1. 325 Original ArticleSIMULTANEOUS ESTIMATION AND VALIDATION OF ATENOLOL,HYDROCHLORO THIAZIDE AND LOSARTAN K IN TABLET DOSAGE FORM BY RP-HPLC METHOD. *,1Parthiban C, 2Bhagavan Raju M, 1Sudhakar M*,1MallaReddy College of Pharmacy, Maissamaguda, Dullapally, Secunderabad,Andhra Pradesh, India - 500 014. Print2231 36482C M College of Pharmacy, Maissamaguda, Dullapally, Secunderabad,ISSN Online 2231 3656Andhra Pradesh, India 500 014. Abstract A simple RP-HPLC method was developed and validated for simultaneous estimation of Atenolol, Hydrochloro thiazide and Losartan K from pharmaceutical dosage forms. A sensitive chromatographic separation was accomplished on thermo scientific C18 column (20 cm4.6 mm, 5) with mobile phase consisting of Acetonitrile : phosphate buffer (pH 3.6 adjusted with anhydrous disodium hydrogen phosphate) in the ratio of 70:30 v/v, at a flow rate of 1.2 ml/min and eluents monitored at 229 nm. The developed method was validated in terms of accuracy, precision, linearity and limit of detection, limit of quantification, robustness and solution stability. The proposed method can be used for the estimation of these drugs in combined pharmaceutical dosage forms. Key words: Simultaneous estimation, Atenolol, Hydrochloro thiazide, Losartan K.IntroductionAtenolol is (RS)-2-{4-[2-hydroxy-3-(propan-2-ylamino)Losartan is in a group of drugs called angiotensin IIpropoxy] phenyl} acetamide. Atenolol is a selectivereceptor antagonists. Losartan is used to treat high1 receptor antagonist, a drug belonging to the groupblood pressure (hypertension). It is also used to lowerof beta blockers. Hydrochlorothiazide is 6-chloro-1, 1-the risk of stroke in certain people with heart disease.dioxo-3, 4-dihydro-2H-1, 2, 4 benzothia-diazine-7- The simultaneous determination of Losartan potassiumsulfonamide1-3. Hydrochlorothiazide belongs to the and Atenolol in tablets by HPLC5. And the Simultaneous thiazide class of diuretics. It reduces blood volume by determination of valsartan and hydrochlorothiazide inacting on kidneys to reduce sodium reabsorption in tablets by first derivative UV spectrophotometry andthe distal convoluted tubule. The major site of action inLC6. And the determined Losartan potassium andthe nephron appears on an electroneutral Na+-Cl- co- Hydrochlorothiazide in tablet dosage form bytransporter by competing for the chloride site on thesimultaneous spectrophotometric estimation9. Literaturetransporter. By impairing Na transport in the distal surveys reveal that no method has been reported forconvoluted tubule, hydrochlorothiazide induces a these combinations. The present manuscript describes anatriuresis and concomitant water loss1-3. Losartan is novel LC method which is simple, rapid, precise,2-Butyl-4-chloro-1-[[2-(1H-tetrazol-5-yl) [1,1-biphenyl] sensitive, selective and accurate isocratic reverse phase-4-yl]methyl]-1H-imidazole-5-methanol monopotassiumHPLC method for simultaneous estimation of Atenolol,salt.Hydrochlorothiazide and Losartan K in tablet dosage form.Author for Correspondence:Parthiban C, Experimental Reagents and MaterialsMalla Reddy College of Pharmacy, Maissamaguda, A tablet Losar-Beta-H (Unichem) contains 50mg ofDullapally, Secunderabad,Atenolol (ATN), 12.5 mg of Hydrochloro thiazide (HCT)Andhra Pradesh, India - 500 014. and 50mg of Losartan K (LSK). Methanol HPLC gradeEmail: parthi1617@gmail.comwas procured from Merck Ltd, Mumbai. Citric acid and Int. J. Pharm & Ind. Res Vol - 01Issue - 04Oct Dec 2011

