paracetamol, diclofenac potassium, and famotidine

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Validated HPTLC Method for Simultaneous Quantitation ofParacetamol, Diclofenac Potassium, and Famotidine in TabletFormulation

LAXMAN D. KHATAL, ASMITA Y. KAMBLE, MAHADEO V. MAHADIK, and SUNIL R. DHANESHWAR1

Bharati Vidyapeeth University, Poona College of Pharmacy, Department of Pharmaceutical Chemistry, Pune, Maharashtra,India 411038

A sensitive, simple, selective, precise, andaccurate HPTLC method of analysis forparacetamol, diclofenac potassium, and famotidine both as a bulk drug and in tablet formulation wasdeveloped and validated. The method used HPTLCaluminum plates precoated with silica gel 60F254 as the stationary phase, and the mobile phaseconsisted of toluene–acetone–methanol–formic acid (5 + 2 + 2 + 0.01, v/v/v/v). Densitometricevaluation of the separated zones was performedat 274 nm. This system was found to give compactspots for paracetamol (Rf value = 0.62 ± 0.03),diclofenac potassium (0.75 ± 0.02), and famotidine(0.17 ± 0.03). The linear regression analysis datafor the calibration plots showed a good linearrelationship over the concentration range of1625–9750 ng/spot for paracetamol,250–1500 ng/spot for diclofenac potassium, and100–600 ng/spot for famotidine. The method wasvalidated for precision, robustness, and recoveryaccording to International Conference onHarmonization guidelines. No chromatographicinterference from the tablet excipients was found.Statistical analysis showed that the method wasrepeatable and selective for the simultaneousquantitation of the three drugs in tablet formulation and for routine quality control of raw materials ofthe drugs.

Acombination of paracetamol (PAR), diclofenacpotassium (DCL), and famotidine (FAM) is used as an analgesic, antipyretic, and antacid for the treatment of

fever, pain, and acidity. PAR (p-hydroxy acetanilide) hasanalgesic and antipyretic effects. The mechanism of action ofPAR is by inhibition of the cyclooxygenase enzyme andprostaglandin synthesis in the central nervous system and itsdirect activity on the center for body temperature regulation in

the hypothalamus (1). Diclofenac, as the potassium salt,is a benzeneacetic acid derivative, designated chemicallyas 2-[(2,6-dichlorophenyl) amino] benzeneacetic acid,monopotassium salt. The mechanism of action of DCL,like that of other nonsteroidal anti-inflammatory drugs, is notcompletely understood but may be related to prostaglandinsynthetase inhibition (2, 3). FAM is chemically3-([2-(diaminomethyleneamino) thiazol-4-yl] methylthio)-N¢sulfamoylpropanimidamide. FAM binds to H2-receptorslocated on the basolateral membrane of the parietal cell,blocking histamine effects (4).

A literature review revealed that methods have beenreported for analysis of PAR, DCL, and FAM inpharmaceuticals, including HPLC and HPLC/MS methods forquantitation in human plasma (5–14), and HPLC and HPTLCmethods for quantitation in formulations (15–33), either aloneor in combination with other drugs. This paper reports for thefirst time the simultaneous quantitation of PAR, DCL, andFAM by HPTLC in bulk drug and pharmaceutical dosage form. The proposed method was validated according to InternationalConference on Harmonization (ICH) guidelines (34).

Experimental

Materials

Working standards of pharmaceutical grade PAR (BatchNo. 260738), DCL (Batch No. 160-2008), and FAM (BatchNo. 0410708) were obtained as generous gift samples fromBal Pharmaceuticals Ltd, Pune, India; Brihans Laboratories,Pune (Maharashtra, India); and Vaasava Pharmaceuticals Pvt. Ltd, Solapur (Maharashtra, India), respectively. Fixed-dosecombination tablets (MAHAGESIC PLUS, Batch No.MHP-24) containing 325 mg PAR, 50 mg DCL, and 20 mgFAM were procured from WINDLABS Biotech Ltd,Deharadun (Uttar Pradesh, India). All chemicals and reagentswere of analytical grade and were purchased from MerckChemicals, Mumbai (Maharashtra, India).

Instrumentation

The samples were spotted in the form of bands of 6 mmwidth with a 100 mL sample syringe (Hamilton, Bonaduz,Switzerland) on silica gel precoated aluminum 60F254 plates,(10 ´ 10 cm with 250 mm thickness; E. Merck, Darmstadt,

KHATAL ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 3, 2010 765

Guest edited as a special report on “New Planar Chromatography–Densitometry Methods for Determination of Drug Substances andFormulations” by Jan Krzek and Joseph Sherma.

