A Simple and Sensitive RP-HPLC Method for Simultaneous Estimation of Atorvastatin Calcium, Ezetimibe and Fenofibrate in Combined Tablet Dosage Form

 

Abhijit D. Dhiware, Padmanabh B. Deshpande*, Santosh V. Gandhi, Sujith Nair, Suvarna Vanjari

Department of Pharmaceutical Analysis, A.I.S.S.M.S. College of Pharmacy, Kennedy Road,

Pune - 411 001, MH, India

*Corresponding Author E-mail: padmanabh77@yahoo.co.in

A simple, accurate, and validated reverse-phase high-performance liquid chromatographic (RP-HPLC) method for the simultaneous determination of Atorvastatin Calcium, Ezetimibe and Fenofibrate in combined tablet dosage form was developed and validated. Separation was carried out on Jasco HPLC system equipped with HiQ sil C18 HS column (250 × 4.6 mm i.d.) and PDA detector using Methanol: Acetonitrile: 0.02M Ammonium acetate buffer pH adjusted to 10 with Ammonia in ratio of (60:30:10, v/v/v) as the mobile phase and detection was carried out at 240 nm. Results were linear in the range of 0.5-10 μg mL−1 for Atorvastatin Calcium and Ezetimibe and 10–60 μg mL−1 for Fenofibrate. The method was successfully applied for the analysis of drugs in pharmaceutical formulation. Results of the analysis were validated statistically and by recovery studies.

 

 

INTRODUCTION:

Atorvastatin Calcium (ATOR), chemically, (3R, 5R)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-yl pyrrol-1-yl]-3, 5-dihydroxyheptanoic acid is used for lowering blood cholesterol¹. Ezetimibe (EZE), (3R, 4S)-1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl) azetidin-2-one lowers cholesterol by decreasing absorption in the intestine². Fenofibrate (FENO), propan-2-yl 2-{4-[(4-chlorophenyl) carbonyl] phenoxy}-2-methyl propanoate is used to reduce cholesterol levels in patients at risk of cardiovascular disease ³.

 

Literature survey reveals spectrophotometric^4-5, reverse phase high-performance liquid chromatographic (RP-HPLC)^6-9, high performance thin layer chromatographic (HPTLC)^10-12, UPLC¹³ methods for determination of ATOR either as a single or in combination with other drugs in pharmaceutical preparations and in human plasma. Analytical methods reported for EZE includes spectrophotometric^{14, 15}, HPLC^16-19 and HPTLC^{20, 21} either as a single drug or in combination with other drugs.

 

Analytical methods reported for FENO includes spectrophotometric^22-25, HPLC^{26, 27} and HPTLC²⁸ either as a single drug or in combination with other drugs.

 

To our best knowledge no reports were found for the simultaneous estimation of ATOR, EZE and FENO in combined dosage form by RP-HPLC method. This paper describes a simple, accurate, and validated reverse-phase high-performance liquid chromatographic (RP-HPLC) method for the simultaneous quantification of these compounds in tablet dosage forms. The proposed method is optimized and validated as per the International Conference on Harmonization (ICH) guidelines ²⁹.

 

MATERIALS AND METHODS:

Chemicals and Reagents

Working standards of pharmaceutical grade ATOR, EZE and FENO were obtained as generous gifts from Emcure Pharmaceuticals Ltd, Pune, India. The pharmaceutical dosage form used in this study was Tritonact tablets (Emcure Pinnacle Pharma Ltd, India) labelled to contain 10 mg of ATOR, 10 mg of EZE and 160 mg of FENO were procured from the local market. Methanol and Acetonitrile (HPLC grade), Ammonium Acetate and Ammonia (AR grade) purchased from Merck specialties Pvt. Ltd.  (Mumbai, India) and double distilled water were used in analysis.

