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A selective and sensitive ultra performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of losartan (LOS), EXP-3174, which is an active metabolite LOS carboxylic acid, and hydrochlorothiazide (HCTZ) in human plasma. Solid-phase extraction was carried out on Oasis HLB cartridges with 100 mu L of plasma to give an extraction recovery in the range of 88.5%-102.5% for the three analytes. Chromatography on a BEH C18 column afforded baseline separation of all the analytes within 2.4 min using 1.0% formic acid in water and acetonitrile (15:85, v/v) as the mobile phase. Quantitation was performed with multiple reaction monitoring in the negative ionization mode. The response of the method was linear over a dynamic range of 0.5-500, 1.0-750, and 0.25-150 ng/mL for LOS, EXP-3174, and HCTZ, respectively. Extent of signal suppression/enhancement was examined through postcolumn infusion. The effect of matrix components was evaluated by postextraction spiking and calculation of the slope of calibration lines. The method was successfully applied to a bioequivalence study of 50 mg losartan and 12.5 mg hydrochlorothiazide tablet formulation in 65 healthy human subjects. Reproducibility of the method was shown by reanalysis of 213 incurred samples.