Development and validation of a dissolution test for Candesartan cilexetil in tablet forms using reverse phase High performance liquid chromatography

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1 Development and validation of a dissolution test for Candesartan cilexetil in tablet forms using reverse phase High performance liquid chromatography R Revathi* 1, T Ethiraj 2, Jhansi L. Marreddy 1, V Ganeshan 2 1 Department of Pharmaceutical Analysis, 2 Department of Pharmaceutics, The Erode College of Pharmacy and Research Institute, Erode , Tamil Nadu, India. * [email protected] Received: October 08, 2011; Accepted: November 15, 2011 ABSTRACT The aim of this study was to develop and validate a dissolution test for the quality control of candesartan cilexetil tablets, labeled as containing 8 mg of active pharmaceutical ingredient (API), using an reverse phase high performance liquid chromatography (RP-HPLC) method. After the determination of solubility, the conditions selected were paddle at 100 rpm, with 1000 ml of 1 % sodium lauryl sulphate (SLS) in water, ph adjusted to 6.8 with 3N hydrochloric acid at 37 C ± 0.5 C. Under these conditions, the in vitro release profiles of candesartan cilexetil uncoated 8 mg tablets shown good results. The drug release was evaluated by reverse phase high performance liquid chromatography method using 0.02M mono potassium phosphate: acetonitrile: triethylamine in the ratio of 40:60:02 and adjusted ph to 6 with phosphoric acid at flow rate of 1 ml/min. The method was validated for specificity, linearity, accuracy, precision and solution stability as per ICH guidelines to meet requirements for a global regulatory filing. Keywords: Dissolution testing, Paddle method, Validation, Reverse phase High performance liquid chromatography, Sodium lauryl sulphate, Candesartan cilexetil tablets. INTRODUCTION Dissolution testing has emerged in the pharmaceutical field as a very important tool to characterize drug product performance. Dissolution tests are used not only for quality control of finished products, but also to assess several stages of formulation development, for screening and proper assessment of different formulations 1. Candesartan cilexetil (Fig. 1) is chemically, 2-Ethoxy- 3-[2-(1H-tetrazol-5-yl)-4-yl methyl]-3h- benzoimidazole- 4-carboxylic acid 1-cyclohexyloxy carbonyl oxy ethyl ester. It has a molecular formula of C 33 H 34 N 6 O 6 and a molecular weight of It is practically insoluble in water and sparingly soluble in methanol 2. Fig. 1. Chemical Structure of Candesartan Cilexetil. The drug is an angiotensin II receptor (type AT1) antagonist and it acts by blocking the vasoconstrictor and aldosterone secreting effects of angiotensin II by selectively blocking the binding of angiotensin II to the AT1 receptor in many tissues such as vascular smooth muscle and the adrenal gland 3. It is used to treat hypertension either alone or in combination with other antihypertensive agents. Few literatures revealed that the RP-HPLC and High Performance Thin Layer Chromatography (HPTLC) method for the estimation of candesartan cilexetil in solid dosage forms Quantitation and dissolution studies were validated with RP-HPLC method for some pharmaceutical dosage forms The method for determination of candesartan cilexetil in solid dosage forms and its dissolution profiles are available only in United States Food and Drug Administration (USFDA) guidelines and United States Pharmacopoeia (USP) 35, National Formulary (NF 30). When compared to USP method, the preparation of dissolution medium in the proposed method is simple and 71

