Development and Validation of Analytical Methods for Simultaneous Estimation of Atorvastatin Calcium and Ezetimibe in Combined Dosage Form

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1 World Applied Sciences Journal 9 (2): , 200 ISSN IDOSI Publications, 200 Development and Validation of Analytical Methods for Simultaneous Estimation of Atorvastatin Calcium and Ezetimibe in Combined Dosage Form V. Rajamanickam, A. Rajasekaran, B. Stephen Rathinaraj and K. Anandarajagopal Department of Pharmaceutical Analysis, A.K.College of Pharmacy, Krishnan koil, Tamilnadu, India 2 Department of Pharmaceutical Analysis, KMCH College of Pharmacy, Coimbatore, Tamilnadu, India 3 Department of Pharmaceutical Analysis, Vaagdevi College of Pharmacy, Warangal, Andhrapradesh, India 4 School of Pharmacy, Masterskill University College of Health Sciences, Batu 9, Cheras-43200, Malaysia Abstract: A simple, rapid and precise method is developed for the quantitative simultaneous determination of Atorvastatin calcium and Ezetimibe a combined pharmaceutical-dosage forms. Three methods are described for the simultaneous determination of Atorvastatin calcium and Ezetimibe in binary mixture. The first method was based on UV-spectrophotometric determination of two drugs, using simultaneous equation method. It involves absorbance measurement at nm (ëmax of Ezetimibe) and 246 nm (ëmax of Atorvastatin calcium) in methanol; For UV spectrophotometric method, linearity was obtained in concentration range of 8 22 µg. ml, for both the drugs; with regression and , intercept and and slope and for Atorvastatin calcium and Ezetimibe, respectively. Recovery was in the range of 99 0%; the value of standard deviation and % R.S.D. were found to be < 2 %; shows the high precision of the method.. The second method was based on HPLC separation of the two drugs in reverse phase mode using these Kromasil C8, 5µ (50 X 4.6 mm). Linearity was obtained in the range of 5-30 µg.ml. The Third method was based on HPTLC method for simultaneous quantification of these compounds as the bulk drug and in tablet dosage forms. Chromatographic separation of the drugs was performed on aluminium plates precoated with silica gel 60 F 254, with Benzene: Methanol: Acetone: Triethylamine (7:2::0.2 v/v/v) as mobile phase. The two drugs were satisfactorily resolved with RF values 0.8 ± 0.0 and 0.39 ± 0.0 for atorvastatin calcium and ezetimibe, respectively. The accuracy and reliability of the method was assessed by evaluation of linearity ( ng/spot for both atorvastatin calcium and ezetimibe), precision (intra-day RSD % and inter-day RSD.00.44% for atorvastatin calcium and intra-day RSD % and inter-day RSD % for ezetimibe), accuracy (99.28 ± 0.23% for atorvastatin calcium and ± 0.5% for ezetimibe) and specificity, in accordance with ICH guidelines. Both these methods have been successively applied to pharmaceutical formulation and were validated according to ICH guidelines. Key words: Reverse phase liquid chromatography HPTLC UV Atorvastatin calcium Ezetimibe ICH. INTRODUCTION hydroxylpropyl]-4(s)-(4-hydroxyphenyl)-2-azetidinone)is another lipid-lowering agent[3]. Literature review reveals Atorvastatin calcium (Fig.) (3R,5R)-7-[2-(4- that methods have been reported for analysis of fluorophenyl)- 3-phenyl-4-(phenylcarbamoyl)-5-(propan- atorvastatin calcium by high-performance liquid 2-yl)- H-pyrrol -yl]-3,5-dihydroxyheptanoic acid, chromatography (HPLC) [4-7]and High-Performance Thincalcium salt (2:) trihydrate) is a synthetic lipid-lowering Layer Chromatography (HPTLC)]8] and for estimation of agent. It is a selective competitive inhibitor of the enzyme ezetimibe by HPLC [9], either alone or in combination with HMG-CoA reductase, which catalyses the conversion of other drugs, but no specific combined analytical methods HMG-CoA to mevalonate, an important rate-limiting step has yet been reported for simultaneous estimation of in cholesterol biosynthesis [, 2]. Ezetimibe (Fig.2) (-(4- atorvastatin calcium and ezetimibe. The purpose of this fluorophenyl)-3(r)-[3-(4-fluorophenyl)-3(s)- research was to establish and validate, in accordance with Corresponding Author: Mr. V. Rajamanicakm, Department of Pharmaceutical chemistry, A.K.College of Pharmacy, Krishnankoil, Tamilnadu, India, Tel: , steaje@gmail.com. 424

