.

Solid Phase Extraction

A SPE sample pretreatment procedure was developed for extraction of

fleroxacin and sparfloxacin from plasma with subsequent analysis by RP-

HPLC.[41] The 6min isocratic separation was performed on a Purospher

RP-18 column (250mm � 4.0mm i.d., 5mm) with 85 : 15 (v/v) 5mM tetra-

butylammonium hydroxide pH 2.97 :methanol mobile phases for fleroxacin

and 73 : 27 (v/v) 50mM ammonium phosphate pH 2.95 (aq) with 25% tetra-

hydrofuran :methanol mobile phases for sparfloxacin. Diode array detection

was performed at 300 and 285 nm for fleroxacin and sparfloxacin, respect-

ively. Fleroxacin, sparfloxacin and the internal standard were well-resolved

and the plasma components did not give any interfering peaks. The average

recoveries of fleroxacin, sparfloxacin were 96% and 92%, respectively. The

limits of detection were 0.041 and 0.06mg/mL for fleroxacin and sparfloxa-

cin, respectively.


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Column Switching

Column switching techniques have also been used as sample pretreatment

methods. Alkyl-diol silica (ADS) stationary phases are used as sample pretreat-

ment for removal of macromolecular compounds from biological samples prior

to HPLC analysis. ADS stationary phases were used for sample pretreatment in

the determination of meloxicam in plasma using a column-switching tech-

nique.[42] Plasma samples were injected onto the LiChrospher RP-18 ADS col-

umn, the buffer was added to remove the plasma matrix, the column valve was

switched and the analyte was transferred to the LiChrocart 125-4 LiChrospher

RP-8 analytical column. The mobile phases used were 0.05M phosphate/25mMTBA(aq), pH7.0 : acetonitrilewithUVdetectionat 364 nm.The recovery

of meloxicam from spiked plasma samples was 99.8%. It was determined that

application ofADScolumnswith conventionalHPLC instrumentation eliminated

the need for more time consuming sample pretreatment techniques such as LLE.

Alternate HPLC Mode

Changing the HPLC mode can help to streamline the assay method for

biologicals. A NP-HPLC assay method was developed by Cho et al.[46] for

determination of 13(S)-hydroxyoctadecadienoic acid (13-(S)-HODE) in rat

tissues. The isocratic normal phase method used a Lichrosorb Si 60 column

(250mm � 4mm i.d., 10mm), mobile phase containing proportions of hex-

ane, isopropanol, acetonitrile, acetic acid, and UV detection at 235 nm. The

limit of quantitation for 13(S)-HODE was 0.5 ng. Rat tissues were subjected

to sample pretreatment prior to analysis, which included LLE. The separation

of 13(S)-HODE and its metabolites was achieved with a single analytical

column. The NP-HPLC method was an improvement compared to previous

methods, which used multiple columns and radioisotopes.

Proteins

Proteins are a class of biopolymers, which are actively involved in cellu-

lar function. Proteins are constructed from one or more unbranched chains of

amino acids and carry out the transport and storage of small molecules and

make up the structural framework of cells and tissues. Examples of proteins

are antibodies, blood clotting factors, hormone receptors, and enzymes. Pro-

teins are non-trivial to analyze due to the complexity of their structure.

Selected new developments in protein analysis are described and advantages

are given.

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A method, which provided quantitative and sensitive analysis of the

isomerized Asp amino acids in proteins, was completed by RP-HPLC by

Sadakane et al.[47] Four types of Asp isomers (L-Asp, D-Asp, L-isoAsp

and D-isoAsp) were quantified individually and isomerization from the

L-form to the D-form was detected at the 0.3% level with UV detection

at 215 nm. The method used a Develosil ODS-UG-5 column (150mm �

4.6mm i.d., 5mm) and 11 : 89 (v/v) acetonitrile : 15mM sodium phosphate

(aq) pH 3.0 mobile phases. The method did not require pre-treatment with

acid hydrolysis and streamlined analysis time since previously a combi-

nation of amino acid sequencing analysis, MS, and enantiomeric analysis

was used.

Anionic thermoresponsive polymer-modified stationary phase was

used for improved separation of bioactive peptides using 100% aqueous

mobile phase conditions.[48] Aminopropyl silica beads were used as the

base matrix for synthesis of the anionic polymer hydrogel of poly(IPAAm-

co-Aac-co-tBAAm) modified stationary phase. The stationary phase

(150mm � 4.6mm i.d., 5mm) provided separation of the bioactive peptides

angiotensins I, II, and III. The method conditions used aqueous phosphate/citrate mobile phases and UV detection at 220 nm. It was found that increasing

the column temperature from 108C to 408C improved resolution of the basic

peptides.

Oligonucleotide profiling by gradient RP-HPLC was used to select the

optimal initial gradient conditions for fast HPLC purification of synthetic

oligonucleotides by Gilar et al.[49] The ion-pair method used a short col-

umn, small sorbent particle size, elevated temperature, and slow flow rate

with ion-pair buffers to improve selectivity. Longer (10–30mer) oligo-

nucleotides were resolved and charge–charge interaction introduced in

the separation mechanism to achieve a regular retention of oligonucleotides

according to their chain length. The ion-pair gradient method used an

XTerra MS C18 column (50mm � 4.6mm i.d., 2.5mm), triethylammonium

acetate (TEAA), triethylamine (TEA), and hexafluoroisopropanol (HFIP)

ion pair aqueous buffers with methanol or acetonitrile mobile phases and

photodiode array UV detection. The chromatograms of the separation of

the 10–30mer heterooligonucleotide ladder using the three different ion-

pairing buffers is shown in Fig. 1. It was found that optimal separation

was obtained using 16.3mM TEA, 400mM HFIP, pH 7.9 ion pair aqueous

buffer : methanol mobile phases [Fig. 1(C)]. It was also found that the

columns packed with small particle size sorbent effectively reduced the

impact of slow diffusion, which reduced peak broadening. A mathematical

model for the prediction of oligonucleotide retention from nucleotide com-

position was developed and found to be useful in choosing initial mobile

phase composition.


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Figure 1. Separation of a 10–30mer heterooligonucleotide ladder using three

different ion-pairing buffers. (A) 0.1M TEAA, pH 7, ion-pairing system. Mobile phase A:

acetonitrile-0.1M TEAA, pH 7 (5 : 95, v/v). Mobile phase B: acetonitrile-0.1M TEAA,

pH 7 (15 : 85, v/v). Gradient starts from 5% acetonitrile; gradient slope was 0.25% aceto-

nitrile/min. (B) 4.1mM TEA, 100mM HFIP ion-pairing buffer. Mobile phase A: 10%

methanol, 90% of aqueous buffer of 4.1mM TEA, 100mM HFIP buffer, pH 8.2. Mobile

phaseB: 40%methanol, 60%of 4.1mMTEA, 100mMHFIP buffer, pH 8.2. Gradient starts

at 10% methanol. Gradient slope was 0.25% methanol/min. (C) 16.3mM TEA, 400mM

HFIP ion-pairing buffer. Mobile phase A: 10% methanol, 90% of 16.3mM TEA,

400mM HFIP buffer, pH 7.9. Mobile phase B: 40% methanol, 60% of 16.3mM TEA,

400mM HFIP buffer, pH 7.9. Gradient starts at 16% methanol and the gradient slope was

0.23%/min. The length of the oligonucleotide fragments is indicated above peak apex

along with 30-terminal nucleotide. All separations were performed using an XTerra MS

C18 50mm � 4.6mm column packed with 2.5m sorbent. Flow rate was 1mL/min, and

the column temperature 608C. (Reprinted from Gilar, M.; Fountain, K.J.; Budman, Y.;

Neue, U.D., Yardley, K.R.; Rainville, P.D.; Russell, R.J.; Gebler, J.C. J. Chromatogr. A.

2002, 958, 167–182. Copyright 2002 with permission from Elsevier.)

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Impurity Profile Method

Impurities may be formed or added during the manufacture of the API.

Any component other than the API is considered an impurity. The impurities

present in the API could be the starting materials, process-related impurities,

synthetic intermediates, or degradation products. The International Confer-

ence on Harmonization (ICH) guidelines on Impurities in New Drug Sub-

stances Q3A(R) for impurities in API indicates that the impurity profile

method should be able to detect impurities (limit of quantitation) at levels

greater than the reporting threshold of 0.05% for drugs with maximum daily

dose of �2 g/day and 0.03% for maximum daily dose .2 g/day.[50] Non-degradate impurities are not monitored or specified in the new drug product

as the levels are quantified in the API and the levels will not increase with

storage at specified long term storage conditions. Therefore, for new drug

product formulations, the detection for limit of quantitation for degradation

products as specified in the ICH guidelines on Impurities in New Drug

Products Q3B(R) is 0.1% for drugs with maximum daily dose of �1 g/dayand 0.05% for drugs with maximum daily dose .1 g/day.[51]

HPLC is the technique of choice for the separation and determination of

related impurities in API and formulations. The HPLC method that is selective

for the API, starting materials, process-related impurities, synthetic intermedi-

ates, and degradation products is the impurity profile method. It is critical to

monitor at a UV wavelength that will be able to detect and separate all poten-

tial impurities. Extensive column screening and pH studies are generally per-

formed as part of the HPLC impurity profile method development studies

to determine the optimum conditions of separation in the shortest time period.

There have been many recent publications of impurity profile methods.[52–61]

Selected examples of recently published impurity profile methods for API and

formulations are described.

A selective RP-HPLC method for sildenafil API was developed by

Nagaraju et al.[52] The 15min isocratic separation was achieved on a

YMC C18 (250mm � 4.6mm i.d., 5mm) analytical column, 70 : 30 (v/v)acetonitrile : 50mM potassium dihydrogenphosphate (aq) mobile phases

with UV detection at 230 nm using a photodiode array detector. The API

was dissolved in the mobile phase and injected. The method was used as

the API impurity profile method and for in-process monitoring of the reactions

used in the synthesis. The impurity profile method was selective for the API,

process related impurities, and intermediates.

A RP-HPLC impurity profile method was developed for diphenytriazol

and its related impurities in an injectable dosage form by Wang et al.[53] A

sample pretreatment procedure used a LLE-centrifugation technique to

remove interferences from the sample matrix prior to analysis. The


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20min isocratic HPLC method used an Agilent ODS (250mm � 4.6mm

i.d., 5mm) analytical column, 70 : 30 (v/v) methanol : 10mM potassium

dihydrogen phosphate (aq) pH 7.5 mobile phases with photodiode array

detection at 235 nm. The method was simple and was applied for routine

QC testing of the formulation.

Hartman et al.[54] developed an impurity profile method for etoricoxib

API. The method resolved 13 process-related impurities and three major

degradant products. The method conditions used a YMC ODS-AQ column

(150mm � 4.6mm i.d., 3mm), 10mM KH2PO4, pH 3.1 (aq) : acetonitrile

mobile phases and UV detection at 220 nm. The method was also found to

be stability-indicating by effective resolution of photolytic and oxidative

degradation products.

A monolith column was used in an impurity profile determination of a

Taxolw sample, which contained 17 impurities.[55] The gradient method con-

ditions used a Chromolith RP18 column (100mm � 4.6mm i.d.), acetonitrile :

watermobile phases andUV detection at 228 nm. The performance of themono-

lith column was found to be comparable to that of a traditional silica particulate

YMCODS-AQ column. Only one impurity, whichwas structurally similar to the

parent compound, could not be separated with the monolith column.

A thermally tuned tandem column approach[56,57] was developed for HPLC

for optimization of an impurity profile method for analysis of antihistamines.

Two columns of significantly different chromatographic selectivity for the ana-

lytes were connected and independent variation of temperature completed for

each column. A Zorbax SB-C18 (50mm � 4.6mm, i.d., 5mm) and PBD-

ZrO2 (50mm � 4.6mm i.d., 3mm) columns were chosen for application of

this method to basic pharmaceuticals due to expected significant differences in

selectivity of each column. The mobile phases were 40 : 60 acetonitrile : 25mM

potassium phosphate (aq) at pH 7.0 with UV detection at 254 nm. Chromato-

grams of an antihistamine separation using the Zorbax SB-C18 column only,

PBD-ZrO2 column only, and using both columns connected with the thermally

tuned tandem column approach are shown in Fig. 2. Separation could not be

achieved using single columns, however, baseline separation of nine antihist-

amines was achieved on the thermally tuned tandem column. The improved sep-

aration on the thermally tuned column system was due to the different retention

mechanisms that each column provided and the ability to tune selectivity further

by adjustment of the individual temperatures of the two columns.

Carbon-clad zirconia columns have been recently developed[58] to extend

conventional silica column lifetimes and improve ruggedness by attaching C18

groups to a carbon surface with stable carbon–carbon bonds. The Diamond-

Bonde-C18 column (100mm � 4.6mm i.d.) was used for a barbiturate sep-

aration. The method used 10 : 15 : 75 (v/v/v) THF : acetonitrile : 20mM

aqueous ammonium phosphate pH 7.0 mobile phases and UV detection at

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254 nm. Additionally, in other experiments, the columns were found to be

stable at high temperatures up to 2008C, low pH using 0.5M HNO3 (aq),

and at high pH using 1M NaOH (aq).

