INTRODUCTION:

Atorvastatin calcium (ATR) is chemically [R-(R*, R*)]-2-(4-fluorophenyl) -β, δ-dihydroxy-5-(1-methylethyl) - 3 - phenyl - 4 - [(Phenylamino) carbonyl] -1H - Pyrrole -1-heptanoic acid, Calcium salt (2:1) trihydrate. Ezetimibe (EZT) is chemically 1-(4-fluorophenyl) 3 (R)-[3-(4-fluorophenyl)-3(S)-hydroxypropyl]-4(S)-(4-hydroxyphenyl) -2-azetidinone. ATR¹ is used as antihyperlipidemic agent and EZT² is a class of lipid lowering compounds. Both of these drugs are available in combined tablet dosage form with the label claim of ATR 10mg and EZT 10mg per tablet. Survey of literature of ATR and EZT revealed few methods based on spectroscopy^3-6 and chromatography^7-18 has been reported for determination of both drugs in single and combined dosage forms. The present work describes the development and validation of reverse phase high performance liquid chromatographic (RP-HPLC) method, which can quantify these components simultaneously.

EXPERIMENTAL:

Reagents and Chemicals:

Acetonitrile, Methanol and Water of HPLC grade were procured from Merck specialties Pvt. Ltd., Mumbai. Orthophosphoric acid was procured from S.D. Fine. Chem. Ltd., Mumbai.

Working standards of ATR and EZT were obtained as gift samples from Dr. Reddys’s Laboratories Pvt. Ltd., Hyderabad.

Equipment:

Chromatographic separation was performed on a Shimadzu chromatographic system equipped with a LC-20AT pump; variable wavelength programmable UV/VIS detector, SPD-20A and Rheodyne injector (7725i) with 20µl fixed loop.

Chromatographic conditions:

Phenomenex C₁₈(250 x 4.6mm) was the column used for separation, mobile phase consisting of a mixture of Acetonitrile and Buffer (0.1% v/v orthophosphoric acid, P^H adjusted to 6 with Triethylamine) in the ratio 60:40 was delivered at a flow rate of 1ml/min with detection at 232nm. The mobile phase was filtered through a 0.45m membrane filter and sonicated for 15 min. Analysis was performed at ambient temperature.

Pharmaceutical formulation:

Commercial tablets were procured randomly from the local market.

Method development:

Acetonitrile, methanol and water in different proportions were tried and finally Acetonitrile: Buffer (0.1%v/v orthophosphoric acid, P^H-6)(60:40v/v) was selected as an appropriate mobile phase which gave good resolution and acceptable system suitability parameters. The chromatogram of working standard solution is shown in fig 1.

Figure 1: Typical Chromatogram of Atorvastatin calcium and Ezetimibe

PROCEDURE:

Preparation of working standard solutions:

A standard stock solution was prepared by accurately weighing about 20 mg of ATR and 20 mg of EZT standard and transferred into 100 ml volumetric flask; added about 25 ml of methanol and sonicated for about 5 minutes to dissolve and made up to volume with mobile phase

Calibration curve:

Accurately measured volumes of working standard solution of ATR and EZT were transferred into a series of 10ml volumetric flasks and diluted appropriately with mobile phase. 20ml of each solution was injected under operating chromatographic conditions described above. Calibration curves were obtained by plotting the response (area of drug peak) versus concentration of drug. Regression equations were calculated. The method was found linear over a concentration range 10mg/ml to 30 mg/ml for both the drugs.

Procedure for analysis of tablets:

20 tablets were weighed and powdered. An accurately weighed portion of this powder equivalent to 100mg of ATR and 100 mg of EZT was transferred to a 100 ml volumetric flask containing 25 ml of methanol. The contents of the flask were allowed to stand for 30 minutes with intermittent sonication to ensure complete solubility of the drugs and made up to volume with mobile phase. Then above solution was filtered through 0.45µm membrane filter. 10 ml of filtrate was diluted to 50 ml with mobile phase. From this solution appropriate dilutions were made with mobile phase to obtain concentration in calibration range for both the drugs and this solution was used for estimation.

