Research Article Available online at www.ijpras.com Volume 3, issue 3 (2014):33-40 ISSN 2277-3657 International Journal of Pharmaceutical Research & Allied Sciences Validated RP-HPLC Method for Simultaneous Determination of Telmisartan and Hydrochlorothiazide in Pharmaceutical Formulation 1 K. Megala1, A. Chenthilnathan1* and A. Sathish babu2 Department of Pharmaceutical Chemistry, Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India 2 The Madras Pharmaceuticals, Chennai – 600 096, Tamil Nadu, India Email: ala.chenthil@gmail.com Subject: Analytical chemistry Abstract This paper deals with a simple, feasible and sensitive reverse-phase high-performance liquid chromatographic method for the simultaneous determination of Telmisartan and Hydrochlorothiazide in bulk and in pharmaceutical formulation. The chromatography was carried out by using HPLC system (Shimadzu LC2010HT) with UV- Visible dual absorbance detector (PDA), using Inertsil 250 x 4.6 mm 5µm packing L11 column. The mobile phase consisting of buffer and mixture, acetonitrile and methanol in the ratio of (50:50) [adjust pH to 3.0 with ortho phosphoric acid] and it was flowed at 1.2 ml/min. The chromatographic detection was made at 298 nm for telmisartan and 270 nm for hydrochlorothiazide. The proposed method was validated by parameters such as suitability, specificity, linearity, accuracy and precision over a linearity range 50–150 µg/ml and stability according to the ICH guidelines (r > 0.9990). The retention times of telmisartan and hydrochlorothiazide were found to be 18.43 min and 8.11 min respectively. Hence, the method could be successfully applied for routine analysis of telmisartan and hydrochlorothiazide in pharmaceutical formulation. Keywords: Telmisartan, Hydrochlorothiazide, Pharmaceutical formulation, RP-HPLC, Validation. Introduction Telmisartan (Fig.1) chemically1, 4'-[(1, 4-dimethyl2'-propyl [2, 6’-1H-benzimidazol]-1'-yl) methyl][1, 1’-biphenyl]-2-carboxylic acid, is a non peptide molecule under the class of angiotensin II receptor antagonist. It is used for the treatment of essential hypertension as alone or in combination with other agents 2. Hydrochlorothiazide (Fig.2) chemically 3, 6-chloro-3, 4-dihydro-2H-1, 2, 4-benzothiadiazine7-sulfonamide 1, 1-dioxide, is a widely used thiazide diuretic. The combination of telmisartan and hydrochlorothiazide is useful mainly in the treatment of mild to moderate hypertension 4. Figure 1: Structure of Telmisartan Figure 2: Structure of Hydrochlorothiazide Literature survey revealed that few analytical methods such as HPTLC 5, LC-MS 6, spectrofluorimetry 7, capillary electrophoresis 8, spectrophotometry 9 and HPLC 10, have been reported for telmisartan as alone and in combination with hydrochlorothiazide. In this present work, an attempt was made to develop a simple, feasible and sensitive reverse-phase highperformance liquid chromatographic method for the quantitative determination of Telmisartan and Hydrochlorothiazide in bulk drug and in pharmaceutical dosage forms. The proposed method was validated according to ICH guidelines. 33 Available online at www.ijpras.com Materials and Methods Experimental Chemicals and reagents Acetonitrile of HPLC grade and methanol were purchased from E.Merck (India) Ltd., Mumbai. Orthophosphoric acid of AR grade was obtained from Qualigens Fine Chemicals Ltd., Mumbai. Telmisartan and Hydrochlorothiazide were a gift sample by The Madras Pharmaceuticals, Chennai – 600 096, Tamil Nadu, India. The commercially available tablets containing Telmisartan and Hydrochlorothiazide were procured from the local market. Instrumentation and chromatographic conditions The chromatography was carried out by using HPLC system (Shimadzu LC2010HT) with UVVisible dual