Department of Studies and Research in Chemistry University of Mysore - PDF document

1 Department of Studies and Research in Ch
Department of Studies and Research in Chemistry, University of Mysore,Corresponding author: Tel: +91 8212419659; Fax: +91 8212516133;E-mail address: basavaiahk@yahoo.co.in A simple, precise and accurate stability-indicating isocratic reverse phase ultra-performance liquidchromatographic (RP-UPLC) method was developed for the determination of repaglinide (RPG) in bulk drugand in its tablets. The method was developed using Waters Aquity BEH C18 (100with mobile phase consisting of a mixture of potassium dihydrogen phosphate buffer of pH 3.2 and acetonitrile(40:60 v/v). The total run time for the assay was only 4 min. The eluted compound was detected at 245 nm withan UV detector. The standard curve of mean peak area versus concentration showed an excellent linearity overRPG with regression coefficient (r) value of 0.9997. The limit of detection3) was 0.03 10) was 0.1 . With three quality control, accuracy and precision of the assay were satisfactory. Both within-day and between-day RSD were less than 1.0%. The method was validated by the determination of RPG intablets and the percent of the label claim was 1002%. The accuracy of the method was further ascertainedby recovery studies standard addition procedure and the recoveries obtained were 96.6-100.9%. Forceddegradation of the bulk sample was conducted in accordance with the ICH guidelines. Acid and base hydrolysis,oxidative, thermal stress and photolytic degradation were used to assess the stability indicating power of themethod. RPG was found to degrade significantly in acidic and basic stress conditions and stable in oxidative,thermal and photolytic conditions. The degradation pr

2 oducts were well resolved from main peak
oducts were well resolved from main peak proving thestability-indicating power of the method. Repaglinide; UPLC; Validation; Stability indicating; Pharmaceutical C. M. Xavier and K.Basavaiahaiah(()butylamino)-2-oxoethyl]benzoic acid (Fig. 1) is a non-class Type 2 anti-diabetic drug [1]. It reduces the fastingby the pancreas. RPG is official in United Statesficial in United Stateswhich recommend non-aqueous titrimetry for its assay.In the literature, two high performance liquidchromatography (HPLC) methods are available for theailable for the 5]. Several methodshave been reported for the determination of RPG inted for the determination of RPG indifferential pulse polarography [11] reversed-phaseersed-phaseThough HPLC is a well-established reliableof active pharmaceutical ingredients (APIs) andcomplexity of the samples; it could still be improved.Ultra performance liquid chromatography (UPLC) is am particles of stationary phase.flow is 15,000 psi which is two times greater than thepressure limit of ordinary HPLC. The elevated mobilephase linear velocity results high resolution, sensitivityelocity results high resolution, sensitivitysensitivity, this technique is gaining considerable attentionin recent years in different fields of pharmaceuticalerent fields of pharmaceuticalDespite these advantages, the UPLC has not beenexpiry date [22]. Stability testing of an active substancesubstance or drug product varies with time under aused in the development of manufacturing process,elopment of manufacturing process, 24]. There isno reported stability-indicating HPLC/UPLC method thattablets. It is, therefore, necessary to develop a stabilityi

3 ndicating method for the quantitative es
ndicating method for the quantitative estimation of RPG.In this piece of work, the authors have made anattempt to develop a faster chromatographic technique,the accuracy, precision and sensitivity. The developedmethod was validated as per the regulations of currentalidated as per the regulations of current 25]. The method was successfullyany additional peaks from the inactive ingredients in thechromatogram and almost zero interference wasobserved.ure active ingredient sample of RPG was kindlysupplied by Torrent Pharmaceuticals Ltd, Ahmadabad, N H O O CH3 COOH N H3C CH3 (2 mg RPG)-both tablets, marketed by TorrentPharmaceuticals, Ahmadabad, India, were purchasedortho phosphate and orthophosphoric acid were fromQualigens-India. Doubly distilled water was usedthroughout the investigation.Analyses were carried out on a Waters Aquity UPLCwith Tunable UV (TUV) detector. The output signalas monitored and processed using Empower software.The Chromatographic column used was Acquity UPLCm particle size.Isocratic elution process was adopted throughout theDissolved 2.0 g of potassium dihydrogenorthophosphate in 500 ml of water and adjusted thepH to 3.0 using 10% ortho phosphoric acid. A 400 mlportion of this resulting buffer was mixed with 600 mlof acetonitrile, shaken well and filtered using 0.22 nylon membrane filter. This solution was also used asThe isocratic flow rate of mobile phase wasC. The injection volume was 1.2 Eluted sample was monitored at 245 nm and the runtime was 4.0 min. The retention time of the sampleA 25 mg of pure RPG was transferred into threedifferent 50 ml volumetric flasks and added 10 ml offlasks were heated for 2 h

