Integrated Molecular Networking and NMR Metabolomics for In-Depth Chemical Profiling and Characteristic Marker...

In-depth chemical characterization provides key fingerprints for honey authentication. An integrated approach combining UHPLC-Q-TOF/MS-based molecular networking (MN) with HSQC NMR-based small molecule accurate recognition technology (SMART) was applied to characterize the chemical profile of Apis cerana honey from the Qinling Mountains. Twenty-eight metabolites were identified, with alkaloids and lipids being the most abundant. Targeted isolation of predominant fractions yielded four alkaloids (lumichrome, chestnutamide, deoxyvascinone, and 2-quinolone), one ionone derivative (plasiaticine I), and one ester (cetyl triacontanoate), all first reported in A. cerana honey as potential markers. ¹H NMR-based metabolomics coupled with OPLS-DA showed 13 unifloral honeys clustered by botanical origin. With this model, characteristic NMR signals of A. cerana honey (δ_H 2.28 [t], 3.45 [s], 4.05 [t] ppm) were identified, among which the two triplets match the oxygenated methylene of the isolated compounds. These findings clarify the chemical basis of A. cerana honey and provide an integrated approach for authentication.