Analytical methods mycotoxins

Tests for mycotoxin contamination can be accomplished both on the finished product and on the raw form. The latter case prevents the manufacture of an unfit product, but it often implies trouble in the evaluation of the contamination in a batch. In this case, for a defined sample size, sample preparation, and analytical method, principles are available to evaluate the accuracy of the aflatoxin determination, depending on the availability of an accurate estimate of the variability associated with each step of the analytical sequence (11,12). A valuable effort to estimate the uncertainty of the analytical sequence as a whole was carried out by the FAO (13). 

The working group CEN TC 275/WG 5 searched for performance criteria to be used in mycotoxin analysis and came up with a document reporting the criteria for the selection of methods (26). The criteria deal with limits of detection, minimum performance characteristics, extraction solvents, and applicability. Criteria for analytical methods in mycotoxin analysis are also included in Directive 98/53/EC (18). 

An excellent review of methods in mycotoxin analysis was published by the FAO (29), and yearly updates on analytical methods are provided by the General Referee Report, Committee on Natural Toxins, published in the Journal of AO AC International. 

European Committee for Standardization CEN/TC 275/WG5. Criteria of Analytical Methods for Mycotoxins (N. 93). Delft The Netherlands, Dutch Standardization Institute, 1993. 

In our research, we proposed a post amplification analytical method to detect F. culmorum, a pathogen causing foot rot and head blight diseases in cereals, and can produce mycotoxins such as zearalenone, deoxynivalenol, and other trichothecenes that can enter the food chain. The early identification of this fungal pathogen is therefore recommended in order to avoid crop losses and protect consumer health... 

Work Item L criteria of analytical methods for mycotoxins - CEN-Report. 

Analytical methods for mycotoxins have historically utilised chemical detection methods which are easily quantifiable, highly sensitive, less subject to interference by co-extractives but can be quite time demanding. Most quantitative methods use a final chromatographic separator followed by a suitable detection... 

Mycotoxins are poorly concentrated in many tissues or biological fluids (e.g., muscle or milk) and the analytical method employed should be sensitive enough to detect down to a few micrograms mycotoxins per kilogram sample or even less (aflatoxins in muscle samples, for instance [2]). 

European Committee for Standardisation, CEN (1999) Food analysis— biotoxins—criteria of analytical methods for mycotoxins, Report Reference no. CR 13505 1999E... 

The assoeiation of fungi with mammalian diseases came to the fore in 1960 with the diseovery of the aflatoxins (1.36). The death of turkeys from liver damage having been fed on groundnuts eontaminated with Aspergillus flavus led to the isolation of the highly carcinogenic aflatoxins. These developments are discussed in Chapter 9. It led to the awareness of the potential human health hazards from microbial metabolites and the implications of the presence of other mycotoxins in foodstuffs such as patulin in apple juice and the tri-chothecenes on corn. The development of analytical methods for the detection of mycotoxins has become an important aspect of food science. 

The separation of trichothecene mycotoxins from biological materials by UV absorption or fluorescence absorption is difficult, but the most suitable analytical methods are gas chromatography mass spectrum analysis. Recently, radioimmunoassay and enzyme linked immunosorbant assay have been developed for T-2 toxin and diacetoxyscirpenol (DAS) and deoxyverrucarol, which are highly sensitive as compared to other biological and chemical methods. 

Bauer J. and Garcis M. (1989) Analytical methods for mycotoxins. DTW-Deutsche Herarztliche Wo-chenschrift., 96, 346-350. 

Screening tests for the trichothecene mycotoxins are generally simple and rapid but, with the exception of the immunochemical methods, are nonspecific. A number of bioassay systems have been used for the identification of trichothecene mycotoxins.73 Although most of these systems are very simple, they are not specific, their sensitivity is generally relatively low compared to other methods, and they require that the laboratory maintain vertebrates, invertebrates, plants, or cell cultures. Thin-layer chromatography (TLC) is one of the simplest and earliest analytical methods developed for myco-toxin analysis. Detection limits for trichothecene mycotoxins by TLC is 0.2 to 5 ppm (0.2 to 5 pg/ mL). Therefore, extracts from biomedical samples would have to be concentrated 10- to 1,000-fold to screen for trichothecene mycotoxins. 

The analytical procedure that is used by this laboratory for the analysis of simple Fusarium mycotoxins will be reported separately. However, the analytical scheme is outlined in Figure 2. The method is very arduous due to several sample clean-up steps which necessitates transfer of the sample between containers. The trichothecenes and their derivatives have a tendency to adhere to glass and can be quantitatively transferred only with numerous methanol washes. While the analytical method is both sufficiently sensitive and definitive for the program requirements, the sheer amount of human manipulation required for the completion of this analysis makes it somewhat unreliable if implemented without a responsible quality assurance and quality control program. 

Gilbert, J. Anklam, E. (2002) Validation of analytical methods for determining mycotoxins in foodstuffs. Trends Anal. Chem. 21,468-486. 

Turner NW, Subrahmanyam S, Piletsky SA (2009) Analytical Methods for Determination of Mycotoxins a Review. Anal Chim Acta 632 168... 

Major problems encountered in the analysis of mycotoxins arise because of their diversity of chemical structure and the wide range of foods and animal feeds in which some of them may occur. This often means the development of quite different analytical methods for each mycotoxin in each food... 

The specificity of monoclonal antibodies make it possible to develop an analytical method for a single mycotoxin, such as aflatoxin Bi in maize and groundnut meal, or aflatoxin Mj in milk and milk products. Even within chemically closely related structures such as the Fusarium trichothecenes there is very little cross-reactivity between a monoclonal raised to a single toxin such as T-2 toxin and other members of the family. Thus, a monoclonal raised against 3-acetyl-deoxynivalenol showed negligible cross-reactivity with deoxynivalenol, nivalenol, or T-2 toxin. 

Piletsky, S. A., Tumera, N. W., and Suhrahmanyamb, S. 2009. Analytical methods for determination of mycotoxins A review. Anal. Chim. Acta 632 168-180. 

Since NIV occurs as a co-contaminant with other trichothecene mycotoxins, it is often analyzed simultaneously with the co-contaminants rather than alone. Analytical methods developed so far include thin layer chromatography (TLC) capillary gas chromatography (GC) with electron-capture detection (BCD), flame ionization detection (FID), or mass spectrometric detection (GC/MS) high-performance liquid chromatography (HPLC) with ultraviolet (UV), fluorescence, or mass spectrometric detection supercritical fluid chromatography (SFC) and time-of-flight mass spectrometry (LC/TOF-MS). 

However, for surveillance studies, trichothecene mycotoxins coexist with other trichothecenes, and a simultaneous analytical method for the determination of some trichothecenes and Fusarium toxins is considered to be more practical than a single method. A decade ago, GC was very popular to analyze some trichothecene mycotoxins in food, but for GC analysis, various derivatives are needed that are sometimes troublesome. 

A selective analytical method based on HPLC, combined with atmospheric pressure photoionization (APPI) mass spectrometry, has been developed for the simultaneous determination of NTV and DON. A liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) method based on time-of-flight MS (TOF/MS) with a real-time reference mass correction technique was also developed for the simultaneous determination of Fusarium mycotoxins (NIV, DON, fusarenon-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, HT-2 toxin, T-2 toxin, diacetoxyscirpenol, ZEN) and Aspergillus mycotoxins (ailatoxin Bl, aflatoxin B2, aflatoxin Gl, aflatoxin G2) in com, wheat, cornflakes, and biscuits [112]. 

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