2. 326anhydrous disodium hydrogen phosphate AR grade ATN, HCT and LSK present in each tablet waswere procured from Qualigens Fine Chemicals, calculated by comparing the peak area of theMumbai. Water HPLC grade was obtained from a standard solution and sample. The amount of the drugsMilli-QRO water purification system. Reference were calculated and tabulated in table 01.standards of Atenolol, Hydrochlorothiazide andLosartan K were gift samples from RanbaxyResults and DiscussionLaboratories Limited.The HPLC procedure was optimized with a view to develop precise and accurate assay method. VariousApparatus and Chromatographic conditions mobile phase systems were prepared and used toThe HPLC system consisted of a separation module provide an appropriate chromatographic separation,(Water Alliance 2695) and Photo Diode Array (PDA)but the proposed mobile phase comprising ofdetector. An isocratic elution was performed on thermo Acetonitrile, phosphate buffer (pH 3.6) (70:30 v/v)scientific C18 column (20cm x 4.6 mm, 5). The mobilegave a better resolution. Using UV-visible PDAphase was degassed and filtered (0.45 , Millipore)detector at 229nm carried out the detection. Amongstmixture of Acetonitrile and phosphate buffer (pH 3.6)the several flow rates tested (0.8 - 2.0 ml/min), theadjusted with anhydrous disodium hydrogen phosphateflow rate of 1.2 ml/min was the best for all the(70:30 v/v). Injection volume was 20l and run timedrugs with respect to location and resolution of peaks.was 12min and flow rate was 1.2ml/min. The columnThe retention time of ATN, HCT and LSK was found towas maintained ambient temperature and the eluents be 2.6, 5.6 and 9.2 min respectively. Thewere detected at 229nm. Quantification was achievedchromatograms of standard and sample solution ofby peak area-ratio method with reference to theATN, HCT and LSK were shown in figure 01 and 02standards. .The asymmetry factor of ATN, HCT and LSK was found to be 0.72, 0.54 and 0.81 respectively, which indicatesPreparation of stock and standard solutions of symmetrical nature of the peak. The percentage labelAtenolol, Hydrochlorothiazide and Losartan K claim of individual drugs found in formulations wereStandard stock solution (1000g/ml) of ATN, HCT andcalculated and presented in table 01.The results ofLSK were prepared separately in Acetonitrile. Theanalysis shows that the amounts of drugs estimatedworking standard solutions were prepared and further were in good agreement with the label claim of thediluted in mobile phase to contain a mixture of ATN, formulations.HCT and LSK in over the linearity range from 10-90g/ml, 5-25g/ml and 10-90g/ml respectively.Estimation of drugs from marketed formulationsTwenty tablets, each containing 50mg of ATN, 12.5 mgof HCT and 50mg of LSK were weighed and tabletcontents are finely powdered. A quantity of powderequivalent to 50mg of ATN, 12.5 mg of HCT and50mg of LSK were weighed and transferred into Figure 01: Chromatogram of standard100ml of standard volumetric flask containing 50 ml ofAcetonitrile. The sample was kept in an ultrasonic bathfor 20 min and further diluted to 100ml by usingmobile phase. Then it is filtered through 0.2membrane filter paper. 10 ml of this solution wasfurther diluted 100ml to get a concentration of 50g/ml of ATN, 12.5 g/ml of HCT and 50 g/ml ofLSK. 20l of this solution was injected into HPLC systemand chromatograms were recorded. The amount of Figure 02: Chromatogram of sampleInt. J. Pharm & Ind. ResVol - 01Issue - 04 Oct Dec 2011 3. 