Abstract

BIOANALYTICAL METHOD DEVELOPMENT, VALIDATION AND STABILITY FOR ESTIMATION OF CHLORTHALIDONE IN HUMAN PLASMA BY USING LC-ESI- MS/MS

Manali Patel*

ABSTRACT

A simple, rapid and accurate bioanalytical method was developed and validated as per US-FDA guidelines for the estimation of Chlorthalidone in spiked human plasma using liquid-liquid extraction technique. Results: Chlorthalidone was well resolved from human plasma interference and internal standard (Chlorthalidone D4) using ZIC®HILIC (100X4.6mm,5μm) column with Methanol: 0.05 Formic acid in water (90:10,v/v) as mobile phase, at a flow rate of 0.900 ml/min. Detection was performed by electrospray ionization (ESI) operated in negative multiple reaction monitoring (MRM) mode. The lower limit of quantitation (LLOQ) of this method was 2071.057 pg/mL and the calibration curves were linear (r(2)≥0.995) over the concentration range of 2000-1200000 pg/mL for Chlorthalidone. The intra- and inter-day precision and accuracy were well within the acceptable limits. The mean extraction recoveries were found to be about 80% and no matrix effect was observed. Chlorthalidone wwsfound to be stable under all relevant storage conditions.

Keywords: Chlorthalidone, Chlorthalidone D4, LC-MS/MS.