Atorvastain calcium trihydrate (CAS No. 344423-98-9; M.W. 1209) is a white powdery substance chemically known as Calcium (3R,5R)-7-(2-(4-fluorophenyl)-5-(1-methylethyl)- phenyl-4-^{(phenylcarbamoyl)}-3,5-dihydroxyheptanoate trihydrate 1. The drug is soluble in methanol having molecular formula of C₆₆H₆₈CaF₂N₄O₁₀,3H₂O.

Atorvastatin is an adjunct to diet for reduction of elevated total cholesterol. Atorvastatin selectively and competitively inhibits hepatic enzyme 3-hydroxyl- 3-methyl glutaryl-coenzyme A (HMGCoA) reductase, lowering plasma cholesterol levels by suppressing hepatic production of very low density lipoprotein and low density lipoprotein cholesterol. The effectiveness of atorvastatin in lowering cholesterol is dose-related. It is available in 10, 20, 40, and 80 mg film coated tablets. In order to ensure quality, safety and efficacy of tablets in formulations, it is important to develop new analytical method for quantitative analysis of drug in pharmaceutical formulations. Azmi and co-workers developed several analytical methods for the estimation and quality control analysis of cefixime 2 and citalopram HBr 3. With increasing regulatory strictness, the quality and safety of atorvastatin calcium is important. Owing to this reason, various analytical methods have been developed in pure and dosage forms such as spectrophotometry 4, high performance thin layer chromatography 56, high performance liquid chromatography 7891011121314151617 and liquid chromatography-mass spectrometry 18. Most of these analytical methods utilize expensive reagents 4, sophisticated instrumentations 789101112131415161718 and more analysis time 56. Published HPLC methods utilized higher column temperature 91214151617 and higher retention time 78910111317. Higher temperature having the possibility of degrading the active drug and higher retention time will delay the analysis process. Therefore, it is required to develop a rapid and accurate high performance liquid chromatographic-UV method with lower retention time at ambient temperature. The main aim of the present work is to develop a new validated reversed phase high performance liquid chromatographic-UV method for the determination of atorvastatin calcium in pharmaceutical formulations. The method was validated as per International Conference on Harmonisation (USA) guidelines 19.

The UV visible absorption spectrum of atorvastatin calcium solution in mobile phases of acetonitrile-dichloromethane-acetic acid (68.6: 30.6: 0.8 v/v/v) and 0.005 M potassium dihydrogen phosphate-acetonitrile-methanol (39: 56: 5 v/v/v; pH 4.6) were recorded in the wavelength range of 190-400 nm. The λ_max of 246 nm was recorded in each mobile phase and thus used as UV detection wavelength in HPLC system for HPLC separation of atorvastatin calcium.

The solution stability of atorvastatin calcium in both mobile phases was checked by UV-visible spectrophotometry for a period of 1 day. The results showed an absorption peak at 240 nm with no change in the absorbance. The solution stability of the standard drug solutions was also tested using TLC plates coated with silica gel G (Merck Limited, Mumbai, India) and developed in acetonitrile: dichloromethane: acetic acid (45:20:0.5 v/v/v) and acetonitrile: 0.005 M KH₂PO_4: methanol (28.5: 5: 2.5 v/v/v) solvent systems. The results showed a single spot with R_fof 0.66 (acetonitrile: dichloromethane: acetic acid - 45:20:0.5 v/v/v) and 0.75 (acetonitrile: 0.005 M KH₂PO_4: methanol - 28.5: 5: 2.5 v/v/v) indicated that the drug solutions were stable for at least 1 day. Hence, the prepared drug can be analyzed within the stability time period of 1 day.

Method validation was performed on the best determined stationary phase i.e. C18 column (250 mm × 4.6 mm; i.d. 5 µm particle size). Separation with good resolution and low tailing factor (less than 2) were advantages of using C18 column.

The basic chromatographic conditions were optimized for developing a new HPLC-UV method for determination of atorvastatin calcium after testing different conditions of HPLC such as column temperature, components of mobile phase, proportion of mobile phase components, detection wavelength, and flow rate.

The mobile phase of acetonitrile-dichloromethane-acetic acid (68.6: 30.6: 0.8 v/v/v) was selected after preliminary trials using different compositions of mobile phase. The chromatogram obtained showed better eluted peak, less retention time and low tailing factor (Figure 1). It is clear from the figure that the retention time of atorvastatin calcium using the said mobile was 2.68 min. The low retention will fasten the analysis, hence selected the said mobile phase for HPLC separation of atorvastatin calcium.

Under theoptimized experimental conditions, peak areas at different concentrations of atorvastatin were obtained. The calibration data were inserted in the Origin Pro 6.1 software and utilized for obtaining linear regression equation with coefficient of correlation. The results are summarized in Table 1. It is evident from the table that the linear regression equation of A= 0.077 + 0.156 C was obtained with high coefficient of correlation (r = 0.9999). With this linear equation, the concentration of atorvastatin calcium was linear in the concentration range of 15-300 µg mL^-1. Analytical parameters and chromatographic system suitability data of proposed and reference methods are given in Table 1. The results in the Table 1 supported the suitability of the proposed and reference methods. The experimental intercept of the proposed method was checked for significance of deviation from the theoretical intercept, zero using t = a / S_a 23 and found to be 0.726, which is less than the tabulated t-value (2.571, =5) at 95% confidence level indicated an acceptable intercept. The acceptable intercept proved that the proposed method was procedurally error free.

