Biomarkers of Deoxynivalenol, Citrinin, Ochratoxin A and Zearalenone in Pigs after Exposure to Naturally Contaminated Feed Close to Guidance Values.

This study applied multi-mycotoxin liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) methods to determine the biomarkers of exposure in urine and serum samples from a dose-response study with pigs. The 24 studied pigs were divided into three groups: a control and two experimental ones (with different levels of feed contamination). They were exposed to feed prepared from cereals contaminated with deoxynivalenol (DON), zearalenone (ZEN), ochratoxin A (OTA) and citrinin (CIT) for 14 days. After that, both experimental groups received the same feed as the control group for the next 14 days to determine the kinetics of the disappearance of mycotoxin biomarkers. Urine samples were collected daily in the morning and blood samples-eight-times during the experiment. The study reported herein was the first prolonged exposure experiment for multiple mycotoxins like OTA and CIT in pigs. The urinary and serum levels of all biomarkers correlated well with the respective toxin intake; thereby demonstrating that they are suitable biomarkers of exposure in pigs. Urine is a good candidate to monitor DON, ZEN, OTA, CIT exposure while serum may be used to monitor DON, OTA and CIT. Additionally, OTA has even been quantified in both matrices in the experimental groups two weeks after changing the contaminated feed back to the control, this result differed from those produced by the other mycotoxins which were only quantified during the first two weeks. Therefore both matrices are suitable candidates to monitor prolonged OTA exposure in pigs.

Mycotoxin Biomarkers in Pigs-Current State of Knowledge and Analytics.

Farm animals are frequently exposed to mycotoxins, which have many adverse effects on their health and become a significant food safety issue. Pigs are highly exposed and particularly susceptible to mycotoxins, which can cause many adverse effects. For the above reasons, an appropriate diagnostic tool is needed to monitor pig' exposure to mycotoxins. The most popular tool is feed analysis, which has some disadvantages, e.g., it does not include individual exposure. In recent years, the determination of biomarkers as a method to assess the exposure to mycotoxins by using concentrations of the parent compounds and/or metabolites in biological matrices is becoming more and more popular. This review provides a comprehensive overview of reported in vivo mycotoxin absorption, distribution, metabolism and excretion (ADME) and toxicokinetic studies on pigs. Biomarkers of exposure for aflatoxins, deoxynivalenol, ochratoxin A, fumonisins, T-2 toxin and zearalenone are described to select the most promising compound for analysis of porcine plasma, urine and faeces. Biomarkers occur in biological matrices at trace levels, so a very sensitive technique-tandem mass spectrometry-is commonly used for multiple biomarkers quantification. However, the sample preparation for multi-mycotoxin methods remains a challenge. Therefore, a summary of different biological samples preparation strategies is included in that paper.

Development of a multi-mycotoxin LC-MS/MS method for the determination of biomarkers in pig urine.

An LC-MS/MS method has been developed for the sensitive and selective determination of 35 mycotoxins (biomarkers of exposure) in pig urine samples. Sample preparation includes creatinine adjustment (with the developed LC-UV method) with enzymatic hydrolysis of pig urine samples followed by liquid-liquid (LLE) extraction. The LLE protocol, as well as enzymatic hydrolysis for indirect mycotoxin glucuronides determination, was optimized in this study. Additionally, two other sample preparation protocols were compared with the developed LLE method: immunoaffinity columns and solid-phase extraction cartridges (Oasis HLB). The detection and quantification of the biomarkers were performed using triple quadrupole mass spectrometry.The method was validated with regard to the guidelines specified by the EMEA (European Medicines Agency). The extraction recoveries were higher than 60% for 77% of the analytes studied, with the intra- and inter-day relative standard deviation being lower than 20% for most of the compounds at four different concentration levels. The limits of quantification ranged from 0.1 ng/mL for zearalenone and sterigmatocystin to 8 ng/mL for nivalenol. To the best knowledge of the authors, the matrix effect was evaluated for the first time in this study for six different urine samples, and the coefficient of variation was found to be lower than 15% for most analytes studied. Finally, the developed method was applied to analyse 56 pig urine samples. Deoxynivalenol (1-20 ng/mL), zearalenone (0.1-1.5 ng/mL) and ochratoxin A (1.5-15 ng/mL) were the main analytes detected in these samples. Moreover, the co-occurrence of alternariol monomethyl ether and alternariol in pig urine is reported herein for the first time.

Dilute-and-Shoot HPLC-UV Method for Determination of Urinary Creatinine as a Normalization Tool in Mycotoxin Biomonitoring in Pigs.

A simple, rapid, and accurate HPLC-UV method was developed for the determination of creatinine in pig urine. Usually, it is determined in urine in biomonitoring of xenobiotics to correct for variations in dilutions of urine samples. The colorimetric method (based on Jaffe reaction), which was mainly used for this purpose in mycotoxin biomonitoring, is not a reliable approach for pig urine. Therefore, a novel and accurate HPLC method for creatinine determination was developed. The sample preparation was based on the dilute and shoot approach. An HPLC separation was performed with a porous graphitic carbon column with an aqueous mobile phase to achieve satisfactory retention time for creatinine. The method has been successfully validated, applied for the determination of creatinine in pig urine, and compared with other methods commonly used for that purpose-a colorimetric method based on Jaffe reaction and commercial ELISA test. The developed HPLC method shows the highest precision and accuracy for pig urine samples. Finally, the method was applied as a normalization tool in LC-MS/MS mycotoxin biomarkers analysis. The standardization to a constant creatinine level (0.5 mg/mL) enables similar matrix effects for eleven mycotoxin biomarkers for pig urine samples with different creatinine levels.