A rapid HPLC assay for zearalenone in laboratory cultures ofFusarium graminearum

A high pressure liquid chromatographic (HPLC) method to determine zearalenone in corn contaminated withFusarium graminearum is described. After extraction with methanol-water and solvent partition, samples were cleaned up by applying the extract to a disposable silica cartridge and by eluting the toxin with a mixture of hexane/dry ethyl ether (5/5). Separation was achieved by a reverse phase Bondapak C₁₈ column followed by fluorescence detection using an excitation wavelength at 274 nm and an emission wavelength at 440 nm. Detection limit was about 5 ng. Recoveries ranging from 85.37 to 100.97%, in standard solutions range 30–0.5 µg/ml, were found.     

Use of thin layer chromatography for detection and high performance liquid chromatography for quantitating gliotoxin from rice cultures ofAspergillus fumigatus Fresenius

Gliotoxin, a mycotoxin with antimicrobial and immunosuppressive capabilities, is produced by several genera of fungi including the pathogenic fungusAspergillus fumigatus. The ability of selected isolates ofA. fumigatus to produce gliotoxin on three different media was tested and a thin layer chromatographic and high performance liquid chromatographic method for quantitation of gliotoxin from rice culture was developed and is described. Rice cultures were extracted with chloroform and the resulting extract was partially purified by precipitation with petroleum ether and cleanup by gel permeation chromatography. Gliotoxin was detected by thin layer chromatography and quantitated by high performance liquid chromatography using a U.V. absorbance detector with a 254 nm filter and a mobile phase of methanol-water 4357 (V/V) with a flow rate of 2.0 ml/min. The retention time for gliotoxin was approximately 4.8 min. From rice samples spiked with gliotoxin concentrations of 0.67, 1.33, 2.67, 4.00 and 5.33g/g the average recovery was 83.8%.     

Chromatographic analysis of Fusarium toxins in grain samples

Summary A method for the analysis of zearalenone, T-2 toxin, neosolaniol and HT-2 toxin from the grains of barley, wheat and oats has been developed. Toxins are extracted with ethyl acetate, purified on a kieselgel TLC-plate and analysed on a HPTLC-plate. The limits of detection are 0.2 mg/kg for zearalenone and T-2 toxin and 5 mg/kg for neosolaniol and HT-2 toxin. For more accurate estimation the purified toxins are analysed as their trimethysilyl derivatives by gas chromatography, in which the detection limit for all toxins in 50 g/kg and the accuracy ±10%–30%. The percentage recovery in both methods is 80%.     

Rapid determination of zearalenone in edible oils by HPLC with fluorescence detection

A fast, cost-efficient, sensitive and accurate assay method for zearalenone in edible oils is described, as an alternative to gel permeation chromatography (GPC). Oil samples were extracted with an alkaline mixture of methanol and water (methanol +10 g/l aqueous ammonium carbaminate solution, pH 9; 9 + 1, v+v). The pH of the extract was neutralized with hydrochloric acid and then concentrated to dryness. The residue was dissolved with HPLC solvent, and zearalenone was determined by high-performance liquid chromatography with fluorometric detection (HPLC-FLD). The method was successfully validated for two matrices, maize oil and rapeseed oil. The recovery rate was 87%, and the coefficient of variation was 2.8% in a rapeseed oil sample contaminated with 27 μg zearalenone/kg. At a signal-to-noise ratio of 3:1, the method detection limit was 10 μg/kg, which was considered to be adequate in view of the present European Union maximum level of 400 μg/kg.     

The fate of the fusarium mycotoxin zearalenone in maize cell suspension cultures

The fusarium mycotoxin zearalenone was transformed in cell suspension cultures of Zea mays giving α- and β-zearalenol and the β-D-glu cos ides of zearalenone and α- and β-zearalenol. The structure of zearalenone-4-β-D-glucopyranoside was determined by liquid — chromatography-mass spectrometry and specific hydrolysis with β-glucosidase. α- and β-zearalenol and their glucosides were identified by co chromatography using tic and HPLC and glucosidase — treatment Up to 50% of the mycotoxin added was bound to a non extractable or “bound” residue fraction. After treating this residue by a sequential cell wall fractionation procedure, zearalenone was found to be bound mainly to starch, hemicellulose, and lignin fractions.     

