Simultaneous Estimation and Validation of Atorvastatin Calcium and Aspirin in Combined Capsule Dosage Form by RP HPLC Method

A new simple, specific, precise and accurate revere phase liquid chromatography method has been developed for estimation of atorvastatin calcium (AST) and ASPIRIN (ASP) simultaneously in a combined capsule dosage forms. The chromatographic separation was achieved on a 5 – micron C 18 column (250x 4.6mm) using a mobile phase consisting of a mixture of Acetonitrile: Ammonium Acetate buffer 0.02M (68:32) pH 4.5. The flow rate was maintained at 0.8 ml / min. The detection of the constituents was done using UV detector at 245 nm for AST and ASP. The retention time of AST and ASP were found be 4.5915 ± 0.0031min and 3.282 ±0.0024 min respectively. The developed method was validated for accuracy, linearity, precision, limit of detection (LOD) and limit of quantification (LOQ) and robustness as per the ICH guidelines.


Introduction
Atorvastatin calcium is chemically a calcium salt of (β R, 8 R)-2-(4 -fluoro-phenyl) -α, δ di hydroxyl 5(1 methyl ethyl) 1, 3, phenyl, 4 (phenyl amino) carbonyl) -1 H pyrroleheptanoic acid tri hydrate used as antihyperlipidaemic.It is official in Indian pharmacopoeia 1 Aspirin is chemically 2-(Acetyloxy) benzoic acid, best known as anti-platelet drug.It is official both in I.P and B.P. 2 Detailed survey of literature for atorvastatin calcium (AST) revealed several methods based on different techniques like extractive Spectrophotometry 3 , HPLC 4-8 , HPLC 9-11 for its determination in human serum, capillary electrophoresis 12 , HPTLC for its determination in pharmaceutical. 13imilarly literature survey for aspirin reveals several methods based on Spectrophotometry 14 , Raman spectroscopy 15 RP-HPLC 16 .This paper describes a simple, precise and accurate RP HPLC method for the estimation of AST and ASP combination in a capsule dosage form.

Chemical and reagents
AST was the generous gifts from Biocon Limited Bangalore, and aspirin was procured from Qualigens Fine Chemicals (Glaxo Ltd).Combination of these drugs was purchased from the local market (Ecosprin AV 75 containing Atorvastatin calcium 10 mg and aspirin 75 mg as per the label claim, marketed by USV limited, India).HPLC grade Acetonitrile, ammonia, glacial acetic acid was procured from Merck.

RP HPLC instrumentation and chromatographic conditions
The following chromatographic conditions were established for the separation of drug and maintained the same parameter throughout the method.Preparation of standard stock solution a) Preparation of standard stock solution of AST: An accurately weighed quantity equivalent to 10 mg AST was dissolved in 25 ml of methanol and then the volume was made up to 50 ml with same solvent.b) Preparation of standard stock solution of AST: An accurately weighed quantity equivalent to 75 mg ASP was dissolved in 25 ml of methanol and then the volume was made up to 50 ml with same solvent.c)

Validation of the method 17
The method was validated as per ICH guide line.The parameters checked were linearity, accuracy, precision, limit of detection, limit of quantification, robustness and specificity.

System suitability testing
A standard solution was prepared using AST and NA working standard as per the test method and was injected nine times into the HPLC system.The parameters like theoretical plates, tailing factor and resolution for the standard solution were calculated.

Calibration curve
From the working standard solution of AST (200 µg/ml) and AST (1500 µg/ ml), appropriate aliquots of AST and ASP stock solution were diluted in methanol to obtain final stock solution of 2, 4, 6, 8 10 µg/ml of AST and 15, 30, 45, 60, 75 µg/ml of ASP.The solutions were injected using a 20 µl fixed loop system and chromatograms were recorded.Calibration curve were constructed and regression equation were computed for AST and AST.

Specificity
A blank solution (mobile phase) was injected and the chromatogram showed no inferring peaks at retention time of the two drugs.The chromatogram of AST and ASP extracted from the tablet dosage form were compared with those acquired from AST and ASP standards, correlation was good (in terms of retention time and area) indicates specificity of method.

Precision: (Reproducibility)
The precision of the method was verified by performing the intraday and interday precision.The intraday and interday precision of the proposed method was determined by estimating the corresponding response three times on the same day and on three different days.

