Development and Validation of RP-HPLC Method for Quantitative Estimation of Atorvastatin Calcium and Metoprolol Succinate in Combined Dose Capsule Formulation

Sagar B. Wankhede*, Nitin R. Dixit, Somnath S. Zambare and Sohan S. Chitlange

Padmashree Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Sant Tukaram Nagar, Pimpri, Pune-411 018. Maharashtra, India.

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

ABSTRACT:

A simple, specific, accurate and precise reverse phase high performance liquid chromatographic method have been developed and validated for simultaneous determination of Atorvastatin calcium and Metoprolol succinate in capsules. Chromatography was carried out by gradient technique on a reversed-phase C-18 column (4.6 mm x 250 mm, 5 μm) using acetonitrile - phosphate buffer (pH 3.5) - methanol (50:40:10, v/v/v) as a mobile phase at a flow rate of 1.0 ml/min and UV detection at 276 nm. The linearity of the calibration curves for each analyte in the desired concentration range was good (r² > 0.99). The method was accurate and precise with recoveries in the range of 99.84 % and 100.62 % for both the drugs and relative standard deviation (R.S.D.) < 2. The proposed method is highly sensitive, accurate and precise and hence was successfully applied for the reliable quantification of API content in the commercial formulation containing these drugs in combination. The different analytical performance parameters such as linearity, accuracy, precision, robustness, limit of detection (LOD) and limit of quantification (LOQ) were determined according to International Conference on Harmonization ICH Q2B guidelines.

KEYWORDS: Atorvastatin calcium, Metoprolol succinate, RP-HPLC

1. INTRODUCTION:

Atorvastatin calcium (ATS), chemically is [R(R*,R*)]-2-(4-flourophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(Phenyl amino) carbonyl]-1H-Pyrrole-1-heptenoic acid, calcium salt (2:1) trihydrate.¹ It is an antihyperlipidemic agent used as HMG-Co-A reductase inhibitor. It is official in IP. Metoprolol succinate (MET), chemically is 1-[4-(2-methoxyethyl)-phenoxy]-3-[(1-methylethylamino]-2-propanol². It is a beta adrenergic blocking agent, which reduces chest pain and lowers high blood pressure. It is official in USP. Literature survey revealed some spectrophotometricand chromatographic methods reported for estimation of ATS^3-6and MET^{7- 9} individually or in combination with other drugs. However, there is no any analytical method reported for the simultaneous determination of these drugs in a pharmaceutical formulation. Present work describes simple, rapid, accurate and precise reversed phase high performance liquid chromatographic method for simultaneous determination of ATS and MET in capsules.

2. MATERIALS AND METHODS:

2.1 Instrumentation:

HPLC system (Merck Hitachi) consisting of quaternary gradient pump (L-7100) autosampler, column oven, and UV detector (L – 7400) was employed for analysis. Chromatographic data was acquired using Winchrome software.

2.2 Sample, Reagents and Chemicals:

Active pharmaceutical ingredient (API) working standards of ATS and MET were obtained from Macleods Pharmaceuticals Limited, Mumbai and Emcure Pharmaceuticals, Pune respectively. Test samples (Capsules Betaone-ATR manufactured by Dr. Reddy’s Laboratories Ltd. with composition 10 mg Atorvastatin calcium and 25 mg Metoprolol succinate) were purchased from local market. HPLC grade acetonitrile and Methanol were obtained from Thermo Fisher Scientific Limited, Mumbai.

2.3 Chromatographic conditions:

Thermo Scientific C18 column (4.6 mm i.d. × 250 mm) was used as stationary phase. Acetonitrile, 0.025 mM phosphate buffer (pH adjusted to 3.5 with orthophosphoric acid) and methanol in the ratio of 50:40:10 % v/v/v was used as mobile phase and was filtered before use through 0.45 μ membrane filter. A constant flow of 1.0 ml/min was maintained throughout the analysis. Detection was carried out using UV detector at 276 nm.

