Electrochemical Reduction of Azo Dyes Mimicking their Biotransformation to More Toxic Products.

INTRODUCTION: Some azo dyes, including Sudans I-IV and Para Red, are genotoxic and may be biotransformed to cancerogenic aromatic amines. They are banned as food and feed additives, but their presence has been detected in food. Aromatic amines are also considered potentially toxic. Online EC-MS is a promising tool to study the transformation mechanisms of xenobiotics such as azo dyes. The aim of the study was to investigate emulation of how azo dyes are enzymatically transformed to amines with EC-MS. MATERIAL AND METHODS: The reduction reactions of five azo dyes (Sudans I-IV and Para Red) were conducted using a glassy carbon working electrode and 0.1% formic acid in acetonitrile. Reduction results were compared with the literature and in silico to select preliminary candidates for metabolites. The LC-MS/MS method was used to confirm results obtained by electrochemical reactor. RESULTS: A limited number of pre-selected compounds were confirmed as azo dyes metabolites - aniline for Sudan I, aniline and 4-aminoazobenzene for Sudan III, o-toluidine for Sudan IV, and 4-nitroaniline for Para Red. No metabolites were found for Sudan II. CONCLUSIONS: Electrochemistry-mass spectrometry was successfully applied to azo dyes. This approach may be used to mimic the metabolism of azo dyes, and therefore predict products of biotransformation.

Pilot Study for The Presence of Fungal Metabolites in Sheep Milk from First Spring Milking.

INTRODUCTION: A mini-study of 20 raw milk samples was conducted to examine the spectrum of fungal metabolites in sheep milk from the first spring milking. MATERIAL AND METHODS: Samples were collected from randomly selected ewes in two animal flocks from the Bieszczady Mountains and analysed using liquid chromatography-tandem mass spectrometry. RESULTS: Out of ~700 bacterial, fungal, and plant metabolites tested for, only one mycotoxin - Enniatin B - was detected in sheep milk samples (18/20; 0.0055-0.0121 µg/kg; 0.0078 µg/kg average). CONCLUSIONS: The results indicated that there was no high-level exposure to fungal metabolites via consumption of raw sheep milk during the sample collection period.

UDP-Glucosyltransferases from Rice, Brachypodium, and Barley: Substrate Specificities and Synthesis of Type A and B Trichothecene-3-O-ß-d-glucosides.

Trichothecene toxins are confirmed or suspected virulence factors of various plant-pathogenic Fusarium species. Plants can detoxify these to a variable extent by glucosylation, a reaction catalyzed by UDP-glucosyltransferases (UGTs). Due to the unavailability of analytical standards for many trichothecene-glucoconjugates, information on such compounds is limited. Here, the previously identified deoxynivalenol-conjugating UGTs HvUGT13248 (barley), OsUGT79 (rice) and Bradi5g03300 (Brachypodium), were expressed in E. coli, affinity purified, and characterized towards their abilities to glucosylate the most relevant type A and B trichothecenes. HvUGT13248, which prefers nivalenol over deoxynivalenol, is also able to conjugate C-4 acetylated trichothecenes (e.g., T-2 toxin) to some degree while OsUGT79 and Bradi5g03300 are completely inactive with C-4 acetylated derivatives. The type A trichothecenes HT-2 toxin and T-2 triol are the kinetically preferred substrates in the case of HvUGT13248 and Bradi5g03300. We glucosylated several trichothecenes with OsUGT79 (HT-2 toxin, T-2 triol) and HvUGT13248 (T-2 toxin, neosolaniol, 4,15-diacetoxyscirpenol, fusarenon X) in the preparative scale. NMR analysis of the purified glucosides showed that exclusively ß-D-glucosides were formed regio-selectively at position C-3-OH of the trichothecenes. These synthesized standards can be used to investigate the occurrence and toxicological properties of these modified mycotoxins.

Absence of evidence or evidence of absence? A transfer and depletion study of Sudan I in eggs.

Sudan I is a carcinogenic industrial azo-dye, forbidden for use in food. However, it has been detected in food on several occasions, such as in paprika, used in animal husbandry to enhance egg yolk colour. Therefore, an animal experiment was designed to simulate the transfer of Sudan I to eggs after its unintentional administration to laying hens. A group of laying hens (n=18) received feed contaminated with Sudan I at the raising concentrations: 0.45mg/kg, 4.97mg/kg and 42.1mg/kg. Residues of Sudan I were detected in egg yolks (0.29±0.03µg/kg, mean±SD) only after the administration of the feed contaminated with the dye at the highest concentration. The determined concentrations were much lower than expected based on the compound's lipophilicity. In conclusion, the transfer of Sudan I to eggs was limited and strongly dependent on its concentration in feed.

Multiresidue determination of veterinary medicines in lyophilized egg albumen with subsequent consumer exposure evaluation.

