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2-(Diphenylacetyl)-1,3-indanedione-1-hydrazone and its derivatives where the NH₂ has been replaced by a substituted NH or an imino group give fluoresence enhancement with trichothecene mycotoxins.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.     

Determination of T-2 Toxin in Traditional Chinese Herbal Medicines by GC-ECD

Gas chromatography with electron capture detection has been applied for the determination of T-2 toxin (T-2) in traditional Chinese herbal medicines (TCHM). The method consists of extracting the sample with aqueous methanol followed by cleanup of the resulting extract with an immunoaffinity column. T-2 was determined as its heptafluorobutyl ester. The reaction temperature and time of derivatization were investigated to obtain the optimum conditions. Recoveries from different TCHMs, spiked with T-2 at levels ranging from 50 to 1,000 μg kg⁻¹, were from 82.2 to 98.6%, with relative standard deviations of less than 7.5%. The limit of detection was 2.5 μg kg⁻¹. Out of 32 commercially available TCHM samples analyzed, none were found to contain any detectable amount of T-2.

     

Bestimmung von T-2-Toxin in pflanzlichen Nahrungsmitteln

Zusammenfassung Ein Verfahren zur quantitativen Bestimmung von T-2-Toxin in verschimmeltem Reis bzw. Mais durch Extraktion, dünnschicht-chromatographische Trennung und Fluorescenzintensitätsmessung der mit H₂SO₄ behandelten DC-Platte wird vorgestellt. Fluorescenzabsorptions- und Fluorescenzemissionsspektrum werden angegeben. Die Abhängigkeit der Mycotoxinkonzentration von der Fluorescenzintensität wird durch eine Gleichung beschrieben. Die analytische Detektion von T-2-Toxin läßt sich durch ein 4-(p-Nitrobenzyl)pyridin-Derivat und dessen Absorptionsspektrum bestätigen. Versuche mit zugesetztem T-2-Toxin zeigten einen Analysenfehler von weniger als 10%.

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Determination of T-2-toxin in vegetable foodstuffs1. T-2-toxin in mouldy rice and maize

Summary A procedure is described for the determination of T-2 toxin in extracts from mouldy rice and maize by thin-layer chromatography (TLC) and subsequent fluorescence intensity measurement of the H₂SO₄-treated TLC plates. The fluorescence absorption and emission spectra are presented. The dependence of the concentration of the mycotoxin on fluorescence intensity is described by an equation. The detection of T-2 toxin can be confirmed by colour reaction with 4-(p-nitrobenzyl)pyridine yielding a blue derivative. Experiments with added toxin revealed an analytical error smaller than 10%.

     

Gas chromatographic assay with pharmacokinetic applications for monitoring T-2 and HT-2 toxins in plasma

A gas-liquid chromatographic (GLC) method for monitoring T-2 and HT-2 toxins in plasma was developed. The procedure involved extraction of the toxins with ethyl acetate, chromatography on a C18 reversed-phase column and derivatization with heptafluorobutyric anhydride (HFBA). The T-2 and HT-2 HFBA derivatives were chromatographed on OV-17 at various temperatures and measured with an electron-capture detector. Iso-T-2 toxin and iso-HT-2 toxin were used as internal standards. Recoveries averaged 95.1 +/- 8.6% for T-2 toxin and 102.1 +/- 5.2% for HT-2 toxin at levels ranging from 40 to 120 ng/ml. The limits of detection were 30 and 5 ng/ml of T-2 and HT-2 toxin, respectively. The range of the assay covers plasma concentrations at which toxicity becomes manifest. The pharmacokinetic application of this GLC method is illustrated by simultaneous monitoring of T-2 and HT-2 toxins levels in plasma obtained after intravenous administration of T-2 toxin to a dog.     

