A NOVEL RP- HPLC METHOD DEVELOPMENT AND VALIDATION OF ATORVASTATIN AND FENOFIBRATE IN BULK AND PHARMACEUTICAL DOSAGE FORMS

ABOUT AUTHORS:
1Vinjam Swathi* 2Nanda Kishore Agarwal
1M pharmacy, Department of Pharmaceutical Analysis and Quality Assurance, Nimra College of Pharmacy, Jupudi, Vijayawada, A.P, India
2Professor and head of the department of pharmaceutical chemistry, Nimra College Of Pharmacy, Jupudi, Ibrahimpatnam, Vijayawada, A.P, India
swathi.vinjam90@gmail.com

ABSTRACT
The present investigation describes about a simple, economic, selective, accurate, precise reverse phase high performance liquid chromatographic method for the simultaneous estimation of Atorvastatin and Fenofibrate in pure and pharmaceutical dosage forms. Atorvastatin and Fenofibrate were well separated using a Thermohypersil BDS C18column of dimension 100 × 4.6, 5µm and Mobile phase consisting of  Methanol: Water (Adjusted with orthophosphoric acid to pH-2) in the ratio of 40:60v/v at the flow rate 1 ml/min and the detection was carried out at 274nm with PDA detector. The Retention time for Atorvastatin and Fenofibrate were found to be 1.438, 2.949 respectively. The developed method was validated for recovery, specificity, precision, accuracy, linearity according to ICH guidelines. The method was successfully applied to Atorvastatin and Fenofibrate combination pharmaceutical dosage form.

REFERENCE ID: PHARMATUTOR-ART-2001

1. INTRODUCTION
Atorvastatin ((3R, 5R)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3, 5-dihydroxyheptanoic acid) is a member of the drug class known as statins. It is used for lowering cholesterol. Atorvastatin is a competitive inhibitor of hydroxymethylglutaryl-coenzyme-A (HMG-CoA) reductase, the rate-determining enzyme in cholesterol biosynthesis via the mevalonate pathway. HMG-CoA reductase catalyzes the conversion of HMG-CoA to mevalonate. It acts primarily in the liver. Decreased hepatic cholesterol levels increases hepatic uptake of cholesterol and reduces plasma cholesterol levels.

Fenofibrate (propan-2-yl 2-{4-[(4-chlorophenyl) carbonyl] phenoxy}-2 methyl propanoate) is a drug of the fibrate class. It is mainly used to reduce cholesterol levels in patients at risk of cardiovascular disease. Like other fibrates, it reduces low-density lipoprotein (LDL) and very low density lipoprotein (VLDL) levels, as well as increasing high-density lipoprotein (HDL) levels and reducing triglycerides level. It is used alone or in conjunction with statins in the treatment of hypercholesterolemia and hypertriglyceridemia. It lowers lipid levels by activating peroxisome proliferator-activated receptor alpha (PPARα). PPARα activates lipoprotein lipase and reduces apoprotein CIII, which increases lipolysis and elimination of triglyceride-rich particles from plasma.

Literature survey revealed that very few methods have been reported for the analysis of Atorvastatin and Fenofibrate combinational dosage forms which include UV spectroscopy, Reverse Phase High performance Liquid Chromatography,  Densitometric method, HPTLC methods. The present study illustrate development and validation of simple, economical, selective, accurate, precise RP-HPLC method for the determination Aof torvastatin and Fenofibrate in bulk and Pharmaceutical dosage forms as per ICH guidelines.

The goal of this study is to develop rapid, economical HPLC method for the analysis of Atorvastatin and Fenofibrate in combined dosage form using most commonly employed column (C18) and simple mobile phase preparation.

In the present proposed work a successful attempt had been made to develop a method for the simultaneous estimation of Atorvastatin and Fenofibrate pharmaceutical dosage form and validate it. From the economical point of view and for the purpose of routine analysis, it was decided to develop a more economical RP-HPLC method with simple mobile phase preparation for the estimation of Atorvastatin and Fenofibrate combinational dosage form. The method would help in estimate of drugs in single run which reduces the time of analysis and does not require separate method for each drug. Thus, the paper reports an economical, simple and accurate RP-HPLC method for the above said pharmaceutical dosage forms.

2. MATERIALS AND METHODS
Quantitative HPLC was performed on a high performance liquid chromatograph -Waters e2695Alliance HPLC system connected with PDA Detector 2998 and Empower2 Software. The drug analysis data were acquired and processed using Empower2 software running under Windows XP on a Pentium PC and Thermohypersil BDS C18column of dimension 100 × 4.6, 5µm particle size. In addition an analytical balance (DENVER 0.1mg sensitivity), digital pH meter (Eutech pH 510), a sonicator (Unichrome associates UCA 701) were used in this study.

