Simultaneous determination of three glucuronide conjugates of scutellarein in rat plasma by LC–MS/MS for pharmacokinetic study of breviscapine

• Three glucuronide conjugates of scutellarein in rat plasma were quantified by LC–MS/MS.
• Baseline separation of scutellarin and its isomer M2 was mandatory for quantification.
• Pharmacokinetics of breviscapine in rats given a dose of 20 mg/kg was demonstrated.
• A low bioavailability of scutellarin and high exposures of M1 and M2 were observed.

A selective and sensitive LC–MS/MS method was developed and validated for simultaneous determination of three glucuronide conjugates of scutellarein in rat plasma. Plasma samples were pretreated by protein precipitation with acetonitrile. The analytes (scutellarin, scutellarein-6,7-di-O-β-d-glucuronide and scutellarein-6-O-β-d-glucuronide), together with internal standard (IS, baicalin) were separated on a Diamonsil C18 column (150 mm × 4.6 mm, 5 μm) with an isocratic mobile phase consisting of methanol–water–formic acid (55:45:0.2, v/v/v). Mass spectrometric detection was performed by selected reaction monitoring (SRM) mode via electrospray ionization source operating in negative ionization mode. The method was linear for all the analytes over the investigated concentration ranges with correlation coefficients greater than 0.9954. The intra- and inter-day precisions were less than 9.1% and the relative error was between −1.7% and 4.2%. The extraction recoveries of the analytes and IS from rat plasma were over 63%. The validated method has been successfully applied to a pharmacokinetic study of breviscapine in rats after intragastric administration at a dose of 20 mg/kg. The pharmacokinetic results would be helpful to better understand the pharmacological actions of breviscapine.