Development of certified calibrants for the analysis of zearalenone and B-trichothecenes

Recent intercomparison studies have clearly shown that the use of common calibrants does considerably improve the between laboratory comparability of measurement results. However, there is still a lack of certified calibrants within the field of mycotoxin analysis. Therefore, two EC-funded projects have been carried out with the aim to investigate the feasibility for the production of calibrants with certified concentrations of B-trichothecenes and for the production of a certified calibrant of zearalenone in acetonitrile. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

DON calibrant: Towards the production of certified B-trichothecene calibrants

Within an EC-funded project calibrants with certified concentrations of Deoxynivalenol (DON), 3-Acetyl-Deoxynivalenol (3-Ac-DON), 15-Acetyl-Deoxynivalenol (15-Ac-DON) and Nivalenol (NIV) in acetonitrile have been produced. So far the project has led to improved isolation and purification of the solid toxins fromFusarium cultures. In addition, conditions for the production, ampouling and transport of the toxin solutions have been optimised. Further investigations should lead to knowledge about storage conditions and internationally accepted molar absorption coefficients for DON, 3-Ac-DON, 15-Ac-DON and NIV in acetonitrile. The intercomparison study which is currently carried out will also help to support knowledge and experience exchange between laboratories in the field ofFusarium mycotoxin analysis.     

Discrepancy between radioimmunoassay and high performance liquid chromatography tandem-mass spectrometry for the analysis of androstenedione

The discrepancy of results for the quantification of androstenedione in human serum between a radioimmunoassay (RIA) method and high performance liquid chromatography tandem-mass spectrometry (LC–MS/MS) was investigated. RIA overestimated concentrations compared to LC–MS/MS on 59 clinical samples (RIA = 1.79 × LC–MS/MS + 0.94). RIA kit and LC–MS/MS calibrants were also determined by both methods. The RIA performed with improved accuracy on the calibrants (RIA = 1.35 × LC–MS/MS − 0.28). Lipid, protein, electrolyte content, and pH of the two sets of calibrants were further investigated. The RIA calibrants contained little lipid material, while the LC–MS/MS calibrant material contained the same levels expected in normal serum/plasma. The pH and sex hormone binding globulin (SHBG) values were different between the RIA calibrants and the LC–MS/MS calibrant material (SHBG, 31 ± 2 and 38 ± 2 nmol/l; pH, 8.27 ± 0.18 and 8.66 ± 0.03, respectively). No correlation was observed between androstenedione RIA and LC–MS/MS discrepancy and lipid or protein. LC–MS/MS sample preparation was tested for the removal of protein-bound material and recovery determined (99–108%). The corresponding RIA results overestimated androstenedione by 52–174% compared to LC–MS/MS. The results here demonstrate that LC–MS/MS is the more accurate method.     

Fusarium mycotoxins in forage maize — Detection and evaluation

The deoxynivalenol concentrations found in forage maize ranged between 0.24 and 14.29 mg/kg DM (detected by ELISA). When highly contaminated samples were analysed for deoxynivalenol by HPLC or LC-MS the resulting concentrations were in the mean about 50% lower. Furthermore, using LC-MS other type-A and type-B trichothecenes, zearalenone and α-zearalenol were found in these samples. The differences between ELISA and HPLC/LC-MS data for deoxynivalenol are assumed to result from cross-reactions of other trichothecenes with the antibodies used in ELISA and toxin losses from sample purification procedures needed for HPLC and LC-MS analysis. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004     

24-hour lock mass protection

The positive role and application of highly accurate mass measurements in proteomics is well documented. The new generation of hybrid FTMS and Q-TOF instruments, including the LTQ-Orbitrap (OT), is remarkable in their ability to routinely produce single-digit to subppm statistical mass accuracy while maintaining high analytical sensitivity. The use of mass calibrants (lock masses) to reduce the systematic error of mass-to-charge measurements has also been reported and, in some cases, incorporated in the instrument control software by the instrument manufacturers. We evaluated the use of one such calibrant in the OT (e.g., polydimethylcyclosiloxane, PCM) to study its impact on the rate of phosphopeptide annotation and found it to lack robustness under normal laboratory conditions. Therefore, we devised a strategy to improve its performance by increasing the external abundance of calibrant molecules in laboratory air. This resulted in a more robust performance of the preprogrammed lock mass recalibration feature as evidenced by improvements in both statistical mass accuracy and peptide annotation rates.     

