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.     

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.     

Metabolism of three trichothecene mycotoxins, T-2 toxin, diacetoxyscirpenol and deoxynivalenol, by bovine rumen microorganisms

The three trichothecene mycotoxins T-2 toxin, diacetoxyscirpenol (DAS) and deoxynivalenol (DON) were incubated in vitro for 12, 24 and 48 h with rumen microorganisms obtained from a fistulated dairy cow. Gas chromatographic and gas chromatographic-mass spectrometric analyses of extracts indicated all three toxins were biotransformed to a variety of deepoxy and deacylated products. DON was partially converted to a product identified as deepoxy DON. DAS was rapidly converted to four products including 15-monoacetoxyscirpenol (MAS), scirpentriol and two new compounds identified as 15-acetoxy-3 alpha,4 beta-dihydroxytrichothec-9,12-diene (deepoxy MAS) and 3 alpha,4 beta,15-trihydroxytrichothec-9,12-diene (deepoxy scirpentriol). T-2 toxin was also completely biotransformed to the products HT-2, T-2 triol and two new metabolites identified as 15-acetoxy-3 alpha,4 beta-dihydroxy-8 alpha-(3-methylbutyryloxy) trichothec-9,12-diene (deepoxy HT-2) and 3 alpha,4 beta,15-trihydroxy-8 alpha-(3-methylbutyryloxy)trichothec-9,12-diene (deepoxy T-2 triol).     

Determination of trichothecenes in cereals

An effective method for the determination of seven trichothecenes-deoxynivalenol (DON), nivalenol (NIV), T-2 tetraol (T-24), fusarenon-X (FUS-X), diacetoxyscirpenol (DAS), T-2 toxin (T-2), HT-2 toxin (HT-2) in cereals is presented. Gel permeation chromatography on Bio-Beads S-X3 was used for clean-up of acetonitrile-methanol extract. GC-ECD was used for identification and quantification of trifluoroacetylated trichothecenes. The limit of quantitation for the method was in the range 40-200 micrograms/kg. Recoveries at a spiking level of 2 mg/kg ranged from 76 to 100%.     

Conversions of 2-thioxo-1,3-dithiol-4,5-dicarboxylic acid dimethyl ester — The path to a new heterocyclic system, (4H,6H)-1,3-dithiolo[4,5-d]pyrimidine-2,5,7-trione and 5,6-dimercaptouracil derivatives

The conversions of 2-thioxo-1,3-dithiol-4,5-dicarboxylic acid methyl ester into anhydride, mono- and diamide, and monoamide methyl ester were demonstrated. The new heterocyclic system, (4H,6H)-1,3-dithiolo[4,5-d]pyrimidine-2,5,7-trione, was obtained by oxidative rearrangement-cyclization of 2-oxo-1,3-dithiol-4,5-dicarboxylic acid diamide with phenyliodosyl(hydroxy)tosylate and used to synthesize previously unknown 5,6-dimercaptouracil derivatives, including nickel dithiolene complex.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 9, pp. 1270–1275, September, 1992.     

Chemical Composition of the Needles Oil of <Emphasis Type="Italic">Pinus canariensis</Emphasis> from Algeria

The essential oil of the needles of Pinus canariensis, cultivated in the Algiers region (Algeria), was obtained by hydrodistillation in a yield of 0.3% and analyzed by capillary GC and GC/MS. More than 46 compounds were identified amounting to ca 92.6% of the total oil. The more important constituents were β-selinene (63.7%), β-caryophyllene (9.9%), and limonene (3.5%).__________Published in Khimiya Prirodnykh Soedinenii, No. 2, pp. 134–135, March–April, 2005.     

