Biosynthesis of seven carbon-13 labeled Alternaria toxins including altertoxins, alternariol, and alternariol methyl ether, and their application to a multiple stable isotope dilution assay

Abstract

An unprecedented stable isotope dilution assay for the genotoxic altertoxins along with exposure data of consumers is presented to enable a first risk assessment of these Alternaria toxins in foods. Altertoxins were produced as the most abundant Alternaria toxins in a modified Czapek–Dox medium with a low level of glucose as the carbon source and ammonium sulfate as the sole nitrogen source. Labeled altertoxins were synthesized in the same way using [¹³C₆]glucose. Moreover, labeled alternariol, alternariol methyl ether, altenuene, and alternuisol were biosynthesized in another modified medium containing [¹³C₆]glucose and sodium [¹³C₂]acetate. A stable isotope dilution LC–MS/MS method was developed and used for food analysis. For altertoxin I, altertoxin II, alterperylenol, alternariol, and alternariol methyl ether, the limits of detection ranged from 0.09 to 0.53 μg kg⁻¹. The inter-/intra-day (n = 3 × 6) relative standard deviations of the method were below 13 %, and the recoveries ranged between 96 and 109 %. Among the various commercial food samples, some of the organic whole grains revealed low-level contamination with altertoxin I and alterperylenol, and paprika powder, which was heavily loaded with alternariol, alternariol methyl ether, and tentoxin, showed higher contamination level of altertoxin I and alterperylenol. Altertoxin II and III and stemphyltoxin III were not detectable. In addition, if the food was contaminated with altertoxins, it was likely to be co-contaminated with the other Alternaria toxins, but not necessarily vice versa. Maximum concentrations of altertoxin I and alterperylenol were detected in sorghum feed samples containing 43 and 58 μg kg⁻¹, respectively. This was significantly higher than that in the measured food samples.