title: High performance liquid chromatography tandem mass spectrometry dual extraction method for identification of green tea catechin metabolites excreted in human urine.
abstract: 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 24h 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-MS(2) 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.
authors.affiliation: School of Food Science and Nutrition, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Kayleigh A
authors.initials: KA
authors.lastname: Clarke
authors.affiliation: School of Food Science and Nutrition, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Tristan P
authors.initials: TP
authors.lastname: Dew
authors.affiliation: Photobiology Unit, Dermatology Centre, Institute of Inflammation and Repair, University of Manchester, Manchester Academic Health Science Centre, Salford Royal NHS Foundation Hospital, Manchester M6 8HD, UK.
authors.forename: Rachel E B
authors.initials: RE
authors.lastname: Watson
authors.affiliation: Photobiology Unit, Dermatology Centre, Institute of Inflammation and Repair, University of Manchester, Manchester Academic Health Science Centre, Salford Royal NHS Foundation Hospital, Manchester M6 8HD, UK.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Farrar
authors.affiliation: Photobiology Unit, Dermatology Centre, Institute of Inflammation and Repair, University of Manchester, Manchester Academic Health Science Centre, Salford Royal NHS Foundation Hospital, Manchester M6 8HD, UK.
authors.forename: Susan
authors.initials: S
authors.lastname: Bennett
authors.affiliation: School of Pharmacy, School of Life Sciences, University of Bradford, Bradford BD7 1DP, UK.
authors.forename: Anna
authors.initials: A
authors.lastname: Nicolaou
authors.affiliation: Photobiology Unit, Dermatology Centre, Institute of Inflammation and Repair, University of Manchester, Manchester Academic Health Science Centre, Salford Royal NHS Foundation Hospital, Manchester M6 8HD, UK.
authors.forename: Lesley E
authors.initials: LE
authors.lastname: Rhodes
authors.affiliation: School of Food Science and Nutrition, University of Leeds, Leeds LS2 9JT, UK. Electronic address: g.williamson@leeds.ac.uk.
authors.forename: Gary
authors.initials: G
authors.lastname: Williamson
mesh_terms: D002392:Catechin / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004311:Double-Blind Method; D006801:Humans; D057230:Limit of Detection; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013662:Tea / Q000737:chemistry*
chemical_list: D013662:Tea; D002392:Catechin
keywords: Conjugates; Green tea catechins; Isolation; Liquid-chromatography mass spectrometry; Metabolites; Urine