A rapid method for extraction of zearalenone and zeraralenols in fermented corn

A rapid extraction method is described for isolation of zearalenone and α- and β-trans-zearalenols from laboratory fermented corn. Corn fermented withFusarium crookwellense at 25°C for 2 weeks was agitated for 5 minutes in acetone. The acetone extract was evaporated to dryness and the remaining residue was chromatographed on a silica gel column with hexane:ethyl acetate (8:2). The products eluted with the hexane:ethyl acetate mixture in 1–1/2 column volumes and were identified by regular phase and C-18 reverse-phase thin layer chromatography. The products were verified by gas chromatography-mass spectroscopy. Product recoveries were 62.5–70% for the zearalenols and 70% for zearalenone in the range 0.5–50 mg/Kg.     

Production and Analytical Properties of Antibodies with High Specificity to Zearalenone

The time course of production, specificity, and analytical potential of antizearalenone polyclonal rabbit antibody synthesized by formaldehyde condensation and conjugated to bovine serum albumin were investigated. The relative cross-reactivities with natural analogues were: zearalenone, 100%; -zearalenole, 0.15%; and -zearalenole, <0.02%. With synthetic analogues: zearalanone, 31.7% and -zearalanone, 0.12%. An enzyme immunoassay for zearalenone with a sensitivity of 0.1 ng/ml was developed on the basis of these antibodies and solid-phase conjugates homologous to the immunogen in the method of synthesis.     

Metabolism of the lignin model compounds veratrylglycerol-β-guaiacyl ether and 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether by Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium metabolized the lignin model compounds veratylglycerol--guaiacyl ether I and 4-ethoxy-3-methoxy-phenylglycerol--guaiacyl ether V in stationary culture under an atmosphere of 100% oxygen and under nitrogen limiting conditions. 2-(o-methoxyphenoxy)-ethanol VII was identified as a product of the metabolism of both substrates. Veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol IV were identified as metabolites of I and V respectively. Metabolites were identified after comparison with chemically synthesized standards by mass spectrometry. These results indicate the existence of an enzyme system capable of directly cleaving the etherated dimers I and V at the , bond. The additional identification of 2-(o-methoxyphenoxy)-1,3 propanediol IX as a metabolic product indicates that cleavage of the alkyl-phenyl bond of these dimers or their metabolites also occurs.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC Thin layer chromatography     

Biosynthesis,isolation, purification and seperation of Zearalenone,deoxynivalenol and 15-Acetyldeoxynivalenol

Fusarium graminearum KF-376 isolate was found to be able to form simultaneously three toxic metabolites: zearalenone (FF-2), deoxynivalenol (DON) and 15-acetyldeoxynivalenol (15-AcDON). Toxins were extracted with methanol — water 3:1 (v/v) and purified by liquid chromatography on charcoal — Kieselgel 60 column (preliminary) and Aluminiumoxid 90 column. Final separation of the metabolites was achived on Kieselgel 60 — Aluminiumoxid 90 column.     

A multicomponent method for Fusarium toxins in cereal based food and feed samples using HPLC-MS/MS

A reliable, sensitive and selective multicomponent method has been developed to determine 12 differentFusarium mycotoxins (trichothecenes type A and B, zearalenone) simultaneously in cereal and grain samples using liquid chromatography tandem mass spectrometry (LC-ESI-MS/MS). The sample preparation based on a standard extraction step followed by two different kinds of solid phase clean-up (multifunctional MycoSep^® material) for trichothecenes, and an immuno-affinity purification which combined antibodies for aflatoxins, ochratoxin A and zearalenone (AOZ-IAC). For quantification of zearalenone (ZON) an internal standard (zearalanone, ZAN) was used, whereas for trichothecenes a recovery standard (verrucarol, VOL) was applied. The average recoveries for the trichothecenes ranged from 65% for nivalenol (NIV) up to 96% for deoxynivalenol (DON) and 89% for zearalenone. The limit of quantification is different for each of the individual trichothecenes and in the range of 1 ppb to 10 ppb.     

