Recent developments in mycotoxin analysis

The mycotoxins of major concern are: the aflatoxins (aflatoxin B1 is the most potent naturally occurring carcinogen known), the trichothecenes (a structurally related family of cytotoxic compounds) including deoxynivalenol (DON), T-2 and HT-2 toxins, the zearalenone (a potent estrogenic mycotoxin), the fumonisins (predominantly fumonisin B1, a possible carcinogen to humans) and the ochratoxins (predominantly ochratoxin A, a potent nephrotoxin). Sensitive, reliable and accurate methods of analysis are hence required to gather adequate information on the levels of exposure to these mycotoxins and to fulfill regulatory requirements.

Analytical methods for mycotoxins in feed and food commodities generally include three steps: i) extraction of the toxin from the matrix with a suitable solvent, ii) purification of extract to eliminate co-extracted interferences, and iii) detection/determination of the toxin by an appropriate analytical instrument/technology. Due to the different chemical structures of mycotoxins and the wide variety of mycotoxincommodity combinations, a wide number of different analytical methods have been developed and validated.

Purification of extracts is essential for the analysis of mycotoxins at regulatory levels, and it usually involves the use of solid phase extraction (SPE), multifunctional (e.g. MycoSep®) or immunoaffinity columns (IACs), the last being the most commonly used in official methods due to the specificity of antibodies providing cleaner extracts with respect to other clean-up methods. Recently, SPE columns based on molecularly imprinted polymers (MIPs) or aptamers have been developed for selective extraction of mycotoxins.

Chromatographic methods are commonly used for the determination of mycotoxins, including gas-chromatography (GC), mainly for type-A trichothecenes, and liquid chromatography (HPLC, UHPLC) coupled with ultraviolet or diode array (UV/DAD), fluorescence (FL) or mass spectrometry (MS) detectors. The method of choice depends on the matrix and the mycotoxin to be analysed. In addition, several commercial immunometric assays, such as enzyme-linked immunosorbent assay (ELISA), are frequently used for screening purposes. Recently, a variety of emerging methods have been proposed for the analysis of mycotoxins in several food matrices, mainly cereals, based on immunochromatography (i.e. lateral flow devices, dipsticks), fluorescence polarisation (FP), infrared spectroscopy (FT-NIR), electronic nose (e-nose) and optical/electrochemical biosensors^{1-5, 9-16}.

A brief overview of recent developments on the determination of mycotoxins by liquid chromatography-mass spectrometry and rapid methods at the Institute of Sciences of Food Production, National Research Council of Italy (ISPA-CNR) is presented. 

Liquid chromatography – mass spectrometry

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is spreading rapidly as a powerful technique for simultaneous screening, identification, characterisation and quantitative determination of a large number of mycotoxins, including their modified forms¹⁷. Just as an example, LC-MS/MS ‘dilute and shoot’ methods have recently been developed for the determination of 191 mycotoxins and 295 bacterial and fungal metabolites including all regulated mycotoxins in food commodities^18,19. Accuracy, precision, and sensitivity of LC-MS methods may vary depending on the mycotoxin, matrix and instrumental sensitivity/selectivity. Quantitative measurement of mycotoxins by LC-MS is often unsatisfactory due to matrix effects and ion suppression. Results of a proficiency test involving 41 participants for the LC-MS/MS determination of up to 11 mycotoxins in maize, showed that laboratories that carried out sample extract clean-up gave acceptable results for the majority of mycotoxins. Purification of extracts by MycoSep^® or IACs is usually needed prior to MS detection^3,20,21.

LC-MS/MS and liquid chromatography high-resolution mass spectrometry (LC-HRMS) based on Orbitrap technology have been applied to investigate the presence of T-2 and HT-2 glucoside derivatives in naturally contaminated cereals and Fusarium langsethiae fungal cultures. Molecular structure details obtained by measuring exact masses of main characteristic fragments with high mass accuracy led to the identification of a monoglucoside derivative of T-2 toxin and two monoglucoside derivatives of HT-2 toxin²². In addition, two mono-glucosyl derivatives of neosolaniol (NEO) and one mono-glucoside derivative of diacetoxyscirpenol (DAS), two type-A trichothecenes, were identified and characterised by LC-MS/MS. These compounds were detected either in fungal cultures or in cereal samples naturally contaminated with the parent toxins²³.