2 World Appl. Sci. J., 9 (2): , 200 Fig. : Structure of Atorvastatin Calcium obtained as generous gifts from Mepro Pharmaceuticals Wadhwan (Gujarat, India) and Hetero Drugs Erragadda (Hyderabad, India), respectively. Fixed-dose combination tablets (Atorec EZ), (Bitorva) containing 0 mg atorvastatin calcium and 0 mg ezetimibe were procured from Hetero Drugs (Solan (H.P.) India). Chemicals and reagents of analytical-grade were purchased from Merck Chemicals, Mumbai, India. All chemicals are of HPLC grade and were purchased from Qualigens fine Chemicals, Mumbai, India. Preparation of Mobile Phase and Stock Solution UV- Spectrophotometry: UV-Vis spectrophotometer 60 (Shimadzu, Japan) with spectral bandwidth of 2 nm and 0 mm matched quartz cells was used. Standard stock Fig. 2: Structure of Ezetimibe solutions of 00 µg.ml were prepared by dissolving 0 mg of each in 00 ml of methanol. From these stock solutions, working standard solutions having concentration 20 µg.ml each were prepared by appropriate dilutions. They were scanned in the wavelength range of nm and the overlain spectrum was obtained (Fig 3). Two wavelengths nm (ëmax of Ezetimibe) and nm (ëmax of Atorvastatin calcium) were selected for the formation of simultaneous equation. The calibration curves were found to be linear in the concentration range of 8-22 µg.ml, for each drug. The absorptivity coefficients of each drug at both Fig. 3: Wavelength Selection of Atorvastatin Calcium and wavelengths were determined. The concentration of two Ezetimibe. drugs in the mixture were calculated using equations, International Conference on Harmonization (ICH) CATV = A2 ay A ay2/ ax2 ay axay2 () guidelines [0], a simple, accurate, economical and reproducible procedure for quantitative analysis of CEZM = A ax2 A2 ax2/ ax2 ay axay2 (2) atorvastatin calcium and ezetimibe as the bulk drug and in tablet dosage forms. Where, A and A2 are absorbance of mixture at Both these drugs are not official in Indian nm and nm; ax and ax2, absorptivities of Pharmacopoeia, British Pharmacopoeia and United States Atorvastatin calcium at nm and nm, and European Pharmacopoeia. At present no HPLC, respectively; ay and ay2 absorptivities of Ezetimibe at HPTLC and UV spectrophotometric methods are reported nm and nm, respectively. CATV and CEZM are for the simultaneous estimation of Atorvastatin calcium concentration of Atorvastatin calcium and Ezetimibe in and Ezetimibe in tablet formulation. Therefore, it was mixture. The absorptivities reported are the mean of six thought worthwhile to develop simple, precise, accurate independent determinations (Table ). UV spectrophotometric, HPLC and HPTLC methods for simultaneous determination of Atorvastatin calcium and HPLC Method: LC system used consisted of pump (model Ezetimibe in tablets. SHIMADZU; LC 0 AT vp) with universal loop injector (Rheodyne 7725 i) of injection capacity 20 µl.detector Experimental consists of Photodiode array detector SPD Avp, Materials and Reagents: Working standards of SHIMADZU; the column used was these Kromasil C8, pharmaceutical grade atorvastatin calcium (batch no. 5µ (50 X 4.6 mm) at ambient temperature. Different ATV ) and ezetimibe (batch no. EZ ) were mobile phases were tested in order to find the best 425

3 World Appl. Sci. J., 9 (2): , 200 Table : Absorptivity Values at nm (?max of Ezetimibe) and nm (?max of Atorvastatin calcium) Absorptivity at nm Absorptivity at nm S. No Atorvastatin Calcium Ezetimibe Atorvastatin Calcium Ezetimibe Mean ax = ay = ax = ay = SD *Absorptivity values are the mean of six determinations.s.d. is Standard deviation.axand ax2 absorptivities of atorvastatin calcium at nm and nm, respectively; ay and ay2 absorptivities of ezetimibe at nm and nm, respectively. Fig. 4: Overlain Spectra of ATV and EZM conditions, for separating both the drugs simultaneously. removed from the chamber and dried in air. Chromatogram The optimal composition of mobile phase was determined was performed at 266 nm (Fig. 4), in reflectance mode, with to be Buffer ph 5.00: Acetonitrile in preparations (55:45, a Camag TLC Scanner 3 using CATS 4 software 50:50, 45:55 % v/v). The flow rate was set to ml.min incorporating track-position optimization. The slit and UV detection was carried out at 240 nm. Stock dimensions were 6.00 mm 0.45 mm. For preparation of a solution was prepared by dissolving 0 mg of calibration plot, ng/ spot standard solutions of Atorvastatin calcium and Ezetimibe in 00 ml volumetric atorvastatin calcium and ezetimibe were