Baokar Shrikrishna et al. / Journal of Pharmacy Research 2011,4(7),2313-2316 Research Article ISSN: 0974-6943 Available online through www.jpronline.info High Performance Liquid Chromatographic Method Development and Validation of Cholesterol Inhibitor Drug. Baokar Shrikrishna 1* , Pawar Vinod 2, Sonawane S.H. 3 Department of Pharmaceutical Analysis, SVPM’s College of Pharmacy, Malegaon (Bk), Tal- Baramati, Dist-Pune, Maharashtra, 413115, India 2 Department of Pharmacology, SVPM’s College of Pharmacy, Malegaon (Bk), Tal- Baramati, Dist-Pune, Maharashtra, 413115, India 3 Department of Pharmaceutics, SVPM’s College of Pharmacy, Malegaon (Bk), Tal- Baramati, Dist-Pune, Maharashtra, 413115, India 1 Received on: 12-04-2011; Revised on: 18-05-2011; Accepted on:21-06-2011 ABSTRACT This study aimed at developing and validating an HPLC method for the assay of Ezetimibe in tablets’ formulation A chromatographic system comprising ODS-3V 4.6 mm x 250mm column, a mobile phase of Buffer and acetonitrile, a flow rate of 1.5 ml/min and a UV detector set at 230 nm has shown good chromatographic separation for Ezetimibe. The degree of linearity of the calibration curves, the percent recoveries of Ezetimibe and related substances, the limit of detection (LOD), and limit of quantization (LOQ) for the HPLC method have been determined. The HPLC method under study was found to be specific, precise, accurate, reproducible indicating stability and robust. Key Words: Ezetimibe, HPLC, validation, I NTRODUCTI ON Ezetimibe[(3R,4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3hydroxypropyl]-4-(4-hydroxyphenyl) - 2-azetidinone] (Fig. 1) is a selective cholesterol absorption inhibitor, which potently inhibits the absorption of biliary and dietary cholesterol1 from the small intestine without affecting the absorption of fat-soluble vitamins (Knoop et.al., ) triglyceride or bile acids. Ezetimibe reduces the small intestinal enterocyte uptake and absorption of cholesterol that keeps the cholesterol in the intestinal lumen for excretion (Davis H.R et al., 2004). Ezetimibe is rapidly absorbed and primarily metabolized in the small intestine and Fig.1. liver to its glucuronide, both of Structure of which undergo enter hepatic reEzetimibe cycling in humans (Heek, M.V.et al., Ballantyne, C.M et al., 2003) Since ezetimibe does not influence the activities of CYP 450 enzymes, significant pharmacokinetic interactions with other medications including stains, fibrates, digoxin and warfarin have not been found. Ezetimibe complements the lipid lowering effects of other therapies, such as statins. Clinical studies have shown that co-administration of ezetimibe with statins could provide significant reductions in both the low-density lipoproteins (LDL) and the total cholesterol with slight increase in the high-density lipoproteins (HDL) (Davidson, M.H. et al., 2008 , Kerzner, B. et al., 2003 , Melani, L et al., 2003 , ) It is a white, crystalline powder that is freely to very soluble in ethanol, methanol, and acetone and practically insoluble in water. It melts at about 163°C and is reported to be stable at ambient temperature (S. S. Dhaneshwar et al., 2007). Experimentation Instruments A High Performance Liquid Chromatography system (Agilent 1100) was used for the analysis, Mettler Tolido as weighing balance and Bronson sonicator, Millipore Milli Q plus purification system, borosil glass apparatus were used for experimental process. Reagents and Chemicals: Ezetimibe was obtained as a gift sample, whereas acetonitrile, methanol, triethanolamine, all were HPLC grade and phosphoric acid were used from Merck specialties Pvt. Limited, Mumbai. Method Development Trial: 1 Mobile phase: Buffer and Methanol were mixed in the ratio of 50:50 Chromatographic conditions : Flow rate : 1.0 ml/min Column : Micro pack Detector wavelength : 252nm Column temperature : Ambient Injection volume : 10µl Run time : 25 min Observation: Retention time was more, peak shape was not good and asymmetry was more than limit According to fig. No. 2 Fig. 2 Trial First Chromatograph Trial: 2 Mobile phase: Buffer and Acetonitrile were mixed in the ratio of 