Development and Validation of HPLC Method for Estimation of Etodolac in Rat Plasma

Research Article

Austin J Anal Pharm Chem. 2016; 3(1): 1061.

Development and Validation of HPLC Method for Estimation of Etodolac in Rat Plasma

Bachhav DG1*, Khadabadi SS2 and Deore LP1

1M.G.V.’s SPH College of Pharmacy, Malegaon, Nashik, India

2Government College of Pharmacy, Amravati, India

*Corresponding author: Bachhav DG, Department of Pharmaceutics, M.G.V.’s S.P.H.College of Pharmacy, Malegaon, Nashik (MS), India

Received: March 20, 2016; Accepted: April 11, 2016; Published: April 14, 2016

Abstract

Etodolac is 1, 8-diethyl-1, 3, 4, 8- tetrahydropyrano [3,4-b] indole-1-acetic acid, used for osteoarthritis. This study was designed to develop and validate high performance liquid chromatography method of etodolac in rat plasma. The samples were analyzed by HiQ Sil C18 HS (250 × 4.6 mm, 5μ.) columns, using Acetonitrile: 0.02M potassium dihydrogen orthophosphate (65: 35 v/v) as a mobile phase. The method showed linearity (r2= 0.9964) over a concentration range of (1-25μg/ml). The method showed good mean recovery (97.53%) for etodolac. The method was found to be accurate, precise, linear, specific, sensitive and stable.

Keywords: Etodolac; HPLC; Rat plasma; Analytical method; Validation

Abbreviations

HPLC: High Performance Liquid Chromatography; ETDO: Etodolac; THIO: Thiocolchicoside; NSAID: Non-steroidal Antiinflammatory Drug; RP-HPLC: Reverse Phase High Performance Liquid Chromatography; GC-MS: Gas Chromatography Mass Spectroscopy; ICH: International Conference on Harmonization; Rpm: Revolution per minute; ISTD: Internal Standard; ACN: Acetonitrile; DMSO: Dimethyl Sulfoxide; LLOQ: Lower Limit of Quantification; CV: Coefficient of Variation; LQC: Low Quality Control; MQC: Middle Quality Control; HQC: High Quality Control

Introduction

Etodolac is 1, 8-diethyl-1, 3, 4, 8 - tetrahydropyrano [3, 4-b] indole- 1-acetic acid. Etodolac contains not less than 98.0% and not more than 102.0% of C17H21NO3, calculated on the anhydrous basis [1]. Etodolac is a nonsteriodal anti-inflammatory drug (NSAID) that exhibits antiinflammatory, analgesic, and antipyretic activities. The mechanism of action of etodolac, like that of other NSAIDs, is not completely understood, but may be related to prostaglandin synthetase inhibition [2]. Well absorbed following oral administration; bioavailability is about 80%. Peak plasma concentration usually attained within about 1.4 hours (conventional capsules and tablets) or 6.7 hours (extendedrelease tablets) [3]. Etodolac is white or almost white crystalline powder [1]. Soluble in water (<1 mg/ml), methanol (9.80-10.20mg/ ml), chloroform, DMSO (58mg/ml), and ethanol (58mg/ml) [4-6]. Several spectroscopic and chromatographic methods are available in literature to determine concentration of Etodolac, individually or in combination with other drugs or metabolites as UV [7-8], HPLC- UV [9], RP-HPLC [10-11], GC-MS [12]. The present article describes a simple HPLC method for estimation of Etodolac and validation of the method as per the guidelines of ICH [13].

Materials and Methods

Reagents and chemicals

Etodolac (Ipca Laboratories, Mumbai), Acetonitrile HPLC grade, Methanol HPLC grade. (Merck Laboratories, Mumbai), Double distilled water, Glacial Acetic Acid, Potassium dihydrogen orthophosphate.

Selection of mobile phase

Different mobile phases like methanol with Phosphate buffer of different molarities at various pH, acetonitrile and Phosphate buffer, methanol and water were tried in different ratio in order to find the best conditions for Etodolac (ETO). After several trials Acetonitrile: 0.02M potassium dihydrogen orthophosphate (65: 35 v/v) was chosen as the mobile phase for analysis in which optimum system suitability parameters were obtained.

Preparation of mobile phase

130 ml of HPLC grade Acetonitrile was added in 70ml of 0.02M potassium dihydrogen orthophosphate i.e. in 65: 35 v/v proportions. The solution was further filtered through 0.45μ membrane filter and sonicated in sonicator bath for 10min.

Preparation of standard stock solutions of Etodolac (100μg/ml)

10 mg of Etodolac was dissolved in 10ml of Acetonitrile and 1ml of this solution was diluted with Acetonitrile to final volume of 10ml in volumetric flask to get concentration 100μg/ml (stock I).

Preparation of intermediate stock solution by using stock solutions of Etodolac (100μg/ml) for plasma calibration curve

Using a calibrated pipette 0.4, 1, 2, 4, 6 and 8 ml of stock solution I was transferred to separate volumetric flasks and then diluted to 10ml with acetonitrile to get concentrations 4, 10, 20, 40, 60 and 80μg/ml.

Preparation of internal standard stock solution of Thiocolchicoside (100μg/ml)

10 mg of Thiocolchicoside was dissolved in 10 ml of Acetonitrile and 1 ml of this solution was diluted with Acetonitrile to final volume of 10 ml in volumetric flask to get concentration 100 μg/ml (stock I). Using a calibrated pipette, 4 ml of ISTD stock solution (100μg/ml) was pipette into a 10.0 ml volumetric flask and made up the volume with the mobile phase to get concentration of 40μg/ml.

Preparation of plasma sample solution

To 0.5ml of rat plasma, 0.5ml of stock solution of Etodolac (Concentrations: 4, 10, 20, 40, 60, 80 and 100μg/ml), 0.5ml of an internal standard solution (Thiocolchicoside, 40μg/ml) and 0.5 ml Acetonitrile were added in a glass tubes (Table 1). Each sample tube was vortex mixed for 10min and centrifuged (3000rpm for 10min). After centrifugation 20μl aliquots of supernatant of each concentration were injected into the HPLC system.