Verfahren zur Bestimmung verschiedener Mykotoxine in Lebensmitteln

Zusammenfassung Es wird ein einfaches Analysenverfahren für die Bestimmung von Aflatoxin B₁, B₂, G₁, G₂, M₁ und M₁, Sterigmatocystin, Penicillinsäure und Patulin in verschiedenen Lebensmitteln beschrieben.Die Mykotoxine lassen sich mit Essigsäureethylester extrahieren. Die Extrakte werden säulenchromatographisch an Mini-Kieselgel-Fertigsäulen vorgereinigt. Die Gehalte der einzelnen Mykotoxine werden nach der dünnschichtchromatographischen Auftrennung fluoro-densitometrisch ermittelt. Eine beträchtliche Erhöhung der Fluorescenzintensitäten kann durch Besprühen der Platten mit Paraffin nach dem Entwickeln erreicht werden. Die Nachweisgrenzen der fluorescierenden Mykotoxine und der fluorescierenden Derivate von Penicillinsäure und Sterigmatocystin können dadurch auf 1/10 bzw. 1/100 herabgesetzt werden.Patulin wird am empfindlichsten durch Messung der Fluorescenzlöschung bestimmt.

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A screening method for the determination of various mycotoxins in food

Summary A simple analytical method for multiple mycotoxins was developed for detecting aflatoxin B₁ B₂, G₁, G₂, M₁, and M₂, sterigmatocystin, penicillin acid, ochratoxin A and patulin. These mycotoxins were extracted with ethyl acetate. The extract was cleaned up by chromatography on silica mini-column. Each fraction was separated by thin-layer chromatography. The final evaluation of the TLC-plates was carried out by fluorodensitometry.A considerable increase in fluorescence intensity was achieved by spraying the plates with paraffin after development. The detection limits of fluorescent mycotoxins and the fluorescent derivates of penicillic acid and sterigmatocystin were lowered to 1:10 up to 1:100.Spots of patulin were measured by fluorescence quenching.

     

Zur Untersuchung von Milch und Milchprodukten auf die Aflatoxine B1, B2, G1, G2 und M1

Zusammenfassung Der vom Bundesgesundheitsamt vorgelegte Entwurf zur Bestimmung der Aflatoxine B₁, B₂, G₁ und G₂ wurde auf die zusätzliche Erfassung des Aflatoxins M₁ überpruft. Es wurde Wert darauf gelegt, die ursprüngliche Methode nur wenig zu ändern, um die zahlreichen Vorschläge zur Aflatoxin-Analytik nicht noch weiter zu vermehren. Mit verhältnisäßig geringen Änderungen können alle Aflatoxine, also B₁, B₂, G₁, G₂ and M₁ in flüissiger Milch, Milchpulver, Butter, Käse, Quark, Saline, Joghurt und Fruchtjoghurt quantitativ erfaßt werden.

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Investigations of aflatoxin B₁, B₂, G₁, G₂, and M₁ in milk and milk products

Summary The method proposed by the Federal Department of Health for the determination of aflatoxin B₁, B₂, G₁, and G₂ was tested for additional determination of aflatoxin M₁. With relatively small changes of the original method, all aflatoxins including. B₁, B₂, G₁, G₂, and M₁ can be determined quantitatively in milk, milk powder, butter, cheese, quark, cream, yoghurt and fruit yoghurt.

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37. Mitteilung: Zur Aflatoxinbildung in Milch und Milchprodukten.     

