Abstract

Mycotoxins are secondary metabolites produced by fungi that can pose a serious threat to human and animal health due to their toxicity. The assessment of human chronic exposure to mycotoxins requires reliable and highly sensitive multi-analyte assay(s) enabling simultaneous measurements of common toxicologically important mycotoxins and their metabolites in human plasma.
The first goal of the thesis was to develop sensitive liquid chromatography – high-resolution mass spectrometry (LC-HRMS) multi-mycotoxin method(s) for the detection and quantification of common toxicologically important mycotoxins frequently occurring in Canada and emerging mycotoxins of interest. Based on the results of extraction recoveries and chromatographic separation, two LC-HRMS methods were required to cover the full mycotoxin panel of interest. The first method combined liquid-liquid extraction with pentafluorophenyl reversed-phase LC-HRMS for the quantification of 17 mycotoxins, aflatoxins B1, B2, G1 and G2, zearalenone, 7-α-hydroxy-zearalenol (α-ZOL), 7-β-hydroxy-zearalenol, zearalanone, 7-α-hydroxy-zearalanol, 7-β-hydroxy-zearalanol, T-2 toxin, HT-2 toxin, deoxynivalenol, nivalenol, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol and fusarenon X. The method was validated using procedures described in the Food and Drug Administration (FDA) guidance for Industry Bioanalytical Method Validation. Lower limits of quantification (LLOQs) ranged from 0.1 to 0.5 ng/ml, except for nivalenol (3 ng/ml). The method (intra-day and inter-day) accuracy and precision ranged from 85.6% to 116.4% and from 1.6% to 15.6% RSD, respectively, excluding α-ZOL for which an accuracy of 72.9 % to 97.2% was observed. The second method covered ten mycotoxins, fumonisin B1, fumonisin B2, ochratoxin α (OTα), citrinin, ochratoxin A, beauvericin, enniatin A, enniatin A1 (ENNA1), enniatin B (ENNB) and enniatin B1, and combined methanol protein precipitation with C18 reversed-phase chromatography and polarity-switching LC-HRMS. LLOQs ranged from 1.25 to 4 ng/ml. Absolute recovery ranged from 86.6% to 127.7% in individual plasma samples. Significant matrix effects were observed for OTα (77.5%) in one out of ten individual plasma samples and fumonisins (134.8% to 167.8%), ENNB (69.7% to 79.4%) and ENNA (69.3% to 79.2%) in all individual plasma samples. The rest of the mycotoxins showed negligible matrix effects ranging from 87.2% to 112.2% in all lots of plasma tested.
Excellent LLOQs, negligible matrix effects and accurate quantitation capability of the first method coupled with the lower cost of analysis per sample make the method suitable for large-scale analysis of human plasma samples. The second method is also simple and low cost but requires additional modification to further improve LLOQs and reduce the matrix effect before full validation and implementation. Both methods are versatile and can be applied for retrospective analysis and other applications such as metabolism studies due to the use of HRMS and superior chromatographic separation. To show this capability, the first method was successfully applied for the in-depth metabolism studies of 17 mycotoxins. The method showed excellent suitability and advantages for the detection of various mycotoxin metabolites from Phase I metabolism and glucuronidation obtained from human microsomal incubations. Two ppm mass accuracy with internal mass calibration reduced the number of possible elemental formulas for a measured m/z value. Data-dependent acquisition in combination with collision-induced dissociation or higher energy collisional dissociation was used to ensure adequate fragmentation and to study the structure of the mycotoxin metabolites. The Compound Discoverer 2.1 software, which contains extensive libraries of common metabolic pathways and mass spectral libraries, was used to streamline the identification and the characterization of the metabolites. In total, 188 mycotoxin metabolites were generated, characterized and used to build an extensive in-house library of human mycotoxin metabolites. One hundred metabolites were reported for the first time, showing the power and sensitivity of the approach. For these 17 mycotoxins, 92 metabolites were previously described in literature, and among these known metabolites only four could not be generated using our approach. Currently, this is the most comprehensive LC-MS library of human mycotoxin metabolites.
In conclusion, both LC-MS methods and the in-house mycotoxin metabolite library will allow the monitoring of 27 mycotoxins and their 188 metabolites in large-scale biomonitoring studies. In the long-term, this will help to prioritize metabolites that should be routinely included during exposure monitoring studies and will provide important new data on mycotoxin exposure of the Canadian population.