A simplified radioimmunoassay of plasma nortriptyline in depressed patients compared with high-pressure liquid chromatography and gas-liquid chromatography

Antiserum to nortriptyline was generated in male New Zealand rabbits innoculated with n-succinylnortriptyline-bovine serum albumin conjugates. The antiserum was used at a final dilution of 14000 and tritiated imipramine was used as the label antigen. An accurate, sensitive, and specific radioimmunoassay of depressed patients' plasma or serum nortriptyline is described. The accuracy was good with a recovery range of 90–100% with a mean of 94%. The method can be used to measure nortriptyline concentration in the range of 0.1 g/liter to 100 g/liter without prior extraction and purification of plasma or serum. Results of this method correlate well with those obtained by high-pressure liquid chromatography (r=0.979) and by gas-liquid chromatography (r=0.98). The specificity of the antiserum was examined by studying the cross-reactivity of 20 different psychopharmacological compounds, including nortriptyline's metabolites.     

Group-Specific Antibodies against Zearalenone and Its Metabolites and Synthetic Analogs

Formation kinetics, specificity, and analytical potential of polyclonal antibodies raised in rabbits against BSA conjugates of zearalanone carboxymethyloxime (CMO-ZAN) and zearalenone carboxymethyloxime (CMO-ZEN) have been studied. Preparation of the conjugates involved conversion of CMO-ZAN and CMO-ZEN into activated esters or carbodiimide condensation. Two versions of a group-specific enzyme immunoassay (for zearalenone/-zearalenol and zearalanone/-zearalanol) based on the heterologous combination of solid-phase antigens are described (sensitivity, 0.01 ng/ml).     

Fumonisin B1: Isolation from corn culture,and purification by high performance liquid chromatography

A method is described to isolate fumonisin B₁ (FB₁) from corn cultured for 18 days at 25°C withFusarium moniliforme. Cultured corn was extracted with aqueous methanol and purified with XAD-2 column chromatography and high performance liquid chromatography (HPLC). About 450 mg of FB₁ were obtained from 800g cultured corn. Its identity was established by fast-atom bombardment (FAB) mass spectrometry, and infrared spectrum and nuclear magnetic spectrum. Its purity was estimated to be 95% by gas chromatography/mass spectrometry (GC/MS).References     

Alkyl-phenyl cleavage of the lignin model compounds guaiacylglycol and glycerol-β-guaiacyl ether by Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized guaiacylglycol--guaiacyl ether (I) in high nitrogen, shaking and stationary cultures. 2-(o-Methoxyphenoxy) ethanol (X), 2-(o-methoxyphenoxy) acetic acid (IX) and methoxy-phydroquinone (MHQ) were identified as products of the metabolism of (I). P. chrysosporium also metabolized guaiacylglycerol--guaiacyl ether (IV) in high nitrogen stationary cultures. 2-(o-Methoxyphenoxy)-1,3 propanediol (XII) and 3-hydroxy, 2-(o-methoxy-phenyxy) propionic acid (XIV) were identified as products of the metabolism of (IV). Finally, P. chrysosporium metabolized -deoxyguaiacylglycol--guaiacyl ether (VI) and -deoxyguaiacylglycerol--guaiacyl ether (VII) in limiting nitrogen cultures. 2-(o-Methoxyphenoxy) ethanol (X) and 2-(o-methoxyphenoxy)-1,3 propanediol (XII) were identified as products of the metabolism of VI and VII respectively indicating hydroxylation of those substrates with subsequent alkyl-phenyl bond cleavage. Metabolites were identified after comparison with chemically synthesized standards by GLC-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography - MHQ methoxyhydroquinone     

Reduction of thymine by leukocytes

The maximal activity of the dihydrothymine dehydrogenase of fractions of human blood was found in leukocytes, especially in the supernatant after centrifugation of homogenates at 100,000 × g in the course of isolation of the cell components. The dihydrothymine dehydrogenase from human and pig leukocytes was purified tenfold with a yield of activity of about 60%. Gel filtration with Sephadex G-200 showed several peaks of enzymatic activity. A quantitative method for the estimation of thymine and dihydrothymine by means of thin layer chromatography and subsequent liquid scintillation counting is described. This method may have application in the study of the genetically determined -aminoisobutyric acid excretion in man.This investigation was supported by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie.     