absorbance detector (PDA), using Inertsil 250 x 4.6 mm 5-µm packing L11 column. The mobile phase consisting of acetonitrile and methanol in the ratio of (50:50) [adjust pH to 3.0 with ortho phosphoric acid] and filtered through 0.45µ membrane filter before use, degassed and were pumped into the column at a flow rate of 1.2 ml/min. The chromatographic detection was made at 298 nm for telmisartan and 270 nm for hydrochlorothiazide. The volume of injection loop was 20 µl prior to the injection of the drug solution; the column was equilibrated for at least 15 min. with the mobile phase following through the system. Sample Preparation Twenty tablets were randomly selected and transferred into 500 ml volumetric flask. 25ml of 0.1N sodium hydroxide solution was added and shake until the tablets have completely disintegrated. Methanol (80% of the total volume of the flask) was added, Sonicated for 10 minutes and stirred vigorously for 30 minutes. Allowed to cool to room temperature, diluted with methanol to volume and mixed well. The concentration of the sample stock solution is about 1.6mg/ml of Telmisartan and 0.5 mg/ml of Hydrochlorothiazide. A portion of the solution was centrifuged at 4000 RPM. 10 ml of above solution was taken in 50 ml volumetric flask and make up the volume with 1:1 solution of buffer and solution contains acetonitrile: methanol (1:1). (320 µg/ml for Telmisartan and 100 µg/ml for Hydrochlorothiazide) Calculation The amount of telmisartan and hydrochlorothiazide present in each tablet formulation was calculated by using the formula: Spl. area ---------- × Std. area Std.dil Purity Avg.wt -------- × -------- × ---------- × 100 Spl.dil 100 L.C The proposed method was validated by parameters such as suitability, specificity, linearity, accuracy and precision (repeatability & reproducibility) and stability according to the ICH guidelines. Preparation of standard stock solution: Results and Discussion Accurately weighed about 320 mg of telmisartan WS and 100 mg of hydrochlorothiazide WS in to a clean 100 ml volumetric flask. 20 ml of standard stock solution (12.50mg of Benzothiadiazine related compound A USP in 500ml of diluents) was added. The volume was finally made up with diluents. 10 ml of above resulting solution was taken in 100 ml volumetric flask and make up the volume with a 1:1 solution of buffer and solution contains acetonitrile : methanol (1:1). (Telmisartan 0.32mg/ml, Hydrochlorothiazide 0.1mg/ml and Benzothiadiazine related compound A 0.5 µg/ml) All of the analytical validation parameters for the proposed method were determined according to International Conference on Harmonization (ICH) guidelines 11 . System Suitability It is essential for the assurance of the quality performance of chromatographic system. Five injections of standard drug solutions, Telmisartan and Hydrochlorothiazide were given separately to the system. The mean area, standard deviation and %RSD were calculated for the standard drug solutions and mentioned in Table 1and 2. It was observed that all the values are with in the limits. 34 Available online at www.ijpras.com Table 1: System suitability for Telmisartan S.No. 1. 2. 3. 4. 5. Standard Standard -1 Standard -2 Standard -3 Standard -4 Standard -5 Peak area response 16068395 15994039 16129546 16034289 16069044 System suitability parameters Number of Retention time theoretical plates (min) 18.606 85986 80611 18.602 80639 18.603 18.617 78931 18.612 88691 Mean 18.608 Standard deviation 0.0063 RSD in % 0.03 Table 2: System suitability for Hydrochlorothiazide S.No. 1. 2. 3. 4. 