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 C 18 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.