Chemical synthesis of Haemophilus influenzae glycopeptide conjugates

A simple procedure for conjugating synthetic fragments of the capsular polysaccharide of Haemophilus influenzae type b, poly-3--D-ribose-(1, 1)-D-ribitol-5-phosphate (sPRP) to linear peptides is described. The procedure consists of (i) reacting the amino group of amino-heptyl sPRP with m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) in phosphate buffer, pH 7.5; (ii) selectively coupling the MBS-modified sPRP to the sulfhydryl group of the cysteine residue of peptides containing functional T-helper cell epitope(s). The glycopeptide conjugates were purified by gel filtration chromatography, biochemically characterized, and elicited protective level of anti-PRP antibody responses in rabbits. Abbreviations: PRP, poly-3--D-ribose-(1, 1)-D-ribitol-5-phosphate; sPRP, synthetic oligo-3--D-ribose-(1, 1)-D-ribitol-5-phosphate; Hib, Haemophilus influenzae type b; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide; PEG, polyethylene glycol monomethyl ether; CRM 197, a non-toxic diphtheria toxin mutant; TT, tetanus toxoid; DT, diphtheria toxoid; OMP, outer membrane protein; RP-HPLC reverse phase high pressure liquid chromatograph     

Nachweis von Zearalenon-4-β-D-Glucopyranosid in Weizen

Maize (Zea mays) cell cultures were used for the production of zearalenone-4-β-D-glucopyranoside as standard compound. Wheat samples were extracted with acetonitrile: water, applied to a florisil column and eluted with methanol:ethyl acetate. For determination and quantification of zearalenone-4-β-D-glucopyranoside and zearalenone a LC-MS method was developed. A concentration of 10 μg/kg zearalenone-4-β-D-glucopyranoside and zearalenone was detectable. The recovery rates were calculated to be 69% and 89% at a concentration level of 100 μg/kg for zearalenone-4-β-D-glucopyranoside and zearalenone, respectively.24 Bavarian wheat samples from harvest 1999 were analyzed. Zearalenone was present in 22 out of 24 field samples, the levels ranged from 11–860 μg/kg. Zearalenone-4-β-D-glucopyranoside was found in 10 out of the zearalenone positive samples (42%) at levels ranging from 17 to 104 μg/kg. The amounts of zearalenone-4-β-D-glucopyranoside were correlated to those of zearalenone (r2=0,86; b=0,10).     

High-performance liquid chromatographic analysis of dipyridamole in plasma and whole blood

A rapid, sensitive, and specific high-performance liquid chromatographic method is described for the quantitative analysis of dipyridamole in plasma and whole blood. The method involves a single extraction of an alkalinized sample with diethyl ether followed by evaporation of the organic solvent and ion-pair chromatography using fluorescence detection. The lower limit of sensitivity for dipyridamole is 1 ng/ml. Concentrations of dipyridamole between 1 and 500 ng per sample are measured with an average coefficient of variation of 4.5% in plasma and 7.4% in whole blood.     

The amino acid sequence of the rabbit lutropin beta subunit

The amino acid sequence of the beta subunit of rabbit lutropin (lLH) has been determined. The amino terminus of about 97% of the beta subunit has a two amino acid extension (pyro-Glu-Pro) compared to other lutropin beta sequences. Overlapping peptides from trypsin and chymotrypsin digestions of the performic acid-oxidized beta subunit and trypsin digestion of the S-aminoethylated cysteine beta subunit were isolated by chromatography on TSK Fractogel 40F and high-pressure liquid chromatography (HPLC). Sequencing was by a combination of the dansyl-Edman method and the direct Edman method. Amide placements were established by HPLC analysis of the PTH amino acid derivatives. The proposed sequence of lLH subunit is: This sequence is highly homologous to the other known lutropin beta subunits, especially rat and pig lutropin beta (91%). Partial cleavage of the peptide bond between Asp-79 and Pro-80 was observed during cyanogen bromide treatment. Rabbit thyrotropin and thyrotropin beta subunit copurified with lLH and lLH except at a final chromatography on Sephadex G-100.     

Separation of o-phthalaldehyde derivatives of amino acids of the internal pool of yeast by reverse-phase liquid chromatography

Summary Derivatization of primary amino acids with orthophthalaldehyde and -mercaptoethanol forms derivatives that can be detected by absorbance at 340 nm. These were separated by reverse-phase high performance liquid chromatography (HPLC). A step- or more complex gradient (acetonitrile/phosphate buffer gradient) was used. The effects of several parameters (pH, ionic strength, etc) were characterized and used to design a rapid separation of the amino acids commonly found in physiological fluids. The method described is rapid, sensitive and precise as sensitivity limits are about 25 pmol and the separation time, injection to injection, is 16 min.