1 Corresponding author’s e-mail: [email protected]

Germany) using a CAMAG Linomat 4 (Muttenz,Switzerland) sample applicator. The plates were prewashedwith methanol and activated at 110°C for 5 min prior tochromatography. A constant application rate of 0.1 mL/s wasused, and the space between two bands was 5 mm. The slitdimension was 5 ´ 0.45 mm, and the scanning speedwas 10 mm/s. The monochromator bandwidth was set at20 nm, each track was scanned three times, and baselinecorrection was used. The mobile phase consisted oftoluene–acetone–methanol–formic acid (5 + 2 + 2 + 0.01,v/v/v/v), and 9 mL mobile phase was used/chromatographicrun. Linear ascending development was carried out in anHPTLC twin-trough glass chamber (CAMAG) saturated withthe mobile phase vapor. The optimized chamber saturationtime was 30 min at room temperature (25 ± 2°C) at a relativehumidity of 60 ± 5%. The length of each chromatogramrun was 8 cm. Following the development, the HPTLC plateswere dried in a current of air using an air dryer in awooden chamber with adequate ventilation. The air flowin the laboratory was maintained unidirectionally(laminar flow, towards the exhaust). Densitometric scanningwas performed using a CAMAG TLC Scanner 3 in thereflectance-absorbance mode at 274 nm and operated by

CATS software (Version 3.15, CAMAG). The radiationsource used was the deuterium lamp emitting a continuousUV spectrum between 190 and 400 nm. Concentrations of thecompound chromatographed were determined from theintensity of the diffused light. Evaluation was based on peakareas with linear regression. In situ spectra were obtainedfrom 200 to 350 nm using the spectral mode of thedensitometer.

Preparation of Standard Stock Solutions

Standard stock solutions of concentration 32.5 mg/mLPAR, 5 mg/mL DCL, and 2 mg/mL FAM were preparedseparately using methanol. From the standard stock solution,the mixed standard solution was prepared using themethanol to contain 3.25 mg/mL PAR, 0.5 mg/mL DCL,and 0.2 mg/mL FAM. The stock solution was stored at 2–8°Cprotected from light.

Optimization of the HPTLC method

The HPTLC procedure was optimized to develop asimultaneous assay method for PAR, DCL, and FAM. Themixed standard stock solution (3.25 mg/mL PAR, 0.5 mg/mLDCL, and 0.2 mg/mL FAM) was spotted onto HPTLC plates

766 KHATAL ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 93, NO. 3, 2010

Figure 1. Overlaid in situ spectra of DCL, FAM, and PAR.

and developed in different mobile phases. Development of a

simultaneous assay method for PAR, DCL, and FAM was

very critical because of the different polarities of these drugs.

FAM is polar, whereas PAR and DCL are nonpolar and their

polarity index is very similar. Initially, toluene–ethyl

acetate–methanol (2 + 6 + 1, v/v/v) was tried, but in this

system PAR and DCL moved and separated from each other,

but FAM did not move from application position. Hence,

in order to move FAM, the polarity of the system was

increased by increasing the ethyl acetate and methanol

concentration, but DCL moved with solvent front. Then,

toluene–n–butanol–formic acid (2 + 5 + 0.01, v/v/v) was

tried; it resulted in movement of FAM, but DCL and

PAR were merged with each other. Finally, a mobile

phase consisting of toluene–acetone–methanol–formic acid

(5 + 2 + 2 + 0.01, v/v/v/v) was found to be optimum.

Densitometric scanning was done at 274 nm, as all drugs

showed maximum response at that wavelength (Figure 1).

In order to reduce the neckless effect (i.e., the effect caused by

improper saturation of the TLC chamber that results in uneven

development of the TLC plate), the HPTLC chamber wassaturated for 20 min using saturation pads. The plate wasdeveloped for a distance of 8 cm, which took approximately20 min.

Validation of the Method

Validation of the optimized HPTLC method was carriedout with respect to the following parameters:

(a) Linearity.—The mixed standard stock solution wasfurther diluted to obtain concentrations of 1.625 mg/mL PAR,0.25 mg/mL DCL, and 0.1 mg/mL FAM. From dilutedmixed standard stock solution, 1 to 6 mL aliquots were spottedon the HPTLC plate to obtain final concentrations of1625–9750 ng/spot for PAR, 250–1500 ng/spot for DCL, and100–600 ng/spot for FAM. Each concentration was appliedsix times on the HPTLC plate. The plate was then developedusing the optimum mobile phase, and the peak areas wereplotted against the corresponding concentrations to obtain thecalibration plots.


