Metabolomic diversity and spatial localization of bioactive compounds in hemp achenes from diverse landraces

Hemp achenes, historically valued as nutritional and medicinal resources, are rich in phytochemicals such as phenethylamines, lignanamides, and flavonoids. However, metabolomic profiles among landraces with diverse size and color phenotypes remain unclear, and the spatial distribution of secondary metabolites within hemp achenes is still largely unexplored. This study conducted a comprehensive phytochemical characterization of 26 polyphenols (6 phenethylamines, 14 lignanamides, and 6 flavonoids) and 29 cannabinoids. Subsequently, a liquid chromatography-mass spectrometry-based targeted metabolomics approach was developed to systematically analyze metabolic differences in kernels and hulls of 20 hemp landraces with varying colors and sizes. Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-MALDI-MSI) was employed to map spatial localization of the 55 compounds in hemp achenes. Differential analysis revealed elevated levels of 4 phenethylamines, 1 lignanamide, and 2 flavonoids in dark achenes. Among 5 differential cannabinoids, 2 enriched in dark achenes and 3 in light achenes. Small achenes accumulated higher levels of 4 phenethylamines, 1 lignanamide, and 5 flavonoids versus large achenes. Among 12 differential cannabinoids, 7 enriched in small and 5 in large achenes. Differentially abundant metabolites predominantly accumulated in hulls, and AP-MALDI-MSI demonstrated distinct spatial compartmentalization of phenethylamines, flavonoids, and cannabinoids within the hull. These findings revealed phenotype-dependent metabolic diversity and compartmentalization in hemp achenes, providing important insights for precision breeding and the development of hemp-derived medicinal products.