A Validated reverse phase HPLC method for simultaneous ...

E- ISSN: 2249 –1929Journal of Chemical, Biological and Physical SciencesAn International Peer Review E-3 Journal of SciencesAvailable online at www.jcbsc.orgResearch ArticleSection A: Biological ScienceA Validated reverse phase HPLC method for simultaneousdetermination of telmisartan and ramipril as bulk drugand in tablet dosage formSantosh V. Gandhi, Padmanabh B. Deshpande*, Varun Godbole, Pankaj Jagdale, SachinKhiste, Sayali KadukarDepartment of Pharmaceutical Analysis, A.I.S.S.M.S. College of Pharmacy, Kennedy Road,Near R.T.O., Pune - 411 001.Received: 30August 2011; Revised: 12 September; Accepted: 15 September 2011ABSTRACTA simple, accurate and sensitive validated Reverse Phase HPLC method for simultaneousdetermination of two-component drug mixture of Telmisartan and Ramipril in combined tabletdosage form has been developed and validated. RP-HPLC separation of drugs was carried out onJasco HPLC system with HiQ-SiL C 8 column (250 mm × 4.6 mm i.d.), using Acetonitrile: 0.01 M 1-Heptane sulphonic acid sodium salt (pH 2.8) i(60: 40, v/v) as mobile phase. Method was developedusing phenylpropanolamine hydrochloride as internal standard and detection was carried out usingUV detector at 216 nm. Telmisartan and Ramipril obeyed Beer’s law in the concentration range of10-50 µg/mL and 2.5-12.5 µg/mL, respectively. The method has been successfully applied for theanalysis of drugs in pharmaceutical formulation. Results of analysis were validated statistically andby recovery studies.Key words: Telmisartan, Ramipril, Phenylpropanolamine Hydrochloride, RP-HPLC, Tablet dosageformINTRODUCTIONTelmisartan, (TELMI), 4’ [(1, 4’-dimethyl-2’-propyl [2, 6’-bi-benzimidazol]-1’-yl) methyl][1,1’-biphenyl]-2-carboxylic acid) used in the treatment of hypertension is a selective AT1 subtypeJ. Chem. Bio. Phy. Sci. 2011, Vol.1, N0.2, Sec.B, 283-288. 283

A Validated High....Padmanabh B. Deshpande et alangiotensin II receptor antagonist. 1 Ramipril, (RAMI), (1S, 5S, 7S)-8-[(2S)-2-[[(1S)-1-ethoxycarbonyl-3-phenyl-propyl] amino] propanoyl] -8-azabicyclo [3.3.0] octane-7-carboxylic acid), isan angiotensin-converting enzyme (ACE) inhibitor, used to treat hypertension and congestive heartfailure. 2 Literature survey reveals spectrophotometric 3-4 , RP-HPLC 5-7 and HPTLC 8 methods for thedetermination of TELMI either as a single or in combination with other drugs. Several methods havebeen reported for determination of RAMI in pharmaceutical dosage form or in biological samples as asingle or in combination with other drugs include spectrophotometry 9-11 , RP-HPLC 12-16 , LC-MS 17-18 .To best of our knowledge no method has been reported for simultaneous determination ofTELMI and RAMI in combined tablet dosage form by RP-HPLC. Aim of present work was to developsimple, accurate and sensitive RP-HPLC method for simultaneous determination of TELMI and RAMIin combined tablet dosage form. The proposed method is optimized and validated as per theInternational Conference on Harmonization (ICH) guidelines 19 .EXPERIMENTALChemicals and Reagents: Pharmaceutical grade working standards TELMI, RAMI andPhenylpropanolamine Hydrochloride (PPA) were obtained from Glenmark Pharmaceuticals Ltd (Nasik,India), Lupin Ltd (Pune, India) and Healing Cross (Daman, India) respectively; used as such withoutfurther purification. Brand of tablets TELMA-R (Glenmark Pharmaceuticals Ltd., India), labeled tocontain 40 mg of TELMI and 5 mg of RAMI were procured from the local market. Acetonitrile (HPLCgrade), 1- heptane sulphonic acid sodium salt (AR grade) purchased from Merck specialties Pvt. Ltd.(Mumbai, India) and double distilled water were used in analysis.INSTRUMENTATION AND CHROMATOGRAPHIC CONDITIONSJasco HPLC system, consisting of Jasco PU-2080 plus HPLC pump, Jasco UV-2075 plusUV/VIS detector and JASCO Borwin Ver 1.50 software was used for analysis. Separation was carriedout on HiQ-SiL C 8 (250 mm × 4.6 mm i.d.) column using acetonitrile: 0.01 M 1-heptane sulphonic acidsodium salt (pH 2.8) (60: 40, v/v) as mobile phase at flow rate of 0.8 mL/min. Samples were injectedusing Rheodyne injector with 20 µl loop, PPA as internal standard and detection was carried out at 216nm. All weighing were done on Shimadzu balance (Model AY-120).Preparation of Standard Stock Solutions: Pure drug samples of TELMI and RAMI weredissolved separately in acetonitrile so as to get stock solution of 0.2 and 0.1 mg/mL, respectively. PPAstock solution (0.2 mg/mL) was prepared by dissolving 5 mg of PPA in 25 mL of acetonitrile.Procedure for Analysis of Tablet Formulation:Twenty tablets were weighed and finely powered.An accurately weighed powder sample equivalent to 10 mg of TELMI (1.25 mg of RAMI) wastransferred to 50 mL volumetric flask; 40 mL of acetonitrile was added and the flask was ultrasonicatedfor 5 min. The volume was then made up to the mark with acetonitrile and solution was filtered throughwhatman filter paper No. 41. One mL of the filtrate was transferred to 10 mL volumetric flak, 0.5 mLPPA standard stock solution was added and volume was made up to the mark using mobile phase to getfinal concentration of 20 µg/mL of TELMI (2.5 µg/mL of RAMI, 10 µg/mL of PPA as internalstandard). After setting the chromatographic conditions and stabilizing the instrument to obtain asteady baseline, the tablet sample solution was injected. Chromatogram was obtained and peak areasJ. Chem. Bio. Phy. Sci. 2011, Vol.1, N0.2, Sec.B, 283-288. 284

