Simultaneous Determination of Epimedin A1, Epimedin B, Epimedin C, Icariin, Icariside I, and Baohuoside I in Rat Plasma by UPLC-MS/MS After Oral Administration of Epimedium Total Flavonoids With Application to Pharmacokinetic Study

Abstract

In this study, a UPLC-Orbitrap-HRMS method was employed to analyze the components of epimedium total flavonoids (ETF), as well as the prototypes and metabolites absorbed into rat plasma following oral administration. Furthermore, a rapid and sensitive UPLC-MS/MS method was developed for simultaneous quantification of epimedin A1, epimedin B, epimedin C, icariin, icariside I, and baohuoside I in rat plasma following oral administration of ETF. Plasma samples were precipitated using methanol-acetonitrile (1:1, v/v), with extraction recovery > 80% for all analytes. Chromatographic separation was achieved on an ACQUITY UPLC BEH C₁₈ column (50 mm × 2.1 mm, 1.7 μm) with gradient elution comprising water containing 0.1% formic acid (A) and methanol-acetonitrile (1:1, v/v, B), delivered at a flow rate of 0.3 mL/min. The method demonstrated excellent linearity (1.0–500 ng/mL) with correlation coefficients (r) more than 0.9950. Method validation revealed satisfactory precision and accuracy: intraday precision (RSD < 12.32%) with accuracy (−8.83% to 10.00%) and interday precision (RSD < 10.30%) with accuracy (−13.00% to 11.09%). The validated method was successfully applied to pharmacokinetic investigations, revealing critical disposition patterns of these flavonoids post-ETF administration. This study provides novel insights into the in vivo disposition of ETF, aiding in explaining the mechanisms underlying its effectiveness and toxicity of this herbal preparation.