Susan Macdonald

Mycotoxin analysis: An update

Mycotoxin contamination of cereals and related products used for feed can cause intoxication, especially in farm animals. Therefore, efficient analytical tools for the qualitative and quantitative analysis of toxic fungal metabolites in feed are required. Current methods usually include an extraction step, a clean-up step to reduce or eliminate unwanted co-extracted matrix components and a separation step with suitably specific detection ability. Quantitative methods of analysis for most mycotoxins use immunoaffinity clean-up with high-performance liquid chromatography (HPLC) separation in combination with UV and/or fluorescence detection. Screening of samples contaminated with mycotoxins is frequently performed by thin layer chromatography (TLC), which yields qualitative or semi-quantitative results. Nowadays, enzyme-linked immunosorbent assays (ELISA) are often used for rapid screening. A number of promising methods, such as fluorescence polarization immunoassays, dipsticks, and even newer methods such as biosensors and non-invasive techniques based on infrared spectroscopy, have shown great potential for mycotoxin analysis. Currently, there is a strong trend towards the use of multi-mycotoxin methods for the simultaneous analysis of several of the important Fusarium mycotoxins, which is best achieved by LC–MS/MS (liquid chromatography with tandem mass spectrometry). This review focuses on recent developments in the determination of mycotoxins with a special emphasis on LC–MS/MS and emerging rapid methods.

Ochratoxin A in dried vine fruit: method development and survey

A method is described for the determination of concentrations of the mycotoxin ochratoxin A in dried vine fruits (currants, raisins and sultanas) using acidic methanolic extraction, immunoaffinity chromatography clean-up and HPLC determination. The limit of detection was estimated as 0.2 microgram/kg, and recoveries of 63-77% were achieved at 5 micrograms/kg. HPLC-mass spectrometric confirmation of the identity of ochratoxin was obtained. Ochratoxin A and aflatoxins were determined in 60 samples of retail dried vine fruits purchased in the United Kingdom. Ochratoxin A was found in excess of 0.2 microgram/kg in 19 of 20 currant, 17 of 20 sultana and 17 of 20 raisin samples examined, an overall incidence of 88%. The maximum level found was 53.6 micrograms/kg. No aflatoxin was found in any sample analysed, using a method with a detection limit of 0.2 microgram/kg for each of aflatoxin B1, B2, G1 and G2.

Assessment of dietary exposure to ochratoxin A in the UK using a duplicate diet approach and analysis of urine and plasma samples

The approach to assess exposure to ochratoxin A from the diet by the analysis of human plasma and urine samples has been developed. Composite duplicate diet samples from 50 individuals and corresponding plasma and urine samples were obtained over 30 days. Samples were analysed using sensitive methods capable of measuring ochratoxin A at 0.001ng g -1 in food, 0.1ng ml -1 in plasma and 0.01ng ml -1 in urine. Analysis of the foods indicated ochratoxin A levels contributing to an average intake in the range 0.26-3.54ng kg -1 bw day -1 over the 30 days. Ochratoxin A was found in all plasma samples and in 46 urine samples. The correlation between the plasma ochratoxin A levels and ochratoxin A consumption was not significant (95% confidence limit). However, a significant correlation was found between ochratoxin A consumption and the urine ochratoxin A concentration expressed as the total amount excreted. This new work offers the possibility of using ochratoxin A in urine as a simple and reliable biomarker to estimate exposure to this mycotoxin.

Fate of ochratoxin A in the processing of whole wheat grains during milling and bread production

Batches of whole wheat contaminated with ochratoxin A were produced by inoculation with Penicillium verrucosum under controlled conditions in the laboratory. The fate of ochratoxin was followed through initial cleaning, abrasive scouring of the outer grain coat, milling into wholemeal wheat or into 10 milled fractions. Bread was baked from both wholemeal flour and straight-run white flour. Concentrations of ochratoxin A in the cleanings, scourings, and the bran and offal fractions were increased, but reduced in the white flour. Scouring removed up to 44% of the ochratoxin A present, but only a small further loss occurred in the bread-making process. An overall reduction of about 75% could be achieved in white bread using a combination of cleaning scouring and removal of the bran and offal fractions. Maximum overall reduction in producing wholemeal bread was about 40%. The reduction in ochratoxin A that can be achieved must be considered in relation to economic constraints concerning the disposal of wasted grain. Appropriate strategies for the use or disposal of potentially highly contaminated cleanings, scourings, bran or offal must be established.