2 less expensive. USFDA guidelines also recommended that the use of surfactant like SLS is suitable for dissolution of water insoluble or sparingly water soluble drug products. No other method validations have been reported for dissolution of candasartan cilexetil in solid dosage forms, so following experiment was performed. This present study describes the development and validation of a simple dissolution test for 8 mg candesartan cilexetil tablets, which was optimized on the basis of solubility and stability considerations. Material and Methods Instrumentation Dissolution test was conducted using an Electro lab TDT-14L dissolution tester using USP Apparatus at a temperature of 37 C ± 0.5 C. A model Agilent Technology-1100 series liquid chromatography equipped with G1311A Quat pump, G1329A Auto injector, G1379A Degasser, G1316A Column oven, G1314 A multi wavelength detector, SCL-10AVP system controller and chem. station manager system software were used. Reagents and materials Milli-Q water was used for preparing both dissolution media and HPLC mobile phase. All other reagents and chemicals were of analytical or HPLC grade. Candesartan cilexetil reference substance (assigned purity, 99.8 %). Tablets containing candesartan cilexetil (8 mg) of brand Ranbaxy Laboratories Ltd., were purchased from the local market. Chromatographic conditions Chromatography was achieved on a C 18 (Prodigy, 150!4.6 mm, 5µ) column. The mobile phase was a mixture of 0.02 M mono potassium phosphate buffer: acetonitrile: triethyl amine in the ratio of 40:60:0.2 and adjusted the ph to 6.0 with phosphoric acid which was filtered (0.45 µm) and degassed before use. All analysis was performed at room temperature at a flow rate of 1 ml/min. Detection was made at 254 nm. Triplicate of 20 µl injections were utilized for each analysis. Dissolution Test Conditions The solubility study and percentage drug release was determined in 1000 ml of 0.1 M hydrochloric acid, sodium phosphate buffer ph 7.0 and 1 % aqueous SLS solution, (ph adjusted to 6.8 with 3N hydrochloric acid). Drug release tests were carried out with paddle method (USP 72 apparatus II) at 75 rpm and 100 rpm. The temperature of the cell was maintained at 37 C ± 0.5 C by using a thermostatic bath. Sampling aliquots of 5.0 ml were withdrawn at 5, 10, 15, 20 and 30 min and replaced with an equal volume of the fresh medium to maintain a constant total volume. After the end of each test time, sample aliquots were filtered and quantified. The percentage content was calculated by validated RP-HPLC method and these contents results were used to calculate the percentage release on each time of dissolution profile. The cumulative percentage of drug released was plotted against time in order to obtain the release profile. Method Validation The dissolution tests were validated to candesartan cilexetil tablets through the determination of specificity, linearity, precision, accuracy and solution stability 17,18. Prior to injecting sample solutions, the column was equilibrated for at least 30 min with the mobile phase flowing through the system. System suitability tests were carried out by making six replicate injections of a standard solution containing 8 µg/ml of candesartan cilexetil and analyzing the chromatograms for candesartan cilexetil peak area, theoretical plates and tailing factor. Specificity The dissolution test specificity was evaluated by preparing sample placebo of the commercial formulation of tablets. These samples were transferred to separate vessels with 1000 ml of dissolution medium at 37 C ± 0.5 C and stirred for 45 min at 100 rpm using a paddle (USP apparatus 2). Aliquots of these solutions were withdrawn, filtered through 0.45 µ membrane filters and analyzed by the RP-HPLC method using candesartan cilexetil standard solution of 8 µg/ml. Linearity To assess the linearity, 50 % % level of concentrated solutions were prepared and standard curve of candesartan cilexetil were constructed, by plotting the concentration (µg/ml) against peak area. The calculation of regression line was employed by the method of least squares. Precision The precision of the method was determined by measuring the intra-day precision and the inter-day