Fluorinated and C8 silica stationary phases were used for the impurity profile

determination of taxanes.[59] The fluorinated stationary phases investigated

included Ethyl-PFH, RP-100, Fluofix 120E, Propyl-PFP, and the PFP-100. All

column dimensions were 150mm � 4.6mm i.d. The gradient separation of tax-

anes was optimized for each column using acetonitrile :water mobile phases and

photodiode array UV detection. It was found that the Propyl-PFP and PFP-100

columns exhibited the highest retention and optimal separation of impurities.

The Ethyl-PFH and Fluofix yielded the lowest retention of impurities. The Betasil

C8 column yielded higher retention of taxanes compared to the fluorinated

stationary phases, however, an unusual temperature effect was observed at con-

ditions where the acetonitrile content was �50%. When the column temperature

was changed from 258C to 558C, the Betasil C8 column revealed changes of

selectivity and retention of the taxanes studied. The same temperature effect

was not observed with the fluorinated stationary phases.

Figure 2. Chromatograms showing the separation of basic drugs on (a) a C18 column at

308C, (b) a polybutadiene-coated zirconia column at 308C, and (c) a thermally tuned tan-

dem column set with a C18 column at 408C and a polybutadiene-coated zirconia column

at 358C. Mobile phase: 40 : 60 acetonitrile–25mM potassium phosphate buffer at pH

7.0; flow rate: 1mL/min; detection wavelength: 254 nm. Peaks: 1, pheniramine; 2, chlor-

pheniramine; 3, thenyldiamine; 4, brompheniramine; 5, cyclizine; 6, pyrrobutamine; 7,

chlorcyclizine; 8, thonzylamine; 9, meclizine; and � ¼ unknown impurities. (Reprinted

with permission from Carr, P.; Mao, Y. LC/GC 2003, 21 (2), 150–167. LC/GC is a

copyrighted publication of Advanstar Communications, Inc. All rights reserved.)


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Stability Methods

It is necessary to conduct API and drug product formulation stability

studies to determine the optimum conditions for long term storage. The

drug should remain within specifications during the time that it is stored.

The stability studies are critical in determining the final container closure of

the formulation used for commercial sale.

Development of a stability-indicating method requires forced or stressed

degradation of the API such as acid hydrolysis, base degradation, oxidation,

photodegradation, and thermal decomposition to generate degradation products

that can be monitored by the HPLC impurity profile method. A stability-

indicating method is an impurity profile method that is selective for the API

and its degradation products. The stability-indicating method is used to

analyze the API and drug product formulation at established time points of

samples stored at various temperatures and humidities to establish the long

term storage conditions and shelf-life. Bakshi and Singh[62] propose a sys-

tematic approach for the method development of validated stability-indicating

assay methods since there are numerous published stability-indicating

methods in which the samples were not subjected to stress conditions and

the definition of a stability indicating method is not clearly defined in the

ICH guidelines.[62] There have been many HPLC stability-indicating methods

published recently.[62–70] Selected examples of deliberate degradation experi-

ments and stability-indicating methods are described.

Stability-indicating HPLC method development for drug candidates was

performed using an automated workstation[63] capable of performing multiple

degradation experiments and transferring the samples onto an HPLC autosam-

pler for analysis with UV detection. The automated degradation HPLC system

streamlined method development time compared to the time taken to perform

manual stress stability experiments. The system could perform injections at

multiple time points for the stability experiments.

Photostability studies are used to force the degradation of API by

exposure to UV, fluorescent, or other light source over time to determine pri-

mary photodegradation products. Pharmaceuticals are typically exposed UV

or visible light. The photochemical degradation of nisoldipine drug solids

was investigated under daylight and UV light conditions by Marinkovic

et al.[64] The main degradation products were identified using retention

times of corresponding standards. The isocratic HPLCmethod used to monitor

the photochemical degradation used a Lichrosorb RP-18 (250mm � 4.0mm

i.d., 5mm) analytical column, 60 : 40 (v/v) methanol : water pH 3.0 mobile

phases, and UV detection at 238 nm. There were degradation products pro-

duced in daylight and under UV illumination. The HPLC method was satisfac-

tory in resolving nisoldipine and its photochemical degradation products.

Natishan1250

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The effect of hydroxyl radical attack on two non-steroidal anti-inflammatory

drugs (NSAIDS) was studied in vitro by Gaudiano et al.[65] Diclofenac and

piroxicam were reacted with hydroxyl free radicals and light and it was

found that several new oxidative or unknown degradation impurities were

formed. The HPLC stability indicating method conditions used a Kromasil

C8KR100 (150mm � 4.6mm i.d., 5mm) analytical column, 58 : 42 metha-

nol : dihydrogen phosphate monohydrate/phosphoric acid (aq) pH 2.5 mobile

phases, and UV detection at 254 nm. The study found that NSAIDS were

modified by direct reaction with hydroxyl radicals in vivo to give degradates.

Variable-parameter kinetic experiments, an alternate approach to study

stability kinetics, were carried out using RP-HPLC-UV by Alibrandi

et al.[66] The kinetic experiments varied the value of physical parameters.

The hydrolysis of aspirin was followed both at variable temperature and at

variable pH conditions. The isocratic HPLC method used to monitor the kin-

etic studies used a Varian Omnispher 5 C18 (150mm � 4.6mm i.d., 5mm)

analytical column, 65 : 33 : 2 (v/v/v) methanol : water : acetic acid mobile

phases, and UV detection at 254 nm. The peak areas relative to salicylic

acid were processed by direct fit to a mathematical model and/or differentialmethod. The values of apparent rate constants were obtained by single exper-

iments in the range of temperatures and pH studied. The results were in agree-

ment with those obtained by constant-parameter kinetics and the experiments

were found to significantly save experimental time compared to spectrophoto-

metric kinetic experiments.

A NP-HPLC method with UV detection to study the dithranol reaction

with nitroxide radicals in dimethylsulfoxide (DMSO) was developed by Fer-

lan et al.[67] The HPLC method conditions used a Nucleosil Si 50-5 analytical

column (250mm � 4.6mm i.d., 5mm), 94 : 3 : 3 (v/v/v) isooctane : dichloro-methane : acetic acid mobile phases, and UV detection at 254 nm. It was found

that the behavior of dithranol in DMSO solution was a useful system for simu-

lating the oxidative transformation of dithranol after topical application. The

HPLC method allowed for simultaneous, quantitative, time dependent moni-

toring of dithranol and its major oxidative products and intermediates. The

rate of oxidation was significantly increased in the presence of nitroxide

with respect to the rates observed in autooxidation.

In-Process Methods

A process monitoring analytical method is an in-processmethod. In-process

methods are essential for the optimization of synthetic and purification pro-

cesses and to maintain processing conditions at the highest level required to

ensure production of high purity API with maximum yield. There have been


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some recent examples in the literature of HPLC methods used to monitor

selected production processes.[71–73] Selected examples of in-process

methods used for monitoring synthetic processes are described.

An HPLC in-process method was developed for the preparation of sulta-

micillin, a b-lactam antibiotic. Laviana et al.[71] developed a rapid, specific,

and sensitive HPLC method for the analysis of sultamicillin and its precursors,

potential impurities, and degradation products. The 25min HPLC gradient

elution method used to monitor the sultamicillin synthesis employed a

Kromasil C18 (150mm � 4.6mm i.d., 5mm) with 25mM phosphate buffer

(aq) pH 7.0 : acetonitrile mobile phases and UV detection at 239 nm. In-situ

reaction analysis using the method was shown to provide real-time monitoring

of the different reagents and products.

A RP-HPLC assay with UV detection was used to monitor the production

of midazolam and its precursors by Laviana et al.[72] Several synthetic routes

were developed for the midazolam synthesis. The HPLC method was devel-

oped to identify and quantify the different synthetic intermediates and analyze

the purity of the isolated API. Different columns, pH, and ionic strength of the

aqueous mobile phase and organic modifier were evaluated during the method

development. The 15min isocratic HPLC method used to monitor the mida-

zolam synthesis employed a Kromasil C8 column (150mm � 4.6mm i.d.,

5mm) with aqueous ammonium chloride, pH 5.5 :methanol : acetonitrile

45 : 22 : 33 (v/v/v) mobile phases, and UV detection at 239 nm. The method

was also used to monitor changes in the synthesis for process optimization.

Cleaning Methods

The manufacture of pharmaceuticals requires proper cleaning of equip-

ment and surfaces to avoid contamination between different production pro-

cesses. Cleaning procedures for the equipment are validated according to

good manufacturing practices (GMP) rules and guidelines.[74] According to

the FDA guideline,[74] verification of the cleaning is completed by using direct

surface sampling using swabbing techniques, which determine the level of

residues on surfaces used in the manufacturing process and indirect sampling

based on the analysis of solutions used for rinsing the equipment. The accep-

table limit of API on surfaces and in the rinse solutions is based on the drug

type and scientific rationale.[75] Several recently published HPLC methods

developed for verification of cleaning are given in Table 2. Selected examples

of cleaning methods are discussed.

Isocratic HPLC methods[75–78] were developed and validated for suma-

triptan succinate, amlodipine, ranitidine hydrochloride, and acetylsalicylic

acid drug substances; the method conditions are shown in Table 2. The solvent

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Table

2.

HPLC-U

Vcleaningmethodsin

pharmaceuticals.

Nam

eof

compound

HPLCmode

Column

Mobilephase

UV

detection

wavelength

(nm)

%recovery,

surfaceused

Detection

limit

Reference

Sumatriptan

succinate

RP-H

PLC

Phenomenex

luna,

150mm

�4.6mm

i.d.,4mm

0.05M

ammonium

phosphate

(aq):acetonitrile

228

95%

stainless

steel,89%

vinyl,94%

glass

3ng/m

L[75]

Amlodipine

RP-H

PLC

LiChroCART

purospher

RP-18e,

125mm

�4.0mm

i.d.,5mm

15:35:50

acetonitrile:

methanol:pH

3.0

triethylamine

solution

237

90%

stainless

steel

0.02mg/m

L[76]

Ranitidine

hydrochloride

RP-H

PLC

Phenomenex

luna,

250mm

�4.6mm

i.d.

40:60

methanol:0.05M

ammonium

acetate

pH

6.7

320

90%

stainless

steel,78%

vinyl,85%

glass

2ng/m

L[77]

Acetylsalicylic

acid

RP-H

PLC

LiChrospher

RP-18,

125mm

�4.0mm

i.d.

790:220:1

water:acetonitrile:

o-phosphoricacid

226

94%

stainless

steel,86%

vinyl,90%

glass

0.04mg/m

L[78]

Ertapenem

sodium

RP-H

PLC

YMCbasic,

250mm

�4.6mm

i.d.

0.05%

H3PO4(aq):

acetonitrile

230

100%

stainless

steel,98%

glass

0.02mg/m

L[79]


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used for moistening the swabs for wiping, swabbing technique and number of

swabs used were optimized to obtain reproducible high recoveries from var-

ious surfaces. The HPLC methods used external standard analysis to deter-

mine the levels of residual drug substance. The methods were sensitive with

limits of detection between 2 ng/mL and 0.04mg/mL and recoveries ranged

between 78–95%.

A sensitive RP-HPLC-UV cleaning method was developed by Sajonz

et al.[79] to detect residual amounts of ertapenem sodium, a 1b-methyl carbape-

nem antibiotic. The method conditions are given in Table 2. Antibiotics are a

significant concern in pharmaceutical production due to the medical impli-

cations of cross-contamination in other production processes or exposure to

employees who are allergic to antibiotic compounds. It is critical to develop a

sensitive cleaning method to confirm the absence of antibiotics from equipment

and processing area surfaces. The gradient elution HPLC cleaning method for

ertapenem sodium was found to be sensitive with a limit of detection of

0.02mg/mL and �98% recoveries from stainless steel and glass surfaces.

Fast HPLC Methods

Screening of thousands of samples in combinatorial chemistry for identi-

fication of lead compounds and analysis for pharmacokinetic studies requires

faster analytical methods than those developed using conventional HPLC

columns to reduce analysis times.

Fast HPLC techniques use HPLC columns operated at high flow rates to

achieve rapid separation without significant loss in resolution. The combined

benefits of using short columns at high flow rates results in analysis times that

are often an order of magnitude shorter than conventional HPLC runs with sat-

isfactory resolution.[80] The analysis time in fast gradient HPLC is reduced by

increasing the gradient rate, decreasing the column length, and increasing

the flow rate. The flow rate is limited by the increased back pressure of par-

ticulate silica columns with increasing flow rate. Monolithic columns offer

a suitable alternative to particulate silica columns as the column is a continu-

ous interconnected skeleton containing bimodal pores, which provide high

permeability and high efficiency. Due to their higher permeability, monolith

columns can be operated at higher flow rates than particulate silica columns

as well as provide comparable chromatographic performance. Recently,

there have been numerous fast HPLC methods published due to new develop-

ments in column technology.[81–90] Selected applications of fast HPLC

method applications are described.

An ultra-fast HPLC separation of anions in aqueous samples, which used

a monolithic stationary phase and indirect UV detection, was completed in

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15 sec by Hatsis and Lucy.[82] The separation was completed using IEX with

95 : 5 (v/v) 1.5mM tetrabutylammonium phthalate/o-phthalic acid (aq) pH

5.5 : acetonitrile, Chromolithw SpeedROD RP-18e (50mm � 4.6mm), flow

rate of 16mL/min and indirect UV detection at 255 nm. The chromatograms

of the anion separation at flow rates of 8 and 16mL/min are shown in Fig. 3.

