researcharticle developmentandvalidationofrp-hplcmethodfor...

Hindawi Publishing CorporationJournal of ChemistryVolume 2013, Article ID 751940, 5 pageshttp://dx.doi.org/10.1155/2013/751940

Research ArticleDevelopment and Validation of RP-HPLCMethod forSimultaneous Estimation of Aspirin and EsomeprazoleMagnesium in Tablet Dosage Form

DipaliPatel,NishitkumarPatel,ReetaVaishy,ViralPatel,ChiragsinhSolanki, andMitulPatel

Department of Quality Assurance, Indubhai Patel College of Pharmacy and Research Centre, Dharmaj, Gujarat 388430, India

Correspondence should be addressed to Dipali Patel, [email protected]

Received 29 June 2012; Revised 5 November 2012; Accepted 12 November 2012

Academic Editor: Nianjun Yang

Copyright © 2013 Dipali Patel et al. is is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A simple, speci�c, precise, and accurate reversed-phase HPLCmethod was developed and validated for simultaneous estimation ofaspirin and esomeprazole magnesium in tablet dosage forms. e separation was achieved by HyperChrom ODS-BP C18 column(200mm × 4.6mm; 5.0 𝜇𝜇m) using acetonitrile: methanol: 0.05M phosphate buffer at pH 3 adjusted with orthophosphoric acid(25 : 25 : 50, v/v) as eluent, at a �ow rate of 1mL/min. Detectionwas carried out at wavelength 230 nm.e retention times of aspirinand esomeprazolemagnesiumwere 4.29min and 6.09min, respectively.e linearity was established over the concentration rangesof 10–70 𝜇𝜇g/mL and 10–30 𝜇𝜇g/mL with correlation coefficients (r2) 0.9986 and 0.9973 for aspirin and esomeprazole magnesium,respectively.emean recoveries were found to be in the ranges of 99.80–100.57% and 99.70–100.83% for aspirin and esomeprazolemagnesium, respectively.e proposedmethod has been validated as per ICH guidelines and successfully applied to the estimationof aspirin and esomeprazole magnesium in their combined tablet dosage form.

1. Introduction

Aspirin (ASP) is chemically 2-(acetyloxy)-benzoic acid(Figure 1). It is nonselective cyclooxygenase inhibitorused as an antipyretic, analgesic, anti-in�ammatory, andantithrombotic agent. Esomeprazole magnesium (ESO) isS-isomer of omeprazole and proton pump inhibitor. It ismag-nesium, bis [5-methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sul�nyl]-1H-benzimidazolato] (Figure 2).It is used in treatment of peptic ulcer disease, NSAIDS-associated ulceration and Zollinger-Ellison syndrome, usedas antiulcerative. ASP and ESO in combined dosage formare used in cardiovascular disorder and cerebrovasculardisorders [1–3].

e review of literature revealed that various analyticalmethods involving spectrophotometry [4–6], HPLC [7–10]and HPTLC [11] have been reported for ASP in single formand in combination with other drugs. Several analyticalmethods have been reported for ESO in single form and incombination with other drugs including spectrophotometry[12, 13], HPLC [14, 15], and HPTLC [16].

e present work describes the development of a simple,precise, accurate, and reproducible HPLC method for thesimultaneous estimation of ASP and ESO in combineddosage form. e developed method was validated in accor-dance with ICH Guidelines [17] and successfully employedfor the assay of ASP and ESO combine dosage form.

2. Experimental Condition

2.1. Materials and Reagents. Analytically pure ASP and ESOwere kindly provided by Baroque Pharmaceuticals, Khamb-hat, Gujarat, India and Osaka Pharmaceuticals, Sakarda,Vadodara, Gujarat, India, respectively, as gi samples. Ana-lytical grademethanol, chloroform, acetonitrile, glacial aceticacid, and ethyl acetate were purchased from SD Fines Chem-icals, Bombay, India. Tablet of ASP and ESO in combineddosage form, AXANUM, with a 20mg ESO and 81mg ASPlabel claim, manufactured by Astrazeneca Pharmaceuticals.

