Determination of Atorvastatin and Fenofibrate in A Fixed Dose Combination by High Pressure Liquid Chromatography

An accurate and reproducible liquid chromatographic assay method was developed and validated for the determination of Atorvastatin and Fenofibrate in capsule formulation. Water (pH 4.0) and acetonitrile (20:80, v/v) was used for reversephase liquid chromatography at detection wavelength 254 nm to determine the contents of Atorvastatin and Fenofibrate in combination capsule dosage form. The method was validated by determining parameters such as specificity, linearity, LOD and LOQ, precision, accuracy, ruggedness and robustness. The method was found to be specific against placebo interference. Linearity was evaluated over the concentration range of 1.0 to 6.0 μg/mL and 5.0-50.0 μg/mL for both Atorvastatin and Fenofibrate (the correlation of coefficient obtained was 0.999 for both). The intraday and interday precision values of the system and method were determined. The accuracy of the method ranged from 99.99 to 102.24% for Atorvastatin from 100.45 to 101.22% for Fenofibrate. The proposed method was found to be robust when slight but deliberate changes were made in analytical conditions. The developed method will be suitable for the assay of Atorvastatin and Fenofibrate in raw materials, capsule formulation as well in other forms of combined dosage forms. Arunadevi S. Birajdar*, S. N. Meyyanathan Department of Pharmaceutical Analysis, J.S.S. College of Pharmacy, Ootacamund, Tamilnadu-643 001 Submission: 29 June 2015 Accepted: 6 July 2015 Published: 25 July 2015 www.ijppr.humanjournals.com Citation: Arunadevi S. Birajdar et al. Ijppr.Human, 2015; Vol. 3 (4): 83-94. 84 INTRODUCTION The combined dosage form of any pharmaceuticals is developed for the synergistic effect or to give longer time effect. In present study Atorvastatin and Fenofibrate combination is used as antihypertensive. Atorvastatin calcium (ATV) is chemically [R-(R*,R*)]-2-(4-flurophenyl)-β, δdihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrole-1-heptanoic acid, calcium salt trihydrate. Atorvastatin calcium is an inhibitor of 3-hydroxy-3methyl glutaryl coenzyme A (HMG-Co A) reductase. This enzyme catalyses the conversion of HMG-CoA to mevalonate, an early and rate-limiting step in cholesterol biosynthesis. Fenofibrate (FEB) is chemically Isopropyl 2,4,4 chlorobenzoyl phenoxy 2 methylpropionate. Fenofibrate a fibric acid derivative is a lipid regulating drug with actions on plasma lipids similar to those of bezafibrate. It is used to reduce low density lipoprotein (LDL) cholesterol, total cholesterol, triglycerides, and apolipoprotein B, and to increase high density lipoprotein (HDL) cholesterol, in the management of hyperlipidaemias, including type IIa, type IIb, type III, type IV, and type V hyperlipoproteinaemias. A literature review reveals that only a few methods have been developed for the quantification of individual drug Atorvastatin and Fenofibrate. The chemical structure is as shown in Figure 2. MATERIALS AND METHODS Experimental HPLC Instrumentation The chromatographic separation was performed on the Waters liquid chromatographic system equipped with (Waters-1515) isocratic solvent delivery system pump with (Waters-2487) dual wavelength absorbance UV-detector and Rheodyne 7725i injector with 50 L loop volume. Breeze 3.3 data station was applied for data collecting and processing. A phenomenox C18 column (25cm x 4.6mm i.d., 5μm particle size) was used for the separation. The mobile phase consisted of a mixture of water (pH 4.0) and acetonitrile in the ratio of (20:80, v/v). The mobile phase was prepared daily, filtered, sonicated before use and delivered at the flow rate of 1.0 mL/min. The detection wavelength fixed at 254 nm an isobestic point for both drugs as shown in Figure 1. www.ijppr.humanjournals.com Citation: Arunadevi S. Birajdar et al. Ijppr.Human, 2015; Vol. 3 (4): 83-94. 85 Chemicals and reagents The pharmaceutical grade gift reference and working standards of Atorvastatin calcium (99.77%) procured from HAB Pharmaceutical & Research Ltd (Thane, India) and Fenofibrate (99.56%) from Cadila pharmaceutical Ltd (Ankleshwer, India). The chemical structure of samples will be as Shown in Figure 2. Internal standard Diclofenac sodium (99.92%), gift sample was procured from Apex pharmaceuticals (Medak, India). The solvents acetonitrile and methanol used were of HPLC grade; all analytical grade chemicals and solvents were obtained from E Merck (India) Ltd, Mumbai. Ammonium sulphate AR grade were procured from Qualigens fine chemicals, Mumbai. Water HPLC grade was obtained from a Milli-QRO water purification system. By UV Spectrophotometer Solvent -Methanol and Water (50: 50 v/v) λ max -Atorvastatin 246 nm Fenofibrate 288.8 nm Overlay -Isobestic point 254 nm Linearity and range -Atorvastatin 1.0 6.0 μg/ml Fenofibrate 5.0 -50 μg/ml Procedure for UV Calibration Above mentioned Spectra shown in Figure 1 and Linearity in Table no. 1. The selection of wavelength is carried out and Linearity is confirmed by UV-Spectrophotometry along with IS selected. Standard solutions and calibration curves by HPLC Standard stock solutions were prepared at concentration of 1 mg/mL of ATV and FEB separately using a mixture of Water (pH 4.0) and acetonitrile (20:80, v/v). The working standard solutions were prepared of different concentrations ranging from 1.0 to 6.0 μg/mL and 5.0 to 50.0 μg/mL for ATV and FEB respectively, by maintaining the concentration of Diclofenac sodium (IS) at a constant level of 50μg/mL. From the above each mixture 50μL was injected in triplicate for the www.ijppr.humanjournals.com Citation: Arunadevi S. Birajdar et al. Ijppr.Human, 2015; Vol. 3 (4): 83-94. 86 estimation of standard drugs, under the optimized chromatographic conditions, a steady baseline was recorded; the typical chromatogram was recorded for standard ATV and FEB with internal standard as shown in Figure 3. The retention times of standard and internal standard were found to be 3.58, 5.68 and 8.85 min, respectively. The calibration curve was obtained by simple linear regression of concentration of drug to the response factor as denoted in Table no. 2. Analysis of formulation by HPLC Twenty tablets each containing 10 mg of Atorvastatin and 200 mg of Fenofibrate were weighed and the amount equivalent to one tablet content was weighed, powdered and dissolved in acetonitrile: water (50:50 v/v) with appropriate amount of IS was added in mixture. The drugs were extracted with same solvent mixture, filtered and further dilutions were made by mobile phase to get a concentration of 2 μg/ml of Atorvastatin, 40μg/ml of Fenofibrate and 50 μg/ml of Diclofenac sodium as internal standard. These solutions were injected for the estimation. The retention times obtained for Atorvastatin, Diclofenac sodium (IS), and Fenofibrate were at 3.56, 4.66 and 8.75 min respectively as in Figure 4. RESULTS AND DISCUSSION