3 precision, both expressed as % RSD. Candesartan cilexetil tablets were subjected to dissolution test conditions (1000 ml of dissolution medium pre-heated at 37 C ± 0.5 C, paddle with stirring rate of 100 rpm, 45 min) in the same day (intra-day precision) and in two different days by different analysts (inter-day precision). Accuracy The accuracy was evaluated for the proposed method by adding known amount of candesartan cilexetil standard drug (50%, 100%, 150% level) to the tablet powder, which were subjected to dissolution test conditions described above. Each solution was analysed in triplicate. The accuracy was calculated as the percentage of the drug recovered from the formulation matrix. Solution stability The solution stability was analyzed over a specified period of time, verifying the response of the sample solution stored at bench top condition (25 C) and refrigeration (5 C). The chromatograms obtained by the RP-HPLC method from freshly prepared solution were compared. Results and Discussion Solubility determination and dissolution test condition When dissolution test is not defined in the monograph of the dosage form, comparison of drug dissolution profiles is recommended on three different dissolution media, in the ph range of The selection of a dissolution medium may be based on the solubility data and dosage range of the drug product. Hydrochloric acid, phosphate buffer and purified water are typical mediums used for dissolution test and these mediums were evaluated. Candesartan cilexetil was insoluble in aqueous medium. For poorly soluble drugs, a percentage of surfactant can be used to enhance drug solubility and it is also recommended by USFDA. Then different concentrations of SLS (0.5 %, 0.75 % and 1 %) were prepared in purified water and used for dissolution study. At 75 rpm, the cumulative percentage drug release was considerably less than that at 100 rpm in above said dissolution medium. It was observed that less than 75 % of drug was dissolved at 30 min in hydrochloric acid and phosphate buffer at a speed of both 75 rpm and 100 rpm (Fig. 2). Fig. 2. Dissolution profiles of candesartan cilexetil in different dissolution medium at 75 rpm and 100 rpm. In 0.1 N hydrochloric acid medium, the drug was not completely soluble; some of the drug particles were detected at the bottom of the dissolution vessel. But at the end of the 45 min, the drug was completely soluble in phosphate buffer ph 7. The cumulative percentage drug release obtained was low when compared to 1 % SLS dissolution medium. At the end of 45 min the 1 % SLS medium showed 100 % cumulative drug release at 75 rpm as well as 100 rpm level. But the candesartan cilexetil drug was completely released from its formulation at the end of the 30 min at a speed of 100 rpm. In 0.5 %, 0.75 %, and 1 % SLS solutions showed more than 87 % of drug release with in 30 min (Fig. 3). But in 1 % SLS medium, % drug dissolved was nearly 100 % with in 20 min at stirring rate of 100 rpm. The cumulative percentages of drug in all the above said solutions were tabulated in Tables 1 and 2. The analysis of variance showed no significant difference between the results obtained at 75 rpm and 100 rpm (p < (or) > 0.05). Fig. 3. Dissolution profiles of candesartan cilexetil in different concentrations of SLS solution at 75 rpm and 100 rpm. 73

4 Table. 1. Cumulative percentage drug release in different media. Time, 75 rpm 100 rpm min 0.1 N HCl Buffer ph 7 1 % SLS solution 0.1 N HCl Buffer ph 7 1 % SLS solution N HCl = 0.1 Normality Hydro Chloric Acid, SLS = Sodium Lauryl Sulphate, rpm = Revolution per minute, min = minute Table. 2. Cumulative percentage drug release in different media. Time, 75 rpm 100 rpm min 0.5% SLS 0.75% SLS 1% SLS 0.5% SLS 0.75% SLS 1% SLS N HCl = 0.1 Normality Hydro Chloric Acid, SLS = Sodium Lauryl Sulphate, rpm = Revolution per minute, min = minute However, it was observed that % drug release was high in 1 % SLS solution at 100 rpm. Based on these results, the selected conditions for dissolution test of candesartan cilexetil tablets were 1000 ml of 1 % SLS solution in water, ph adjusted to 6.8 with 3 N hydrochloric acid using paddle apparatus at stirring rate of 100 rpm. In the present study, the % drug dissolved for candesartan cilexetil tablet was > 60 % in 30 min in all the condition. But % drug release showed > 85 % within 30 min for 0.5 %, 0.75 %, and 1 % SLS and the suggested acceptance criteria could be 85 %. The specificity of the dissolution test was evaluated through the analysis of placebo tablets from a dissolution test using the HPLC method (Fig. 4). The specificity test by HPLC demonstrated that the excipients from tablets do not interfere in the drug peak. Thus, the HPLC method is useful to quantify candesartan cilexetil in pharmaceutical formulation by comparing standard drug with sample (Fig. 5 and 6). The stability of candesartan cilexetil was satisfied up to 24 hours at bench top condition (25 C) and refrigeration (5 C) and results were shown in Table 3. Fig. 4. Chromatogram for placebo (Specificity study) 74 Fig. 5. Chromatogram for candesartan cilexetil standard drug

