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

• Published in: Analytical biochemistry Vol. 601; p. 113723
• Main Authors: Tu, Yifan, Zhou, Lei, Li, Li, Wang, Lu, Gao, Song, Hu, Ming
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.07.2020
• Subjects: acetonitrile; ammonium acetate; Animals; Apigenin - analysis; Apigenin-7-O-Glucuronide; Baicalin; Bile; Bile - chemistry; biochemical pathways; Blood; blood sampling; chemical species; Chromatography, High Pressure Liquid; Flavanones - analysis; Flavonoids - analysis; Glucosides - analysis; Glucuronide quantification; high performance liquid chromatography; LC-MS/MS; Male; monitoring; portal vein; Portal Vein - chemistry; Rats; Rats, Wistar; solid phase extraction; SPE; spectrometers; Tandem Mass Spectrometry; taurocholic acid; Wogonoside; LC-MS/MS; Apigenin-7-O-Glucuronide; SPE; Glucuronide quantification; Wogonoside; Baicalin; Blood; Bile
• ISSN: 0003-2697; 1096-0309; 1096-0309
• DOI: 10.1016/j.ab.2020.113723

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. [Display omitted] •Directly quantify flavonoid glucuronides in bile and blood matrix without hydrolysis.•Require only 5 μl of bile and 10 μl of blood samples to finish the analysis.•Test 5 commonly used SPE cartridges and remove 90% of bile acids without significant loss of analytes.•Apply successfully the validated method to the quantification of selected flavonoid glucuronides.