applied to the flask with methanol. TLC plates. From the above stock solutions, dilutions HPTLC: Analysis was performed on 20 cm 20 cm were made in the concentration range of 8 22 µg.ml of aluminium plates pre-coated with silica gel 60 F 254. Before Atorvastatin calcium and Ezetimibe, respectively. A use, plates were washed with methanol, activated in an volume of 20 µl of each sample was injected into column. oven at 05 C for 20 min, then left to cool at room All measurements were repeated three times for each temperature. Standard solutions of atorvastatin calcium concentration and calibration curve was constructed by and ezetimibe (00ng /Spot ) were applied to pre-washed plotting the peak area ratios of analyte to the activated plates, as 6-mm bands, 6 mm apart, under a corresponding drug concentration. stream of nitrogen, by means of a Camag Linomat IV automated spray-on and applicator equipped with a Analysis of Pharmaceutical Dosage Forms: To determine Hamilton 00-µL syringe. The plates were developed, with the content of Atorvastatin calcium and Ezetimibe Benzene: Methanol: Acetone: Triethylamine (7:2::0.2 simultaneously in tablets (label claim: 0 mg Atorvastatin v/v/v) as mobile phase, in a Camag twin-trough chamber calcium and 0 mg Ezetimibe, film coated); twenty tablets previously saturated with mobile phase vapour for 20 min. were weighed; their average weight determined and The development distance and time were 5 cm and 25 were finely powdered. The correct amount of min, respectively. After development the plates were powder was dissolved in methanol by stirring for 30 min. 426

4 World Appl. Sci. J., 9 (2): , 200 Table 2: Analysis data of tablet formulations UV spectrophotometry HPLC HPTLC S.No Parameters Atorvastatin Calcium Ezetimibe Atorvastatin Calcium Ezetimibe Atorvastatin Calcium Ezetimibe. Label Claim(mg) *Drug content ± S.D %R.S.D * Value for Drug content ( mg) is the mean of five estimations; S.D. is standard deviation and R.S.D. is Relative standard deviation Table 3: Recovery studies UV-Spectrophotometry HPLC HPTLC Excess drug *Recovery % R.S.D Excess drug *Recovery % R.S.D Excess drug *Recovery % R.S.D Atorvastatin Calcium Ezetimibe * Recovery is mean of three estimations The excipients were separated by filtration. Appropriate In HPLC method, HPLC conditions were optimized to aliquots were subjected to above methods and the obtain, an adequate separation of eluted compounds. amount of Atorvastatin calcium and Ezetimibe were Initially, various mobile phase compositions were tried, to determined. The results are reported in Table 2. separate drugs and internal standard. Mobile phase and flow rate selection was based on peak parameters (height, Recovery Studies: To check the accuracy of the tailing, theoretical plates, capacity factor), run time etc. developed methods and to study the interference of The system with 20 mm ammonium acetate buffer ph 5.0: formulation additives, analytical recovery experiments acetonitrile: triethylamine (50:50:02 v/v) with ml.min were carried out by standard addition method, at 80, 00 flow rate is quite robust. A typical chromatogram for and 20 % level. From the total amount of drug found, the Atorvastatin calcium, Ezetimibe is shown in Fig 4. The percentage recovery was calculated. The results are optimum wavelength for detection was 240 nm at which reported in Table 3. better detector response for drugs was obtained. The average retention times for Atorvastatin calcium and RESULTS AND DISCUSSION Ezetimibe was found to be 5.23 ± 0.03 and.822 ± 0.02 min, respectively. According to USP XXIV (62), system Both, UV spectrophotometric, HPLC and HPTLC suitability tests are an integral part of chromatographic methods were found to be simple, accurate, economic and method. rapid for routine simultaneous estimation of Atorvastatin In HPTLC method, Chromatographic separation of calcium and Ezetimibe, in tablet dosage forms. For UV the drugs was performed on aluminium plates precoated spectrophotometric method, linearity was obtained in with silica gel 60 F 254, with Benzene: Methanol: Acetone: concentration range of 8 22 µg. ml, for both the Triethylamine (7:2::0.2 v/v/v) as mobile phase. drugs; with regression and , intercept Chromatographic evaluation of the separated zones was and and slope and for performed at 266 nm. The two drugs were satisfactorily Atorvastatin calcium and Ezetimibe, respectively. resolved with RF values 0.8 ± 0.0 and 0.39 ± 0.0 for Recovery was in the range of 99 0%; the value of atorvastatin calcium and ezetimibe, respectively. The standard deviation and % R.S.D. were found to be < 2 %; accuracy and reliability of the method was assessed by shows the high precision of the method. evaluation of linearity ( ng/spot for both 427

