Targeted and untargeted characterisation of free and glycosylated simple phenols in cocoa beans using high resolution-tandem mass spectrometry (Q-Orbitrap).

Free simple phenols have positive effects on health and influence the organoleptic profile of cocoa products, contributing towards defining their aroma and nutritional properties. Glycosidically bound simple phenols can be hydrolysed during the production phase to the corresponding free forms, and thus potentially contribute to the final sensory profile. In this work, 60 samples of Forastero cocoa beans from all over the world were analysed, combining on-line solid phase extraction (SPE) clean-up with ultra-high performance liquid chromatography (UHPLC), coupled with high resolution mass spectrometry. Operating in negative ion mode and with a heated electrospray, 62 simple phenols were measured, of which four were glucosidic precursors, with quantification limits ranging from 0.04 to 40mgkg, calibration R of 0.99 for over 93% of compounds, and precision (R.S.D.%) always lower than 12%. On the basis of accurate mass, isotope pattern and MS/MS spectrum, 32 monoglycosylated simple phenols such as hexoside and pentoside precursors, and 14 diglycosylated simple phenols such as hexoside-hexoside, hexoside-pentoside and pentoside-hexoside precursors, were tentatively identified. The untargeted approach was validated using 3 glucosidic precursors synthesized by an external supplier. Honestly Significant Difference Tukey's test (p<0.05) and Discriminant Analysis showed it was possible to distinguish the geographical origin of cocoa beans. In particular, the absolute free phenol profile made it possible to characterise 4 out of 5 production macro-areas well, while an untargeted approach based on the ionisation profiles of glycosylated forms allowed complete characterisation of all the 5 macro-areas.