An UPLC–MS/MS method for simultaneous quantitation of two coumarins and two flavonoids in rat plasma and its application to a pharmacokinetic study of Wikstroemia indica extract

• A sensitive, selective and fast UPLC–ESI–MS/MS method has been developed for the simultaneous determination of two coumarins (umbelliferone and daphnoretin) and two flavonoids (apigenin and genkwanin) in rat plasma for the first time.
• The method was fully validated and successfully applied to the pharmacokinetic study of four ingredients after oral administration of Wikstroemia indica extract (WIE) to rats.
• The results of this study may provide some references for the apprehension of the action mechanism and safe clinical application of W. indica.

In this study, an ultra performance liquid chromatography tandem mass spectrometry method (UPLC–MS/MS) was developed for simultaneous determination of umbelliferone, apigenin, daphnoretin and genkwanin in total (free and conjugated) forms in rat plasma using psoralen as internal standard. Plasma samples were protein precipitated with acetonitrile followed by liquid–liquid extracted with ethyl acetate. Four ingredients were separated on an Acquity UPLC® BEH C18 column using gradient elution with the mobile phase consisting of 0.1% formic acid aqueous solution and acetonitrile, and detected by positive ion electrospray ionization (ESI) in multiple reaction monitoring mode (MRM). The method was linear for all analytes over investigated ranges with all correlation coefficients greater than 0.99. The validated lower limit of quantification was 3 ng/mL for umbelliferone, 3 ng/mL for apigenin, 12 ng/mL for daphnoretin and 2 ng/mL for genkwanin, respectively. Intra- and inter-day precisions (RSD%) were less than 15% and accuracy (RE%) ranged from −1.1% to 15%. The mean absolute extraction recoveries of analytes and IS from rat plasma were all more than 75%. The validated method was firstly and successfully applied to investigate the pharmacokinetics of four chemical ingredients after oral administration of Wikstroemia indica extract (WIE) to rats.