A sensitive LC–MS/MS assay for the simultaneous analysis of the major active components of silymarin in human plasma

Silymarin, an extract of crushed achenes of the milk thistle plant Silybum marianum is a multi-constituent mixture, 70–80% of which consists of a complex assortment containing the flavonolignans silybin A and B, isosilybin A and B, silydianin, and silychristin, and the flavonoid taxifolin. To date, numerous pharmacological actions of the silymarin extract have been documented in the biomedical literature, including hepatoprotective, anti-inflammatory, anti-tumor, and anti-fibrotic activities. The present study describes a novel liquid chromatographic–tandem mass spectrometric method for simultaneous analysis of silychristin, silydianin, silybin A and silybin B, isosilybin A and isosilybin B, and taxifolin in human plasma employing liquid–liquid extraction. This assay provides excellent resolution of the individual silymarin constituents via utilization of a 100 A 250 mm × 2 mm, 5 μm C18 column with the mobile phase consisting of 51% methanol, 0.1% formic acid, and 10 mM ammonium acetate. The lower limit of quantification was 2 ng/ml for each constituent. Calibration curves were linear over the range from 2 ng/ml to 100 ng/ml for all analytes (r2 > 0.99). The intra- and inter-day accuracies were 91–106.5% and 95.1–111.9%, respectively. The intra- and inter-day precision was within 10.5%. Additionally, recovery, stability, and matrix effects were fully validated as well. This method was successfully applied to human plasma samples from subjects treated with the milk thistle extract Legalon®.