Chemical Standardization of Milk Thistle (Silybum marianum L.) Extract Using UHPLC-MS/MS and the Method of Standard Addition

Abstract

Extracts prepared from the seeds of the medicinal plant milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)] are widely used as dietary supplements due to anti-inflammatory, antitumor, and hepatoprotective effects. Called silymarin, the main components of lipophilic extracts of milk thistle seeds are flavonoids and flavonolignans including silybin A, silybin B, isosilybin A, isosilybin B, silydianin, silychristin, taxifolin, and 2,3-dehydrosilybins. The aim of this study was to develop a method based on UHPLC-MS/MS for the chemical authentication and standardization of milk thistle silymarin. Validation included the method of standard addition to account for the lack of a blank matrix. Potential matrix effects were investigated by analyzing silymarin standards dissolved only in the initial UHPLC mobile phase. Measurements of six flavonolignans and taxifolin in the milk thistle extract using UHPLC-MS/MS with standard addition or external standard calibration produced similar results for all analytes except silydianin and 2,3-dehydrosilybin B, which showed significant peak enhancement during negative ion electrospray due to botanical matrix effects. The UHPLC-MS/MS-based method of standard addition requires <10 min per injection and is suitable for the standardization of silymarin from milk thistle in support of preclinical and clinical studies of safety and efficacy.

Figure 1

Chemical structures of common milk thistle constituents in silymarin and deuterated internal standards.

Figure 2

High-resolution negative ion electrospray UHPLC-MS total ion chromatogram survey of milk thistle extract (silymarin) showing separation and detection of major silymarin constituents plus the internal standard daidzein-d₄. The corresponding high-resolution Q-ToF positive ion electrospray chromatogram is shown in Supplemental Figure 2. The most abundant milk thistle constituents (labeled in red font) were then measured using a faster UHPLC-MS/MS method on a triple quadrupole mass spectrometer.

Figure 3

Negative ion electrospray UHPLC-MS/MS selected reaction monitoring chromatograms of flavonolignans, flavanonol taxifolin, and deuterated internal standards daidzein and genistein. (A) Standards at 256 ng/mL in 50% aqueous methanol; (B) internal standards at 100 ng/mL; and (C) silymarin (10 μg/mL) extracted from milk thistle seed.

Figure 4

Using the method of standard addition, the concentration of the analyte silychristin was determined by extrapolating the calibration curve to 0 on the negative side of the x-axis (in the equation, solve for x when y = 0). A constant amount of internal standard was added to each aliquot of milk thistle extract immediately before UHPLC-MS/MS analysis. The peak area of silychristin, spiked with different amounts of silychristin standard, was normalized by dividing with the internal standard peak area. This area ratio was plotted versus the amount of silychristin spiked into the extract.