DETERMINATION OF ATORVASTATIN CALCIUM IN PHARMACEUTICAL FORMULATIONS BY REVERSE PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A simple, sensitive and reproducible reverse-phase high performance liquid chromatographic (RP-HPLC) method has been developed for the quantitative estimation of Atorvastatin calcium in the pharmaceutical formulations. Chromatographic separation was achieved on a 250 × 4.6 mm, 5µ, Waters symmetry column. The flow rate was 1ml/min and eluent was monitored by absorbance at 246 nm using a mixture of Methanol and Acetonitrile (pH 3.0±0.01) in the ratio of 25:75 (v/v). The retention times of Atorvastatin calcium was found to be 5.5 min. Calibration plots were linear in the concentration range of 5-25 µg/ml for Atorvastatin calcium. The total run time is 12 min. The proposed method was validated by testing its linearity, recovery, specificity, system suitability, precision (Interday, intraday, analyst and instrument precision), robustness and LOD/LOQ values and it was successfully employed for the determination of Atorvastatin calcium in pharmaceutical tablet formulations.


Introduction:
Atorvastatin calcium (ATOR) (Fig. 1) is the calcium salt (2:1) trihydrate of [R-(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methyl ethyl)-3-phenyl4 [ (phenylamino) carbonyl] lH pyrrole-heptanoic acid. It is an inhibitor of 3hydroxy-3-methyl glutaryl-coenzyme A (HMGCoA) reductase. Atorvastatin is the most efficacious of the currently available HMG-CoA reductase inhibitors in terms of lowering plasma cholesterol levels by suppressing the hepatic production of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) cholesterol 1 . A novel formulation Atorvastatin calcium commercially available, benefits for antihyperlipidemic action. This provides powerful efficacy for day long control of BP and has proven evidence in cardiovascular (CV) outcomes of Atorvastatin calcium 2 . Literature review revealed that there are various methods for determination of Atorvastatin calcium, individually and in combination with other drugs. A variety of analytical methods are reported such as, estimation of enantiomeric of Atorvastatin 3 , in human serum 4 and its impurity in bulk drugs 5 . The majority of methods reported are liquid chromatography in which atorvastatin was estimated simultaneously with ezetimibe 1, 6, 7 , fenofibrate 8 , aspirin 9, 10 , ramipril 11,12 , nicotinic acid 13 and amlodipine 14,15,16,17,18 . Some stability indicating RP-HPLC methods of atorvastatin and amlodipine 19 was also reported. Some triple combination of atorvastatin was reported with aspirin and pioglitazone 20  (G1315C), Quaternary pump (G1311A) and column thermostat (G1316A). Data acquisition was made with Chemstation software. The peak purity was evaluated with DAD detector. c. Liquid chromatographic conditions: Chromatographic conditions were obtained using a stainless steel column (Waters symmetry C 18 250mm x 4.6mm 5µm), which was maintained at 40⁰C. The analytical wavelength was set at 246 nm and samples of 20µl were injected to HPLC system. The mobile phase was Potassium dihydrogen phosphate (10mM, pH 3.0 adjusted with ortho-phosphoric acid) and acetonitrile in ratio of 60:40 (v/v) at a flow rate of 1ml/min. The mobile phase was filtered through 0.45µm filter and degassed for 10 minutes by sonication. Accurately weighed quantities of ATOR (≈10 mg) was transferred into a 100 ml volumetric flask, than dissolved with 25 ml of methanol and diluted to required volume with mobile phase, having the concentration of 100 µg/ml of ATOR. An accurately measured 1.0 ml portion of the resultant solution was diluted to 10.0 ml with diluent to obtain a laboratory mixture having concentration similar to marketed formulation.

