Chromatography of Trichothecene Mycotoxins

Abstract

Trichothecene mycotoxins occur in agricultural commodities and can cause problems from feed refusal to death in animals. This paper describes chromatographic methods for selective analysis for trichothecene mycotoxins. These methods include gas chromatography (GC), thin layer chromatography (TLC), and high pressure liquid chromatography (HPLC). The trichothecene analysis methods by GC and TLC are shown to have a greater sensitivity than in HPLC for the underivatized mycotoxins.     

2-(Diphenylacetyl)-1,3-Indanedione-1-Hydrazone (Dipain) Derivatives for Detection of Trichothecene Mycotoxins

Abstract

2-(Diphenylacetyl)-1,3-indanedione-1-hydrazone and its derivatives where the NH₂ has been replaced by a substituted NH or an imino group give fluoresence enhancement with trichothecene mycotoxins. Absolute amounts of T-2 toxin as low as 50 ng are detectable. Detection limits for HT-2, Diacetoxyscirpenol, Neosolaniol, and Fusarenon-X range from 0.1 to 4μg.

The advantage in using 2-(diphenylacetyl)-1,3-indanedione-1-hydrazone (DIPAIN) derivatives for the detection of trichothecene mycotoxins is that response time is based on the rate of formation of a molecular association complex between the toxin and the detector reagent rather than on the chemical reactivity of the toxin. Hence, sensitive detection or mycotoxins can be achieved simply and rapidly at 25°C.

Since DlPAlN derivatives have been found to be direct-acting reagents capable of detecting droplets of dissolved trichothecenes, it is speculated that they may be useful as coatings in optical waveguide devices or in other devices that are developed as field detectors for aerosols that contain trichothecene mycotoxins. The DlPAlN reagents may also be used in kits that are designed to detect surface contamination by trichothecene mycotoxins.     

Thermospray high performance liquid chromatographic/mass spectrometric analysis of some fusarium mycotoxins

Thermospray high performance liquid chromatography/mass spectrometry (TSP HPLC/MS) was used to analyze five Fusarium mycotoxins in porcine plasma and urine. Four cytotoxic trichothecene mycotoxins, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), T2 tetraol, and the fungal estrogen zearalenone (F-2 toxin) were analyzed. The thermospray mass spectrum contained molecular weight information with few, if any, fragment signals. Detection limits ranging from 1 to 10 ng of mycotoxin injected onto the HPLC column were obtained using selected ion monitoring (SIM) HPLC/MS. Neither the plasma nor the urine matrix interfered with TSP HPLC/MS analysis of these mycotoxins and no sample derivatization was necessary for the analysis. The TSP HPLC/MS technique appears to be ideal for very sensitive analysis of mycotoxins in biological samples.     

Spectrophotometric and fluorimetric determinations of trichothecene mycotoxins with reagents for formaldehyde

Several trichothecene mycotoxins, such as fusarenone-X (F-X) and T-2 toxin (T-2), readily liberate formaldehyde on heating with sulfuric acid. Spectrophotometric and fluorimetric methods for the determination of trichothecenes with reagents for formaldehyde are therefore possible. F-X (or T-2) can be determined in the 50–1000 mg l^-1 (or 50–1500 mg l^-1) range by the chromotropic acid method, in the 30–1200 mg l^-1 (or 50–2000 mg l^-1) range by the phenyl J acid method, and in the 1.25–25 mg l^-1 (or 2–40 mg l^-1) range by the J acid method. Other trichothecenes, neosolaniol, nivalenol, tetraacetylnivalenol, diacetoxyscirpenol and HT-2 toxin, etc. also give positive reactions but trichothecin and dihydronivalenol do not.     

