Asian J. Research Chem. 1(1): July-Sept. 2008, ,
www.ajrconline.org
ISSN 0974-4169
RESEARCH ARTICLE
RP-HPLC Method for Simultaneous Estimation of Simvastatin and Ezetimibe in Bulk Drug and its Combined Dosage Form. Nilesh Jain*1, Ruchi Jain1, Hemant Swami2 and Deepak Kumar Jain3 *1Shri Ravi Shankar College of Pharmacy, Bhanpura Road, Bhopal, (M.P), India. 2 IPS College of Pharmacy, Gwalior, (M.P), India. 3 Truba Institute of Pharmacy, Karond Bypass, Bhopal (M.P), India. *Corresponding Author E-mail:
[email protected]
ABSTRACT This work is concerned with application of simple, accurate, precise and highly selective reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous estimation of simvastatin and ezetimibe in combined dosage form. Chromatographic separation was achieved isocratically at 25°C ± 0.5°C on Luna C18 column (250 х 4.6 mm i.d.) with a mobile phase composed of methanol: water: acetonitrile in the ratio of 75: 18.75: 6.25 % v/v/v at flow rate of 1.8 ml/min. Detection is carried out using a UV-PDA detector at 231 nm. The retention time of simvastatin and ezetimibe was found to be 13.5 + 0.5 min and 4.02 + 0.3 min. respectively. The method was found to be linear in the range of 1-50 μg/ml with mean recovery of 99.21% for simvastatin and 99.50% for ezetimibe. The correlation coefficients for all components are close to 1. The developed method was validated according to ICH guidelines and values of accuracy, precision and other statistical analysis were found to be in good accordance with the prescribed values. Thus the proposed method was successfully applied for simultaneous determination of simvastatin and ezetimibe in routine analysis.
KEY WORDS
Simvastatin, Ezetimibe, R P- H PLC
INTRODUCTION: Simvastatin (SIM) butanoic acid, 2, 2-dimethyl-1, 2, 3, 7, 8, 8a-hexahydro-3, 7-dimethyl-8- [2 (tetrahydro-4hydroxy-6-oxo-2H-pyran-2-yl) -ethyl]-1-naphthalenyl ester, is a lipid-lowering agent that is derived synthetically from fermentation products of Aspergillus terreus1. After oral ingestion SIM, which is an inactive lactone, is hydrolyzed to the corresponding β-hydroxy acid that is an inhibitor of 3-hydroxy 3-methyl glutaryl – coenzyme A. (HMG- CoA) reductase. This enzyme catalyzes the conversion of HMG CoA to mevalonate, which is an early and rate limiting step in cholesterol biosynthesis2. Ezetimibe (EZ), 1- (4-Fluorophenly) – 3 (R)- [3-(4fluorophenyl)- 3 (S) hydroxy propyl]-4 (S)–(4-hydroxy phenyl) – 2 azetidinones, is a therapeutically beneficial drug that works by inhibiting the protein transporters on small intestinal brush border, which brings about this active transport of cholesterol. In addition, it also inhibits phytosterol absorption3.
Received on 01.09.2008 Accepted on 16.10.2008
Modified on 24.09.2008 © AJRC All right reserved
Asian J. Research Chem. 1(1): July-Sept. 2008;Page 29-31
EZ has no inhibitory effect on absorption of lipid soluble vitamins, triglycerides or bile acids, as do statins. This distinct mechanism of action results in a synergistic cholesterol lowering effect when used together with statins that inhibits cholesterol synthesis by liver4. Recently a combination of SIM and EZ has been launched in market. In this combination, EZ shows a synergistic effect with SIM. SIM was determined by several methods including gas chromatography–mass spectrometry (GC–MS)5, liquid chromatography with UV detection (LC–UV)6-8. EZ was determined with or without combination of several drugs by HPLC and spectrophotometrically9, 10. Literature survey revealed that no HPLC method has been reported yet for the analysis of these two drugs in combination without preliminary separation that makes it worthwhile to pursue the present work.
