Development of a sensitive, high‐throughput UPLC‐MS/MS method for detecting both native flavonoid compounds from Vitaflavan grape seed extract and their gut microbial metabolites

Abstract

Flavonoids found in Vitaflavan grape seed extract and their gut microbial metabolites are of interest due to the possible health benefits they may provide. While there are numerous examples of chromatographic methods to measure both groups of compounds separately, there is limited methodology to evaluate them together in one analysis. This increases analysis time and required resources, and decreases data throughput. A single UPLC‐MS/MS method was developed to measure both native monomers and procyanidins as well as predominant microbial metabolites simultaneously. The UPLC‐MS/MS method developed was able to baseline resolve 38 standard peaks within 10.0 min using an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8μm particle size) and a mobile phase gradient containing 0.1% formic acid in water and 0.1% formic acid in acetonitrile. MS was run in negative ESI with capillary voltage of −4.25 kV. Compounds measured included native GSE monomers (catechin, epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin gallate), native GSE procyanidins (dimers B1, B2, B5, and B2‐gallate, trimers C1 and T2, tetramer cinnamtannin A2, pentamers, and hexamers), as well as predominant colonic microbial metabolites including phenylacetic acids, phenylpropionic acids, and hydroxybenzoic acids. Values for the lower limit of detection were in the range of 0.0006–0.02 ng/mL for monomers and procyanidins and 0.0002–0.02 ng/mL for microbial metabolites, while values for the lower limit of quantitation were in the range of 0.0008–0.06 ng/mL for monomers and procyanidins and 0.0008–0.2 ng/mL for microbial metabolites. This method can be used for complex polyphenol and metabolite analyses in biological samples in a single chromatographic run. This method will facilitate future research to discover when, where, and to what scale these polyphenols are metabolized in the colon.