2.2. Instrumentation. An isocratic HPLC system (Analyt-ical Technologies Limited) consisted of P2230 plus HPLC

2 Journal of Chemistry

COOH

O

O

CH3

F 1: Structure of aspirin.

Me

Me

Me

Me

MeO

MeO

CH2

CH2

S

S

N

N

N

N

N

−

O

O

OMe

OMeN−

Mg2+

F 2: Structure of esomeprazole magnesium.

pump, variable wavelength programmable UV 2230 plusdetector system, Rhenodyne valve with 20 𝜇𝜇L �xed loopand Analchrom 2006 as operating soware. e chromato-graphic column used was HyperChromODS-BP C18 column(200mm × 4.6mm i.d, particle size 5 𝜇𝜇m). Analytical balanceK-EA 210 (K-Roy Instrument Pvt. Ltd) was used forweighingpurpose.

2.3. Chromatographic Condition. A mixture of Acetoni-trile :Methanol : 0.05M Phosphate Buffer at pH 3 adjustedwith orthophosphoric acid (25 : 25 : 50 v/v) was used asmobile phase andwas �ltered through 0.45𝜇𝜇membrane �lterprior to use. e �ow rate of mobile phase was maintainedat 1mL/min. Detection was carried out at 230 nm at theambient temperature. e total run time 10min was usedwith injection volume of 20𝜇𝜇L.

2.4. Preparation of Mobile Phase and Standard Stock Solu-tions. Accurately weighed potassium dihydrogen phosphate(3.4 g) was dissolved in 500mL of water. is solution wasmixed with 250mL of acetonitrile and 250mL of methanol.Finally the pH was adjusted to 3.0 with orthophosphoricacid. e solution was sonicated for 10 minutes and �lteredthrough 0.45 𝜇𝜇membrane �lter. 100mg of standard ASP andESO were accurately weighed and transferred separately toa 100mL volumetric �ask and dissolved in 50mL mobilephase. e �ask was sonicated for 10min. e �ask wasshaken and volume was made up to the mark with mobilephase to give a solution containing 1000𝜇𝜇g/mL ASP andESO, respectively. Appropriate volume of aliquot from ASP

75645036228

−6

0 1 2 3 4 5 6 7 8 9 10

(Minutes)

ASP ESO

(AW)

F 3: Chromatogram of mixed standard solution containing10 𝜇𝜇g/mL ESO and 10 𝜇𝜇g/mL ASP using mobile phase acetoni-trile :methanol : 0.05M phosphate buffer at pH 3 adjusted withorthophosphoric acid (25 : 25 : 50 v/v).

0.608

0.4

0.2

0

200 250 300 350 400

(nm)

Abs.

230

ASP

ESO

−0.072

F 4: Overlain spectrum of 10 𝜇𝜇g/mL ASP and 5 𝜇𝜇g/mL ESO inmethanol.

T 1: Summary of validation parameters.

Parameters ASP ESO

Recovery % 99.80–100.57 99.70–100.83

Repeatability (C.V., 𝑛𝑛 = 6) 0.205123 0.537209

Precision (% RSD)Intra-day (𝑛𝑛 = 3) 0.14–0.38 0.22–0.49Inter-day (𝑛𝑛 = 3) 0.38–0.83 0.22–0.86

Limit of detection (𝜇𝜇g/mL) 3.25067 2.09632

Limit of quantitation(𝜇𝜇g/mL) 9.85051 6.35250

Speci�city Speci�c Speci�c

Robustness Robust Robust

Solvent stability Solvent stablefor 24 hrs.

Solvent stablefor 24 hrs.

T 2: Calibration data for ASP and ESO.

