Objective: To develop and validate a simple, selective, rapid, precise, and accurate high performance liquid chromatographic (HPLC) method fordetermination of atorvastatin in bulk and its pharmaceutical formulation product.Method: Reversed phase-HPLC (RP-HPLC) method was performed by a mobile phase consisting mixture of methanol and ammonium acetate buffer(pH 4.5) in the proportion 60:40 v/v. A ZORBAX Eclipse plus C(4.6 mm Ã— 100 mm, 3.5 Î¼) column was used as a stationary phase. HPLC analysis ofatorvastatin was carried out at a wavelength of 241 nm with a flow rate of 1 ml/minute.18 Results: The linear regression analysis data for the calibration curve showed a good linear relationship with a correlation coefficient 0.9984. Thelinear regression equation was y=3726540.2x+27390388.1. This was found to give a sharp peak of atorvastatin at a retention time of 2.77 minutes.Validation parameters were evaluated for the method according to the ICH (Q2R1) guidelines. The limit of detection and limit of quantification for themethod were 0.6721 Î¼g/mL and 1.9989 Î¼g/mL, respectively. The % relative standard deviation values for intra-day precision and inter-day precisionwere found to be 0.31% and 0.30%, respectively. An accuracy of the method was determined through recovery studies which were found to be within97.57-102.22%.Conclusion: The method was validated for system suitability, accuracy, precision, robustness, and ruggedness. The precision, accuracy, sensitivityshort retention time and composition of the mobile phase indicated that this method is better than the earlier methods developed for the quantificationof atorvastatin.Keywords: Atorvastatin, Reversed phase-high performance liquid chromatographic method development, Validation