5. Standard Standard -1 Standard -2 Standard -3 Standard -4 Standard -5 Peak area response 6289389 6293528 6309963 6319474 6278727 Specificity The specificity of the HPLC method is illustrated in Fig. 3, where a complete separation of Telmisartan and Hydrochlorothiazide were noticed in presence of other inactive excipients used in tablets. In addition, there was no any interference at the retention time of in the chromatogram of placebo solution. In peak purity analysis with PDA, purity angle was always less System suitability parameters Number of Retention time theoretical plates (min) 8.666 6247 8.700 6464 8.653 6416 6454 8.654 6608 8.664 Mean 8.667 Standard deviation 0.0191 RSD in % 0.22 than purity threshold for the analyte. This shows that the peaks of analyte were pure and excipients in the formulation does not interfere the analyte. The data were presented in the Table 3 and 4. It was observed that all the values are within the limits. Table 3: Specificity for Telmisartan S.No. 1. 2. 3. 4. Name Blank Placebo Standard Sample No. of Injections 1 1 1 1 Area Nil Nil 16129546 16129550 Table 4: Specificity for Hydrochlorothiazide S.No. 1. 2. 3. 4. Name Blank Placebo Standard Sample No. of Injections 1 1 1 1 Area Nil Nil 6289389 6289392 35 Available online at www.ijpras.com Figure 3 : Typical HPLC Chromatogram of Sample Tablets (Telmisartan and Hydrochlorothiazide) Linearity The Linearity of this method was determined at five levels from 50%– 150% of operating concentrations for Telmisartan and Hydrochlorothiazide and it was shown in Table 5 and 6 . The plots of peak area of each sample against respective concentrations of Telmisartan and Hydrochlorothiazide were found to be linear (Figure 4 and 5) in the range of 50%– 150% of operating concentrations. Beer’s law was found to be obeyed over this concentration range. The . linearity was evaluated by linear regression analysis using least square method. The regression equations were found to be Y= 46366x+1E+06 and Y= 61940x+57848, for Telmisartan and Hydrochlorothiazide respectively and correlation coefficient of the standard curves were found to be 0.9995 and 1.000 for Telmisartan and Hydrochlorothiazide respectively. It observed that correlation coefficient and regression analysis are within the limits. Table 5: Linearity of response for Telmisartan S.No Target Level (%) 1. Linearity Level Linearity -1 2. 50 Concentration (µg/ml) 160 Area 8398067 Linearity -2 75 240 12167558 3. Linearity -3 100* 320 16034230 4. Linearity -4 125 400 19730215 5. Linearity -5 150 480 23163318 Table 6: Linearity of response for Hydrochlorothiazide S.No Target Level (%) 1. Linearity Level Linearity -1 2. 50 Concentration (µg/ml) 25 Area 1614164 Linearity -2 75 37.5 2370008 3. Linearity -3 100* 50 3156705 4. Linearity -4 125 62.5 3925706 5. Linearity -5 150 75 4707534 * Operating concentration 36 Available online at www.ijpras.com y = 46366x + 1E+06 R2 = 0.9995 25000000 Area 20000000 15000000 10000000 5000000 0 0 100 200 300 400 500 600 Concentration (µg/ml) Figure 4: Linearity of response for Telmisartan Figure 5: Linearity of response for Hydrochlorothiazide Accuracy Accuracy of the method was found out by recovery study by standard addition method. The known amounts of standards, Telmisartan and Hydrochlorothiazide were added to pre-analysed samples at a level from 80% up to 120% and then subjected to the proposed HPLC method individually. The results of recovery studies were shown in Table 7 and 8. It was observed that the mean percentage recoveries were found to be for Telmisartan and Hydrochlorothiazide which demonstrated that the method was highly accurate. Table 7: Accuracy for Telmisartan S.No. 1. 2. 3. 4. 5. 6. 7. 8. 9. Target level 80% 80% 80% 100% 100% 100% 120% 120% 120% Mean Standard deviation RSD % Area 13694177 13751455 13729102 16881622 17236505 16804965 19734447 19856807 21008516 Drug Recovery (%) 101.59 101.81 101.35 100.19 101.71 99.35 98.58 98.57 101.66 100.53 1.380 1.37 37 Available online at www.ijpras.com Table 8: Accuracy for Hydrochlorothiazide S.No. 1. 