Simple, accurate, precise and economical HPLC method was developed for estimation of ezetimibe in tablet dosage form. Liquid chromatography method was extensively validated for linearity, range, accuracy and precision (intermediate precision, repeatability)
Simple, accurate, precise and economical HPLC method was developed for estimation of ezetimibe in tablet dosage form. Liquid chromatography method was extensively validated for linearity, range, accuracy and precision (intermediate precision, repeatability)
Simple, accurate, precise and economical HPLC method was developed for estimation of ezetimibe in tablet dosage form. Liquid chromatography method was extensively validated for linearity, range, accuracy and precision (intermediate precision, repeatability)
Article History: Int.J.Ph.Sci, May-August 2009; 1(1):176-181 Received on: 17-08-09 Available online from: www.ijps.info Accepted on: 07-09-09 Copyright 2009 www.ijps.info
VALIDATED RP- HPLC METHOD FOR ESTIMATION OF EZETIMIBE IN DIFFERENT TABLET DOSAGE FORM
PRABHAT K. SHRIVASTAVA 1* , PAWAN K. BASNIWAL, SUSHANT K. SHRIVASTAVA 1 , DEEPTI JAIN 2 ,
1 Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi (U.P.) -221 005 2 School of Pharmaceutical Sciences, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Airport By pass Road, Gandhi Nagar, Bhopal (M.P.) 462 036. L.B.S. College of Pharmacy, Tilak Nagar, Jaipur 302 004, Rajasthan ABSTRACT Simple, accurate, precise and economical HPLC method was developed for estimation of ezetimibe in tablet dosage form. To achieve the separation, the mixture of solvent acetonitrile and methanol in ratio of 50:50 v/v was selected as mobile phase. This mixture was found to be appropriate allowing good separation of the ezetimibe at retention time 4.9 minutes at flow rate of 1.0 ml/min and detection wavelength 245.0 nm. The linearity was found in the concentration range 0 50.0 !g/ml. Linearity was determined by the linear regression equation for the drug and correlation coefficient was found to be 0.998 indicating good linearity. The liquid chromatography method was extensively validated for linearity, range, accuracy and precision (intermediate precision, repeatability). All these analytical validation parameters were observed and the RSD was found to be less than one, which indicates the validity of method. Key-words- Reverse Phase High Performance Liquid Chromatography (RP-HPLC); Ezetimibe; Method Validation; ICH Guidelines
INTRODUCTION Ezetimibe, 1- (4-fluorophenyl)-3 (R) [3 (4 fluorophenyl) -3 (S) hydroxyl propyl]- 4 (S) (4 hydroxyphenyl)-2- azetidione 1 , a new lipid-lowering agent, which inhibits the absorption of cholesterol from * For Correspondence Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi, U.P., India-221 005 Phone No- 0542-2307049, Fax No- 0542-2368428 E-mail: sushant_itbhu@rediffmail.com sprabhats@rediffmail.com
intestine 2-3 . Literature survey reveals that few chromatographic methods are reported for estimation of ezetimibe in tablet dosage from 4-5 . The objective of present communication is to developed simple, rapid (less run time), and economical in case of mobile phase composition, precise RP-HPLC method for estimation of ezetimibe in tablet dosage form.
Prabhat K Shrivastava et al Int.J.Ph.Sci, May-August 2009; 1(1):174-181 www.ijps.info 177 EXPERIMENTAL Materials and method Ezetimibe working standard was obtained as generous gift from Torrent Pharmaceuticals, Baroda. Ezetimibe tablets were procured from market [Zetia Torrent Pharmaceutical, Ezta Zydus Cadila and Ezedoc Pinnacle Lupin]. HPLC grade acetonitrile and methanol were obtained from Merck (India) Limited. Instrumentation and chromatograph The HPLC system consisted of a solvent delivery module LC-10ATvp Shimadzu Liquid chromatograph pump equipped with 20 !l loop and model SPD M10Avp Shimadzu UV/VIS diode array detector. Integration was achieved by using the software LC-10 7
. Separation was carried out on a Phenomenex, (250x4.6mm) Luna 5! C-18 (2) 100A column. The chromatographic analysis was performed at ambient temperature. The mobile phase consisted of the mixture of solvents, acetonitrile: methanol in the ratio 50:50; v/v. The prepared mobile phase was filtered through ultipore N
66 nylon, 6,6 membrane (0.45!m) filter and ultrasonically degassed prior to use. The detection wavelength was set 245.0 nm and the peak area was recorded using chromatographic data system. The flow rate and runtime was set to 1.0 ml/min and 10.0 minute respectively. Preparation of solution All the system suitability parameters capacity factor, plate number, tailing factor and retention time was optimized by freshly prepared standard solution of 10.0 !g/ml. To develop a suitable method accurately weighed 10.0 mg of ezetimibe working standard was transferred into 50 ml volumetric flask, dissolved in 5.0 ml of mobile phase than volume was made up to 50.0 ml with mobile phase to get concentration of solution 200 !g/ml (Stock-A). The 12.5 ml volume of stock-A was taken and diluted up to 25.0 ml to get concentration of 100 !g/ml (Stock-B). Further dilution were made from stock-B to get a series of concentration 10, 20, 30, 40, and 50 !g/ml and before the injection the solution was filter through 0.45 micron HPLC filter and subjected for analysis. Peak area under the curve of mixed standard were observed and plotted against respective concentration. The representative chromatogram of ezetimibe was given in figure no. 1. Prabhat K Shrivastava et al Int.J.Ph.Sci, May-August 2009; 1(1):174-181 www.ijps.info 178