Identification and characterization of new Fusarium masked mycotoxins,T2 and HT2 glycosyl derivatives,in naturally contaminated wheat and oats by liquid chromatography–high‐resolution mass spectrometry

The presence of glucoside derivatives of T‐2 and HT‐2 toxins (type A trichothecene mycotoxins) in naturally contaminated wheat and oats is reported for the first time. The use of advanced high‐resolution mass spectrometry based on Orbitrap technology allowed to obtain molecular structure details by measuring exact masses of main characteristic fragments, with mass accuracy lower than 2.8 ppm (absolute value). A monoglucoside derivative of T‐2 toxin and two monoglucoside derivatives of HT‐2 toxin were identified and characterized. The analysis of their fragmentation patterns provided evidence for glucosylation at C‐3 position for T‐2 toxin and at C‐3 or C‐4 position for HT‐2 toxin. A screening for the presence of these new masked forms of mycotoxins was carried out on a set of naturally contaminated wheat and oats samples. On the basis of peak area ratio between glucoside derivatives and free T‐2 and HT‐2 toxins, the presence of glucoside derivatives was more likely in wheat than in oats samples. The present work confirms the widespread occurrence of trichothecene glucosides in cereal grains naturally contaminated with the relevant unconjugated toxins, thus suggesting the importance of developing suitable analytical methods for their detection. Besides toxicity studies, tracking down these new masked forms of trichothecenes along the food/feed chain would enable to collect information on their relevance in human/animal exposure to mycotoxin risk. Copyright © 2012 John Wiley & Sons, Ltd.     

New, sensitive thin-layer chromatographic-high-performance liquid chromatographic method for detection of trichothecene mycotoxins

Diphenylindenone sulphonyl (Dis) esters of trichothecene mycotoxins when sprayed with sodium methoxide showed fluorescent spots on a thin layer of silica gel when viewed under long-wavelength UV light. The detection limit for trichothecene esters in thin-layer chromatography (TLC) was 20-25 ng per spot for T-2 toxin, HT-2 toxin, diacetoxyscirpenol, T-2 triol, T-2 tetraol and iso-HT-2 toxin. A quantitative high-performance liquid chromatographic (HPLC) analysis of Dis-trichothecene esters was also developed using UV detection at 278 nm. The detection limit for the above esters varied between 30 and 50 ng per injection. This sensitive TLC-HPLC method is very useful for in vivo pharmacokinetic analyses of trichothecenes.     

Volatilization of trichothecene mycotoxins for analysis

T-2 toxin and diacetoxyscirpenol (DAS), two trichothecene mycotoxins containing one hydroxy group, have been volatilized by induction heating, revolatilized, and analyzed by gas chromatography (GC) and/or GC mass spectroscopy. Seventy to eighty percent of DAS was recovered by this system; 60–70% T-2 toxin was recovered. When the hydroxy group is derivatized by acetate, 90–100% recovery is obtained. Other trichothecenes of the macrocyclic ester type (e.g., Roridan A) were also tried. Ten to twenty percent of the macrocyclic ester was obtained without derivatization.     

N,N-dimethyl-trimethylsilyl-carbamate as a derivatizing agent in gas chromatography of trichothecene mycotoxins

N,N-dimethyl-trimethylsilyl-carbamate, a commercially available silylating agent, has been tested in the derivatization of trichothecenes for the first time. Its reaction with alcohols is an autocatalytic, non-equilibrium process with volatile by-products. As a consequence of these advantageous characteristics N,N-dimethyl-trimethylsilyl-carbamate proved to be easily utilizable and effective for the derivatization of all studied trichothecenes, namely deoxynivalenol (DON), nivalenol (NIV) and 4,15-diacetoxyscirpenol (DAS). During testing also, optimum conditions (temperature: 15 °C, reaction time: 35 min) were established for the trimethylsilylation and for the gas chromatographic determination of the above-mentioned three trichothecenes. The linearity of the detector response was found to be proper in the 5-1100 ng range. (Given in the grams of trichothecene equivalent to the derivatives injected). The limit of detection is less than 1 ng for each of the studied trichothecenes. Relative standard deviations of the peak heights ranged from 0.75 to 4.32%.     

