HPLC-UV and LC-MS-MS Characterization of Silymarin in Milk Thistle Seeds and Corresponding Products

A HPLC analytical protocol for the separation of taxifolin, silychristin, silydianin, silybinin A, silybinin B, isosilybinin A, and isosilybinin B was developed. The gradient method used a combination of water and methanol as the eluent, and the separation was monitored at 290 nm. The authenticity of the reference compounds was correctly analyzed by liquid chromatography-mass spectrometry-mass spectrometry and by spiking HPLC experiments. The dihydroflavonol taxifolin and the flavanolignans silychristin, silydianin, and silybinin A and B were extracted from two milk thistle (Silybum marianum(L.) Gaertn.) seed batches using organic solvents, and subsequently quantified by the developed method. Both seed batches differed in their silydianin, silybinin A, and silybinin B contents. Taxifolin and the flavanolignans were also quantified in finished products, where the ratio of the silybinin compounds was similar in tested Brands A and B. Brand A was first extracted in a Soxhlet with ethanol. Under these conditions, Brand A was short of the silymarin content on its label. The same product was then extracted in room temperature ethanol, and was shown to contain 152 mg of silymarin, 12 mg more than was indicated on the products' label. Analysis of Brand B, a tincture, showed that it contained almost half of the silymarin content found in Brand A capsules. Significant variations in the ratios of the flavanolignan constituents among products and seeds, as well as variation between product and product label were detected.