Development and Validation of Analytical Method for Estimation of Leflunomide in Bulk and their Pharmaceutical Dosage Form

Research Article

Austin J Anal Pharm Chem. 2015;2(4): 1046.

Development and Validation of Analytical Method for Estimation of Leflunomide in Bulk and their Pharmaceutical Dosage Form

Patel SK¹*, Patel KH¹, Karkhanis VV² and Captain AD³

¹Department of Quality Assurance, AR College of Pharmacy and GH Patel Institute of Pharmacy, India

²Department of Pharmaceutical Chemistry, AR College of Pharmacy and GH Patel Institute of Pharmacy, India

³Department of Pharmaceutical Analysis, AR College of Pharmacy and GH Patel Institute of Pharmacy, India

*Corresponding author: Shraddha K Patel, Department of Quality Assurance, AR College of Pharmacy and GH Patel Institute of Pharmacy, India.

Received: April 30, 2015; Accepted: July 07, 2015; Published: July 09, 2015

Abstract

The analytical method was developed for estimation of Leflunomide in bulk and their pharmaceutical dosage form. Stability indicating HPTLC method was developed and validated. A simple and accurate High Performance Thin Layer Chromatography method was developed using aluminium sheet precoated with silica gel 60 F254 & mobile phase n-Hexane: Ethyl Acetate (7:3 v/v). The detection wavelength was 271 nm. The method was validated as per ICH guidelines. The linearity was found to be 75-450 ng/band. The correlation coefficient of calibration curve was found to be 0.9985. The method was found to be accurate, precise, specific and robust according to ICH guidelines. The limit of detection (LOD) for Leflunomide was found to be 3.604 ng/band and the limit of quantification (LOQ) for Leflunomide was found to be 10.923 ng/ band. The proposed HPTLC method was successfully applied for the forced degradation study of Leflunomide in bulk and tablet dosage form. Forced degradation study was carried out in acidic condition- 1N HCl at (80±2°C) for 3 hrs, in basic condition- 0.01 N NaOH at room temperature for 3 hrs, in oxidative condition- 6% w/v H2O2 at (60±2°C) for 1 hour, in thermal condition at 80°C for 24 hours and in photolytic condition for 24 hours in UV light. Degradation products were well separated by proposed HPTLC method and the method was found to be specific according to ICH guidelines. The developed method can be used in routine analysis for estimation and for stability study assessment of Leflunomide in Pharmaceutical dosage form.

Keywords: HPTLC; Leflunomide; Forced degradation; Validation

Abbreviation

API: Active Pharmaceutical Ingredient; AR: Analytical Reagent Grade; B.P: British Pharmacopoeia; CDSCO: Central Drug Standard Control Organization; DMARDS: Disease Modifying Antirheumatic Drug; HPLC: High Performance Liquid Chromatography; HPTLC: High Performance Thin Layer Chromatography; ICH: International Conference on Harmonization; I.P: Indian Pharmacopoeia; IUPAC: International Union of Pure & Applied Chemistry; LC: Liquid Chromatography; LOD: Limit Of Detection; LOQ: Limit Of Quantification; MS: Mass Spectrometry; Rf: Retention Factor; RPC: Reverse Phase Chromatography; RSD: Relative Standard Deviation; SD: Standard Deviation; UPLC: Ultra Performance Liquid Chromatoraphy; UV: Ultra Violet; U.S.P: United States Pharmacopoeia; USFDA: United States Food and Drug Administration.

Introduction

Leflunomide is chemically 5-methyl-N-(4-(trifluromethylphenyl)- 4-isoxazolecarboxamide has empirical formula C12H9F3N2O2 with molecular weight 270.21 (g/mol) (Figure 1) [1,2,3]. Leflunomide is a pyrimidine synthesis inhibitor belonging to the DMARD (diseasemodifying antirheumatic drug) class of drugs, which are chemically and pharmacologically very heterogeneous. Leflunomide was first approved by FDA for treatment of active rheumatoid arthritis on 11 September, 1998. Leflunomide was approved by CDSCO on 1st October, 2001. Leflunomide is one of the new drugs used in the treatment of rheumatoid arthritis. It works by suppressing the immune system because rheumatoid arthritis is caused by damage from an overacting immune system [5]. After oral doses Leflunomide undergoes first-pass metabolism to teriflunomide, which is responsible for the majority of the in vivo activity. The bioavailability of Leflunomide after oral doses ranges from 82 to 95%. Peak plasma concentrations of the active metabolite may occur from 1 to 24 hours after a dose. About 43% of a dose is eliminated in the urine, mainly as tablets. This research article reports a precise, accurate and sensitive HPTLC method with UV detection, useful for routine quality control of Leflunomide and its degradation products in solid oral formulation. The method was validated by parameters such as linearity, accuracy, precision and robustness. Leflunomide is official in IP-2014, BP-2010 and USP-2011. Literature review reveals that the methods like HPLC, UV-Spectroscopic, LC-MS/MS, RP-HPLC, and UPLC have been reported for estimation of Leflunomide in bulk and tablet dosage form. But, HPTLC method has not been reported for estimation of Leflunomide. So, aim of dissertation work is to develop and validate precise and accurate HPTLC method. HPTLC method is widely used for routine drug analysis. Less amount of mobile phase is required which is one of the advantages of this method. Solvents need no prior treatment like filtration and degassing. High sample throughout of similar or different nature of samples. Low cost precoated HPTLC plates are available. Hence, this method is more economic and lower analysis time as compare to reported HPLC methods [7]. Our work deals with the forced degradation of Leflunomide under stress condition like acid hydrolysis, base hydrolysis, and oxidation, thermal and photolytic stress. The developed method is useful for routine quality control analysis and determination of stability.