Page 1© 2016 Veeragoni Anil Kumar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).Journal of Applied Pharmaceutical Science Vol. 6 (03), pp. 071-076, March, 2016Available online at http://www.japsonline.comDOI: 10.7324/JAPS.2016.60312ISSN 2231-3354

Stability Indicating RP-HPLC Method for the Simultaneous Estimationof Pyrimethamine and Sulphadoxine in Bulk and Tablet Dosage FormVeeragoni Anil Kumar1*, Vasudeva Murthy Sindgi2, Shoba Rani Satla3, Manish Kumar Thimmaraju41Department of Pharmaceutical Analysis, Pathfinder institute of Pharmacy Education and Research, Warangal-Telangana.  2Jayamukhi College of Pharmacy,Narsampet, Warangal, Telangana. 3Center for Pharmaceutical Sciences, Institute of Science and Technology, JNTUH, Hyderabad, Telangana. 4CentralAnalytical Laboratory, Balaji Institute of Pharmaceutical Sciences, Narsampet, Warangal-Telangana.ARTICLE INFOABSTRACTArticle history:Received on: 24/12/2015Revised on: 15/01/2016Accepted on: 08/02/2016Available online: 30/03/2016A stability indicating simple, selective, accurate high Performance Liquid Chromatographic (HPLC) methodwas developed and validated for the combined tablet formulation of pyrimethamine & sulphadoxine.Chromatographic separation was optimized by gradient HPLC on a C18 column [Inertsil Silica, 250 x 4.6 mm,5µ] utilizing a mobile phase of potassium dihydrogen phosphate and acetonitrile taken in the ratio 70:30 at aflow rate of 1.0 ml/min with UV detection at 221nm. The retention time of pyrimethamine and sulphadoxinewas 2.77 and 6.57 min respectively. The developed method was validated in terms of accuracy, precision,linearity, limit of detection, limit of quantitation, robustness and stress degradation studies. Validation of themethod was done in accordance with ICH guidelines for the assay of active ingredients. Thus validated methodcan be recommended for the routine laboratory analysis.Key words:Pyrimethamine,sulphadoxine, ICH guidelinesand stress degradationstudies.INTRODUCTIONPyrimethamine is an anti-malarial drug which inhibitsthe dihydrofolatereductase of plasmodia and there by blocks thebiosynthesis of purines and pyrimidines, which are essential forDNA synthesis and cell multiplication. Sulfadoxine is a sulfadrug, often used in combination with pyrimethamine to treatmalaria. Review of literature for pyrimethamine & sulphadoxinegave information regarding the various studies conducted andanalytical methods established for the drugs alone, incombination and in combination with other drugs inpharmaceutical dosage forms and in biological fluids (Minzi etal., 2013, Sinnaeve et al., 2005). There are few methods reportedin the literature for analysis of pyrimethamine & sulphadoxinealone or in combination with other drugs in the pure form,pharmaceuticals formulations and biological fluids by UV-spectrophotometer (Onah and Odeiani, 2002, Meena and.

* Corresponding AuthorVeeragoni Anil Kumar, Department of Pharmaceutical AnalysisPathfinder Institute of Pharmacy Education And Research, Warangal-Telangana. Email: v_aneel@yahoo.co.inSandhya, 2013), HPTLC (Meena and Sandhya, 2013), Capillaryzone electrophoresis (Amin et al., 2012), RP-HPLC (Green et al.,2002, Bergqvist et al., 1991; Astier et al., 1997; Bergqvist et al.,1985; SaeedArayne et al., 2010), LC-MS (Sinnaeve et al., 2005).There was no stability indicating HPLC methodsestablished for the simultaneous estimation of pyrimethamine &sulphadoxine in formulation. The aim of the present work is todevelop a stability indicating analytical method for the combinedtablet formulation of pyrimethamine & sulphadoxine. Validation ofthe method was done in accordance with ICH guidelines for theassay of active ingredients. Thus validated method can berecommended for the routine laboratory analysis.MATERIALS AND METHODSPyrimethamine (PYR) and Sulphadoxine (SUL) wereprocured as gift samples from Taj pharmaceuticals, Mumbai.REZIZ (Pyrimethamine -25mg and Sulphadoxine - 500mg) tabletsmanufactured by Shreya life sciences pvt. Ltd. India was procuredfrom a local pharmacy. Acetonitrile (HPLC grade), orthophosphoric acid, Potassium dihydrogen ortho phosphate, Methanol(HPLC grade), Tri ethyl amine and TDW (Triple Distilled Water).

