Development and validation of TOF/Q-TOF MS/MS, HPLC method and in vitro bio-strategy for aflatoxin mitigation

Some secondary metabolites produced by fungi are carcinogenic, hepatotoxic, and/or cause birth defects in humans and animals. We developed and optimised bio-analytical tools for detection of metabolites, aflatoxins and evaluated the effectiveness of the methods in co-infected maize tissues. Isolate KSM012 (atoxigenic) demonstrated no peaks and no blue fluorescence on HPLC and TLC plates respectively confirming non-toxicity. AFB1 and AFB2 were produced by Isolate KSM015 in addition to AFG1 and AFG2, which is an indication of possible S_BG morphotype. The limits of quantification and detection ranged from 0.02 to 35.81 µg/mL and 0.01-6.8 µg/mL, respectively. The best mass spectrum with lowest noise was obtained at 100% ACN and sterile water spiked with 0.1% formic acid at a flow rate of 0.3 mL/min. The positive ion mode with electrospray ionisation application exhibited better fragmentation for mycotoxins. In total 17 metabolites were detected by targeted and formula mass. KDVI maize line exhibited high fungal colonisation in comparison to GAF4 at equal co-infection ratio 50:50. AFB1 and AFG2 were remarkably higher in GAF4 in comparison to sensitive KDV1 (p ˂ 0.05). The detection limits, linearity and sensitivity showed the method developed was suitable for the determination of mycotoxin in comparisons to the guidelines of European Commission 657/EC 2002.