Parameter ASP ESOLinear range (𝜇𝜇g/mL) 10–70 10–30Slope 89.00785 93.47274Intercept −237.786 23.3054Standard deviation of slope 1.93647 2.79913Standard deviation of intercept 87.67731 59.37857

and ESO standard stock solution was further diluted withmobile phase to obtain �nal concentration of 100 𝜇𝜇g/mL and100 𝜇𝜇g/mL, respectively.

Journal of Chemistry 3

T 3: Robustness study.

Condition varied Changed condition Area (𝑛𝑛 = 3) % AssayESO ± S.D. ASP ± S.D. ESO ASP

Change in mobile phase ratio (v/v) 28 : 22 : 50 1299.76 ± 4.90 4797.53 ± 12.33 98.01 99.9122 : 28 : 50 1310.43 ± 5.60 4782.56 ± 15.12 100.82 99.68

T 4: System suitability test parameter.

System suitabilityparameters

Proposed MethodESO ASP

Retention times (𝑅𝑅𝑇𝑇)(min) ± S.D.

6.088 ± 0.10740 4.286 ± 0.03983

eoretical plates(𝑁𝑁) ± S.D.

3063.4 ± 25.8438 2535.4 ± 5.8242

Resolution (𝑅𝑅𝑆𝑆) 4.62Tailing factor(𝐴𝐴𝑆𝑆) ± S.D.

1.364 ± 0.07197 1.412 ± 0.05167

Capacity factor 5.088 3.286

2.5. Determination of ESO and ASP fromCombined Dosage Form

2.5.1. Sample Preparation. A powder quantity equivalent to40mg ESO and 162mg ASP was accurately weighed andtransferred to volumetric �ask of 100mL capacity. 60mLof solvent (acetonitrile : methanol : water (25 : 25 : 50)) wastransferred to this volumetric �ask and sonicated for 15min.e �ask was shaken and volume was made up to the markwith methanol. e above solution was �ltered throughmembrane �lter (0.45 𝜇𝜇). From this solution 3.5mL wastransferred to volumetric �ask of 100mL capacity. �olumewas made up to the mark to give a solution containing14 𝜇𝜇g/mL of ESO and 56.7𝜇𝜇g/mL of ASP. e resultingsolution was analyzed by proposed method. e preparedsample solution was chromatographed for 10 minutes usingmobile phase at a �ow rate of 1.0mL/min. From the peak areaobtained in the chromatogram, the amounts of both the drugswere calculated.

3. Method Validation

e proposed method has been extensively validated interms of speci�city, linearity, accuracy, precision, limits ofdetection (LOD) and quanti�cation (LOQ), robustness, andsystem suitability. e accuracy was expressed in terms ofpercent recovery of the known amount of the standarddrugs added to the known amount of the pharmaceuticaldosage forms. e precision (% RSD) was expressed withrespect to the repeatability, intraday, and interday variationin the expected drug concentrations. Aer validation, thedeveloped methods have been applied to pharmaceuticaldosage form.

3.1. Speci�city. Commonly used excipients (starch, micro-crystalline cellulose, and magnesium stearate) were spiked

into a preweighed quantity of drugs. e chromatogram wastaken by appropriate dilutions and the quantities of drugswere determined.

3.2. Linearity. Appropriate volume of aliquot from ASPand ESO standard stock solution was transferred to samevolumetric �ask of 10mL capacity. e volume was ad�ustedto the mark with mobile phase to give a solution containingASP (10, 25, 40, 55, and 70𝜇𝜇g/mL) and ESO (10, 15,20, 25, and 30 𝜇𝜇g/mL). e mixed standard solution waschromatographed using above chromatoghraphic condition(𝑛𝑛 = 6). All solutions were �ltered through 0.45 𝜇𝜇m �lterprior to use. Calibration curves were constructed by plottingaverage peak area versus concentrations for both drugs.Straight line equations were obtained from these calibrationcurves.