G, PAVANI

LATHA N, MADHAVI

P, SUPRIYA

RAMANA G, VENKATA

English

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Vol 8, Issue 6, 2015 ISSN - 0974-2441REVERSED PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD DEVELOPMENT AND VALIDATION OF ATORVASTATIN IN BULK DRUG AND FORMULATIONVENKATA RAMANA G*, PAVANI G, SUPRIYA P, MADHAVI LATHA NDepartment of Pharmaceutical Analysis, A. U. College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, Andhra Pradesh, India. Email: venkataramana1214@gmail.comReceived: 22 June 2015, Revised and Accepted: 03 August 2015ABSTRACTObjective: To develop and validate a simple, selective, rapid, precise, and accurate high performance liquid chromatographic (HPLC) method for determination of atorvastatin in bulk and its pharmaceutical formulation product.Method: Reversed phase-HPLC (RP-HPLC) method was performed by a mobile phase consisting mixture of methanol and ammonium acetate buffer (pH 4.5) in the proportion 60:40 v/v. A ZORBAX Eclipse plus C18 (4.6 mm × 100 mm, 3.5 μ) column was used as a stationary phase. HPLC analysis of atorvastatin was carried out at a wavelength of 241 nm with a flow rate of 1 ml/minute.Results: The linear regression analysis data for the calibration curve showed a good linear relationship with a correlation coefficient 0.9984. The linear regression equation was y=3726540.2x+27390388.1. This was found to give a sharp peak of atorvastatin at a retention time of 2.77 minutes. Validation parameters were evaluated for the method according to the ICH (Q2R1) guidelines. The limit of detection and limit of quantification for the method were 0.6721 μg/mL and 1.9989 μg/mL, respectively. The % relative standard deviation values for intra-day precision and inter-day precision were found to be 0.31% and 0.30%, respectively. An accuracy of the method was determined through recovery studies which were found to be within 97.57-102.22%.Conclusion: The method was validated for system suitability, accuracy, precision, robustness, and ruggedness. The precision, accuracy, sensitivity, short retention time and composition of the mobile phase indicated that this method is better than the earlier methods developed for the quantification of atorvastatin.Keywords: Atorvastatin, Reversed phase-high performance liquid chromatographic method development, Validation.INTRODUCTIONThis compound belongs to the class of organic compounds known as diphenylpyrroles. It is aromatic heterocyclic compounds with a structure based on a pyrrole ring linked to exactly two phenyl groups. It is chemically known as 7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-(propan-2-yl)-1H-pyrrol-1-yl]-3,5-dihydroxyheptanoate. The chemical structure is given in Fig. 1. It may be used to reduce the risk of cardiovascular events in patients with acute coronary syndrome and may be used in the treatment of primary hypercholesterolemia and mixed dyslipidemia, homozygous familial hypercholesterolemia, primary dysbetalipoproteinemia, and/or hypertriglyceridemia as an adjunct to dietary therapy to decrease serum total and low-density lipoprotein cholesterol (LDL-C), apolipoprotein B, and triglyceride concentrations, while increasing high-density lipoprotein cholesterol levels. Atorvastatin selectively and competitively inhibits the hepatic enzyme 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase [1,2].As HMG-CoA reductase is responsible for converting HMG-CoA to mevalonate in the cholesterol biosynthesis pathway, these results in a subsequent decrease in hepatic cholesterol levels. Decreased hepatic cholesterol levels stimulate up regulation of hepatic LDL-C receptors which increases hepatic uptake of LDL-C and reduces serum LDL-C concentrations. Atorvastatin is rapidly absorbed after oral administration with maximum plasma concentrations achieved in 1-2 hrs. Atorvastatin is extensively metabolized to ortho-and para-hydroxylated derivatives and various beta-oxidation products. Literature survey revealed that several high performance liquid chromatographic (HPLC) methods for determination of atorvastatin were reported but with longer retention time. Since the retention time is one of the important factors for the separation of drugs using HPLC, the present work reports the development and validation of HPLC method for the estimation of atorvastatin in bulk and formulation with shorter retention time, and the method has been highly used for its sensitivity and reproducibility.METHODSInstrumentsHPLC Agilent 1260 with PDA detector and auto sampler, Zorbax eclipse Plus C18 (4.6 × 100 mm, 3.5 μ) equipped with Open lab EZ Chrome software, Lab India – T60 UV/Vis double beam spectrophotometer, Mettler Toledo ME 204 balance, Eutech pH 700 pH meter.Chemicals and reagentsThe sample of atorvastatin standard was procured as gift sample from MSN Laboratories Ltd, Hyderabad, India. HPLC-grade methanol, water, and ammonium acetate were procured from Merck Pharmaceuticals Private Ltd., Mumbai, India. Glacial acetic acid was also used.Selection of detection wavelengthThe UV spectrum of diluted solutions of various concentrations of atorvastatin in mobile phase was recorded using UV spectrophotometer. The wavelength of maximum absorbance was observed at 241 nm. This wavelength was used for detection of atorvastatin.Research ArticleAsian J Pharm Clin Res, Vol 8, Issue 6, 2015, 