Abstract
An untargeted metabolomics approach based on ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) fingerprinting was applied to investigate the metabolic differences of black pepper among three geographical origins (Sri Lanka, Vietnam, and Brazil) and two post-harvest processing (sterilized and non-sterilized spice). Principal component analysis (PCA) was employed to assess the overall clustering of samples, whereas supervised orthogonal partial least squares discriminant analysis (OPLS-DA) was effectively used for discrimination purposes. OPLS-DA models were fully validated (R2Y and Q2 values > 0.5) and the variable importance in projection (VIP) approach was employed to provide valuable data about differential metabolites with high discrimination potential (8 markers were putatively identified). For origin differentiation, three markers were highlighted with VIP values > 1.5 (i.e. reynosin, artabsinolide D, and tatridin B). Fatty acid derivates were the most frequent markers within the metabolites annotated for processing discrimination (e.g. 10,16-dihydroxyhexadecanoic acid and 9-hydroperoxy-10E-octadecenoic acid). Additionally, different combinations of mid-level data fusion of chromatographic-mass spectrometric techniques (UHPLC and gas chromatography coupled to HRMS) and proton nuclear magnetic resonance spectroscopy (1H NMR) were evaluated for the first time for geographical and processing discrimination of black pepper. The NMR-UHPLC-GC mid-level fused model was preferred among the tested fusion approaches since good sample clustering and no misclassification were achieved. Enhanced correct classification rate was achieved by mid-level data fusion compared with the findings obtained for one of the individual techniques (1H NMR fingerprinting) (from 92% to 100% of samples correctly classified). This study opens the path to new metabolomics approaches for black pepper authentication and quality control.
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