Research Article PRAJAPATI ARUN M., PRAJAPATI RAHUL B. *

Transcription

1 Research Article Vol: 2; Issue: 3 DETERMINATION OF GLIMEPIRIDE & EZETIMIBE IN COMBINE TABLET DOSAGE FORM BY RATIO DERIVATIVE SPECTROSCOPY AND Q-ABSORBANCE METHOD PRAJAPATI ARUN M., PRAJAPATI RAHUL B. * Department of Pharmaceutical Quality Assurance, Shree S. K. Patel College of Pharmaceutical Education & Research, Ganpat University, Kherva , Mehsana, Gujarat, India. Date Received: 4-Mar-2014 Date of Accepted: 22-Mar-2014 Date Published: 27-Mar-2014 Abstract: Glimepiride is antidiabetic drug and Ezetimibe is cholesterol absorption inhibitor.the combination of this two drug is used for the treatment of hypercholesterolemia.two new,simple,accurate and precise uv spectrophotometric methods have been developed amd validated for the simultaneous determination of Glimepiride (GLM) and Ezetimibe (EZE) in their combine dosage forms. The first method is ratio derivative spectroscopy method (Method A) in which ratio derivative amplitudes were measured at selected wavelengths. The amplitudes at nm and nm in the ratio derivative spectra were selected to determine GLM and EZE, respectively. The second method is Q-absorbance method. For Q-absorbance method the absorbances of the standard solutions were taken at two wavelengths nm (max of glimepiride) and 23.0 nm (Isobestic point), in methanol. Beer s law is obeyed in the concentration ranges of -1 µg/ml for GLM and EZE in methanol for both the methods. Both methods were validated statistically and recovery studies carry out.the suitability of this method for the quantitative determination of Glimepiride and Ezetimibe was proved by validation. The proposed method was found to be simple and sensitive for the routine quality control application of Glimepiride and Ezetimibe in combination. The results of analysis have been validated statistically and by recovery studies.the validation study is statistically significant as all statistical parameter are within the acceptance range ( % RSD < 2.0 and S.D. < 2.0) for both accuracy and precision. Keywords: Ezetimibe, Glimepride, Q-absorbance, Ratio spectra, Method validation. Introduction Glimepiride(GLM) is chemically 3-ethyl-4-methyl-N- (4-[N-((1r,4r)-4 methylcyclohexylcarbamoyl)sulfamoyl]phenethyl)-2- oxo-2,5-dihydro-1h-pyrrole-1 carboxamide(figure 1) well known Anti diabetic drug. [1] It is official in Indian Pharmacopoeia (IP), British Pharmacopoeia (BP), European Pharmacopoeia (EP), and United States Pharmacopoeia (USP). IP [2], BP [3], USP [4] and EP [5] describe Liquid chromatographic method for estimation. Literature survey reveals HPLC,spectrophotometric method for estimation Glimepiride in single dosage form. [,7,8] Literature survey also reveals spectrophotometric [9],HPTLC [10] and HPLC [11] methods for determination of glimepiride with other drugs in combination. Ezetimibe (EZT) is (3R,4S)-1-(4-fluorophenyl)-3- [(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4 hydroxy phenyl) azetidin-2-one(figure 2). It is not official in any pharmacopeia. Literature survey reveals spectrophotometric [12] and HPLC [13] methods for determination of Ezetimibe in single dosage form. Literature survey also reveals spectrophotometric [14], HPLC [15] and HPTLC [1] methods for determination of Ezetimibe with other drugs in combination. The combination of these two drugs is not official in any pharmacopoeia; hence no official method is available for the simultaneous estimation of glimepiride and ezetimibe in their 239

