Characterization and application of isotope-substituted (13C15)-deoxynivalenol (DON) as an internal standard for the determination of DON

The powerful combination of liquid chromatography and mass spectrometry (MS) is often limited by matrix effects during ionization in the MS ion source. The use of fully isotope-substituted (¹³C₁₅)-deoxynivalenol ((¹³C₁₅)-DON) as an internal standard (IS) corrects matrix effects and improves the accuracy of analytical methods using mass spectrometry for the quantitative determination of the Fusarium mycotoxin deoxynivalenol (DON). The IS was characterized with respect to its chromatographic purity by liquid chromatography-ultraviolet light and its isotope distribution by time-of-flight mass spectrometry. Its low-energy collision-induced dissociation behaviour was compared with DON. Moreover, this work describes the successful application of (¹³C₁₅)-DON as IS for the determination of DON in maize using high-performance liquid chromatography (HPLC) electrospray (ESI) with tandem mass spectrometry. The results demonstrate that the IS can successfully correct for fluctuations during extraction and clean-up of the sample as well as the ionization of DON in the MS ion source. Random variations in ionization affect the IS in the same way as the analyte. Recoveries for DON in maize of 76% ± 1.9% (external calibration) or 101% ± 2.4% (internal calibration) were reached, respectively, after sample clean-up.     

Occurrence of deoxynivalenol and T-2 toxin in bread and pasta commercialised in Spain

Deoxynivalenol and T-2 toxin were extracted from wheat-based bread (n = 75) and pasta (n = 75) samples using a mixture of acetonitrile:water (86:14 v/v); for analysis, gas chromatography/mass spectrometry after derivatisation with trifluoroacetic anhydride was utilised. The recovery of deoxynivalenol and T-2 toxin from both food matrixes ranged from 90.1 to 94.0%. The occurrence of these mycotoxins in bread was 28.0% and 2.6% for deoxynivalenol and T-2 toxin, respectively, whereas in pasta, the occurrence of both mycotoxins was higher, varying from 9.3 to 62.7%. The mean content of deoxynivalenol (42.5 μg/kg) in bread was lower than the content of T-2 toxin (68.37 μg/kg), while in pasta the content of deoxynivalenol (137.1 μg/kg) was superior. The estimated daily intake of deoxynivalenol and T-2 toxin from the consumption of these products represents 8.4% and 0.2% of the tolerable daily intake, respectively. These results back up the necessity to take a vigilant attitude in order to prevent human intake of trichothecenes. This information is necessary and of high priority in order to protect the consumer’s health from the risk of exposure to these toxins.     

Stability of the mycotoxin deoxynivalenol (DON) during the production of flour-based foods and wheat flake cereal

Deoxynivalenol (DON) is a mycotoxin found in cereal grains and cereal-based foods. DON concentrations in finished products are reduced under some processing conditions, but not others. DON concentrations in flour, wheat and selected foods made from them under commercially relevant conditions were compared by GC with electron capture detection. Average concentrations (n?=?9/item) in cookies, crackers and pretzels ranged from 61% (cookies) to 111% (pretzels) compared with flour (100%?=?0.46?µg?g^?1). Lesser amounts were found in donuts and bread: their respective DON concentrations were 44% and 30% that of flour. Mass balance estimates for DON (µg?g^?1 flour equivalents) ranged from 50% (bread?=?0.23?µg?g^?1 flour equivalents) to 120% (donuts), indicating that dilution by recipe ingredients contributed to DON reductions in bread and accounted for all of the apparent reduction in donuts. Mass balance estimates averaged 76% (crackers) to 107% (pretzels) for the other flour products. DON concentrations were higher in cereal flakes (0.55?µg?g^?1 in the finished product and 0.58?µg?g^?1 on a mass balance basis) than in wheat (0.40?µg?g^?1), suggesting that DON concentrations might increase during processing of wheat cereals under some conditions. In summary, DON concentrations of finished food products were reduced?≥50% only in bread and donuts. Reduction in bread resulted from a combination of DON ‘loss’ and dilution by recipe ingredients whereas the reduction in donuts was due entirely to dilution. These results are further evidence of DON stability during the preparation of popular flour or wheat-based products.     