Standards and chemicals used:Pharmaceutical grade Atorvastatin and Fenofibratewere kindly supplied as a gift sample by Dr.Reddy’s Laboratory, Hyderabad, and Andhra Pradesh, India. Methanol was of HPLC grade and Purchased from E. Merck, Darmstadt, Germany. Ortho Phosphoric Acid was analytical reagent grade supplied by Fischer Scientific Chemicals. Water HPLC grade was obtained from a Milli-QRO water purification system. Atorvastatin and Fenofibrate Tablets available in the market as Lipicure-TG (intas pharmaceuticals, Sikkim, India.)  in composition of Atorvastatin (10mg), Fenofibrate (150mg).

Preparation of mobile phase: Transfer water into 1000ml of beaker dissolve and diluted volume with water. Then adjust its pH to 2 with Ortho Phosphoric Acid (OPA). The Water adjusted pH to 2 with OPA: Methanol (60:40 v/v) and filtered through 0.45µ membrane filter and degassed by sonication.

Preparation of calibration standards:10mg Atorvastatin and 16mg Fenofibrate was taken into a 10, 50 ml of volumetric flask and add 10ml ofDiluent and sonicated for 10 minutes and made up with Diluent. It was further diluted to get stock solution of Atorvastatin and Fenofibrate (To get 0.02 ppm and 0.32 ppm solution respectively). This is taken as a 100% concentration. Working standard solutions of Atorvastatin and Fenofibrate was prepared with mobile phase. To a series of 10 ml volumetric flasks, standard solutions of Atorvastatin and Fenofibrate in the concentration range of 0.01-0.03µg/ml and 0.16-0.48µg/ml were transferred respectively.

System suitability: System suitability are an integral part of chromatographic system. To ascertain its effectiveness, certain system suitability test parameters were checked by repetitively injecting the drug solutions at 100% concentration level for Atorvastatin and Fenofibrate to check the reproducibility of the system. At first the HPLC system was stabilized for 40 min.One blank followed by six replicate analysis of solution containing 100% target concentration of Atorvastatin and Fenofibrate were injected to check the system suitability. To ascertain the system suitability for the proposed method, a number of parameters such as theoretical plates, peak asymmetry, and retention time were taken and results were presented in Table 1.

Recommended procedure:

Calibration curves for Atorvastatin and Fenofibrate: Replicate analysis of solution containing 0.01-0.03µg/mL, 0.16-0.48µg/mL of Atorvastatin and Fenofibrate sample solutions respectively were injected into HPLC according to the procedure in a sequence and chromatograms were recorded. Calibration curves were constructed by plotting by taking concentrations on X-axis and ratio of peak areas of standards on Y-axis and regression equation were computed for both drugs and represented in Table .6

Analysis of marketed formulation: The content of ten tablets was weighed accurately. Their average weights were determined. Powder of tablets equivalent to one tablet weight (521.1mg) were weighed and taken in a 50 ml volumetric flask, dissolved in diluents, shaken and sonicated for about 20 minutes then filtered through 0.45µ membrane filter. The filtered solution was further diluted (5 to 50ml) in the diluents to make the final concentration of working sample equivalent to 100% of target concentration.The prepared sample and standard solutions were injected into HPLC system according to the procedure. from the peak areas of Atorvastatin and Fenofibrate the amount of the drugs in the sample were computed. The contents were calculated as an average of six determinations and experimental results were presented in Table 4. The representive standard and sample chromatograms were shown in fig.2 and fig.3.

Validation study of Atorvastatin and Fenofibrate: An integral part of analytical method development is validation. Method validation is the process to confirm that the analytical procedure employed for a specific test is suitable for its intended use. The newly developed RP-HPLC method was validated as per International Conference on Harmonization (ICH) guidelines for parameters like specificity, system suitability, accuracy, linearity, precision (repeatability), limit of detection (LOD), limit of Quantification (LOQ) and robustness.

Specificity: The effect of wide range of excipients and other additives usually present in the formulation of Atorvastatin and Fenofibrate in the determination under optimum conditions were investigated. The specificity of the RP-HPLC method was established by injecting the mobile phase and placebo solution in triplicate and recording the chromatograms. The common excipients such as lactose anhydrous, microcrystalline cellulose and magnesium state have been added to the sample solution injected and tested.

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