327 Table 01: Table for Assaybeing d one i n replicate. The %RSD was within t h e Label*Amount *Percentagea c c e p t a b l e limit ( 2%). It is evident fromS.No.Tablet sample claim PresentLabel claim the results of accuracy study, reported in table (mg/tablet) (mg/tablet) (%w/w) 02, that the proposed method enables very 1Atenolol 5049.7299.44 accurate quantitative simultaneous estimation ofHydrochloro ATN, HCT and LSK. 2thiazide 12.512.3899.04 3Losartan K50 50.20100.40The Precision of the method was demonstrated byMethod Validation system precision and method precision studies. In theThe proposed method was validated with respect to system precision studies, six replicate injections of theaccuracy, precision, linearity and range, limit ofworking standard solution prepared as per thedetection, limit of quantification, robustness andproposed method and chromatograms were recorded.stability of analytical solutions following the guidelinesStandard deviation and relative standard deviationof International Conference on Harmonization. for the area was calculated and presented in [Table3]. In the method precision studies, six replicateAccuracy and Precisioninjections of the analyte solution prepared as per theThe accuracy of the method was determined byproposed method and chromatograms were recorded.recovery experiments. It was confirmed by studyingStandard deviation and relative standard deviationthe recovery at three different concentrationsfor the area was calculated and presented in table 04.50%, 100%, and 150 % of those expected by From the data obtained, the developed RP-HPLCspiking a previously analyzed test solution withmethod was found to be precise.additional s t a n d a r d drug solutions, t h e analysis Table 02: Accuracy of the method %ConcentrationDrug Amount Added (mg) Amount Found (mg) % Recovery Mean % Recovery (at specification Level) 50% 2524.82 99.28 Atenolol 100% 5050.16 100.3299.69150% 7574.62 99.49 50%7.5 7.42 98.93 Hydrochlorothiazide100% 1514.92 99.46 99.58150%22.5 22.58 100.35 50% 2525.24 100.96 Losartan K 100% 5050.19 100.38100.21150% 7574.47 99.29 Table 03: System precision ReportParametersArea of AtenololArea of Hydrochloro thiazideArea of Losartan K Trial 1237653 69334 219243 Trial 2235497 68948 221435 Trial 3238129 68974 222857 Trial 4239258 69163 221984 Trial 5236385 69356 223124 Trial 6234781 69034 221678 Average 236950.569134.83 221720.2 Standard deviation1693.878179.0625 1382.187 % Relative standard deviation 0.7148660.259005 0.623392Linearity and Range thiazide and 10-90g/ml for Losartan K Table 05. TheA linear relationship was observed between thelinearity was expressed as R2, which was 0.999 forabsorbance and concentration over the range from 10-Atenolol, 0.999 for Hydrochloro thiazide and 0.999 for90g/ml for Atenolol, 5-25g/ml for Hydrochloro Losartan K. Values o f R 2 shown in figures 3, 4 & 5.Int. J. Pharm & Ind. Res Vol - 01 Issue - 04 Oct Dec 2011 4. 328Table 04: Method precision Report Parameters Area of Atenolol Area of Hydrochloro thiazide Area of Losartan KTrial 1 23542368358225823Trial 2 23934568698225628Trial 3 23653468936223649Trial 4 23762968269223517Trial 5 23745969065225372Trial 6 23361368703224812Average236667.2 68671.5 224800.2Standard deviation 1980.734 312.021 1003.028% Relative standard deviation0.836928 0.4543680.446187Table 5: Linearity ReportResults Limit of Detection (LOD) and Limit of Quantification (LOQ) Parameters Hydrochloro Limit of detection (LOD) and limit of quantification Atenolol Losartan K thiazideLinearity(R2) 0.999 0.999 0.999(LOQ) were calculated as 3.3 /S and 10 /S,RSD,%222 respectively as per ICH guidelines, where is theMean Recovery,% 99.69 99.58100.21 standard deviation of the response (y-intercept) and Sis the slope of the calibration plot. The LOD is thesmallest concentration of the analyte that gives ameasurable response (signal to noise ratio of 3).TheLOD for ATN, HCT and LSK was found to be0.50g/ml, 0.20g/ml and 0.54g/ml, respectively.The LOQ is the smallest concentration of the analytewhich gives response that can be accurately quantified(signal to noise ratio of 10).The LOQ of ATN, HCT andLSK was found to be 4.50g/ml, 3.20g/ml andFigure 03: Linearity of Atenolol5.60g/ml respectively.Ruggedness and RobustnessThe ruggedness of the method was determined bycarrying out the experiment on