• Sample preparation:
Twenty tablets (ATOCOR, Dr. Reddy's) were weighed and ground to a fine powder. An amount of powder equivalent to 10mg of Atorvastatin calcium was weighed accurately and transferred into a 100 ml A-grade volumetric flask containing 25 ml of methanol and sonicated for 30 min to effect complete dissolution of the Atorvastatin and diluted upto 100 ml with diluent, then the solution was filtered through 0.45 µm membrane filter and 10 ml of filtrate taken into 100 ml volumetric flask. The aliquot portion of the filtrate was further diluted to get final concentration of 10 µg/ml of Atorvastatin calcium. e. Linearity study and Calibration curve: To study the linearity range of component, serial dilutions were made to obtain working standards in the concentration range of Atorvastatin calcium (5-25 µg/ml). A graph was plotted as concentration of drugs versus peak area response and results found linear for analytes. From the standard stock solution, a mixed standard of working concentration was prepared containing Atorvastatin calcium (10 µg/ml). The system suitability test was performed from five replicate injection of mixed standard solution. f. Analysis of Laboratory Mixture: In order to establish suitability of the proposed method for quantative estimation of Atorvastatin calcium in the pharmaceutical formulations, the method was first tried for the estimation of the component in a standard laboratory mixture of two drugs by using eq. 1and 2. h. Recovery study: Recovery studies were performed to validate the accuracy of developed method. For recovery study different concentrations (50%, 100% and 150%) of standard drug was prepared and then its recovery was analyzed. i. Method validation: The HPLC method was validated in terms of precision, accuracy, specificity and linearity according to ICH guidelines 22 . Accuracy: The accuracy of the assay method was evaluated with the recovery of the standards from excipients. Three different quantities (low, medium and high i.e. 50%, 100% and 150%) of the authentic standards were added to the placebo. The mixtures were extracted as described in section 2d, and were analyzed using the developed HPLC method. Precision: Assay method precision was determined using nine-independent test solutions (3 concentration/3 replicates). To study precision 80%, 100% and 120% concentration was prepared and three replicate of each concentration was injected. The intermediate precision of the assay method was also evaluated using different analyst different days. Specificity: Accurately weighed quantities of the tablets powder equivalent to about 10 mg of ATOR was taken in a dry 50.0 ml volumetric flask. Each sample solution was stored under following different relevant small stress conditions (light, heat, acid/base hydrolysis and oxidation) for sufficient time (24 hrs) to achieve 10 to 30% degradation of the initial sample.
1. Addition of small amount of alkali solution (0.1 N NaOH). 2. Addition of small amount of acid solution (0.1 N HCl). 3. Addition of small amount of oxidative agent (3% H 2 O 2 ). 4. Sample solution was heated 50 0 C on water bath for a sufficient time 5. Sample solution was exposed 600 footcandle of UV light for a sufficient time.
After 24 hr each treated sample was analyzed and percent labeled claims were calculated by the method using formula under estimation of ATOR by proposed method. Linearity: Solutions for linearity study were prepared as described in Section 2e. Six replicates of each concentration were injected and results are examined and it was found that calibration curve was linear in the concentration range of 5-25 µg/ml for TELM with correlation coefficient (R 2 ) 0.999. LOD and LOQ: The LOD and LOQ for analytes were estimated by SD of injecting a series of dilute solutions of known concentrations. Ruggedness: Ruggedness was ascertained by getting the sample analyzed from different analysts and carrying out analysis on different days by proposed method.

Robustness:
To determine the robustness of the method, the final experimental conditions were altered and the results were examined. The ratio of mobile phase was varied.

Results and Discussion:
Optimization of the chromatographic conditions: In order to develop RP-HPLC method for antihyperlipidemic drug Atorvastatin in formulation. The chromatographic conditions were optimized for better resolution by using different buffers like phosphate, acetate and citrate for mobile phase preparation. After a series of screening experiments, it was concluded that Phosphate buffer (10mM Phosphate buffer pH at 3.0) gave better peak shapes than their acetate and citrate counterparts. With methanol as solvent both the peaks shows less theoretical plates and bad peak shapes, on changing to acetonitrile the peak shape improved along with theoretical plates. Further optimization experiments were carried out 30% and 40% of acetonitrile in mobile phase. The best peak shape and maximum separation was achieved with mobile phase composition consisting acetate buffer-acetonitrile (60:40 v/v). The best separation, peak symmetry and reproducibility were obtained on Waters symmetry C 18 , 250 mm x 4.6 mm, 5 µm column compared to Hypersil ODS C 18 , 250 mm x 4.6 mm, 5 µm. The optimum wavelength for detecting the analytes was ascertained and found to be 246 nm. The specificity of the HPLC method is illustrated in Fig. 2 and Fig. 3, where complete separation of Atorvastatin was noticed in presence of tablet excipients and its impurities produced by alkali and thermal degradation. There were no interfering peaks of endogenous compounds observed at the retention time of the analytes. Accuracy of the method was calculated by recovery studies at three levels by standard addition method ( Table 1). The mean percentage recoveries obtained for Atorvastatin were 100.23. Precision is the degree of repeatability of an analytical method under normal operational conditions. The system precision is a measure of the method variability that can be expected for a given analyst performing the analysis and was determined by performing 80%, 100% and 120% analyses of the working solution.
The intra-day, inter-day, analyst and instruments variability or precision data are summarized in Table 3. The R.S.D of the assay results, expressed as percentage of the label claim, was used to evaluate the method precision. The inter-day precision was also determined by assaying the tablets in triplicate per day. The results indicated the good precision of the developed method.
The developed method was applied to the analysis of Atorvastatin in tablet dosage from marketed as ATOCOR (Label claim 10 mg strength, Dr. Reddy's). The results of analysis are given in Table 5 and fig. 4. The contents of marketed tablet dosage form were found to be in the range of 100±2% with RSD less than 2% which indicate suitability for routine analysis of Atorvastatin in tablet dosage form.
The robustness of an 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. Robustness of the method was investigated under a variety of conditions including changes of pH of the mobile phase, flow rate, percentage of acetonitrile in the mobile phase. The mixed standard solution is injected in five replicates and sample solution of 100% concentration is prepared and injected in triplicate for every condition and % R.S.D. of assay was calculated for each condition. The degree of reproducibility of the results obtained as a result of small deliberate variations in the method parameters has proven that the method is robust (Table 4).

Conclusion:
A simple, specific, linear, precise and accurate RP-HPLC method has been developed and validated for quantitative determination of Atorvastatin tablet formulation. The method is very simple and specific as both peaks are well separated from its excipient peaks and with total runtime of 12 min, makes the developed method it's suitable for routine quality control analysis work.