Trichothecenes analytical methods

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... 

Detection methods for T-2 toxin and other Fusarium toxins have been recently reviewed (Krska et al., 2007 Ler et al., 2006). Trichothecene analysis can be done by screening methods such as thin layer chromatography (TLC) and ELISA or analytical methods such as gas chromatography (GC) and high performance hquid chromatography (HPLC). GC instrumentation has been the most frequently used method for experimental work with trichothecenes. Newer methodologies, such as GC-MS and LC-MS, have an excellent lowest level of detection (LOD) of 5 ng/g for T-2 toxin in cereals and food, and wheat flour respectively (Ler et al., 2006). Improved sensitivity for... 

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. 

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. 

Recently, few topics in analytical chemistry have occupied the scientific community more than the ability of chemical laboratories to reliably determine at the low parts-per-billion level the presence of Fusarium trichothecenes in environmental and toxicological samples. This paper provides a systematic approach for developing and implementing a quality assurance and quality control program for a complex analytical method in which human error and system failure can occur. The application of this approach to the problem of determining the presence of nine naturally... 

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. 

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. 

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. 

Gas chromatography and capillary column GC represent by far the most developed and most used analytical techniques for the determination of multiple trichothecenes, especially in view of the possibility of employing MS for confirmation of the peaks. The available methods, such as the Scott and Ware methods (90,91), differ in sample extraction, sample cleanup, and the derivatization step. Heptafluorobutyryl (HFB), trimethylsilyl (TMS), and trifluoroacetyl derivatives are frequently used, coupled with electron capture detection. A GC method (92) has recently been collaboratively tested in barley and malt and accepted by the American Society of Brewing... 

Trichothecene mycotoxins are secondary metabolites of various fungal species. Structures of some trichothecene mycotoxins of interest to the US ARMY are given in Figure 1. Several methods have been reported for the analysis of these toxins (1-11, 15). Of these, mass spectrometry techniques are both sensitive and definitive when applied to toxicologic and environmental samples. With current technology, the most sensitive and qualitatively definitive analytical technique for the determination of these toxins is derivatization with an electron deficient moiety followed by analysis with negative ion chemical ionization gas chromatography-mass spectrometry (NICI-GC/HS). 

Sulyok et al. [113] reported the first validated method for the determination of 39 mycotoxins in wheat and maize by liquid chromatography with electrospray ionization-triple quadmpole mass spectrometry (LC/ESI-MS/MS) without the need for any cleanup. The 39 analytes included A and B trichothecenes (including deoxynivalenol-3-glucoside), ZEN and related derivatives, fumonisins, enniatins, ergot alkaloids, ochratoxins, aflatoxins, and moniliformin, and six trichothecene mycotoxins (NIV, DON, fusarenon-X, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, and T-2 toxin). 

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