LC/MS/MS in Detecting and Quantifying Mycotoxins

The European Union (EU) has comprehensive regulations that are referenced by several other countries for establishment of their own limits. Commission Regulation (EC) No. 1881/2006 and its amendments set out specific rules in relation to mycotoxins and other contaminants. It includes specific maximum levels for 11  mycotoxins, including aflatoxins, ochratoxin A, type A and B trichothecenes, fumonisins, and zearalenone. This regulation applies to all food business operators involved, for example, in the import, production, processing, storage, distribution, and sale of food.

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Figure 2: Extracted quantifier ion peak, qualifier to quantifier ion ratios, triggered spectra library matching (upper panel) and calibration curve and structure for aflatoxin M1 (lower panel), using the UHPLC/MS/MS method for infant formula. An Agilent 1290 Infinity LC system and ­Agilent 6490 Triple Quadrupole LC/MS with triggered MRM was used.

Efficient Testing

Most traditional methods for the determination of mycotoxins in food or feed have been single-analyte methods, and few of them used liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) until a few years ago. However, tandem mass spectrometry is a powerful tool capable of accurately detecting and quantitating the levels of mycotoxins that are dictated by the regulations. Several LC/MS/MS methods have been developed that enable high throughput analysis of food products for accurate and reproducible quantitation of very low levels of several mycotoxins at once. A few are presented here.

Accurate quantitation in complex food matrices can be hampered by suppression or enhancement of the analyte signal due to matrix effects during the mass spectrometry ionization process. Differences in the degree of matrix effects cannot only be expected between different commodities but, to a lesser extent, also between individual samples of one matrix type.

There are different strategies to compensate for matrix effects such as the dilution of the sample, matrix-matched calibrations, standard addition, or the use of internal standards. For busy routine testing laboratories, the use of internal standards which behave exactly like the target compounds but are still distinctive, is most attractive. In the past, internal standards have often been analogs of a single compound or group of compounds. However, this has limited value when the intention is to compensate for matrix effects, since such effects are retention time dependent and target compounds rarely elute concurrent with such analogs.

Stable isotopically-labeled compounds are ideally suited as internal standards since they share the same physicochemical properties (meaning they elute together with the target compound) but are still distinguishable by MS due to their different molecular mass. In addition, they are not present in naturally contaminated samples. Since the naturally abundant isotopic distribution of the analyte is diluted by the addition of stable isotopically labeled compounds, this procedure is often referred to as stable isotope dilution assay (SIDA).

A SIDA LC/MS/MS assay has been developed for the analysis of the 11 mycotoxins regulated by the EU in maize. To assure accurate quantitation, a uniformly (13C)-labeled homolog for each target analyte was used as the internal standard (Figure 1). A two-step extraction without further cleanup was combined with ultra high performance liquid chromatography (UHPLC) separation and highly sensitive MS/MS detection using Dynamic Multiple Reaction Monitoring (dMRM). This method was successfully validated for maize based on method performance parameters including linearity of response, the limit of quantitation (LOQ) based on the signal-to-noise (S/N) ratio, and repeatability. The accuracy and reliability of the method were proven by analyzing several test materials with well-characterized concentrations. The key benefits of this method are the simple and complete extraction, the improved accuracy for a wide variety of matrices enabled by efficient compensation of all matrix effects, and high sensitivity.