Development and Application of HPLC Methods with Tandem Mass Spectrometric Detection for the Determination of Hydrochlorothiazide in Human Plasma and Urine Using 96‐Well Liquid‐Liquid Extraction

Abstract

Sensitive and selective methods for the determination of hydrochlorothiazide (HCTZ) in human plasma and urine were developed. Both methods were based on high performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection. HCTZ and hydroflumethiazide (HFTZ), used as an internal standard, were isolated from human plasma and urine by liquid‐liquid extraction in the 96‐well plate format using a Tomtec Quadra 96 workstation. The MS/MS detection was performed on an Applied Biosystems‐Sciex API 3000 tandem mass spectrometer interfaced with a heated nebulizer probe and operated in the negative ionization mode. The developed methods were validated in the concentration ranges of 1–100 ng/mL and 0.05–20 µg/mL in human plasma and urine, respectively. The precision of both methods, as expressed by the coefficients of variation (C.V.), was less than 5.6% at all concentrations within the standard curves with acceptable accuracy. The plasma and urine methods provided sufficient sensitivity and high sample throughput to map out the pharmacokinetics of HCTZ in human subjects following an oral dose of HCTZ/Losartan, in support of a human clinical trial designed to evaluate the bioequivalence of HCTZ between two formulations.

Keywords:

• HPLC‐MS/MS determination
• Hydrochlorothiazide
• Hydroflumethiazide
• 96‐Well liquid‐liquid extraction
• Human plasma
• Urine

Acknowledgment

The authors would like to thank Dr. Robert Alexander for directing and monitoring the clinical program from which the human plasma and urine samples were obtained for analyses.