Analysis of cocoa pulp and the formulation of a standardised artificial cocoa pulp medium

Results of analyses of cocoa pulp from the Ivory Coast, Nigeria and Malaysia are reported. These include pH, viscosity and a_w measurements and concentration of soluble sugars, vitamins and anions. Pulp from Malaysian cocoa pods had a higher water content, lower citrate, hemicellulose, lignin and pectin concentration and a higher pH than Ivorian or Nigerian cocoa pods. The composition of a defined cocoa pulp medium which has the same overall composition, pH, a_w and viscosity as cocoa pulp is described. The defined cocoa pulp medium supported the growth of yeasts, lactic acid bacteria and acetic acid bacteria.     

Occurrence of deoxynivalenol (vomitoxin) in processed cereals and pulses in Turkey

The aim was to investigate the occurrence of deoxynivalenol (DON) in cereal and pulse products in Turkey. DON was detected using high-performance liquid chromatography (HPLC) with ultraviolet detection at 220 nm and positive results greater or equal to 0.60 ppm were confirmed by thin layer chromatography (TLC). An acetonitrile-water (21:4 v/v) extract of the sample was cleaned up on a column packed with alumina-Celite-charcoal (0.35 + 0.25 + 0.40 g). The detection limits for DON were 3 ng/injection (0.10 ppm) and 50 ng/spot (0.60 ppm) for HPLC and TLC, respectively. Eighty-three commercially available cereal and pulse product samples collected from markets and street bazaars were analysed. The recovery rates for boiled, pounded wheat and rice spiked with added DON (1 ppm) were 80.9% (SD 8.37, n =5) and 72.3% (3.85, n =5), respectively. DON was detected in six (8.82%) of 68 cereal and in none of 15 pulse products. The maximum detected amount was 2.67 ppm in a corn flour sample.     

The advantages of solid fat content determination in cocoa butter and cocoa butter equivalents by the Karlshamns method

This paper describes the preparation of samples and the evaluation techniques applied to determine the Solid Fat Content (SFC) by means of pulsed nuclear magnetic resonance (NMR) in confectionery fats characterized by distinct polymorphism (cocoa butter, cocoa butter equivalents and their mixtures). Due to the deviation of SFC values in certain softer cocoa butter equivalents at the temperature of 20 °C, an internal method devised at Karlshamns Oils & Fats AB is applied as well. When compared to the official IUPAC 2.150 (b) method, Karlshamns' method involves a lower temperature and a prolonged period of time at the stage of the final SFC stabilization in the pre-treatment of fat samples. The calculation of SFC in the analysed samples for the purposes of this paper was carried out by employing both methods (the indirect method and a serial procedure). The results are presented graphically. Differences were reflected in the SFC of the fats and their mixtures that were under observation depending on the method and temperatures applied. The obtained results indicated a considerable advantage of employing the Karlshamns method since the results yielded by this method are more lucid. Moreover, the behaviour of fats in the production process can be more easily predicted on the basis of these results.     

Quantification of cocoa butter equivalents in mixtures with cocoa butter by chromatographic methods and multivariate data evaluation

Received: 25 August 1999     

粮油食品中呕吐毒素危害及风险分析

该文分析呕吐毒素性质,在自然界和粮油食品中存在情况,及对人类和动物危害,并进行风险评估,同时提出预防呕吐毒素危害建议。     

Occurrence and risk assessment of population exposed to deoxynivalenol in foods derived from wheat flour in Brazil

Deoxynivalenol (DON) is the most important of the trichothecenes in terms of amounts and occurrence in wheat. This compound was shown to be associated with a glomerulonephropathy involving an increase of immunoglobulin A in humans. This study assessed the occurrence of DON in wheat flour and the exposure of Brazilian teenagers, adults and elderly to this mycotoxin due to intake of wheat flour-based products. DON extraction in wheat flour was carried out by solid phase extraction and the quantification was performed by ultra-high proficiency liquid chromatography with diode-array detection. A total of 77.9% of all samples were positive for DON, with concentrations ranging from 73.50 to 2794.63 µg kg^?1. The intake was calculated for the average and 90th percentile of the contamination levels of DON in foods based-wheat for teenagers, adults and elderly in Brazil, and compared with the provisional maximum tolerable daily intakes (PMTDI). Females of all age groups were exposed to DON at higher levels when compared to males in regard of consumption of breads and pastas. Teenagers were the main consumers of foods derived from wheat flour, with maximum probable daily intakes of 1.28 and 1.20 µg kg^?1 b.w. day^?1 for females and males, respectively. This population is at an increased risk of exposure to DON due to consumption of wheat flour-based foods in Brazil.     

