A High-Performance Liquid Chromatography/Negative Ion Electrospray Tandem Mass Spectrometry Method for the Determination of Hydrochlorothiazide in Human Plasma: Application to a Comparative Bioavailability Study

Abstract

A sensitive, selective and rapid liquid chromatography/tandem mass spectrometric (LC–MS/MS) method was developed and validated for the determination of hydrochlorothiazide (HCTZ) in human plasma. The plasma samples were prepared by solid-phase extraction using Oasis HLB 30 mg/1CC cartridges. Chromatographic separation was accomplished on a Thermo Hypurity Advance (50 mm × 4.6 mm i.d., 5 μm) column. The mobile phase consisted of HPLC-grade acetonitrile: 2 mM ammonium acetate (90: 10 v/v) at a flow rate of 0.5 mL min−1. Detection of hydrochlorothiazide and the internal standard (IS) zidovudine was achieved by electrospray ionization (ESI) MS/MS in the negative ion mode. The total chromatographic run time was 2.5 min. The linear range of the method was from 2.036–203.621 ng mL−1. The mass transition ion pair has been followed as m/z 296.10/205.00 for HCTZ and m/z 266.10/223.10 for zidovudine. The mean overall recovery of HCTZ was 66.40% with a precision of 2.44%. The mean recovery of the internal standard (zidovudine) was 63.62% with a precision ranging from 2.06% to 5.40%. The method was successfully applied to the evaluation of pharmacokinetics of hydrochlorothiazide after single oral dose of 25 mg hydrochlorothiazide to healthy volunteers.