Fingerprinting Chemical Markers in the Mediterranean Orange Blossom Honey: UHPLC-HRMS Metabolomics Study Integrating Melissopalynological Analysis, GC-MS and HPLC-PDA-ESI/MS

Abstract

(1) Background: Citrus honey constitutes a unique monofloral honey characterized by a distinctive aroma and unique taste. The non-targeted chemical analysis can provide pivotal information on chemical markers that differentiate honey based on its geographical and botanical origin. (2) Methods: Within the PRIMA project "PLANT-B", a metabolomics workflow was established to unveil potential chemical markers of orange blossom honey produced in case study areas of Egypt, Italy, and Greece. In some of these areas, aromatic medicinal plants were cultivated to enhance biodiversity and attract pollinators. The non-targeted chemical analysis and metabolomics were conducted using ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS). (3) Results: Forty compounds were disclosed as potential chemical markers, enabling the differentiation of the three orange blossom honeys according to geographical origin. Italian honey showed a preponderance of flavonoids, while in Greek honey, terpenoids and iridoids were more abundant than flavonoids, except for hesperidin. In Egyptian honey, suberic acid and a fatty acid ester derivative emerged as chemical markers. New, for honey, furan derivatives were identified using GC-MS in Greek samples. (4) Conclusions: The application of UHPLC-HRMS metabolomics combined with an elaborate melissopalynological analysis managed to unveil several potential markers of Mediterranean citrus honey potentially associated with citrus crop varieties and the local indigenous flora.

Keywords: HRMS; aromatic medicinal plants; citrus; honey; mass spectrometry; melissopalynology; metabolomics; pollen.

Figure 3

Heatmap analysis in honey extracts from Greece, Italy, and Egypt derived from UHPLC-HRMS analysis in the negative (A) and positive (B) ion mode. The heatmaps indicate the relative content of secondary metabolites and compounds among the different countries.

Figure 4

PCA (Ai,Bi,Ci), OPLS-DA (Aii,Bii,Cii) score plots and permutation tests (Aiii,Biii,Ciii) after the comparison of honey extracts from Greece (blue) and Italy (red) (A), Greece (red) and Egypt (blue) (B), and Italy (red) and Egypt (blue) (C), in the negative ion mode. The goodness of fit and prediction of the models were for PCA analysis (Ai) R2X = 0.585, (Bi) R2X = 0.656, (Ci) R2X = 0.533 and for OPLS-DA analysis (Aii) R2X = 0.745, R2Y = 0.992, Q2Y = 0.838, (Bii) R2X = 0.814, R2Y = 0.996, Q2Y = 0.873, (Cii) R2X = 0.692, R2Y = 0.994, Q2Y = 0.851.

Figure 5

PCA (Ai,Bi), OPLS-DA (Aii,Bii) score plots and permutation tests (Aiii,Biii) after the comparison of honey extracts from Greece (blue) and Italy (red) (A) and Italy (red) and Egypt (blue) (Β), in the positive ion mode. The goodness of fit and prediction of the models were for PCA analysis (Ai) R2X = 0.576, (Bi) R2X = 0.538 and for OPLS-DA analysis (Aii) R2X = 0.774, R2Y = 0.994, Q2Y = 0.792, (Bii) R2X = 0.676, R2Y = 0.992, Q2Y = 0.665.

Figure 7

Indicative chemical markers characteristic of Mediterranean citrus honey.

Figure 8

Principal component analysis of honey samples (n = 40) using melissopalynological parameters (listed in detail in Table S5A). Score plots of the samples in the space of the two first principal components.

Figure 1

Representative chromatograms of citrus honey extracts from Greece (i), Italy (ii), and Egypt (iii) after UHPLC-HRMS analyses in negative (A) and positive (B) ionization modes.

Figure 2

HS-SPME GC-MS full scan chromatogram of a Greek citrus honey extract (selected chemicals’ peaks pointed out).

Figure 6

Hesperidin annotation based on MS/MS fragmentation pattern using mzCloud database. The top panel is the MS/MS pattern of the raw file from a Greek citrus honey sample, whereas the bottom panel is the MS/MS pattern of Hesperidin from the mzCloud database.

Figure 9

Indicative pollen grains in Greek (EL, a,b), Italian (IT, c,d), and Egyptian (EG, e,f) honey.