Rapid and high resolution capillary supercritical fluid chromatography (SFC) and SFC/MS of trichothecene mycotoxins

The potential application of capillary column supercritical fluid chromatography (SFC) and SFC/mass spectrometry (SFC/MS) for the separation and analysis of mycotoxins of the trichothecene group was examined. Trichothecenes present significant analytical problems for both gas and liquid chromatography with a major difficulty for the latter being the lack of sufficiently sensitive and selective detectors. Supercritical carbon dioxide mobile phases at temperatures up to 100 degrees C were used with deactivated fused silica columns coated with crosslinked stationary phases. Separations were obtained under pressure ramped conditions using long (15 m) 50-micron i.d. columns for several trichothecenes (diacetoxyscirpenol, deoxynivalenol, and T-2 toxin) and related higher molecular weight macrocyclic (roridin and verrucarin) trichothecenes. In addition, new rapid pressure programming techniques with short (less than 2m) 25- to 50-micron i.d. capillary columns were used to obtain fast separations in as little as 1 min. SFC/MS with ammonia chemical ionization provided high selectivity and sensitive detection (with approximately 1-pg detection limits) for trichothecene mixtures. The extension to complex sample matrices is discussed and the application of selective MS/MS detection is demonstrated.     

Analysis of trace levels of trichothecene mycotoxins in Austrian cereals by gas chromatography with electron capture detection

Summary Various analytical methods developed for trichothecene determination, including TLC, HPLC, GC, supercritical fluid chromatography (SFC) and enzyme immuno assay (EIA) are reviewed. In addition a new method is described for the simultaneous determination of the trichothecene mycotoxins deoxynivalenol (DON), nivalenol (NIV), 3-acetyldeoxynivalenol (3-ADON), diacetoxyscirpenol (DAS), T-2 toxin (T-2), HT-2 toxin (HT-2) and T-2 triol (TRIOL), in Austrian wheat and corn samples by GC-ECD. A clean-up procedure has been developed using a combination of liquid-liquid and liquid-solid extraction. Trichothecenes were detected as their heptafluorobuturyl esters or alternatively as trimethylsilyl ethers (only sensitive for deoxynivalenol and nivalenol) using nandrolone or chloramphenicol as internal standard. Four derivatization techniques using HFBI, HFBA+DMAP on polystyrene, TMSI and TMSI+BSA+TMCS have been studied and the advantages and disadvantages of each are discussed. Quantification of trichothecenes from 10 to 1000 ppb in cereals could be accomplished routinely.Presented at the 19th ISC, Aix-en-Provence, France, September 13–18, 1992.     

Isolation,chemical characterization and hydrolysis of the trichothecene 7α-hydroxy, 15-deacetylcalonectrin (3ANX) from Fusarium graminearum DAOMC 242077

Fusarium graminearum and related species produce regulated mycotoxins that contaminate important food commodities resulting in serious agricultural and animal productivity losses. F. graminearum DAOMC 242077 was isolated from soft wheat in Eastern Canada and an investigation of this strains secondary metabolites confirmed it produced the recently discovered type-A trichothecene 7α-hydroxy, 15-deacetylcalonectrin (3ANX; 1). In an effort to prepare large amounts of its deacetylated product 7α-hydroxy, 3, 15-dideacetylcalonectrin (NX; 2), F. graminearum DAOMC 242077 was grown using conditions favoring the biosynthesis of trichothecenes to enable large amounts of 3ANX (1) to be purified. The structure of 3ANX (1) was confirmed by interpretation of spectroscopic data, physical properties and LC-HRMS. A simple hydrolysis scheme was developed to convert 3ANX (1), to NX (2). The objective of this study was to generate and chemically characterize 3ANX (1) and NX (2) reference materials for the scientific community for toxicology studies, analytical method development and exposure assessments.     

Electron ionization mass spectral fragmentation of deoxynivalenol and related tricothecenes

Careful analysis of the electron impact (EI) mass spectral data obtained for the trimethylsilyl (TMS) ethers of known trichothecene mycotoxins of the deoxynivalenol group permitted the construction of a database useful for the identification of these mycotoxins directly from a gas chromatography/mass spectrometry (GC/MS) run. Structures of the ions at m/z 103, 117, 147 and 191 were elucidated by high-resolution mass spectrometry (HRMS) and a fragmentation scheme was suggested. The relative abundances of these ions in the mass spectra of the trichothecenes allowed a fast structural diagnosis during analysis of biological matrices. A new mycotoxin of this group, 3-acetylnivalenol, was tentatively identified by using MS data interpretation only.     

