The analysis of CGJ-derived polyphenols and their microbial phenolic metabolites was conducted as previously reported by Ho et al.27 (link) with modifications. Two internal standards (ISs), trans-cinnamic acid-d7 and 4-hydroxybenzoic-2, 3, 5, 6-d4, were spiked into an aliquot (200 μL) of thawed serum. After purging with nitrogen, the mixture was incubated with β-glucuronidase (2000 U, in contamination with sulfatase) at 37°C for 45 minutes. Enzymatic reaction was stopped by adding ethyl acetate (500 μL) and extracted twice. The upper organic phase was pooled with addition of 20 μL of 2% ascorbic acid before being dried under a gentle stream of nitrogen. The residue was reconstituted in 100 μL of 60% methanol containing 0.1% formic acid and centrifuged at 16,500 xg for 10 min before targeted metabolomics analysis.
For each sample extract, 3.5 μL was injected into an Agilent UPLC-QqQ/MS system for chemical and metabolite analysis under dynamic multiple reaction monitoring (dMRM) mode. For analytes without reference standards, we referred to earlier publications for their multiple reaction monitoring (MRM) transitions. MRM parameters were set according to other optimized analytes with a similar molecular structure, and concentrations were calculated based on the correction factor of molecular weight (MW) ratio of the compound of interest to that of its corresponding analogue.
For each sample extract, 3.5 μL was injected into an Agilent UPLC-QqQ/MS system for chemical and metabolite analysis under dynamic multiple reaction monitoring (dMRM) mode. For analytes without reference standards, we referred to earlier publications for their multiple reaction monitoring (MRM) transitions. MRM parameters were set according to other optimized analytes with a similar molecular structure, and concentrations were calculated based on the correction factor of molecular weight (MW) ratio of the compound of interest to that of its corresponding analogue.