2.4 Preparation of solutions:

2.4.1 Standard stock solution:

Standard stock solution of ATS and MET were prepared separately by transferring 25 mg of each working standard in a 25 ml volumetric flask. A 20 ml portion of mobile phase was added, sonicated and remaining volume was made up to the mark with mobile phase.

2.4.2 Sample solution:

Twenty capsules were weighed and the capsule powder was removed. The weight of empty capsule shells was then recorded. Difference in the weight of filled capsules and empty capsule shells was calculated to know the weight of powder present in twenty capsules. Average weight was then calculated. Capsule powder equivalent to about 10 mg ATS and 25 mg of MET was transferred to 25.0 ml volumetric flask; 20 ml portion of mobile phase was added and sonicated for 20 min. and then volume was made up to the mark with mobile phase. The resulting solution was mixed and filtered through Whatmann filter paper and filtrate was appropriately diluted to get approximate concentration of 40 μg/ml of ATS and 100 μg/ml of MET. The diluted solutions were filtered through 0.20 μ filter.

2.5 Chromatography:

Twenty microlitres of standard and sample solutions were injected and chromatographed under above mentioned chromatographic conditions. A typical chromatogram of ATS and MET is shown in fig.1. The calibration curves for ATS and MET were prepared in the concentration range of 10 - 60 µg/ml and 25 - 150 µg/ml, respectively at 276 nm by plotting concentration against peak area. The concentrations of both ATS and MET in the sample solutions were determined by comparing peak area of sample with that of standard at 276 nm. Respective peak areas, dilution factors, sample and standard weights were taken into account to quantitate the amounts of ATS and MET in mg per tablet. The results of capsule analysis are shown in table 2.

Figure 1: Typical Chromatogram of ATS and MET

MET - Metoprolol succinate: Retention time = 3.99 min

ATS - Atorvastatin calcium: Retention time = 8.28 min

2.6 Validation procedure:

The accuracy of the method was carried out by standard addition method, adding known amount of pure drug to the preanalysed capsule powder at three levels 80%, 100% and 120% of the analytical test concentration. The samples were given the same treatment as described in Section 2.4.2

Precision of the method was checked by analyzing the samples at three different time intervals of the same day (intraday precision) as well as on three different days (interday precision).

Robustness was performed by deliberately changing the chromatographic conditions. The flow rate of the mobile phase was changed from 1.0 mL/min to 0.9 mL/min and 1.1 mL/min while composition of the mobile phase was changed by ± 1 ml

LOD and LOQ are calculated by using the values of slopes and intercepts of the calibration curves for both the drugs.

3. RESULTS AND DISCUSSION:

3.1. Optimization of chromatographic conditions:

In order to achieve simultaneous elution of the two components, different chromatographic conditions were attempted. Under the optimized conditions, the analyte peaks were well resolved and free from tailing. The tailing factors were <1.5 for both the peaks. The elution order was MET (RT = 3.99 min) and ATS (RT = 8.28 min), at a flow rate of 1.0 mL/min. The chromatogram was recorded at 276 nm as the overlaid UV spectrum of ATS and MET showed maximum response at this wavelength.

3.2 Method validation:

The newly developed method was validated according to the ICH guidelines with respect to linearity, accuracy, precision, and robustness.[10] System suitability was established by injecting standard solution and results are shown in table 1.

3.2.1 Linearity:

ATS and MET showed linearity in the range of 10 - 60 µg/ml and 25 - 150 µg/ml, respectively. Linear regression equations and correlation coefficient (R²) are: Y_ATS = 410488 x − 71858 (R² = 0.9964) and Y_MET = 70254 x + 18071 (R² = 0.9992).

3.2.3. Accuracy:

The accuracy was expressed as the percentage of analytes recovered by the assay method. It was confirmed from results that the method is highly accurate (Table 3).

