An improved ultra-high performance liquid chromatography-tandem mass spectrometry method determining hispidulin and homoplantaginin in rat plasma and associated pharmacokinetic studies

Flavonoids are the most abundant components in Salvia plebeia, with significant pharmacological antioxidant and hepatoprotective properties. Hispidulin and homoplantaginin are the main flavonoid components in S. Plebeia. In this study, we established an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to determine hispidulin and homoplantaginin in rat plasma samples, which were precipitated using acetonitrile-methanol (9:1, v/v). We used a UPLC HSS T3 (100 mm × 2.1 mm, 1.7 μm diameter) chromatographic column, an acetonitrile-water (containing 0.1% formic acid) mobile phase, and a gradient elution flow rate of 0.4 mL min−1 in an elution time of 4 min. We used electrospray ionization (ESI) detection in positive ion mode, and multiple reaction monitoring mode (MRM) for quantitative analysis: m/z 301 → 286 for hispidulin, m/z 463 → 301 for homoplantaginin, and m/z 465 → 303 for internal standard (IS). In pharmacokinetic studies, 24 rats were orally administered hispidulin and homoplantaginin (5 mg kg−1) and received sublingual intravenous injections (1 mg kg−1) at two different doses, four groups with six rats/group. Differences in hispidulin and homoplantaginin pharmacokinetics in rat plasma were evaluated. The calibration curve showed good linearity in the 0.5–1,000 ng mL−1 range, with r > 0.99. Precision, accuracy, recovery, matrix effects, and stability results all met standard biological sample detection requirements. Our pharmacokinetic studies showed hispidulin bioavailability was much higher than homoplantaginin at 17.8% and 0.1%, respectively.