Simultaneous determination of major components of Huangqi-Honghua extract in rat plasma using LC-MS/MS and application to a pharmacokinetic study.

Abstract

A sensitive and reliable LC-MS/MS method was developed and validated for simultaneous quantification of the major components of Huangqi-Honghua extact in rat plasma, including hydroxysafflor yellow A (HSYA), astragaloside IV (ASIV), calycosin-7-O-β-d-glucoside (CAG), calycosin, calycosin-3'-O-glucuronide (C-3'-G) and calycosin-3'-O-sulfate (C-3'-S). After extraction by protein precipitation with acetonitrile and methanol from plasma, the analytes were separated on a Hypersil BDS C18 column by gradient elution with acetonitrile and 5 mM ammonium acetate. The detection was carried out on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization source switched between negative and positive modes. HSYA was monitored in negative ionization mode from 0 to 4.9 min, and ASIV, CAG, calycosin, C-3'-G and C-3'-S were determined in positive ionization mode from 4.9 to 10 min. The lower limits of quantification of the analytes were 6.25 ng/mL for HSYA, 0.781 ng/mL for CAG and 1.56 ng/mL for ASIV and calycosin. The intra- and inter-assay precision (RSD) values were within 13.43%, and accuracy (RE) ranged from -8.75 to 9.92%. The validated method was then applied to the pharmacokinetic study of HSYA, ASIV, CAG, calycosin, C-3'-G and C-3'-S in rat after an oral administration of Huangqi-Honghua extract.