Occurrence of mycotoxins in feedstuffs of dairy cows and estimation of total dietary intakes

A survey was conducted to determine the occurrence of mycotoxins in feedstuffs of dairy cows in the Netherlands and to estimate total dietary intakes of these compounds. Twenty-four dairy farms were visited twice and samples taken of all diet ingredients. Feed intake data were collected by means of questionnaires. A total of 169 feed samples were collected and analyzed for 20 mycotoxins using a liquid chromatography tandem mass spectrometry multimethod. Silage and compound feed were the main diet ingredients, representing on average 67 and 23% of dry matter intake, respectively. Deoxynivalenol (DON), zearalenone, roquefortine C, and mycophenolic acid were the mycotoxins with the highest incidence. The incidence of DON in silage, compound feed, and feed commodity samples was 38 to 54%. The incidence of zearalenone in silage, compound feed, and feed commodity samples was 17 to 38%. The DON and zearalenone had a low incidence in forage samples and were not detected in ensiled by-product samples. Roquefortine C and mycophenolic acid were only detected in silage and ensiled by-product samples (incidence 7 to 19%). Fumonisins B₁ and B₂ were detected in 2 compound feed samples and one feed commodity sample. Aflatoxins B₁, B₂, G₁, and G₂, ochratoxin A, T-2 and HT-2 toxin, 3-acetyl-DON, 15-acetyl-DON, diacetoxyscirpenol, sterigmatocystin, fusarenon-X, ergotamine, and penicillinic acid were not detected in any of the samples. Average concentrations of DON, zearalenone, roquefortine C, and mycophenolic acid in complete diets were 273, 28, 114, and 54 μg/kg, respectively. Maximum concentrations were 969, 203, 2,211, and 1,840 μg/kg, respectively. Calculated average daily intakes of these mycotoxins were 5.0, 0.5, 2.0, and 0.9 mg/animal, respectively, and maximum daily intakes 19.3, 3.5, 38.9, and 32.3 mg/animal, respectively. Corn silage was the major source of all 4 of these mycotoxins in the diet. Extremely high concentrations of roquefortine C and mycophenolic acid (up to 45 and 25 mg/kg, respectively) were detected in visibly molded areas in surface layers of corn silage. These areas appeared to be the main source of roquefortine C and mycophenolic acid in the diet. Because carry-over of DON, zearale-none, roquefortine C, and mycophenolic acid into milk is negligible, their occurrence in feedstuffs is not considered of significant concern with respect to the safety of dairy products for consumers. Potential implications for animal health are discussed.     

Occurrence of fumonisins and aflatoxins in cereals from markets of Hebei province of China

Fumonisins B₁, B₂ and B₃ (FB₁, FB₂ and FB₃) and aflatoxins B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁) and G₂ (AFG₂) are both major mycotoxins of food concern, because of their wide range of concentration and possible co-occurrence. Therefore, a contamination survey in corn and wheat flour by liquid chromatography–tandem mass spectrometry was carried out. Quantification of fumonisins and aflatoxins was based on internal calibration (by the use of ¹³C₃₄-fumonisin) and external calibration, respectively. Fumonisins were detected in 95% of corn samples and in 7% of wheat flour samples, with the mean level (FB₁?+?FB₂?+?FB₃) of 441?µg?kg^?1 and 0.09?µg?kg^?1, respectively. Low levels of aflatoxins were detected in 37% of the samples with a mean level (B₁?+?B₂?+?G₁?+?G₂) of 0.12?µg?kg^?1. Fumonisins and aflatoxins were not detected in 29% of the samples analysed. Simultaneous occurrence of fumonisins and aflatoxins was observed in 12% of samples.     

SCREENING AND QUANTIFICATION OF AFLATOXINS AND OCHRATOXIN A IN DIFFERENT CEREALS CULTIVATED IN ROMANIA USING THIN-LAYER CHROMATOGRAPHY-DENSITOMETRY

