A sensitive and selective method for the quantitative determination of fatty acid tryptamides as shell indicators in cocoa products

Tetracosanoyl-2-(3-indolyl)ethane amide (lignocerinic acid tryptamide; LAT) and docosanoyl-2-(3-indolyl)ethane amide (behenic acid tryptamide; BAT) were identified as the most prominent tryptamides in cocoa shells based on electrospray ionisation mass spectrometry and ¹H NMR measurements. The structure of LAT, which is reported for the first time in cocoa shells, and also that of BAT were confirmed by synthesis. By using synthesised heptadecanoyl-2-(3-indolyl)ethane amide as the internal standard, a sensitive and reproducible method was developed for the quantification of LAT and BAT in the picogram range by means of HPLC/fluorescence detection. The detection limit was determined to be 30 pg/run. In authentic shell samples, 50-fold higher concentrations of both tryptamides were determined compared to the cocoa cotyledons. In 15 commercial chocolate samples, concentrations of 23.1-63.0 μg of the tryptamides (sum of both) per gram of fat were found. A first experiment attempted to correlate the tryptamide content with the amounts of shells in a model chocolate showed that the method is a promising tool to determine the shell content in the quality assessment of cocoa products.