Liquid Chromatography–Tandem Mass Spectrometry Determination and Pharmacokinetic Analysis of Amentoflavone and Its Conjugated Metabolites in Rats

Amentoflavone (AMF) is a biflavone found in many herbal dietary supplements. To investigate the pharmacokinetic profile of AMF in rats, a sensitive, simple, and accurate liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and used to monitor AMF and its conjugated metabolites in plasma. AMF was administered to rats by oral gavage (po), or by intravenous (iv) or intraperitoneal (ip) injection. Plasma samples (with apiolin as an internal standard) were liquid/liquid extracted after hydrolysis with β-glucuronidase/sulfatase in vitro. Following chromatographic separation on a C18 column with a methanol:water:formic acid (70:30:0.1, v/v/v) mobile phase, AMF and internal standard were determined by electrospray ionization in negative ion mode and their precursor–product ion pairs (<i>m</i>/<i>z</i> 537.1 → 374.9 and <i>m</i>/<i>z</i> 269.2 → 224.9, respectively) were used for measurement. This bioanalytical method was fully validated and showed good linearity (<i>r</i>² > 0.99), wide dynamic range (0.93–930 nmol/L), and favorable accuracy and precision. After iv or ip AMF (10 mg/kg) injection, 73.2% ± 6.29% and 70.2% ± 5.18% of the total AMF detected in plasma was present as conjugated metabolites. Furthermore, AMF and AMF conjugates showed similar time courses with no significant differences in the time to reach the maximum plasma concentration (<i>t</i>_max) and terminal half-life (<i>t</i>_1/2) (<i>p</i> > 0.05). Following po AMF administration (300 mg/kg), 90.7% ± 8.3% of the total AMF was circulating as conjugated metabolites. When compared with iv administration (with dose correction), the bioavailability of po AMF was very low (0.04% ± 0.01% for free AMF; 0.16% ± 0.04% for conjugated AMF). Collectively, these data provided a preliminary pharmacokinetic profile for AMF that should inform further evaluations of its biological efficacy and preclinical development