Validation of a quantitation method for conjugated quercetin in human plasma

Since the type of glycoside affects the pharmacokinetic profile of the aglycon after oral ingestion of quercetin glycosides, clinical studies on the pharmacokinetics of quercetin glycosides are required. However, a suitable method to determine the concentrations of quercetin phase II metabolites in human plasma and urine is lacking. Therefore, we developed and validated an LC-MS method for the quantitation of conjugated quercetin using relevant reference standards, including hetero-conjugates with glucuronic acid and sulfonic acid (QC-GA/S). Quercetin hetero-conjugates extracted from rat serum were used for the method development, and reference standards were biosynthesized for the quantitation. The use of a solid-phase extraction (SPE) column in a 96 well format enabled high-throughput analysis of up to 96 tests in a day, without compromising recovery and sensitivity. The SPE column with a weak anion exchange group contributed to the high recovery of QC-GA/S. The method was then validated, and its usefulness was confirmed using clinical samples. QC-GA/S was the predominant phase II quercetin metabolite after the ingestion of quercetin glucoside or quercetin supplements. Moreover, the two peaks of QC-GA/S found in human plasma and urine were isomers of QC-7GA/4'S, which has been reported as the predominant peak in rat plasma. If QC-GA/S in plasma is responsible for a physiological activity of quercetin, it is important to determine the concentration of each QC-GA/S isomer.