High performance liquid chromatography/negative ion electrospray tandem mass spectrometry method for the measurement of hydrochlorothiazide in human plasma: application to a comparative bioavailability study.

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 x 4.6mm i.d., 5 microm) 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 ESI MS/MS in the negative ion mode. The total chromatographic runtime was 2.5 minutes. 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 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 internal standard (Zidovudine) was 63.62% with a precision ranging from 2.06% to 5.40%. The method was successfully applied for the evaluation of pharmacokinetics of hydrochlorothiazide after single oral dose of 25 mg hydrochlorothiazide to healthy volunteers.