45:55 Chromatographic conditions: Flow rate : 1.0 ml/min Column : Eclipse Detector wave length : 240nm Column temperature : Ambient Injection volume : 10µl Run time : 30 mins Observation: The retention time was more, peak shape was not good and theoretical plates are less. According to fig. No.3 * Corresponding author. Shrikrishna Babahari. Baokar, Department of Pharmaceutical Analysis, SVPMS College of Pharmacy, Malegaon (Bk), Tal- Baramati, Dist-Pune, Maharashtra, 413115, India Mobile No. 09960225455 Email: krishnabaokar@gmail.com Fig. 3 Trial Second Chromatograph Journal of Pharmacy Research Vol.4.Issue 7. July 2011 2313-2316 Baokar Shrikrishna et al. / Journal of Pharmacy Research 2011,4(7),2313-2316 Trial: 3 Mobile phase: Buffer and Acetonitrile were mixed in the ratio of 45:55 Chromatographic conditions: Flow rate : 1.5 ml/min Column : ODS-3V 4.6 mm x 250mm Detector wavelength : 230nm Column temperature : Ambient Injection volume : 10µl Run time Amount of ezetimibe in each tablet = Sample Area Standard dilution Potency —————x ———————— x ——— x Average weight of tablet Standard area Sample dilution 100 Amount present % content = ———————— x 100 Label claim : 25 mins Observation: Retention time was more. According to fig. No.4 Specificity The specificity of the method was evaluated by analyzing the sample solution added (known amount) with excipients at appropriate levels that the assay result is unaffected by the presence of extraneous materials. Fig. No. 4 Trial Third Chromatograph Optimized Method Finally 230nm wavelength was selected for final analysis as it gives maximum absorbance at particular wavelength. Buffer: Acetonitrile (50:50) solvent system was selected with 1.5 ml/min flow rate for final analysis because it gives best results. Described in table no.1 Table No. 1 Mobile phase Flow rate Column Detector wavelength Floe Rate Injection volume Validation Validation of an analytical method is a process to establish that the performance characteristics of the developed method meet the requirement of the intended analytical application. The method was validated for linearity, accuracy and specificity, intra-day and inter-day precision, repeatability of measurement of peak area, and repeatability of sample application, in accordance with ICH guidelines. Placebo Method: Specificity of the method was established by demonstrating that there is no interferences from the excipients. This was demonstrated by preparing the placebo containing all excipients except the drug and also the sample prepared from the same. Chromatograph for placebo observed in fig No. 6 and described in table no. 2 Buffer : acetonitrile 1.5 ml/min ODS-3V 4.6 mm x 250mm column, 230 nm 1.5 ml/min 20 ul Quantitative Determination of The Drugs Using The Developed Method Standard Solution: Accurately 20 mg Ezetimibe was weighed and it was diluted with 100 ml Acetonitrile. Fig. No. 7 Chromatograph of Placebo Table 02 .Data For Specificity: Sr. No. Parameters Acceptance criteria Inference 1 2 Blank Placebo No interference peak at the retention time of the analyte peak. No interference peak at the retention time of the analyte peak Passes Passes Blank interference : Chromatograph for Blank observed in fig No. 7 and described in table no. 2 Fig. 5 Chromatograph of Standard Sample Solution: 20 tablets were weighed and powdered. From this, powder equivalent to 20 mg of Ezetimibe was taken and it was extracted with acetonitrile. Procedure: 10µl of the blank, Standard and sample solution were injected separately into the chromatographic system and chromatographs were recorded and the peak areas were measured. This is observed in fig No.6 and 7 Fig. No.6 Chromatograph of Sample Fig. No. 7 Chromatograph Of Blank Linearity First the System suitability was checked with five replication of standard of 20ppm. Then the different concentrations of sample solution were injected from concentration range of 10ppm to 50ppm of the target analyte concentration including highest and lowest concentration i.e. 10ppm & 50ppm. And the dilutions were made with methanol and the solutions were injected. The correlation co-efficient and