Preparative chromatographic purification of diastereomers of silybin and their quantification in human plasma by liquid chromatography–tandem mass spectrometry

A novel preparative HPLC method separating silybin has been developed to meet the need for both silybin A and silybin B standard. After the preparation of silybin A and silybin B standard, a simple, sensitive, selective and reproducible liquid chromatography–tandem mass spectrometry (LC–MS–MS) method with negative electrospray ionization (ESI) was developed for the quantification of silybin A and silybin B in human plasma. Following rapid sample preparation, silybin A, silybin B and naringin (internal standard, ISTD) were separated on a Zorbax Eclipse XDB-C18 column, using methanol–water containing 0.1% formic acid (48:52, v/v) as the mobile phase. The mass spectrometer was operated in selected reaction monitoring (SRM) mode using the transition m/z 481.1 → 300.9 for both silybin A and silybin B and m/z 579.2 → 271.1 for naringin, respectively. Linear calibration curves were obtained in the concentration range of 2–5000 ng/ml with a lower limit of quantitation (LLOQ) of 2 ng/ml for both silybin A and silybin B, respectively. The intra- and inter-day precision values were below 7.5% and accuracy was within ±4.9% at all three quality control (QC) levels, for both silybin A and silybin B, respectively. This method was successfully applied to the stereospecific analysis of silybin in plasma samples from a pharmacokinetic study of silybin A and silybin B in 22 healthy male Chinese volunteers after a single oral dose of silybin–phosphatidylcholine complex (equivalent to 280 mg silybin, including 133 mg silybin A and 147 mg silybin B).