LC–MS/MS method for the simultaneous determination of icariin and its major metabolites in rat plasma

A rapid and sensitive method using liquid chromatography–tandem mass spectroscopy (LC–MS/MS) was developed and validated for the simultaneous quantitative determination of icariin and its two major metabolites, icariside I and icariside II in rat plasma. The analytes were extracted by liquid–liquid extraction with ethyl acetate after internal standard (daidzein) spiked. The separation was performed by a ZORBAX SB-C18 column (3.5 μm, 2.1 mm × 100 mm) and a C18 guard column (5 μm, 4.0 mm × 2.0 mm) with an isocratic mobile phase consisting of acetonitrile–water–formic acid (50:50:0.05, v/v/v) at a flow rate of 0.25 mL/min. The Agilent G6410A triple quadrupole LC–MS system was operated under the multiple reaction monitoring (MRM) mode using the electrospray ionization technique in positive mode. The nominal retention times for icariin, icariside I, icariside II and daidzein were 1.21, 1.88, 2.34 and 1.35 min, respectively. The lower limits of quantification (LLOQ) of icariin, icariside I and icariside II of the method were 1.0, 0.5 and 0.5 ng/mL, respectively. The method was linear for icariin and its metabolites with correlation coefficients >0.995 for all analytes. The intra-day and inter-day accuracy and precision of the assay were less than 12.5%. This method has been applied successfully to a pharmacokinetic study involving the intragastric administration of icariin to rats.