Page 2072Anil Kumar et al. / Journal of Applied Pharmaceutical Science 6 (03); 2016: 071-076InstrumentationShimadzu gradient HPLC MODEL NO (JAPAN), HPLCcolumn Inertsil (250 x 4.6mm, 5μm), Mobile phase filtration unit(Pall Life sciences, Mumbai, India), LAB-INDIA U.V with UVWin software, Sonicator, PH meter (LAB-INDIA), digital balance(Denver).METHOD DEVELOPMENTPreparation of standard solutionsAccurately weighed and transferred 10 mg ofPyrimethamine and 10 mg of Sulphadoxine working Standardsinto two separate 100 ml clean dry volumetric flasks, add 30ml ofdiluent , sonicated for 5 minutes and make up to the final volumewith diluent.Chromatographic ConditionsThe HPLC system consisted of Shimadzu gradient HPLC(JAPAN) with dual λ Absorbance UV detector. The wavelength ofdetection as set at 221nm. Separation was carried out in gradientmode on inertsil C18 column (4.6x250mmx5µm) and the retentiontime of pyrimethamine and sulphadoxime was found to be 2.952and 6.832 respectively (figure 1), using 70:30 v/v dihydrogenorthophosphate : acetonitrile as mobile phase at a flow rate of 1ml/min. The mobile phase filtered through nylon milli pore(0.2µm) membrane filter, purchased from pall life sciences,

Mumbai and degassed with Ultrasonicator prior to use.Chromatography was carried out at room temperature 250c andmaintains the column temperature at 320c.Fig. 1: Chromatogram of pyrimethamine and sulphadoxime.Preparation of Standard SolutionsStock solutions of pyrimethamine (0.5mg/ml) andsulphadoxime (1mg/ml) were prepared in methanol. Furtherdilutions were carried out in 60% acetonitrile and calibrationstandards were prepared freshly with pyrimethamine andsulphodoxime stock solutions to give the concentrations of 5, 10,15, 20, 25 and 30 μg/ml.Sample Preparation (Assay)5 tablets were weighed and calculate the average weightof each tablet then the weight equivalent transferred into a 100 mLvolumetric flask, 60mL of diluent added and sonicated for 25 min,further the volume made up with diluent and filtered. From thefiltered solution 0.2ml was pipetted out into a 10 ml volumetricflask and made upto 10ml with diluent.RESULTS & DISCUSSIONSMethod ValidationMethod validation was performed as per the ICHguidelines Q2 (R1) Validation of Analytical Procedure. Thedeveloped method was validated for the following parameters.LinearityLinear concentrations of both drugs were prepared andthe best fit line was calculated. Wide range calibration wasdetermined by solutions containing 5μg/ml to 30μg/ml (table 1).Correlation coefficient was found to be 0.999 & 0.997 forPyrimethamine & Sulphadoxine respectively (shown in fig 2&3).Table 1: Linearity results of pyrimethamine & sulphadoxine.SnoPyrimethamineSulphadoxineConcentration(µg/ml)Peak areaConcentration(µg/ml)Peak area15329102522799232108766841058057643151225460158254245420175356120118705625252109537251448153063026144203018076428PrecisionThe intraday precision was demonstrated by injecting sixtest solutions at 25 µg/ml concentration as per the test procedure(shown in table no 2&3) & recording the chromatograms of sixtest solutions. The % RSD of pyrimethamine and sulphodiximewas found to be 0.207 and 0.324 respectively.Table 2: Method Precision of Pyrimethamine.SnoPyrimethamine (25µg/ml)Retention time(Rt)Peak area% Assay12.902109429100.0122.930210983799.9932.839210994199.9842.9142107535100.0952.8452108530100.0562.872211952899.53Mean211080099.94SD4371.180.206RSD0.2070.206Table 3: Method Precision of SulphadoxineSnoSulphadoxine (25µg/ml)Retention time(Rt)Peak area% Assay16.8281448253699.9926.82314481829