Table 1. Linear regression data for calibration plots

Parameters PAR DCL FAM

Linearity range, ng/spot 1625–9750 250–1500 100–600

r2 0.992 0.988 0.995

Slope 326 1642 1394

Intercept 973 527 438

Table 2. Precision studies

Repeatabilitya Intermediate precisiona

Concentration, ng/spotMeasured concentration

± SD RSD, %Measured concentration

± SD RSD, %

PAR

1625 1606 ± 21 1.30 1598 ± 29 1.814

4875 4852 ± 47 0.968 4864 ± 16 0.328

9750 9732 ± 18 0.184 9724 ± 25 0.257

DCL

250 242 ± 4.3 1.77 246 ± 3.5 1.422

750 738 ± 6.9 0.934 732 ± 5.1 0.696

1500 1483 ± 7.1 0.478 1478 ± 6.8 0.460

FAM

100 97.3 ± 1.4 1.44 98.1 ± 0.8 0.876

300 288 ± 1.7 0.59 293 ± 2.6 0.883

600 586 ± 3.3 0.56 591 ± 3.8 0.635

a n = 6.

(b) Precision.—The precision of the method was verifiedby repeatability and intermediate precision studies.Repeatability studies were performed by analysis of threedifferent concentrations (1625, 4875, and 9750 ng/spot forPAR; 250, 750, and 1500 ng/spot for DCL; and 100, 300, and600 ng/spot for FAM) of the drug six times on the same day.The intermediate precision of the method was checked byrepeating the studies on 3 different days.

(c) LOD and LOQ.—LOD and LOQ representthe concentration of the analyte that would yield an S/N of3 and 10, respectively. LOD and LOQ were determined bymeasuring the magnitude of the analytical background byspotting a blank and calculating the S/N for a series of spottedPAR, DCL, and FAM solutions until the S/N was 3 for LODand 10 for LOQ. To determine the LOD and LOQ, serialdilutions of mixed standard solution of PAR, DCL, and FAMwere made from the standard stock solution in the range of10–100 ng/spot. The samples were applied to the HPTLCplate, the chromatograms were run, and the measured signalsfrom the samples were compared with those of blank samples.

(d) Robustness.—Following the introduction of smallchanges in the mobile phase composition (±0.1 mL foreach component), the effects on the results were examined.Mobile phases having different compositions, e.g.,toluene–acetone–methanol–formic acid (5.1 + 2 + 2 + 0.01,

v/v/v/v), (4.9 + 2 + 2 + 0.01, v/v/v/v), (5 + 2.1 + 2 + 0.01,v/v/v/v), and (5 + 1.9 + 2 + 0.01, v/v/v/v), were used todevelop chromatograms. The amount of mobile phase wasvaried over the range of ±5%. The plates were prewashed with methanol and activated at 110°C for 2, 5, and 7 min prior tochromatography. The time from spotting to chromatographyand from chromatography to scanning was varied by ±10 min. The robustness of the method was determined at threedifferent concentration levels: 1625, 4875, and 9750 ng/spotfor PAR; 250, 750, and 1500 ng/spot for DCL; and 100, 300,and 600 ng/spot for FAM.

(e) Specificity.—The specificity of the method wasdetermined by analyzing standard and drug samples. Thespots for PAR, DCL, and FAM in the samples were confirmed by comparing the Rf and spectrum of the spot with that of astandard. The peak purity of PAR, DCL, and FAM wasdetermined by comparing the spectrum at three differentregions of the spot, i.e., peak start, peak apex, and peak end.

(f) Accuracy.—Accuracy was measured by applying themethod to preanalyzed drug sample (PAR, DCL, and FAMcombination tablet) to which known amounts of PAR, DCL,and FAM standard powder corresponding to 80, 100, and120% of the label claim had been added (standard additionmethod). The drug sample and spike were mixed, and thepowder was extracted and analyzed by the optimized method.


Table 3. Robustness testinga

ParameterSD of peak area

for PAR RSD, %SD of peak area

for DCL RSD, %SD of peak area

for FAM RSD, %

Mobile phase composition (±0.1 mL) 7.22 0.46 3.25 0.67 3.84 1.07

Amount of mobile phase (±5%) 2.13 0.31 4.37 1.12 4.08 0.863

Time from spotting to chromatography (±10 min) 1.26 0.631 2.69 2.33 5.14 1.42

Time from chromatography to scanning (±10 min) 3.28 1.17 1.66 1.02 2.72 1.17

a n = 6.

Table 4. Recovery studiesa

DrugLabel claim,

mg/tabletAmount added,

%Total amount,

mgAmount recovered,

mg ± RSD, %Recovery,

%

PAR 325 80 585 564 ± 1.27 96.41

100 650 638 ± 0.614 98.15

120 715 690 ± 0.452 96.50

DCL 50 80 90 87.6 ± 0.527 97.33

100 100 96.3 ± 1.14 96.30

120 110 105 ± 0.748 95.45

FAM 20 80 36 34.5 ± 1.02 95.83

100 40 38.3 ± 1.32 95.75

120 44 42.1 ± 0.851 95.68

a n = 6.