Furthermore, a preliminary screening of naturally contaminated cereal samples (i.e. wheat, oats and barley) showed a widespread occurrence of type-A trichothecene glucosides in cereal grains naturally contaminated with the relevant unconjugated toxins. A chemically synthesised T-2 toxin-β-glucoside and a T-2 toxin-α-glucoside obtained by Blastobotrys muscicola cultures were characterised and compared with T-2 toxin-glucoside found in naturally contaminated oats and wheat samples. The study revealed the presence of the α-linked form of T-2 toxin-glucoside in naturally contaminated plants, showing MS behaviour identical to the yeast biotransformation product²⁴. The availability of a reference standard made possible the collection of a first survey data aimed at obtaining more comprehensive information on the co-occurrence and contamination levels of T-2 and HT-2 toxins and their glucosylated derivatives in naturally contaminated barley samples²⁵.

Rapid methods

Immunological assays, such as ELISA, have become very popular in mycotoxin screening since many years ago. Screening methods are intended to be rapid and easy-to-use and do not require skilled operators and expensive instrumentations. In the last years, several rapid immunoassay-based tests have been developed for the analysis of mycotoxins in food/feed commodities^9-16.

Fluorescence polarisation immunoassay (FPIA) is a homogenous assay based on the competition between the antigen and a fluorescently labeled antigen (tracer) for a specific antibody. The binding of the tracer to the antibody affects the rate of rotation of the tracer and increases the fluorescence polarisation value. The amount of bound tracer is inversely proportional to the amount of free analyte in the sample, as a result the polarisation value is inversely related to the analyte concentration. Recently, reliable FPIAs have been developed for the rapid determination of T-2 and HT-2 toxins in wheat, ochratoxin A (OTA) in wheat and deoxynivalenol (DON) in wheat bran and whole-wheat flour^26-28. A preliminary treatment with activated charcoal was used to eliminate the strong matrix effect due to highly coloured interfering compounds present in raw wheat bran extracts. The overall time of analysis ranged from 10 to 15 minutes. The immunoassays were validated for comparison with widely used HPLC-IAC methods by analysing naturally contaminated samples showing accuracy and precision values similar to those obtained with HPLC methods.

Lateral flow devices (LFDs), also called immunochromatographic strip tests, are rapid immunoassays based on the interaction between specific antibodies and antibody-coated dyed receptors, e.g., colloidal gold, that react with the analyte to form an analyte-receptor complex. Generally, LFDs have been developed for the determination of a single mycotoxin. A multiplex dipstick immunoassay for the simultaneous determination of zearalenone (ZEA), T-2 and HT-2 toxins, deoxynivalenol (DON) and fumonisins (sum of FB1 and FB2) in wheat, oats and maize has been recently developed²⁹. Analysis of naturally contaminated samples and the comparison with an LC-MS confirmatory method showed how the developed multiplex immunoassay can provide a reliable tool for rapid and simultaneous assessment of the presence/absence of the six major Fusarium toxins at levels close to the EU regulatory levels within 30 min. A collaborative study involving 12 laboratories for evaluating the performances of the multiplex dipstick immunoassay showed the test to be able to differentiate blank samples from samples contaminated at target mycotoxin levels with a false positive rate lower than 10% for ZEA, DON and fumonisins. Unsatisfactory results were obtained for the sum of T-2 and HT-2 toxins³⁰. The assay is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins in cereals.

In recent years, near-infrared (NIR) and mid-infrared (MIR) spectroscopy associated to principal component analysis (PCA) have been proven to be promising tools to detect fungal contamination and to estimate mycotoxin contamination in cereals³¹. Among mycotoxins, the most investigated one was DON, mainly in Fusarium-damaged wheat kernels. The analysis is non-destructive, rapid and requires minimal or no sample preparation. The feasibility of using Fourier Transform-NIR (FT-NIR) spectroscopy for the qualitative and quantitative prediction of DON at levels up to about 2,000µg/kg in unprocessed ground durum and common wheat was reported for the first time by De Girolamo et al³². Recently, Partial Least-Squares (PLS) regression analysis was used by the same research group to determine DON in 464 naturally contaminated durum wheat samples in the range of <50–16,000μg/kg DON. Results indicated a very poor prediction ability of the quantitative PLS model. On the contrary, the classification model based on Linear Discriminant Analysis (LDA) successfully distinguished wheat samples based on their DON content with high overall classification rates and low misclassification. The classification model fulfilled the requirement of the European official guidelines for screening methods (Commission Regulation No. 519/2014) when a cut-off level of 1,400μg/kg of DON was used³³.

