Simultaneous quantification of Akebia saponin D and its five metabolites in human intestinal bacteria using ultra-performance liquid chromatography triple quadrupole mass spectrometry

A rapid and sensitive ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-TQ/MS) method was developed for simultaneous quantification of Akebia saponin D (ASD) and its five metabolites in intestinal mixtures of bacteria from human feces. After protein precipitation, the analytes and internal standard (IS), glycyrrhetinic acid, were determined in selected ion recording (SIR) mode with negative ion ESI source. Chromatographic separation was carried out on an ACQUITY UPLC™ BEH C18 column (100 mm × 2.1 mm, 1.7 μm) using gradient elution. The mobile phase consisted of solvents A (acetonitrile) and B (0.1% aqueous formic acid) at the flow rate of 0.4 mL/min. Each sample was chromatographed within 10.5 min including equilibration time. The linearity ranged from 0.1 to 100 μg/mL for ASD, and 2-1000 ng/mL for five metabolites, Dipsacus saponin A (M1), HN-saponin F (M2), hederagenin-28-O-β-d-glucopyranoside (M3), Akebia saponin PA (M4), hederagenin (M5). The limits of detection (LOD) were 0.41, 0.59, 0.61, 0.55, 0.52 and 0.31 ng/mL for ASD, M1, M2, M3, M4 and M5, respectively. The intra- and inter-day precision was all within 11.1% and accuracy ranged from −8.33% to 12.47%. The conversion rate of five metabolites was 41.21% in 24 h. The method was validated and successfully applied to quantification of ASD and its five metabolites in human intestinal bacteria.