 

Instrumentation and Chromatographic Conditions

Jasco HPLC system consisting of Jasco PU-2080 plus HPLC pump and MD 2010 PDA detector and JASCO Borwin- PDA software (version 1.5) was used for analysis. Separation was carried out on HiQ Sil C₁₈ HS (250 x 4.6 mm i.d.) column using methanol: acetonitrile: 0.02M ammonium acetate buffer pH adjusted to 10 with ammonia in ratio of (60:30:10, v/v/v) as mobile phase at flow rate of 1 mL min^-1. Samples were injected using Rheodyne injector with 20 µL loop and detection was carried out at 240 nm.

 

Preparation of Standard Stock Solutions

Standard stock solutions of pure drugs were prepared separately by dissolving 10 mg of each drug in 10 mL mobile phase to get concentration of 1000 μg mL⁻¹. For ATOR and EZE, 1 mL of the above solution was further diluted to 10 ml with mobile phase to get working standard solution having concentration 100 μg mL⁻¹.

 

Procedure for Analysis of Tablet Formulation 

Contents of twenty tablets were weighed accurately and powdered. A quantity of powder equivalent to 1 mg of ATOR (1 mg of EZE and 16 mg of FENO) was transferred to 10 mL volumetric flask containing 6 mL of mobile phase and ultrasonicated for 10 min. The volume was made up to the mark with the mobile phase and filtered through Whatmann paper No. 41. One mililitre of filtrate was further diluted with mobile phase to get solution having concentration 1 μg mL⁻¹ for ATOR, 1 μg mL⁻¹ for EZE and 16 μg mL⁻¹ for FENO. After setting the chromatographic conditions and stabilizing the instrument to obtain a steady baseline, the sample solution was injected, chromatogram was obtained and the peak areas were recorded. The procedure was repeated six times for homogeneous sample and the peak areas were recorded.

 

System Suitability

The system suitability was assessed by six replicate injections of the mixture containing 10 μg mL⁻¹ of each drug. The resolution, peak asymmetry, number of theoretical plates, and HETP were calculated as represented in Table 1.

 

Table 1: System suitability parameters for RP-HPLC method

Sr. No.	Parameters	ATOR	EZE	FENO
1	Theoretical Plates	4894	6425	7245
2	HETP (cm)	0.00510	0.00389	0.003450
3	Resolution a	--	8.96*	13.45*
4	Asymmetry Factor	1.28	1.14	1.33

^a With respect to previous peak.

 

The values obtained demonstrated the suitability of the system for the analysis of these drugs in combination. Mean retention time and standard deviation for ATOR, EZE and FENO were found to be 2.120 ± 0.078, 3.427 ± 0.0049 and 6.680 ± 0.0015 respectively. Representative chromatogram obtained from a mixed standard solution of ATORVA, EZE and FENO is shown in Figure 1.

 

Method Validation

The method was validated for linearity, accuracy and intra-day and inter-day precision, repeatability of measurement of peak area, and repeatability of sample application, in accordance with ICH guidelines.

 

Linearity

Aliquots of 0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 mL of standard stock solution of ATOR and EZE (100 μg mL⁻¹) and 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mL of standard stock solution of FENO (1000 μg mL⁻¹) were transferred in a series of 10 mL volumetric flasks and the volume was made up to the mark with the mobile phase. Six replicates per concentration were injected and chromatograms were recorded. The peak areas were recorded and calibration curve was plotted of peak area against concentration of drug.

 

 

Figure 1: Representative chromatogram obtained for standard mixture of ATOR (10 μg mL^-1, 2.120 min), EZE (10 μg mL^-1 , 3.427   min) and FENO (10 μg mL^-1, 6.680 min)

 

Table 2: Recovery Studies of ATOR, EZE and FENO

^aAverage of three determinations

 

Precision

One set of three different concentrations of mixed standard solutions of ATOR, EZE and FENO were prepared. All the solutions were analyzed thrice, in order to record any intraday variations in the results. For Inter day variations study three different concentrations of the mixed standard solutions in linearity range were analyzed on three consecutive days. The peak areas were recorded and Relative standard deviation (RSD) was calculated for both series of analyses.

