Quantification of Silymarin Compounds via LC-MS

Silymarin compounds were quantified using a LC-MS analysis that was performed on a Water 2695 Alliance (Waters-Micromass, Manchester, UK) coupled with a single quadrupole mass spectrometer ZQ (Waters-Micromass, Manchester, UK). LC-ESI-MS data were collected in the positive and negative modes. Data acquisition and processing were performed with MassLynx 4.0 software (Waters-Micromass, Manchester, UK). The separation was performed at 35 °C on a core-shell column (Kinetex 5 µm XB-C18, 100 Å, LC Column 150 × 4.6 mm, C18 with iso-butyl side chains, and with TMS endcapping, core-shell silica, Phenomenex Le Pecq France). The mobile phase was composed with a mixture of methanol (solvent A) and HPLC grade water (solvent B), both being acidified with 0.05% formic acid. A linear gradient was applied for the mobile phase variation, ranging from a 5:95 (v/v) to 100:0 (v/v) mixture of solvents A and B, respectively, with a flow rate of 1.30 mL/min. The injection volume was 3 µL, the maximum back pressure was 110 bar and the detection was performed at 280 nm. Flavonolignans were identified by comparison with authentic standards (Sigma Aldrich). The limits of detection (LOD) and the limits of quantification (LOQ) were determined based on the signal-to-noise ratio (S:N) of 3:1 and 10:1, respectively.