Fungal volatile metabolites can be used as an indicator of mycotoxins occurrence in cereals. Electronic nose technology has been shown to be able to determine the mycological quality of cereals as well as to predict the content of some mycotoxins. A rapid, easy to-perform and non-invasive method using an electronic nose based on metal oxide semiconductor (MOS) sensors has been recently developed to distinguish ground durum wheat samples in three classes based on the content of DON: class A ([DON] < 1,000µg/kg), class B (1,000 ≤ [DON] ≤ 2,500µg/kg) and class C ([DON] > 2,500µg/kg)³⁴. Both the classification models (based on FT-NIR and e-nose) could be used as useful tools for high throughput screening of a large number of wheat samples for DON contamination. This would mean a reduction in the number of analysis to be carried out by HPLC of samples contaminated at levels closer or higher than the maximum permitted limit set by the EU for unprocessed durum wheat (i.e. 1,750µg/kg).

Future prospects

LC-MS/MS is presently the technique most widely used for the simultaneous determination of mycotoxins, although it generally suffers from matrix effects. Matrix-assisted calibration, isotopically labelled internal standards and improved sample preparation are essential for an accurate multi-mycotoxins determination. At present, no LC-MS methods are recognised as standard or official methods for mycotoxin detection, although LC-MS methods validated by interlaboratory studies for the simultaneous determination of mycotoxins are highly required. Proficiency tests for multi-mycotoxin LC-MS methods are a useful tool to provide insights on the used methodologies and related performances and could provide useful information for the optimisation and selection of methods to be used in interlaboratory validation studies. An emerging issue in the area of mycotoxins is represented by modified mycotoxins. LC-MS/MS has been shown to be a reliable technique for their detection and characterisation, as well as for the identification of new secondary metabolites of toxigenic fungi. Standards of identified modified mycotoxins are necessary for their quantification in naturally contaminated samples. Reference materials for quality control of mycotoxin methodologies are commercially available for the major mycotoxins, although they are quite limited. Certified Reference Materials of complex matrices, as well as multi-mycotoxin standards and multi-mycotoxin reference materials are highly necessary to assess quality of methods, especially when LC-MS methods are used.

Rapid methods for mycotoxin analysis are increasing in the last years. The advantages of these methods, with respect to conventional methods, are the easiness of operations and the rapidity of analysis together with their low cost. However, the analytical performances of these rapid methods should meet defined criteria and performance parameters. Harmonised validation guidelines for rapid methods are not always available and do certainly have a highly priority. Recently, the European Commission has established criteria with which screening methods for mycotoxins have to comply with when they are used for regulatory purposes (Commission Regulation No. 519/2014). Moreover, reliable multiplex screening assays for simultaneous determination of mycotoxins are highly required.

References

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About the author

Dr. Michelangelo Pascale graduated in Chemistry at the University of Bari, Italy. He is Senior Researcher at the Institute of Sciences of Food Production (ISPA), National Research Council of Italy (CNR), and since 2006 is responsible of the CNR project ‘Innovative methods for food characterisation and control of mycotoxins, toxigenic fungi and allergens’. Michelangelo has scientific responsibility for several national/international projects of ISPA relevant to food safety. His specific expertise is in toxigenicity of fungi, occurrence of mycotoxins in food and feed, effect of fungicides on cereal diseases and mycotoxin accumulation, fate of mycotoxins during food processing, development and validation of analytical methods for mycotoxins in food, feed and biological fluids, and organisation of collaborative studies for method validation. Nowadays he is engaged in the development of rapid analytical methods based on UHPLC, immunoassays and biosensors for the detection of mycotoxins in agro-food products. He is also (Co)-author of over 100 papers in peer reviewed journals.