Impurity profile fast HPLC methods for five pharmaceuticals were separ-

ated on a monolith column with fast HPLC by van Nederkassel et al.[83] and

compared to separations using conventional C-18 columns with the same

Figure 3. Separation of common anions, (A) in 30 sec (8mL/min), (B) in 15 sec

(16mL/min). Experimental conditions: speed ROD, 1.5mM TBA–1.1mM phthalate

with 5% (v/v) acetonitrile, 20mL injection. Eluent flow for 15 sec separation with a

Waters 590 pump. Analyte concentration approximately 25 times detection limit.

(Lucy, C.A; Hatsis, P. Analyst 2002, 127, 451–454. Reproduced with permission

from The Royal Society of Chemistry.)


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mobile phases. The pharmaceuticals studied were alkylbenzene, nimesulide,

phenoxymethylpenicillin, erythromycin, and tetracycline. The monolith column

methods used Chromolith SpeedROD RP-18e (50 � 4.6mm) and Chromolith

Performance RP-18e (100 � 4.6mm), 30 : 70 (v/v) acetonitrile : NH4H2PO4

(aq) pH 7.0 mobile phases, flow rates between 1 and 9mL/min and UV detec-

tion. The chromatogram of nimesulide and impurities is shown in Fig. 4. The

nimesulide analysis time was reduced up to 40 times using a monolith column

with increased flow rates while maintaining separation of impurities. Analysis

of tetracycline and alkylbenzenes using the monolith column was found also to

be significantly shortened without loss of resolution of impurities compared to

the C18 stationary phase. However, it was found that fast HPLC monolith

methods for phenoxymethylpenicillin, erythromycin were unsuccessful, this

was attributed to lack of theoretical plates or selectivity differences between

the monolith and C18 stationary phases.

Additional fast HPLC comparisons of silica based monolith columns with

conventional particulate silica C18 or C8 columns was studied by Smith and

McNair.[84] A Chromolith SpeedROD RP18e (50 � 4.6mm) monolith col-

umn, acetonitrile : water mobile phases, UV detection at 220 nm, 8.0mL/min flow rate conditions were used for analysis of a seven component mixture

containing benzamide, N-methylbenzamide, benzyl alcohol, acetophenone,

ethyl paraben, propyl paraben, and biphenyl. The results of the evaluation

indicated that monolith columns performed similarly to a hybrid column for

analytes in the mid-polarity range.

Figure 4. Chromatogram of the nimesulide mixture on the SpeedROD column.

Mobile phase ACN/NH4H2PO4 1.15 g/L; pH 7.0 (30/70 v/v); T ¼ 308C;(l ¼ 230 nm; flow rate ¼ 9mL/min. Elution order of the peaks: impurity A, nime-

sulide, impurity B, impurity C, impurity D, impurity E. (Reprinted from van Nederkas-

sel A. M.; Aerts, A.; Dierick, A.; Massart, D. L.; Vander Heyden, Y. J. Pharm. Biomed.

Anal. 2003, 32, 233–249. Copyright 2003 with permission from Elsevier.)

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Short columns have been recently developed for fast HPLC methods. The

Intelligent Speed (IS) columns with 2.1mm � 20mm, 3.0 � 20mm,

3.9 � 20mm and 4.6mm � 20mm all with 2.5mm particles were shown to

separate atenolol, pindolol, and metoprolol in a spiked protein precipitated

plasma sample with total run time of less than 4min.[85] The chromatograms

yielded similar chromatography initially and after 1000 injections indicating

good column reproducibility and ruggedness for analysis of biological

samples.

Rapid Resolution HT HPLC columns contain particles ,2mm and have

been shown to be applicable for fast HPLC.[86] The analysis of the antibiotics

clindamycin and lincomycin was demonstrated using a Rapid Resolution SB-

C18 HT HPLC column (4.6mm � 30mm, 1.8mm), flow rate of 1.0mL/min,

isocratic 85 : 15 (v/v) 20mM Na2HPO4 (aq) pH 2.8 : acetonitrile mobile

phases, and UV detection at 210 nm. The method run time was 6min. When

a linear gradient of 10–40% acetonitrile in 2min was used, the run time

was shortened to ,2min using the same column and mobile phases.

Micro-HPLC Methods

Miniaturized separation techniques offer a number of advantages over

conventional chromatographic methods. These advantages include reduced

solvent consumption, applicability to small sample sizes, higher mass sensi-

tivity, separation improvements with long packed columns, and flexibility

of coupling the column with MS as well as other types of detectors.[91] Several

micro-HPLC methods have been recently published for analysis of biological

samples due to the small sample sizes that can be analyzed.[91–97] Some

recently published micro-HPLC methods are given in Table 3. Selected

micro-HPLC methods given in Table 3 are discussed.

Semi-micro HPLC was used for the determination of triazolam in rat

plasma and brain microdialysate.[92] The sample was extracted by LLE and

than analyzed by HPLC-UV; method conditions are given in Table 3. The

method was selective for triazolam and sensitive with limits of detection

between 0.7 and 2.1 ng/mL for triazolam in the biological samples.

A micro-HPLC method was used for analysis of amitriptyline, imi-

pramine, nortriptyline, and desipramine in biological samples.[93] A polymer-

coated fibrous material was used as an extraction medium for the on-line

sample preparation technique and then analysis performed by HPLC-UV,

method conditions are given in Table 3. The on-line extraction method

coupled with micro-HPLC allowed for rapid determination of the analytes

without large sample and solvent consumption. The method was sensitive

with limits of quantitation between 0.5 and 1.8 ng/mL.


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Table

3.

Micro-H

PLC-U

Vmethodsusedforanalysisofpharmaceuticals.

Analyte,sample

matrix

LCmethod

Column

Mobilephase

UV

detector

wavelength

(nm)

Detectionlimit

Reference

Triazolam

inratplasm

aand

brain

microdialysate

Sem

i-micro

DevelosilODS-5

(250mm

�1.5mm

i.d.,5mm)

62:38water:

acetonitrile,

isocratic

222

2.1ng/m

L(rat

plasm

a);

0.7ng/m

L

(cerebrospinal

fluid)

[92]

Amitriptyline,im

ipramine,

nortiptyline,desipramine

inurine

Micro-H

PLC

CapCellPak

C18MG

(150mm

�1.0mm

i.d.,5mm)

Acetonitrile:

water

254

Lim

itof

quantitation:

0.5–1.8ng/m

L

[93]

Difluprednateandmetabolite

inaqueoushumor

Sem

i-micro

with

column

switching

CapcellPak

C18UG120

S5(250�

1.5mm

i.d.,5mm)

10mM

phosphate

buffer

(aq),pH

3.0:acetonitrile

240

Notreported

[94]

3a-H

ydroxyglycyrrheticacid;

3-dehydroglycyrrheticacid

inratfeces

Sem

i-micro

TSKgel

ODS-80TsQ

A

(150mm

�2.0mm

i.d.,5mm

0.017%

aqueous

phosphoric

acid:acetontirile

254

0.2pmol

[95]

Tofisopam

inhuman

serum

Sem

i-micro

CapcellPak

C18

UG120S5

(250mm

�1.5mm

i.d.,5mm)

0.1%

phosphoric

acid/5

mM

sodium

octanesulfonate

(aq):acetonitrile

310

2ng/mL

[97]

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A specific and sensitive semi-micro HPLC method for the determination

for difluprednate and its metabolite in aqueous humor was developed.[94]

Difluprednate and its metabolite were first adsorbed on a Pinkerton-type

column and then by column switching analyzed by HPLC-UV, method con-

ditions are given in Table 3. The method was sensitive with detection of

0.5 ng/mL difluprednate.

Molecular Imprinted Polymers

MIPs are highly cross-linked synthetic polymers, which exhibit mole-

cular recognition properties towards template molecules with specificity and

binding selectivity similar to naturally occurring binding polymers such as

antibodies and enzymes.[98] MIPs have been used for chromatographic separa-

tion, SPE, membranes, and sensors.[99] Selected methods used for preparation

of MIPs and subsequent application to HPLC-UV analysis are described in

these references.

A cortisol-template imprinted polymer MIP HPLC column was prepared

and used to analyze a mixture of cortisol, cortisone, corticosterone, proges-

terone, 11-keto-progesterone, 11a-hydroxyprogesterone, 17a-hydroxy-

progesterone, cortisol 21-hemisuccinate, and cortisol 21-acetate.[98] The

method used chloroform containing 0.5% acetic acid as the mobile phase

and UV detection at 265 nm. The method revealed good selectivity for cortisol

and recognition of the column for cortisol was confirmed by the HPLC separa-

tion of the steroid mixture.

Uniformly sized MIPs were imprinted with bisphenol A and b-estradiol

target molecules and used as HPLC analytical columns by Sanbe and

Haginaka.[99] The imprinted and non-imprinted MIP 4-vinyl pyridine mono-

mer/ethylene glycol dimethacrylate cross-linker (4-VPA EDMA) columns

were evaluated using bisphenol A, b-estradiol, and other structurally related

steroidal and non-steroidal estrogen analytes. The HPLC method used

50 : 50 (v/v) 20mM sodium dihydrogen phosphate/disodium hydrogen phos-

phate (aq) pH 5.1 : acetonitrile mobile phases and UV detection at 200 nm. The

chromatograms of b-estradiol, estrone, estriol, testosterone, and corticoster-

one on the non-imprinted and b-estradiol-imprinted columns are shown in

Fig. 5. The non-imprinted column exhibited overlapping of b-estradiol with

estrone and estriol with testosterone, however, the b-estradiol-imprinted

column revealed complete separation of all analytes. The MIP column more

efficiently separated the bisphenol A and b-estradiol target molecules from

the other structurally related compounds.

A chlorphenamine-imprinted polymer MIP was prepared by

Chen et al.[100] The selectivity of the MIP column was evaluated using acetic


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acid :methanol mobile phases and UV detection at 254 nm. The specificity

was confirmed by the separation of chlorphenamine and diphenhydramine,

two structurally similar compounds. It was found that chlorphenamine-

MIP could be used to concentrate solutions of chlorphenamine 50-fold with

recoveries .90% at pH 5.

Moring et al.[101] synthesized a MIP column, which used 2,6-pyridinedi-

carboxylic acid (DPA) as the template molecule. Chromatographic capacity

factors and selectivities of a series of structural analogs of DPA were

compared using the DPA-MIPs prepared with different cross-linkers. HPLC

analysis was completed with 40 : 60 (v/v) methanol : 0.1% trifluoroacetic

acid (aq) mobile phases and UV detection at 270 nm. Selectivity appeared

to be dependent on a combination of ion-pairing and hydrogen bonding of

the template to both functional monomer and cross-linker.

Figure 5. Separation of steroids on the non-imprinted 4-VPA-co-EDMA (A) and

b-estradiol-imprinted 4-VPY-co-EDMA polymers (MIP 8) (B). Peak assignments: 1,

corticosterone; 2, testosterone; 3, estriol; 4, estrone; 5, b-estradiol; HPLC conditions

in text. Loaded amounts: corticosterone, 580 ng; testosterone, 880 ng; estriol, 80 ng;

estrone and b-estradiol, 230 ng. (Reprinted from Sanbe, H.; Haginaka, J. J. Pharm.

Biomed. Anal. 2002, 30, 1835–1844. Copyright 2002 with permission from Elsevier.)

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Ionic Liquids

Room temperature ionic liquids (RTIL) are novel salts that are non-

volatile and non-flammable liquids at or below room temperature, which

have been recently used in chromatography as GC stationary phases[102] and

in MS.[103,104] RTIL are creating increasing interest in synthetic chemistry

due to unique chemical and physical properties.[105] The most common

RTIL are composed of a large organic cation, typically an unsymmetric sub-

stituted nitrogen-containing cation such as imidazolium with a weakly coordi-

nating inorganic anion such as Cl2, PF62, or BF4.

[106]

An HPLC-UV method, which used an ionic liquid as an aqueous mobile

phase modifier, was described by Jiang et al.[107] The authors used 1-butyl-3-

methylimidazolium tetrafluoroborate ionic liquid ([bmim]BF4) aqueous sol-

utions as the mobile phase for a separation of ephedrines by HPLC-UV.

The method used a Chromatorex ODS column (100mm � 4.6mm i.d.,

5mm), 2.6–62.4mM [bmim]BF4 (aq) pH 3.0 mobile phase, flow rate

1.0mL/min, and UV detection at 252 nm. Chromatograms of the separation

of ephedrines using aqueous mobile phase compared to varied concentrations

of [bmim]BF4 ionic liquid aqueous mobile phases are shown in Fig. 6. It was

found that concentration of the [bmim]BF4 in the mobile phase affects the

retention and separation of the analytes. The separation was improved with

addition of the [bmim]BF4 to the mobile phase and initially an increase of

the concentration of [bmim]BF4 in the mobile phase gave an increase in reten-

tion, but then a decrease in retention. This was attributed to competition

between the ionic liquid imidazolium cations and the polar groups of the ana-

lytes for silanol groups on the column and formation of a weak bilayer.[107]

The results of the study revealed that ionic liquids can be used as modifiers

in HPLC for separation of polar pharmaceuticals without using organic

solvents in the mobile phases.

