An LC-MS/MS method for simultaneous determination of four flavonoids from Semen Oroxyli in rat plasma and its application to a pharmacokinetic study.

Semen Oroxyli, a Traditional Chinese Medicine, has many significant pharmacological activities such as analgesic, apoptotic, anti-inflammatory, anticancer, and immunostimulant activities. A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantitation of four flavonoids (oroxin A, oroxin B, baicalin, and chrysin) of Semen Oroxyli in rat plasma. After the addition of internal standard, plasma samples were pretreated with acetonitrile via a single-step protein precipitation. Chromatographic separation was performed on a Capcell Pak C18 column (100 mm × 2.0 mm, 5 μm particles) with isocratic elution using a mobile phase of methanol and 2mM ammonium acetate buffer solution (75:25, v/v) at a flow rate of 0.45 mL/min. The analytes were detected without interference in the selection reaction monitoring mode with negative electrospray ionization. The validated method exhibited good linearity over a wide concentration range (r ≥ 0.9958), and the lower limits of quantification were 1.0-5.5 ng/mL for all the analytes. The mean extraction recoveries of the analytes from rat plasma exceeded 80.6%. The intra- and inter-day precisions at three QC levels were both less than 11.5%, and the accuracies ranged from -6.2% to 10.3%. The LC-MS/MS method was successfully applied to a pharmacokinetic study of the four flavonoids in rat plasma after oral administration of Semen Oroxyli extract.