Determination of T-2 Toxin and HT-2 Toxin in Milk: A Comparison of Three Formats of Immunoassays

This research paper presents a comparative study of three formats of immunoassays, including conventional enzyme-linked immunosorbent assay (ELISA), one-step ELISA, and reverse enzyme-linked immunosorbent assay (reELISA), for the determination of T-2 toxin (T-2) and HT-2 toxin (HT-2). This comparative study was performed with regard to specificity, sensitivity, matrix effect, accuracy, and precision. Among the three procedures, reELISA exhibited the best sensitivity with limits of detection (LOD) at 3.04 and 2.79 ng mL^?1 for T-2 and HT-2. For the other parameters, there were no apparent differences. Good recoveries in negative spiked samples at concentrations of 50, 200, and 500 ng mL^?1 were obtained, and the values were 67.9–112.9% for conventional ELISA, 60.8–108.8% for one-step ELISA, and 62.7–88.1% for reELISA. The relative standard deviation (RSD) for each procedure was less than 10.3%, 14.0%, and 11.5%, respectively. Among all three methods, the one-step protocol was the most time efficient, and the reELISA procedure exhibited the best sensitivity.     

A rapid fluorescence polarization immunoassay for the determination of T-2 and HT-2 toxins in wheat

A rapid fluorescence polarization (FP) immunoassay has been developed for the simultaneous determination of T-2 and HT-2 toxins in naturally contaminated wheat samples. Syntheses of four fluorescein-labelled T-2 or HT-2 toxin tracers were carried out and their binding response with seven monoclonal antibodies was evaluated. The most sensitive antibody-tracer combination was obtained by using an HT-2-specific antibody and a fluorescein-HT-2 tracer. The developed competitive FP immunoassay in solution showed high cross-reactivity for T-2 toxin (CR% = 100%) while a very low CR% for neosolaniol (0.12%) and no cross-reactivity with other mycotoxins frequently occurring in wheat. A rapid extraction procedure using 90% methanol was applied to wheat samples prior to FP immunoassay. The average recovery from spiked wheat samples (50 to 200 μg kg⁻¹) was 96% with relative standard deviation generally lower than 8%. A limit of detection of 8 μg kg⁻¹ for the combined toxins was determined. Comparative analyses of 45 naturally contaminated and spiked wheat samples by both the FP immunoassay and high-performance liquid chromatography/immunoaffinity clean-up showed a good correlation (r = 0.964). These results, combined with the rapidity (10 min) and simplicity of the assay, show that this method is suitable for high throughput screening as well as for quantitative determination of T-2 and HT-2 toxins in wheat.     

Enzyme-linked immunosorbent assay for the determination of T-2 toxin in cereals and feedstuff

A reliable enzyme-linked immunosorbent assay method was developed for the assay of T-2 toxin in cereals and feedstuff. A hapten of the T-2 toxin was synthesized, and a polyclonal antibody with high affinity and specificity was obtained after immunization of rabbits. Compared to the other ELISA methods, the assay is simple, rapid and affordable. The concentration of T-2 causing 15% inhibition is 0.01?±?0.001 ng mL^?1, which makes the method more sensitive than others. The cross-reactivity against other mycotoxins is low, except for the HT-2 toxin. Sample extraction was achieved within 3 min. The recoveries from samples including barley, wheat, corn, oat, rice and feedstuff were between 75% and 102%, and the detection limit for T-2 toxin was lower than 4 ng g^?1. The method was validated by high-performance liquid chromatography with tandem mass spectrometry detection.     

Determination of type A and type B trichothecenes in paprika and chili pepper using LC-triple quadrupole-MS and GC-ECD