ABSTRACT

The main focus of our study was to implement a rapid, inexpensive and reliable method that could be utilized to check the cereals for safety (i.e., screening for total aflatoxins, as well as individual B₁, B₂, G₁, G₂ aflatoxins and ochratoxin A). We developed a protocol by which we were able to isolate mycotoxins from cereals collected from different regions of Romania. After extraction in chloroform, the mycotoxins were separated by thin‐layer chromatography (TLC) and quantified using densitometry. Forty‐three samples of different cereals (wheat, maize, rye and Triticale) were analyzed. Twenty‐five of the 43 samples (58.14% of the total number) were found to be contaminated with different mycotoxins in various concentrations: aflatoxin B₁ (1.6–5.7 µg/kg), aflatoxin B₂ (0.89–4 µg/kg), aflatoxin G₁ (1.2–5.76 µg/kg), aflatoxin G₂ (0.96–3.4 µg/kg) and/or 4.3–30 µg/kg ochratoxin A. The concentration of total aflatoxin contamination ranged from 11.2 to 10.8 µg/kg. Among the different cereals, the highest number of contaminated samples was found to be in the wheat samples (62.5%). The method outlined in this study has been adopted already by our laboratory for current analyses of mycotoxins. The results obtained using this method is in compliance with the strict regulatory guidelines developed both in Romania, as well as in the European Union.

PRACTICAL APPLICATIONS

Thin‐layer chromatography (TLC) is a rapid, inexpensive and convenient method that can be used routinely to screen for mycotoxins. This article describes the detailed procedures for the extraction, purification and quantification of aflatoxins and ochratoxin A, using TLC. Using this method one can identify concomitantly five different mycotoxins and by coupling it with densitometry a quantitative determination is also possible. Therefore, this could become a routine technique in regional laboratories responsible for checking agrifood safety.     

Identification and quantification of fungi and mycotoxins from Pu-erh tea

Pu-erh tea originates from the province of Yunnan in south-western China. As this tea is produced by so called Aspergillus post-fermentation the question arises which molds and mycotoxins may be found in this tea. In total 36 samples of Pu-erh tea were investigated for their content of filamentous fungi and the mycotoxins aflatoxins B₁, B₂, G₁, and G₂, fumonisins B₁, B₂, and B₃, and ochratoxin A. Fungi were isolated from all samples in a concentration of 1.0 × 10¹ to 2.6 × 10⁶ colony forming units (cfu)/g tea, all together 19 fungal genera and 31 species were identified. The most prevalent species were Aspergillus acidus and Aspergillus fumigatus, followed by Zygomycetes and Penicillium species. Aflatoxins and fumonisins were not found in the samples investigated, ochratoxin A was detected in 4 of 36 teas (11.1%).     

Aflatoxins in Foods and Feedstuffs. Part 3. Fluorodensitometric method for determination of aflatoxins M1 and M2 in presence of aflatoxins B1, B2, G1 and G21

The method of aflotoxins M₁ and M₂ determination in presence of aflatoxins B₁, B₂, G₁ and G₂ is presented. The fluorescence properties of aflatoxin M₁ and M₂ are discribed. A simple method of aflatoxin M extraction is proposed. Results of different tests for confirmation of aflatoxins M are discussed. During control of powdered milk from three factories aflatoxins M were not detected in final products.     

Multi-mycotoxins Analysis in Dried Fruit by LC/MS/MS and a Modified QuEChERS Procedure

A sensitive and reliable multi-mycotoxin method was developed for the simultaneous determination of 16 toxicological important mycotoxins, such as aflatoxins B₁, B₂, G₁, and G₂; enniatins A, A₁, B, and B₁; beauvericin; ochratoxin A; fumonisin B₁, B₂, and B₃; diacetoxyscirprenol; HT-2; and T-2 toxin in dried fruits using liquid chromatography combined with electrospray ionization-triple quadrupole tandem-mass spectrometry. Mycotoxins have been extracted from the samples using a modified quick, easy, cheap, effective, rugged, and safe procedure. The method was based on a single extraction with acidified acetonitrile, followed by partitioning with salts, avoiding any further clean-up step. Limits of detections ranged from 0.08 to 15 μg kg^?1 and limits of quantification ranged from 0.2 to 45 μg kg^?1, which were below the legal limit set by the European Union for the legislated mycotoxines. The recoveries in spiked samples ranged from 60 to 135 % except for beauvericin using matrix-matched calibration curves for quantification, with good inter- and intraday repeatability (respective relative standard deviation ≤20 and 9 %). The developed method was applied to 15 commercial dried fruits: raisins, figs, apricots, plums, and dates purchased in local markets from Spain. Among the mycotoxins studied, enniantins and aflatoxins were the most predominant mycotoxins.     