ITS-RFLP characterization of black <Emphasis Type="Italic">Aspergillus</Emphasis> isolates responsible for ochratoxin A contamination in cocoa beans

In the present study, ochratoxigenic mycobiota in cocoa beans was identified at species level by digestion of the ITS products using the endonucleases HhaI, NlaIII and RsaI. Of the 132 isolates of Aspergillus section Nigri collected from cocoa beans, 89 were identified as A. tubingensis, 27 as A. niger, 10 as A. tubingensis-like and 6 as A. carbonarius. No variation was observed between RFLP patterns (C, N, T1 and T2) described previously for grape isolates and those of the cocoa isolates analysed. With respect to OTA-producing fungi, a high percentage of black aspergilli (50.7%) was able to produce OTA. Additionally, most of the OTA-producing isolates were of moderate toxigenicity, producing amounts of OTA from 10 μg g⁻¹ to 100 μg g⁻¹. Percentages of OTA-producing isolates in the A. niger aggregate were higher than in other substrates, ranging from 30% to 51.7%. Furthermore, the detected levels of OTA production in the A. niger aggregate, particularly in A. tubingensis species was higher than in A. carbonarius, ranging from 0.7 μg g⁻¹ to 120 μg g⁻¹ (mean 24.55 μg g⁻¹). Due to the high occurrence, percentage of ocratoxigenic isolates and their ability to produce OTA, isolates belonging to the A. niger aggregate could be considered as the main cause of OTA contamination in cocoa beans used for manufacturing cocoa products.     

Effects of oral exposure of pigs to deoxynivalenol (DON) sulfonate (DONS) as the non-toxic derivative of DON on tissue residues of DON and de-epoxy-DON and on DONS blood levels

The Fusarium toxin deoxynivalenol (DON) is of outstanding importance in pig nutrition because of its frequent occurrence in cereal grains at levels high enough to cause adverse effects such as a decrease in feed intake and impairment of the immune system. Thus, simple decontamination procedures would be useful. The present study aimed to examine the effects of wet preservation of triticale contaminated with DON and zearalenone (ZON) with sodium metabisulphite (SBS) on the treatment-related non-toxic derivative of DON (DON-sulfonate, DONS), and on ZON and its metabolites in blood and various physiological specimens of piglets. The uncontaminated control triticale (CON) and the DON-contaminated triticale (FUS) were included in the diets either untreated or SBS treated (CON-SBS, FUS-SBS) and fed to piglets for 28 days starting from weaning. The diet concentrations for DON were 0.156, 0.084, 2.312 and 0.275 mg kg^?1, for DONS were <0.05, <0.05, <0.05 and 1.841 mg kg^?1, and for ZON were <0.001, 0.006, 0.017, and 0.016 mg kg^?1 for each of CON, CON-SBS, FUS and FUS-SBS, respectively. DONS was present in the blood of piglets fed the FUS-SBS at a median concentration of 15.5 ng ml^?1 (3–67 ng ml^?1), while the median DON concentration amounted to 2 ng ml^?1 (0–5 ng ml^?1) at the same time. The median DON concentration in the blood of piglets fed the FUS diet reached a median concentration of 10.5 ng ml^?1 (5–17 ng ml^?1). Moreover, the relative differences between the DON concentrations in other physiological specimens (muscle, liver, kidney, bile and urine) in piglets fed the FUS-SBS and the FUS diet were comparable with the blood DON concentration differences. Although these differences can be taken as an indication for DONS stability after absorption and distribution further studies examining DONS in these other physiological specimens directly are necessary to substantiate this conclusion. Moreover, ZON and α-zearalenol could only be detected in bile and urine where their levels were not influenced by the SBS treatment.     

Identification of cocoa butter equivalents added to cocoa butter. III. Stereospecific analysis of triacylglycerol fraction and some its subfraction

In previous papers, six cocoa butter equivalent (CBE) samples were considered to carry out a study about the detection of their presence in cocoa butter (CB). In this work the % fatty acid (FA) compositions of triacylglycerol (TAG) subfractions separated by Ag⁺-High Performance Liquid Chromatography (Ag⁺-HPLC) from CB, CBE and CB/CBE mixtures were investigated. The fully saturated (SatSatSat) TAG subfractions isolated from CB, CBE and CB/CBE mixtures were the most suitable for the aim of the research. In fact, the multiple regression models obtained using the % FA composition of SatSatSat subfractions permitted to carry out effective CBE detections. In the present work the attempt to improve the statistical approach using as variables the results of the stereospecific analysis of the TAG fraction and of its main subfractions from CB, CBE and CB/CBE mixtures is described.     

