Development and Validation of a HPLC method for Simultaneous Analysis of Aspirin and Atorvastatin Calcium as the Bulk Drugs and in the capsule Dosage Form

R.K. Nanda¹*, S.E. Potawale¹, V.V. Bhagwat¹, S.C.Hamane¹ and R. S. Deshmukh²

¹D.Y.Patil Pratishthan’s Padmashree Dr. D.Y.Patil Institute of Pharmaceutical, Sciences and Research, Pimpri, Pune-411018, India.

²Sinhgad College of Pharmacy, Vadgaon, Pune-411041, India.

*Corresponding Author E-mail: rabindrananda@rediffmail.com

ABSTRACT:

A simple, precise, accurate and rapid HPLC method has been developed, and validated for the determination of Aspirin, Atorvastatin Calcium simultaneously, in combined dosage form. Acetonitrile: 0.02 M Phosphate buffer with pH 6.1 (50 %: 50 % v/v) is used as the mobile phase, pH adjusted with Sodium Hydroxide (1% aqueous). Mobile phase was delivered at the flow rate of 1.0 ml/min., 239 nm is the detection wavelength for this study. The applicability of the method for simultaneous determination of Aspirin, Atorvastatin Calcium was verified by the determination of these compounds in marketed capsules. Results of the analysis were validated statistically, and by recovery studies. Linearity for Aspirin And Atorvastatin Calcium was in the range of 5-35 μg/ml and 1-7 μg/ml, respectively and reproducibility was found to be satisfactory. The % assay (Mean ± S.D.) was found to be 99.33± 0. 501 and 101.4± 0.18 for Aspirin and Atorvastatin Calcium respectively. The recovery and RSD values are within the limits given in ICH guide lines. Method development indicates the suitability of proposed method for the routine determination of these compounds in capsule dosage form. The proposed method can be successfully used to determine the drug contents of marketed formulation.

KEYWORDS: RP-HPLC; validation; Aspirin; Atorvastatin

INTRODUCTION:

Aspirin (ASP) the well known antithrombotic, antipyretic, analgesic agent^1-2, official in USP-NF³, BP⁴ and IP⁵. Atorvastatin Calcium (ATO) ([R-(R*, R*)]-(2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4(phenyl amino) carbonyl]-1H-pyrrole-1-heptanoic acid, calcium salt, (2:1) trihydrate), a synthetic lipid-lowering agent is official in IP⁶. It is a selective competitive inhibitor of the enzyme HMG-CoA reductase, which catalyses the conversion of HMG-CoA to mevalonate, an important rate-limiting step in cholesterol biosynthesis⁷.

Literature review reveals that variety of methods have been reported for analysis of ASP by high-performance liquid chromatography (HPLC)^8-12, high-performance thin-layer chromatography (HPTLC)^13,14 and spectroscopy¹⁵.

Chromatographic^16-22 and spectroscopic^{23, 24} methods have been reported for determination of ATO, in combination with other drugs, in bulk and pharmaceutical dosage forms. The purpose of this study was to develop and establish a simple, accurate, economical, and reproducible procedure for the simultaneous HPLC analysis of Aspirin, Atorvastatin Calcium as the bulk drug and in their combined dosage forms and validate it, in accordance with ICH guidelines²⁵.

EXPERIMENTAL:

Chemicals and Reagents:

Working standards of pharmaceutical grade Aspirin, Atorvastatin Calcium were obtained from Torrent Pharmaceuticals (Gujarat, India). Commercially available Ecosprin AV capsules [Aspirin (75mg), Atorvastatin Calcium (10 mg)] were used. Chemicals and reagents of HPLC-grade were purchased from Merck Chemicals, Mumbai, India.

Instrument used:

Agilent technologies-1120 Compact LC, UV detector and EZchrom Elite software.

