Development of a Stable Isotope Dilution LC–MS/MS Method for the Alternaria Toxins Tentoxin, Dihydrotentoxin, and Isotentoxin

Abstract

For the Alternaria toxins tentoxin, dihydrotentoxin, and isotentoxin, a stable isotope dilution LC-MS/MS method was first developed. Triply deuterated internal standards were prepared via total synthesis and introducing the labels in the last step before cyclization. Method validation was carried out by using potato starch, tomato puree, and white pepper powder as blank matrices. For the three toxins the limits of detection ranged from 0.10 to 0.99 μg/kg. The inter-/intraday relative standard deviations of the method were below 8.8%, and the recoveries ranged between 98 and 115%. Although cyclic peptides are known to show only negligible fragmentation, a low limit of detection was achieved with the optimization of mass spectrometry parameters and cleanup on C18-phenyl SPE columns providing a more selective binding of these phenyl-containing cyclic peptides. The method was applied to 103 food samples including bread, cereals, chips, juice, nuts, oil, sauce, seeds, and spices. Of these, 85% were contaminated with tentoxin and 55% were contaminated with dihydrotentoxin, whereas isotentoxin was not quantifiable. Maximal concentrations of tentoxin and dihydrotentoxin were 52.4 and 36.3 μg/kg, respectively, and were both detected in paprika powder.