2013—2015年广州市市售普洱茶真菌毒素污染调查

目的 了解广州市市售普洱茶真菌毒素污染状况及评估健康风险。方法 2013—2015年,在广州市批发市场、零售店、超市、餐饮单位、网店和茶博会6类场所采集不同生产工艺、包装形态、生产年份、原料产地、价格的普洱茶样品432份,使用多功能净化柱净化-柱后衍生高效液相色谱法测定黄曲霉毒素(B₁、B₂、G₁、G₂),高效液相色谱-质谱法测定雪腐镰刀菌烯醇、脱氧雪腐镰刀菌烯醇和玉米赤霉烯酮。结果 432份普洱茶样品黄曲霉毒素(B₁、B₂、G₁、G₂)、雪腐镰刀菌烯醇、脱氧雪腐镰刀菌烯醇、玉米赤霉烯酮7个项目检测结果均低于所用检测方法的检出限。结论 本次对广州市市售普洱茶的调查研究,未检出真菌毒素。     

LC–MS/MS multi-method for mycotoxins after single extraction,with validation data for peanut,pistachio, wheat,maize, cornflakes,raisins and figs

Mycotoxin analysis is usually carried out by high performance liquid chromatography after immunoaffinity column cleanup or in enzyme-linked immunosorbent assay tests. These methods normally involve determination of single compounds only. EU legislation already exists for the aflatoxins, ochratoxin A and patulin in food, and legislation will come into force for deoxynivalenol, zearalenone and the fumonisins in 2007. To enforce the various legal limits, it would be preferable to determine all mycotoxins by routine analysis in different types of matrices in one single extract. This would also be advantageous for HACCP control purposes. For this reason, a multi-method was developed with which 33 mycotoxins in various products could be analysed simultaneously. The mycotoxins were extracted with an acetonitrile/water mixture, diluted with water and then directly injected into a LC–MS/MS system. The mycotoxins were separated by reversed-phase HPLC and detected using an electrospray ionisation interface (ESI) and tandem MS, using MRM in the positive ion mode, to increase specificity for quality control. The following mycotoxins could be analysed in a single 30-min run: Aflatoxins B₁, B₂, G₁ and G₂, ochratoxin A, deoxynivalenol, zearalenone, T-2 toxin, HT-2 toxin, α-zearalenol, α-zearalanol, β-zearalanol, sterigmatocystin, cyclopiazonic acid, penicillic acid, fumonisins B₁, B₂ and B₃, diacetoxyscirpenol, 3- and 15-acetyl-deoxynivalenol, zearalanone, ergotamin, ergocornin, ergocristin, α-ergocryptin, citrinin, roquefortin C, fusarenone X, nivalenol, mycophenolic acid, alternariol and alternariol monomethyl ether. The limit of quantification for the aflatoxins and ochratoxin A was 1.0 µg kg^?1 and for deoxynivalenol 50 µg kg^?1. The quantification limits for the other mycotoxins were in the range 10–200 µg kg^?1. The matrix effect and validation data are presented for between 13 and 24 mycotoxins in peanuts, pistachios, wheat, maize, cornflakes, raisins and figs. The method has been compared with the official EU method for the determination of aflatoxins in food and relevant FAPAS rounds. The multi-mycotoxin method has been proven by the detection of more than one mycotoxin in maize, buckwheat, figs and nuts. The LC–MS/MS technique has also been applied to baby food, which is subject to lower limits for aflatoxin B₁ and ochratoxin A, ergot alkaloids in naturally contaminated rye and freeze-dried silage samples.     