different instrumentslike Shimadzu HPLC (LC 10 AT), Water Alliance 2695by different operators using different columns.Robustness of the method was determined by makingslight changes in the chromatographic conditions. It wasobserved that there were no marked changes in the Figure 04: Linearity of Hydrochloro thiazide chromatograms, which demonstrated that the RP- HPLCmethod developed is rugged and robust.Solution StabilityIn order to demonstrate the stability of both standardand sample solutions during analysis, both solutionswere analyzed over a period of 6 h at roomtemperature. The results show that for both solutionsthe retention time and peak area of ATN, HCT and LSKremained unchanged (percentage RSD less than 2.0),Figure 05: Linearity of Losartan Kthus indicated that both solutions were stable for 24h,Int. J. Pharm & Ind. ResVol - 01 Issue - 04Oct Dec 2011 5. 329which was sufficient to complete the whole analytical derivative UV spectrophotometry and LC. J Pharmprocess.Biomed Anal 2001, 25, 009-13.7.Charles J, Brault J, Boyer S, Langlois C, CabreroSystem suitability studiesMS, Dubost L. Simultaneous determination ofThe column efficiency, resolution and peak asymmetryIrbesartan and Hydrochlorothiazide in tablets bywere calculated for the standard solutions (Table derivative spectrophotometry. Anal Lett 2003, 36,No.6). The values obtained, demonstrated the2485-95.suitability of the system for the analysis of this drug 8.Huang T, He Z, Yang B, Shao L, Zheng X, Duan G.combinations. Simultaneous determination of captopril andhydrochlorothiazide in human plasma by reverse-Table 06: System suitability Report phase HPLC from linear gradient elution. J PharmHydrochloro LosartanBiomed Anal 2006, 41, 644-8. ParametersAtenolol thiazideK9.Lande NR, Shektar BM, Kadam SS, Dhaneshwar Retention time, min2.6 5.6 9.2 Tailing factor 0.56 0.38 0.72SR. Simultaneous spectrophotometric estimation of Number ofLosartan potassium and Hydrochlorothiazide in 12672 394111927 theoretical plates tablet dosage form. Indian drugs 2000, 37, 577- Resolution6.928.43 81.10. Lastra OC, Lemus IG, Sanchez HJ, Perez RF.ConclusionDevelopment and validation of an UV derivativeThe proposed RP-HPLC method is precise, rugged, spectrophotometric determination of Losartanrobust, simple and rapid. Hence the present RP-HPLC potassium in tablets. J Pharm Biomed Anal 2003,method is suitable for the simultaneous estimation of 33, 175-80.Atenolol, Hydrochloro thiazide and Losartan K in tablet 11. Prasad CV, Parihar C, Sunil K, Parimoo P.dosage form.Simultaneous determination of amiloride HCl,hydrochlorothiazide and atenolol in combinedReference formulations by derivative spectroscopy. J Pharm1. Indian Pharmacopoeia - Addendum, The IndianBiomed Anal 1998, 17, 877-84. Pharmacopoeia Commission, 6thed Ghaziabad, 12. Ulu ST, Saglik S. Comparison of UV and Second 2010: 847, 1451, 1452. derivative spectrophotometric and HPLC methods2. Martindale, Pharmaceutical Press, 34th edition,for the determination of Losartan in tablets. Turk J 2005: 865, 933, 947. Pharm Sci 2004, 1, 165-75.3. The Merck Index, Merck & Co., Inc., 14th edition, Whitehouse Station. NJ, 2006: 859, 4781, 5583.4. Prasad CV, Parihar C, Sunil K, Parimoo P. Simultaneous determination of amiloride HCl, hydrochlorothiazide and atenolol in combined formulations by derivative spectroscopy. J Pharm Biomed Anal 1998, 17, 877-84.5. Sivakumar T, Venkatesan P, Manavalan R, Valliappan K. Development of a HPLC method for the simultaneous determination of Losartan potassium and Atenolol in tablets, Indian J Pharm Sci 2007, 69, 154-57.6. Atana ES, Altmay SA, Goger NG, Ozkanab SA, Senturk Z. Simultaneous determination of valsartan and hydrochlorothiazide in tablets by firstInt. J. Pharm & Ind. ResVol - 01 Issue - 04Oct Dec 2011