HPLC WITH MS DETECTION

HPLC coupled with mass spectrometric detection (LC/MS) is widely

employed in all stages of drug development. This is due to innovations in

MS source design, improvements in mass accuracy, and computer-controlled

automation.[108] The high selectivity of LC/MS, high proficiency for struc-

tural elucidation of unknown impurities, and increased need for rapid, high-

throughput analysis in drug discovery has greatly increased the use of the

technique in pharmaceuticals. The general principles of LC/MS and the vari-

ations of instrumentation have been discussed in detail elsewhere.[109–113]

Most commonly, MS is performed using electrospray ionization (ESI),


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Figure 6. Chromatograms of ephedrines with a mobile phase containing different

concentrations of [bmim]BF4 at pH 3.0. (a) 0, (b) 2.6, (c) 5.2, (d) 20.8, and (e)

62.4mM. Chromatographic conditions: column: C18 (5mm, 100mm � 4.6mm i.d.);

flow rate: 1.0mL/min; detection: 252 nm. Peaks: 1, norphedrine; 2, ephedrine; 3,

pseudoephedrine; and 4, methylephedrine. (Reprinted from He, L.; Zhang, W.; Zhao,

L.; Liu, X. J. Chromatogr. A. 2003, 1007, 39–45. Copyright 2003 with permission

from Elsevier.)

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or atmospheric-pressure chemical ionization (APCI) coupled with single (MS)

or triple (MS/MS) quadrupoles. There has been a tremendous growth of appli-

cations of HPLC-MS in the pharmaceutical industry.[114–140]

Selected applications of LC/MS to the analysis of pharmaceuticals are

described.

Biologicals

HPLC with tandem mass spectrometric (MS/MS) detection has been

demonstrated to be a powerful analysis tool for the quantitative determination

of drugs and metabolites in biological fluids. Analysis of biological samples

by HPLC-MS/MS can be subject to lack of selectivity due to ion suppression

or matrix effects.[111] The effects can be reduced by changing or improving the

sample extraction procedure, obtaining a better separation of analytes and

changing the HPLC-MS interface and mechanism of ionization of the ana-

lytes. Reviews of LC-MS of biologicals have been completed by Watson

et al.[112] and Gelpi.[113] Some recently published methods in LC-MS and

LC-MS/MS of biologicals are given in Table 4.

Guan et al.[114] developed a LC-ESIþ-MS method, which simultaneously

detected and quantified glutathione, glutathione disulfide, cysteine, homocys-

teine, and homocystine in biological samples, see method conditions in

Table 4. The method required little sample treatment and was found to provide

higher sensitivity and selectivity than previous methods used, which did not

use MS detection.

Selective ion monitoring LC-ESIþ-MS was used to detect analytes

extracted from complex biological matrixes. Nelson et al.[117] coupled selec-

tive ion monitoring with a stable isotope dilution scheme, which provided high

accuracy, excellent precision, and specific analyte confirmation for homocys-

teine, method conditions are shown in Table 4.

A novel stable isotope dilution LC-ESI-MS method for cis-amminedi-

chloro(2-methylpyridine)platinum(II) determinations in human plasma ultra-

filtrates was developed by Oe et al.,[119] method conditions are shown in

Table 4. The method was unique, in which provides conversion of aquated

forms of the molecule that may be present in plasma samples back to the

parent drug. The method was used for quantitative analysis of the intact plati-

num anticancer drug, which allowed accurate and precise pharmacokinetic

parameters to be obtained.

A highly sensitive LC-APCIþ-MS/MS method for the determination of

buprenorphine and its primary metabolite norbuprenorphine was developed

by Ceccato et al.,[123] the method conditions are shown in Table 4. An

automated SPE on disposable extraction cartridges was used to isolate and


ORDER REPRINTS

Table

4.

HPLC-M

SandHPLC-M

S/M

Sapplicationsforquantitativedeterminationofpharmaceuticalsin

biologicals.

Analyte

Sam

ple

matrix

LC-M

S

instrumentation

HPLCcolumn

HPLCmobilephase

Lim

itofdetection

Reference

Glutathione,

glutathione

disulfide,cysteine,

homocysteine,

homocystine

Rat

brain,lung,

liver,heart,

kidneys,

erythrocytes,

plasm

a

LC-ESIþ-M

SAdsorbosilC18

(250mm

�3.2mm

i.d.,

5mm)

0.1%

trifluoroacetic

acid(aq):acetonitrile,

gradient;15min

3.3–29.6pmol

[114]

Oxybutynin,

desethyloxybutynin

Dogplasm

aLC-ESIþ-

MS/MS

XTerra

MSC18

(30mm

�2.1mm

i.d.,

3.5mm)

90:10(v/v)methanol:

water;2min

0.1ng/mL

[115]

30 -C-ethynylcytidine

Human

plasm

a

andurine

LC-ESIþ-

MS/MS

InertsilODS-3

(150mm

�2.1mm

i.d.,

5mm)

95:5

(v/v)10mM

ammonium

acetate:m

ethanol;

6min

LOQ:1ng/mLin

plasm

a

and10ng/mLurine

[116]

Homocysteine

Human

plasm

aLC-ESIþ-M

S,

isotope

‘dilution

SupelosilLC-CN

(250mm

�4.6mm

i.d.,

5mm)

0.1%

form

icacid

(aq)and

0.1%

form

icacid

in

methanol,gradient;

15min

0.06mmol(0.12ngon

column)

[117]

Alprazolam,

estazolam,

midazolam,and

metabolites

Rat

hairand

plasm

a

LC-ESIþ-M

S,

MightysilRP-18

(100mm

�2.0mm

i.d.,

3mm)

Water:acetic

acid:acetonitrile,

gradient;35min

Notreported

[118]

Cis-ammine-

dichloro(2-

methylpyridine)

platinum(II)

Human

plasm

aLC-ESI-MS

YMCODS-A

Q

(150mm

�2.0mm

i.d.,

3mm)

5mM

ammonium

acetate,

0.1%

acetic

acid

(aq);

5mM

ammonium

acetate,

0.1%

acetic

acid

inmethanol;

19min

LOQ:10ng/mL

[119]

Natishan1264

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Amoxycillin

and

majormetabolites

Anim

altissues

LC-ESIþ-

MS/MS

Hypersil(100mm

�3mm

i.d.,5mm)

9.6mM

pentafluoropropionic

acid

(aq):

cetonitrile:w

ater,

gradient;20min

Amoxycillin:

2.3–12.0ng/g;

amoxycilloic

acid:

1.1–15.1ng/g;

amoxycillinpiperazine-

20 ,50 -dione:

0.2–2.4ng/g

[120]

Sodium

borocaptate

Human

plasm

aLC-ESIþ-

MSQTOF

NucleosilC18

(250mm

�2.1mm

i.d.,

5mm)

1:1

(v/v)

methanol:5mM

tetrabutylammonium

acetate(aq),pH

�8.5,

isocratic;10min

LOQ:0.5mg/mL

[121]

Stavudine

Human

serum

LC-ESIþ-

MS/MS

LiChrospher

100RP-18,

(125mm

�4mm

i.d.,

5mm)

75:25(v/v)10mM

ammonium

acetate/1%

acetic

acid

(aq):m

ethanol/

1%

acetic

acid;4min

4ng/mL

[122]

Buprenorphine,

norbuprenorphine

Human

plasm

aLC-A

PCIþ-

MS/MS

Purospher

STARRP-18e

(55mm

�4mm

i.d.)

50:50(v/v)

methanol:50mM

ammonium

acetate

(aq),pH

4.5;12min

2pg/mL,12pg/mL

[123]

Sufentanil

Human

plasm

aLC-A

PIþ-

MS/MSwith

ionspray

interface

SupelcosilLC-C

18-D

B

(300mm

�1mm

i.d.,

5mm)

20:80(v/v)0.2%

trifluoroacetic

acid

(aq):0.2%

trifluoroacetic

acid

in

acetonitrile;15min

LOQ:0.3ng/mL

[124]

Ambroxol

Human

plasm

aLC-M

S/MS

withturbo

electrospray

XTerra

MSC18

(30mm

�2.1mm

i.d.,

3.5mm)

20mM

ammonium

acetatein

90%

acetonitrile,pH

8.8;

2min

LOQ:0.2ng/mL

[125]

(continued

)


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Table

4.

Continued.

Analyte

Sam

ple

matrix

LC-M

S

instrumentation

HPLCcolumn

HPLCmobilephase

Lim

itofdetection

Reference

Epherenoneand

hydrolyzed

metabolite

Human

urine

LC-A

PI-

MS/MSwith

both

negative

andpositive

ionization

Zorbax

XDB-C8

(50mm

�2.1mm

i.d.,

5mm)

60:40(v/v)

acetonitrile:w

ater

containing10mM

ammonium

acetate

(aq),pH

7.4;5min

LOQ:50ng/mL

[126]

Nitronemetabolite

ofsubstance

P

antagonist

Rat

anddog

plasm

a

LC-A

PIþ-

MS/MSwith

TurboIonSpray

interface

InertsilODS-2

(250mm

�2mm

i.d.,

5mm)

0.1%

trifluoroacetic

acid

(aq):0.1%

trifluoroacetic

acid

in

acetonitrile,gradient;

36min

Notreported

[127]

Digoxin

Rat

plasm

aLC-A

PIþ-

MS/MSwith

TurboIonSpray

interface

YMCODSAQ

(50mm

�2.0mm

i.d.,

3mm)

50:50(v/v)

acetonitrile:5

mM

ammonium

form

ate,

pH3.4;isocratic;4min

LOQ:0.1ng/mL

[128]

Paclitaxel

Human

plasm

aLC-A

PIþ-

MS/MSwith

TurboIonSpray

interface

Zorbax

SB

C18(150mm

�4.6mm

i.d.,5mm)

65:35(v/v)

acetonitrile:2

mM

ammonium

acetate,

pH

5;gradient;5min

LOQ:1ng/mL

[129]

Nevirapine

Human

plasm

aLC-A

PIþ-

MS/MSwith

TurboIonSpray

interface

Zorbax

XDB-C8

(50mm

�2.1mm

i.d.,

5mm)

10mM

ammonium

form

ate(aq),pH

4.1:0.1%

form

icacid

inacetonitrile;

gradient;5min

LOQ:25ng/mL

[130]

Brostallicin

Human

plasm

aLC-A

PIþ-

MS/MSwith

TurboIonSpray

Platinum

Cyano

(100mm

�4.6mm

i.d.,

3.6mm)

70:30(v/v)

acetonitrile:20mM

ammonium

form

ate

(aq),pH

3.5;8min

LOQ:0.12ng/mL

[131]

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Loratadine,

descarboethoxy-

loratadine

Human

plasm

aLC-M

S/MSwith

TurboIonSpray

Betasilsilica

(50mm

�3.0mm

i.d.,

5mm)

90:10:0.1

(v/v/v)

acetonitrile:

water:trifluoroacetic

acid,isocratic;3min

LOQ:loratadine,

10pg/

mL;descarboethoxy-

loratadine,

25pg/mL

[132]

Roxifiban

metabolites

Human

plasm

aLC-A

PI-

MS/MSwith

TurboIonSpray

interface

Phenomenex

LunaC18

(50mm

�2.0mm

i.d.,

3mm)

0.01%

form

icacid

(aq):0.01%

form

ic

acid

inmethanol;

gradient;23min

Notreported

[133]

Terpenelactones

Anim

alplasm

aLC-A

PCI2-

ITMS

HypersilC18

(100mm

�3mm

i.d.,

5mm)

Methanol:water,

gradient;10min

2ng/mL

[134]

Rofecoxib

Human

plasm

aLC-A

PCI2-

MS/MS

YMCODSAQ

(100�

3.0mm

i.d.,3mm)

50:50(v/v)

acetonitrile:w

ater;

7.5min

LOQ:,

1ng/mL

[135]

Methadoneand

metabolites

Oralfluid

LC-A

PCIþ-M

S/

MS

SynergiPolarRP

(150mm

�2.0mm

i.d.,

4mm)

10mM

ammonium

form

ate,

0.001%

form

icacid

(aq):acetonitrile;

gradient;26min

0.25–5ng/mL

[136]

8-H

ydroxy-2

0 -

deoxyhuanosine

Urine

LC-A

PCI-

MS/MS

Symmetry

C18

(250mm

�4.6mm

i.d.)

Methanol:water:

trifluoroacetic

acid

10:90:0.025(v/v/v),

isocratic;16min

�1ng/mL

[137]

Clozapineandother

new

chem

ical

entities

Rat

plasm

aLC-A

PIþ-

MS/MS

Chromolith

SpeedROD

(50mm

�4.6mm

i.d.)

4mM

ammonium

acetate

(aq)/acetonitrile;

gradient;1.4min

Notreported

[138]

Note:LOQ,limitofquantitation.


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preconcentrate the analytes from the biological matrix prior to injection onto

the LC-MS/MS. The SPE was coupled at-line to the LC-MS/MS and stream-

lined sample pretreatment procedures as well as shortened overall analysis

times.

Shi et al.[133] developed a LC-API-MS/MS with TurboIonSpray interface

to selectively extract and simultaneously quantify seven active metabolites of

roxifiban from human plasma. The method conditions are shown in Table 4.

The HPLC method separated two pairs of epimers and one pair of geographic

isomers. The LC-API-MS/MS method was successfully applied in roxifiban

clinical studies to evaluate the pharmocokinetics of the seven metabolites.

