Development and validation of an LC-MS/MS method for the quantification of flavonoid glucuronides (wogonoside, baicalin, and apigenin-glucuronide) in the bile and blood samples: Application to a portal vein infusion study.

Glucuronidation is one of the major metabolic pathways for flavonoids. However, quantification of flavonoid glucuronides in biological samples, especially in the bile, is sometimes challenging due to signal suppression by bile acids. The purpose of this study is to establish a robust LC-MS/MS method for directly measuring flavonoid glucuronides in bile and blood. Wogonoside (wogonin-7-O-glucuronide), baicalin (baicalein-7-O-glucuronide) and apigenin-7-O-glucuronide were used as the model compounds and taurocholic acid (T-CA) were used as the model bile acid to establish the method. Bile samples were processed using solid phase extraction (SPE) and blood samples were prepared using protein precipitation method. The analytes were separated on a Resteck HPLC (50 mm × 2.1 mm ID, 1.7 μm) column using acetonitrile and 0.1% formic acid in water as the mobile phases. The mass analysis was performed in an AB Sciex 5500 Qtrap mass spectrometer via multiple reaction monitoring (MRM) in the positive mode. The results showed that the linear range of the above three analytes were 10 nM-5000 nM in the bile and 1.56 nM-4000 nM in the blood, respectively. The recoveries of three glucuronides were >85% and the matrix effects were <20% at low, medium and high concentrations in the bile and the blood. The results also showed that >90% of these bile acids were removed by the selected SPE procedure to facilitate glucuronide analysis. The validated method was successfully applied to a portal vein infusion study using rats to quantify baicalin, wogonoside, and apigenin-glucuronide in bile and blood samples.