Stereochemistry of nitrogen heterocycles. 62. Conformational analysis of isomers of 2-methyl-cis-decahydro-5-quinolinol

Isomers of 2-methyl- and 1,2-dimethyl-cis-decahydro-5-quinolinol with a syn orientation of the hydroxy and amino groups and different orientations of the methyl group relative to the methylene group at C₍₈₎H₂ were subjected to conformational analysis. In the case of a cis orientation of the methyl and methylene groups the equilibrium is shifted completely to favor the conformation with an intramolecular hydrogen bond, whereas in the case of their trans orientation the mole fraction of this conformation amounts to 21–24% for the secondary amino alcohol and 18–21% for the tertiary amino alcohol. The energies of the hydrogen bonds were determined from the intensities of the absorption bands of the free and associated hydroxy groups in the IR spectra: for the secondary hydroxy amine, according to the band of the free hydroxy group, G⁰ _OH/N is –0.8 kcal/mole, whereas, according to the band of the associated hydroxy group, it is –0.9 kcal/mole; the values for the tertiary hydroxy amine are, respectively, –0.7 and –0.8 kcal/mole.See [1] for Communication 61.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 11, pp. 1514–1519, November, 1987.     

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%.     

A conformational analysis of the methyl ester of O-acetyl-L-lactyl-L-alanine and the methylamide of N-acetyl-L-alanyl-L-lactic acid

Summary 1. The most preferred extended conformations for Ac-L-Lac-L-Ala-OMe and Ac-L-Ala-L-Lac-NHMe in a polar medium are R-R, R-B, B-R, and B-B. Of the conformations with a hydrogen bond of the 1–4 type, the most stable are R-R and R-B.2. There is an interrelationship between the conformational states of neighboring hydroxy and amino acid residues.3. The stereochemical characteristics of the ester group are similar to those of the amide group.M. M. Shemyakin Institute of the Chemistry of Natural Compounds, Academy of Sciences of the USSR. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 191–194, March, 1971.     

Pyrroles from ketoximes and acetylene. 19. Regioselectivity of the reaction of alkyl benzyl ketoximes with acetylene

Alkyl benzyl ketoximes react with acetylene at 60–150 °C in MOH-dimethyl sulfoxide (M = Li, K) primarily by means of the methylene group of the benzyl grouping, which is primarily anti-oriented with respect to the hydroxy group, to give 2-alkyl-3-phenylpyrroles in greater than 70% yields. The specific participation of the antimethylene group in the construction of a pyrrole ring constitutes evidence against mechanisms that include a [3,3]-sigmatropic shift in O-vinyl oximes. It follows from the results obtained that the reaction proceeds through an anti-dianion of the benzyl type stabilized by conjugation with the aromatic ring.See [1] for communication 18.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 2, pp. 193–198, February, 1982.     

Carbon dioxide extract from woody verdure of the Scotch pine. Group composition and acids

The group and individual compositions of the acids of an extract from the woody verdure of Scotch pine obtained on a pilot plant have been studied. The yield of extract amounted to 4.0% on the weight of the raw material, and 88% of the extract dissolved in petroleum ether. The petroleum-ether-soluble substances of the extract contained 32.8% of free acids and 55.4% of neutral substances. The compositions of the higher fatty acids, the diterpene acids, and the bicyclic acids have been determined. Pinifolic acid and its monomethyl ester, 18-acetoxy- and 18-hydroxyanticopalic acids, an acid of the 4-epiimbricatic series, and oxo and hydroxy acids of the abietic type have been isolated and identified.S. M. Kirov Leningrad Academy of Wood Technology. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 529–534, July–August, 1988.     

O-acylated lignans from the wood of Abies species

Summary From the wood of the Siberian firAbies sibirica Ledeb. and the Khingan firA. nephrolepis Maxim., five new compounds have been isolated which are ester derivatives of the lignans lariciresinol, olivil, and hydroxymatairesinol.It has been established that olivil is esterified at the primary alcoholic hydroxyl by hydroxycinnamic acids — p-coumaric or ferulic — and lariciresinol is esterified by vanillic acid. A representative of the guaialignanolides — hydroxymatairesinol — is esterified at the secondary aliphatic hydroxy group with p-hydroxybenzoic or vanillic acid.Institute of Organic Chemistry, Siberian Branch of the Academy of Sciences of the USSR. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 337–341, May–June, 1977.     