Development of a new method for the simultaneous determination of 21 mycotoxins in coffee beverages by liquid chromatography tandem mass spectrometry

A new method for the simultaneous detection of 21 mycotoxins (ochratoxin A, aflatoxin B₁, aflatoxin B_2, aflatoxin G₁, aflatoxin G₂, sterigmatocystin, nivalenol, deoxynivalenol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, diacetoxyscirpenol, neosolaniol, HT-2 toxin, T-2 toxin, fumonisin B₁, fumonisin B₂, enniatin A, enniatin A₁, enniatin B, enniatin B₁, and beauvericin) in coffee beverages was internally validated. The method is based on liquid/liquid extraction with a mixture of ethyl acetate/formic acid (95:5 v/v) and detection using triple quadrupole (QqQ) and ion trap (IT) liquid chromatography tandem mass spectrometry. The limits of detection and quantification were 0.02 to 39.64 μg/kg, respectively, and the correlation coefficients were optimal for all mycotoxins (R² ≥ 0.992). The recovery values ranged from 72% to 97%. The developed method was demonstrated in six real samples of roasted and instant coffee, caffeinated and decaffeinated coffee, and coffee with sugar added. The analyses indicate the presence of the studied mycotoxins in coffee beverages at μg/kg concentrations. Ochratoxin A, a mycotoxin that is regulated in coffee, was detected in two samples under the maximum limit established by a European legislation (CE1881/2006).     

Aflatoxins and cyclopiazonic acid in feed and milk from dairy farms in São Paulo,Brazil

The occurrence of aflatoxins (AF) B₁, B₂, G₁, G₂ and cyclopiazonic acid (CPA) in feeds, and AFM₁ and CPA in milk was determined in dairy farms located in the northeastern region of São Paulo state, Brazil, between October 2005 and February 2006. AF and CPA determinations were performed by HPLC. AFB₁ was found in 42% of feed at levels of 1.0–26.4 µg kg^?1 (mean: 7.1 ± 7.2 µg kg^?1). The concentrations of AFM₁ in raw milk varied between 0.010 and 0.645 µg l^?1 (mean: 0.104 ± 0.138 µg l^?1). Only one sample was above the tolerance limit adopted in Brazil (0.50 µg l^?1) for AFM₁ in milk. Regarding CPA in feed, six (12%) samples showed concentrations of 12.5–153.3 µg kg^?1 (mean: 57.6 ± 48.7 µg kg^?1). CPA was detected in only three milk samples (6%) at levels of 6.4, 8.8 and 9.1 µg l^?1. Concentrations of aflatoxins and CPA in feed and milk were relatively low, although the high frequency of both mycotoxins indicates the necessity to continuously monitor dairy farms to prevent contamination of feed ingredients.     

Mycotoxins in cereal grain. Part XI. Simple multidetection procedure for determination of 11 mycotoxins in cereals

A simple method for the simultaneous detection of the 11 mycotoxins aflatoxins (B₁, B₂, G₁, G₂), ochratoxin A, zearalenone, sterigmatocystin, citrinin, penicillic acid, T-2 toxin and rubratoxin B is reported. The elaborated method was tested for all cited mycotoxins extracted from 5 cereal species (rye, barley, wheat, oats and corn) spiked with mycotoxins standards. Different chromatoplates, developing solvents and spraying reagents were tested. New tests and modification of known confirmatory tests, recovery and detection limits are reported.     

Multiclass mycotoxin analysis in edible oils using a simple solvent extraction method and liquid chromatography with tandem mass spectrometry