2 combined dosage forms. Literature survey does not reveal any simple spectrophotometric method for simultaneous estimation of glimepiride and ezetimibe in synthetic mixture or dosage forms. The present communication describes simple, sensitive, rapid, accurate, precise and cost effective spectrophotometric method based on simultaneous equations for simultaneous estimation of both drugs in their tablet dosage form. MATERIALS & METHODS Apparatus A shimadzu model 1700 (Japan) double beam UV/Visible spectrophotometer with spectral width of 2 nm, wavelength accuracy of 0.5 nm and a pair of 10 mm matched quartz cell was used to measure absorbance of all the solutions. Spectra were automatically obtained by UV-Probe 2.42 system software. A Sartorius CP224S analytical balance (Gottingen, Germany), an ultrasonic bath (Frontline FS 4, Mumbai, India) was used in the study. Reagents and materials Glimepiride bulk powder was kindly gifted by Cadila Pharmaceuticals Ltd., Ahmedabad, Gujarat, India. Ezetimibe bulk powder was kindly gifted by Medwin pharmaceutical Ltd., Ahmedabad, Gujarat, methanol as Solvent and Whatman filter paper no. 41 (Millipore, USA) were used in the study. PREPARATION OF STANDARD STOCK SOLUTIONS An accurately weighed quantity of standard Glimepiride (10 mg) and Ezetimibe (10 mg) powder were weighed and transferred to 100 ml separate volumetric flasks and dissolved in methanol. The flasks were shaken and volumes were made up to mark with methanol to give a solution containing 100 µg/ml each of Glimepiride and Ezetimibe. METHODOLOGY: METHOD I:RATIO DERIVATIVE SPECTROSCOPY METHOD : The working standard solutions of Glimepiride and Ezetimibe were prepared separately in methanol having concentration range of -1 µg/ml. They were scanned in the wavelength range of nm against methanol as blank. The method involves dividing the spectrum of mixture by the standardized spectra of each of the analyte and deriving the ratio to obtain spectrum that is dependent of concentration of analyte used as a divisor. Using appropriate dilutions of standard stock solution,the two solutions were scanned separately. The ratio spectra of different GLM standards at increasing concentrations were obtained by dividing each with the stored spectrum of the standard solution of EZE (1 µg ml-1) and the first derivative of these spectra traced, illustrated in Fig 1. Wavelength nm was selected for the quantification of GLM in GLM+EZE mixture. The ratio and ratio derivative spectra of the solutions of EZE at different concentrations were obtained by dividing each with the stored standard spectrum of the GLM (1 µg/ml ) [Fig. 2]. Wavelength nm was selected for the quantification of EZE in GLM+EZE mixture. Measured analytical signals at these wavelengths were proportional to theconcentrations of the drugs. Calibration curves were prepared from the measured signals at this selected wavelength and concentration of the standard solutions. The amount of GLM (CGLM) and EZE (CEZE) in tablets was calculated by using equations 1 and 2, respectively. METHOD II:Q-ABSORBANCE METHOD In this method, solutions of GLM and EZE ( -1 µg/ml, each),were prepared separately by appropriate dilution of standard stock solution and scanned in the spectrum mode from 400 nm to 200 nm against distilled water as blank. The sampling wavelengths selected for the estimation of GLM and EZE were nm and 23.0 nm. Suitable dilutions of standard stock solution of the GLM and EZE were prepared and scanned in the spectrum mode from the wavelength range nm and their overlay spectra were obtained. The isobestic point was obtained at 23.0 nm. Analytical wavelengths selected were the isobestic point and other being the wavelength of maximum absorption of one of the two components nm (λmax of GLM). GLM and EZE exhibited linearity in the range of -1 µg/ ml at the selected wavelengths. Mixed standard solutions of both the drugs in the ratio of 1:10 were prepared and the absorbances of the mixed standard solutions were measured at the selected wavelengths. (Fig.2) The concentration of CGLM and CEZE in mixed standard solutions obtained by solving equation (3) and (4). CX = QM-QY / QX - QY x A1 /ax (3) CY= QM-QX / QY - QX x A2 /ax (4) Where QX = ax2 /ax1 QY = ay2 /ay1 QM = A2 / A1The absorption spectra thus obtained were derivatized for first order (Fig. 240