Trichothecenes and Mycoflora in Wheat Harvested in Nine Locations in Buenos Aires Province, Argentina

A total of 120 freshly harvested wheat samples from the 2004 season in nine locations from Northern Buenos Aires Province, Argentina, were analysed for trichothecene natural occurrence and associated mycoflora, and for determining the influence of commonly used fungicide field treatment and the cultivar type on trichothecene contamination. The trichothecenes T-2 tetraol, T-2 triol, HT-2 and T-2 toxin (HT-2, T-2), diacetoxyscirpenol (DAS), nivalenol (NIV), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) were analysed by gas chromatography and electron capture detection. Detection limits ranged from 4 to 20 μg/kg. The isolation frequencies of species were calculated. Alternaria alternata, Fusarium graminearum, Fusarium poae and Fusarium semitectum were the predominant fungal species identified as endogenous mycoflora. The type of cultivar and the fungicide field treatment did not affect significantly the trichothecene contamination. The trichothecenes type A detected were HT-2 and T-2 triol toxins and the type B were DON, NIV and 3-ADON. Based on 120 samples the incidences were 21.7% for 3-ADON, 22.5% for HT-2, 27.5% for T-2 triol and 85% for DON. NIV was confirmed in one sample. Mean levels of trichothecene positive samples were between 7 and 2788 μg/kg.     

Sources of variation in the analysis of trichothecenes in cereals by gas chromatography-mass spectrometry

The sources of variation In the analysis of trichothecenes in cereals by gas chromatography mass spectrometry (GC-MS) were studied and Identified. Ways to decrease some of the variations identified are presented and discussed. The method Is validated for deoxynivalenol, nivalenol, 3-acetyldeoxynivalenol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin and T-2 toxin. The general variability in the performance of the GC-MS apparatus itself, as well as derivatisation, matrix effect, injection and quantification were identified to be the sources of variation. The use of internal standards was studied in order to decrease the variation due to problems with derivatisation and the overall sample preparation. Two derivatisation reagents were compared, one of which was found to be more effective for decreasing this variation. The calibrants prepared in the cereal extract reduced the variation due to the matrix effect and thus improved the quantification. “Constant Standards” were introduced in order to detect and decrease the variation caused by injection and the apparatus itself. The validation study proved that this analytical method for trichothecenes was adequately reliable and sensitive. The relative standard deviation varied between 3–9% and the recovery between 46–90% for the different trichothecenes determined. The limit of detection for a range of trichothecenes varied from 5 μg/kg to 15 μg/kg.     

Development and evaluation of multiplex PCR for detection of T-2 and zearalenone producing Fusarium spp

The study aimed to develop and evaluate a multiplex polymerase chain reaction assay (mPCR) for the concurrent detection of three major mycotoxin metabolic pathway genes, namely tri8 (T-2 toxin), tri6 (trichothecene) and pks4 (zearalenone), along with competitive internal amplification control. Specific primers for each of the aforementioned genes were optimized and validated using 14 reference strains and 10 pure culture isolates. The optimized mPCR assay detected the three metabolic pathway genes in artificially contaminated maize samples with a sensitivity of 2 × 10³ CFU per g for tri6 and pks4 positive Fusarium strains, whereas 2 × 10⁴ CFU per g for tri8 positive Fusarium strains. Application of the developed mPCR assay to 30 cereal and 20 feed samples revealed 24% (12 of 50) contamination with either one or more mycotoxins. The results of mPCR assay were further evaluated with high performance liquid chromatography (HPLC), and both methods provided unequivocal results. This mPCR assay might be a supplementary tool to conventional mycotoxin analytical techniques like thin-layer chromatography, HPLC, etc. The current mPCR assay is a rapid and reliable tool for simultaneous, sensitive and specific detection of T-2, zearalenone and trichothecene producing Fusarium spp. from naturally contaminated foods and to monitor them during the processing steps of food and feed commodities.     