With the optimized chromatographic conditions, a steady baseline was recorded, the mixed working standard solution was injected and the chromatogram was recorded. The retention times of ATR and EZT were found to be 3.7 and 6.1 min respectively. The proposed method was found to be specific and no interference from common tablet excipients like lactose, starch etc. was observed. The response factors of the standard solutions and sample solutions were calculated. The assay was calculated from the equation of regression line for each drug. The assay procedure was repeated for 6 times, the percentage of individual drug in the formulation was calculated. The results of analysis shows that the amount of drug was in good agreement with the label claim of formulation. (Table 1)

Table 1: Analysis of tablet formulation

Formulation

Analyte

Label claim (mg)

% label claim estimated*

Tablet

Atorvastatin calcium

Ezetimibe

100.25

99.20

*mean of six determinations

METHOD VALIDATION:

Linearity:

The method was linear in the range of 10mg/ml to 30 mg/ml for both ATR and EZT standards. Linear regression data was given in Table 2.

Table 2: Linear regression data for calibration curves

Parameter	Atorvastatin calcium	Ezetimibe
Linearity range (µg/ml)	10 – 30	10 – 30
Correlation coefficient	0.9998	0.9999
Slope	30.727	39.524
Intercept	- 0.1187	- 5.8813

Precision:

The precision of the method was demonstrated by inter day and intraday variation studies. In the intraday studies, solutions of standard and sample were repeated thrice in a day and percent relative standard deviation (%RSD) for response factor was calculated. The intraday %RSD of ATR and EZT were found to be 0.282 and 0.162 respectively.

In the interday variation studies, injections of standard and sample solutions were made on three consecutive days and %RSD was calculated. The interday %RSD for ATR and EZT were found to be 0.269 and 0.153 respectively. From the data obtained the developed RP-HPLC method was found to be precise.

Accuracy:

The accuracy of the method was determined by recovery experiments. Known concentration of working standard was added to the fixed concentration of the pre-analyzed tablet solution. Percent recovery was calculated by comparing the area before and after the addition of working standard. For both the drugs, recovery was performed in the same way. The recovery studies were performed in triplicate. This standard addition method was performed at 50%, 100%, 150% level and the percentage recovery was calculated. Percent recovery was within the range of 99.65 to 100.5 for ATR and 99.2 to 100.1 for EZT which indicates that the method was accurate.

Limit of detection and limit of quantification:

The Limit of detection and quantification were calculated using standard deviation of the response and slope of calibration curve. The LOD for ATR and EZT was found to be 0.39 mg/ml and 0.25 mg/ml respectively. The LOQ is the smallest concentration of the analyte, which gives response that can be accurately quantified. The LOQ was 1.22 mg/ml and 0.80 mg/ml for ATR and EZT respectively.

Robustness:

Robustness of the method was checked by making slight deliberate changes in chromatographic conditions like mobile phase ratio, pH of buffer, flow rate. It was observed that there were no marked changes in chromatograms, which demonstrated that the developed RP-HPLC method is robust.

Solution stability:

In order to demonstrate the stability of both standard and sample solutions, the solutions were analyzed over a period of 12 hours at room temperature. The results show that, the retention time and peak area of ATR and EZT remained unchanged (%RSD less than 0.2) and no significant degradation within the indicated period was observed. This indicates that both solutions were stable for at least 12hours, which was sufficient to complete the analytical procedure.

RESULTS AND DISCUSSION:

The proposed method was found to be linear in the concentration range of 10 to 30 mg/ml for both ATR and EZT. The method was specific since excipients in the formulation did not interfere in the estimation of ATR and EZT. Accuracy of the method was indicated by recovery values from 99.2 to100.5%. Precision is reflected by %RSD values less than 2. The LOQ for ATR was found to be 1.22 mg/ml and for EZT, it was 0.80 mg/ml. These low values suggest sensitivity of the developed method. Validation parameters were summarized in Table 3

Table 3: Summary of validation parameters

Parameter	Atorvastatin calcium	Ezetimibe
LOD (µg/ml)	0.39	0.25
LOQ (µg/ml)	1.22	0.80
Mean % recovery	100.01	99.8
Precision (% RSD)
Interday (n=3)	0.269	0.153
Intraday (n=3)	0.282	0.162
Robustness	Robust	Robust
Retention time (min)	3.7	6.1
Theoretical plates	5912	6983
Tailing factor	1.08	1.89

CONCLUSION:

The developed RP-HPLC method was simple, sensitive, precise and accurate and hence can be used in routine for the simultaneous determination of ATR and EZT in bulk as well as in pharmaceutical preparations.

ACKNOWLEDGEMENTS:

The authors are thankful to Hindu college of pharmacy, Guntur for providing necessary facilities to carry out present study.

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Received on 05.01.2010 Modified on 15.01.2010

Asian J. Research Chem. 3(1):Jan.-Mar. 2010 page 225-228