3 VALIDATION OF THE PROPOSED METHOD The proposed method was validated according to the International Conference on Harmonization (ICH) guidelines. [1] Figure 3- Chemical structure of glimepiride LINEARITY (CALIBRATION CURVE) The calibration curves were plotted over a concentration range of -1 µg/ml for Glimepiride and Ezetimibe. Accurately measured standardd solutions of Glimepiride and Ezetimibe (0., 0.8, 1.0, 1.2, 1.4, 1. ml) were transferred to a series of 10 ml of volumetric flask and diluted to the mark with methanol. The absorbances of the solutions were measured at 228 nm and 233 nm against methanol as blank. The calibration curves were constructed by plotting absorbances versus concentrations and the regression equations were calculated. METHOD PRECISION (REPEATABILITY) Figure 4 -chemical structure of ezetimibe The precision of the method was checked by repeated scanning and measurement of absorbance of solutions (n = ) for Glimepiride ( µg/ml) ) and Eeztimibe ( µg/ml) without changing the parameter of the proposed Spectrophotometry method. INTERMEDIATE PRECISION (REPRODUCIBILITY) The intraday and interday precision of the proposed method was determined by analyzing the corresponding responses 3 times on the same day and on 3 different days over a period of 1 week for 3 different concentrations of standard solutions of Glimepiride and Ezetimibe (, 8, 10 µg/ml for Glimepiride and, 8, 10 µg/ml for Ezetimibe). The result was reported in terms of relative standard deviation (% RSD). ACCURACY (RECOVERY STUDY) Figure 5- overlay absorbance spectra of Glimepiride ( µg/ml) and Ezetimibe ( µg/ml) in methanol. The accuracy of the method was determined by calculating recovery of Glimepiride and Ezetimibe by the standard addition method. Known amounts of standard solutions of Glimepiride and Ezetimibe were added at 50, 100 and 150 % level to prequantified sample solutions of Glimepiride and Ezetimibe ( µg/ml EZE, respectively for ratio derivative spectroscopy 241

4 GLM and µg/ml EZT). The amounts of GLM and EZT were estimated by applying obtained values to the respective regression line equations. The experiment was repeated for five times. LIMIT OF DETECTION AND LIMIT OF QUANTIFICATION The limit of detection (LOD) and the limit of quantification (LOQ) of the drug were derived by calculating the signal -to-noise ratio (S/N, i.e., 3.3 for LOD and 10 for LOQ) using the following equations designated by International Conference on Harmonization (ICH) guidelines. [20] LOD = 3.3 σ/s LOQ = 10 σ/s Where, σ = the standard deviation of the response S = slope of the calibration curve ANALYSIS OF GLM AND EZT FROM TABLET DOSAGE FORM Twenty tablets were weighed individually and powdered. Quantity of the powder equivalent to 1 mg GLM & 10 mg EZT was transferred in 100 ml volumetric flask separately and powder was dissolved in 50 ml of methanol with sonication to dissolve drug as completely as possible. The solution was filtered through whatman filter paper No. 41. Than the volume was adjusted up to mark with methanol. Take ml of GLM & Take 1 ml EZT in 10 ml volumetric Flask From the stock solution. The volume was adjusted up to the mark with nethanol to get a final concentration of GLM ( µg/ml) and EZT (10 µg/ml). The proposed ratio derivative method were then followed to determine concentration of analytes in the sample solutions.in which absorbance take at nm and nm for GLM and EZE respectively. The same sample solutions