Evaluation of SPE as Preparative Technique for the Analysis of Phenolic Metabolites in Human Feces

Abstract

Solid phase extraction (SPE) methodology has been evaluated as a cleanup strategy prior to the analysis of phenolic metabolites in fecal samples by UPLC–DAD–ESI–TQ MS. Among the sorbents tested, Oasis® HLB led to the higher phenolic standard recoveries. Sample acidification (0.4 M HCl, final concentration) before SPE considerably improved standard recoveries. Values of the process efficiency (C_SPE/C_{Without SPE}) for a standard solution containing gallic acid, protocatechuic acid, caffeic acid, benzoic acid, 3-phenylpropionic acid, (+)-catechin, (−)-epicatechin, procyanidin B2, and 4-hydroxybenzoic 2,3,5,6 d₄ acid were acceptable (>90 %) for all compounds, except for procyanidin B2 (26 %). The developed SPE methodology was applied to fecal samples of individuals subjected to a wine intervention study. Phenolic metabolites, including intermediate metabolites (phenyl-γ-valerolactones and phenylvaleric acid derivatives) and end products (simple phenols, hydroxyphenylpropionic, hydroxyphenylacetic, hydroxycinnamic, and hydroxybenzoic acids) were identified. Most of the compounds (n = 14) exhibited values of process efficiency between 85 and 115 %. Although some compounds (n = 4) showed process efficiency>115 %, there was a group of metabolites (4-O-methylgallic acid, syringic acid, and 4-hydroxy-5-(3′,4′-dihydroxyphenyl)-valeric acid) whose process efficiency was <85 %, which represented a serious limitation and made us to discard SPE as a preparative technique for the analysis of these phenolic metabolites. Finally, the paper reports the concentrations of phenolic metabolites in a randomized set of human fecal samples from healthy volunteers (n = 15) without any previous SPE application. Large inter-individual variability was observed, which was attributed to differences in human gut microbiota composition.