84-87 Ramana et al. 85Preparation of mobile phaseThe content of the mobile phase was prepared from filtered and degassed mixture in the ratio 60:40 v/v of methanol and ammonium acetate buffer (1.54 g of ammonium acetate in 1.0 liter distilled Water) and pH was adjusted to 4.5 with 0.2 M glacial acetic acid.Preparation of atorvastatin standard stock solution100 mg of Atorvastatin powder was weighed and transferred into 100 ml volumetric flask, and 30 ml of diluent was added to it, sonicated and was further filtered through 0.45 μ filter paper and the volume was made up with diluent. Then 100 µg/mL working standard solution was prepared by pipetting out 10 ml of 1000 µg/mL solution into the 100 ml volumetric flask, and the remaining volume was made up with diluent.Preparation of sample solution10 tablets, each containing 500 mg of atorvastatin, were weighed and crushed into fine powder and amount of powder equivalent to 100 mg of atorvastatin was weighed and transferred into 100 ml dried volumetric flask. The content was dissolved by adding 30 ml of diluent and rapidly shaking for few minutes. The volume was made up with diluent, mixed well, and injected immediately.Optimized method for atorvastatinChromatographic conditionsColumn: Zorbax Eclipse Plus C18 (4.6 mm × 100 mm, 3.5 μ)Mobile phase: Methanol:ammonium acetate buffer - pH 4.5 (60:40)Flow rate: 1 ml/minutesWavelength: 241 nmColumn temperature: 25°CInjection volume: 15 μLRun time: 5 minutesDiluent: Methanol:water (60:40)Elution: IsocraticNeedle wash: MethanolMETHOD VALIDATION [5-11]LinearityThe linearity of an analytical procedure is its ability to obtain test results, which are directly proportional to the concentration of an analyte in the sample. A linear relationship should be evaluated across the range of the analytical procedure. It is demonstrated directly on the drug substance by dilution of a standard stock solution of the drug product components, using the proposed procedure. For the establishment of linearity, minimum of five concentrations are recommended by ICH guideline. The value of correlation coefficient should fall around 0.99. The regression equation and correlation coefficient was calculated and found to be within the required limits as shown in Tables 1 and 2, respectively.PrecisionThe precision of the analytical procedure expresses the closeness of agreement among series of measurements obtained from multiple sampling of the same homogeneous sample. The precision of the analytical procedure is usually expressed as the variance, standard deviation, or coefficient of variation of series of measurements. The intra-day and inter-day precision results were shown in Tables 3 and 4, respectively.Accuracy/recoveryThe accuracy of the analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. The evaluation of accuracy has got very prime importance as it deliberately forces the method to extract the drug and impurities at higher and lower level. The recovery results for accuracy study of atorvastatin were represented in Table 5.Limit of detection (LOD) and limit of quantification (LOQ)LOD is the lowest concentration in a sample that can be detected but not necessarily quantified under the stated experimental conditions. The LOQ is the lowest concentration of the analyte in a sample that can be determined with acceptable precision and accuracy. LOD and LOQ were calculated using following formula:LOD = 3.3 SD/S and LOQ = 10SD/SWhere, SD = Standard deviation of response (peak area) and S = Slope of the calibration curve.RobustnessThe robustness of the analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. The results for robustness study were shown in Tables 6-8, respectively.RESULTS AND DISCUSSIONSLinearityThe standard calibration curve was constructed between concentration Vs peak area and linearity was found in the range from 20 to 100 µg/mL. The regression equation and correlation coefficient were calculated and found to be within the required limits as specified in ICH guidelines. The calibration curve was given in Fig. 2.PrecisionIntra-day precision was investigated by replicate applications and measurements of peak area for atorvastatin for six times on the same Table 1: Linearity resultsS. No Concentration (µg/ml) Peak area1 20 9790047652 40 1795043903 60 2570389204 80 3203270535 100 400143166Table 2: Linearity parameters and their valuesS. No Parameters Values1 Concentration range 20-100 µg/mL2 Regression equation (Y) Y=3726540.2X+27390388.13 Correlation coefficient (r2) 0.9984 Slope (m) 3726540.25 y-intercept (c) 27390388.1Table 3: Intra‑day results for precisionConcentration (µg/mL) Injection number Area % RSD60 (µg/ml) 1 257038920 0.312 2582124713 2561265104 2572015465 2570034816 256021567Table 4: Inter‑day results for precisionConcentration (µg/ml)Injection numberArea % RSD60 (µg/ml) 1 257124852 0.302 2585698423 2565214364 2572542165 2570421576 256367421Asian J Pharm Clin Res, Vol 8, Issue 6, 2015, 84-87 Ramana et al. 86day under similar conditions. Inter-day precision was obtained from % relative standard deviation (RSD) values obtained by repeating the assay 6 times on 2 different days. The % RSD was calculated which was within the acceptable limits of not more than 2.0.AccuracyThe accuracy of the method was tested by the triplicate sample at 3 different concentrations equivalent to 75%, 100%, and 125% of the active ingredient, by adding a known amount of atorvastatin standard to a sample with a pre-determined amount of atorvastatin. The recovered amount of atorvastatin, % RSD of recovery, and % recovery of each concentration were calculated to determine the accuracy.RobustnessRobustness is the ability to provide accurate and precise results under a variety of conditions. To measure the extent of method robustness, the Fig. 1: Structure of atorvastatin [3,4]Fig. 2: Calibration curve of atorvastatinFig. 