Fate of the fusarium mycotoxins, deoxynivalenol, nivalenol and zearalenone, during extrusion of wholemeal wheat grain

In the European Union, deoxynivalenol in cereals and cereal products is controlled by recent legislation with the objective of minimizing consumer exposure to this mycotoxin. Relatively few studies have examined the loss of Fusarium mycotoxins during processing and whether this is accurately reflected by the processing factors. The behaviour of deoxynivalenol, nivalenol and zearalenone during extrusion of naturally contaminated wholemeal wheat flour has been examined using pilot-scale equipment. Factors examined were temperature and moisture content. Concentrations of the three mycotoxins were little changed by extrusion although the amount of deoxynivalenol decreased at the lowest moisture content. However, this effect did not appear to be temperature-dependent, suggesting that the apparent loss is either due to binding or inability to extract the residue. Under some conditions, concentrations of the mycotoxins, particularly nivalenol, were higher after extrusion.

Survey of ochratoxin A and deoxynivalenol in stored grains from the 1999 harvest in the UK

Three hundred and twenty samples from the 1999 UK harvest comprising wheat (201 samples), barley (106) and oats (13) were analysed for ochratoxin A and deoxynivalenol. A small number of organic samples was also obtained. Samples were collected from farms, central stores, mills, maltings and ports from across the UK from February to April 2000. Ochratoxin A and deoxynivalenol analysis was by affinity column clean up and high-performance liquid chromatography with fluorescence and ultraviolet light detection, respectively, with limits of detection of 0.2 and 20 μg kg−1. The survey found ochratoxin A at below 5 μg kg−1 in 97% of the samples indicating satisfactory storage conditions. The remaining 3% of the samples contained ochratoxin A at levels between 5.2 and 231 μg kg−1, but none of these samples was intended for human consumption. Deoxynivalenol was detected in 88% of all samples, with 83% below 100 μg kg−1; the maximum level was 600 μg kg−1. Twenty samples containing deoxynivalenol at or above 150 μg kg−1 by high-performance liquid chromatography were all confirmed by gas chromatography/mass spectrometry. Nivalenol was also detected by gas chromatography/mass spectrometry at levels of 50 μg kg−1 or higher in 18 of 20 samples where deoxynivalenol was confirmed.

Fate of Fusarium mycotoxins in maize flour and grits during extrusion cooking

Extrusion technology is used widely in the manufacture of a range of breakfast cereals and snacks for human consumption and animal feeds. To minimise consumer exposure to mycotoxins, the levels of deoxynivalenol (DON) and zearalenone (ZON) in cereals/cereal products and fumonisins B1 and B2 (FB1 and FB2) in maize are controlled by European Union legislation. Relatively few studies, however, have examined the loss of Fusarium mycotoxins during processing. The behaviour of FB1, FB2 and fumonisin B3 (FB3), DON and ZON during extrusion of naturally contaminated maize flour and maize grits is examined using pilot-scale equipment. DON and ZON are relatively stable during extrusion cooking but the fumonisins are lost to varying degrees. There is some loss of ZON when present in low concentrations and extruded at higher moisture contents. The presence of additives, such as reducing sugars and sodium chloride, can also affect mycotoxin levels. Moisture content of the cereal feed during extrusion is important and has a greater effect than temperature, particularly on the loss of fumonisins at the lower moistures. The effects are complex and not easy to explain, although more energy input to the extruder is required for drier materials. However, on the basis of these studies, the relationship between the concentration of Fusarium toxins in the raw and finished product is toxin- and process-dependent.

Statistical modelling as an aid to the design of retail sampling plans for mycotoxins in food

A study has been carried out to assess appropriate statistical models for use in evaluating retail sampling plans for the determination of mycotoxins in food. A compound gamma model was found to be a suitable fit. A simulation model based on the compound gamma model was used to produce operating characteristic curves for a range of parameters relevant to retail sampling. The model was also used to estimate the minimum number of increments necessary to minimize the overall measurement uncertainty. Simulation results showed that measurements based on retail samples (for which the maximum number of increments is constrained by cost) may produce fit-for-purpose results for the measurement of ochratoxin A in dried fruit, but are unlikely to do so for the measurement of aflatoxin B1 in pistachio nuts. In order to produce a more accurate simulation, further work is required to determine the degree of heterogeneity associated with batches of food products. With appropriate parameterization in terms of physical and biological characteristics, the systems developed in this study could be applied to other analyte/matrix combinations.

Chapter 2:Natural Occurrence of Masked Mycotoxins

This chapter describes recent knowledge of the occurrence of the major masked mycotoxins, principally those derived from deoxynivalenol, zearalenone and fumonisin. The occurrence of several minor masked mycotoxins is described and summaries of quantitative data from some large-scale surveys are provided. Some factors that might influence the formation of masked mycotoxins are discussed.