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 (r(2)>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.