percentage curve fitting were calculated. Linearity curve is observed in fig No. 8 and described in table no. 6 Journal of Pharmacy Research Vol.4.Issue 7. July 2011 2313-2316 Baokar Shrikrishna et al. / Journal of Pharmacy Research 2011,4(7),2313-2316 in table no. 11 Linearity Table No. 10 Data Of System-To-System Variability (Samples) 3000000 2500000 Area 2000000 Sample. No Spl.area/ Std.area Std.Dilution factor Spl.Dilutin factor Avg.wt/ Label claim Std. Purity % Assay 1 1604662 1592053 1621047 1592053 1598257 1592053 1645215 1592053 1622955 1592053 1655810 1592053 20.11 200 20.11 200 20.11 200 20.11 200 20.11 200 20.11 200 100 77.13 100 77.68 100 76.1 100 79.95 100 77.48 100 80.24 76.42 10 76.42 10 76.42 10 76.42 10 76.42 10 76.42 10 99.5 99.9 99.5 100.2 99.5 100.9 99.5 98.8 99.5 100.6 99.5 99.1 Mean SD %RSD 99.9 0.81 0.81 1500000 2 1000000 3 500000 4 0 0 20 40 60 80 100 120 140 160 Concentration 5 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Fig. No. 8 Linearity Curve Accuracy The accuracy of the method was determined by recovery experiments. Known concentration of working standard was added to the fixed concentration of the pre-analyzed tablet solution. Percent recovery was calculated by comparing the area before and after the addition of working standard 13 mg found Percentage Recovery = ———————— x 100 mg added Table No. 11 Column To Column Variability: Accuracy data is described in Table .no. 7 4 1 2 3 4 5 Spike level 50% 75% 100% 125% 150% Spl.area/ Std.area Std. Dilution factor Spl. Dilution factor Avg.wt/ Label claim Std. Purity % Assay 1 1595182 1586847 1611452 1586847 1592006 1586847 1624027 1586847 1613343 1586847 1642949 1586847 20.06 200 20.06 200 20.06 200 20.06 200 20.06 200 20.06 200 100 77.23 100 77.27 100 75.73 100 78.69 100 76.65 100 78.72 76.42 10 76.42 10 76.42 10 76.42 10 76.42 10 76.42 10 99.5 99.9 99.5 100.2 99.5 101.0 99.5 99.2 99.5 101.2 99.5 100.3 Mean SD %RSD 100.2 0.84 0.84 2 3 5 Table No 07 Accuracy Data Sample No. Sample. No µg/ml added 50.05 75.07 100.10 125.12 150.15 Mean of µg/ml found Mean of % Recovery 49.66 74.29 99.23 124.09 148.75 99.2 98.96 99.13 99.2 99.06 Precision The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Repeatability data is described in table no. 8 intermediate precision that is analyst to analyst is described in table no. 9 Table No. 08 Repeatability Data For Ezetimibe Tablets Sample ID Wt.Taken (mg) Area mg found % assay Sample 1 Sample2 Sample3 Sample4 Sample5 Sample6 84.60 83.93 81.50 87.59 86.97 78.11 1803605 1738459 1735044 1850236 1852101 1632030 10.04 9.76 10.03 9.95 10.04 9.85 Mean SD %RSD 100.4 97.6 100.3 99.5 100.4 98.5 99.45 1.17 1.18 6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Robustness Robustness of the method was checked by making slight deliberate changes in chromatographic conditions like mobile phase ratio, pH of buffer, flow rate. It was observed that there were no marked changes in chromatograms, which demonstrated that the developed RP-HPLC method is robust 12 In the case of liquid chromatography, examples of typical variations are:# Influence of variations in flow rate (+/- 10%) described in table no. 12 Table No 12 Effect of Variation Of Flow Rate: Flow Rate Std Area 1.3ml/min 1826746 1829561 1829568 1828169 1825555 Avg. 1827920 SD 1764 %RSD 0.10 Tailing factor Flow Rate Std Area Tailing factor 1.230 1.240 1.250 1.250 1.240 1.242 0.01 0.67 1.7ml/min 1395305 1393670 1394399 1392830 1394936 1394228 995 0.07 1.220 1.240 1.240 1.240 1.230 1.234 0.01 0.72 Flow Rate 1.5ml/min Std Area Tailing factor 1580480 1581003 1580748 1578147 1579953 1580066 1141 0.07 1.230 1.240 1.250 1.250 1.250 1.244 0.01 0.72 # Influence of variations in column temperature (+/-5) described in table no. 13 Table No. 13 Effect Of Variation Of Temperature: Temp Std Area Tailing factor Temp Std Area Tailing factor Temp Std Area Tailing factor 1391339 1399008 1393262 1391650 1391142 1393280 3309 0.24 250 C 1575947 1577645 1580281 1576538 1576566 1394228 995 0.07 300 C 1390785 1392867 1391585 1389770 1391737 1391349 1153 0.08 Table No 09 Intermediate Precision (Analyst To Analyst Variability): Sample ID Wt. taken (mg) Area mg found % assay 200 C Sample 1 Sample2 Sample3 Sample4 Sample5 Sample6 81.63 77.04 82.51 84.48 85.40 78.43 1714642 1635618 1775848 1792983 1823389 1660627 9.87 9.98 10.11 9.97 10.03 9.95 Mean SD %RSD 98.7 99.8 101.1 99.7 100.3 99.5 99.9 1.17 1.18 Avg. SD %RSD Ruggedness The ruggedness of an analytical method is degree of reproducibility of test results obtained by the analysis of the same samples under a variety of normal test conditions, such as different laboratories, different analysts, different instruments, different lots of reagents, different elapsed assay times, different assay temperatures, different days etc. To carryout ruggedness experiment, analysis was repeated with different column, different day, different analyst and different systems. Observed data for intermediate precision that is analyst to analyst is described in table no. 10 and system to system variability is described 1.250 1.240 1.260 1.260 1.240 1.250 0.01 0.80 1.200 1.230 1.230 1.240 1.250 1.230 0.02 1.52 1.190 1.210 1.210 1.220 1.210 1.208 0.01 0.91 RESULTS AND DISCUSSION Specificity of the method was found out through non-interference of the placebo in identical conditions of assay. This confirms the specificity of the developed method. Linearity of the drug was obtained in the range of 10ppm to 50ppm of ezetimibe In the linearity correlation coefficient was found to be 1.000 Accuracy of the method was determined through the recovery studies of the drugs. Recovery of the drugs was well within the acceptance limit (98% to102%) Precision of the method was determined by analyzing the drug formulation by replicate injections and precision of the system was determined by mixed standard solutions. Percent RSD of the analyte was found to be within the Journal of Pharmacy Research Vol.4.Issue 7. July 2011 2313-2316 Baokar Shrikrishna et al. / Journal of Pharmacy Research 2011,4(7),2313-2316 limit of 2%, thus the developed method was found to provide high degree of precision and reproducibility. Ruggedness was determined by checking system to syster variability and column to column variability studies. The test results were found within limit. Robustness was determined by carrying out the assay during change in column Temp, and Flow Rate. Percent RSD was found to be within the limit NMT 2%. System suitability was determined by performing the assay with the same sample repeatedly. The number of theoretical plates was found to be > 7500for ezetimibe and the Tailing factor was found to be 1.138. The result for the developed method, quantitative determination of the formulation using the developed method & validation of the developed method are shown in following table . CONCLUSION The run time of the HPLC procedure is only 25 minutes. Good agreement was seen in the assay results of pharmaceutical formulation by developed method. We concluded that the proposed method is a good approach for obtaining reliable results and found to be suitable for the routine analysis of Ezetimibe in pharmaceutical formulation. REFERENCES 1. 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Kerzner, B., Corbelli, J., Sharp, S., Lipka, L.J., Melani, L., LeBeaut, A. Suresh, R. Mukhopadhyay, P. and Veltri, E.P. Am. J. Cardiol. 91, 2003, 418–424. Melani, L., Mills, R., Hassman, D., Lipetz, R., Lipka, L., LeBeaut, A., Suresh, R., Mukhopadhyay, P. and Veltri, E.P. Eur. Heart J. 24, 2003, 717–728. S. S. Dhaneshwar, S. R. Dhaneshwar, P. Deshpande, and M. Patil, development and validation of a method for simultaneous densitometric estimation of atorvastatin calcium and ezetimibe as the bulk drug and in tablet dosage forms, , acta chromatographica, no. 19, 2007 Fegade, J. D., Bhole, R. P., Shaikh I., Chaudhari, R. Y. , Patil, V. R. Development and Validation of Reverse Phase High Performance Liquid Chromatographic Method for Simultaneous Estimation of Paracetamol and Piroxicam in Tablet, International Journal of PharmTech Research Vol.1, No.2, pp 184-190 , April-June 2009. Source of support: Nil, Conflict of interest: None Declared Journal of Pharmacy Research Vol.4.Issue 7. 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