There is a need to develop sensitive and accurate analytical methods for determining deoxynivalenol (DON), HT-2 toxin and T-2 toxin in paprika to properly assess the relevant risk of human exposure. An optimized analytical method for determination of HT-2 toxin and T-2 toxin using capillary gas chromatography with electron capture detection and another method for determination of DON by liquid chromatography-mass spectrometry in paprika was developed. The method for determination of HT-2 toxin and T-2 toxin that gave the best recoveries involved extraction of the sample with acetonitrile-water (84:16, v/v), clean-up by solid-phase extraction on a cartridge made of different sorbent materials followed by a further clean-up in immunoaffinity column that was specific for the two toxins. The solvent was changed and the eluate was derivatized with pentafluoropropionic anhydride and injected into the GC system. The limits of detection (LOD) for T-2 and HT-2 toxins were 7 and 3 μg/kg, respectively, and the recovery rates for paprika spiked with 1000 μg toxin/kg were 71.1% and 80.1% for HT-2 and T-2 toxins, respectively. For DON determination, the optimized method consisted of extraction with acetonitrile-water (84:16, v/v) solution followed by a solid-phase extraction clean-up process in a cartridge made of different sorbent compounds. After solvent evaporation in N₂ stream, the residue was dissolved and DON was separated and determined by LC-MS/MS. The LOD for this method was 14 μg DON/kg paprika sample and the DON recovery rate was 86.8%.     

Analysis of T-2 and HT-2 toxins in cereal grains by immunoaffinity clean-up and liquid chromatography with fluorescence detection

A sensitive, precise and accurate method has been developed for the simultaneous determination of T-2 and HT-2 toxins in cereal grains at ppb levels using high-performance liquid chromatography (HPLC) with fluorescence detection and 1-antroylnitrile (1-AN) as labeling reagent after immunoaffinity clean-up. Cereal samples were extracted with methanol/water (90:10, v/v), and the extracts were cleaned-up through commercially available immunoaffinity columns containing monoclonal anti-T-2 antibodies (T-2 test HPLC, Vicam). T-2 and HT-2 toxins were quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 381 nm, emission wavelength 470 nm) after derivatization with 1-AN. The monoclonal antibody showed 100% cross-reactivity with both T-2 and HT-2 toxin, and the immunoaffinity column clean-up was effective up to 1.4 microg of both toxins. The method was successfully applied to the analysis of T-2 and HT-2 toxins in wheat, maize and barley. Recoveries from spiked samples with toxin levels from 25 to 500 microg/kg ranged from 70% to 100%, with relative standard deviation generally lower than 8%. The limit of detection of the method was 5 microg/kg for T-2 toxin and 3 microg/kg for HT-2 toxin, based on a signal-to-noise ratio 3:1. HT-2 toxin was detected in ten naturally contaminated wheat samples out of 14 samples analyzed, with toxin levels ranging from 10 to 71 microg/kg; three of them contained also T-2 toxin up to 12 microg/kg.     

Characterization of (<Superscript>13</Superscript>C<Subscript>24</Subscript>) T-2 toxin and its use as an internal standard for the quantification of T-2 toxin in cereals with HPLC–MS/MS

In this paper, the structure and the identity of fully ¹³C-substituted T-2 toxin were confirmed using high-resolution mass spectrometry, ¹H-NMR, ¹³C-NMR, tandem mass spectrometry and HPLC–DAD. The purity of this compound was estimated to be at least 98.8% according to UV data. The isotopic distribution of (¹³C₂₄) T-2 toxin indicated a total isotopic enrichment of 98.2 ± 1.0 atom% ¹³C, and the application of different MS measurement modes revealed the MS/MS fragmentation pattern of T-2 toxin. Furthermore, a stable isotope dilution mass spectrometry method for the quantification of T-2 toxin was developed using (¹³C₂₄) T-2 toxin as internal standard. The method was evaluated with and without conventional clean-up and validated for maize and oats. Both cereals showed strong matrix enhancement effects, which could be compensated for through the application of the isotope-substituted internal standard.     

免疫亲和柱净化/柱前衍生化-高效液相色谱荧光检测法测定粮谷中的T-2毒素

建立了免疫亲和柱净化/柱前衍生化-高效液相色谱荧光检测器测定粮谷中T-2毒素含量的方法。样品经甲醇-水(体积比为80∶20)混合溶剂提取,通过免疫亲和柱(IAC)净化,以氰酸蒽(1-AN)为衍生化试剂、4-二甲基氨基吡啶(DMAP)为催化剂进行衍生,以ZORBAX Eclipse XDB-C18 柱为分离柱,乙腈-水(体积比为80∶20)为流动相进行高效液相色谱分离及荧光检测，荧光检测的激发波长为381 nm，发射波长为470 nm。T-2毒素的质量浓度为0.01~1.5 mg/L时与峰高呈良好的线性,相关系数为0.9985。在0.01~1.5 μg/g添加水平下，回收率为79.7%~94.5%,相对标准偏差小于7%；检出限（S/N＝3）为0.01 μg/g。该方法净化效果好,灵敏度高,操作简便快速。     

