Abstract

Mycotoxins are toxic secondary metabolites produced by filamentous fungi. These toxins contaminate foods such as corn, nuts, wine, oats, milk, and pasta. Mycotoxins pose a wide range of acute and chronic health risks to human and animals including carcinogenicity, hepatotoxicity, and estrogenicity. Mycotoxins can undergo both phase I metabolism (oxidation, reduction, hydrolysis) and phase II metabolism (conjugation reactions such as glucuronidation, glutathione conjugation, sulphation, acetylation, and methylation). Currently, most liquid chromatography-mass spectrometry methods for the biomonitoring of mycotoxins do not include mycotoxin metabolites due to the lack of commercial synthetic standards and lack of mass spectral libraries. Therefore, the aim of this study was to characterize sulphate and glutathione metabolites of 17 mycotoxins from aflatoxin, zearalenone, and trichothecene classes. These mycotoxins were chosen based on high prevalence in the food supply and/or high toxicity. The metabolites obtained from human liver microsomal incubations were characterized using pentafluorophenyl liquid chromatography-high resolution mass spectrometry on ion trap – Orbitrap instrument. In total, 15 phase I oxidation metabolites, 42 sulphate and 17 glutathione metabolites were characterized using accurate mass, MS/MS, and/or MS3 fragmentation. Among these, 20 sulphates of phase I metabolites, 2 sulphates, and 14 glutathione conjugates were reported for the first time. Following 1-hour incubations, the major sulphate metabolites were detected. However, in order to detect all minor sulphate metabolites, 24-hour incubations were required. Zearalenones generally formed sulphates (36) and phase I metabolites, with only one glutathione conjugate observed for ZEN. Trichothecenes formed sulphates and unexpectedly also exhibited extensive glutathione conjugations. Aflatoxins underwent phase I metabolism and glutathione conjugation. Multiple sulphate conjugation on single mycotoxin was not observed for any of the mycotoxin classes studied. Furthermore, sulphate metabolism of 12 mycotoxins belonging to the enniatin, fusarium, alternariol, ochratoxin, and citrinin class was also characterized using reversed-phased C18 LC-HRMS on quadrupole time-of-flight instrument. In total, six sulphates were characterized, of which two were novel sulphates. All metabolite data was added to an extensive LC-MS/MS library of human mycotoxin metabolites to support future biomonitoring studies by increasing the efficiency of identification of mycotoxins and their metabolites.