A monolithic HPLC-MS/MS method was developed by Hsieh et al.[138]

for high speed direct determination of clozapine and other new chemical enti-

ties in rat plasma samples. The chromatographic separation was completed in

1.3min using a Chromolith SpeedROD, RP-18e (50mm � 4.6mm), 4mM

ammonium acetate (aq) : acetonitrile : 0.015% TFA (aq) mobile phases,

internal standard, flow programing and detection using a mass spectrometer

in the positive ion mode. The monolithic column served a dual role to both

remove matrix macromolecules and to provide chromatographic efficiency

comparable to microparticulate silica for small molecules. The large macro-

pores of the monolith column allow the large protein molecules to pass

through the column while the drug molecules are retained on the bonded

reverse phase for chromatographic interaction. The study also demonstrated

that little or no matrix ion suppression was observed using the direct mono-

lithic HPLC-MS/MS method.

Proteomics

Proteomics is the study of protein location, interaction, structure, and

function. The object of proteomic studies is to identify and characterize the

proteins present in the normal vs. diseased state in biological samples.

Abnormalities in protein production or function have been connected to

many diseases and health conditions, and therefore, the ability to modulate

proteins represents an attractive target for drug design in pharmaceuticals.[141]

Righetti et al.[142] reviewed the definitions related to functional and structural

proteomics and the human proteome.

The use of HPLC coupled with MS is developing rapidly for analysis of

complex mixtures of proteins and peptides. The focus of recent developments

is the emergence of multidimensional techniques[143] for separation of pro-

teins. Andrews et al.[144] reviewed the analysis of DNA adducts, specifically

modified nucleosides, nucleotides, and oligonucleotides using HPLC-MS-

ESI multidimensional methods. Yates and co-workers[145,146] developed an

Natishan1268

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on-line two-dimensional (2-D) IEX column coupled to a RP-HPLC column

called multidimensional protein identification technology (MudPIT) for separ-

ating tryptic digests of 80S ribosomes from yeast. The acidified peptide mix-

ture was loaded onto a strong cation exchanger (SCX) column, eluate fractions

injected onto a RP-HPLC column, and effluent coupled to a mass spectrometer.

Microtechnology in chromatography is playing a major role in protein

research[147,148] and the methods are serving as alternatives to 2-D gel electro-

phoresis methods, which have been used previously for protein analysis.

Nano-HPLC-ESI-FTICR-MS has been used with monolith columns for

rapid peptide screening by Leinweber et al.[149] The 100mm i.d. � 50 cm

monolith capillary column using gradient elution with 0.1% formic acid aqu-

eous and acetonitrile mobile phases, 20 nL injection and 20mL/min flow rate

was found to provide a fast, efficient elution, and high loading capacity for the

separation of synthetic libraries of peptides and amino acids.

Recent examples of proteomic LC/MS multidimensional capillary

methods[150–164] are given in Table 5. Selected applications are discussed in

further detail below.

A multidimensional chromatographic 2-D liquid phase method for separ-

ation of proteins from whole-cell lysates was developed using analytical-scale

chromatofocusing (CF) and RP-HPLC[150] with subsequent protein identifi-

cation by ESI-TOF, MALDI-TOF MS, and MALDI-TOF-MS/MS. The 2-D

liquid mapping technique was used for fractionating and comparing protein

expression using HCT-116 human colon adenocarcinoma cancer cell line trea-

ted with drug to the same untreated cell line. CF used a HPCF-1D column

(250mm � 2.0mm i.d.) and pH gradient with UV detection at 280 nm. CF

fractions were pooled according to the pH change and subsequently ana-

lyzed by the RP-HPLC method. The RP-HPLC method used a HPCF2D

(33mm � 4.6mm i.d.) NPS column and 0.1% trifluoroacetic acid (aq) :

0.1% trifluoroacetic acid in acetonitrile mobile phases, and UV detection of

proteins at 214 nm. Eluent fractions were collected and subsequently analyzed

by ESI-TOF-MS, MALDI-TOF-MS, and MALDI-TOF-MS/MS for identifi-

cation of proteins. It was found that the comparison between drug-treated

and untreated colon cancer cells showed distinct changes in protein expression

as a function of drug treatment. The method was more reproducible than the

2-D gel electrophoresis method and was effective in isolation and purification

of proteins in the liquid phase.

The rapid analysis of low resolution three-dimensional protein structure

of Cytochrome C and lysozyme was performed with minimal protein sample

by a combination of chemical cross-linking, enzymatic digestion, and

high resolution Fourier transform ion-cyclotron resonance (FTICR) mass

spectrometric analysis.[151] A nano-HPLC system was used with column

switching, directly coupled to an ESI-FTICR-MS, with a C18 separation


ORDER REPRINTS

column of dimensions 150mm � 75mm i.d., aqueous formic acid : acetoni-

trile mobile phases and flow rate at 200 nL/min. Computer software was

used for assignment of cross-linking products.

LC-MS has been used for identification of binding sites of covalent

acylglucuronide–albumin complexes using delta bilirubin as a model com-

pound.[162] A rapid LC-TOF/MSmethod in the ESI mode was used to identify

sequencing peptide fragments digested with trypsin. The LC/MS method

demonstrated that TOF/MS was useful for identifying peptide fragments

containing covalently bound drug.

RP-HPLC/Ion Mobility/TOF-MS was used to characterize a combina-

torial peptide library, which contained 4000 peptides.[163] Incorporation of

ion mobility allowed for separation of mixture components into distinct

charge-state families. This made it possible to obtain integrated mass spectra

for specific families of ions and isomeric library peptides, which have

identical LC retention times and could be separated on the basis of differences

in gas-phase mobilities.

Table 5. LC/MS multidimensional proteomic methods.

Sample LC/MS multidimensional method Reference

Whole-cell lysates HPLC-HPLC, MALDI-TOF MS [150]

Cytochrome C, lysozyme Nano-RP-HPLC FTICR-MS [151]

Human plasma Capillary RP-HPLC ESI-MS/MS [152]

Human plasma Capillary RP-HPLC Ion Trap-MS/MS [153]

Human plasma Capillary-RP-HPLC FTICR-MS/MS,

Capillary HPLC Ion Trap-MS/MS

[154]

Plasma membrane Capillary-RP-HPLC/MS [155]

Cell lysate RP-HPLC, ESI-TOF-MS,

MALDI-QTOF-MS

[156]

Human K562 cells Capillary and nano RP and SCX-

HPLC ESI-MS/MS

[157]

Bovine serum albumin Nano-RP-HPLC FTICR-MS [158]

Ovarian surface epithelial

cells, ovarian

carcinoma-derived

cell line

2-D liquid phase separation:

isoelectric focusing, RP-HPLC ESI-

TOF-MS

[159]

Breast cell cancer line Capillary RP-HPLC ESI-MS/MS [160]

Breast epithelial cell line RP-HPLC-MALDI-QTOF-MS/MS [161]

Bilirubin RP-mHPLC-TOF-MS ESI mode [162]

Combinatorial library

peptide mixture

RP-HPLC-IMS-TOF-MS [163]

Antisense oligonucleotides RP-HPLC-ESI2-MS and LC/MS [164]

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Structural characterization of antisense oligonucleotide–peptide conju-

gates using LC-ESI-MS and ion trap ESI2-MS was performed by Tengvall

et al.[164] The LC-ESI-MS method used a Luna C18 column (50mm �

2mm i.d.) and aqueous TEAA and triethylammonium formate pH 7 : aceto-

nitrile mobile phases. The LC-ESI-MS method provided a fair resolution

of similar compounds. Molecular weight determination with mass errors of

0.1–3.1 amu could be assigned.

Impurity Identification

LC/MS plays a critical role in the pharmaceuticals to characterize and iden-

tify impurities in API, drug product, and in chemical processes.[165] The ICH

regulatory requirements[50] indicate that impurities need to be identified at levels

at or exceeding 0.10% for drugs with maximum daily dose of �2 g/day and

0.05% for drugs with maximum daily dose.2g/day. Additionally, active phar-maceutical compounds are continuing to evolve to larger andmore complex mol-

ecules. The need for improved highly selective analytical methods is increasing

to determine structurally or closely related impurities is growing as well. LC-MS

has been widely employed in the pharmaceutical industry due to its high selec-

tivity. Complete structure elucidation may require not only MS but also MS/MS

or MS techniques coupled with NMR. There have been some recently published

applications of LC/MS for impurity identification.[165–169] Selected LC/MS

applications used for impurity identification are discussed.

Li et al.[167] used LC-MS in combination with direct infusion MS, NMR,

and organic synthesis to identify an unknown observed in a TLC separation of

cyproheptadine HCl tablets. The LC-MS conditions used a Zorbax SB-C8

column (150mm � 4.6mm i.d., 5mm), 0.1% trifluoroacetic acid (aq) : aceto-

nitrile mobile phases, and LC-ESIþ-MS. The results of the experiments indi-

cated that the unknown was two impurities, one an N-oxide degradate and the

other an adduct that was an artifact of the sample preparation procedure used

for the TLC method.

A new polar impurity present in a sample of mosapride was analyzed by

LC-MS/MS to obtain the identity and structural information.[168] The method

used a Symmetry Shield column (250mm � 4.6mm, 5mm), KH2PO4 (aq),

pH 4.0/TEA/acetonitrile mobile phases, and LC-MS/MS in the positive

mode with Turbo ion spray interface. The impurity identification was obtained

by the LC-MS/MS method and confirmed by independent synthesis of the

impurity and NMR determination.

A LC-MS method to determine the amounts of the meso-isomer impurity

in a 99mTc-d,l-HMPAO preparation was developed by Vanderghinste et al.[169]

using a radio-HPLC-MS method. The method used a XTerra MS C18 column


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(50mm � 2.1mm, 3.5mm), 0.1% formic acid : acetonitrile mobile phases and

TOF-MS with an orthogonal ESI probe. The HPLC method conditions gave

baseline separation between 99mTc-d,l-HMPAO and the 99mTc-meso-

HMPAO impurity and there was no decomposition of 99mTc-d,l-HMPAO

during the HPLC analysis.

Assay Methods

LC/MS is also used to quantify levels of minor components present in

pharmaceutical samples.[170–177] Selected recently published LC/MS assay

methods are given in Table 6. The LC-MS methods allowed quantitative

determination of the analyte with enhanced selectivity in the selective ion

monitoring mode (SIM) or by using tandem MS.

Stability Methods

LC/MS is also used for structural elucidation of impurities formed in API

and drug product formulation stability studies.[178–181] Recent publications in

LC/MS stability applications are discussed.

A review of LC-MS for identification of drug degradation products in

pharmaceutical formulations was completed by Wu.[178] Several examples

of LC-MS investigations are presented for formulations, which contained

oxidative, hydrolysis, dehydration, dimerization, rearrangement, or excipient

reaction degradates. LC-MS and LC-MS/MS methods were shown to be

powerful techniques for drug degradate elucidation, however, it may be

required to perform different MS ionization techniques and LC-NMR elucida-

tion for confirmation of the degradate structure.

An automated workstation for forced degradation of API was described

by Sims et al.[179] An Anachem SK233 XL autosampler with a reaction station

was used with HPLC-ESI-MS. The autosampler removed aliquots of API

diluted in acidic, basic, or oxidative solutions at programed time points, pre-

pared the analytical samples by neutralization and dilution, and injected the

samples onto the HPLC-ESI-MS. The automated workstation configuration

and fast HPLC method conditions allowed the reactions to be followed over

time since multiple timepoints can be taken. The automated system allowed

for the stress stability experiments to be completed in a significantly shorter

time compared to manual degradation techniques. Coupling the HPLC system

to an ESI-MS detector allowed faster identification of drug degradates.

A LC-MS approach for short-term stability studies of drug candidates as

an alternative to HPLC-UV stability assessments was reported by Simmonds

Natishan1272

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Table

6.

LC/M

Sassaymethodsusedforpharmaceuticals.

Analyte

LC-M

S

instrumentation

HPLCcolumn

HPLCmobilephase

Reference

Choline,acetylcholinein

lyophilized

powder

LC-A

PIMS/M

S

inESImode

LunaC18column

(150mm

�2.0mm

i.d.,3mm)

0.1:90:10(v/v/v)

heptafluorobutyricacid

(aq):w

ater:m

ethanol;

isocratic

[170]

Atorvastatin,lovastatin,

pravastatin,simvastatin

inaqueoussolution

LC-ESI-MS/M

SGenesisC18column

(50mm

�2.1mm

i.d.,3mm)

2mM

methylamine(aq)

2mM

inacetonitrile;

gradient

[171]

Aceminophen

gluronide

conjugatein

API

LC-A

PCIþ-M

SShim

-packCLCODScolumn

(150mm

�4.6mm

i.d.)

90:10(v/v)

methanol:water;

isocratic

[172]

Quinupristin,dalfopristin

inparenteralinjection

LC-ESIþ-M

S/MS

XTerra

MSC18column

(30mm

�2.1mm

i.d.,2.5mm)

70:30(v/v)

acetonitrile:w

ater;

isocratic

[173]

Carbam

azepinein

aqueous

solution

LC-ESIþ-M

S/MS

GenesisC8column

(150mm

�2.1mm

i.d.,3mm)

Acetonitrile/

methanol:10mM

ammonium

acetate/0.1%

form

icacid;gradient

[174]


ORDER REPRINTS

et al.[180] using LC-ESIþ-MS. The drug candidates were placed in each in one

of three commonly used preclinical study vehicles: 0.9% saline, Labrasolw, or

methyl cellulose and subjected to stability stress conditions (acid, base, or oxi-

dation) and analyzed by both HPLC-UV and HPLC- ESIþ-MS. It was found

that the LC-ESIþ-MS analysis revealed greater specificity in that co-eluting

impurities with similar UV chromophores as well as impurities that did not

have chromophores were detected.

