simultaneous determination of lafutidine and domperidone in ... - IJPI

INTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-1031SIMULTANEOUS DETERMINATION OF LAFUTIDINE ANDDOMPERIDONE IN TABLET DOSAGE FORM BY HPLCRay Chaudhury Dipanjan*, Chakraborty Mithun, Chakraborty Susanto, Saha NanditaCentral Drugs Laboratory, Govt. of India, 3 Kyd Street, Kolkata-700016Abstract:A simple, precise and rapid isocratic reverse phase high performance liquid chromatographicmethod was developed for the simultaneous determination of Lafutidine and Domperidone intablet dosage form. The chromatographic separation was performed on µ-Bondapack C18column (250 mm × 4.6 mm I.D. 5 µm particle size). Mobile phase consisted of a mixture ofphosphate buffer of pH 7.0 and acetonitrile in the ratio of 60: 40 at a flow rate of 1.5 mL/minand with the detection wavelength of 220 nm. The proposed method was validated for linearity,accuracy, precision, robustness LOD and LOQ. The calibration curve was linear over the rangeof 20-80 µg/mL for Lafutidine and 60-240 µg/mL for Domperidone. The retention times werefound as 4.8 min for Domperidone and 6.1 min for Lafutidine. The method can be easilyadopted for routine analysis of the same type of combination products.Keywords: Lafutidine, Domperidone, RP-HPLC, ValidationIntroductionLafutidine (LAF) is 2-(furan-2-ylmethylsulfinyl)-N [4-[4-(piperidin-1-ylmethyl) pyridin-2-yl] oxybut-amide [1](structure in Fig 1a). It belongs to the classof H 2 receptor antagonists. Used in thetreatment of peptic ulcer and gastrooesophagealreflux disease (GERD) [1]. Itis freely soluble in methanol, whereas it ispractically insoluble in water. It is asecond generation histamine H 2 -receptorantagonist used as an antiulcerative agent[2]. Structures are shown in fig 1a. This isan antisecretory drug. Antisecretory drugsare used in the treatment and prophylaxisof peptic ulcer disease some are alsoemployed in other disorders associatedwith gastric hyperacidity such as gastro-oesophageal reflux disease and dyspepsia.They may be divided into Histamine H2-receptor antagonist (H2-antagonists),which act by blocking histamine H2-receptors on gastric parietal cells, therebyantagonizing the normal stimulatory effectof endogenous histamine on gastric acidproduction. Literature survey reveals thatmany methods are reported for itsestimation alone in biological fluids bytandem mass spectrometry [3-5] and chiralseparations by HPLC and LC-MS [6].Domperidone (DOM) chemicallyCorresponding Author*Ray Chaudhury DipanjanVolume 2, Issue 6, Nov. − Dec. 20121 | P a g ehttp://www.ijpi.org

INTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-1031(5-chloro-1-{1-[3(2,3-dihydro-2-oxo-1Hbenzimidazol-1-yl)propyl] -4-piperidinyl} benzimidazolin-2-one)(Structure in Figure 1b) used as anantiemetic drug [7,8]. It stimulates gastrointestinalmotility an disused as anantiemetic forth short term treat men t ofnausea and vomiting.A combination of these drugs, DOM andLAF is available as tablets for clinicalpractice. This unique combination hascomprehensive acid control and prokineticaction which ensures better control andrelief from reflux, gastric ulcers, andassociated gastrointestinal (GIT) disorders.Many methods like HPLC [9],[10],HPTLC [11], and LCMS [12] have beendescribed in the literature for thedetermination of DOM and LAFindividually or in combination with others.aim of present work is to develop andvalidate the rapid and sensitive highperformance liquid chromatography(HPLC) method for simultaneousdetermination of Lafutidine andDomperidone in tablets.Experimental:Instrumentation:HPLC (Agilent 1200 series) equipped withQuaternary pump, Auto sampler, UltravioletDetector and EZ-Chrome EliteSoftware. Waters Spherisorb ODS2 (4.0 x250 mm, 5 µm) column was used.Chemicals and ReagentsThe solvents used were of Mili-Q water(18.2 MΩ-cm conductivity) forpreparation of buffer of mobile phase,HPLC grade Acetonitrile and Orthophosphoricacid (AR-Grade). ReagentPotassium-di-hydrogen phosphate (ARgrade)was used for preparation of buffer.Lafutidine (Potency: 99.85% as such) andDomperidone (Potency: 99.1% as such)reference standard of a renowned companywere used and samples was obtainedfrom market.Fig 1a: Structure of LafutidineFig 1b: Structure of DomperidoneAccording to literature study, so manyanalytical methods are reported viz. UVspectrophotometry[13-16], HPLC [17-18]and drugs in human plasma [19- 21]. TheVolume 2, Issue 6, Nov. − Dec. 2012Chromatographic ConditionsWhile several mobile phases were tried,the mobile phase containing buffer (0.01M KH 2 PO 4 , pH adjusted to 7.0 using diluteorthophosphoric acid) and acetonitrile inthe ratio of 60: 40 was used. The mobilephase was filtered through 0.45µmmembrane filter and sonicated for 10minutes. After several trials flow rate wasfixed at 1.5 ml/min, column temperature at30 degree-C and injection volume 20 µl.The proposed method was validated forlinearity, accuracy, precision, LOD and2 | P a g ehttp://www.ijpi.org

INTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-1031LOQ. The calibration was linear over therange of 20-80 µg/mL for Lafutidine and60-240 µg/mL for Domperidone. Theretention times were found as 4.8 min forDomperidone and 6.1 min for Lafutidine.The method can be easily adopted forregular routine analysis.Preparations of Standard Solutions:Weigh accurately about 10.1 mg of LAFand 30.4 mg of DOM working standardsinto 100 ml volumetric flask. Dissolve inmethanol, sonicate for 15 minutes andmake up the volume in methanol. Dilute 5ml of this stock solution in to 10 mlvolumetric flask with Methanol. Theconcentration of LAF is about 50.3 µg/ mland DOM is about 150 µg/ ml.Preparations of Sample SolutionsTake the weight of 20 tablets; take about500 mg powdered, equivalent to about 10mg LAF and 30 mg DOM in a 100 mlvolumetric flask. Add about 60 ml ofMethanol, sonicate for 30 minutes anddilute up to the mark with Methanol.Filter and discard first few ml and use thisfiltered as sample solution. Dilute 5 ml ofthis stock solution in to 10 ml volumetricflask with Methanol. The concentration ofLAF is about 50 µg/ ml and DOM is about300 µg/ ml.Method ValidationThe method was validated for linearity,precision and repeatability, accuracy androbustness.Result and discussionLinearity of Lafutidine andDomperidoneTo prepare the calibration curve forLafutidine a stock solution of Lafutidine ofconc. about 1mg/ml was prepared. From itVolume 2, Issue 6, Nov. − Dec. 20121.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4 ml ofstandard stock solution of LAF wastransferred to a series of seven individual10 ml volumetric flasks. The volume ofeach flask was adjusted to 10 ml withMethanol. (Table-1). Similarly a stocksolution of concentration of 1 mg/mlDomperidone was prepared in methanol.Form it takes, 0.6, 0.9, 1.2, 1.5, 1.8, 2.1and 2.4 ml solution was transferred to each10 ml volumetric flasks. The volume ofeach flask was adjusted to 10 ml withMethanol. (Table-2). Detection responsefor each LAF and DOM were found to belinear in concentration range of 20.0 – 80.0mcg/ml and 60 – 140 mcg/ml ( fig-1 ,fig-2).The slope, regression equation andcoefficient of correlation for DOM andLAF were found to be y = 67859 x +28173, R 2 = 0.999 and y = 76331x +12902, R 2 = 0.999.PRECISIONa) System precision for LAF and DOM:The system precision was evaluated bymeasuring the peak response of LAF andDOM for six replicate injections ofstandard solutions, prepared as theproposed method. The result shown in theTable- 3 indicates that the precision of thesystem is within acceptance criteria( i.e.RSD not more than 2%).b) Method precision LAF and DOM:The method precision was determined bypreparing a sample solution of a singlebatch LAF and DOM tablets six times andanalyzing as per the proposed method. Theresult shown in Table- 4 indicates that theproposed method is precise. (Acceptancecriteria: % RSD not more than 2%).3 | P a g ehttp://www.ijpi.org

INTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-1031A representative chromatogram of mixedstandard solution of LAF (mcg/ml) andDOM (mcg/ml) are shown in figure-3.ACCURACY (recovery study) for LAFand DOM:Known amount of LAF and DOM werespiked to placebo at 80%, 100% and 120%of specification in triplicate and analyzedas per the proposed method to determinethe accuracy of the method. Percentage ofrecovery was calculated from the amountfound and amount added. The results areshown in Table- 5 & 6. The percentagerecovery is within the acceptance criteria,which indicates the accuracy of themethod.(Acceptancecriteria: % recovery between 98 to 102).Limit of detection (LOD):The limit of detection (LOD) is thesmallest concentration that can be detectedbut not necessarily quantified as an exactvalue. LOD can be calculated as:LOD = 3.3 σ / SWhere,σ = Standard deviation of the response[22]S= Slope of the calibration curveLimit of quantitation (LOQ):The limit of quantitation (LOQ) is thelowest amount of analyze in the samplethat can be quantitatively determined withsuitable precision and accuracy. LOQ canbe calculated as:LOQ = 10 σ / SWhere,Volume 2, Issue 6, Nov. − Dec. 2012σ = Standard deviation of the responseS= Slope of the calibration curveThe limit of detection (LOD) was found tobe 16.46 mcg/ml for LAF and 15.09mcg/ml for DOM whereas the limit ofquantitation (LOQ) was found to be 49.89mcg/ml for LAF and 45.73 mcg/ml forDOM respectively.RobustnessThe robustness of a method is its ability toremain unaffected by small deliberatechange in parameter. To evaluate therobustness, the developed method wassubjected to small deliberate variation inthe optimized method parameters likevariation of flow rate 1.5 ± 0.2 ml/min,detection wavelength i.e. 220 ± 2.0 nmand pH 7.0 ± 0.2 in the buffer of mobilephase.The mixed standard solution containing150 mcg/ml of DOM and 50.3 mcg/ml ofLAF was injected in replicate at variedchromatographic condition and thestandard deviation of the retention time ofeach analyte were calculated. The methodwas found to be robust as the slightdeliberate variations in detectionwavelength and flow rate did not lead tochanges in retention time of the peaks ofinterest. While evaluating the robustnessdata it was observed that system suitabilityparameters (e.g. tailing factor, plate count,resolution etc.) were found to be within thespecified limit under those deliberatelyvaried conditions, which ensures that thevalidity of the analytical procedure wasmaintained whenever used. The result ofrobustness studies is summarized in table7.System Suitability4 | P a g ehttp://www.ijpi.org

INTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-1031System suitability testing is used to verifythat the resolution and reproducibility ofthe system are adequate for the analysis tobe performed. System suitability wasassessed by injecting DOM and LAFmixed standard preparation in replicate.Parameter such as theoretical plate, tailingfactor, resolution were determined. Thesystem suitability parameters for themethod are listed in table-8.Conclusions:The developed method is better thanprevious methods and valid for capsuleand tablet dosage also. The mobile phase issimple to prepare. The developed methodfulfilled all the requirements includingaccuracy, linearity, recovery and precisiondata. Therefore the RP-HPLC methodfound to be simple, accurate, economical,and rapid and can be used as a routinesample analysis.Acknowledgement:The authors express their gratitude to theDirector, Central Drugs Laboratory,Kolkata and all the staff member ofCentral Instrument Room of Central DrugsLaboratory, Kolkata for providingnecessary facilities in the department.References:1. The Merck Index - Anencyclopedia of chemicals,drugs and biologicals,published by Merck ResearchLaboratories, 14th edition,Lafutidine, (5347), 9262. Y.AkibaandJ.D.Kaunitz,“Lafutidine, aprotective H2 receptorantagonist, enhances mucosaldefense in ratesophagus,”DigestiveDiseasesaVolume 2, Issue 6, Nov. − Dec. 2012ndSciences,vol.55, no. 11, pp.3063–3069, 2010.3. Dong Chen, W., Y. Liang, H.Li, Y. Xiong, X. Dong Liu, G.Ji Wang and L. Xie, 2006.Simple, sensitive and rapid LC-ESI-MS method for thequantitation of lafutidine inhuman plasma-Application topharmacokinetic studies. J.Pharmaceutical and BiomedicalAnalysis, 41(1): 256-260.4. Wu, L., Z. Zhang, Y. Tian, W.Li, F. Xu, Y. Chen and H. Wei,2005. Determination oflafutidine in human plasma byhigh-performance liquidchromatographyelectrosprayionization mass spectrometry:application to a bioequivalencestudy. J. Mass Spectrometry,40(12): 1637-1643.5. Sun, X., Y. Tian, Z. Zhang andY. Chen, 2009. A single LCtandemmass spectrometrymethod for the simultaneousdetermination of four H2antagonists in human plasma. J.Chromatography B,877(31):3953-3959.6. Xiu Pan, C., X. Zhu Xu, H. MeiHe, X. Jun Cai and X. JunZhang, 2005. Separation andidentification of cis and transisomers of 2-butene-1,4-dioland lafutidine by HPLC andLC-MS. J. Zhejiang UniversityScience B., 6(1): 74-78.7. L. Swann, E. N. Thompson,and K. Qureshi, “Domperidoneor metoclopramide in5 | P a g ehttp://www.ijpi.org

INTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-1031preventingchemotherapeutically inducednausea and vomiting,” BMJ,vol. 2, no. 6199, article 118,1979.8. Karthik, G. Subramanian, A.Ranjith Kumar, and N.Udupa,“Simultaneousestimation of paracetamol anddomperidone in tablets byreverse phase HPLC method,”Indian Journal ofPharmaceuticalSciences, vol.69, no. 1, pp. 142–144, 2007.9. Karthik, G. Subramanian, A.Ranjith Kumar, and N.Udupa,“Simultaneousestimation of paracetamol anddomperidone in tablets byreverse phase HPLC method,”Indian Journal ofPharmaceutical Sciences,vol.69, no. 1, pp. 142–144, 2007.10. M. Kobyli´nska and K.Kobyli´nska, “Highperformanceliquidchromatographic analysis forthe determination ofDomperidone in humanplasma,” Journal ofChromatography B, vol.744,no. 1, pp. 207–212, 2000.11. B.H.Patel, B.N.Suhagia,M.M.PatelandJ.R.Patel,“HPTLCdetermination of rabeprazoleand Domperidone in capsulesand its validation,” Journal ofChromatographic Science, vol.46, no.4, pp. 304–307, 2008.Volume 2, Issue 6, Nov. − Dec. 201212. C.X.Pan,X.Z.Xu,H.M.He,X.J.Cai,andX.J.Zhang, “Separationand identification of cis andtrans isomers of 2butene-1,4-diol and lafutidine by HPLCand LC-MS,”PharmaceuticalJournal, vol. 6, 2003.13. Salem, M.Y., E.S. El Zanfaly,M.F. El Tarras and M.G. ElBardicy, 2003. Simultaneousdetermination of domperidoneand cinnarizine in a binarymixture using derivativespectrophotometry, partial leastsquares and principlecomponent regressioncalibration. Analytical andBioanalytical Chemistry,375(2): 211-216.14. Ravi kumar, P., P. BhanuPrakash, M. Murali Krishna,M.Santha Yadav and C. Ashadeepthie, 2006.SimultaneousEstimation of Domperidoneand Pantoprazole in SolidDosage Form by UVSpectrophotometry. E-Journalof Chemistry, 3(12): 142-145.15. Ramesh, S., B. Lokesh, J.Rupali and L. Prashant, 2010Validated spectrophotometricmethods for simultaneousestimationofParacetamol,Domperidone andTramadol HCl in pure andtablet dosage form, J. ChemicalMetrol., 4(1): 21-27.16. Sherje, A.P., A.V. Kasture,K.N. Gujar and P.G. Yeole,2008. SimultaneousSpectrophotometric6 | P a g ehttp://www.ijpi.org

INTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-1031determination of Lansoprazoleand Domperidone in capsuledosage form. Indian J.Pharmaceutical Sci., 70(1):102-105.17. Patel, B.H., B.N. Suhagia,M.M. Patel and J.R. Patel,2007. Simultaneous estimationof pantoprazole anddomperidone in pure powderand a pharmaceuticalformulation by highperfomanceliquidchromatography andhigh-performance thin-layerchromatography methods. J.AOAC International,18. Michaud, V., C. Simard and J.Turgeon, 2007. AnimprovedHPLC assay with fluorescencedetection for the determinationof domperidone and threemajor metabolites forapplication to in vitro drugmetabolism studies, J.Chromatography B,AnalyticalTechnologies in Biomedicaland Life Sci.,852(1-2): 611-616.19. Imran, A., V.K. Gupta, S.Prashanth and H.V. Pant, 2006.Screening of domperidone inwastewater by highperformanceliquidchromatography and solidphase extraction methods,Talanta, 68(3): 928-931.20. Dong Hang, X., L. Hong Gang,Y. Hong, B. Jiang, Q. Zhou, Z.Miao Zhang and Z. Rong Ruan,2008. Quantitativedetermination of domperidonein human plasma by ultraperformanceliquidchromatography with electrospray ionization tandem massspectrometry, BiomedicalChromatography,22(4): 433-440.21. Smit, M.J., F.C.W. Sutherland,H.K.L. Hundt, K.J. Swart, A.F.Hundt and J. Els, 2002. Rapidand sensitive liquidchromatography-tandem massspectrometry method for thequantitation of domperidone inhuman plasma, Journal ofChromatography A., 949(1-2):65-70.22. Vogel’s Textbook ofQuantitative ChemicalAnalysis, 6 th edition, 163-165.Volume 2, Issue 6, Nov. − Dec. 20127 | P a g ehttp://www.ijpi.org

