Rp-HPLC Method Development and Validation for Simultaneous Estimation of Atorvestatin Calcium and Fenofibrate in Bulk and Formulation

A reverse phase high performance liquid chromatographic method was developed for the simultaneous determination of atorvastatin and Fenofibrate in bulk and pharmaceutical dosage forms. The determination was performed by using Waters Symmetry C18 (250×4.6mm×5μ) as column stationary phase and Methanol: Acetonitrile: Water in the ratio of (70:10:20 %v/v) as mobile phase. The flow rate of mobile phase was optimized as 1mL/min and effluents were monitored at 274 nm. The retention time of atorvastatin and Fenofibrate were found as 4.15 min and 8.10 min respectively. The method shows linearity concentration ranges between 4-30 μg/mL and 80-400 μg/mL respectively. The developed method was validated for specificity, precision, linearity, accuracy, robustness, Ruggedness, LOD and LOQ. Recovery of atorvastatin and Fenofibrate in formulations was found to be 100.52% and 99.92% respectively which conforms the noninterferences of the excipients in the formulation. the proposed RPHPLC method can be used for the simultaneous determination of these two drugs in bulk and formulation.


MATERIALS AND METHODS
Quantitative HPLC was performed on a highperformance liquid chromatograph -Waters HPLC system connected with PDA Detector and Empower 2 Software. The drug analysis data were acquired and processed using Empower2 software running under Windows XP on a Pentium PC and C18column of dimension 250 × 4.6, 5µm particle size.

Preparation and Selection of Mobile Phase
The preliminary isocratic studies on a reverse phase C18 column with different mobile phases like Acetonitrile, Methanol and Distilled water different buffers were tried. After some trials the mobile phase was optimized as follows.
A mixture of 70 volumes of Methanol, 10 volumes of Acetonitrile and 20 volumes of Water was used as mobile phase. The mobile phase was filtered through 0.45µ membrane filter to remove all fine particles and ultra-sonicated for 10min to remove dissolved gases [17].

Preparation of Standard Solution
Preparation of standard stock solutions of Fenofibrate and atorvastatin: Weigh accurately 10 mg of Fenofibrate and atorvastatin (Working standard) in to a 100 ml volumetric flask and dissolved in Methanol and then dilute up to the mark with methanol this solution was further diluted to get desired concentration range.

Preparation of Sample Solution
Weigh accurately powder equivalent to 16 μg/ml of Fenofibrate and 1mg of atorvastatin and transfer to 10ml volumetric flask. Then add enough mobile phase to dissolve the content by vigorous shaking or subjected to sonication and make up the final volume up to the mark with mobile phase. Then filter the resulting solution through whattman filter paper.
From the above solution pipette out required aliquots and dilute with mobile phase to prepare the solution containing the concentration of 160 μg/ml of Fenofibrate and10μg/ml of atorvastatin. This solution was used for recording the chromatogram.

Chromatographic Conditions
The mobile phase methanol: Acetonitrile : Water in the ratio of 70: 10: 20 v/v was pumped at a flow rate of 1 mL /min through the waters symmetry (250×4.6×5µ) column at room temperature. The mobile phase was degassed prior to use under vacuum by filtration through a 0.45μ membrane filter. Both drugs showed high absorbance values at 274 nm, which was selected as wavelength for further analysis.

System Suitability Study
System suitability study of the method was carried out by six replicate analysis of solution containing 100% target concentration of atorvastatin and Fenofibrate. Various chromatographic parameters such as retention time, peak area tailing factor, theoretical plates (Tangent) of the column and resolution between the peaks were determined and the method was evaluated by analysing these parameters ( Table 2).

Validation of Developed Analytical
Method [18][19][20][21][22] Linearity: Linearity of the method was determined by constructing calibration curves. Standard solutions of atorvastatin and Fenofibrate of different concentrations level were used for this purpose. Each measurement was carried out in six replicates and the peak areas of the chromatograms were plotted against the concentrations to obtain the calibration curves and correlation coefficients.

Accuracy (Recovery Studies):
To check the degree of accuracy of the method, recovery studies were performed in triplicate by standard addition method at 80%, 100% and 120%. Known amounts of standard drugs were added to pre-analyzed samples and were subjected to the proposed HPLC method. The measured value was obtained by recovery test. Spiked amount of both the drugs were compared against the recovery amount. % recovery was 100.52% for atorvastatin and 99.92% for Fenofibrate (Table 3 and 4).
Precision: Precision was evaluated by carrying out six independent sample preparation of a single lot of formulation. The sample solution was prepared in the same manner as described in sample preparation

Robustness of Method:
To evaluate the robustness of the developed RP-HPLC method the prepared solution as per the test method was injected at different variable conditions like using different conditions flow rate and wavelength. (Table 5)

Limit of detection and limit of quantification:
The limit of detection (LOD) and limit of quantification (LOQ) of the drug were calculated using the following equations as per International Conference of Harmonization (ICH) guidelines. (Table 6) LOD = 3.3 X α /S LOQ = 10 X α /S

RESULTS AND DISCUSSION
Development and optimization of the method Column chemistry, solvent type, solvent strength, detection wavelength and flow rate were varied to determine the chromatographic conditions giving the best separation. Separation with good resolutions was studied on different type's columns (C8 and C18). The mobile phase conditions were optimized so that the components were not interfered from the solvent and excipients. Several buffer systems at different pH values were trailed in various ratios with methanol, and acetonitrile as mobile phase. Mobile phase and flow rate selection was based on peak parameters like area, tailing, theoretical plates, capacity factor and resolution. Decisively after several experimental trials, the best result was obtained by use of phase methanol: Acetonitrile: Water in the ratio of 70: 10: 20 v/v with 1.0 mL/min flow rate.
System suitability parameters like number of plates, asymmetry factor, resolution was found within the specified values suggested by regulatory guidelines seen in Table 1.
The developed method was validated as per the parameters like System suitability parameters, linearity, precession, accuracy, robustness, and the values all above parameters are within the limit so the developed method was validated according to ICH guidelines. Linearity was determined based on value of the correlation coefficient for standard preparations of atorvastatin and Fenofibrate which was found to be 0.999 and 0.999 respectively. The relationship between the concentration of atorvastatin and Fenofibrate and area atorvastatin and Fenofibrate was linear in the range examined since all points lie in a straight line and the correlation coefficient was well within limits. Limit of Detection and limit of quantitation was calculated based on the values of slope obtained from calibration curve. (Table 2).
Precision of the method for intra-day study was evaluated based on relative standard deviation and Percentage relative standard deviation (%RSD) which was found to be less than 2% for within a day variation, which proves that method is precise. ( Table 3).
Accuracy of the developed method was evaluated based on recovery studies and mean percentage recovery value for each drug was found within 98-102% range which shows that there is no interference of excipients on the results. The mean percentage recovery of Atorvastatin and Fenofibrate was 100.53% and 99.92% respectively. (Tables 4 and 5).
Robustness of the method was evaluated by some small and deliberate changes in the operating conditions and effect was evaluated with relative standard deviation. Values of relative standard deviation shown in Table 5 suggests that there is no major effect of change in operating conditions. Developed method was used for analysis of commercial formulation and amount of drug obtained by the method was checked with the label claim amount and assay value was found to be 99.50 and 99.87% for atorvastatin and fenofibrate respectively. (Table 6)

CONCLUSION
The developed method gave good resolution between atorvastatin and fenofibrate with short analysis time and high efficiency and complies with all system suitability test specifications of USP. The use of C18 column in the present work has indicated better elution of analytes with good resolution, improved plate count and capacity factor.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.