ABSTRACT

A simple and rapid method for the simultaneous determination of 11 mycotoxins – aflatoxins B₁, B₂, G₁ and G₂; fumonisins B₁, B₂ and B₃; ochratoxin A; zearalenone; deoxynivalenol; and T-2 toxin – in edible oils was established using liquid chromatography tandem mass spectrometry (LC-MS/MS). In this study, QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe), QuEChERS with dispersive liquid–liquid microextraction, and solvent extraction were examined for sample preparation. Among these methods, solvent extraction with a mixture of formic acid/acetonitrile (5/95, v/v) successfully extracted all target mycotoxins. Subsequently, a defatting process using n-hexane was employed to remove the fats present in the edible oil samples. Mass spectrometry was carried out using electrospray ionisation in polarity switching mode with multiple reaction monitoring. The developed LC-MS/MS method was validated by assessing the specificity, linearity, recovery, limit of quantification (LOQ), accuracy and precision with reference to Commission Regulation (EC) 401/2006. Mycotoxin recoveries of 51.6–82.8% were achieved in addition to LOQs ranging from 0.025 ng/g to 1 ng/g. The edible oils proved to be relatively uncomplicated matrices and the developed method was applied to 9 edible oil samples, including soybean oil, corn oil and rice bran oil, to evaluate potential mycotoxin contamination. The levels of detection were significantly lower than the international regulatory standards. Therefore, we expect that our developed method, based on simple, two-step sample preparation process, will be suitable for the large-scale screening of mycotoxin contamination in edible oils.     

Comparison of two Analytical Methods (ELISA and LC-MS/MS) for Determination of Aflatoxin B1 in Corn and Aflatoxin M1 in Milk

The aim of this paper is to assess the closeness of agreement between results of ELISA and LC-MS/MS methods for determination of aflatoxin B₁ in corn and aflatoxin M₁ in milk. Samples of corn (n=100) and milk (n=250) were simultaneously analyzed using ELISA and LC-MS/MS methods, after the severe drought that affected Serbia in summer 2012 resulting in occurrence of aflatoxin B1 in corn and aflatoxin M₁ in milk. Regression analysis showed higher level of agreement between aflatoxin B₁ samples (R²=0.994), compared to aflatoxin M₁ samples (R²=0.920). However, both techniques were satisfactory in meeting the requirements for official control purposes.     

Screening for mycotoxins in the inoculum used for production of attiéké, a traditional Ivorian cassava product

For the production of attiéké, a fermented staple food from the Ivory Coast, a traditional inoculum consisting of mould-covered cassava tubers is used. Nineteen inoculum and five attiéké samples of different provenance were tested for contamination with the major mycotoxins ochratoxin (OTA), aflatoxin (AF) B₁, B₂, G₁, G₂ and deoxynivalenol (DON). Selected samples were also subjected to an effect-based bioassay visualising noxious impact on a test organism, the bacterium Vibrio fischeri. Neither AF nor DON were detected and only trace amounts of up to 0.2 μg/kg OTA were present in some samples, but extract fractions inhibiting the biological test system were discovered through the bioassay. Our findings suggest that the most potent fungal toxins do not present a general health hazard to attiéké consumers. Unidentified bioactive microbial metabolites, however, might still be present and affect the human or animal organism in an unknown manner.     

Effects of chronic low-dose aflatoxin B1 exposure in lactating Florida dairy goats

In the past few years there has been a growing trend in the prevalence of aflatoxins, attributable to climate change, in substances destined for animal feeding, together with an increase in dairy product consumption. These facts have triggered great concern in the scientific community over milk pollution by aflatoxin M₁. Therefore, our study aimed to determine the transfer of aflatoxin B₁ from the diet into milk as AFM₁ in goats exposed to different concentrations of AFB₁, and its possible effect on the production and serological parameters of this species. For this purpose, 18 goats in late lactation were divided into 3 groups (n = 6) and exposed to different daily doses of aflatoxin B₁ (T1 = 120 µg; T2 = 60 µg, and control = 0 µg), during 31 d. Pure aflatoxin B₁ was administered 6 h before each milking in an artificially contaminated pellet. The milk samples were taken individually in sequential samples. Milk yield and feed intake were recorded daily, and a blood sample was extracted on the last day of exposure. No aflatoxin M₁ was detected, either in the samples taken before the first administration, or in the control group ones. The aflatoxin M₁ concentration detected in the milk (T1 = 0.075 µg/kg; T2 = 0.035 µg/kg) increased significantly on a par with the amount of aflatoxin B₁ ingested. The amount of aflatoxin B₁ ingested did not have any influence on aflatoxin M₁ carryover (T1 = 0.066% and T2 = 0.060%), these being considerably lower than those described in dairy goats. Thus, we concluded that the concentration of aflatoxin M₁ in milk follows a linear relationship with respect to the aflatoxin B₁ ingested, and that the aflatoxin M₁ carryover was not affected by the administration of different aflatoxin B₁ doses. Similarly, no significant changes in the production parameters after chronic exposure to aflatoxin B₁ were observed, revealing a certain resistance of the goat to the possible effects of that aflatoxin.     