Enantioselective separation and degradation of the herbicide dichlorprop methyl in sediment

Chiral pesticides currently constitute about 50% of all pesticides dosage used in China, and this ratio is increasing as more complex structures are introduced. Dichlorprop methyl (DCPPM) is a chiral herbicide consisting of a pair of enantiomers. In this study, the enantiomeric separation of DCPPM was investigated by gas chromatography (GC) and high-performance liquid chromatography (HPLC) using chiral stationary phases (CSPs), and its enantiomeric degradation was characterized using a DCPPM-degrading bacterial strain isolated from an activated sludge from a textile-printing wastewater treatment plant. Baseline separation by both GC and HPLC was achieved. Incubation with DCPPM-degrading bacteria in different pH solutions showed that the R enantiomer was preferentially degraded over the S enantiomer of DCPPM. The degradation rate constant decreased with increasing pH in the order of k(pH5) approximately k(pH7) >k(pH9). In comparison, the enantioselectivity as indicated by EF followed the order of pH 7 > pH 9-pH 5.     

Production of neosolaniol byFusarium tumidum

Extracts from autoclaved maize culture ofFusarium tumidum strain R-5823 were toxic towardsArtemia salina. Bioassay-guided fractionation of the organic extract led to the isolation of the toxic compound that was identified as the trichothecene toxin neosolaniol (NEOS) by¹H,¹³C nuclear magnetic resonance spectroscopy and low-resolution electronic impact mass spectrometry. The amount of NEOS produced by the strain R-5823 was 300 mg/kg maize culture. NEOS was also detected by HPLC in cultures of four out of seven additional strains ofF. tumidum andGibberella tumida with different origin, in amounts ranging from 1 to 311 mg/kg. This is the first report on the production of a trichothecene toxin byF. tumidum.     

银耳多糖单糖组成分析的三种色谱方法比较

为选择一种准确快捷的方法测定银耳多糖的单糖组成,对薄层色谱法(TLC)、气相色谱法(GC)、高效液相色谱法(HPLC)三种色谱方法进行比较。结果表明,前两种方法的测定结果均不理想,而HPLC法,操作简便,灵敏度高,分离效果好,信息完整。测定结果为由葡萄糖、甘露糖、葡萄糖醛酸、木糖、岩藻糖组成,其摩尔比为0.24∶1.00∶0.06∶0.29∶0.25。HPLC法对酸性杂多糖组成糖分析是一种比较理想的选择。     

Trichothecene production and the relationship to vegetative compatibility groups in shape Fusarium poae

Twenty-two Norwegian and two Polish isolates of Fusarium poae were cultured on rice and in two liquid media, MOSS and MYRO. All samples were analysed for trichothecenes by gas chromatography mass-spectrometer. Association of trichothecene production with vegetative compatibility groups was studied in the F. poae isolates. Twenty of the isolates produced the type A trichothecenes diacetoxyscirpenol or monoacetoxyscirpenol in at least one of the media, in the concentration range of 0.01 to 65 μg ml^-1 in the liquid culture and 0.1 to 67 μg g^-1 on rice. The other group of trichothecenes detected were of type B; nivalenol and its two acetyl-derivatives 4-acetylnivalenol and diacetylnivalenol. Twelve of the strains produced at least one of these metabolites in the concentration range of 0.01 to 7 μg ml^-1 in the liquid culture and 0.1 to 18 μg g^-1 on rice (sum of nivalenol and its acetylated derivatives). A significant correlation was observed between the two groups of toxins (logarithm). None of the isolates produced T-2 or HT-2 toxin. In a previous study these isolates were divided into 13 vegetative compatibility groups. Significant variation in the trichothecene production was observed between different vegetative compatibility groups, but also to some extent within the same group. This revised version was published online in August 2006 with corrections to the Cover Date.     

Verification of the final anion exchange chromatography in the r-hGH manufacturing process