A phage-displayed chicken single-chain antibody fused to alkaline phosphatase detects Fusarium pathogens and their presence in cereal grains

Fusarium and its poisonous mycotoxins are distributed worldwide and are of particular interest in agriculture and food safety. A simple analytical method to detect pathogens is essential for forecasting diseases and controlling mycotoxins. This article describes a proposed method for convenient and sensitive detection of Fusarium pathogens that uses the fusion of single-chain variable fragment (scFv) and alkaline phosphatase (AP). A highly reactive scFv antibody specific to soluble cell wall-bound proteins (SCWPs) of F. verticillioides was selected from an immunized chicken phagemid library by phage display. The antibody was verified to bind on the surface of ungerminated conidiospores and mycelia of F. verticillioides. The scFv–AP fusion was constructed, and soluble expression in bacteria was confirmed. Both the antibody properties and enzymatic activity were retained, and the antigen-binding capacity of the fusion was enhanced by the addition of a linker. Surface plasmon resonance measurements confirmed that the fusion displayed 4-fold higher affinity compared with the fusion's parental scFv antibody. Immunoblot analyses showed that the fusion had good binding capacity to the components from SCWPs of F. verticillioides, and enzyme-linked immunosorbent assays revealed that the detection limit of the fungus was below 10⁻² μg mL⁻¹, superior to the scFv antibody. The fusion protein was able to detect fungal concentrations as low as 10⁻³ mg g⁻¹ of maize grains in both naturally and artificially contaminated samples. Thus, the fusion can be applied in rapid and simple diagnosis of Fusarium contamination in field and stored grain or in food.     

Simultaneous Determination of Trichothecenes A, B, and D in Maize Food Products by LC–MS–MS

Two new methods of analysis, based on liquid chromatography–tandem mass spectrometry, for simultaneous determination of trichothecenes A, B, and D in maize flour and oil have been developed and validated in accordance with European Commission decision 2002/657/EC (recovery, CCα, CCβ, and precision). The trichothecenes were extracted from maize flour by matrix solid-phase dispersion, with recoveries ≥79%, and from maize oil by liquid–liquid extraction, with recoveries ≥78%. Limits of quantitation ranged between 0.03 and 50 μg kg^?1, depending on the electrospray response to each analyte and on the matrix. Monitoring of flour and oil samples with this HPLC–MS–MS method revealed the presence of deoxynivalenol, T-2 toxin, and diacetoxyscirpenol at very low concentrations.     

Regulations relating to mycotoxins in food

Regulations relating to mycotoxins have been established in many countries to protect the consumer from the harmful effects of these compounds. Different factors play a role in the decision-making process of setting limits for mycotoxins. These include scientific factors, for example the availability of toxicological data and occurrence data, detailed knowledge about possibilities for sampling and analysis, and socio-economic issues. By the end of 2003, approximately 100 countries (covering approximately 85% of the world’s inhabitants) had specific regulations or detailed guidelines for mycotoxins in food. The regulations were related to aflatoxins (B₁, B₂, G₁ and G₂), aflatoxin M₁, trichothecenes (deoxynivalenol, diacetoxyscirpenol, T-2 toxin and HT-2 toxin), fumonisins (B₁, B₂, and B₃), agaric acid, ergot alkaloids, ochratoxin A, patulin, phomopsins, sterigmatocystin, and zearalenone. In Europe, and in particular in the EU, regulatory and scientific interest in mycotoxins has undergone a development in the last decade from autonomous national activity towards more EU-driven activity with a structural and network character. Harmonized EU limits now exist for 40 mycotoxin–food combinations. It is expected this number will grow in 2007 to approximately 50. The direct or indirect influence of European organizations and programs on the EU mycotoxin regulatory developments is significant. They include the European Food Safety Authority, the Scientific Cooperation on Questions relating to Food, the Rapid Alert System for Food and Feed, the creation of an EU Community Reference Laboratory for Mycotoxins and a mandate of the EC to the European Standardization Committee in methods for analysis for mycotoxins in food. Large pan-European research and networking projects as “BioCop” and “MoniQA” are also important.     