 

Accuracy

To check the accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre-analyzed sample solution at three different levels 50, 100 and 150 %. The resulting sample solutions were injected and chromatogram was recorded. The results of recovery studies are shown in Table 2.

 

Robustness

In the robustness study, the influence of small, deliberate variations of the analytical parameters on retention time of the drugs was examined. The following two factors were selected for change: flow rate of the mobile phase (1 ± 0.05 mL min^-1), a wavelength at which the drugs were recorded (240 ± 1 nm). One factor at the time was changed to estimate the effect. The sample solutions containing 1 μg mL⁻¹ of ATOR and EZE and 16 μg mL⁻¹ of FENO were applied onto the column. A number of replicate analyses (n = 3) were conducted at 3 levels of the factor (-, 0, +).

 

RESULTS AND DISCUSSION:

Different mobile phases containing methanol, water and acetonitrile in different proportions were tried and finally methanol: acetonitrile: 0.02M ammonium acetate buffer pH adjusted 10 with Ammonia (60:30:10, v/v/v) was selected as an appropriate mobile phase which gave good resolution. The optimum wavelength for detection and quantitation used was 240 nm.

 

Results were found to be linear in the concentration range of 0.5-10 μg mL⁻¹ for ATOR and EZE and 10-60 μg mL⁻¹ for FENO with high correlation coefficient > 0.999 (Figure 1). The proposed method was also evaluated by the assay of commercially available tablets containing ATORVA, EZE and FENO. The % assay (mean ± S.D.) was found to be 100.82 ± 0.52 for ATOR, 100.11 ± 0.29 for EZE and 99.80 ± 0.61 for FENO. For ATOR, the percentage of recovery study ranged from 99.58 to 100.71 with % RSD values ranging from 0.21 to 0.93. For EZE, the percentage of recovery results ranged from 99.83 to 100.60 with % RSD values ranging from 0.34 to 0.75. FOR FENO, the percentage recovery results ranged from 100.07 to 100.48 with % RSD values ranging from 0.45 to 0.82. The method was found to be accurate and precise, as indicated by recovery studies as recoveries were close to 100% and % RSD not more than 2. Robustness of the method checked after deliberate alterations of the analytical parameters showed that areas of peaks of interest remained unaffected by small changes of the operational parameters (% RSD < 2). The summary of validation parameters of proposed RP-HPLC method are given in Table 3.

 

Table 3: Summary of validation parameters of proposed RP-HPLC method

Parameters	ATOR	EZE	FENO
Linearity range (μg mL-1)	0.5-10	0.5-10	10-60
Correlation co-efficient	0.995	0.999	0.995
Slope (m)	37459	38435	18378
Intercept (c)	6097.7	3472.8	15987
Accuracy (% Recovery)	99.58-100.71	99.83-100.60	100.07-100.48
Precision (% R.S.D.)a
Intraday (nb = 3)	0.39-0.76	0.29-0.87	0.35-0.96
Inter day (n = 3)	0.22-0.68	0.26-0.93	0.41-0.82

 ^aR.S.D. = Relative standard déviation

^bn = Number of determinations

 

CONCLUSION:

The validated HPLC method employed proved to be simple, fast, accurate, precise and robust, thus can be used for routine analysis of ATOR, EZE and FENO in combined tablet dosage form.

 

ACKNOWLEDGEMENTS:

The authors express their gratitude to Emcure Pharmaceuticals Ltd, Pune, India for the gift sample of pure ATOR, EZE and FENO. Thanks are also extended to Dr. Ashwini R. Madgulkar, Principal, A.I.S.S.M.S. College of Pharmacy for providing necessary facilities and her constant support.

 

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Accepted on 21.05.2013         © RJPT All right reserved