were subjected to analysis by the Q- absorbance method where absorbances of sample solutions were recorded at nm and 22.0 nm. The concentration of each drug was determined by using equation (3) and ( 4). a RSD = Relative standard deviation. b LOD = Limit of detection. c LOQ = Limit of quantification d S. D. is standard deviation RESULTS The standard solutions of GLM and EZE were scanned separately in the UV range and zero-order spectra for GLM and EZE were recorded. Maximum absorbance was obtained at nm and nm for GLM and method(method I).In Q-absorbance method(method II) quantification were carried out at 228 nm for GLM and EZE and 23 nm for isobestic point.a critical evalution of this two method is also carried out and slope,intercept,correlation coefficient was measured by statistical method.it is shown in table 3&4.As per ICH guideline the method validation parameter were checked linearity.precision,accuracy,lod and LOQ.Overlain First derivative of the ratio spectra of GLM solution ( 1 µg ml-1)when 1µg ml-1 solution of EZE is used as divisor (figure 3).Overlain first derivative of the ratio spectra of EZE solution ( 1 µg ml-1)when 1µg ml- 1 solution of GLM is used as divisor(figure 4).For both the drug linearity range is -1 µg/ml.by both method the percent recovery is in the range of 97.32% to % with standard deviation well below 2 indicating accuracy of the methods.percent label claim was found to be in the range of 99.41% to % for GLM and EZE by both the method.the percent RSD for intraday and interday precision for bothe drugs were found in the acceptable range by both method.so the both method have good repetability and reproducibility.lod and LOQ value for GLM were found to be and 0.21 at nm and for EZE it is 1.5 and 5.00 at nm by ratio derivative spectroscopic method.by Q-absorbance method LOD and LOQ for GLM were found to be 0.02and 0.081and for EZE it was 0.48 and 1.4 µg/ml. DISCUSSION: The proposed spectrophotometric method (simultaneous equation and first order derivative method) was found to be simple, sensitive, accurate and precise for determination of GLM and EZE in tablet dosage form. The method utilizes easily available and cheap solvent like methanol for analysis of GLM and EZE hence the method was also economic for estimation of GLM and EZE in tablet dosage form. The common excipients and other additives are usually present in the tablet dosage form do not interfere in the analysis of GLM and EZE in methanol, hence it can be conveniently adopted for routine quality control analysis of the drugs in combined pharmaceutical formulation. ACKNOWLEDGEMENT The authors are thankful to cadila Pharmaceutical Ltd, Ahmedabad, India and Medwin pharmaceutical Ltd. Ahmedabad, India for providing gift sample of GLM and EZE, respectively for carry out the research work. The authors are highly thankful to Shree S. K. Patel College of Pharmaceutical Education and Research, Ganpat University, Ganpat Vidyanagar , Mehsana, Gujarat, India for providing all the facilities to carry out the research work. 242

5 Drug GLM Amount present in mixture (µg/ml) TABLE 1 RECOVERY DATA OF GLM AND EZT % recovery ± S. D. (n = 3) Amount added % METHOD I ± ±1.48 METHOD II 99.38± ±0.38 EZE ± ± ± ± ± ± ±1.35 S. D. = Standard deviation. n = Number of determinations ±0.99 Ratio method TABLE 2 ANALYSES OF GLM AND EZE IN TABLET DOSAGE FORM Method Label Claim (mg) Amount found (mg) % Label claim ± S. D. mixture(n = 3) GLM EZT GLM EZT GLM EZT spectra ± ±1.01 Q-Absorbance method ± ±1.20 S. D. = Standard deviation. n = Number of determination TABLE 3 REGRESSION ANALYSIS DATA AND SUMMARY OF VALIDATION PARAMETERS FOR GLM AND EZT RATIO DERIVATIVE SPECTROSCOPY METHOD PARAMETERS GLM EZE Wavelength (nm) Beer s law limit (µg /ml) -1-1 Regression equation (y = mx + c) Slope (m) Intercept (c) Y=0.00x Correlation coefficient (r 2 ) LOD a (µg/ml) LOQ b (µg /ml) %Repeatability RSD (n=) Y=0.020x Precision Intraday %RSD n=3 Interday Accuracy± S. D. d (% Recovery, n = 3) ± ±1.07 Assay ± S. D. (n = 3) 98± ±