VoltsDiluent1.547Domperidone4.867Lafutidine6.120VoltsINTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-10312000000015000000y = 67859x + 28173R² = 0.99910000000500000000 50 100 150 200 250Fig -1: Linearity study of Domperidone70000006000000500000040000003000000200000010000000y = 76331x + 12902R² = 0.9990.000 20.000 40.000 60.000 80.000 100.000Fig 2: Linearity study of Lafutidine500500400400300300200200100100000 1 2 3 4 5 6 7 8 9 10MinutesFig 3: The representative chromatogram for mixed standard of Domperidone andLafutidineVolume 2, Issue 6, Nov. − Dec. 20128 | P a g ehttp://www.ijpi.org

INTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-1031Table – 3:System precision for Lafutidine and DomperidoneInjection No RT of DOM Area of DOM RT of LAF Area of LAF1 4.867 9610163 6.12 38796562 4.867 9678066 6.127 39105193 4.867 9721358 6.127 39167494 4.867 9701590 6.133 39048005 4.86 9701318 6.127 39160226 4.852 9701236 6.123 3915893Average 9685621.8 3907273STD0.003 39429.314 0.005 14282.82DEVIATION% RSD 0.45 0.39Table – 4: Method precession for DOM and LAFUAssay ResultsLafutidineDomperidoneFound/Tab %Found Found/Tab %FoundSET1 29.670 98.901 9.925 99.254SET2 29.871 99.571 10.032 100.319SET3 30.363 101.211 10.062 100.621SET4 29.587 98.623 9.987 99.870SET5 29.930 99.767 10.012 100.120SET6 29.980 99.933 10.010 100.100Average Found 29.9 99.67 10.00 100.05(mg) =%RSD = 0.91 0.91 0.46 0.46Table – 5: Recovery study of LafutidineLevel of standardLafutidineadded added sample added std Recovered %wt. (mg) wt. (mg) mg recovery80% 34.91 26.6 26.746 100.55100% 34.17 32.16 31.884 99.14120% 33.63 39.31 39.606 100.75Volume 2, Issue 6, Nov. − Dec. 201210 | P a g ehttp://www.ijpi.org

INTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-1031Table – 6: Recovery study of DomperidoneLevel of standardDomperidoneadded added sample added std Recovered % ofwt. (mg) wt. (mg) mg recovery80% 26.85 20.29 20.629 101.67100% 26.28 25.37 25.524 100.61120% 25.87 30.24 29.901 98.88Table- 7 Robustness Data in terms of Retention Time for Lafutidine and DomperidoneLevel Wave Length a* Flow Rate b* pH c*DOM LAFU DOM LAFU DOM LAFU- 4.835±0.066.125±0.0234.835±0.0026.125±0.0134.835± 0.02 6.125±0.033330 4835 6.125 4.835 6.125 4.835 6.125+ 4.835 ±0.0276.125 ±0.0354.835 ±0.0476.125±0.0054.835 ± 0.04 6.125±0.015a* Wavelength 220± 2 nm, b* Flow Rate 1.5 ± 0.2 ml/min, c* pH 7.0±0.2Table- 8: System Suitability parametersParameterResults for Results for LafutidineDomperidoneCalibration Range (mcg/ml) 60-240 20 – 80Retention Time (min) 4.835 6.125Theoretical Plate / meter 8245 5429Tailing factor 0.965 0.983Resolution 3.34 ---LOD (mcg/ml) 15.09 16.46LOQ (mcg/ml) 45.73 49.89Volume 2, Issue 6, Nov. − Dec. 201211 | P a g ehttp://www.ijpi.org