Method:

Preparation of stock solutions:

About 10 mg of Aspirin and Atorvastatin Calcium weighed and transferred to 100 ml volumetric flasks respectively. It was dissolved and diluted to volume with mobile phase consisting of Acetonitrile: 0.02 M Phosphate buffer with pH 6.1 (50 %: 50 % v/v). The pH of the mixed solvents was adjusted to 6.1 using Sodium Hydroxide (1% aqueous). An Ultrasonic bath (Art No.400014CL) was used for sonicating the stock solution, and then filtered through 0.2 micron nylon membrane filter.

Selection and optimization of mobile phase:

Pure drug of Aspirin and Atorvastatin Calcium were injected into the HPLC system and run in different solvent systems. Different mobile phases containing various proportions of Methanol, Water, Acetonitrile, Toluene, Acetone were tried (Data not shown) in order to determine the best conditions for the effective separation of Aspirin and Atorvastatin Calcium. Hence the mobile phase consisting of Acetonitrile: 0.02M Potassium dihydrogen ortho phosphate (KH₂PO₄) (50:50v/v) pH 6.1 with Sodium Hydroxide (1% aqueous) was selected as it gave high resolution of Aspirin and Atorvastatin Calcium with improved peak shapes under selected chromatographic conditions (Fig.1).

Chromatographic conditions:

HPLC Column : Agilent TC- C18 (2) column (5 µm, 250 X 4.6 mm ID)

Column temperature : Ambient temperature

Mobile Phase : Acetonitrile: 0.02M Potassium dihydrogen ortho phosphate

(KH₂PO₄) (50:50v/v) pH 6.1 with Sodium Hydroxide

(1% aqueous)

UV detection : 239 nm

Injection volume : 20 μL.

Run time : 10 minutes.

Procedure:

Preparation of standard calibration curves:

Appropriate dilutions of the standard stock solutions of each drug were prepared in triplicate. From these triplicate solutions, 20 μL injections of each concentration of the drugs were injected into the RP-HPLC system two times separately. Evaluation of the drugs was performed with the UV detector set at 239 nm and the peak areas were recorded. Calibration plots were constructed by plotting peak areas against the concentration (µg mL^-1) of the drugs. For Aspirin response was linear in the concentration range 5-35 µg mL^-1 and 1-7 µg mL^-1 for Atorvastatin Calcium. The regression coefficient (r²) for Aspirin and Atorvastatin Calcium were found to be 0.998. (Table 2)

Analysis of mixed standards:

The proposed method was employed for the analysis of mixed standards of Aspirin and Atorvastatin Calcium in the concentrations ratio of 1:7.5 as in the marketed formulation. The analysis was performed under the optimized chromatographic conditions.

Assay of the Marketed Formulation:

For analysis of the capsule dosage form, twenty capsules were weighed and their average weight was calculated. A powder sample equivalent to 10 mg of Atorvastatin Calcium was weighed, transferred to a 100 mL volumetric flask and dissolved in mobile phase. The solutions were sonicated for 30 minutes to allow for dissolution of the active components and the volume was made up to the mark with the mobile phase. Solution was mixed and filtered through Whatman filter paper No.42. Appropriate dilutions were made and the concentrations of Aspirin and Atorvastatin Calcium in the sample solutions were determined by the proposed method (Table 3).

RESULTS AND DISCUSSION:

Method Validation:

System suitability parameters:

System suitability tests are used to verify that resolution and reproducibility of the chromatographic system are adequate for the analysis to be done. Resolution is a measure of quality of separation of adjacent bands; obviously overlapping bands have small Rs values. The tailing factor T, a measure of peak symmetry, is unity for perfectly symmetrical peaks and its value increases as tailing becomes more pronounced. As peak asymmetry increases, integration, and hence precision becomes less reliable. The developed chromatogram showed good resolution of peaks with Rs value of 5.13 between Aspirin and Atorvastatin Calcium with minimal tailing (Table 1).