Effects of sampling and extraction on deoxynivalenol quantification

Deoxynivalenol was extracted from wheat grain and quantified by GC-ECD. The quantification of deoxynivalenol can be critical, for example in certifying the amount of the mycotoxin in a lot and determining if this amount is over or under a fixed limit. Thus, an objective was to obtain representative samples to monitor DON quantification variability. We show that among the different steps of analysis the critical one is grain sampling. We also show that we were able to significantly improve the extraction rate without increasing the variability by using a longer extraction time with a magnetic agitator, and a grinding process which takes into account both the heterogeneous repartition of deoxynivalenol within wheat grains and different extraction rates according to the size of the flour powder particles. Thus, it could be of interest to use this methodology to determine if a lot is above or below a maximum limit because it enables the detection limit to be lowered, thereby simplifying the subsequent analysis.     

Influence of temperature and pH on the degradation of deoxynivalenol (DON) in aqueous medium: comparative cytotoxicity of DON and degraded product

Deoxynivalenol (DON), a toxic fungal metabolite, is stable under different processing conditions; however, its stability in aqueous medium at different temperatures and low pH (1–2) (present in the gastrointestinal tract) has not been investigated. In the present study, DON standard was used to study the influence of temperature and pH on DON stability in aqueous medium, the characterisation of the degraded product, and the comparative toxicity profile of the degraded and the parent compound. The results suggest that standard DON was unstable at 125–250°C showing 16–100% degradation whereas DON at pH 1–3 had 30–66% degradation, with a concomitant increase in the formation of a degraded product. Further ESI-MS characterisation of the dominant precursor ion of the HPLC eluate of the DON-degraded product was found to be m/z 279, resembling the known metabolite DOM-1. The degraded product of DON was reconfirmed as DOM-1 by comparison with standard DOM-1 and both gave a similar λ_max at 208 nm. Comparative studies of both standard DOM-1 and the degraded product of DON showed no cytotoxicity up to 6400 ng ml^–1 while significant cytotoxicity was observed for DON (400 ng ml^–1). The results suggest that a highly acidic environment (pH 1–2) could be responsible for the de-epoxydation of DON leading to the formation of DOM-1.     

Effect of fermentation on naturally occurring deoxynivalenol (DON) in Argentinean bread processing technology.

The stability of naturally occurring DON was evaluated during the fermentation stage of the bread-making process on a pilot scale. Two different products, French bread and Vienna bread, were prepared with naturally contaminated wheat flour (150 mg kg(-1)) under controlled experimental conditions. Dough was fermented at 30, 40 and 50 degrees C according to standard procedures employed in Argentinean low-technology bakeries. When the dough was fermented at 50 degrees C, the maximum reduction was 56% for the Vienna bread, with French bread being reduced by 41%. DON reduction during bread-making occurs not only in the baker due to thermal decomposition, but also during the fermentation step. The Argentinean traditional bread-making process might reduce DON levels during the fermentation stages if the dough is leavened at temperatures > 30 degrees C.     

Investigation of aromatic compounds in roasted cocoa powder

Most of the volatile compounds identified are highly significant in determining cocoa powder flavour, and this paper demonstrates that basic sensory perceptions (undesirable, bitter pungent, repulsive, fruity, nutty, floral, vegetal, and sweet chocolate) can be totally explained by aroma compounds with R²-adjusted values of 0.85 and greater. Samples from five geographical origins of cocoa bean were characterized by chemical compounds and sensory attributes. The aroma extracts were obtained by a two-step procedure involving (1) preliminary steam distillation under reduced pressure to evaluate the methylpyrazines generated in roasted cocoa powder by spectrophotometry (flavour index), and (2) Likens-Nickersons simultaneous steam distillation and solvent extraction method with added NaCl. The distilled compounds were separated by adsorption chromatography in six fractions depending on the polarity. A combined total of 114 compounds were detected by gas chromatography/mass spectrometry, 110 of which were identified. About 15 components in the mean milligrams per kilogram range (1.09–4.67 mg kg^–1) and 95 components in the mean micrograms per kilogram range (12–980 µg kg^–1) were quantified. The major components of cocoa aroma were 2,3,5,6-tetramethylpyrazine, benzaldehyde, 2-phenylacetaldehyde, acetophenone, 3-methylbutyric acid, 5-methyl-2-phenyl-2-hexenal, ethyl phenylacetate, and 3-hydroxy-2-methyl-4-pyrone (mean greater than 1.30 mg kg^–1).     

