Masked Mycotoxin: Using Mass Spectrometry to Uncover Hidden Contaminants in Corn

While the impact of mycotoxin on the corn supply has been a concern for producers and regulators of corn products for food and feed for a long time, researchers have only recently been able to use high-resolution mass spectrometry to detect and identify glucosylated derivatives of so-called “masked mycotoxins.” These subspecies of Fusarium may evade detection through conventional methods and, thus, may be considered a particular threat for consumer safety. In this study, two of these contaminants in particular, T2 and HT2, as well as MAS, were detected with MS, and aspects of their structure were elucidated.

Nakagawa et al. used a high-resolution LC-Orbitrap MS instrument (Thermo Scientific) to search for T2GlcGlc and HT2GlcGlc. For the former, the [T2GlcGlc + H]+ ion was found to exhibit three peaks at 12.17, 12.73, and 12.94 min, while the [T2Glc + H]+ ion and [T2 + NH4]+ ion evidenced two peaks at 13.45 min and 15.85 min. During a second scan, peaks were discovered at 12.92, 13.44, and 15.83 min for the monitor ion [T2GlcGlc-GlcGlc-C5H10O2-2C2H4O2 + H]+ after the expected loss of isovaleric acid, acetic acid, and formaldehyde from the T2 molecule. The researchers observed low spectral intensity and, based on elution times, greater hydrophilicity in T2GlcGlc as compared to T2Glc and T2. The peak at 12.94 min was identified to be T2GlcGlc, while the other two peaks are likely isomers.

Based on previous studies, HT2GlcGlc was evaluated in the negative polarity with the [HT2 + CH3COO]− ion. Two peaks were apparent at 12.36 and 12.70 min for the first scan and 12.36 min for the second scan after the expected loss of isovaleric acid, acetic acid, and formaldehyde. The researchers also noted that the peak associated with HT2GlcGlc was low intensity and that the molecule is more hydrophilic than HT2Glc and HT2 based on its earlier elution. The researchers positively identified the compound at peak 12.36 min to be HT2GlcGlc. For tri-glucosides, neither ion ([T2GlcGlcGlc + H]+ or [HT2GlcGlcGlc + CH3COO]−) was detected in the search for T2GlcGlcGlc or HT2GlcGlcGlc.

MASGlc was also searched for in the negative polarity with [MASGlc + CH3COO]− and [MASGlc − H]− exhibiting peaks abundantly at 10.65-66 min and less abundantly at 10.43-44 min. During a second scan after the expected loss of acetic acid and formaldehyde, a 10.64 min peak was identified for [MASGlc-Glc − H]−. These spectra were also low intensity, and elution times suggest greater hydrophilicity for MASGlc in comparison with MAS. The researchers positively identified the compound at peak 10.65 min to be MASGlc and the one at 10.44 min to be an isomer.

This study represents a first identification of di-glucosylated derivatives of the masked mycotoxin T2, as well as di-glucosylated derivatives of HT2 and a mono-glucosylated derivative of MAS, through the novel use of high-resolution MS using the proton adduct rather than the ammonium adduct for screening of T2GlcGlc and the preferential use of negative scan mode for HT2GlcGlc and MASGlc. It is believed that the ammonium adducts were diminshed by the glucosylating event. The researchers assert the possibility that these masked mycotoxins may represent potential hazard for consumers since it is possible that triggering events may transform them to their corresponding aglycons. The researchers also point positively to the specifics of their MS instrument and its novel ability to detect and identify masked mycotoxins, such as Fusarium, in the absence of discrete chemical standards for identification. Although absolute identification of the specific structure of the di-glucosylated derivatives can not be specified through this means and the amount of glucosides present can not currently be measured due to a dearth of appropriate chemical standards, the sensitivity of the instrument and nature of the approach represents a breakthrough in research potential for the detection of species that elude identification through conventional means. Further research into the development of standards for accurate measurement and confirmation studies with regard to mycotoxin isomers may prove useful for future research.

References

Nakagawa, H., et al. (2013) ‘Detection of Type A Trichothecene Di-Glucosides Produced in Corn by High-Resolution Liquid Chromatography-Orbitrap Mass Spectrometry‘, Toxins, 5 (3), (pp. 590-604)