PRiME pass-through purification of lignans in Silybum marianum and UPLC-MS/MS analysis

A PRiME (process, robustness, improvements, matrix effects, ease of use) pass-through cleanup procedure was developed for the extraction and purification of silychristins A and B, silybins A and B, isosilybins A and B, and silydianin in Silybum marianum. After optimizing the extracting solvent types and the sample loading volume, the crude extract was diluted to 3 mL with 95% acetonitrile and then loaded on the PRiME cartridge. The eluate was analyzed by ultra-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS). All the target analytes were deprotonated as [M-H]^- at m/z 481 by conducting collision-induced dissociation (CID), and the major fragment ions were m/z 463 ([M-H₂O-H]^-), 453 ([M-CO-H]^-), 355 ([M-C₆H₆O₃-H]^-), 301 ([M₃₅₅-CO₂-H]^-), and 179 ([C₁₀H₁₁O₃]^-). Afterwards, this method was validated in terms of linearity (R² > 0.9990), intra-day precision (1.02%-3.79%), inter-day precision (1.59%-4.87%), sensitivity (LOD ≤ 0.45 μg·kg^-1 and LOQ ≤ 1.50 μg·kg^-1), and recovery (76.9-103.4%, RSD < 8.90%). Finally, the proposed protocol was successfully applied to eight batches of S. marianum samples. The total content of the seven active compounds varied amongst the batches from different places of origin.