Quantitation of estragole by stable isotope dilution assays

Various calibration strategies for the quantitation of the phenylpropane estragole by gas chromatography-mass spectrometry were developed and compared. For application in stable isotope dilution assays, two deuterium labelled estragole isotopologues were synthesized. Of these, [3′,3′-²H₂]estragole was prepared by Wittig reaction of 4-methoxy-phenylacetaldehyde with [²H₃]methyl-triphenyl-phosphonium bromide, whereas [1″,1″,1″-²H₃]estragole was obtained by demethylation of estragole and deuteromethylation of the resulting 4-allylphenole. Besides estragole isotopologues, 1,2,4-trimethoxybenzene and 4-propylanisole were also tested as internal standards (I.S.) for the determination of estragole in fennel tea. [1″,1″,1″-²H₃]Estragole, 1,2,4-trimethoxybenzene, and 4-propylanisole revealed linear calibration functions and, therefore, were suitable for estragole quantitation. In contrast to this, [3′,3′-²H₂]estragole could only be applied as I.S. if it was added to the extracts in stoichiometric deficiency compared to unlabelled estragole. Moreover, due to its different chemical and physical properties, 1,2,4-trimethoxybenzene showed a recovery as low as 77%, whereas the other I.S. revealed recovery rates close to 100%. Considering the "real" values of estragole in fennel tea, the choice of the I.S. obviously is less important than the way of preparing the tea. In contrast to the common method for tea preparation, squeezing of the teabags increased the estragole content significantly by 50%.