Determination of type A trichothecenes by high-performance liquid chromatography with coumarin-3-carbonyl chloride derivatisation and fluorescence detection

A method for the analysis of type A trichothecenes T-2 toxin, HT-2 toxin, neosolaniol and diacetoxyscirpenol by high-performance liquid chromatography with fluorescence detection using coumarin-3-carbonyl chloride has been developed. Different parameters concerning the analytical procedure such as stability of both the reagent and derivatised analytes, time and temperature of the derivatisation reaction, were studied and optimised. Three different clean-up procedures (solid-phase extraction with silica gel or C-18 cartridges, and liquid–liquid partition between toluene and dihydrogen phosphate buffer) were tested in order to remove the excess reagent peaks. The last procedure gave the best results when the buffer pH was 3–5.5, and is therefore recommended. Separations were performed on a stainless steel LiChrospher 100 C-18 reversed-phase column with pre-column of the same phase. The mobile phase was acetonitrile/water (65:35, v/v) containing 0.75% acetic acid at a flow-rate of 1.0 ml/min. The proposed method provides good separation between the four trichothecenes and good reproducibility (RSD of calibration standards <5%). The limits of detection of the studied trichothecenes at a signal-to-noise ratio of 3:1, with an injection volume of 20 μl were 10 ng/g sample for T-2 toxin and about 15 ng/g sample for the remaining mycotoxins. The calibration curve was linear between 10 and 2000 ng for the four trichothecenes assayed. The method was applied to the analysis of these mycotoxins in fungal cultures (corn and rice) of Fusarium sporotrichioides, and is also perfectly suitable for the quantification of type A trichothecenes in contaminated cereals.     

Effect of Selected Microorganisms on Fusarium Toxins Production

The aim of this study was to examine the effect of selected microorganisms on mycotoxins production by molds of the genus Fusarium, namely HT-2 and T-2 toxins. Appropriate nutritive media were inoculated with test microorganisms (Rhodotorula spp., Leuconostoc spp., Pantoea agglomerans), subsequently inoculated with Fusarium molds, then incubated under various conditions. Content of Fusarium mycotoxins in individual samples was determined using HPLC/MS/MS. Separation of mycotoxins was performed on a C₁₈ stationary phase column using gradient elution. Total analysis time was less than 20 minutes. In examining the effect of accompanying microflora on the production of HT-2 and T-2 toxins, a decrease in production of both mycotoxins was observed under various experimental conditions. Greatest inhibitory effect was observed in the presence of Pantoea agglomerans CCM 298 bacteria. It was found that the amount of HT-2 and T-2 toxins produced by the examined mold strains also depends on cultivation conditions and the nutritive medium used.     

Species-specific optical genosensors for the detection of mycotoxigenic Fusarium fungi in food samples

Plant-pathogenic Fusarium species, Fusarium verticillioides and Fusarium proliferatum, are the major producers of fumonisins which are one of the most common mycotoxins found in maize. Herein, we report the development of specific and sensitive genosensors for detecting these two closely related Fusarium species in food samples. The sensors are based on species-specific capture and detection probes, which bind to the intergenic spacer region of rDNA (IGS). Oligonucleotide functionalized magnetic microbeads are used to capture the target DNA which is then detected using biotinylated detection probes and a streptavidin-coupled label. The developed genosensors had detection limits of 1.8 pM and 3.0 pM for F. proliferatum and F. verticillioides, respectively, using synthetic DNA targets. Furthermore, the biosensors were used to analyze natural fungal contamination of commercial maize samples. After amplification of the genomic DNA the sensors detected the presence of the fungi, in accordance with previous results obtained with PCR. No cross-reactivity between F. verticillioides and F. proliferatum, or other fungi species tested, was observed. The developed biosensors can provide a valuable tool to evaluate the potential for mycotoxin contamination in conditions where detection of mycotoxins directly is challenging.     

Use of Mycosep multifunctional clean-up columns for the determination of trichothecenes in wheat by electron-capture gas chromatography

A quick and reliable method is described for the determination of trichothecene mycotoxins employing gas chromatography with electron capture detection. Using the Mycosep® multifunctional clean-up columns the clean-up procedure is reduced to a 1-step extract purification, thus saving considerable time in comparison with classical clean-up methods, without sacrificing the purity of the extract. The method was tested and validated for five trichothecenes (deoxynivalenol, nivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol and fusarenon X). In addition, the performance of this new clean-up technique was compared with an already well established method.