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Single Laboratory Validation of UHPLC-MS/MS Assays for Six Milk Thistle Flavonolignans in Human Serum
Ruth N Muchiri1,2, Richard B van Breemen1,2
1Linus Pauling Institute, Oregon State University, 2900 SW Campus Way, Corvallis, OR 97331, USA.
Background:
Extracts of milk thistle, Silybum marianum (L.) Gaertn., are used as dietary supplements for their hepatoprotective, anti-inflammatory, and anti-tumor activities.
Objective:
An assay based on UHPLC-MS/MS was developed and validated for the quantitative analysis of six major milk thistle flavonolignans extracted from human serum.
Methods:
Ethyl acetate containing 0.1% formic acid was used to extract flavonolignans from human serum. A 10-min UHPLC-MS/MS method using selected reaction ion monitoring was developed for measuring extracts for silybin A, silybin B, isosilybin A, isosilybin B, silychristin, and silydianin.
Results:
The quantitative method was validated with respect to selectivity, specificity, accuracy, linearity, precision, LOD, and LLOQ. Extraction efficiency for the quality control standards at LLOQ, low, medium, and high concentrations ranged between 81% and 109%, and the calibration curves were linear (R2 > 0.997) for all flavonolignans. The method precision was determined using coefficients of variation, which were <15%. The method accuracy was assessed using percent relative error which was <15%.
Conclusions:
The UHPLC-MS/MS assay is fast, precise, sensitive, selective, accurate, and useful for the analysis of milk thistle flavonolignans in human serum.
Highlights:
The UHPLC-MS/MS assay is suitable for rapid quantitative analysis of milk thistle flavonolignans in human serum.
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