3: Blank chromatogram of atorvastatinTable 5: Results for accuracySample number % level Standard amount Spiked amount % recovery Mean recovery1 75 80 60 102.20 Mean=102.2212 80 60 102.22 SD=0.01803 80 60 102.24 % RSD=0.02%1 100 80 80 99.98 Mean=99.992 80 80 100 SD=0.01153 80 80 100 % RSD=0.01%1 125 80 100 97.57 Mean=97.572 80 100 97.58 SD=0.00573 80 100 97.57 % RSD=0.01%SD: Standard deviation, RSD: Relative standard deviationTable 6: Results for robustness flow rate variationS. No Concentration (µg/mL)Area0.8 (ml/minute) 1.2 (ml/minute)1 (60 µg/mL) 257038916 2571359152 254217540 2572175403 257982451 2568245244 25824503 2562145005 255124901 2555249076 257412031 257612034% RSD 0.63 0.30RSD: Relative standard deviationTable 7: Results for robustness pH variationS. No Concentration (µg/mL)AreapH 4.3 pH 4.71 (60 µg/mL) 257412820 2548712302 254871245 2587469503 256485124 2574812454 251538546 2541325495 252487120 2531572456 259124850 259854721% RSD 1.15 1.06RSD: Relative standard deviationTable 8: Results for robustness mobile phase composition variationS. No Concentration (µg/mL)Area (Methanol:Buffer)65:35 v/v 75:25 v/v1 (60 µg/mL) 254745147 2574812472 256487120 2574215103 258694721 2568542144 257124573 2531426585 257654801 2584512706 256124523 256140124% RSD 0.53 0.72RSD: Relative standard deviationAsian J Pharm Clin Res, Vol 8, Issue 6, 2015, 84-87 Ramana et al. 87most critical parameters were interchanged while keeping the other parameters unchanged and in parallel, the chromatographic profile was observed and recorded. The studied parameters were the composition of flow rate, pH, and mobile phase. The results for robustness study Fig. 4: Chromatogram of placeboFig. 5: Standard chromatogram of atorvastatinFig. 6: Formulation chromatogram of atorvastatinindicated that the small change in the conditions did not significantly affect the determination of atorvastatin.LOD and LOQThe LOD was found to be 0.6721 µg/mL.The LOQ was found to be 1.9989 µg/mL.CONCLUSIONThe proposed method for the assay of atorvastatin was rapid, accurate, precise, and sensitive for the quantification of atorvastatin from its pharmaceutical dosage forms. The method was validated for linearity, accuracy, precision, LOD, LOQ, and robustness. The method was free from the interference of other active ingredient and excipients. Hence, it can be concluded that this method may be employed for routine quality control analysis of atorvastatin in active pharmaceutical ingredient and formulation product. The chromatograms for blank, placebo, standard and formulation product were given in Figs. 3, 4, 5 and 6.ACKNOWLEDGMENTWe are thankful to MSN laboratories ltd, Hyderabad, India for providing sample of Atorvastatin. We also thank Dr. A.K.M. Pawar, Asst. Professor, A.U College of pharmaceutical sciences, Visakhapatnam for providing necessary facilities to carry out the research work.REFERENCES1. Lakshmi surekha M, Kumara swamy G, Vinay kumar D. Development and validation of RP-HPLC method for the estimation of atorvastatin in bulk and Tablet dosage form. Int J Pharm Sci 2012;2(4):91-3.2. Anjum F, Radhika T, Kumari KR, Ajitha M. Int Res J Pharm 2012;(2230-8407):177-180.3. Available from: https://www.google.co.in/webhp?sourceid=chrome-instant&ion=1&espv=2&ie=UTF-8#q=atorvastatin+drug+bank. [Last cited on 2015 Jun 20].4. Nash RA. Pharmaceutical Process Validation, An International 3rd ed. Revised and Expanded. Vol. 129. New York: Eastern Hemisphere Distribution, Marcel Dekker, Inc; p. 507-22.5. Chatwal GR. Instrumental Methods of Chemical Analysis. Mumbai: Himalaya Publishing House; 1979. p. 2.566-2.699.6. ICH, Q2B, Harmonised Tripartite Guideline, Validation of Analytical Procedure: Methodology, IFPMA. In: Proceedings of the International Conference on Harmonisation. Geneva: March, 1996.7. Snyder LR. Practical HPLC Method Development. 2nd ed. New York: Wiley-Interscience; 1997.8. Kumar P, Ghosh A, Chaudhary M. Stability indicating method development for simultaneous estimation of ezetimibe and atorvastatin in pharmaceutical formulations by RP-HPLC. Pharm Anal Acta 2012;3:6.9. Ahmed M, Manohara YN, Ravi MC. RP-HPLC method development and validation for simultaneous estimation of atorvastatin calcium and amlodipine besylate. Int J ChemTech Res 2012;4(1):337-345.10. Surekha ML, Swamy GK, Kumar DV. Development and validation of RP-HPLC method for the estimation of atorvastatin in bulk and tablet dosage form. Int J Pharma Sci 2012;2(4):91-3.

REVERSED PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD DEVELOPMENT AND VALIDATION OF ATORVASTATIN IN BULK DRUG AND FORMULATION

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REVERSED PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD DEVELOPMENT AND VALIDATION OF ATORVASTATIN IN BULK DRUG AND FORMULATION

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