6 TABLE 4 REGRESSION ANALYSIS DATA AND SUMMARY OF VALIDATION PARAMETERS FOR GLM AND EZT Q-ABSORBANCE METHOD PARAMETERS GLM EZE ISOBESTIC POINT Wavelength (nm) Beer s law limit (µg /ml) Regression equation (y = mx + c) Slope (m) Intercept (c) Y=0.052x Y=0.040x Y=0.044x Correlation coefficient (r 2 ) LOD a (µg/ml) LOQ b (µg /ml) %Repeatability RSD (n=) Precision Intraday %RSD n=3 Interday Accuracy± S. D. d (% Recovery, n = 3) 99.72± ± Assay ± S. D. (n = ± ±1.2 - REFERENCES: 1. K.D.Tripathi, Essential of medical pharmacology, 5 th edition, Jaypee brothers publisher, New delhi;24 2. Indian Pharmacopoeia. Vol. II. The Controller of Publication. th edition. Govt. of India. New Delhi, , British Pharmacopoeia. Vol. I. Stationary office. London Medicines and Healthcare product regulatory agency, 979, United states pharmacopoeia.the united states pharmacopoeial convention Inc, Rockville,2497, European Pharmacopoeia.0. Vol. II. th edition. Starboary: Council of Europe, 115, Bhargavi S.,Suryasaga G.,Sowmy D.K.,Ashok K. and Nama S. UV-spectrophotometric method for determination of glimepiride in pharmaceutical dosage form.international journal of pharmaceutical science and research,23: , Asha Ranjani V.,Abigna C.,Akhilesh D.,Prashanthi K.,and Sindhuja M.Analytical method development and validation of glimepiride in bulk and tablet dosage form. International journal of pharmacy and analytical research, IJPAR 2(4); Mastiholimath V.S.,Patel A.P.,Shah B.,Gadad A.P.and Manur V.S.Rp-hplc method development and validation for the estimation of glimepiride in tablet dosage form.inventi impact:pharma Analysis & Quality Assurance,19(11); Patel A.P.,Analytical method development and validation of glimepiride and extended release metformin hydrochloride tablet.dspace at KLE university,belgaum dissertation.pharmacy quality assurance; Singh D.,Dwivedi S.C.,Kushnoor A.Development and validation of a HPTLC method for simultaneous estimation of pioglitazone and glimepiride in bulk and tablet dosage form.international journal of biomedical and advance 2(9); El-Enany N.M.,Abdelal A.A.,Belal F.F.,Itoh Y.I.,and Nakamura M.N.Devlopment and validation of a rp-hplc method for simultaneous determination of rosiglitazone and glimepiride in combine dosage forms and human plasma.chemistry central journal :9, Shravya A.,Chandan S.,Gurupadayya B.M.,Sireepha M.,Spectrphotometric determination of ezetimibe using MBTH reagent in pharmaceutical dosage form.international journal of research in ayurveda & formula;2(2); ; Sistla R.,Tata V.S.,Kashyap Y.V.,Chandrasekar D.and Diwan P.V.Development and validation of a rp-hplc method for the determination of ezetimibe in pharmaceutical dosage forms.journal of pharmaceutical science biomedical analysis.39(3-4); ; Ramchandran S.,Kumar B.,Gnavalgund S.Simultaneous spectrophotometric estimation of valsartan and ezetimibe in pharmaceuticals.tropical journal of pharmaceutical research,10(); ; Krishnaveni G.,Satyanarayana PVV.Method development and validation for simultaneous determination of ezetimibe and simvastatin in combined pharmaceutical dosage form by RP-HPLC 244

7 method.international journal of pharmaceutical and life science,2(2);0; Rathinaraj S.Devlopment and validation of HPTLC method for determination of simvastatin and ezetimibe in combine dosage form.international journal of pharmaceutical and biological archive,1(4);32; The International Conference on Harmonization, Q2 (R1), Validation of Analytical Procedure, Text and Methodology, Avalabile online at 245