100.0036.7291459925999.1946.7991449974399.8756.71214481539100.0066.72214481645100.00Mean1450442599.84SD47005.990.3220RSD0.3240.3225

Page 3Anil Kumar et al. / Journal of Applied Pharmaceutical Science 6 (03); 2016: 071-076073Fig. 2: Linearity of pyrimethamine.Fig. 3: Linearity of sulphadoxine.Intermediate PrecisionIntermediate precision of the analytical method wasdetermined by performing method precision on in three successivedays by different analysts under same experimental condition.Assay of all six replicate sample preparations was determined andthe mean % RSD of pyrimethamine and sulphodixime was foundto be 0.259 and 0.353 respectively (shown in table no 4).Table 4: Intermediate Precision of pyrimethamine & sulphadoxine.S.NoParameter%RSDMeanRSDDay-1Day-2Day-3Pyrimethamine(25µg/ml)Peak Area0.1990.2640.3180.260% Assay0.1980.2630.3170.259sulphadoxine(25µg/ml)Peak Area0.3170.2880.4620.356% Assay0.3150.2870.45840.353AccuracyAccuracy of the method was established by performingrecovery studies according to the ICH guidelines. Spiked sampleswere prepared by spiking pre-analyzed sample solutions with puredrug at three different concentration levels each in triplicate. Meanpercentage recovery values at three different concentrations of thetwo drugs was calculated. The % recovery of Pyrimethamine(98.02-100.45%) & Sulphadoxine (99.79-100.20%) at each levelwas within the limits of 98% and 102% (shown in table no 5&6).Hence, accuracy was established for the present work and themethod was said to be accurate.Table 5: % recovery of Pyrimethamine.S.noConc(µg/ml)Conc(µg/ml)found%recoveryMeanaccuracy%RSD11514.8101.35100.451.02421515.199.3431514.9100.6742020.398.5298.021.53052019.7101.5262020.199.5072525.299.21100.140.8328

2524.9100.4092524.8100.81Table 6: % Recovery of Sulphadoxine.SnoConc(µg/ml)Conc(µg/ml)found%recoveryMeanaccuracy%RSD11514.91100.60100.200.81121515.1199.2731514.89100.7442020.1299.4099.790.66452019.89100.5562020.1299.4072525.0999.6499.870.42882525.199.6092524.91100.36Limit of Detection & QuantificationIn the present study, the LOD and LOQ were calculatedaccording to the standard deviation of the response and the slopeof the calibration curve i.e., 3.3σ/S and 10σ/S criterions,respectively; where σ is the standard deviation of y-intercepts ofregression lines and S is the slope of the calibration curve.Thelowest possible concentration of Pyrimethamine that can bedetected and quantified by the present method was found to be0.264 µg/ml and 0.800 µg/ml respectively and that ofSulphadoxine was found to be 0.53 µg /ml and 1.62µg/mlrespectively.RobustnessRobustness of the proposed method was determined byvarying various parameters, the %RSD reported was found to beless than 2 %.As the system suitability parameters for the standardand test chromatograms of Pyrimethamine & Sulphadoxine werewithin limits for variation in flow rate (±0.1ml) and mobile phasecomposition, the allowable variation in flow rate, organic solventratio in mobile phase composition and column temperature shouldbe 1±0.1ml/min, 65±2ml and 30±50c respectively (shown in tableno 7 & 8).STRESS DEGRADATION STUDIESStress degradation studies were performed as per the ICHguidelinesQ1A (R2) Stability Testing of New Drug Substancesand Products, using the proposed validated analytical method andthe results were shown in table no 9 & 10.y = 70181x-5618R² = 0.99905000001000000150000020000002500000300000005101520253035y = 24199x-13401R² = 0.9970200000040000006000000800000010000000120000001400000016000000180000002000000005101520253035

