First Quantitation of Key Odor-Active Thiols in Flowers of Different Hemp Cultivars Using HPLC-MS/MS

This study introduces an HPLC-MS/MS method to quantify four key odor-active compounds─3-methylbut-2-ene-1-thiol, 4-methyl-4-sulfanylpentan-2-one, 3-sulfanylhexyl acetate, and 3-sulfanylhexan-1-ol─in hemp extracts obtained by solvent-assisted flavor evaporation, after derivatization with 4,4'-dithiodipyridine, using a stable isotope dilution assay. Five different hemp cultivars were investigated, and the quantitative results were compared with those obtained with a heart-cut 2D GC-MS method. This work presents the first quantitation of 4-methyl-4-sulfanylpentan-2-one in hemp flowers, as well as the first example of derivatization of 3-methylbut-2-ene-1-thiol and its isotopically substituted standard with 4,4'-dithiodipyridine. The use of isotopically substituted standards ensured robustness and accuracy. The limit of quantification ranged between 97 ng/kg for 3-sulfanylhexan-1-ol and 130 ng/kg for 3-methylbut-2-ene-1-thiol. The quantitative data obtained emphasize the importance of thiols in the overall complex aroma of dried hemp flowers and demonstrate the utility of the developed HPLC-MS/MS methodology for quantifying these odorants at trace levels in complex plant matrices.