Simultaneous Determination of Olmesartan and Hydrochlorothiazide in Human Plasma by UPLC-MS/MS Method and Its Application to a Bioequivalence Study.

A simple, sensitive and efficient ultra performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for simultaneous quantitative determination of olmesartan and hydrochlorothiazide in human plasma has been developed and validated. Using olmesartan-d4 and hydrochlorothiazide-13C-d2 as stable isotope-labeled internal standard (SIL-IS). Plasma samples were processed by protein precipitation. Separated on a ZORBAX Eclipse XDB-Phenyl column (150 mm × 4.6 mm, 5 μm) using a gradient elution with mobile phases methanol and 5 mM ammonium acetate. Detected by electrospray ionization (ESI) in negative ion mode of multiple reaction monitoring (MRM), mass transition ion pairs were m/z 445.1 → 149.0 for olmesartan, m/z 295.9 → 204.9 for hydrochlorothiazide, m/z 449.1 → 149.1 for olmesartan-d4, and m/z 301.0 → 272.0 for hydrochlorothiazide-13C-d2. Linear ranges were 10-2000 ng/mL for olmesartan and 1.5-300 ng/mL for hydrochlorothiazide. Mean recoveries of olmesartan, hydrochlorothiazide, olmesartan-d4, and hydrochlorothiazide-13C-d2 were 91.31%, 99.34%, 92.62%, and 98.61%, respectively. Our method was well validated in selectivity, carryover, lower limit of quantification (LLOQ), calibration curve, accuracy, precision, dilution effect, matrix effect (normal, hyperlipidemic, and hemolyzed matrices), stability, recovery, and IRS. It was successfully applied in a bioequivalence study of olmesartan medoxomil and hydrochlorothiazide tablets (20/12.5 mg) in healthy Chinese volunteers.