Method development using LC-MS/MS and LC-IMS-QTOF MS to determine mycotoxins in fish feed samples

Abstract Mycotoxin contamination is an increasing concern in aquafeed industry due to the replacement of marine-based ingredients to plant-based ingredients and climate changes associated with growing mycotoxin contamination in plant ingredients. Consequently, the health and growth of aqua animals could be affected by contaminated fish feeds, and finally influence human health. Therefore, it is necessary to develop an effective screening method for mycotoxin determination in fish feed. This master project develops two screening methods (using low resolution UHPLC-QqQ-MS/MS and high resolution UHPLC-IMS-QTOF MS instruments) for the determination of 18 mycotoxins: aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), beauvericin (BEA), deoxynivalenol (DON), diacetoxyscirpenol (DAS), enniatin A (ENA), enniatin A1 (ENA1), enniatin B (ENB), enniatin B1 (ENB1), fumonisin B1 (FB1), fumonisin B3 (FB3), moniliformin (MON), ochratoxin A (OTA), T2-Toxin (T2), HT-2 Toxin (HT2) and neosolaniol (NEO) and 6 metabolites: 15-Acetyldeoxynivalenol (15-ADON), 3-Acetyldeoxynivalenol (3-ADON), alpha-zearalenol (αZEL), beta-zearalenol (βZEL), deoxynivalenol-3-glucoside (D3G) and deepoxy deoxynivalenol (DOM-1). In addition to screening method, a quantitative method was developed by UHPLC-QqQ-MS/MS for all compounds except AFs, MON and DAS. The developed method was validated in terms of linearity. ENA, ENA1, ENB, ENB1, BEA, NEO, HT-2, T-2 and αZEL had a R2 > 0.99; DON, 3-ADON, OTA and βZE had a R2 >0.95, showing good linearity performance. Recovery (95%-111%) and intra-day precision (RSD of 4%-18%) for ENA, ENA1, ENB, ENB1 and BEA using T-2 as IS, demonstrated excellent validation performances. Both isotopic IS and structural analogue IS were used during the validation. T-2 was confirmed as a good alternation to substitute expensive isotopic IS for the validation of ENA, ENA1, ENB, ENB1 and BEA. Ten fish feeds including one fish feed ingredient collected in the market were analyzed using the screening method. Results show considerable mycotoxin contamination for all samples. The developed method by UHPLC-QqQ has shown great promise in quantification of mycotoxins. The other method was developed by UHPLC-IMS-QTOF MS for all compounds with CCS value as extra identification point. The developed method was applied to ten commercial fish feeds including one fish feed ingredient and results reveals considerable mycotoxin contamination for almost all samples. Mycotoxins detected in the fish feed samples were ENNs (including ENA, ENA1, ENB and ENB1) and BEA, while FUMs (FB1 and FB3) were detected in the fish feed ingredient sample. A chemical degradation experiment for two frequently occurred mycotoxins (BEA and ENB) was also performed. The transformation products (TPs) of both compounds are tentatively predicted