In this study, final anion exchange chromatography in the recombinant human growth hormone (r-hGH) manufacturing process was validated using a validation protocol that was consistent with both policy and standard operation procedure (SOP). Two buffer solutions used in chromatography were first validated and were found to satisfy pre-established acceptance criteria as follows: pH: 8.2, endotoxin: < 0.6 EU/mL, bioburden test: negative. Final anion exchange chromatography was conducted using a DEAE Sepharose FF Resin and eluted with a linear gradient of 30 to 110 mM NaCl in 50 mM Tris-HCl buffer at a flow rate of 15 L/h. Three consecutive batches of hGH solutions were generated via anion exchange chromatography, which was performed within pre-established operating parameters determined through in-process control. When all three batches were assessed by the pre-established sampling plan and tested for quality control, this purification process was shown to satisfy pre-established acceptance criteria; endotoxin: ≤ 0.5 EU/mg, ECP: ≤ 1.4 ppm, IEF: same removal distance, hGH content by Native-PAGE: 100%, purity by HPLC: ≥ 99%, yield by UV scanning: 87 to 89%, hGH monomer protein content by HPLC: 99%. Therefore, the final anion chromatography process was successfully validated in this study, and this method consistently yielded hGH solutions that satisfied pre-established criteria for subsequent processing.     

A chromatographic and mass-spectrometric approach for the analysis of lipase-produced thioester derivatives

The synthesis of thioethyl 2-methylpropanoate, butanoate, 3-methylbutanoate, hexanoate, and of thiobutyl propanoate, butanoate, and pentanoate was achieved via esterification of ethanethiol or butanethiol with short-chain acids using an immobilised lipase from Rhizomucor miehei. High acid conversions were obtained (47% for thiobutyl pentanoate). The analysis of educts and products was carried out by reverse-phase liquid chromatography (HPLC), gas chromatography (GC), GC/mass spectrometry, and GC/olfactometry. All of the thioesters imparted a similar, onion-like smell, which remained unchanged on dilution. The thresholds of thioesters derived from the same thiol were loosely correlated with the size of the acid moiety: the larger the molecular mass the higher the threshold. Surprisingly, higher odour thresholds were obtained for the branched-chain thioesters than for their linear analogues. Received: 23 June 1997 / Received revision: 7 October 1997 / Accepted: 14 October 1997     

A colorimetric technique for detecting trichothecenes and assessing relative potencies

We tested a novel colorimetric toxicity test, based on inhibition of beta-galactosidase activity in the yeast Kluyveromyces marxianus, for sensitivity to a range of mycotoxins. A variety of trichothecene mycotoxins could be detected. The order of toxicity established with this bioassay was verrucarin A > roridin A > T-2 toxin > diacetoxyscirpenol > HT-2 toxin > acetyl T-2 toxin > neosolaniol > fusarenon X > T-2 triol > scirpentriol > nivalenol > deoxynivalenol > T-2 tetraol. The sensitivity of detection was high, with the most potent trichothecene tested, verrucarin A, having a 50% effective concentration (concentration of toxin causing 50% inhibition) of 2 ng/ml. Other mycotoxins (cyclopiazonic acid, fumonisin B1, ochratoxin A, patulin, sterigmatocystin, tenuazonic acid, and zearalenone) could not be detected at up to 10 micrograms/ml, nor could aflatoxins B1 and M1 be detected at concentrations up to 25 micrograms/ml. This test should be useful for trichothecene detection and for studies of relevant interactions-both between trichothecenes themselves and between trichothecenes and other food constituents.     

TRI12, a trichothecene efflux pump from Fusarium sporotrichioides : gene isolation and expression in yeast

Many of the genes involved in trichothecene toxin biosynthesis in Fusarium sporotrichioides are present within a gene cluster. Here we report the complete sequence for TRI12, a gene encoding a trichothecene efflux pump that is located within the trichothecene gene cluster of F. sporotrichioides. TRI12 encodes a putative polypeptide of 598 residues with sequence similarities to members of the major facilitator superfamily (MFS) and is predicted to contain 14 transmembrane-spanning segments. Disruption of TRI12 results in both reduced growth on complex media and reduced levels of trichothecene production. Growth of tri12 mutants on trichothecene-containing media is inhibited, suggesting that TRI12 may play a role in F. sporotrichioides self-protection against trichothecenes. Functional analysis of TRI12 was performed by expressing it in yeast strains that were co-transformed with a gene (TRI3) encoding a trichothecene 15-O-acetyltransferase. In the presence of the TRI3 substrate, 15-decalonectrin, cultures of yeast strains carrying TRI12 and TRI3 accumulated much higher levels of the acetylated product, calonectrin, than was observed for strains carrying TRI3 alone. PDR5, a transporter of the ABC superfamily, which is known to mediate trichothecene resistance in yeast, increased calonectrin accumulation in TRI12/TRI3 yeast strains but not in TRI3 strains. These results confirm the involvement of TRI12 in the trichothecene efflux associated with toxin biosynthesis, and demonstrate the usefulness of yeast as a host system for studies of MFS-type transporters. Received: 4 September 1998 / Accepted: 14 April 1999     