Organotin analysis by gas chromatography–pulsed flame-photometric detection (GC–PFPD)

Monobutyltin (MBuT), dibutyltin (DBuT), and tributyltin (TBuT) mixtures have been separated and quantified by gas chromatography with pulsed flame-photometric detection (GC–PFPD). The compounds were first derivatized with NaBEt₄, then extracted with hexane and injected into the GC in splitless mode. Optimum GC and detector conditions were established. For GC, various injector temperatures and oven temperature programs were tested. For the PFPD detector, gate settings (gate delay and gate width) and detector temperature were optimized. A very good linearity was obtained up to 100–150 ppb for all organotin compounds. The detection limits obtained were: MBuT (0.7 ppb), DBuT (0.8 ppb), and TBuT (0.6 ppb). RSD for repeatability and reproducibility were well below 20% when the instrument was in routine operation. A biological sample (CRM 477) was also analyzed for organotins. Extraction from the biological matrix was performed with TMAH. Besides the increased risk of contamination, the derivatization step seemed to be critical. pH and amount of derivatizing agent were tested. When using an internal standard (TPrT) between 90% and 110% of the certified amounts of organotin were recovered.     

Luminescence of dihydro derivatives of benzo[f]quinoline

The spectral luminescence properties of 3-aryl-3,4-dihydrobenzo[f]quinolines containing alicycles and ester groups in the 1 and 2 positions were investigated. It is shown that compounds of this type have intense luminescence (52–76%). The quenching effect of an ester group in the heterocyclic ring was established. The spectral luminescence characteristics of compounds with an ester group in the alicycle are determined by the proton-acceptor properties of the carbonyl function of this group. The effect of the solvent on the spectral luminescence characteristics of the investigated compounds is discussed.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 6, pp. 810–813, June, 1977.     

Oxidation of l-methionine with aqueous chromic acid: a kinetic study

The kinetics of oxidation of l-methionine by chromic acid in acidic medium (pH=0.83–2.2) has been studied spectrophotometrically. The effect of l-methionine and chromium(VI) concentrations on the rate of the reaction was determined. The reaction rate decreases with increasing the pH of the medium. The kinetics of the formation of a chromium(III) complex conform to the rate law:with k1=7.5×10–2s–1 and Kes1=43.85 at constant [H+]=1.9×10–2moldm–3 and [l-methionine]T= 2.0×10–2moldm–3. The same values were found with [l-methionine]T variation at constant [H+]. The reaction proceeds through formation of chromium(VI)-l-methionine ester in a rapid pre-equilibrium step, followed by a slower redox reaction of the ester. The present study provides kinetic evidence for formation of a complex ion (ester). One mole of Cr2O72– oxidizes four moles of l-methionine, which acts as a monodentate ligand and binds to chromium(VI) through the sulfur atom. The coordinated sulfur atom of l-methionine–chromium(VI) ester is responsible for the oxidative degradation (breaking of the C1-C2 bond) of l-methionine. Coordinated oxygen of the carboxylate group inhibits the cleavage of the C1-C2 bond.     

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.     

Three New Resin Glycosides and a New Tetrahydropyran Derivative from the Seeds of <i>Quamoclit pennata</i>

Three new resin glycosides, quamoclins V (1), VI (2), and VII (3) and a new tetrahydropyran derivative, quamopyran (4), were isolated from the seeds of Quamoclit pennata BOJER (Convolvulaceae). The chemical structures of these compounds were determined primarily on the basis of spectroscopic data. The carboxyl group of the aglycone, 11S-convolvulinolic acid, of 1 and 2 was linked intermoleculary with a hydroxy group of the sugar moiety to form a macrocyclic ester structure, as in already known jalapins, and 3 was an acylated glycosidic acid methyl ester. All of the sugar moieties of 1-3 were acylated by one 2S-methylbutyric acid. Compound 4 was a diketone having a tetrahydropyran ring.     

Stereochemistry of latilobinol

The results are given of a study of the stereochemistry of a coumarin terpenoid derivative — latilobinol — by nuclear magnetic resonance spectroscopy using lanthanoid shift reagents. On the basis of the results obtained, it has been established that the hydroxy group in the cyclohexane ring occupies the equatorial position.Leningrad Sanitary-Rygienic Medical Institute. Translated from Khimiya Prirodnykh Soedinenii, No.6, pp. 712–716, November–December, 1984.     

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 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.