Simultaneous determination of Triamterene and Hydrochlorothiazide in human plasma by liquid chromatography tandem mass spectrometry and its application to a bioequivalence study

A rapid and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the simultaneous determination of Triamterene and Hydrochlorothiazide in human plasma was established. The analytes and the internal standards were separated on a Zorbax Eclipse Plus RRHD C18 column (2.1 mm × 50 mm, 1.7 μm) using isocratic elution with a mobile phase of 0.1% formic acid:methanol:acetonitrile 5:4:1 and 0.1% formic acid in water at a flow rate of 0.4 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode to monitor the precursor-to-product ion transitions of m/z 254.0 → 237.1, 259.1 → 242.2 for Triamterene and Triamterene D5 in positive electrospray ionization mode, m/z 295.9 → 269.0, 300.9 → 271.0 for Hydrochlorothiazide and Hydrochlorothiazide 15N2-13C-D2 using a negative electrospray ionization mode. The method was validated over a concentration range of 0.5–200 ng/mL for Triamterene and 2.5–400 ng/mL for Hydrochlorothiazide. Total time for each chromatogram was 5.2 min (after 1.5 min switching to positive mode). The method was successfully applied to a bioequivalence study of Triamterene and Hydrochlorothiazide in healthy human volunteers.