HPLC WITH FL DETECTION

FL detectors in HPLC offer higher sensitivity and selectivity compared to

UV detection. The compound analyzed can be naturally fluorescent, subjected

to a chemical reaction or photoirradiation that converts the non-fluorescent

compound into a strongly fluorescent species. The FL detector in HPLC is

selective for fluorescent compounds only, non-fluorescent analytes, which

could be potential interferences, will not be detected. The high specificity of

the HPLC FL methods is especially useful in the analysis of biological

samples. A summary of some recently published methods[182–189] used for

analysis of biological samples using HPLC with FL detection are given in

Table 7. Advantages and different strategies to obtain fluorescent analytes

in selected methods given in Table 7 are described.

A simple and rapid fully automated method for the RP-HPLC determi-

nation of sotalol in human plasma was performed by Crommen et al.[184]

Sotalol is a naturally fluorescent compound. The method was performed by

coupling a precolumn packed with cation exchange restricted access material

(RAM) to the analytical column by means of a column switching technique for

sample pretreatment prior to HPLC analysis. FL detection improved the

method selectivity and sensitivity compared to the methods used previously.

A sensitive RP-HPLC method was developed for taurine in human

plasma.[185] Taurine is a free b-amino acid, which is not naturally fluorescent.

The HPLC method was performed using an N-methyltaurine internal standard

and derivatization with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxy-

phenylsulfonyl chloride (DMS-Cl). DMS-Cl reacts quantitatively with

amino acids to form stable and highly fluorescent sulfonamides with labeling

yield of about 100%. The method was improved in sensitivity and highly

reproducible for determination of low levels of taurine in human plasma.

A fluorometric method was developed for determination of khellin in human

urine and serum by RP-HPLC with post-column photoirradiation prior to FL

detection.[188] A post-column reagent was mixed with the mobile phase and irra-

diated with UV light to induce FL. The biological sample pretreatment steps

included dilution or de-proteinization with aqueous perchloric acid. The method

Natishan1274

ORDER REPRINTS

Table

7.

HPLCmethodsofbiological

samplesusingFLdetection.

Analyte,biological

matrix

HPLCmode

Column

Mobilephase

Fluorescence

wavelength,l

Lim

itofdetection

(LOD)

Reference

Telmisartanin

urine

RP-H

PLC

Nova-Pak

C18

(150mm

�3.9mm

i.d.,4mm)

55:45(v/v)5mM

phosphate

(aq):acetonitrile,

pH

6.0

Excitation

l¼

305nm,

emission

l¼

365nm

LOQ:

1.0mg/L

[182]

Aspartate,

glutamatein

rat

brain

micro-

dialysates

RP-H

PLC

HypersilC18

(150mm

�3.2mm

i.d.,5mm)

0.05M

sodium

acetatepH

7:m

ethanol

Excitation

l¼

330nm,

emission

l¼

440nm

Asp:0.12�

1026

mol/LGlu:

0.18�

1026

mol/L

[183]

Sotalolin

human

plasm

a

RP-H

PLC

LiChrospher

60

RP-SelectB

(125mm

�4mm,

5mm)

20:80(v/v)

methanol:50mM

potassium

dihydrogen

phosphate

pH

7/1

mM

octanesulfonicacid

Excitation

l¼

235nm,

emission

l¼

300nm

LOQ:5ng/mL

[184]

Taurinein

human

plasm

a

RP-H

PLC

Nova-Pak

C18,

(150mm

�3.9mm

i.d.,4mm)

Phosphate

(aq):acetonitrile

Excitation

l¼

318nm,

emission

l¼

392nm

3fm

ol

[185]

Nabumetonein

human

and

minipig

plasm

a

RP-H

PLC-

APCI-MS

LiChrospher

100

RP-C18,

(100mm

�4mm

i.d.,5mm)

45:55:1

(v/v/v)

acetonitrile:

water:aceticacid

Excitation

l¼

230nm,

emission

l¼

350nm

,0.002nmol/mL

[186]

(continued

)


ORDER REPRINTS

Table

7.

Continued.

Analyte,biological

matrix

HPLCmode

Column

Mobilephase

Fluorescence

wavelength,l

Lim

itofdetection

(LOD)

Reference

Nabumetonein

human

plasm

a

RP-H

PLC

Nova-Pak

C18,

(150mm

�3.9mm

i.d.,5mm)

50:50(v/v)

acetonitrile:

0.02M

TEA,pH

7

Excitation

l¼

230nm,

emission

l¼

356nm

0.05ng

[187]

Khellinin

human

urineandserum

RP-H

PLCwith

post-column

Photoirradiation

CapcellPak

C8,

(250mm

�4.6mm

i.d.)

40:60(v/v)

ethanol:75mM

H2O2in

water

Excitation

l¼

378nm,

emission

l¼

480nm

,1.3ng

[188]

Note:LOQ,limitofquantitation.

Natishan1276

ORDER REPRINTS

yielded improved sensitivity compared to previous methods and a simplified

procedure to determine khellin levels in human urine and serum samples.

HPLC WITH CL DETECTION

CL detection in HPLC may be used when the analyte lacks a chromo-

phore and/or improvements in selectivity and sensitivity are required. CL is

the generation of an excited species from a chemical reaction, which then

emits light upon relaxation.[1] Recent examples of HPLC with chemilumines-

cent detection are given in Table 8. Selected HPLC methods with CL detec-

tion given in Table 8 are discussed.

Isocratic RP-HPLC with coupled CL and FL detection was developed by

Kai et al.[190] for determination of cefaclor, a b-lactam antibiotic in blood

serum. The samples were first deproteinized by denaturation, derivatized with

4-(20-cyanoisoindolyl) phenylisothiocyanate for FL detection, injected into the

HPLC, which contained the analytical column, FL detector, on-line post-column

reactor, and CL detector in series. After detection by the FL detector, the column

eluate was mixed with the post-column reaction system. The cefaclor CL and

fluorescent derivatives were separated from other interfering compounds, the

separation is shown in the chromatograms in Fig. 7. The CL detector

[Fig. 7(B)] was found to be 10-fold more sensitive than by FL detection

[Fig. 7(A)]. The limits of detection were 1 and 10pmol for the CL and FL

detectors, respectively.

Pharmaceuticals containing a nitrogen atom, which lack a suitable chro-

mophore for HPLC analysis with UV detection, can be detected by an alternate

detector such as the chemiluminescent nitrogen detector (CLND).[191] The

HPLC effluent is oxidized and subjected to combustion in a high temperature

furnace. All nitrogen containing compounds (excluding N2) are converted to

nitric oxide gas. The gas is transferred to a membrane dryer and subjected

to a reaction with ozone that converts nitric oxide to excited state nitrogen

dioxide, which produces a photon emission upon relaxation. The signal of

the analyte is proportional to the moles of nitrogen present in the molecule.

A disadvantage of the CLND is that mobile phases for the HPLC separation

must not contain nitrogen; therefore, acetonitrile is unsuitable for use.

HPLC-CLND was evaluated by Allgeier et al.[191] A study was com-

pleted, which compared the CLND with the ELSD for pharmaceuticals that

lacked UV chromophores. The results of the detector evaluation revealed

that the CLND exhibited a more linear response and was 4-fold more sensitive

than the ELSD. The CLND, however, yielded poorer precision and required

more maintenance than the ELSD.


ORDER REPRINTS

Table

8.

HPLC-CLanalysisin

pharmaceuticals.

Analyte

Matrix

CLreagent

HPLCcolumn

HPLCmobile

phase

Lim

itofdetection

Reference

Cefaclor

Human

serum

CIPIC

a,post-

columnreaction

TSKgel

ODS-80TM

(150mm

�4.6mm

i.d.,5mm)

RP-H

PLC;

acetonitrile:

0.1%

triethylamine,

pH

8.5

1pmol

[190]

3APIclinical

candidates,

whichlacked

chromophore

andcontained

nitrogen

Powder

None,CLND

detectorused

GLSciencesIntersil

C4

(150mm

�2.1mm

i.d.)orZorbax

Rx

(150mm

�2.1mm

i.d.)columns

RP-H

PLC;0.1%

trifluoroacetic

acid

(aq):

methanol

5.4–9ng

[191]

Atenolol

metoprolol

Human

urine

[Ru(bpy) 32þ]2

b

post-column

addition

XterraRP18

(150mm

�4.6mm

i.d.,5mm)

RP-H

PLC;17:83

(v/v)

acetonitrile:

50mM

phosphatebuffer

(aq),pH

7.5

Atenolol:50pmol,

metoprolol:

8pmol

[192]

Lipid hydroperoxides

Human

plasm

a

Luminol,

post-column

addition

SupelcosilLC-N

H2

(250mm

�4.6mm

i.d.,5mm)

NP-H

PLC

9:1

:2:0.1,

(v,v,v,v)

methanol:

chloroform

:

1-propanol:

water

Notreported

[193]

Natishan1278

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Disopyramide

Human

serum

[Ru(bpy) 32þ]2

post-column

addition

TSKgel

ODS-80TS

(150mm

�4.6mm

i.d.,5mm)

RP-H

PLC;60:40

(v/v)methanol:

50mM

phosphate

buffer,pH

6.8

1.5ng

[194]

Erythromycin

Human

urineand

plasm

a

[Ru(bpy) 32þ]2

mobilephase

addition

BioanalyticalUnijet

C18(150mm

�

1mm

i.d.,5mm)

Micro-RP-H

PLC;

85:15(v/v)

100mM

phosphatebuffer

(aq)pH

7.0:

acetonitrile

50fm

ol

[195]

Dansylam

ino

acidsand

oxalate

Human

urineand

plasm

a

[Ru(bpy) 32þ]2

mobilephase

addition

Zorbax

ODS

(250mm

�4.6mm

i.d.,5mm)and

Microsorb-M

VC18

(100mm

�4.6mm

i.d.,3mm)

RP-H

PLC;0.1M

phosphate(aq)

with10%

methanol,

pH

7.0

Dansylam

ino

acids:0.1mM,

oxalate:

,0.1mM

[196]

a4-(20 -cyanoisoindolyl)phenylisothiocynate.

b[Ru(bpy) 32þ]2

¼tris(2,2

0 -bipyridyl)ruthenium(II).


ORDER REPRINTS

Electrogenerated chemiluminescence (ECL) of secondary or tertiary

amines has been performed by addition of tris(2,20-bipyridyl)ruthenium(II)

([Ru(bpy)32þ]2) to the mobile phase or adding the reagent by post-column

addition.[192,195–198] [Ru(bpy)32þ]2 based ECL has been used frequently for

CL determinations of secondary and tertiary amines due to simplicity of the

method, high sensitivity and selectivity. A micro-HPLC technique by Ridlen

et al.[195] used [Ru(bpy)32þ]2 ECL for determination of low level erythromycin

in urine and plasma samples. The ECL method permitted minimal sample

preparation (no extraction required) and yielded higher sensitivity than pre-

vious methods used.

HPLC WITH ELECTROCHEMICAL DETECTION

There has been new developments recently in the development of

ECD.[199] There have been advances in design of measuring systems and

new materials developed for working electrodes. ECD in HPLC is generally

used when the electroactive analyte of interest lacks a chromophore and

Figure 7. FL (A) and CL (B) detections in HPLC of a reaction mixture of cefaclor

and 4-(20-cyanoisoindolyl)phenylisothiocynate. 1.0mM cefaclor was used for the deri-

vatization reaction, and its 10 pmol amount was injected onto the chromatograph. (Rep-

rinted from Kai, M.; Kinoshita, H.; Ohta, K.; Hara, S.; Koo Lee, M.; Lu, J. J. Pharm.

Biomed. Anal. 2003, 30, 1765–1771. Copyright 2003 with permission from Elsevier.)

Natishan1280

ORDER REPRINTS

improved selectivity and sensitivity is required. Dual electrode systems offer

additional flexibility by operating in different modes for measurement.[200]

There have been numerous recently published applications using ECD.[199–208]

Selected examples of HPLC with voltammetric detection are described.

A sensitive and rapid isocratic RP-HPLC method with dual channel ECD

for determination of oxidized (GSSG) and reduced glutathione (GSH) in

dosage forms was developed by Manna et al.[200] A Supelcosil LC-18 DB

column (100mm � 4.6mm i.d., 5mm), 98 : 2 (v/v) 0.1% trifluoroacetic

acid (aq) : acetonitrile mobile phases, ECD working parameters of þ0.450V

for the first electrode and þ0.750V for the second electrode. The first elec-

trode potential was selective for GSH and the second potential for GSSG.

The detection limits were 0.60 ng for GSH and 0.15 ng for GSSG. The method

eliminated the need for pre-column derivatization, was more sensitive than

UV detection methods, allowed for quantitation of GSH and its oxidized

impurity GSSG, and was suitable for routine analysis.

Legorburu et al.[201] developed an HPLC method with amperometric

detection for determination of bumetanide in urine and pharmaceuticals.

The HPLC method uses a m-Bondapak column (300 nm � 3.9mm i.d.,

10mm), 50 : 50 (v/v) acetonitrile : 5mM KH2PO4–K2HPO4 (aq), pH 4.0

mobile phases, and amperometric glassy carbon working electrode set at

þ1350mV. The urine samples were subjected pretreatment using SPE or

LLE prior to analysis. The detection limit was 0.25 ng/mL. The method

was found to be more sensitive than previously reported fluorimetric and

diode array UV methods.

