Cocoa powder is a widely used food and ingredient, and is vulnerable to adulteration by cheaper ingredients. A non-targeted metabolomics approach based on the use of reversed-phase liquid chromatography coupled to a high-resolution mass spectrometer (HPLC-HRMS) was developed to carry out the characterisation of cocoa powder samples adulterated at two different levels, with carob flour, soya flour, and chicory. The extraction procedure and chromatographic parameters were optimised to obtain the highest number of molecular markers. After chemometric analysis of the data, 21 and 37 metabolites in positive and negative ionisation modes, respectively, were found as potential authenticity and adulteration markers in the cocoa powder samples and simulated samples of adulterated cocoa powder. Of these, the identity of only 16 compounds in negative mode and 4 in positive mode could be determined, and most of these compounds belong to the groups of flavonoids, fatty acids, terpenoids, and lysophospholipids, which were potential markers for cocoa adulteration.
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