Stable Isotope Dilution Assays of Alternariol and Alternariol Monomethyl Ether in Beverages

Stable isotope dilution assays (SIDAs) for the determination of the most important mycotoxins of the black mold Alternaria, namely, alternariol and alternariol monomethyl ether, have been developed. For this purpose, deuterated alternariol and alternariol methyl ether were synthesized by palladium catalyzed protium-deuterium exchange from the unlabeled toxins. Reaction conditions were chosen in such a manner that the formation of the [H-2(4)]-isotopologues was favored. The synthesized products were characterized by LC-MS, NMR, and UV-spectroscopy. On the basis of the use of [H-2(4)]-alternariol and [H-2(4)]-alternariol methyl ether as internal standards, SIDAs were developed and applied to the determination of alternariol and alternariol methyl ether in beverages using LC-MS/MS. Method validation revealed a high sensitivity, i.e., low limits of detection (alternariol, 0.03 mu g/kg; alternariol methyl ether, 0.01 mu g/kg) and limits of quantitation (alternariol, 0.09 mu g/kg; alternariol methyl ether, 0.03 mu g/kg), respectively. Recovery from spiked apple juice was 100.5 +/- 3.4% for alternariol (range 0.1-1 mu g/kg) and 107.3 +/- 1.6% for alternariol methyl ether (range 0.05-0.5 mu g/kg). Interassay precision (expressed as coefficient of variation, CEV) for alternariol was 4.0% (7.82 +/- 0.31 mu g/kg; vegetable juice, naturally contaminated) and 4.6% (1.04 +/- 0.05 mu g/kg; grape juice, naturally contaminated). For alternariol methyl ether, a CEV of 2.3% (0.79 +- 0.02 mu g/kg; vegetable juice, naturally contaminated) was obtained. Analysis of fruit juices showed low contamination with alternariol and alternariol methyl ether in general, but higher values of both toxins were found in wine and vegetable juices. The values for alternariol were higher than those for alternariol methyl ether in nearly any case. However, the developed SIDA has proven to be optimally suited for further studies on alternariol and alternariol methyl ether content in food samples to obtain further insight into possible health hazards for the consumer.