LC/MS/MS in Detecting and Quantifying Mycotoxins

Providing feed to cows that is contaminated with mycotoxins can result in the contamination of products processed from their milk, including infant formula. The EU regulation for the presence of mycotoxins in formula is quite stringent, limiting the maximum concentrations of aflatoxin M1, aflatoxin B1 and ochratoxin A, for example, to 0.025, 0.1, and 0.5 microgram/kilogram, respectively. Most current methods for this analysis involve labor intensive and time consuming sample purification and concentration steps required to achieve these detection levels using liquid chromatography with fluorescence detection or LC/MS.

A UHPLC/MS/MS assay for the EU regulated mycotoxins in baby formula has been developed that uses a simple extraction without a concentration step to attain the sub-part per billion detection limits required by the regulation. This method utilizes triggered MRM acquisition (tMRM) for ultimate confidence in the identification of the mycotoxins. Pre-selected MRM transitions trigger the collection of additional MS/MS transitions, each with optimized collision energy and maximized dwell time to enable the highest sensitivity. The collected ions are formulated into a spectrum, which is compared to a triggered MRM library spectrum for confirmation. This method enables the detection of the regulated mycotoxins in infant formula at levels below the maximum allowable limits, as is demonstrated by the results for aflatoxin M1, which is typically associated with mycotoxin contamination of milk (Figure 2). In addition to the ideal sensitivity and precision of the method, its key benefit is the high confidence in the result due to the availability of high quality spectra down to very low concentration levels, which is only possible with triggered MRM.

Expanding Detection Capabilities

A method for the analysis of mycotoxins in nuts exploits the power of UHPLC and tandem mass spectrometry by enabling the detection and semi-quantitation of 191 mycotoxins and other fungal metabolites, in just two chromatographic runs per sample. UHPLC allows better separation of the analytes from the matrix, when compared to other LC/MS/MS methods, and the overall repeatability is superior to other published methods. This method features fast and easy sample preparation that includes only a single extraction step before injection of the diluted raw extract into the UHPLC/MS/MS. The multiplex analysis capability of the method enables a throughput of 25 samples per day.

This method has been utilized to survey 53 different nut samples for the presence of the 191 fungal compounds (Figure 3). The importance of using multi-mycotoxin methods was demonstrated by the detection of 40 different analytes in the nut samples. The key benefit of this method is the ability to detect mycotoxins in unlikely matrices. By applying comprehensive screening methods, the availability of occurrence data is greatly improved. In addition, this method is a good repository of MRM transitions for method extension of, for example, one of the two methods mentioned previously.

Although aflatoxins are the only mycotoxins regulated in nuts in the EU, these results suggested that other toxins may also be relevant. Major mycotoxins found in more than 50 percent of the samples were beauvericin, enniatin B, macrosporin, 3-nitropropionic acid, emodin, and alternariol methyl ether. These results also confirmed for the first time the presence of HT-2 and T-2 toxins in hazelnuts. Analysis of such a large number of fungal toxins might be useful in the future since possible toxic effects on humans are still not fully evaluated and additive or synergistic effects of such toxins are largely unknown.