Determination of deoxynivalenol, T-2 and HT-2 toxins in a bread model food by liquid chromatography-high resolution-Orbitrap-mass spectrometry equipped with a high-energy collision dissociation cell

A sensitive and accurate method employing a single stage high resolution mass spectrometer equipped with a high-energy collision-dissociation cell (HCD) for the simultaneous determination of deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT-2) in a processed bread model food has been developed. Two sample pre-treatment routes for the extraction of these mycotoxins were investigated, based on Mycosep^® column clean up or QuEChERS-like procedure, respectively. The former approach suffered less from matrix effects and allowed to achieve in bread samples LODs of 7, 12 and 17 ng/g for T-2, HT-2 and DON, respectively, with 0.5 ppm mass accuracy. Two acquisition modes, full scan MS and all ion fragmentation, exploiting the fragmentation features offered by an HCD chamber and integrated within the Orbitrap analyser, were compared for quantitative purposes. The method was applied to investigate the degradation of these mycotoxins during bread processing using a bread model food. Most T-2 hydrolyzed to HT-2 during dough preparation, and about 20–30% of HT-2 and DON was degraded during bread baking.     

Determination of trichothecenes in cereals by gas chromatography with ion trap detection

Summary A method for determination of the trichothecene toxins, deoxynivalenol, 3α-acetyl-deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin and diacetoxyscirpenol in cereals (wheat, barley, oats, corn) is described. Extraction was performed according to Tanaka et al. (J. Chromatogr.328, 271 (1985)) [33], derivatization by trifluoroacylation with trifluoroacetic acid anhydride. For quantitation and confirmation a capillary gas chromatograph combined with a selective mass detector (ion trap) working in CI-mode with methanol as reagent gas was used. The quantitation limit for the complete method is 1–5 μg/kg, depending on the chemical characteristics of each toxin and cleanness of the extracts. Recoverics from spiked cereals were 78–89%.     

Use of the modified quick easy cheap effective rugged and safe sample preparation approach for the simultaneous analysis of type A- and B-trichothecenes in wheat flour

A suitable extraction and purification method for the simultaneous liquid chromatography–mass spectrometry (LC–MS) determination of five mycotoxins, three type A, diacetoxyscirpenol (DAS), T-2 toxin (T-2) and HT-2 toxin (HT-2), and two type B-trichothecenes, deoxynivalenol (DON) and nivalenol (NIV), has been optimised using a modified “Quick Easy Cheap Effective Rugged and Safe” (QuEChERS) method. Different solvents were studied in the extraction procedure to obtain better recoveries, which ranged from 86 to 108%, using a 85/15 (v/v) mixture of methanol/acetonitrile. The values obtained for recovery, repeatability and reproducibility of the optimized method are in agreement with Commission Directive 2005/26/EC for methods of analysis of Fusarium toxins. Finally, this optimized procedure was applied in wheat flour samples commercialized in Spain.     

Analysis of trichothecene mycotoxins in human blood by capillary column gas chromatography-ammonia chemical ionization mass spectrometry

Capillary column gas chromatography-ammonia chemical ionization mass spectrometry was found to be an excellent technique for the trace detection and identification of underivatized trichothecene mycotoxins. Abundant (M + H)+ and/or (M + NH4)+ pseudo-molecular ions were observed for T-2 toxin, HT-2 toxin, T-2 triol, diacetoxyscirpenol, deoxynivalenol and verrucarol under the conditions developed. This method was successfully applied to the analysis of human blood samples spiked with mycotoxins in the 0-500 ng/g range during a recent interlaboratory exercise. T-2 toxin and diacetoxyscirpenol were detected in these samples in the 2-180 ng/g range. Detection limits of 0.7 and 3.6 ng/g for T-2 toxin and diacetoxyscirpenol, respectively, were possible owing to the specificity of the method.     