Figure-1 HPLC chromatogram of Working Standard of Ezetimibe
Method validation The LC Method for estimation of ezetimibe was extensively validated as per ICH guideline for "analytical method validation", for linearity, range, stability, accuracy and precision (repeatability and intermediate precision) 8 . Different system suitability parameters were observed is retention times, capacity factor, tailing factor and plate number. For linearity, series of dilutions were prepared and response ratios were determined respectively. The range was determined by preparing a series of dilutions from 80 % to 120 % of test concentration in six replicate. For the stability of the drugs, solutions were stored at room temperature and analysed on HPLC at different time intervals. Accuracy of methods was determined by recovery studies in which known amounts of standard drug were added to the previously analysed tablet sample and mixture were analysed by the proposed method. Precision was studied for repeatability and intermediate precision (days, and analysts). TABLE 1:
RESULTS OF SYSTEM SUITABILITY PARAMETER* Retention time (min) Capacity Factor Plate Number Tailing Factor 4.959 0.86 8913 1.24 *Mean values of six replicates
Prabhat K Shrivastava et al Int.J.Ph.Sci, May-August 2009; 1(1):174-181 www.ijps.info 179 Tablet analysis For analysis of tablet sample, twenty tablets were accurately weighed and than average weight of the tablet were determined. The tablets were powdered and powder equivalent to 10.0 mg of ezetimibe accurately weighed and dissolved in mobile phase and volume was made-up to 50 ml. Resulted solution was sonicated for 10 min and filtered through Whatman filter paper no. 42 and diluted to get the concentration in the range of linearity. Before the injection solution was filter through 0.45 micron HPLC filter and subjected for analysis. The peak area under the curve of tablet solution was observed and plotted against respective concentration. RESULTS AND DISCUSSION: All the system suitability parameters plate number, tailing factor and retention time was optimized before starting the experiment and this parameter were found with in the limit that shows the stability of method (table 1). For standard drug, linearity was observed by linear regression equation method. Equation is AUC = 0.423 x 10 6 X 0.0111 x 10 6 and correlation coefficient for drug was found to be more than 0.998, indicating good linearity (table 2). TABLE 2:
RESULTS OF LINEAR REGRESSION ANALYSIS Regression Parameter Observation Regression equation Slope of curve (m) Intercept (C) Correlation Coefficient (r 2 ) Linearity range (!g/ml) Absorption maxima (nm) AUC = mx + C 0.423 x 10 6
0.0111 x 10 6
0.998 0-50 245.0 The LC Method has been extensively validated for estimation of ezetimibe using linearity, range, accuracy and precision. All these analytical validation parameters were observed and the RSD was found to be less than one, which indicates the validity of method (table 3). The concentration of ezetimibe was estimated in the three different branded tablet dosage from. Sample
were found to be in the range 99.21-101.58 %, while the % relative standard deviation values obtained from replicate analysis of tablet were found to be in the range of 0.135-0.269, which indicates satisfactory applicability and reproducibility of the method (table 4).
Prabhat K Shrivastava et al Int.J.Ph.Sci, May-August 2009; 1(1):174-181 www.ijps.info 180 TABLE 3:
RESULTS OF VALIDATION PARAMETER Validation Parameter Mean a SD a %RSD a
0.328 0.463 a Maximum values of SD and RSD of six replicates, SD = Standard deviation RSD = Relative standard deviation TABLE 4:
RESULTS OF TABLET ANALYSIS FOR EZETIMIBE Tablet formulation Label Claim (mg/tab) Percentage of label estimated* mg/tab SD RSD SE! A
B
C 10
10
10 99.21-100.35
99.54-101.23
100.56-101.58 0.204
0.228
0.134
0.269
0.234
0.135
0.091
0.053
0.054 Maximum and minimum values of three different concentration level in three replicates, A Zetia, 10mg tablet of Torrent Pharmaceutical, B- Ezta, 10mg tablets of Zydus Cadila, and C- Ezedoc, 10mg tablets of Pinnacle Lupin. This method was found to be simple, economical, rapid, accurate and precise for estimation of ezetimibe in different tablet dosage form and as the method was extensively validated therefore has routine industrial application. ACKNOWLEDGEMENTS The authors wish to thanks, Head of the Department, School of Pharmaceutical Sciences, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, for providing necessary facilities and Torrent Pharmaceuticals, Baroda, for supplying ezetimibe as a gift sample. The financial assistance received from AICTE in the form of fellowship for postgraduate studies in Engineering and Technology is being gratefully acknowledged by one of the author.
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