EXPERIMENTAL: Instrumentation: The present work was carried out on gradient high pressure liquid chromatograph (Shimadzu HPLC class VP series) with double reciprocating plunger pump LC-10ATVp pumps for constant flow and constant pressure delivery. Photodiode array detector SPD-M10 Avp connected to
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software Class M10A for controlling the instrumentation as well as processing the data generated was used. The reverse phase luna C18 column (250 × 4.6 mm i.d. particle size 5 μ) was used for all chromatographic separation. Tablets, Simvotin ez-10 containing SIM and EZ in ratio of 10mg: 10mg respectively was purchased from local market. Reagents and chemicals: SIM and EZ were obtained as gift sample from Ranbaxy Lab, Dewas, India. All solvents were of HPLC grade obtained from Merck Research Laboratory, Mumbai, India. Experimental condition: The HPLC system was operated isocratically at flow rate of 1.8 ml/min. at 25°C ± 0.5°C. The mobile phase found to be most suitable for analysis was methanol: water: acetonitrile in the ratio of 75:18.75: 6.25%v/v/v, detection was carried out at 231 nm. FIG. 1: Representative chromatogram of Simvastain and Ezetimibe.
42, finally different concentrations of tablet sample were prepared by serial dilution technique. PROCEDURE Chromatographic condition: Chromatographic separation was achieved by using mobile phase consisted of methanol: acetonitrile: water in the ratio of 75:18.75:6.25 v/v/v, flowing through Luna C18 column at a constant flow rate of 1.8 ml/min. A Luna C18 column was used as the separation phase. Detection was carried out using a photo diode array detector at 231 nm. Linearity: To establish the linearity a series of dilutions ranging from 1-50 μg/ml for SIM and 1-50μg/ml for EZ were prepared separately and calibration graph was plotted between the mean peak area Vs respective concentration and regression equation was derived. Method validation: The accuracy, precision and robustness were determined by analyzing a set of laboratory sample (n=3) with each of the five concentrations ranging from 10-50 μg/ml for both drugs.
RESULTS AND DISCUSSION:
Preparation of standard solution: Standard stock solution of 1000 μg/ml of each SIM and EZ were prepared by dissolving 100 mg of each drug in 70% methanol. Sub stock solution was prepared from stock solution by diluting each standard stock solution (10ml) up to 100 ml to get 100 μg/ml of both drugs. The nominal concentration in range of 10, 20, 30, 40, 50 μg/ml were prepared for calibration. Tablets of SIM and EZ combination are available in 1:1, 2:1, 4:1 & 8:1 ratio. Working standards were prepared in the ratio of 2: 1 from standard sub stock solution. TABLE 1: SYSTEM SUITABILITY PARAMETERS Parameter SIM EZ Tailing factor* 1.08 1.07 No. of theoretical plate* 4615 7747 Resolution factor 10.5 Calibration range 1-50 μg/ml 1-50 μg/ml * Each value is the Mean of six determinations
Sample preparation: Twenty tablets were weighed and crushed to fine powder. Powder equivalent to 10 mg of SIM was weighed and dissolved in 70% methanol, sonicated for 10 min and filtered through whatmann filter paper No.
Chromatographic method: Initially methanol: water was tried as mobile phase, in which SIM and EZ shows greater peak asymmetry which was not satisfying the system suitability criterion and the resolution requirement for the simultaneous estimations of these two drugs. The tailing for both the peaks was reduced considerably by adding acetonitrile in mobile phase and brought close to 1, which is ideal requirement for chromatographic analysis. Acetonitrile can help in reducing the viscosity of the mobile phase and hence reduce the backpressure and increase the column life. A representative graph of this is shown in Fig.1. System suitability: The system suitability test was applied to a representative chromatogram to check the various parameters such as column efficiency, resolution, precision and peak tailing. The result obtained is shown in Table 1. The resolution between SIM and EZ was 10.5. The number of theoretical plate for SIM and EZ were 4615 and 7747 respectively. All these parameters were evaluated with the background of regulatory requirements, which also suggests good chromatographic condition. Linearity: Simvastain and ezetimibe showed a linearity of response between 1 to 50 μg/ml. This linearity was represented by a linear regression equation as follows. YSIM =40223.7 conc. + 19264.9 YEZ = 50511.1 conc. + 9696.8