Intra (within day) and inter day (between days) precisions were tested by determining the concentration of atorvastatin calcium at lower and upper concentration levels for 5 repeated times within the same day and on five consecutive days, respectively. The results are summarized in Table 2. It can be seen from the table that % relative standard deviation (RSD) values were in the range of 0.143-0.162 % for intraday and inter day precisions. The low % RSD values (intraday and interday precisions) at different concentrations showed that the proposed method was precise and can be used to analyze atorvastatin calcium in pharmaceutical formulations.

The selectivity of the proposed method was investigated by testing the interference from common excipients such as glucose, fructose, lactose, sodium benzoate, starch, povidone, methyl cellulose and micro crystalline cellulose at 60 mg mL^-1 of atorvastatin calcium. The peak area was recorded at varying concentrations of excipients and the results are summarized Table 3. Microcrystalline cellulose was tested and found to be insoluble in distilled water whereas methyl cellulose gave a viscous solution in distilled water. Therefore, both excipients were removed as added with drug in mobile phase and hence did not interfere in the determination process of atorvastatin calcium. The proposed method tolerated larger amount of excipients indicated that the proposed method was specific and selective, thus can be used to determine atorvastatin calcium in pharmaceutical formulations.

The ruggedness of the proposed method was examined with a small change in the optimized data in the following manner.

volume of acetonitrile-dichloromethane-acetic acid (68.6: 30.6: 0.8 v/v/v, pH 4.5) (± 0.2 mL)

column temperature, 25 ˚C (± 1.0 ˚C)

flow rate, 1 mL min-1 (± 0.2 mL min-1)

In the above conditions, tablet solution of atorvastatin calcium (Torvast) at 60.0 µg mL^-1 atorvastatin calcium was analyzed by the proposed method. The results showed mean % recovery of 100.04. Hence the proposed method was rugged and considered to be reliable for determination of active drug in tablet formulations.

The accuracy of the proposed method was tested by performing recovery experiments through standard addition technique. For this purpose, known portions of pure atorvastatin calcium were spiked with definite amount of Torvast solution (unknown) and the peak area was recorded. Standard addition graph was obtained by plotting the peak area versus concentration of added standard drug solution (Figure 2). The linearity of the regression line for Torvast tablet solution was good (r = 0.999). The intercept and slope of 9.414 and 0.157, respectively were obtained. The amount of atorvastatin calcium in Torvast tablet solution was calculated and found to be 59.86 µg mL^-1 with % recovery of 99.76. The amount of drug in tablet solution was subjected to standard deviation, , and calculated using the following expression:

The value of was found to be 1.038 µg mL^-1. The confidence limit for the concentration of atorvastatin calcium in Torvast was calculated by at n - 2 degrees of freedom and found to be 59.86 ± 1.038 µg mL^-1. The most attractive feature of the proposed method by standard addition technique was its relative freedom from pharmaceutical additives and excipients and hence did not interfere with the determination process.

The applicability of the proposed method for the determination of atorvastatin calcium in Torvast and Atorlip has been tested. The results of the proposed method were statistically compared with those of the reference method using point and interval hypothesis tests. t- and F-values at 95% confidence level were calculated using point hypothesis test and found to be in the range of 0.769-1.581 and 1.617-2.275, respectively. Both cases, t and F values were found to be less than the theoretical t and F values at 95% confidence level and hence proved that both methods were accurate and having no significant difference between them. The results are summarized in Table 4. Interval hypothesis test was utilized to calculate bias (lower limit, θ_L and upper limit, θ_U) in two methods and found to be in the range of 0.98-1.02 indicated the compliance of regulatory guidelines 21.

The performance of the proposed HPLC method was compared with other existing HPLC methods (Table 5). As can be seen from the table, the linearity range was higher from the existing HPLC methods 789. Ambient temperature was used for preventing the possible degradation of active atorvastatin. Retention time was reasonably low as compared to the reported HPLC methods 78910 providing fast analysis of active atorvastatin in pharmaceutical preparations. The volume of acetonitrile in mobile phase was reasonable low as compared to other HPLC methods 89. Hence, the overall performance of the proposed method was quite appreciable, and hence can be used as alternate method routine quality control analysis of active atorvastatin in commercial dosage forms.

The proposed method was successfully applied for the determination of atorvastatin calcium in pharmaceutical formulations in the presence of excipients. The separation of the drug was achieved in 2.68 min. The proposed method was more specific, selective and rapid. It followed system suitability parameters such as tailing factor of 1.81 and theoretical plate of 4418. The ease of operation, sensitivity and reproducibility of the proposed method made it more suitable for routine quality control analysis of drug in research laboratories, hospitals and pharmaceutical industry.