4 on a water bath maintainedC. Then the s
on a water bath maintainedC. Then the solutions were cooled and neutralizedby adding base or acid, the volume in each flask wasappropriate volume (1.2 l) was injected for analysis.Solid state thermal degradation was carried out byC for 3 h. Forphase and the chromatographic procedure was followed.as prepared by dissolving an accurately weighed 250mg of pure drug in 50 ml volumetric flask using theRPG were prepared by serial dilutions of aliquots of thereported chromatographic conditions. The average peakarea versus the concentration of RPG in wasplotted. Alternatively, the corresponding regressionequation was derived using mean peak area-enty five Eurepa-2contained 2.0 mg RPG) were weighed and transferredin to a clean, dry mortar and powdered. The entirematerial was transferred in to a 100 ml volumetric flaskand 60 ml of the mobile phase was added. The solutionas sonicated for 20 min to achieve complete dissolutionof RPG, and the solution was then diluted to volumefilter. The solution obtained was analyzed by UPLC.The same procedure was repeated with fifty Eurepa-11.Accuracy and precision C. M. Xavier and K.Basavaiahpure RPG solutions at three different concentrationsMobile phase was injected as blank solution beforesample injection and the RSD (%) values of peak areaand retention time were calculated.2.Limits of detection (LOD) and quantificationThe LOD and LOQ were obtained by signal tonoise (S/N) ratio method. LOQ and LOD were obtainedPrecision study was performed at LOQ level also. LOQsolution was injected six times (n=6) and calculatedthe %RSD values for the obtained peak area and3.LinearityLinearity solutions were prepared f

5 rom LOQ levelRPG). A total of eight conc
rom LOQ levelRPG). A total of eight concentrations of the solutions levels).4.Robustness and ruggednessexperimental conditions were deliberately changed.column oven temperature (40composition (66:34 and 54:36; acetonitrile: buffer v/v)and detection wavelength (2451 nm) were the variedparameters. In each case the %RSD values wereThe number of theoretical plates and tailing factors wereconditions. Three different columns of same dimensionsere used for the analyses. The study was performed ina same day and three different days by three differentanalysts for three different concentrations of RPGconcentration was compared with that of the optimizedone. The relative standard deviation values wereevaluated for each concentration.5.Solution stability and mobile phase stabilityStability of RPG solution was performed by injectingarea was recorded in the time intervals of 0, 12 and24 h and the RSD values were calculated. The mobilephase stability was studied by injecting a freshly preparedsample solution at the same time intervals (0, 12 and24 h) and RSD values of the peak areas were calculated.In order to achieve the better efficiency of thesuch as mobile phase composition, detection wavelength,diluent were optimized by varying one parameter andkeeping other constant at a time. Several proportions ofbuffer, water, acetonitrile and methanol were evaluatedplates and run time were the major tasks while developingthe method. Several combinations of gradient methodsere also performed. Isocratic method was found betterand the resultant peak showed tailing factor more thanm column was also found to give inconsistent resultwith fronting of the peak

6 . The column temperature wassame column,
. The column temperature wassame column, the peak shape was found unaltered.Buffer and acetonitrile solvents ratio were changed andended up with less number of theoretical plates. Differentbuffers like sodium dihydrogen othophospahe,dipotassium hydrogen orthophosphate and disodiumhydrogen orthophosphate were tried and the resultsrevealed that the use of potassium dihydrogenorthophosphate was most suitable. The pH of the mobilephase was varied from 2 to 6. At pH greater than 3.2,the peak eluted very early and resulted in less numberof theoretical plates. At lower and higher flow rates,non-elution of peak and inefficiency of the system15000 psi), respectively, were found. buffer of pH 3.2 v/v, Acquity BEH C18, (100found more suitable for the validation study with theconditions are depicted in Fig. 2.All forced degradation samples were analyzed atconcentration level. The observation wasmade based on the peak area of the respective sample.Degradation was not observed when RPG was subjectedand oxidative (10% Hfor 2 h) conditions (observeddegradation was less than 0.08%). Significant degradationas observed when the drug was subjected to acid and35.8% and 41.9%, respectively. The chromatogramspresented in Fig. 3a-3e. Assay study was carried outhas been validated as per the current ICH Q2 (R1)alidated as per the current ICH Q2 (R1)Analytical parametersA calibration curve was obtained for RPG fromas obtained between the peak area and thewhich the linear regression equation was computed and0.9999)(1)correlation coefficient. The LOD and LOQ values, slopeevaluated and presented in Table 1. These results confirmthe linear relation between conc