The process of lyophilization causes that the veterinary drugs residues present in egg albumen do not decompose, as it takes place during the process of high-temperature drying. Thus, lyophilized albumen may be a potential source of their residues for consumers. As a consequence, reliable methods for the determination of veterinary medicinal products from egg albumen are needed. The method for the determination of 85 analytes in lyophilized egg albumen was developed and successfully validated. The recoveries were between 84 and 110%, within laboratory repeatability and reproducibility - in the range of 3.29-16.8% and -5.93 to 19.3%. The presence of enrofloxacin and doxycycline was confirmed in real egg albumen samples. The concentrations ranged from 5.65-596µg/kg for doxycycline to 0.89-134µg/kg for enrofloxacin. Nevertheless, the evaluated daily intake and % of the ADI (Acceptable Daily Intake) received by the consumers' were at a toxicologically accepted level.

Simultaneous Determination of Ten Illegal Azo Dyes in Feed by Ultra-high Performance Liquid Chromatography Tandem Mass Spectrometry.

INTRODUCTION: The paper presents the method of simultaneous determination of 10 illegal azo dyes in feed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry technique. MATERIAL AND METHODS: The dyes were extracted with hexane, evaporated to dryness, and analysed. Separation was achieved in 7 min in a gradient elution using acetonitrile (A) and 0.1% formic acid (B) as a mobile phase. RESULTS: The validation results showed the repeatability of the method, which was evaluated at three levels (50, 500, and 5,000 µg/kg). All the matrix calibration curves for the working ranges were linear (R2 0.9904 to 1.0), the repeatability was between 2.1% and 24%, and recoveries ranged from 77.9% to 120%. The LOD and LOQ were at 1-2 and 5-10 µg/kg for different dyes, respectively. Furthermore, the method was applied in the homogeneity tests of the in-house prepared feed containing Sudan I at the levels of 0.5, 5, and 50 mg/kg. CONCLUSIONS: A sensitive, selective, and fast multiresidue method was successfully developed and validated. Its robustness was confirmed by the analysis of an experimental feed containing Sudan I.

Occurrence of Ochratoxin a in Animal Tissues and Feeds in Poland in 2014-2016.

INTRODUCTION: Ochratoxin A (OTA) is a toxic metabolite mainly produced by Aspergillus spp. and Penicillum spp. fungi. Research on the contamination of cereals, complete feeds, and tissues with this mycotoxin has indicated that it can be a toxicological problem impacting animal health and food safety in temperate climes. OTA contamination mainly besets the global pig industry, necessitating the monitoring of feeds and animal tissues. The aim of the study was to present the results of the official monitoring of OTA in animal tissues and feeds in Poland in 2014-2016 and determine the possible correlation between the presence of OTA in different types of samples. MATERIAL AND METHODS: The presence of ochratoxin A was determined using accepted procedures based on liquid chromatography with fluorescence detection after immunoaffinity column clean-up. Determination of OTA was afforded in the range of 0.3 µg/kg to 300 µg/kg in complete feeds and from 0.2 µg/kg to 150 µg/kg in the kidneys, liver, and muscles. RESULTS: Over the three year span, about 23.5% of the animal tissues samples were contaminated by ochratoxin A. In the 2014 survey, 10% of the sample tissues contained 5-10 µg/kg (only one sample above 10 µg/kg), and in 2015 and 2016, 24% of samples showed levels above the limit of quantification 0.2 µg/kg, while none of the samples exceeded the established provisional action level of 5 µg/kg for animal tissues. The animal feed analysis showed that 9% was contaminated with ochratoxin A above the limit of quantification of 0.3µg/kg. In 2% of feed samples the OTA concentration was greater than 50 µg/kg. CONCLUSION: The results confirm the appropriacy of OTA contamination monitoring and help to increase food safety.

Multiresidue method for the simultaneous determination of veterinary medicinal products, feed additives and illegal dyes in eggs using liquid chromatography-tandem mass spectrometry.

A multiclass method was developed for the simultaneous determination of 120 analytes in fresh eggs. The method covers the analytes from the groups of tetracyclines (6), fluoroquinolones (11), sulphonamides (17), nitroimidazoles (9), amphenicols (2), cephalosporins (7), penicillins (8), macrolides (8), benzimidazoles (20), coccidiostats (14), insecticides (3), dyes (12) and others (3). Samples were extracted using 0.1% formic acid in acetonitrile:water (8:2) with the addition of EDTA and cleaned using solid phase extraction with Hybrid SPE cartridges. The chromatographic separation was achieved on C8 column using mobile phase consisting of (A) methanol:acetonitrile (8:2) - (B) 0.1% formic acid in a gradient mode. Validation results according to the Commission Decision 2002/657/EC are as follows: linearity (r⩾0.99), recovery (75-108%), repeatability (CV 1.60-15.9%), reproducibility (CV 2.60-15%), decision limit (CCα 2.25-1156 µg/kg) and detection capability (CCß 2.04-1316 µg/kg). The presented method was used for analysis of 150 real eggs samples taken from monitoring control program.