Survey of the enniatins and beauvericin in raw and UHT cow's milk in Poland.

Introduction: The enniatins A, A1, B and B1 (ENNs) and beauvericin (BEA) are structurally related compounds produced by Fusarium species. They occur as contaminants in cereals, such as wheat, barley and maize. They are called "emerging mycotoxins", because they have been reported in feed and food and their toxic effects are not fully known. Data on their levels in food (especially in milk) are limited. The study aimed to evaluate the occurrence of ENNs and BEA in milk. Material and Methods: A total of 103 bovine milk samples (76 of raw milk and 27 of UHT milk) were collected from different parts of Poland and analysed using liquid chromatography-tandem mass spectrometry. Results: Among the 76 raw milk samples, 31 (41%) and 15 (20%) samples were contaminated with ENN B and with BEA, respectively. No contamination with other enniatins was found. The highest concentration of BEA was found in raw milk and was 6.17 µg kg-1. Out of the 27 samples of UHT milk, 16 (59%) were contaminated with ENN B at concentrations ranging from 0.157 µg kg-1 to 0.587 µg kg-1 (limit of quantification (LOQ) 0.098 µg kg-1). Beauvericin was detected in 9 UHT milk samples (33%) at concentrations ranging from 0.101 µg kg-1 to 1.934 µg kg-1 (LOQ 0.095 µg kg-1). Conclusion: This study demonstrated constant but low milk contamination in Poland with ENN B and BEA. The analysis of milk samples revealed that the emerging mycotoxins ENN B and BEA were measured in trace amounts. It does not suggest any immediate risk to milk consumers; however, it is unknown whether long-term exposure to low levels of toxins may be harmful.

Inter-laboratory validation of liquid chromatography-tandem mass spectrometry multi-mycotoxin determination in animal feed - method transfer from the reference laboratory to regional laboratories.

Introduction: The results are presented of the inter-laboratory validation of a liquid chromatography-tandem mass spectrometry method for the determination of eight mycotoxins (aflatoxin B1, deoxynivalenol, fumonisin B1, fumonisin B2, ochratoxin A, toxin T-2, toxin HT-2 and zearalenone) in animal feeds. Material and Methods: This study was an essential part of the method's transfer from the National Reference Laboratory to six regional laboratories in Poland working in the official survey of mycotoxins in feed. The laboratories received a batch of standard solutions, blank samples and quality control materials on which to perform analysis with one procedure and different liquid chromatography-tandem mass spectrometry conditions. Results: The validation results show good precision (reproducibility coefficient of variation 3.7-20.5%) and accuracy of the method (recovery 89-120% and trueness 94-103%) and sufficient skills of the laboratory personnel. Conclusion: The study is an example of the successful transfer of the method among laboratories.

Simultaneous Determination of Deoxynivalenol, Its Modified Forms, Nivalenol and Fusarenone-X in Feedstuffs by the Liquid Hromatography-Tandem Mass Spectrometry Method.

A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3Ac-DON), 15-acetyldeoxynivalenol (15Ac-DON), DON-3-glucoside (DON-3Glc) nivalenol and fusarenone-X in feedstuffs. Different techniques of sample preparation were tested: solid-liquid-extraction, QuEChERS, solid phase extraction with OASIS HLB columns or immunoaffinity columns and a Mycosep 225 Trich column. None of the six immunoaffinity columns tested showed cross-reactivity to all of the mycotoxins. Surprisingly, the results show that if the immunoaffinity columns bound 3Ac-DON, then they did not bind 15Ac-DON. The most efficient sample preparation was achieved with a Mycosep 225 Trich column clean-up. The chromatography was optimised to obtain full separation of all analytes (including 3Ac-DON and 15Ac-DON isomeric form). The validation results show the relative standard deviations for repeatability and reproducibility varied from 4% to 24%. The apparent recovery ranged between 92% and 97%, and the limit of quantification described a 1.30 to 50 µg/kg range. The method trueness was satisfactory, as assessed by a proficiency test and analysis of reference material. A total of 99 feed samples were analysed by the developed method, revealing the presence of DON and DON-3Glc in 85% and 86% of examined animal feeds, respectively at concentrations between 1.70 and 1709 µg/kg. The ratios DON-3Glc to DON in the surveyed feedstuffs were from a low of 3% to high of 59%.

Could mycotoxigenic Fusarium sp. play a role in ulcerative dermal necrosis (UDN) of brown trout (Salmo trutta morpha trutta)?

Fusarium infections have been reported in aquatic animals, but are still poorly investigated in wild salmonids. The aim of the study was to determine the impact of the fungi and their toxins on the health status of brown trout (Salmo trutta morpha trutta) migrating from the Baltic Sea to the freshwater. Individuals from the wild brown trout population exhibiting ulcerative skin lesions were collected from the Slupia River in Poland and subjected to microbiological, histopathological, and hematological examinations, as well as toxicological analysis for a presence of mycotoxins. The results of microflora isolation from the brown trout skin samples revealed the presence of conditionally pathogenic bacteria and fungi classified by molecular techniques as Fusarium spp. Toxicological analysis allowed for detection of zearalenone (ZEN) in the liver, kidney, and gastrointestinal tract of the fish. In several cases, there was α-zearalenone (α-ZEL) identified at trace levels in the liver, as well as sterigmatocystin and enniatin B at low levels in the kidney and the liver. Histopathological examination revealed the presence of fungal hyphae disrupting the epidermis and penetrating into the necrotic dermis and hypodermis. The decreased values of the blood parameters, i.e., hemoglobin concentration (HGB), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and white blood cell count (WBC), were indicative of osmoregulation failure being a consequence of the skin damage. The results of the study provide new information regarding Fusarium sp. infection in brown trout and serve as the basis for further research on the potential impact of the fungi and their mycotoxins on the Baltic salmonid population, including their role in ulcerative dermal necrosis.

Multiple mycotoxins analysis in animal feed with LC-MS/MS: Comparison of extract dilution and immunoaffinity clean-up.

The aim of this study was a performance comparison of two clean-up procedures (dilutions versus immunoaffinity columns) in the simultaneous determination of eight mycotoxins (aflatoxin B1, deoxynivalenol, fumonisin B1 & B2, ochratoxin A, toxin T-2 & HT-2 and zearalenone) in the animal feed. After extraction the analytes were separated on a Kinetex Biphenyl column with a gradient elution using methanol/0.01 M ammonium acetate as a mobile phase and analyzed with the LC-MS/MS technique. Both of the procedures were validated by analysis of a series of spiked feed samples (n = 6) at three different concentration levels. Better signal to noise ratios were observed for immunoaffinity clean-up. The recoveries of analyses were in the range 88-110% for the dilution procedure and 78-120% for the immunoaffinity clean-up. The dilution procedure was more precise (coefficient of variation of the within-laboratory reproducibility for it was 7.8-22.4% in comparison to 12-35.5% for the immunoaffinity clean-up. The results show that both procedures fulfilled the requirements for mycotoxin analysis and can be used successfully in multi-analyte determination. Although the dilution procedure shows better precision and trueness, the immunoaffinity clean-up procedure can have advantages in more complex feed samples thanks to lower matrix effect and limits of detections.

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.