Accuracy: (% Recovery)
For accuracy of method, recovery studies were carried out by applying a known amount of standard AST and ASP at a level of 80,100,120 % to the sample solution (standard addition method).Three determinations were performed at each level, using same chromatographic condition as describe above.

Limit of Detection (LOD) and Limit of quantification (LOQ):
The LOD and LOQ were calculated using following equations as per International conference on Harmonization guide line LOD= 3.3 x σ/S LOQ= 10 x σ/S Where σ is standard deviation of the response and S is the standard deviation of y intercept of regression lines.

Robustness
Robustness was checked by making a slight deliberate change in the experimental procedure like slight change in the temperature, flow rate and pH and the data are expressed in terms of relative standard deviation.

Analysis of the marketed products
To find the content of the marketed formulation, (Ecosprin AV 75 containing Atorvastatin calcium 10 mg and aspirin 75 mg as per the label claim, marketed by USV limited, India), twenty tablets were weighed and average weight was determined, powdered, from this equivalent weight of 10 mg for AST and 75 mg of ASP was transferred into a 50 ml volumetric flask, containing 15 ml of methanol and sonicated for 30 minutes, filtered through Whatmann filter paper No.41and then volume was made up to 50 ml with methanol.

Validation
To optimize the HPLC parameters, several mobile phases were tried and satisfactory results were obtained by using the mobile phase consisting of Acetonitrile: ammonium acetate 0.02M (68:32) at of P H of 4.5, with a flow rate of 0.8ml / min.UV detection was carried out at 245nm and run time was 15min.The selection of the mobile phase was made on the basis of resolution, asymmetric factor, and theoretical plates.The retention time of AST and ASP was found to 4.5915 ± 0.0031min and 3.282 ±0.0024 min respectively.A typical chromatogram of the test is shown in Figure 1.

System suitability parameters
System suitability parameters proved that the proposed method suits for the simultaneous estimation of AST and ASP.After various trials performed, chromatogram for AST and ASP was found satisfactory on, using mobile phase composition of Acetonitrile: Ammonium acetate 0.02M (68:32) pH 4.5.Drug peak was found to be symmetrical as observed from asymmetry factor of 1.4315 for AST and 1.4455 for ASP.Resolution of the proposed method was found to be satisfactory and the results are given in table 1.

Linearity
The linear regression data revealed a good linear relationship over the concentration range of 2-10 µg/ ml for AST and 15-75 µg/ ml for ASP with correlation coefficient (r 2 = 0.9998) for AST and correlation coefficient(r 2 =0.9994)forASP respectively.The results are showed in table 2 and Figure 2 and Figure 3.

Specificity
The method was found to be specific since no interferences chromatograms were seen when carried out in presence of additives, when only mobile phase was injected.

Precision
The proposed method was found to be precise as indicated by percent RSD not more than 2% as per ICH guidelines for interday and intra day determination.The results are shown in the table 2.

Limit of Detection (LOD) and Limit of quantification (LOQ)
The limit of detection was found to be 0.088ng/ml and limit of quantification was found to be 0.266ng/ml for AST and similarly for ASP is 1.31ng/ml and 3.98ng/ml respectively.The results are shown in table 2.

Robustness
Robustness was checked by making a slight deliberate change in the experimental procedure by slight change in the temperature, flow rate and pH and no significant change in area and retention time was found in all the parameters and all the values were found to be with in 2% relative standard deviation.The results of robustness are given in the table 5, 6, 7.

Analysis of the marketed products
The proposed method was applied successfully to determine the content of AST and ASP in pharmaceutical product.The results are expressed in terms of percentage in Table 8.

Conclusion
The proposed HPLC method is simple and the total run time for the two components is less than 9 min.The quantitation of each component was not affected by any of the possible interfering substances included during tablet manufacturing.The method is accurate and precise as indicated from the recovery study.It can be concluded that the proposed HPLC method has great promise for the simultaneous determination of two components in pharmaceutical formulations.

Table : 1
. Study of System Suitability Parameters.
* Mean of nine trials.

Table 3 .
Recovery studies of Atorvastatin Calcium Excepted concentration.

Table 4 .
Recovery studies of Aspirin Excepted concentration.

Table 5 .
Robustness for flow rate studies (temperature).

Table 6 .
Robustness for pH studies.

Table 7 .
Robustness for Flow rate Studies.

Table 8 .
Analysis of tablet formulation.