3.3. Accuracy. Accuracywas assessed by determination of therecovery of the method by addition of standard drug to pre-analyzed test sample preparation at 3 different concentrationlevels 80, 100, and 120%, taking into consideration percent-age purity of added bulk drug samples. Each concentrationwas chromatographed 3 times and average recoveries weremeasured.

3.4. Precision. e repeatability was evaluated by assaying6 times of test samples prepared for assay determination.eintraday and interday precision study of ASP and ESO wascarried out by estimating different concentrations of ASP andESO, 3 times on the same day and on 3 different days and theresults are reported in terms of% RSD.

3.5. Detection Limit and Quantitation Limit. ICH guidelinedescribes several approaches to determine the detection andquantitation limits. ese include visual evaluation, signal-to-noise ratio, and the use of standard deviation of theresponse and the slope of the calibration curve. In the presentstudy, the LOD and LOQ were based on the third approachand were calculated according to the 3.3𝜎𝜎/s and 10𝜎𝜎/scriterions, respectively, where 𝜎𝜎 is the standard deviationof 𝑦𝑦-intercepts of regression lines and 𝑠𝑠 is the slope of thecalibration curve.

3.6. Robustness. e robustness of the method was evaluatedby assaying test solutions aer slight but deliberate changesin the analytical conditions. For the proposed method itwas done by changing the mobile phase composition (ace-tonitrile : methanol : buffer, 28 : 22 : 50 and 22 : 28 : 50, v/v byobserving the stability of the drugs for 24 hr in the mobilephase.

4 Journal of Chemistry

T 5: Assay results of marketed formulation.

Formulation Actual concentration 𝜇𝜇g/mL Amount obtained 𝜇𝜇g/mL% ESO % ASPESO ASP ESO ASP

Tablet 14 56.7 13.92 56.55 99.42 99.73

3.7. System Suitability. e suitability of the chromato-graphic system was tested before each stage of validation.Five replicate injections of standard preparationwere injectedand resolution, asymmetry, number of theoretical plates, andrelative standard deviation of peak area were determined.

3.8. Determination of ASP and ESO from Combined DosageForm. Sample solution was injected 6 times at above chro-matographic conditions. An average peak area was measuredfrom chromatograms. e quantitation was carried out bykeeping these values to the straight line equation of calibra-tion curve.

4. Results and Discussion

Optimizations of chromatographic conditions were per-formed to obtain the best resolution and peak parameter(asymmetry, theoretical plates). For the selection of mobilephase initially methanol-water and acetonitrile-water havebeen tried in different ratio which gave poor peak shape.en acetonitrile :methanol : 0.050M potassium dihydrogenphosphate buffer adjusted to pH 3.0 in different ratiohave been tried. Finally, acetonitrile : methanol : 0.05Mphos-phate buffer at pH 3 adjusted with orthophosphoric Acid(25 : 25 : 50 v/v) was found to be satisfactory and gave twosymmetrical peaks with good resolution (4.62) for ASP andESO at �ow rate of 1mL/min.e average retention times forASP and ESOwere 4.286 ± 0.03983 and 6.088 ± 0.10740min-utes, respectively. e asymmetric factors for ASP and ESOwere 1.412 ± 0.05167 and 1.364 ± 0.07197, respectively. Forthe selection of detection wavelength overlain UV spectrumof ASP and ESO was taken which revealed that at 230 nmboth the drugs possess signi�cant absorbance (Tables 3 and4; Figures 3 and 4).

Summary of validation parameters for proposed methodwas given in Table 1.

e developed HPLC method was validated. e linearrange, correlation coefficient, detection limit and standarddeviation for ASP and ESO by HPLC method are shownin Table 2. Accuracy was determined by calculating therecovery. e method was found to be accurate with %recovery 99.80–100.57% for ASP and 99.70–100.83% for ESOrespectively.