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.     

T-2 Toxin—The Most Toxic Trichothecene Mycotoxin: Metabolism,Toxicity, and Decontamination Strategies

Among trichothecenes, T-2 toxin is the most toxic fungal secondary metabolite produced by different Fusarium species. Moreover, T-2 is the most common cause of poisoning that results from the consumption of contaminated cereal-based food and feed reported among humans and animals. The food and feed most contaminated with T-2 toxin is made from wheat, barley, rye, oats, and maize. After exposition or ingestion, T-2 is immediately absorbed from the alimentary tract or through the respiratory mucosal membranes and transported to the liver as a primary organ responsible for toxin''s metabolism. Depending on the age, way of exposure, and dosage, intoxication manifests by vomiting, feed refusal, stomach necrosis, and skin irritation, which is rarely observed in case of mycotoxins intoxication. In order to eliminate T-2 toxin, various decontamination techniques have been found to mitigate the concentration of T-2 toxin in agricultural commodities. However, it is believed that 100% degradation of this toxin could be not possible. In this review, T-2 toxin toxicity, metabolism, and decontamination strategies are presented and discussed.     

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.     

Determination of T-2 toxin in cereal grains by liquid chromatography with fluorescence detection after immunoaffinity column clean-up and derivatization with 1-anthroylnitrile

1-Anthroylnitrile (1-AN) has been shown to be an efficient labelling reagent for the determination of T-2 toxin (T-2) by high-performance liquid chromatography (HPLC)-fluorescence detection. This reaction has been used to develop a sensitive, reproducible and accurate method for the determination of T-2 in wheat, corn, barley, oats, rice and sorghum. The method uses immunoaffinity columns containing antibodies specific for T-2 for extract clean-up, pre-column derivatization with 1-AN and HPLC with fluorescence detection for toxin determination. Ground cereal samples were extracted with methanol-water (80:20, v/v), the extracts were purified by immunoaffinity columns and the toxin was quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 381 nm, emission wavelength 470 nm) after derivatization with 1-AN. Recoveries from the different cereals spiked with T-2 at levels ranging from 0.05 to 1.5 microg/g were from 80 to 99%, with relative standard deviations of less than 6%. The limit of detection was 0.005 microg/g, based on a signal-to-noise ratio of 3:1.     

High-Performance Liquid Chromatography of Domoic Acid,a Marine Neurotoxin,with Application to Shellfish and Plankton

Abstract

A recent outbreak of poisoning resulting from the consumption of cultured blue mussels (Mytilus edulis L.) from a localized area in Eastern Canada has been attributed to the presence of domoic acid (1), a relatively rare neurotoxic amino acid, previously found only in some algae of the family Rhodomelaceae. Studies on aqueous extracts of shellfish tissue indicated that the toxin and several of its isomers could be separated (and isolated in sufficient amounts for subsequent structural identification) by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) diode array detection (DAD). Aqueous acetonitrile containing 0.1% v/v trifluoroacetic acid was used as mobile phase. As the retention time and characteristic UV absorption spectrum of 1 (λ_max = 242 nm) permit unequivocal identification, the HPLC-DAD procedure was refined with a microbore column to provide a rapid (5 min), sensitive (0.3 ng detection limit) and reproducible assay method for the determination of 1 in shellfish tissue. Extraction was accomplished by boiling homogenized shellfish tissue for 5 min with distilled water. Extracts were taken through an octadecylsilica solid phase extraction clean-up prior to HPLC. This method has been applied to a variety of shellfish and phytoplankton samples.

BRIEF

Reversed-phase HPLC with ultraviolet diode array detection was used to analyze shellfish tissue and phytoplankton extracts for domoic acid. A rapid (5 min) and sensitive (0.3 ng detection limit) assay is presented.