Characterization of the constituents and antioxidative activity of cocoa tea (Camellia ptilophylla)

Three main types of cocoa tea derived from Camellia ptilophylla, white cocoa tea (WCT), green cocoa tea (GCT) and black cocoa tea (BCT) were prepared. A comparison between cocoa tea and commonly consumed tea Biluochun cha (BLC), Longjing cha (Lj), Qimenhong cha (Qmh) and Yinghong 9^# (Yh9) was studied. Levels of catechins and purine alkaloids were determined by HPLC. Total polyphenol contents were determined by ferrous tartrate assay. Antioxidative activity was evaluated using ABTS decolorization assay, DPPH radical assay and ferric thiocyanate (FTC) method. The results showed that WCT contained the highest polyphenol content and showed the highest antioxidative activity. The radical scavenging ability of the tea extracts followed this order: WCT > GCT > Blc > Lj > BCT > Yh9 > Qmh. Tea extracts measured by the linoleic acid system also showed the same patterns of activity as the ABTS and DPPH method. A high correlation between polyphenol content and antioxidative activity of tea extracts was observed.     

Effect of sugar, cocoa particles and lecithin on cocoa butter crystallisation in seeded and non-seeded chocolate model systems

The effect of major chocolate ingredients (sugar, cocoa particles and lecithin), in combination with the two pre-crystallization techniques, seeding and non-seeding, was investigated with respect to the kinetics of cocoa butter crystallisation and the resulting microstructure. Confocal laser scanning microscopy (CLSM) was used to monitor microstructural evolution under dynamic thermal conditions. DSC measurements and image analysis were also applied in order to quantify the impacts of processing and formulation on microstructure. All ingredients and pre-crystallisation techniques considered proved to have a large impact on fat crystallisation kinetics and the resulting microstructure. Seeded samples tended to form multiple nucleation sites, inducing rapid growth of a crystal network. The non-seeded samples showed an altering structure, with some domains developing large spherical crystals while in other domains a more heterogeneous microstructure resulted. Lecithin showed a remarkable impact on crystallisation kinetics in both the seeded and non-seeded samples. For the seeded samples, the effect was most noteworthy in samples containing cocoa butter and sugar, where lecithin significantly reduced the induction time. In the absence of seeds, lecithin itself acted as the nucleation site for fat crystallisation.     

Analysis of deoxynivalenol and deoxynivalenol-3-glucosides content in Canadian spring wheat cultivars inoculated with Fusarium graminearum

Contamination of wheat grains with Fusarium mycotoxins and their modified forms is an important issue in wheat industry. The objective of this study was to analyse the deoxynivalenol (DON) and deoxynivalenol-3-glucosides (D3G) content in Canadian spring wheat cultivars grown in two locations, inoculated with a mixture of 3-acetyldeoxynivalenol (3-ADON)-producing Fusarium graminearum strains and a mixture of 15-acetlyldeoxynivalenol (15-ADON)-producing F. graminearum strains. According to the analysis of variance, significant differences were observed among the cultivars for Fusarium head blight (FHB) disease index, Fusarium-damaged kernel percentage (%FDK), DON content and D3G content. When the effect of chemotype was considered, significant differences were observed for FHB disease index, FDK percentage and DON content. The D3G content and D3G/DON ratio were not significantly different between the chemotypes, except for D3G content at the Winnipeg location. The Pearson correlation coefficient between DON and D3G was 0.84 and 0.77 at Winnipeg and Carman respectively. The highest D3G/DON ratio was observed in cultivars Carberry (44%) in Carman and CDC Kernen (63.8%) in Winnipeg. The susceptible cultivars showed lower D3G/DON ratio compared with the cultivars rated as moderately resistant and intermediate. The current study indicated that Canadian spring cultivars produce D3G upon Fusarium infection.