SIMULTANEOUS DETERMINATION OF AMLODIPINE AND ATORVASTATIN IN CADUET® TABLETS USING HPLC

A simple, selective, sensitive and precise, simulta neous high performance liquid chromatographic analysis of tablets containing amlodipine and atorv astatin was described. Good chromatographic separation was achieved using a Perfectsilr Target ODS-3 (4.6 cm × 250 mm, 5 μm) and a mobile phase consisting of acetonitrile-phosphate buffer pH 4.5 (5 :45, v/v) at a flow rate 1ML min . The ultraviolet detector was set at wavelength 237 nm. Amlodipine a d torvastatin were measured at 1.071 and 3.765 min, respectively. The linear ranges for amlodipine and atorvastatin were 1-10 and 5-50 μg mL , respectively. The recoveries of amlodipine and ator vastatin in pharmaceutical preparation were all gre ater than 98.5% and their relative standard deviations w ere less than 2.0%. The limits of detection were 0. 19 and 1.25 μg mL −1 for amlodipine and atorvastatin, respectively.


INTRODUCTION
Caduet is a combination of amlodipine ( Fig. 1) and atorvastatin ( Fig. 2) that is administered orally. They work together to control the blood pressure in patients with cardiovascular and hyperlipidemia problems.
Amlodipine is a calcium ion influx inhibitor (slow channel blocker or calcium ion antagonist) and inhibits the trans membrane influx of calcium ions into cardiac and vascular smooth muscle (O'Neil, 2006).
Atorvastatin is a competitive inhibitor of HMG-CoA reductase. It is a completely synthetic compound, which is the rate-limiting step in hepatic cholesterol biosynthesis (Elobieta et al., 2012).
Several methods have been reported for the analysis of atorvastatin either in bulk powder, different dosage forms or in biological fluids. These methods include spectrophotometric (Darwish et al., 2011) and Chromatographic (Shariati et al., 2013;Pilli et al., 2011) methods.

Chemicals
Amlodipine besylate and atorvastatin calcium were kindly supplied by PFIZER Company.

Pharmaceutical Preparation
Caduet ® tablets; B.N. 1127020 (labeled to contain 5 mg amlodipine besylate and 10 mg atorvastatin calcium per each tablet) were kindly supplied from PFIZER Company.

. Chromatographic Conditions
The analytical column was a Perfectsilr Target ODS-3 (4.6 cm × 250 mm, 5 µm) and a mobile phase consisting of acetonitrile-phosphate buffer pH 4.5 (55:45, v/v) at a flow rate 1 mL min −1 and at room temperature. The ultraviolet detector was set a wavelength of 237 nm. Solutions and mobile phase were freshly prepared at the time of use.

Standard Solution Preparation
Stock solutions of amlodipine besylate and atorvastatin calcium were prepared daily by dissolving the appropriate amount of drug standards in mobile phase to yield a final concentration of 5.0 and 10.0 mg mL −1 , respectively. Separate stock solutions were prepared for the calibration standards and quality control samples. Further, solutions were obtained by serial dilutions of stock solutions with mobile phase.

Preparation of Pharmaceutical Dosage Sample
The contents of twenty tablets were weighed and finely powdered and transferred to 100 mL volumetric flasks. Each 10-mL equivalent to 5 mg amlodipine besylate and 10 mg atorvastatin calcium. Working solutions were prepared individually by diluting the stock solutions with mobile phase to obtain concentration range of 1-10 µg mL −1 for amlodipine and 5-50 µg mL −1 for atorvastatin.

Chromatograms of Samples
The aim of this research was to develop a new, simple, accurate, reproducible, sensitive HPLC method for the simultaneous determination of amlodipine besylate and atorvastatin calcium. A satisfactory separation of each drug from pharmaceutical excipients was obtained. To

Science Publications
AJAS optimize the appropriate HPLC conditions for separation of the examined drugs, various reversedphase columns, isocratic and gradient mobile phase systems were tried. The optimum wavelength for detection was 237 nm at which much better detector responses for the three drugs were obtained. The mobile phase was found to be suitable to improve the sharpness and thinness of the amlodipine besylate and atorvastatin calcium. The retention times for the investigated drugs were found to be 1.071 min and 3.765 min, respectively. No pharmaceutical excipients eluted at the retention times of the peaks of interest.

Calibration and Linearity
Calibration curves were constructed in the ranges of 1-10 and 5-50 µg mL −1 for amlodipine besylate and atorvastatin calcium, respectively. The slope, intercept and regression coefficient for each compound were estimated.

Accuracy
Absolute recoveries of six different authentic concentrations of amlodipine besylate and atorvastatin calcium ( Table 1) and the studied drugs in tablets ( Table  2) were determined by assaying the samples as described above. Mean recoveries, standard deviations and the relative standard deviations were calculated by standard method (Table 1 and 2).

Precision
Intra-day precisions were assessed injecting standard solution four to five times during a day (this solution was extracted via the same procedure as the capsules) of each analyte at two different concentrations (a low and a high concentration). The resultant standard deviations were less than 2% for all ( Table 3). Inter-day precision experiments were done after treatment of the standard solution in the same method of tablets extraction and then analyzed every day over 5 days ( Table 3). All RSD% were lower than 2%.

Method Validated
The method was validated with regard to specificity, linearity, Limit of Detection (LOD), Limit of Quantification (LOQ), precision, accuracy and robustness.
Peak areas of amlodipine besylate and atorvastatin calcium of calibration standards were proportional to the concentration in dosage forms over the ranges tested 1-10 and 5-50 µg mL −1 , respectively. Each concentration was tested in triplicate. The slope values for amlodipine besylate and atorvastatin calcium were calculated with intercept values. The standard deviations of slope were calculated and similarly standard deviations of intercept. The calibration curves were fitted by linear least-square regression and showed correlation coefficients not less than 0.9998.    Table 3. Reproducibility and precision Intra-day (n = 4-5) Inter-day (n = 5)

AJAS
The LODs and LOQs of thioctic acid, benfotiamine and cyanocobalamin were calculated on the peak area using the following equations: LOD = 3.3 × σ/S, LOQ = 10 × σ/S, where σ, is the standard deviation of the intercept of regression line of the drugs and S is the slope of the corresponding calibration curve. Determination of authentic samples of amlodipine besylate and atorvastatin calcium and statistical analysis of the results obtained for the proposed method (

Application to Pharmaceutical Dosage form
The proposed method were successfully applied for the simultaneous determination of amlodipine besylate and atorvastatin calcium in Caduet tables ® without interference of the excipients present and without prior separation ( Table 2). The utility of the method was also verified by applying the standard addition technique.

CONCLUSION
The chromatographic method described is adequate for quantitation of amlodipine besylate and atorvastatin calcium in pharmaceutical dosage forms at different concentration levels. It is very simple, accurate and effective and provided no interference peaks for endogenous components and pharmaceutical excipients. Acceptable values of precision and accuracy have been obtained all levels by this method regarding the guidelines for assay validation. The separation of these drugs takes 5 min in one chromatogram, so a large number of samples can be analyzed in a short period of time. The method uses simple mobile phase and is very beneficial for column life. In summary, the method can be successfully applied to samples of pharmaceutical dosage form.