High performance liquid chromatography tandem mass spectrometry dual extraction method for identification of green tea catechin metabolites excreted in human urine

• A new method is reported for analysis of free-form and mono-conjugated metabolites together.
• A good recovery of multiple urinary catechin metabolites was obtained.
• A double-blind intervention study on volunteers consuming green tea was performed to obtain suitable samples for analysis.

The simultaneous analysis of free-form and conjugated flavonoids in the same sample is difficult but necessary to properly estimate their bioavailability. A method was developed to optimise the extraction of both free and conjugated forms of catechins and metabolites in a biological sample following the consumption of green tea. A double-blind randomised controlled trial was performed in which 26 volunteers consumed daily green tea and vitamin C supplements and 24 consumed a placebo for 3 months. Urine was collected for 24 h at 4 separate time points (pre- and post-consumption) to confirm compliance to the supplementation and to distinguish between placebo and supplementation consumption. The urine was assessed for both free and conjugated metabolites of green tea using LC–MS2 analysis, after a combination extraction method, which involved an ethyl acetate extraction followed by an acetonitrile protein precipitation. The combination method resulted in a good recovery of EC-O-sulphate (91 ± 7%), EGC-O-glucuronide (94 ± 6%), EC (95 ± 6%), EGC (111 ± 5%) and ethyl gallate (74 ± 3%). A potential total of 55 catechin metabolites were investigated, and of these, 26 conjugated (with methyl, glucuronide or sulphate groups) and 3 free-form (unconjugated) compounds were identified in urine following green tea consumption. The majority of EC and EGC conjugates significantly increased post-consumption of green tea in comparison to baseline (pre-supplementation) samples. The conjugated metabolites associated with the highest peak areas were O-methyl-EC-O-sulphate and the valerolactones M6/M6′-O-sulphate. In line with previous studies, EC and EGC were only identified as conjugated derivatives, and EGCG and ECG were not found as mono-conjugated or free-forms. In summary, the method reported here provides a good recovery of catechin compounds and is appropriate for use in the assessment of flavonoid bioavailability, particularly for biological tissues that may contain endogenous deconjugating enzymes.