3.3.3 Precision:

The relative standard deviations (R.S.D) values for intraday and interday precision for ATS were 0.2918 % and 0.3030 % respectively and that for MET were 0.1849 and 0.1008 respectively which are well within the acceptable limit of 2 %

Table 1: System Suitability Parameters

Component	Retention time (min)	Resolution	Tailing Factor	Theoretical plates	LOD (µg/ml)	LOQ (µg/ml)
ATS	8.28	3.55	1.01	4579.15	0.0316	0.0960
MET	3.99	-	1.33	3680.51	0.0277	0.0841

Table 2: Analysis of Capsule Formulation

Component	Label claim (mg/capsule)	*Amount found (mg/capsule)**	Percent label claim *	S.D.	R.S.D. %
ATS	10.0	9.88	99.23	0.4412	0.4446
MET	25.0	25.37	101.28	0.9343	0.9224

* Average of six determinations, SD-Standard Deviation, RSD-Relative Standard Deviation

Table 3: Results of Recovery Studies

Level of Recovery	Amt. of pure drug added (mg)		Percent recovery*
	ATS	MET	ATS	MET
80 %	8.0	20.0	100.62	100.16
100 %	10.0	25.0	99.93	100.27
120 %	12.0	30.0	99.84	100.24
Mean % recovery			100.13	100.22
S.D.			0.4267	0.0568
R.S.D. %			0.4261	0.0566

* Average of three determinations, SD-Standard Deviation, RSD-Relative Standard Deviation

3.3.4 Robustness:

In all deliberately varied conditions, the RSD of contents of ATS and MET were found to be well within the acceptable limit of 2%. The tailing factor for both the peaks was found to be <1.5.

4. CONCLUSION:

Proposed RP-HPLC method is simple, accurate and precise for the simultaneous determination of ATS and MET from pharmaceutical dosage form. The described method is suitable for routine analysis and quality control of pharmaceutical preparations containing these drugs either alone or in combination.

ACKNOWLEDGEMENT:

The authors are thankful to Dr. Avinash D. Deshpande, Director of Pharmacy, Padm. Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune for providing necessary facilities. Authors are also thankful to Mcleods Pharmaceuticals, Mumbai and Emcure pharmaceuticals, Pune for providing gift sample of pure drugs.

REFERENCES:

1. Indian Pharmacopoeia, Vol. II, Government of India, The Controller of Publication, New Delhi. 2007: 749

2. United States Pharmacopoeia, XXVI, National Formulary XXI, Asian edition, US Pharmaceutical Convention, Inc; Rockville, MD 2003: 1220

3. Godse VP et al. Simultaneous spectrophotometric estimation of Ezetimibe and Atorvastatin in pharmaceutical dosage form. Asian J Research Chem. 2009; 2(1): 86-89.

4. Dhaneshwar SS et al. Development and validation of a method for simultaneous densitometric estimation of Atorvastatin calcium and Ezetimibe as the bulk drug and in tablet dosage forms. Acta Chrom. 2007; (19) : 141-148

5. Jamshidi A, Nateghi R. HPTLC determination of Atorvastatin in plasma. Chromatographia. 2007; (65) : 763–766

6. Chaudhari BG, et al. Development and validation of a HPTLC method for the simultaneous estimation of Atorvastatin calcium and Ezetimibe. Indian J. Pharm Sci. 2006; 68(6): 793-796.

7. Kulkarni MN, Kshirsagar RV, Sakarkar DM. Development and validation of spectrophotometric method for determination of Metoprolol succinate. Int. J Chem. Research. 2009; 1(4): 1273-1277.

8. Chitlange SS, Mohammed I, Sakarkar DM, RP-HPLC method for simultaneous estimation of amlodipine and metoprolol in tablet formulation, Asian J Pharm. 2008 : 232-234

9. Phale MD, Hamrapurkar PD. A validated and simplified RP-HPLC of Metoprolol Succinate from bulk drugs Asian J Research Chem. 2009; 2(2): 119-122

10. Validation of Analytical Procedures: Text and Methodology, Proceedings of the International Conference on Harmonization (ICH). Geneva, 2005