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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Vol. 902; pp. 1 - 9
• Main Authors: Brinda, Bryan J., Zhu, Hao-Jie, Markowitz, John S.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2012
• Subjects: ammonium acetate; Assaying; Chromatography; Chromatography, Liquid - methods; Constituents; Drug Stability; Extraction plants; Female; formic acid; fruits; Human; Humans; LC–MS/MS; Liquid-Liquid Extraction; Liquids; Male; mass spectrometry; methanol; Methyl alcohol; Milk; Milk thistle; Plant Extracts; Reproducibility of Results; Sensitivity and Specificity; Silybin; Silybum marianum; Silymarin; Silymarin - blood; Silymarin - chemistry; Silymarin - pharmacokinetics; Tandem Mass Spectrometry - methods; Taxifolin; Silybin; Milk thistle; Taxifolin; Silymarin; LC–MS/MS
• ISSN: 1570-0232; 1873-376X; 1873-376X
• DOI: 10.1016/j.jchromb.2012.06.003

► Silymarin extract contains numerous bioactive flavonolignans and flavonoids. ► Analysis of silychristin, silydianin, silybin A and B, isosilybin A and B, and taxifolin. ► Pure standard compounds were utilized for absolute quantification. ► The lower limit of quantification was 2ng/ml for each analyte. ► Method was applied to human plasma samples from subjects treated with Legalon®. 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 100A 250mm×2mm, 5μm C18 column with the mobile phase consisting of 51% methanol, 0.1% formic acid, and 10mM ammonium acetate. The lower limit of quantification was 2ng/ml for each constituent. Calibration curves were linear over the range from 2ng/ml to 100ng/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®.