Table 1: System Suitability Studies

Parameter with Standard Values	Aspirin	Atorvastatin Calcium
Retention time(min)±RSD	2.807±0.02	7.937±0.02
Resolution NLT 2	5.13
Tailing factor NMT 2	1.20	1.35
Asymmetry factor	1.3	1.37
Theoretical Plates	>2500	>2000

NMT – Not more than, NLT – Not less than

Precision:

Precision of the method was determined with the marketed formulation. The repeatability of sample application and measurement of peak area were expressed in terms of % R.S.D. and were found to be less than 2% for Aspirin and Atorvastatin Calcium indicating that the proposed method provides acceptable intra-day and inter-day precision (Table 2).

Table 2: Validation data of Aspirin and Atorvastatin Calcium.

Parameter	Aspirin	Atorvastatin Calcium
Linearity range µg/ml	5-35	1-7
Correlation coefficient	0.998±0.6	0.998±0.26
Limit of detection	1.41	0.0950
Limit of quantitation	4.277	0.294
Recovery (%)	99-100.5	98-100.7
Precision (%RSD)
Inter- day (n=6)	1.14	1.21
Intra-day (n=6)	1.47	1.18
Robustness	Robust	Robust

* Denotes average of six estimations

Accuracy (Recovery study):

The accuracy of the method was evaluated by determination of recovery at three different concentrations, equivalent to 80, 100, and 120% of the amount in the dosage form. The results of recovery indicated that the method is accurate for estimation of these drugs in the combined capsule dosage form (Table 3).

Table 3: Assay and recovery data of capsule formulation

Component	Level of % recovery	% Recovery**	Relative standard Deviation**
Aspirin	80	98.90	0.805
	100	99.40	0.357
	120	99.71	0.341
Atorvastatin Calcium	80	100.05	0.146
	100	99.55	0.230
	120	100.21	0.238

* Denotes average of six estimations

** Denotes average of three estimations at each level of recovery.

Limit of detection (LOD) and limit of quantitation (LOQ):

The LOD and LOQ were separately determined based on the standard deviation of the y- intercept and slope of the calibration curves with RSD values less the 2%. The LOD were found to be 1.41 µg mL-1 for Aspirin and 0.0950 µg mL-1 for Atorvastatin Calcium. Similarly, the LOQ were 4.277 µg mL-1 and 0.294 µg mL-1 for Aspirin and Atorvastatin Calcium respectively (Table 2).

Robustness:

The robustness of the developed RP-HPLC method was evaluated by making deliberate variations in the method parameters such as change in flow rate, ratio of aqueous: organic composition and pH of the mobile phase. Flow rate was changed to 1± 0.1ml/min. The mobile phase was changed to ± 2% for both components and pH was changed to 6.1± 0.1. (Table 4).

CONCLUSION:

It is shown above that the accuracy, precision, reproducibility, repeatability, linearity, and selectivity of this new HPLC method compare favorably with those of HPLC, HPTLC, spectrophotometry, and other methods reported regularly in the literature. The results also meet ICH guidelines for validation of pharmaceutical HPLC methods. The proposed method is less expensive, simpler, more rapid, and more flexible.

ACKNOWLEDGEMENTS:

The authors express their gratitude to Dr. A. D. Deshpande, Director, Padm. Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pune, Maharashtra, India, for providing necessary facilities. The authors are also thankful to Torrent Pharmaceuticals Pvt. Ltd. (Gujarat) India for providing Aspirin, Atorvastatin Calcium as gift samples .

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Received on 26.05.2010 Modified on 11.06.2010

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 961-964

*% RSD Found For Robustness Study**
	Flow Rate (1mLmin-1)		pH (6.1)		Mobile phase ratio (50:50)
Drugs used	0.9	1.1	6.0	6.2	+2%	-2%
Aspirin	1.31	1.29	1.12	0.72	1.02	0.93
Atorvastatin Calcium	1.57	1.50	0.98	1.21	1.32	1.46

INTRODUCTION:

Parameter