7 entration of RPG andthe peak areas as we
entration of RPG andthe peak areas as well as the sensitivity of the method.The percent relative error which is an index of1.5 and is indicative of high accuracy. Thecalculated percent relative standard deviation (%RSD)based and retention time based RSD values were The results obtained for the evaluation of precision andaccuracy of the method are compiled in Table 2. Pure repaglinide overlay with blank 0.000.501.001.502.002.503.003.504.00 C. M. Xavier and K.Basavaiah 0.000.501.001.502.002.503.003.504.00 0.000.501.001.502.002.503.003.504.00 0.000.501.001.502.002.503.003.504.00 90Thai J. Pharm. Sci. 37 (2013) 84-94 Figure 3Chromatograms of repaglinide (RPG) after (a) forced degradation with 1 N HCl at 80°C for 2 h (pure RPG, 500 µg ml-1); (b)forced degradation with 1 N NaOH at 80°C for 2 h (pure RPG, 500 µg ml-1); (c) forced degradation with 10% H2O2 at 80°Cfor 2 h (pure RPG, 500 µg ml-1); (d) thermal treatment at 105°C for 3 h (pure RPG, 500 µg ml-1); (e) exposure to UV at1.2 million lux hrs (pure RPG, 500 µg ml-1) 0.000.501.001.502.002.503.003.504.00 0.000.501.001.502.002.503.003.504.00 Table 1 Linearity and regression parameters with precision data ParameterValue)0.10)0.03Slope (b)3195.5786Intercept (a)-8145.7707Correlation coefficient0.9999Regression0.9997Residual sum of squares1154120143.61539 Standard deviation of a, (Sa) C. M. Xavier and K.Basavaiah Results of accuracy and precision study (n=6) Concentration ofIntra-dayInter-dayConcentration ofRE (%)Concentration ofRE (%) 10098.741.26100.680.68200201.880.92101.700.85 300297.750.75303.101.04 Concentration of RPGIntra-day precisionInter-day precision SDRSDSDRSD10029276113360.4

8 60.1829454919220.650.642005870349690.170
60.1829454919220.650.642005870349690.170.1558478244180.760.30 30088342420930.240.1188658044650.500.29Relative errorRelative standard deviation based on peak areaRelative standard deviation based on retention time Results of robustness and ruggedness (n=3) ConditionModifica-Mean peakRSDMean RtRSDTheoreticalRSDTailingRSD tionarea(%)(%)plates(%)factor(%)Actual---14643620.012.1220.3149511.611.010.10emperature3514987640.032.2210.4049711.991.040.1914578610.121.9980.2548241.171.000.20Mobile phase66:3414687980.011.9980.3051222.051.020.49composition54:3614987990.072.1220.2345982.130.990.50buffer)Flow rate0.4515067450.132.2160.2351251.961.020.680.5514678540.081.9980.1551371.971.030.87elength (nm)24414797530.372.1060.2948092.151.010.3024614687930.672.1120.3347341.891.050.38Analyst,Analyst-1,column,column-1,14698990.272.1210.0948132.271.010.10dayday-1column-2,14618960.082.0230.3549192.851.050.38column-3,14719340.222.1280.4248921.611.050.29 measure of its capacity to remain unaffected by small,but deliberate variations in method parameters andusage. At the deliberate varied chromatographictheoretical plates were remained near to the actual values.The RSD values ranged from 0.2 to 0.7% resumes theruggedness, different columns (same lot), at differentday by different analyst were performed. The resultsare summarized in Table 3.At the specified time interval, %RSD for the peakphase stability were within 1%. This shows no significantdrug and mobile phase were stable at least for 24 hduring the assay performance.Selectivity of the method was evaluated by injectingand tablet extract. No peaks were observed for mobilephase and placebo blank and no e

9 xtra peaks wereobserved for tablet extra
xtra peaks wereobserved for tablet extracts (Fig. 4).solution of tablets was prepared aspeak area of the tablets was found to be equivalentto the pure drug and the results were compared withere compared withinvolves the titration of the tablet extract in 1:6 (methanol:and precision of the proposed method was furtherevaluated by applying Studentûs t-test and varianceratio F-test, respectively. The t-and F-values at 95%confidence level did not exceed the tabulated valuesand this further confirms that there is no significantdifference between the reference and proposed methods.A standard addition procedure was followed toevaluate the accuracy of the method. The solutionspre-analyzed tablet extract at three different levels andinjected to chromatographic column. The recovery ofthe known amount of added analyte was computed.The percentage recovery of RPG from pharmaceuticalresults shown in Table 5 revealed good accuracy of 0.000.501.001.502.002.503.003.504.004.505.00 Figure 4 Chromatogram of tablet extract (1-mg and 2-mg tablets) C. M. Xavier and K.Basavaiah A rapid, isocratic RP-UPLC method developedfor quantitative analysis of repaglinide in pharmaceuticalspecific. Satisfactory results were obtained from validationenables rapid determination of the drug which is importantperformance in terms of sensitivity and speed.method reveals that the product is highly unstable inAuthors thank Torrent Pharmaceuticals Ltd,Authors are thankful to the University of Mysore forto pursue Ph.D degree programme. We do not have anymentioned in the article that might lead to a conflict ofticle that might lead to a conflict ofThe Merck Index (13rd ed.), Wh