5 World Appl. Sci. J., 9 (2): , 200 Fig. 4: Chromatogram of Standard Atorvastatin calcium (0 µg.ml ); (Rt 5.23) and Ezetimibe (0 µg.ml ); (Rt.82) measured at 240 nm, mobile phase Ammonium acetate buffer (ph 5): Acetonitrile: Triethylamine (50:50:0.2 v/v) Fig. 5: HPTLC Chromatogram obtained from Atorvastatin calcium and Ezetimibe atorvastatin calcium and ezetimibe), precision (intra-day concentrations analyzed on three different days, RSD % and inter-day RSD.00.44% for over a period of one week. These results show the atorvastatin calcium and intra-day RSD % and accuracy and reproducibility of the assay. Thus, it was inter-day RSD % for ezetimibe), accuracy (99.28 concluded that there was no significant difference ± 0.23% for atorvastatin calcium and ± 0.5% for on the assay, which was tested on an intra day and ezetimibe) and specificity, in accordance with ICH inter day basis. The % R.S.D. values reported in guidelines. They are used to verify the reproducibility of Table 5, shows that proposed methods provides the chromatographic system. To ascertain its acceptable intra day and inter day variation of effectiveness, system suitability tests were carried out on Atorvastatin calcium and Ezetimibe. Ruggedness of the freshly prepared stock solutions. The parameters obtained proposed methods was determined by analysis of aliquots are shown in Table 4. The calibration was linear in from homogeneous slot in different laboratories, by concentration range of 2 22 µg. ml, with regression different analysts, using similar operational and and , intercept 0.54 and and environmental conditions; the % R.S.D. reported in Table slope and for Atorvastatin calcium and 5 was found to be less than 2 %. The proposed methods Ezetimibe, respectively. The low values of % R.S.D. are accurate, simple, rapid and selective for the indicate the method is precise and accurate. The mean simultaneous estimation of Atorvastatin calcium and recoveries were found in the range of %. Ezetimibe in tablet dosage forms. Hence, it can be Sample to sample precision and accuracy were conveniently adopted for the routine quality control evaluated using, three samples of three different analysis in the combination formulations. As the drug concentrations, which were prepared and analyzed on combination is available in market, hence, work is toward same day. Day to day variability was assessed using three development of an analysis. 428

6 World Appl. Sci. J., 9 (2): , 200 Table 4: System suitability parameters Parameters Atorvastatin Calcium Ezetimibe Tailing Factor Theoretical Plates Resolution Factor Capacity Factor Table 5: Summary of Repeatability, precision and ruggedness UV-Spectrophotometry HPLC HPTLC Parameter Atorvastatin Calcium Ezetimibe Atorvastatin Calcium Ezetimibe Atorvastatin Calcium Ezetimibe Repeatability Precision Intra-day Inter-day Ruggedness Analyst Analyst n is the number of repetitions ACKNOWLEDGEMENT 4. Erturk, S., E. Sevinc, L. Erosy and S. Ficicioglu, J. Pharm. Biomed. Anal., 33: 07. The authors are thankful to Blue Cross Labs. Ltd. 5. Manoj, K., P. Shanmugapandiyan and (Nasik), for providing drug samples and A.K.College of S. Anbazhagan, Indian Drugs., 4: 284. Pharmacy, Krishnankoil, Tamilndau, India, for providing 6. Rajeswari, K., G. Sankar and J. Seshagirirao, facilities to carry out this work. Indian J. Pharm., Sci., 68: Havaldar, F. And V. Chaudhari, Asian J. Chem., REFERENCES 7: Yadav, S., D. Mhaske, A. Kakad and S. Dhaneshwar,. O Neil, M.J., The Merck Index, 4th edn, Indian J. Pharm., Sci., 67: 82. Merck Research Laboratories, Whitehouse Station, 9. Sistla, R., V. Tata, Y. Kashyap and D. Chandrasekar, NJ, J. Pharm. Biomed. Anal., 39: Beringer, P., Remington s-the Science and 0. ICH., Guidance on Analytical Method Practice of Pharmacy, 2st edn, Vol. II, Mack Validation. In: Proc. Int. Convention on Quality for Publishing, Easton, PA, 368. the Pharmaceutical Industry, Toronto, Canada. 3. Budawari, S., editor, 200. In, The Merck index. th 3 ed. Whitehouse Station, (NJ): Merck and Co, Inc.,