Liquid chromatography and tandem mass spectrometry method for quantitative determination of pioglitazone and its metabolite 5-hydroxy pioglitazone in human plasma

Abstract

A liquid chromatography tandem mass spectrometry (LC-MS/MS) based method was developed for the simultaneous estimation of pioglitazone and its active metabolites in human plasma for applicability to pharmacokinetic studies. The chromatographic separation was carried on the reversed phase Peerless Basic C18, column (100×4.6mm, 5μm) at column temperature of 40°C using a binary mobile phase consisting of methanol: 5mM ammonium acetate in 0.1% formic acid (80:20, v/v). The mobile phase was run at a flow rate of 1mL/min and the sample injection was 10μL. The method utilized pioglitazone D4 (IS1) and 5-hydroxyl pioglitazone M-IV D4 (IS2) as an internal standard. The linearity of the method was validated over the range of 6.04-1503.21ng/mL for pioglitazone and 6.01-1496.28ng/mL for 5-hydroxyl pioglitazone. The mean extraction recovery of PIO & HPIO from the spiked plasma was found to be 94.92% for pioglitazone and 96.13% for 5-hydroxy pioglitazone. The developed method can be successfully employed in healthy human volunteers to monitor the pharmacokinetics profile of pioglitazone.

Keywords: Hydroxypioglitazone; LC-MS; LC-MS validation; LCMS; LCMS validation; Pioglitazone.