2. 3. 4. 5. 6. 7. 8. 9. Target level 80% 80% 80% 100% 100% 100% 120% 120% 120% Mean Standard deviation RSD % Area 4898396 4884222 4884780 6349232 6472426 6301825 7370832 7464171 7768496 Precision The precision of an analytical procedure expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the homogenous sample under the prescribed conditions. Drug Recovery (%) 99.34 98.75 99.51 100.87 100.50 100.51 99.32 100.68 101.28 100.08 0.8663 0.87 Reproducibility Examines the precision between laboratories and is often determined in collaborative studies. Reproducibility data for Telmisartan and Hydrochlorothiazide were shown in Table 9 and 10. This indicated that method was highly precise. Table 9: Precision - Reproducibility for Telmisartan S.No. Sample Name Area 1. Sample -1 15771133 2. Sample -2 15883503 3. Sample -3 16017056 4. Sample -4 15588983 5. Sample -5 15756517 6. Sample -6 15910440 Mean Standard deviation RSD % Drug Recovery (%) 99.26 99.96 100.80 100.00 99.16 100.13 99.88 0.6053 0.61 Table 10: Precision - Reproducibility for Hydrochlorothiazide S.No. Sample Name 1. Sample -1 2. Sample -2 3. Sample -3 4. Sample -4 5. Sample -5 6. Sample -6 Mean Standard deviation RSD % Area 5938671 5927322 5960768 5925739 5952645 5910516 Drug Recovery (%) 98.58 98.39 98.95 98.37 98.81 98.11 98.535 0.3094 0.31 38 Available online at www.ijpras.com Stability Standard and sample solutions to be used in the analytical method were scrutinized for their solution’s stability. This study was performed by injecting standard and sample solution for the period of 24 hours and results were presented in the Table 11 and 12. It was found that there were no remarked changes in the system suitability parameters. Table 11: Stability results obtained for Telmisartan standard solution Condition Average standar response Response Average Response %Recovery % Difference from initial 15739257 Initial 16059062 12th hr 16469413 24th hr 15850204 15803009 16300189 16315144 15834255 15815221 15771133 99.26 - 16307666 99.65 0.39 15824738 100.05 0.79 Table 12: Stability results obtained for Hydrochlorothiazide standard solution Condition Average standar response Response Average Response %Recovery % Difference from initial 5938578 Initial 6298216 12th hr 6533690 24th hr 6271969 5938765 6031753 6015389 5862660 5860338 Conclusion The Proposed study describes a simple, feasible and sensitive reverse-phase high-performance liquid chromatographic method for the quantitative determination of Telmisartan and Hydrochlorothiazide in bulk and in pharmaceutical formulations. The method was validated as per ICH guidelines and found to be simple, sensitive, accurate and precise. Therefore the proposed method can be successfully used for the routine analysis of Telmisartan and Hydrochlorothiazide in solid dosage form without interference. 98.58 - 6023571 98.96 0.38 5861499 98.08 0.50 “Cite this article” K. Megala, A. Chenthilnathan, A. S. Babu “Validated Rp-Hplc Method for Simultaneous Determination of Telmisartan And Hydrochlorothiazide In Pharmaceutical Formulation” Int. J. of Pharm. Res. & All. Sci. 2014;3(3):33-40 References 1. 2. Acknowledgements The authors are thankful to the management of The Madras Pharmaceuticals, Chennai – 600 096, Tamil Nadu, India for providing the necessary facilities to carry out for the research work. 5938671 3. 4. Indian Pharmacopoeia; 1996. p. 371-372. Sharpe MB. Jarvis, and Karen L. Telmisartan a review of its use in hypertension. Drugs. 2001; 61(10): 1501–29. Indian Pharmacopoeia; 2010. p. 2187-2188. Rane VP, Sangshetti JN and Shinde DB. 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