5 The precision results of the dissolution method were evaluated by analyzing intra-day precision and inter-day precision (Table 5). The % RSD values were obtained at 0.3 % for intra-day precision and 0.5 % for inter-day precision. % RSD values not more than 2 % indicated the good precision of the method. Table. 5. Precision of the assay method: intra-day precision and interday precision. Fig. 6. Chromatogram for sample preparation Table. 3. Stability study data. Time,h Bench top (at 25ºC) Mean % Assay Refrigerator (at 5ºC) Initial 98.7 % 96.3 % % 96.5 % % 96.1 % h = hour Linearity of the method was evaluated at five concentration levels by diluting the standard stock solution to give solutions in the range of ìg/ml. The calibration curve for candesartan cilexetil was prepared by plotting the graph with area versus concentration. Calibration data for candesartan cilexetil was shown in Table 4. The representative linear equation was Y=321.1X and correlation coefficient for candesartan cilexetil. Linearity observed in the expected concentration range demonstrated the suitability of the method for analysis. This indicated that the method is linear in the specified range for the analysis of candesartan cilexetil in solid dosage form. S.No Intra-day precision Inter-day precision % Assay % Assay Mean %RSD The recovery experiments were carried out by the standard addition method (Fig.7 and 8). The method was found to be accurate with % recovery of 98.71% % and has found with acceptable % RSD of not more than 2% at each level. The recoveries obtained by the dissolution method for candesartan cilexetil were shown in Table 6. Table. 4. Regression analysis of calibration graphs for candesartan cilexetil. Parameters Candesartan cilexetil Linearity range (µg/ml) 4-12 Correlation co-efficient (R 2 ) Regression equation Y=321.1X Slope Intercept µg/ml = micro gram/milli litre Fig. 7. Accuracy 50 % level 75

6 Table 6. Accuracy data for the dissolution method. Concentration of Amountadded Amountfound % Recovery* % RSD spike level (mg/ml) (mg/ml) % % % % % % *(n=3), mg/ml = milli gram/milli litre, %RSD= Percentage Relative Standard Deviation REFERENCES 1. The United States Pharmacopoeia. 31 st Ed., United States Pharmacopoeial Convention. Rokville, O Neil MJ. The Merck Index. 14 th Ed., Merck & Co, Inc. Whitehouse Station. NJ. 2006; Laurence LBJ. Goodman and Gilman s. The Pharmacological basis of therapeutics. 11 th Ed. Medical Publishing division (McGraw-Hill). 2006; Conclusions Fig. 8. Accuracy 150 % level The dissolution test developed and validated for candesartan cilexetil tablets was considered satisfactory. The conditions that allowed the dissolution determination were 1000 ml of 0.1 % SLS (ph 6.8) at 37 C ± 0.5 C, paddle apparatus, 100 rpm stirring speed and filtration with quantitative filter. In these conditions, the candesartan cilexetil was more stable. It can be concluded that the proposed method was fully validated and it was found to be simple, sensitive, accurate, precise, reproducible and relatively inexpensive and they gave an acceptable recovery of the analyte. Hence, the developed method can be recommended for routine quality control analysis of candesartan cilexetil in tablet formulation. 4. Ganesh A, Kandikonda S, Saikumar B, Rasapally RK, Santhosh KK.RP-HPLC method development and validation of candesartan cilexetil in bulk and their pharmaceutical dosage forms. Inter J Pharml Sci Res. 2010; 1(12): Kamalakkannan V, Puratchikody A, Masilamani K, Saraswathy T. Analytical method development and validation for Candesartan Cilexetil as bulk and pharmaceutical dosage forms by HPLC. Der Pharmacia Lett. 2011; 3(3): Patil BS, Rao Raghavendra NG, Jadhav S, Kulkarni U and Gada MM. Estimation of Candesartan Cilexetil in bulk and tablet dosage forms by UV spectrophotometric method. Inter J Res Ayurveda Pharmacy. 2011; 2(1): Patel J, Dave JB, Patel CN and Patel D. Q-Analysis Spectrophotometric methods for estimation of Candesartan Cilexetil and Hydrochlorothiazide in tablet dosage form. J Chem Pharma Res. 2010; 2(3): Balamuralikrishna K and Syamasundar B. Development and validation of High Performance Liquid Chromatographic method for the simultaneous estimation of Candesartan Cilexetil and Hydrochlorothiazide in combined tablet dosage form. Der Pharma Chemica. 2010; 2(6):

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