ECD was also used in a RP-HPLC method for determination of lercani-

dipine in tablets.[202] The ECD detector was equipped with a glassy carbon

working electrode, Ag/AgCl/NaCl reference electrode, platinum rod as the

auxiliary electrode and operated at 1000mV. The isocratic method used a

Symmetry C-18 column (150mm � 4.6mm i.d., 5mm) and 45 : 55 (v/v)acetonitrile : 0.01M phosphate buffer pH 4.0 mobile phases. The detection

limit of the method was 7.5 � 1027mol/L. The method was found to be sen-

sitive and selective to distinguish the parent compound from its hydrolysis,

photolysis, and chemical degradation products and excipients, which are not

electrochemically active.

Wyszecka-Kaszuba et al.[203] used HPLC with amperometric detection

for determination of 4-aminophenol impurities in multicomponent analgesic

preparations. It was necessary to develop a more sensitive method for the ana-

lytes. The analysis was performed using a Phenomenex Luna C-18 column

(100mm � 4.6mm, 5mm), isocratic 0.05M LiCl (aq) pH 4.0 :methanol

mobile phases, and the amperometric detector glassy carbon electrode

potential set at þ325mV vs. the Ag/AgCl reference electrode. The limit of

detection of the 4-aminophenol impurities was 1 ng/mL for API and 4 ng/


ORDER REPRINTS

mL for tablets. It was found that 4-aminophenol impurities could be selec-

tively detected by the HPLC method with amperometric detection in analgesic

preparations containing many other components, which could interfere

in the analysis, such as paracetamol, pseudoephedrine, dextromethorphan,

chlorpheniramine, codeine, mepiramine, propyphenazone, and caffeine.

A simple and sensitive HPLC method with ECD for determination of

creatine and its degradation product creatinine in rat plasma was developed

by Mo et al.[205] The method used a pulsed ECD interfaced to a thin-layer

amperometric with a gold working electrode, pH-Ag/AgCl combination

reference electrode and titanium counter electrode. The HPLC conditions

used a Jordi Glucose–DVB (250mm � 4.6mm i.d.) analytical column

and 2.5 : 2.5 : 90 : 5 (v/v/v/v) water : acetonitrile : 0.01M sodium acetate

(aq) : 1.0M sodium hydroxide (aq) mobile phases. The biological samples

were vortexed and diluted in water prior to injection; no other sample pretreat-

ment was required. The limit of detection was 0.0134mg/mL for creatine and

0.0113mg/mL for creatinine. The method offers the advantages of increased

sensitivity and selectivity for the electroactive analytes and the biological

samples did not have to be subjected to SPE or LLE prior to injection.

HPLC WITH ELSD

ELSD is also useful for the analysis of pharmaceuticals, which lack

chromophores. In ELSD, the HPLC effluent is nebulized and then vaporized

in a heated drift tube containing a nebulizer that disperses the vapor of par-

ticles through the light source.[191] The analyte particles scatter the light

beam and generate a signal at the photomultiplier tube that is proportional

to the amount of light scattered. The light scattered is proportional to the par-

ticle size and quantity of particles. ELSD is limited to the use of volatile

mobile phase components and relatively non-volatile analytes. A requirement

for ELSD detection is that the analytes detected should be less volatile than the

mobile phases. Advantages are that the detector can be used at high flow rates,

yield stable baselines with rapid changes in mobile phase composition, so suit-

able for gradient elution methods, has high sensitivity (low ppm), low dis-

persion, and is complementary to MS detection.[209] Table 9 gives some

recent published applications of HPLC with ELSD. Selected methods from

Table 9 are discussed in more detail.

Polyethylene glycols (PEGs) in dosage forms were effectively detected by

gradient HPLC with ELSD detection,[209] the HPLC method conditions are

given in Table 9. PEGs lack a chromophore for UV detection. PEG 400 and

PEG 1080 were analyzed by HPLC-ELSD with ELSD conditions of nebulizer

temperature at 508C and evaporator temperature at 708C. The HPLC-ELSD

Natishan1282

ORDER REPRINTS

Table

9.

HPLC-ELSD

applicationsin

pharmaceuticals.

Analyte

HPLCmode

Method

type

Column

Mobilephase

Reference

PEGsin

dosageform

sRP-H

PLC

Assay

PLRP-S

(150mm

�4.6mm

i.d.,5mm)

Acetonitrile:w

ater

[209]

Inulinin

biological

fluids

IEX

Assay

Aminex

HPX87P

(300mm

�7.8mm

i.d.,9mm);

BioradCarbohydrate

deashing

guardcolumn:cation-exchange

resin(30mm

�4.6mm

i.d.)

andanion-exchangeresin

(30mm

�4.6mm

i.d.)

Water

[210]

Phospholipid

based

derivatives

ofvalproic

acid

RP-H

PLC

Stability

Zorbax

Eclipse

XDB-C18

(250mm

�4.6mm

i.d.,5mm)

85:15:5

(v/v/v)

methanol:

acetonitrile:w

ater

[211]

Ibuprofenand

hydroxypropylm

ethylcellulose

inform

ulation

RP-H

PLC

Assay

GlycoSep

N(250mm

�4.6mm

i.d.)

60:40(v/v)

methanol:water

[212]

Sim

ethiconein

tabletand

suspensionform

ulations

RP-H

PLC

Assay

AlltimaC8(250mm

�4.6mm

i.d.,5mm)

Acetonitrile:

chloroform

gradient

[213]

Polysorbate80in

parenteral

form

ulations

RP-H

PLC

Assay

AlltimaC18(250mm

�4.6mm

i.d.,5mm)

Methanol:water

gradient

[214]

Midecam

ycin

RP-H

PLC

Impurity

profile,

assay

DiamonsilC-18(250mm

�4.6mm

i.d.,5mm)

Acetonitrile:0.2M

ammonium

form

ate

(aq),pH

7.3

[215]


ORDER REPRINTS

method was found to be more sensitive when compared to previous methods

used.

A method of simultaneously quantifying hydroxypropylmethylcellulose

(HMPC) and ibuprofen was developed by Whelan et al.[212] HMPC is an addi-

tive, which does not contain a chromophore and is used to modify crystal

structure during crystallization processing. The applied HPLC-ELSD method,

description given in Table 9, was advantageous in allowing simultaneous

analysis of HMPC and ibuprofen.

Polysorbate 80, which lacks a suitable chromophore for UV detection,

was quantified in parenteral formulations using HPLC-ELSD,[214] the HPLC

method conditions are given in Table 9. The Polysorbate 60 could be quanti-

fied as a single peak, which streamlined the quantification and was an advan-

tage of the methods compared to previous methods.

HPLC WITH RI DETECTION

RI detection in HPLC is a universal detection method. The analysis is

performed at a wavelength at which the analytes do not have absorptivity.[216]

Some conventional HPLC-RI pharmaceutical applications, which have been

published recently are given in Table 10. Selected examples from Table 10

are discussed in more detail.

Erlandsson et al.[217] included the developed an aqueous size exclusion

chromatography (SEC) HPLC method for determination of the molar mass

of poloxamers 188 and 407. Poloxamers are used in pharmaceuticals as deter-

gents and as dispersing, emulsifying, gelling, and solubilizing agents. The

HPLC method conditions are given in Table 10. RI detection provided a suit-

able alternative to the USP method for the molar mass determination as the

method could differentiate between polxamers 188 and 407.

Conventional HPLC-RI has also been used as an in-process method to

monitor the formation of a-, b- and g-cyclodextrin during the cyclodextrin

glycosyltransferease enzymatic synthesis from starch,[219] the method condi-

tions are given in Table 10. The method was also used for purity determination

and estimation of process yield.

A HILIC method with RI detection for analysis of tromethamine in API

was developed by Guo and Huang[221] Tromethamine is a polar compound,

lacks a UV chromophore, could not retained by typical RP-HPLC method

conditions and has limited solubility in aqueous solutions, which made it a

suitable candidate for HILIC with RI detection. The limit of detection was

0.03mg/mL. The HILIC approach exhibited advantages compared to conven-

tional RP-HPLC methods since tromethamine was retained, the large API

peak eluted at the solvent front, which did not interfere with the tromethamine

Natishan1284

ORDER REPRINTS

Table

10.

Conventional

HPLC-RIpharmaceuticalapplications.

Analyte

HPLCmode

Method

type

Column

Mobilephase

Reference

Poloxam

ers188

and407

SEC

Assay

TSKgel

3000PW

(300mm

�7.5mm

i.d.)

90:10(v/v)0.01M

(aq)NaC

l:

methanol

[217]

PEG/P

EO

SEC

Assay

Watersstyragel

HR3

andHR4

(300mm

�7.8mm

i.d.)

DMF:0.01M

LiBr

(aq):0.05M

acetic

acid

(aq)

[218]

a-,b-,and

g-Cyclodextrin

RP-H

PLC

In-process,

assay

Finepak-N

H2

(250mm

�4.6mm

i.d.,

5mm)

Acetonitrile:w

ater

mobilephases

and

pHbetween5.5and

6.0.

[219]

L-K

etohexoses

SEC

In-process

MetaC

arb87C

(300mm

�7.8mm

i.d.)

calcium

exchange

column

100%

water

[220]

Trometham

ine

HILIC

Assay

Zorbax

NH2

(150mm

�4.6mm

i.d.,

5mm)

80:20(v/v)

acetonitrile:w

ater

mobilephases

[221]


ORDER REPRINTS

determination, and higher organic mobile phase content effectively solubil-

ized tromethamine in solution.

Refractive index backscattering (RIBS) detection is a laser-based mode of

RI developed to be used with micro-separation systems.[216] The detector is in

early stages of development. The pathlength is very short in the RIBS detec-

tors, which is more appropriate for micro-HPLC methods rather than conven-

tional HPLC. The detector is based on backscatter interferometry,[222] uses a

simple optical train, produces higher sensitivity RI measurements in a small

diameter capillary with minimal path length sensitivity and requires no modi-

fications to the capillary tube.[222] The system has been used to separate stan-

dard test mixtures of salt, phenol, toluene, and ethylbenzene[222] and to test

mixtures of fructose, glucose, and sucrose.[223] Bornhop et al.[224] developed

a method, which resulted in 2.5-fold increase in sensitivity. The detection

limit was 7 � 1028 RIU in a 40-nL probe volume.

HPLC HYPHENATED WITH NMR

NMR spectroscopy is very useful for structural elucidation. MS with

HPLC does not always provide unequivocal structural identification and

NMR spectroscopic data is often needed. HPLC-NMR and HPLC-NMR-

MS save time and resources by avoiding the time consuming isolation of

unknown analytes for off-line NMR structural elucidication. By means of a

95 : 5 (v/v) post-column split to the NMR and MS detectors, NMR and MS

can be determined in the same analytical run.[225] Direct on-line coupling

of an NMR spectrometer as a detector to an HPLC required the develop-

ment of flow-probe hardware, solvent suppression pulse sequences and

software.[226]

There are currently five main operational modes, which can be used for

HPLC-NMR operation for isocratic or gradient elution HPLC methods.

They are on-flow, stop-flow, “time-sliced” stop-flow, peak collection into

capillary loops for post-chromatographic analysis and automatic peak detec-

tion with UV-detected triggered NMR acquisition.[226] Due to the develop-

ment of NMR resonance solvent suppression, it is not necessary to use

deuterated solvents, but in practice, D2O is often used instead of H2O because

it makes multiple solvent suppression easier.

A drawback of NMR is that the technique is not as sensitive as HPLC with

UV detection. The sensitivity of the on-flow LC-NMR experiment run at typi-

cal magnetic field strengths of 500MHz is limited to the residence time of

the analyte flowing at the HPLC flow rate through the NMR cell. Strategies

to increase the analyte residence time have been to reduce the flow rate

to 0.1–0.4mL/min, perform sample pretreatment techniques (lyophilization

Natishan1286

ORDER REPRINTS

or SPE) to concentrate the analyte or overload the chromatographic

column.[225] Additionally, excellent sensitivity of NMR has been achieved

in the stop-flow mode.[226]

NMR coupled with HPLC and/or MS has been used for structure

identification in combinatorial chemistry, synthetic chemical impurities,

natural products, and drug metabolism areas in the pharmaceutical industry.

Lindon et al.[226] have reviewed directly coupled HPLC-NMR and HPLC-

NMR-MS in pharmaceutical research and development. Some recently pub-

lished examples of HPLC-NMR and HPLC-NMR-MS applications[225–235]

are given in Table 11. Selected applications given in the table are described in

more detail.

Spraul et al.[225] reported an advance in increasing the sensitivity of a

hyphenated LC-NMR-MS system by using a cryogenic probe built in flow

configuration: the method conditions are given in Table 11. The probe was

found to provide significantly higher sensitivity over conventional non-

cryogenic flow NMR probes. The cryo probe allowed identification of pre-

viously undetected metabolites of acetaminophen (APAP) to be identified

and simultaneous MS data provided information concerning analytes that

are not detected by the NMR. The results revealed that there were co-eluting

components (undetected previously) in the chromatographic peaks. Figure 8

illustrates typical on-flow and stopped-flow 1H NMR spectra from the

LC-NMR-MS experiments. Figure 8(c) and (d) exhibited good quality spectra

for APAP phenolic glucuronide and phenolic sulfate with only 16 free-induc-

tion decays (FID) during the on-flow experiment. Stopped-flow analysis was

carried out to achieve higher sensitivity and stop-flow spectra are shown in

Fig. 8(e)–(g), which indicate that there were six co-eluting components in

the HPLC peaks. The experiments permitted direct injection of whole

untreated urine with LC-NMR-MS. Definitive assignment of metabolites

was possible with the NMR and MS data.

