A tandem mass spectrometry method based on selected ions detects low-abundance phenolics in black tea – theatridimensins as products of the oxidative cascade

Rationale: Mixtures of phenolics are widespread in plant-derived food products, for instance black tea. Detailed compositional analysis of phenolics present is important for quality control. Characterization of low-abundance compounds often requires extensive purification; hence, the need for rapid screening methods to annotate compounds in complex mixtures without extensive sample preparation. Opportunities of ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS) as tool in a rapid screening method are discussed for black tea analysis, with the two-step-oxidation product theatridimensin (T3D) as example. Methods: Three MS screening methods were compared for their ability to tentatively annotate two-step-oxidation products in black teas without the need for prior fractionation: (i) full MS; (ii) tandem mass spectrometry (MS/MS) on selected ions; and (iii) selected reaction monitoring (SRM), in combination with post-analysis extracted ion chromatography. A model system of theaflavin (TF), epicatechin (EC) and tyrosinase was used to prepare the two-step-oxidation product T3D, consisting of three oligomerized catechin subunits. Commercial teas were screened for the occurrence of T3Ds. Results: The MS² fragmentation pattern of T3D was compared with that of an isomeric catechin trimer from black tea, TFsEC. MS² signature fragments were found to distinguish the two isomers, i.e. m/z 617 for T3D and m/z 563 for TFsEC. The MS screening methods, MS/MS on selected ions and SRM, both enabled monitoring MS² data of compounds present in low abundance. The former provided the most complete MS² data set, which facilitated the discovery of another isomer, i.e. theaflavate A. T3Ds, TFsECs, and theaflavate A could be tentatively annotated in all tested tea samples. Conclusions: When exploring black tea for the occurrence of two-step-oxidation products, the use of MS/MS on selected ions combined with extracted ion chromatography proved to be the most suitable. The occurrence of T3Ds and T3Dgs in various black teas was shown for the first time and the ‘oxidative cascade hypothesis’ was extended with novel oxidation products.