10 itehouse station, NJ, USA,2003.[2]US Pha
itehouse station, NJ, USA,2003.[2]US Pharmacopeia-National formulary, USP-29, United StatesPharmacopeial Inc., Rockville MD, 2006, pp. 2780-2785.[3]European Pharmacopeia (6th ed.), European directorate forthe quality of medicines & health care, 2009, p. 2813.[4]A.B. Ruzilawati, M.S.A. Wahab, A. Imran, Z. Ismail, andS.H.Gan. Method development and validation of repaglinide[5]P. Venkatesh, T. Harisudhan, H. Choudhury, R.M. Ramesh,and N.R. Srinivas, Simultaneous estimation of six anti-pioglitazone, repaglinide and rosiglitazone: development ofa novel HPLC method for use in the analysis of pharma- FormulationNominalRPG foundSDt-valueF-valueamount, mgReferenceProposed methodmethod1.099.510.62100.100.212.356.13 2.0101.20.8599.940.782.441.19Marketed by Torrent pharmaceuticals, India;Mean value of five determinations. Tabulated t-value at 95% confidence level is 2.78; Tabulated F-value at 95% confidence level Results of recovery study by standard addition method TabletTablet (Pure RPGMean peakTotal RPG foundPercent recovery ofstudied(area( Eurepa 1200.2100943398.85297.7797.57200.22001256749.77395.8397.82200.23001593299.71501.14100.30Eurepa 2199.9100948452.85299.3599.45199.92001248399.77393.2196.66 199.93001598765.71502.85100.98 J.R. Patel, B.N. Suhagia, and B.H. Patel. Simultaneous[7]B.A. Alkhalidi, M. Shtaiwi, H.S. Alkhatib, M. Mohammad,and Y. Bustanji. A comparative study of first-derivativespectrophotometry and column high-performance liquid[8]S.K. Jain, G.P. Agrawal, and N.K. Jain. Spectrophotometrical, and N.K. Jain. SpectrophotometricA. Goyal, and I. Singhvi. Visible spectrophotometric[10]R.M. Singh, H.R. Khan, S. Talegaonkar, S.C.

11 Mathur, andalegaonkar, S.C. Mathur, and
Mathur, andalegaonkar, S.C. Mathur, andM.A.N. El-ries, G.G. Mohamed, and A.K. Attia. Electrochemicaldetermination of the antidiabetic drug repaglinide, YakugakuZasshi 128: 171-177 (2008).[12]G. Anna, B. Anna, and H. Hanna. Quantitative analysisof repaglinide in tablets by reversed-phase thin-layer[13]M. Gandhimathi, T.K. Ravi, and S.K. Renu. Determination of[14]B. Anna, G. Anna, and H. Hanna. Development and validationof a new high-performance liquid chromatography methodfor the determination of gliclazide and repaglinide inJ. AOAC Int.[15]P.A. Rani, C. Balasekaran, N. Archana, P.S. Teja, and B.[16]A.D. Jerkovich, J.S. Mellors, and J.W. Jorgenson. The useof micrometer-sized particles in ultrahigh pressure liquidLCGC North Americath AmericaV.G. Dongre, P. Karmuse, P.P. Rao, and A. Kumar. Developmentand validation of UPLC method for determination of[18]C. Krishnaiah, A.R. Reddy, R. Kumar, and K. Mukkanti.alsartan and their degradation products in active pharma-[19]R. Plumb, J.C. Perez, J. Granger, I. Beattie, K. Joncour, andA. Wright. Ultra-performance liquid chromatography coupledto quadrupole-orthogonal time-of-flight mass spectrometry,[20]S.A.C Waren, and P. Tchlitcheff. Use of ultra-performanceliquid chromatography in pharmaceutical development, [21]R. Li, L. Dong, and J. Huang. Ultra performance liquid[22]FDA. Centre for Drug Evaluation and Researchaluation and ResearchInternational Conference on Harmonization (ICH). Guidancefor Industry Q1A (R2): Stability Testing of New Drug IFPMA, Geneva. 2003.[24]J.T. Cartensen, and C.T Rhodes, Marcel Dekker, New York, 2000.ork, 2000.ICH Q2 (R1), Validation of Analytical Procedure: Text a