Precision was calculated as repeatability and intra andinterday variation for both the drugs.emethodwas precisewith % RSD 0.14–0.38 for intraday (𝑛𝑛 = 3) and % RSD0.38–0.83 for interday (𝑛𝑛 = 3) for ASP and 0.22–0.49 forintraday (𝑛𝑛 = 3) and % RSD 0.22–0.86 for interday (𝑛𝑛 = 3)for ESO, respectively.

e method was speci�c as no interference observedwhen the drugs were estimated in presence of excipients.

e method was also robust as there was no change inarea up to 24 hours of preparation of solution in acetoni-trile : methanol : 0.05M phosphate buffer at pH 3 adjustedwith orthophosphoric acid (25 : 25 : 50 v/v). e LOD forASP and ESO was found to be 3.25 𝜇𝜇g/mL and 2.09𝜇𝜇L/mL,respectively. Summary of validation parameters is tabulatedin Table 1. Marketed formulation was analyzed by theproposed method and assay result of marketed formulationwas shown in Table 5.

5. Conclusion

e proposed HPLC method provide simple, speci�c, pre-cise, accurate, and reproducible quantitative analysis forsimultaneous analysis of ASP and ESO in combined dosageform. e method was validated as per ICH guidelinesin terms of speci�city, linearity, accuracy, precision, limitsof detection (LOD) and quanti�cation (LOQ), robustness,and reproducibility. e proposed method can be used forroutine analysis and quality control assay of ESO and ASP incombined dosage form.

Acknowledgments

e authors are thankful to Osaka Pharmaceuticals (Vado-dara, India) for providing gratis sample with great pleasureand a note of thanks to Baroque Pharmaceuticals (Khambhat,India) for providing gratis sample with great pleasure. eauthors are also thankful to Indubhai Patel College of Phar-macy and Research Centre (Dharmaj, India) for providingthe necessary facilities for research work.

References

[1] Martindale-the Complete Drug Reference, Pharmaceutical Press,London, UK, 34th edition, 2005.

[2] M. J. O’Neil, Ed., e Merk Index- An Encyclopedia of Chemi-cals, Drugs and Biological, Merck Research Laboratories, 14thedition, 2006.

[3] Antithrombotic Trialists’ (ATT) Collaboration, “Aspirin in theprimary and secondary prevention of vascular disease: collab-orative meta-analysis of individual participant data from ran-domised trials,” e Lancet, vol. 373, no. 9678, pp. 1849–1860,2009.

[4] M. D. Game, K. B. Gabhane, and D. M. Sakarkar, “Quantitativeanalysis of clopidogrel bisulphate and aspirin by �rst derivativespectrophotometric method in tablets,” Indian Journal of Phar-maceutical Sciences, vol. 72, no. 6, pp. 825–828, 2010.

[5] Z. Kokot and K. Burda, “Simultaneous determination of sal-icylic acid and acetylsalicylic acid in aspirin delayed-releasetablet formulations by second-derivative UV spectrophotome-try,” Journal of Pharmaceutical and Biomedical Analysis, vol. 18,no. 4-5, pp. 871–875, 1998.

Journal of Chemistry 5

[6] P. Mishra and A. Dolly, “Simultaneous determination of clopi-dogrel and aspirin in pharmaceutical dosage forms,” IndianJournal of Pharmaceutical Sciences, vol. 68, no. 3, pp. 365–368,2006.

[7] D. Shah, K. Bhatt, R. Mehta, M. Shankar, S. Baldania, and T.Gandhi, “Development and validation of a RP-HPLC methodfor determination of atorvastatin calcium and aspirin in acapsule dosage form,” Indian Journal of Pharmaceutical Sciences,vol. 69, no. 4, pp. 546–549, 2007.

[8] E. R. Montgomery, S. Taylor, J. Segretario, E. Engler, andD. Sebastian, “Development and validation of a reversed-phase liquid chromatographic method for analysis of aspirinand warfarin in a combination tablet formulation,” Journal ofPharmaceutical and Biomedical Analysis, vol. 15, no. 1, pp.73–82, 1996.

