A novel method based on volumetric absorptive micro-sampling (VAMS) combined with UPLC-MS/MS was developed and validated to determine the principal (poly)phenolic metabolites in human blood following the consumption of 140 g of purple whole-grain barley (WGB) biscuits. Finger-prick blood samples were collected from 11 healthy volunteers at multiple time points up to 48 h post-ingestion. To extract (poly)phenolic metabolites efficiently, various extraction parameters were optimized. Then, the method was successfully applied and five colonic (poly)phenolic metabolites from the main (poly)phenolic families from barley were detected: benzene-1,2-diol-O-sulphate, 3-(4'-hydroxy-3'-methoxy)propanoic acid and its sulphated form, 5'-(3',4'-dihydroxyphenyl)-γ-valerolactone-O-sulphate, and methyl luteolin-O-glucuronide. Maximum absorption occurred at 12 h for most metabolites, while luteolin-O-glucuronide showed two distinct peaks at 2 and 6 h, indicating its dual-phase absorption. Comparison with venous plasma samples collected during the 0-6 h period showed no significant differences (p > 0.05), validating the statistical reliability of VAMS as an alternative to venipuncture. Thus, VAMS emerges as a less invasive and statistically robust means for analyzing the pharmacokinetic profile of (poly)phenols, particularly those arising from colonic metabolism.
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