An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for aflatoxin M1 in milk and infant milk products

A sensitive and specific monoclonal antibody (Mab) against aflatoxin M₁ (AFM₁), named as 2C9, was selected by semi-solid HAT medium. It exhibited high affinity for AFM₁ of 1.74 × 10⁹ L/mol and no cross-reactivity to aflatoxin B₁, B₂, G₁ and G₂. Based on the antibody, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for AFM₁ in milk and infant milk products. Assays were performed in the AFM₁-BSA coated (0.0625 μg/mL) ELISA format in which the antibody was diluted 1:10,000. Several physicochemical factors (pH, ionic strength and blocking solution) that influence assay performance were optimised. Finally, the limits of detection were 3 ng/L for milk and 6 ng/L for milk-based cereal weaning food, inter-assay and intra-assay variations were less than 10%, and the recovery ranged from 91% to 110%. Thirty samples were analysed, and concordant results were obtained when the data were compared with a reference high-performance liquid chromatography method.     

Behavior of Aflatoxin during the Manufacture, Ripening and Storage of Manchego-type Cheese

The behavior of aflatoxins B₁, B₂, G₁, G₂, and M₁ was investigated during manufacture, ripening, refrigeration, and frozen storage of Manchego-type cheese. More aflatoxins (per weight or volume) occurred in curd than in whey, although total quantity was greater in whey. Aflatoxins B₁ B₂, and M₁ remained stable during ripening, but G₁ and G₂ levels increased. Concentration of B₁, B₂, and M₁ decreased after 15 and 30 days refrigeration storage, but after 60 days 100% recovery occurred in the inner portion and only 60% in the outer. Concentrations of G₁ and G₂ also decreased during the first 30 days, but on day 60 an increase was observed, 200% in the inner and 120% in the outer portions. Aflatoxins were stable after 90 days in frozen storage.     

Quantitative determination of mycotoxins in urine by LC-MS/MS

Naturally occurring mycotoxins are responsible for a wide array of adverse health effects. The measurement of urinary mycotoxin levels is a useful means of assessing an individual's exposure, but the development of sensitive and accurate analytical methods for detecting mycotoxins and their metabolites in urine samples is challenging. Urinary mycotoxins are present in low pg ml^–1 concentrations, and the chromatographic identification of their metabolites can be obscured by other endogenous metabolites. We developed an analytical method focused on the selection of two appropriate multiple-reaction monitoring transition for unambiguous identification and quantification of carcinogenic aflatoxin M₁ (AFM₁), ochratoxin A (OTA) and fumonisin B₁, B₂ (FB₁, FB₂) in urine samples from a small volunteer group in a pilot study. AFM₁, OTA, FB₁ and FB₂ were concentrated selectively, interfering substances were removed using an immunoaffinity column (IAC), and mycotoxins were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in combination with a stable-isotope standard-dilution assay (SIDA). The method was sensitive enough to measure mycotoxins and their metabolites at pg ml^–1 levels in urine. The combination of LC-MS/MS and SIDA was critical to distinguishing pseudo-OTα interference from genuine OTα. Twelve urine samples contained OTA ranging from 0.013 to 0.093 ng ml^?1 (mean = 0.031 ng ml^?1). AFM₁ were detected in one sample at a 0.002 ng ml^?1 level, while FB₁ and FB₂ were undetectable in all 12 samples. None of the samples in this pilot study contained a detectable level of OTα, despite the presence of OTA, and this may suggest the need for further epidemiological investigation of OTA exposure in the Korean population.