Simple and sensitive liquid chromatography–tandem mass spectrometry assay for simultaneous measurement of five Epimedium prenylflavonoids in rat sera

A rapid and sensitive method to separate and quantify icariin, icariside I, icariside II, icaritin and desmethylicaritin in rat sera was developed using liquid chromatography–tandem mass spectrometry. Serum samples were extracted with ethyl acetate without further derivatization. Using coumestrol as an internal standard, calibration curves with good linearity (r2 > 0.99) within the concentration range of 0.78–12.5 nM for icariin, icaritin and desmethylicaritin, and 0.78–100 nM for icariside I and II, were obtained in the multiple reaction monitoring mode. For all analytes, the limits of detection and quantification were <1 nM and 1–2 nM, respectively. Inter- and intra-assay variabilities were <15% and accuracies were between 94% and 114%, respectively. This method was successfully applied to quantify levels of icariin, icariside I, icariside II, icaritin and desmethylicaritin in rat sera after oral administration of an Epimedium preparation.