Analysis of a Marketed Formulation

To determine the content of PAR, DCL, and FAM in apharmaceutical tablet (brand name: MAHAGESIC PLUS;label claim: 325 mg PAR, 50 mg DCL, and 20 mgFAM/tablet), 20 tablets were weighed, their mean weight wasdetermined, and they were finely powdered. The weight of the tablet triturate equivalent to 325 mg PAR, 50 mg DCL, and20 mg FAM was transferred into a 50 mL volumetric flaskcontaining 20 mL methanol. The solution was sonicated for30 min, and diluted to 50 mL with methanol. The resultingsolution was centrifuged at 3000 rpm for 5 min, and the drugcontent of the supernatant was determined (6.5, 1.0, and0.4 mg/mL for PAR, DCL, and FAM, respectively). Then5 mL of the above filtered solution was diluted to produce aconcentration of 3250, 500, and 200 mg/mL for PAR, DCL,and FAM, respectively, and 1 mL of this solution (3250, 500,and 200 ng/spot for PAR, DCL, and FAM, respectively) wasapplied to an HPTLC plate that was developed in theoptimized mobile phase. The analysis was repeated intriplicate. The possibility of excipient interference with theanalysis was examined.

Results and Discussion

Validation

The results of validation studies on the simultaneousHPTLC determination method developed for PAR, DCL,and FAM with toluene–acetone–methanol–formic acid

(5 + 2 + 2 + 0.01, v/v/v/v) as the mobile phase are givenbelow.

Linearity.—The drug response was linear over theconcentration range between 1625–9750 ng/spot for PAR,250–1500 ng/spot for DCL, and 100–600 ng/spot for FAM(Table 1).

Precision.—The results of the repeatability andintermediate precision experiments are shown in Table 2. Thedeveloped method was found to be precise, with RSD valuesfor repeatability and intermediate precision studies below 2%, as recommended by ICH guidelines.

LOD and LOQ.—S/N of 3:1 and 10:1 were obtained forthe LOD and LOQ, respectively. The LOD and LOQ werefound to be 50 and 100 ng/spot for PAR and DCL,respectively, and 10 and 50 ng/spot for FAM.

Robustness.—The RSD of peak areas was calculated foreach parameter and was found to be less than 2%. The lowvalues of the RSD, as shown in Table 3, indicated robustnessof the method.

Specificity.—The peak purity of PAR, DCL, and FAMwas assessed by comparing their respective spectra at thepeak start (S), peak apex (M), and peak end (E) positionsof the spot. It was found that r (S, M) = 0.9971 and r (M,E) = 0.9987. A good correlation (r = 0.9982) was alsoobtained between the standard and sample spectra of PAR,DCL, and FAM.

Recovery studies.—As shown from the data in Table 4,good recoveries of the PAR, DCL, and FAM in the range from 95 to 98% were obtained for the various added concentrations.


Figure 2. Densitogram of FAM (Rf = 0.17), PAR (Rf = 0.62), and DCL (Rf = 0.75) of formulation (MAHAGESIC PLUS)showing no interference of excipients in analysis.

Analysis of a Formulation

Experimental results for the amounts of PAR, DCL, andFAM in the tablets were in good agreement with the labelclaims, thereby proving that there was no interference fromany of the excipients that were present (Figure 2). The drugcontent was found to be 97.5% for PAR, 96.3% for DCL, and97.1% for FAM. Two different lots of PAR, DCL, and FAMcombination tablets were analyzed using the proposedprocedure.

Conclusions

Introducing HPTLC into pharmaceutical analysisrepresents a major step in terms of quality assurance. TodayHPTLC is rapidly becoming a routine analytical techniquedue to its advantages of low operating costs, high samplethroughput, and the need for minimal sample preparation.The major advantage of HPTLC is that several samples canbe run simultaneously using a small quantity of mobilephase—unlike HPLC—thus reducing the analysis time andcost/analysis.

The developed HPTLC technique is precise, specific, andaccurate. Statistical analysis proved that the method is suitable for the analysis of PAR, DCL, and FAM as bulk drugs and in a pharmaceutical formulation without any interference from the excipients. It may be extended to study the degradationkinetics of PAR, DCL, and FAM and also for theirdetermination in plasma and other biological fluids. Theproposed HPTLC method is less expensive, simpler, and more flexible than HPLC.

Acknowledgments

We would like to thank Bal Pharmaceuticals Ltd, BrihonsLaboratories, and Vaasava Pharmaceuticals Pvt. Ltd forproviding gift samples of PAR, DCL, and FAM, respectively.We would also like to thank Kakasaheb R. Mahadik, PoonaCollege of Pharmacy, Pune, India, for providing the necessary facilities to carry out the work, and All India Council forTechnical Education for providing financial support.

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paracetamol, diclofenac potassium, and famotidine

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