A Validated High....Padmanabh B. Deshpande et alwere recorded. The peak area ratio of each of the drug to the internal standard was calculated and theamount of each drug present per tablet was estimated from the respective calibration curve.METHOD VALIDATIONLinearity:For each drug different aliquots were pipetted out from standard stock solutions into seriesof 10 mL volumetric flask, 0.5 mL of PPA standard stock solution was added to each flask and volumewas made up to the mark using mobile phase. Each solution was injected and a chromatogram wasrecorded. The peak area ratios of TELMI to PPA and RAMI to PPA were calculated and respectivecalibration curves were plotted of response factor against concentration of each drug.System Suitability: The system suitability was assessed by six replicate injections of the mixturecontaining 10 µg/mL of TELMI, 10 µg/mL of RAMI and 10 µg/mL of PPA as internal standard. Theresolution, peak asymmetry, number of theoretical plates and HETP were calculated as represented inTable 1.Table 1: System suitability parameters of proposed RP-HPLC methodSr. No. Parameters TELMI RAMI PPA1 Theoretical plates 4657.61 2188.70 3134.932 Resolution 3.19 4.50 0.003 Asymmetry factor 1.78 1.35 1.78The values obtained demonstrated the suitability of the system for the analysis of these drugs incombination. Mean retention times for PPA, RAMI and TELMI were found to be 4.033 min, 5.792 minand 7.258 min, respectively. The typical chromatogram of the standard solution of mixture is shown inFigure 1.Figure 1: Representative chromatogram obtained for standard mixture of PPA (10 µg/ mL,4.033 min), RAMI (10 µg/ mL, 5.792 min), TELMI (10 µg/ mL, 7.258 min)J. Chem. Bio. Phy. Sci. 2011, Vol.1, N0.2, Sec.B, 283-288. 285

