Development and Validation of RP-HPLC Method with Diode Array Detection for Estimation of Metaxalone in Rat Plasma

Abstract

Purpose: To develop a simple, highly sensitive, precise and accurate high-performance liquid chromatographic method with photodiode array detection and validated for the rapid quantification of metaxalone in rat plasma samples. Method: Following Liquid-Liquid Extraction (LLE), metaxalone and the internal standard Phenytoin (PHY) were extracted from an aliquot of 200 mL of plasma. Chromatographic separation was carried out using Phenomenex Luna C 8 column (250 mmμ 4.6 mmμ 5 mm) with mobile phase composed of phosphate buffer, pH 7 and acetonitrile in 35:65, v/v ratio. The analyte was monitored with UV detector at 219 nm. The developed method was validated with respect to linearity, accuracy, precision, specificity and stability. The peak area ratio of MET to that of internal standard, PHY was used for the quantification of samples. Results: The retention time of MET and PHY were found to be 2.30 and 3.02 min respectively. The calibration curve was linear (r 2 > or=0.99) ranging from 1.505-538.254 ng/ml and the lower limit of quantification was 1.505 ng/mL. Interday and Intraday precision were lower than 5% (CV) and accuracy ranged from 95 to 105% in terms of percent accuracy. Mean extraction recovery was found to be above 94%. Conclusion: A simple, alternative, reproducible and sensitive HPLC-DAD method was developed for MET that can be used in preclinical pharmacokinetics. Key words: Ammonium formate buffer, Acetonitrile, Metaxalone, Phenytoin, Preclinical pharmacokinetics.