(r2 = 0.9996) (r2 = 0.9999)
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Asian J. Research Chem. 1(1): July-Sept. 2008, ,
TABLE 2: RECOVERY STUDIES DATA SHOWING AMOUNT OF DRUG RECOVERED FROM SAMPLE SOLUTION AND AVERAGE RECOVERY. Drug Tablet Amount (μg/ml) Amount added (μg/ml) Amount Recovered (μg/ml)* Recovery (%) Coefficient of variation Atorvastatin 10 10 10.04 103.8 Calcium 10 20 20.01 100 30 30.1 102.3 0.017 10 10 40 40.1 101.5 10 50 50.1 101.2 Fenofibrate 10 10 10.05 100.2 10 20 20.3 101.5 10 30 30.06 100.2 0.0038 10 40 40.82 102.05 10 50 50.30 100.6 * Each value is the mean of three determinations
Accuracy and Precision: The recovery experiment was carried out by spiking the already analyzed sample of the tablets with their different known concentration of standard SIM and EZ. The result is summarized in Table 2.The percent recovery for SIM ranges from 99.33 to 99.93% and EZ ranges from 99.36 to 99.46%. Assay: The content of SIM and EZ found in the tablets by the proposed method are shown in Table 3. The low R.S.D indicates that the method is precise and accurate. Stability of sample solution: The sample solution injected after 12 h did not shows any appreciable changes. TABLE 3: DOSAGE FORM ANALYSIS TABLET SR. NO. SIM (%) Simvotin EZ- 1 96.1 10 2 95.3 3 96.9 4 96.6 5 96.8 6 95.3 96.16 MEAN R.S.D 0.74
EZ (%) 104.1 102.6 102.3 102.2 101.9 101.8 102.48 0.82
CONCLUSIONS: The proposed RP-HPLC method allows for accurate, precise and reliable measurement of SIM and EZ simultaneously in combined dosage form. The developed RP-HPLC method was found to be simple, rapid, selective, accurate and precise for the concurrent estimation of drugs in respective two-component tablet dosage form of SIM and EZ. The RSD for all parameters was found to be less than one, which indicates the validity of method and assay results obtained by this method are in fair agreement. The developed method can be used for routine quantitative simultaneous estimation of SIM and EZ in multicomponent pharmaceutical preparation.
REFERENCES: Budawari S. editor, In; The Merck index. 13th ed. Whitehouse Station, (NJ): Merck &Co., Inc., 2001; 868. 2. Ochiai H. et al. Determination of Simvastatin and Its Active Metabolites in Human Plasma by Column-Switching High Performance Liquid Chromatography with Fluorescence Detection after Derivatization with 1-Bromoacetylpyrene. J Chromatogr B Biomed Sci. 1997: 694:211-217. 3. Budawari S. editor, In; The Merck index. 13th ed. Whitehouse Station, (NJ): Merck &Co., Inc., 2001; 148. 4. Darkes MJ, Poole RM, Goa KL, Ezetimibe, Am J. Cardio Vasc. Drugs. 2003: 3: 67-76. 5. Morris MJ. et al. Determination of the HMG-CoA Reductase Inhibitors Simvastatin, Lovastatin, and Pravastatin in Plasma by Gas Chromatography/Chemical Ionization Mass Spectrometry. Biol Mass Spectrom. 1993:22:1-8. 6. Tan L, Yang LL, Zhang X, Yuan YS and Ling SS. Determination of Simvastatin in Human Plasma by High Performance Liquid Chromatography. Se Pu. 2000:18:232234. 7. Curlucci G, Mazzeo P, Biordi L, and Bologna M. Simultaneous Determination of Simvastatin and its Hydroxy Acid Form in Human Plasma by High Performance Liquid Chromatography with UV Detection. J Pharm Biomed Anal. 1992: 10:693-7. 8. Wang L and Asgharnejad M. Second- Derivative UV Spectrometric Determination of Simvastatin in Tablet Dosage Form. J Pharm Biomed Anal. 2000:21:1243-1248. 9. Chaudhari BG. et al. Stability-Indicating Reversed-Phase Liquid Chromatographic Method for Simultaneous Determination of Atorvastatin and Ezetimibe from Their Combination Drug Products. J. AOAC Int. 2007: 90:1539-46. 10. Imran M, Singh RS and Chandran S. Stability Indicating Ultraviolet Spectroscopic Method for the Estimation of Ezetimibe and Carvedilol. Pharmazie 2006: 61:766-9. 1.
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