Fusarium graminearum Tri12p influences virulence to wheat and trichothecene accumulation

The gene Tri12 encodes a predicted major facilitator superfamily protein suggested to play a role in export of trichothecene mycotoxins produced by Fusarium spp. It is unclear, however, how the Tri12 protein (Tri12p) may influence trichothecene sensitivity and virulence of the wheat pathogen Fusarium graminearum. In this study, we establish a role for Tri12 in toxin accumulation and sensitivity as well as in pathogenicity toward wheat. Tri12 deletion mutants (tri12) are reduced in virulence and result in decreased trichothecene accumulation when inoculated on wheat compared with the wild-type strain or an ectopic mutant. Reduced radial growth of tri12 mutants on trichothecene biosynthesis induction medium was observed relative to the wild type and the ectopic strains. Diminished trichothecene accumulation was observed in liquid medium cultures inoculated with tri12 mutants. Wild-type fungal cells grown under conditions that induce trichothecene biosynthesis develop distinct subapical swelling and form large vacuoles. A strain expressing Tri12p linked to green fluorescent protein shows localization of the protein consistent with the plasma membrane. Our results indicate Tri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta.     

Natural multi-occurrence of mycotoxins in rice from Niger State,Nigeria

Twenty-one rice samples from field (ten), store (six) and market (five) from the traditional rice-growing areas of Niger State, Nigeria were analysed for aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisin B₁ (FB₁) and B₂ (FB₂), and patulin (PAT) by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) respectively. T-2 toxin was determined using TLC only. AFs were detected in all samples, at total AF concentrations of 28–372 μg/kg. OTA was found in 66.7% of the samples, also at high concentrations (134–341 μg/kg) that have to be considered as critical levels in aspects of nephrotoxicity. ZEA (53.4%), DON (23.8), FB₁ (14.3%) and FB₂ (4.8%) were also found in rice, although at relatively low levels. T-2 toxin was qualitatively detected by TLC in only one sample. Co-contamination with AFs, OTA, and ZEA was very common, and up to five mycotoxins were detected in a single sample. The high AF and OTA levels as found in rice in this study are regarded as unsafe, and multi-occurrences of mycotoxins in the rice samples with possible additive or synergistic toxic effects in consumers raise concern with respect to public health.     

Immunochemical analysis of trichothecenes produced by various fusaria

The production of type A trichothecene mycotoxins by 19 Fusaria, including 12Fusarium sporotrichioides, 4F. chlamydosporum and 3F. graminearum at 15°C and 25°C over a 35-day period was analyzed by ELISA using antibodies cross-reactive with most type A trichothecenes after conversion to T-2 tetraol tetraacetate. The toxin production peaked at 20–25 days of incubation with maximum yield between 4–6 mg type A trichothecene/ml of culture medium for 5F. sporotrichioides cultures and between 1 to 2 mg/ml for 6F. sporotrichioides cultures. OneF. sporotrichioides produced 700 µg type A trichothecenes/ml of culture medium. Detectable type A trichothecene was also found in the culture extracts ofF. chlamydosporum andF. graminearum, but the yield was very low (less than 100 µg/ml). Quantitative determination of individual trichothecenes was achieved by separation of different toxin in HPLC and followed by ELISA analysis. Eight to 10 immunoreactive peaks, corresponding to various type A trichothecenes, were detected in all the fungal extracts. T-2 tetraol (T-2-4ol), 4-acetyl-T-2 tetraol (4-Ac-T-2-4ol), neosolaniol (NEOS), diacetoxyscirpenol (DAS), HT-2 and T-2 toxin accounted for more than 85% of the total toxins. In general, low temperature was preferred for total type A trichothecene production. More T-2-4ol, 4-Ac-T-2-4ol, HT-2 and DAS were produced at 25°C. In contrast, more T-2 toxin and NEOS were produced at 15°C. Transformation of T-2 toxin and NEOS to polar metabolites such as T-2-4ol, 4-acetyl-T-2-4ol and HT-2 by various strains were observed at both temperatures after 25 days incubation.