•Simultaneous determination of eight active constituents in rat plasma.•Pharmacokinetic study of eight active constituents by UHPLC-MS/MS.•The first report on pharmacokinetic study of Hypericum japonicum Thunb by UHPLC-MS/MS.A rapid and sensitive assay based on ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was established and validated for the simultaneous determination of gallic acid, protocatechuic acid, vanillic acid, caffeic acid, epicatechin, isoquercitrin, vincetoxicoside B and quercetin in rat plasma using catechin and daidzein as the internal standards (IS). Plasma samples added internal standards were acidified with formic acid then pretreated by direct protein precipitation with acetonitrile. The separation of eight constituents was achieved on a C18 column with gradient elution using methanol and 0.2% acetic acid aqueous solution as the mobile phase and detected by multiple reaction monitoring using electrospray ionization source in the positive–negative ionization mode. The method was validated for sufficient specificity, precision, accuracy, and sensitivity over the concentration range of 10–6000 ng mL−1 for gallic acid, 1.5–3000 ng mL−1 for protocatechuic acid, 10–15000 ng mL−1 for vanillic acid, 2–3600 ng mL−1 for caffeic acid, 1.5–3600 ng mL−1 for epicatechin, 4–6000 ng mL−1 for isoquercitrin, 2–9000 ng mL−1 for vincetoxicoside B, and 20–18000 ng mL−1 for quercetin. The overall intra‑run precision and the inter‑run precision were showed in the range of 1.0–14.2% and 2.8–12.9%, respectively, and the accuracy was no more than 12.8%. This analytical method was successfully applied to investigate the pharmacokinetics of eight ingredients in rats after oral administration of Hypericum japonicum Thunb extract.

•Using two internal standards to quantify.•Simultaneous determination of ten active constituents in rat plasma.•Pharmacokinetic study of ten active constituents by UPLC–MS/MS.•The limit of quantifications is low enough to detect analytes.•The first report on pharmacokinetic study of Yankening Capsule.An ultra high performance liquid chromatography with tandem mass spectrometry (U-HPLC–MS/MS) method was developed for simultaneous determination and pharmacokinetic study of ten active constituents, phellodendrine, coptisine, jatrorrhizine, berberine, palmatine, baicalin, wogonoside, baicalein, wogonin and emodin in rat plasma after oral administration of Yankening Capsule. After mixing with two internal standards tetrahydropalmatine and rutin, plasma samples were pretreated by protein precipitation with anhydrous ethanol–acetonitrile (9:1, v/v). The U-HPLC separation was carried on a ZORBAX RRHD Eclipse Plus C18 column (2.1 mm × 50 mm, 1.8 μm) with gradient elution using a mobile phase composed of methanol and water (containing 0.3% formic acid) at a flow rate of 0.3 mL min−1. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via electrospray ionization source with positive–negative ionization mode. The calibration curves of ten analytes showed good linearity (r > 0.9979). The lower limits of quantification of phellodendrine, coptisine, jatrorrhizine, berberine, palmatine, baicalin, wogonoside, baicalein, wogonin and emodin were 0.50, 0.50, 0.30, 0.30, 0.30, 10, 3.0, 8.0, 1.0, 8.0 μg L−1, respectively. The relative standard deviation of intra-day precision and inter-day precision were in the range from 1.13% to 5.96% and from 0.65% to 8.85%, respectively. The matrix effects of all analytes were found to be within the acceptable range with a range of 89.99–109.3%. The method is reliable and rapid and has been applied successfully to pharmacokinetic study of the ten active constituents in rat plasma after oral administration of Yankening Capsule.