Development and Validation of a HPLC-MS/MS Method for Simultaneous Determination of Twelve Bioactive Compounds in Epimedium: Application to a Pharmacokinetic Study in Rats

A rapid and reliable HPLC-MS/MS method has been developed and validated for the simultaneous quantification of twelve bioactive compounds (baohuoside II, baohuoside I, sagittatoside A, sagittatoside B, magnoflorine, epimedin A, epimedin B, epimedin C, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid and icariin) in rat plasma. The collected plasma samples were prepared by protein precipitate with acetonitrile. The twelve compounds were separated on a CORTECS®C18 column (4.6 mm × 150 mm, 2.7 μm) with a gradient mobile phase system of 0.1% (v/v) formic acid and acetonitrile at a flow rate of 0.3 mL/min. All of the analytes were quantitated using electrospray ionization (ESI) in negative ion mode with selected reaction monitoring (SRM). The intra- and inter-day accuracy ranged from -5.6% to 13.0%, and the precisions of the analytes were less than 10.9%. The mean recoveries of the analytes were in the range of 60.66% to 99.77% and the matrix effect ranged from 93.08% to 119.84%. Stability studies proved that the analytes were stable under the tested conditions, with a relative standard deviation (RSD) lower than 11.7%. The developed method was successfully applied to evaluating the pharmacokinetic study of twelve bioactive compounds after oral administration of Epimedium extract in rat.