Quantification of free coumarin and its liberation from glucosylated precursors by stable isotope dilution assays based on liquid chromatography-tandem mass spectrometric detection

A stable isotope dilution assay for the quantification of free coumarin and glucosylated coumarin precursors has been developed using [ ¹³C ₂]-coumarin as the internal standard. The doubly labeled coumarin was synthesized by reacting [ ¹³C ₂]-acetic anhydride with salicylic aldehyde and characterized by means of mass spectrometry and nuclear magnetic resonance (NMR) experiments. The specifity of liquid chromatography-tandem mass spectrometry enabled unequivocal determination and sensitive quantitation of the odorant. Because of the very simple extraction procedure, free coumarin could be analyzed within 1h. For quantification of total coumarin, the odorant was liberated from its precursors by an incubation with hydrochloric acid or β-glucosidase. In analyses of breakfast cereals, the intra- assay coefficient of variation was 9.9% (n = 5) for total coumarin. When coumarin was added to butter cookies at a level of 10 μg/kg, a recovery of 94.1% was found. Further addition studies revealed a detection limit of 2.9 μg/kg and a quantification limit of 8.6 μg/kg. Application of the stable isotope dilution assay to several plants, foods, and essential oils revealed high contents in cassia products and those foods in which cassia has been used as an ingredient. In contrast to this, Ceylon cinnamon contained much less coumarin. The odorant was also quantified in woodruff, clover seeds, and the essential oils of lavender, citron, and chamomile. Only trace amounts were detected in carrots and the essential oils of peppermint and dill, whereas in bilberries, black raspberries, and Angelica roots, coumarin was below detectable levels. In Ceylon cinnamon and cassia, the odorant occurred mainly in its free form, whereas in fenugreek seeds and woodruff, 68 and 88% of the total coumarin content was liberated from glucosylated precursors, respectively.