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074Anil Kumar et al. / Journal of Applied Pharmaceutical Science 6 (03); 2016: 071-076Acid degradation studiesTo 1ml of stock solution pyrimethamine andsulphadoxine, 1ml of 2N HCl was added and refluxed for 30min at600c. From the above solution10µl was injected into the systemand the chromatograms were recorded to detect the stability ofsample (figure 4).Alkali Degradation StudiesTo 1ml of stock solution of of standard drug and samplepyrimethamine and sulphadoxine, 1ml of 2N NaOH was addedand refluxed for 30min at 60 °C. From the above solution10 µlwas injected into the system and the chromatograms were recordedto detect the stability of sample (figure 5).OxidationTo 1ml of stock solution of standard drug and sample ofpyrimethamine and sulphadoxine, 1ml of 20% H2O2was added andrefluxed for 30min at 600c. From the above solution10 µl wasinjected into the system and the chromatograms were recorded todetect the stability of sample (figure 6).Photo Stability StudiesThe photochemical stability of the drug was also studiedby exposing the 25 µg/ml solution to UV Light by keeping thebeaker in UV Chamber for 7days or 200 Watt hours/m2 in photostability chamber. . For HPLC study, from the above solution10 µlwas injected into the system and the chromatograms were recordedto detect the stability of sample (figure 7).Pyrimethamine and Sulphadoxine undergoes degradationin acidic, oxidation, alkaline, and UV. More degradation wasfound for oxidation. As per ICH guidelines peak purity angleshould be less than peak purity threshold. Hence, method of theanalysis of PYR and SUL in tablet dosage form shows that thedegradation product doesn’t interfere with the analyticaldetermination. The stress degradation studies showed that the drugformulation containing pyrimethamine and sulphadoxineundergoes degradation in acidic, oxidation, alkaline, and UV(7.29% ,15.01% ,16.13% , 7.88% , 5.51% and 12.92% , 10.72% ,7.13% , 13.32% , 6.01%). hence the proposed analytical method isalso useful for the determination of pyrimethamine andsulphadoxine stability in sample of pharmaceutical dosage form.Table 7: Robustness of Sulphadoxine.ParameterVariation in flowVariation in Mobile phaseVariation in column tempflow rateflow rateBuffer:Acetonitrile (75:25)Buffer:Acetonitrile (80:20)30-50

30+50

Stds(0.9ml/mim)(1.1ml/mim)19709676889403089907089020771896277489215432972115188627828982909902566389653008925743Mean971541488784068986809902321789655378922143SD8114.722095.755152044.63163.42262.4%RSD0.10.20.10.10.10.1Retention time7.092.7156.037.076.476.37Tailing factor1.331.331.321.31.321.43Theoretical plates521750825089530652535346Table 8: Robustness of Pyrimethamine.ParameterVariation in flowVariation in Mobile phasevariation in column tempflow rateflow rateBuffer:Acetonitrile (75:25)Buffer:Acetonitrile (80:20)30-50

30+50

Stds(0.9ml/mim)(1.1ml/mim)1

1588766148537514358451539898145422714349072160392014841121425867154919814808621440355Mean159634314847431430856154454814675451437631SD10715892.87055.56576.418833.73852.6%RSD0.70.10.40.41.30.3Retention time2.952.7152.433.0762.712.66Tailing factor1.471.441.421.391.441.43Theoretical plates367936653797382437463981Table 9: Results of stress degradation studies of Sulphadoxine.SnoStress conditionsTime% Assay% Degradation1Acid Degradation30 min88.08211.9182Base Degradation30 min87.28112.7193Peroxide Degradation30 min93.2146.7864UV Degradation7 days92.8877.113Table 10: Results of stress degradation studies of Pyrimethamine.SnoStress conditionsTime% Assay% Degradation1Acid Degradation30 min92.7137.2872Base Degradation30 min85.98514.0153Peroxide Degradation30 min82.79817.2024UV Degradation7 days93.5026.498

Page 5Anil Kumar et al. / Journal of Applied Pharmaceutical Science 6 (03); 2016: 071-076075Fig. 4: Chromatogram for Sample acid degradation.Fig. 5: Chromatogram for Sample Alkali degradation.Fig. 6: Chromatogram for Sample Peroxide degradation.

Page 6076Anil Kumar et al. / Journal of Applied Pharmaceutical Science 6 (03); 2016: 071-076CONCLUSIONThe proposed HPLC method was found to be simple,specific, precise, accurate, rapid and economical for simultaneousestimation of pyrimethamine & sulphadoxine in bulk and tabletdosage form and was found to be suitable for the routine analysisand quality control and percentage degradation of pharmaceuticalpreparations containing these drugs either individually or incombination.REFERENCES

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