Simultaneous determination of four flavonoids in rat plasma after oral administration of Malus hupehensis (Pamp.) Rehd. extracts by UPLC-MS/MS and its application to a pharmacokinetics study

A rapid, sensitive, selective, and accurate UPLC–MS/MS method was developed and fully validated for the simultaneous determination of quercitrin, phloridzin, quercetin, and phloretin in rat plasma after oral administration of Malus hupehensis (Pamp.) Rehd extracts. The pharmacokinetic parameters of oral phloridzin monomer and phloridzin in the extract were also compared. Plasma samples were processed with a simple protein precipitation technique using methanol, followed by chromatographic separation using a Sun Fire TMC18 column. Bergenin was used an internal standard (IS). A 15.0 min linear gradient elution was used at a flow rate of 0.8 mL/min with a mobile phase of 0.1% formic acid in water and acetonitrile. The analytes and IS were detected using negative ion electrospray ionization in multiple reaction monitoring mode. The developed method exhibited good linearity (r ≥ 0.9911), and the lower limits of quantification ranged from 0.2 to 0.8 ng/mL for the four analytes. Intra-day and inter-day precision were both less than 8.5% and were within the acceptable limits. Matrix effect and recovery efficiency of all analytes were found to be >76.2% and >71.4%, respectively. Stability results showed that the analytes were stable at all conditions. Additionally, the carry-over effect and dilution effect were within the acceptance range. The developed method was successfully applied to a pharmacokinetic study of four analytes in rats after oral administration of Malus hupehensis (Pamp.) Rehd. extracts.