A strategy for rapid isolation and structural identification of the meta-

bolites of two API’s using HPLC-MS/MS and HPLC-NMR was completed

by Kim et al.,[227] see method conditions in Table 11. Various in-vitro biologi-

cal systems were evaluated for biosynthesis of the metabolites. Isolation of the

metabolites from rat liver microsomes and rat bile was completed by HPLC-

MS, pretreatment with SPE and then subsequent structural elucidation by

HPLC-NMR. Combining the HPLC-MS/MS and HPLC-NMR results

allowed exact hydroxylation sites to be unambiguously assigned.

Dear et al.[233] used directly coupled IEX-HPLC-NMR-MS for polar

metabolite identification. A novel N-acetyl metabolite of a highly polar

drug candidate [2-(ethanimidoylamino)ethyl]sulfonyl alanine, which lacks

a UV chromophore was identified, see method conditions in Table 11.

Urine samples were pretreated by SPE prior to analysis with recoveries of


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Table

11.

HPLC-N

MRandHPLC-N

MR-M

Sapplicationsin

pharmaceuticals.

Analyte,matrix

HPLCcolumn

HPLCmobilephase

NMRconditions

MS

Reference

Acetaminophen

metabolites

inurine

YMC-PackFL-O

DS

column

(50mm

�4.6mm

i.d.,5mm)

0.05%

trifluoroacetic

acid

(D2O):0.05%

trifluoroacetic

acid

(acetonitrile-d

3);

gradient

1H

NMRspectroscopy

at500.17MHz,

cryogenic

probe,

stop-flow

Positiveion

electrosprayMS

[225]

Metabolitesoftwonew

drugcandidates

Eclipse

XDB-C18

column

(150mm

�4.6mm

i.d.,5mm),

Symmetry

C-18

(150mm

�3.9mm

10mM

ammonium

acetate

with0.6%

acetic

acid

(aq):10mM

ammonium

acetatewith0.6%

acetic

acid

(acetonitrile);

gradient

1H

NMRspectroscopy

at500MHz,

stop-flow

withUV

detector

TurboIonSprayin

positivemode

MS

[227]

Compoundswithno

chromophores:taurine,

hydroxyproline,aspartic

acid,glycinein

plasm

a

Purospher

RP-18e

column(150mm

�

4mm

i.d.,5mm)

0.5mM

pentadecafluorooctanoic

acid

(D2O);isocratic

1H

NMRspectroscopy

at400MHz,

stop-flow

n/a

[228]

2-Trifluorm

ethylaniline,

2-trifluoromethyl

acetanilidein

urine

Spherisorb

ODS2C-18

(250�

4.6mm

i.d.,

5mm)

0.1M

ammonium

acetatein

D2O,pH

5.2:

acetonitrile;gradient

On-flow

19FNMRat

400MHz,stop-flow

1H

NMRat

500MHz

Triple

quad

with

positiveand

negativeFIB

[229]

4-Trifluorm

ethoxyaniline,

[13C]-4-trifluoro-

methoxyacetanilide

metabolitesin

urine

Spherisorb

ODS2C-18

(250�

4.6mm

i.d.,

5mm)

0.1M

ammonium

acetatein

D2O,pH

5.2:

acetonitrile;gradient

On-flow

19FNMRat

400MHz,stop-flow

1H

NMRat

500MHz

Triple

quad

with

positiveand

negativeFIB

[230]

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Steroidsin

gel

form

ulation

ProntoSilC30column

(250mm

�4.6mm

i.d.,3mm)

Methanol:D2O;gradient

Stop-flow

1H

NMRat

600MHz,LC

inverse

probe

n/a

[231]

2,3,5,6-Tetrafluoro-4-

trifluoromethylaniline

metabolitesin

urine

HypersilBDSC18

(250mm

�4.6mm

i.d.,5mm)

0.01%

ammonium

form

ate

(D2O),pH

7:acetonitrile;gradient

Stop-flow

19FNMRat

564.62MHz,stop-

flow

1H

NMRat

500.13MHz

LC-ESI-MSiontrap

[232]

[2-(Ethanim

ido-

ylamino)ethyl]sulfonyl

alanineN-acetylmetabolite

inurine

Dionex

PCX-100

(250mm

�

4mm

i.d.)

Acetonitrile:aqueous

50mM

form

ic

acid:100mM

aqueous

ammonium

form

ate;

gradient

Loopstorageorstop-

flow

1H

NMRat

600.13MHz,with

dual

1H-19FLC-flow

probe

Off-line

LC-A

PI-MSwith

electrospray

interface

[233]

HIV

-1RTinhibitor

BW935U83metabolitesin

urine

WatersSpherisorb

ODS-2

(250mm

�

4.6mm

i.d.)

0.1%

trifluoroacetic

acid

(D2O):acetonitrile;

gradient

Stop-flow

1H

NMRat

500.13MHz,on-

flow

19FNMRat

470.59MHz,with

dual

1H-19FLC-flow

probewithUV

detector

HPLC-ESIþ-M

S[235]


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Figure 8. (a, b, h) Individual 1H NMR spectra extracted from the on-flow LC-NMR-

MS experiment. Each extracted experiment corresponds to one NMR increment of 16

accumulated FIDs. (c–g) Stopped-flow spectra of a repeated injection of the same

sample with the flow stopped at the indicated retention times, with 16 (c, d), 128 (e),

and 256 (f, g) accumulated FIDs for improved signal/noise ratio. The residual HDO

signal was attenuated by filtering the d 4.5–5.2 region post-acquisition. (Reprinted

with permission from Spraul, M.; Freund, A. S.; Nast, R. E.; Withers, R. S.; Maas,

W. E.; Corcoran, O. Anal. Chem. 2003, 75, 1546–1551. Copyright 2003 American

Chemical Society.)

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approx. 80%. The method was successful in determination of the polar meta-

bolite in [2-(ethanimidoylamino)ethyl]sulfonyl alanine.

HPLC HYPHENATED WITH ICP-MS

HPLC has been used for separation and speciation of metal compounds.

ICP is a very sensitive techniques for detection of metals. ICP-MS offers the

capabilities of speciation with multi-element detection, of isotope measure-

ments to improve precision and accuracy, excellent sensitivity and detection

limits, and a wide dynamic range.[236] As a result, the coupling of LC and

ICP-MS has become one of the most popular techniques for elemental specia-

tion studies due to high versatility, robustness, sensitivity, and multi-elemental

capabilities. Coupling with HPLC is accomplished by connection of the nebu-

lizer to the exit of the column. Conventional HPLC and ICP flow rates are

comparable. Several reviews have been written about LC-ICP and LC-ICP-

MS.[237,238] The modes of HPLC that are the most compatible with ICP are

SEC, MLC, and IEX since the mobile phases used are predominantly aqueous.

RP-HPLC methods are limited since methods containing high ratios of organic

mobile phase are not ICP-MS compatible.

HPLC-ICP-MS has been used for the analysis of biological fluids to help elu-

cidate metabolic and detoxification pathways and also to help in the identification

and characterization of proteins.[239] The speciation of Fe, Cu, and Zn in human

serum was completed using LC-ICP-MS and on-line isotope dilution. An ion

exchange separation with a Mono-Q HR 5/5 anion exchange column with aqu-

eous ammonium acetate at pH 7.4was used for the analysis.[240] LC-ICP-MSwas

also used for quantitative determination of cis-[amminedichloro(2-methylpyri-

dien)]-platinum(II) (ZD0473) drug substance in dog plasma.[241] The method

used a Phenomenex Synergi Polar RP (150mm � 4.6mm) and 20 : 80 (v/v)methanol :water mobile phase containing 0.1% formic acid and 0.15mM

ammonium acetate pH 3. The chromatograms of the blank, 10pg ZD0473 and

the dog plasma sample are shown in Fig. 9. The LC-ICP-MS method was instru-

mental in providing a quantitative novel assaymethod for the drug substance that

could distinguish between the parent compound containing Pt, two Pt “aqua”

species and inactive metabolites with a limit of quantification of 0.1 ng/mL.

HPLC HYPHENATED WITH IR

HPLC has been coupled with IR as a means to provide detection and

useful spectral information of unknown impurities in API and drug product.

LC-IR analysis can be valuable in that there are numerous spectra available


ORDER REPRINTS

in spectral libraries where characteristic bands in the IR spectrum can be used

to identify functional groups of unknowns, identification of unknowns can be

performed by peak matching methods, and different isomers can be distin-

guished. Additionally, LC-IR is a non-destructive technique and the same

sample can be used for further analysis.

The interfaces that have been used for LC-IR are a flow-through cell or

solvent-elimination.[242] The simplest approach is the flow-through cell in

which the mobile phase from the HPLC column flows through the IR cell and

spectra are continuously reported. The solvent-elimination interface evaporates

the solvent from the HPLC eluent and deposits the analytes onto a substrate.

Most commonly for identification of minor components or unknown impurities,

the solvent-elimination interface is used to increase sensitivity because most

Figure 9. Example chromatograms for HPLC-ICP-MS. (A) blank, (B) limit of

quantification 10 pg on column, and (C) 0.5 hr sample. (Reprinted with permission

from Smith, C. J.; Wilson, I. D.; Abou-Shakra, F.; Payne, R.; Parry, T. C.; Sinclair,

P.; Roberts, D. W. Anal. Chem. 2003, 75, 1463–1469. Copyright 2003 American

Chemical Society.)

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common solvents used in HPLC absorb IR radiation at wavelengths in the mid-

infrared region and water has intense absorption across most of the spectrum,

which obscures sample bands in several spectral regions and significantly limits

sensitivity. Volatile modifiers should be used for solvent elimination interface

techniques and the analyte should be less volatile than the mobile phases.[242]

Selected applications of LC-IR of pharmaceuticals are described.

LC-IR with a solvent-elimination interface was applied to the analysis of

analgesics and antibiotics.[243] The LC-IR method used a Jordi OVB reverse

phase column (250mm � 4.6mm, 5mm) and 100% methanol mobile phases.

The LC-IR analysis gave separation and clean spectra of the acetaminophen,

acetylsalicylic acid, and caffeine components present in the analgesic. The

chromatogram and spectra generated from a separation of an analgesic

Figure 10. Chromatographic and spectral data from LC-IR analgesic separation.

Conditions-column: Jordi OVB reverse phase (250mm � 4.6mm, 5mm); mobile

phase: 100% methanol; flow rate: 0.8mL/min total, 0.2mL/min split to IR detector;

injection volume: 25mL; drift tube temperature: 858C; column temperature: 508C;collection rate: 20 spectra/min; IR band: 1750 cm21 with a range of 50 cm21 to maxi-

mize the absorbance from the carbonyl peaks in the aspirin and caffeine spectra.

Analytes: (a) acetaminophen, (b) acetylsalicylic acid (aspirin), (c) caffeine. (Geldart,

S. Am. Lab. 2000, 32 (Jan), 32–37. Reprinted with permission from International

Scientific Communications, Inc.)


ORDER REPRINTS

tablet are shown in Fig. 10. The IR was used for identification of the

acetaminophen, acetyl salicylic acid, and caffeine peaks present in the

HPLC chromatogram. LC-IR was also shown to be useful for analysis of

compounds that do not have a UV chromophore such as erythromycin. Ery-

thromycin was subjected to acid hydrolysis and the LC-IR technique helped a

determine the identity of the degradate by providing functional group spectral

information.

The on-line detection of water soluble vitamins in multivitamin tablet for-

mulations was completed by LC-IR with a solvent elimination interface.[244]

The HPLC method used a C18 Microsorb column (250mm � 4.6mm,

5mm), 0.01M ammonium acetate : methanol mobile phases and gradient

elution. The vitamin analytes were deposited on a moving zinc selenide

plate and FTIR spectra of the solids was taken. The vitamins in the multivita-

min tablet formulation were identified by retention and IR spectral matching

with standards.

CONCLUSION

This review has summarized recent developments in pharmaceuticals

using achiral HPLC methodology with different detectors for the analysis of

API, formulated drug product, biologicals and protein samples. HPLC is a

powerful technique used throughout the pharmaceutical industry during all

stages of drug development. While RP-HPLC is used most commonly in

pharmaceutical analysis, alternative HPLC modes such as normal phase, ion

exchange, size exclusion, MLC, hydrophilic interaction, and MIP have been

used when RP-HPLC was not acceptable for the separation. Recent develop-

ments in column technology, fast-LC, capillary, and micro-HPLC have signi-

ficantly shortened analysis times to assist with the increasingly fast pace of

drug development. There is a wide variety of HPLC detectors, which are

very useful for pharmaceutical compounds that lack a chromophore or require

high sensitivity and selectivity. The hyphenation of HPLC with MS, NMR,

ICP, and IR spectroscopic detectors has become critical in identification of

unknown chemical compounds.

ACKNOWLEDGMENTS

I would like to gratefully acknowledge Nelu Grinberg for his valuable

technical discussions. I would also like to acknowledge and thank Vincent

Antonucci, Xiaoyi Gong, Marie Achalabun, and Angela Pinto for their kind

support and review of the document.

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Received December 1, 2003

Accepted December 9, 2003

Manuscript 6284

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