[9] E. Deconinck, P. Y. Sacré, S. Baudewyns, P. Courselle, and J.De Beer, “A fast ultra high pressure liquid chromatographicmethod for quali�cation and quanti�cation of pharmaceuticalcombination preparations containing paracetamol, acetyl sal-icylic acid and/or antihistaminics,” Journal of Pharmaceuticaland Biomedical Analysis, vol. 56, no. 2, pp. 200–209, 2011.

[10] S. L. Yang, L. O. Wilken, and C. R. Clark, “A high performanceliquid chromatographic method for the simultaneous assay ofaspirin, caffeine, dihydrocodeine bitartrate and promethazinehydrochloride in a capsule formulation,”DrugDevelopment andIndustrial Pharmacy, vol. 11, no. 4, pp. 799–814, 1985.

[11] P. K. Sinha, M. C. Damle, and K. G. Bothara, “A validatedstability indicatingHPTLCmethod for determination of aspirinand clopidogrel bisulphate in combined dosage form,” EurasianJournal of Analytical Chemistry, vol. 4, no. 2, pp. 152–160, 2009.

[12] S. S. Patil, P. N. Dhabale, and B. Kuchekar, “Development andstatistical validation of spectrophotometric method for estima-tion of esomeprazole in tablet dosage form,” Asian Journal ofResearch in Chemistry, vol. 2, no. 2, pp. 154–156, 2009.

[13] V. V. Gawande and A. V. Chandewar, “Spectroscopic esti-mation of esomeprazole magnesium in solid dosage form,”International Journal of Pharmacy & Technology, vol. 2, no. 3,pp. 617–622, 2010.

[14] A. Önal and A. Öztunç, “Development and validation of highperformance liquid chromatographic method for the determi-nation of esomeprazole in tablets,” Journal of Food and DrugAnalysis, vol. 14, no. 1, pp. 12–18, 2006.

[15] P. Sripal Reddy, S. Sait, G. Vasudevmurthy, B. Vishwanath, V.Prasad, and S. Jayapal Reddy, “Stability indicating simultaneousestimation of assay method for naproxen and esomeprazolein pharmaceutical formulations by RP-HPLC,” Der PharmaChemica, vol. 3, no. 6, pp. 553–564, 2011.

[16] S. Sharma and M. C. Sharma, “Densitometric method for theQuantitative determination of Esomeprazole and Domperi-don,” American—Eurasian Journal of Toxicological Science, vol.3, no. 3, pp. 143–148, 2011.

[17] ICHHarmonized Tripartite Guidelines,Validation of AnalyticalProcedures: Text and Methodology, Q2 (R1), Geneva, Switzer-land, 2005.

Submit your manuscripts athttp://www.hindawi.com

Chromatography Research International

Hindawi Publishing Corporationhttp://www.hindawi.com Volume 2013


Carbohydrate Chemistry

International Journal of

Hindawi Publishing Corporationhttp://www.hindawi.com

International Journal of

Analytical ChemistryVolume 2013

ISRN Chromatography


Hindawi Publishing Corporation http://www.hindawi.com Volume 2013Hindawi Publishing Corporation http://www.hindawi.com Volume 2013

The Scientific World Journal

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttp://www.hindawi.com Volume 2013


CatalystsJournal of

ISRN Analytical Chemistry


ElectrochemistryInternational Journal of

Hindawi Publishing Corporation http://www.hindawi.com Volume 2013


Advances in

Physical Chemistry

ISRN Physical Chemistry


SpectroscopyInternational Journal of


ISRN Inorganic Chemistry



Journal of

Chemistry


Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation http://www.hindawi.com Volume 2013

International Journal ofPhotoenergy

Hindawi Publishing Corporationhttp://www.hindawi.com

Analytical Methods in Chemistry

Journal of

Volume 2013

ISRN Organic Chemistry



Journal of

Spectroscopy

researcharticle developmentandvalidationofrp-hplcmethodfor...

Documents