Determination of Irbesartan and Hydrochlorothiazide in Human Plasma by LC-MS/MS and Its Application

OBJECTIVE To establish an LC-MS/MS method for simultaneous determination of irbesartan(IBST) and hydrochlorothiazide(HCZ) concentrations in human plasma and to be used for a bioequivalence study of IBST/HCZ tablets. METHODS Plasma samples were pre-treated by protein precipitation. IBST-d4 and chlorothiazide(MCTZ) were used as the internal standard. The analytes were retained and eluted by using a Waters ACQUITY UPLC^®XTERRA RP₁₈(4.6 mm×100 mm, 3.5 μm) column, with a binary mobile phase system consisting of acetonitrile(A) and 5 mmol·L^-1 ammonium acetate solution(B) at a flow rate of 0.85 mL·min^-1. The analytes were detected in an electrospray ionization source under negative ion mode, using the ion transitions of m/z 427.1→193.1(IBST), m/z 431.2→193.1(IBST-d4), m/z 295.9→268.9(HCZ) and m/z 357.8→322.0 (MCTZ). The quantitation method was applied to a bioequivalence study of IBST/HCZ tablets(150 mg/12.5 mg) in healthy subjects. RESULTS Endogenous substances in plasma did not interfere with the quantification of the substance to be measured, and no interference and transformation on and from IBST, HCZ, IBST-d4 and MCTZ. The quantitative ranges of IBST and HCZ in human plasma were 10-4 000 ng·mL^-1 and 1-400 ng·mL^-1, respectively, with good linearity. The extraction recoveries of IBST and IBST-d4 were between 95.7% and 105.4%, HCZ and MCTZ were between 95.0% and 106.4%; the internal standard normalization matrix effect factors of IBST and HCZ were recorded as 101.2%-102.6%, and 79.8%-93.6% all with coefficient of variation(CV)<5%, respectively. Intra- and inter-batch accuracy and precision were all within 15%, CV<10%. The concentrations of IBST and HCZ determined by the method and the pharmacokinetic parameters calculated thereafter were in accordance with those found in previous reports, and the difference of concentrations generated in the incurred sample reanalysis assay and the original measurement was <10%. CONCLUSION The method is rapid, specific, sensitive, accurate, and reproducible requiring a small sample volumn, and fully meets the requirements of high-throughput quantitative analysis of IBST/HCZ tablets bioequivalence studies.