A Validated High....Padmanabh B. Deshpande et alPrecision: The precision of the method was demonstrated by inter day and intra day variation studies.To study intra-day variation, six mixed standard solutions containing TELMI (10 µg/mL), RAMI (10µg/mL) and PPA (10 µg/mL) were injected. All the solutions were analyzed on the same day to recordany intra-day variation in the results. To study inter-day variation, analysis of three mixed standardsolutions of the same concentration was performed on different days.Accuracy: To study accuracy of the proposed method, recovery studies were carried out by additionof standard drug solution to pre-analyzed sample at three different levels 50 %, 100 % and 150 %. Thepercentages of recoveries were calculated, results of which are represented in Table 2.Level of% RecoveryTable 2: Recovery studies of TELMI and RAMI% Mean Recovery ∗ Standard Deviation % R.S.D.TELMI RAMI TELMI RAMI TELMI RAMI50 99.96 100.17 0.571 0.706 0.571 0.705100 100.27 100.40 0.494 0.484 0.492 0.482150 100.11 99.87 0.563 0.462 0.563 0.462LOD and LOQLOD and LOQ were calculated as 3.3 σ /S and 10 σ /S, respectively, where σ is the standarddeviation of the response (y-intercept) and S is the slope of the calibration plot.Robustness: To determine the robustness of the developed method, slight deliberate changes inchromatographic conditions was carried out. The factors varied one at a time were change in flow rate(± 0.02 mL/min), detection wavelength (±1 nm) and ratio of the mobile phase constituents (± 2 %). Itwas observed that there were no marked changes in the chromatograms, which demonstrated that theRP-HPLC method developed is robust.Solution Stability: In order to demonstrate the stability of both standard and sample solutions, thesolutions were analyzed over a period of 5 hours at room temperature. The results show that retentiontime as well as peak area of TELMI and RAMI remained almost unchanged (% RSD less than 1.5) andno significant degradation within the indicated period was observed, this indicates that both solutionswere stable for at least 5 hours, which was sufficient to complete the whole analytical process.RESULTS AND DISCUSSIONResults were found to be linear in the concentration range of 10-50 µg/mL for TELMI and 2.5-12.5 µg/mL for RAMI with high correlation coefficient. The detection wavelength 216 nm was selectedas RAMI have negligible absorbance at higher wavelengths. The proposed method was also evaluatedby the assay of commercially available tablets containing TELMI and RAMI. The % assay was foundto be 99.77 ± 0.084 for TELMI and 99.00 ± 0.688 for RAMI (mean ± S.D., n = 6). The method wasfound to be accurate and precise, as indicated by recovery studies and % RSD not more than 1.5. Thesummary of validation parameters of proposed HPLC method is given in Table 3.J. Chem. Bio. Phy. Sci. 2011, Vol.1, N0.2, Sec.B, 283-288. 286

A Validated High....Padmanabh B. Deshpande et alTable 3: Summary of validation parameters of proposed HPLC methodParameters TELMI RAMILinearity range (µg/mL) 10-50 2.5-12.5Correlation co-efficient 0.9997 0.9989LOD a (µg/mL) 2.599 0.067LOQ b (µg/mL) 7.877 0.20Accuracy (% recovery) 99.96-100.27 99.87-100.40Precision (% RSD) cIntra day (n d =3) 0.084 1.10Inter day (n=3) 0.94 0.42a LOD = Limit of detection;b LOQ = Limit of quantitation;c R.S.D.= Relative standard deviation; d n = Number of determinationCONCLUSIONA simple, accurate and sensitive RP-HPLC method for simultaneous determination of twocomponentdrug mixture of TELMI and RAMI in combined tablet dosage form has been developed andvalidated. The method may be recommended for routine and quality control analysis of the investigateddrugs in pharmaceutical formulations.ACKNOWLEDGEMENTSThe authors are thankful to Glenmark Pharmaceuticals Ltd (Nasik, India), Lupin Ltd (Pune,India) and Healing Cross (Daman, India) for providing the pure drug samples of TELMI, RAMI, andPPA respectively.REFERENCES1. http://www.pharm-marketing.com/bulk_drug_hp_eng.htm#telmisartan (accessed 25/08/07)2. http://en.wikipedia.org/wiki/Ramipril, (accessed 25/08/07)3. M. S.Palled, M. Chatter and A. R. Bhat, Ind. J. Pharm. Sci., 2006, 68, 685.4. I. B. Lories, S. A. Saman, A. F. Laila and H. R. Heba, Farmaco II, 2005, 60, 859.5. V. Kumer and P. R. Muley, Indian Pharmacist, 2005, 6, 39.6. S. B. Wankhede, M. R. Tajne, K. R. Gupta and S. G. Wadodkar, Ind. J. Pharm. Sci., 2007, 69, 298.7. L. R. Bhat, R. K. Godge, A. T. Vora and M. C. Damle, J. Liq. Chrom. Relat. Tech., 2007, 30, 3059.8. N. J. Shah, B. N. Suhagia, R. R. Shah and P. B. Shah, Ind. J. Pharm. Sci., 2007, 69, 202-2059. A. A. Al-Majed, F. Belal and A. A. Al-Warthan, Spectrosc. Lett., 2001, 34, 211.10. H. E. Abdellatef, Spectrochimica Acta A, 2007, 66, 701.11. N. Rahman, Y. Ahmad and S. N. H. Azmi, AAPS Pharm. Sci. Tech., 2005, 6, 543.J. Chem. Bio. Phy. Sci. 2011, Vol.1, N0.2, Sec.B, 283-288. 287

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