Metabolites Reflects Regional Differences, Mycotoxin Intake and Feed Contaminations
4. Materials and Methods 1. Collection of Samples
The sampling of pig urine, collected from slaughterhouses, was conducted from January–December 2012 by the Swedish National Food Agency. The age of the pigs at slaughter were, on average, 6 months with a range of 5–8 months. The West, South and East regions of Sweden were represented by Västra Götaland county (n=74), Skåne county (n=83) and Kalmar county (n=38), respectively (Figure2).
Soon after collection, urine samples were frozen and sent from the National Food Agency (Uppsala, Sweden) to the Institute of Sciences of Food Production (Bari, Italy) for UPLC-MS/MS analysis of biomarkers of the five agriculturally important mycotoxins, i.e.; DON and DOM-1 for DON, AFM1for AFB1, FB1for FB1, ZEA,α-ZOL andβ-ZOL for ZEA, and OTA for OTA.
4.2. Chemicals and Reagents
Standard solutions of mycotoxins and their key metabolites were purchased from Romer Labs Diagnostic (Tulln, Austria). In particular, solutions of DON (100μg/mL), DOM-1 (50μg/mL), AFM1
(0.5μg/mL), ZEA (100μg/mL),α-ZOL (10μg/mL),β-ZOL (10μg/mL) and OTA (10μg/mL) were prepared in acetonitrile (ACN), whereas FB1solution (50μg/mL) was prepared in acetonitrile–water (50:50v/v). The enzymatic solutionβ-glucuronidase/sulfatase type H-2 fromHelix pomatia(specific activity 130,200 units/m Lβ-glucuronidase, 709 units/mL sulfatase) was purchased by Sigma Aldrich (Milan, Italy). Chromatography-grade methanol (MeOH) and glacial acetic acid were obtained from Carlo Erba (Milan, Italy). Ultrapure water was obtained from a Milli-Q system (Millipore, Bedford, MA, USA). Myco6in1+TM immunoaffinity columns were purchased from Vicam L.P (Watertown, MA, USA).
OASIS HLB®columns (60 mg, 3 mL) were purchased from Waters (Milford, MA, USA) and regenerated cellulose filters (0.45μm) were purchased from Sartorius Stedim Biotech (Goettingen, Germany).
4.3. Urine Analysis
The analyses of pig urinary mycotoxin biomarkers (DON, DOM-1, AFM1, FB1, ZEA,α-ZOL, β-ZOL and OTA) were performed using an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method reported elsewhere [6,7]. Briefly, 5 mL of pig urine was treated with the aqueous solution ofβ-glucuronidase/sulfatase type H-2 fromHelix pomatiato hydrolyze glucuronide- and sulfate- conjugates of mycotoxins and their key metabolites. The digested sample was diluted with water (1:1,v/v) and purified on a Myco6in1+TM multi-antibody immunoaffinity column and OASIS HLB®column connected in tandem. The purified urine was dried down and reconstituted in 200μl of mobile phase (MeOH:H2O, 20:80,v/v) and analyzed by UPLC-MS/MS with a triple quadrupole API 5000 mass spectrometer (Applied Biosystems, Foster City, CA, USA), equipped with an ESI interfaced to an Acquity UPLC system comprising a binary pump and a microautosampler (Waters, Milford, MA, USA). Data acquisition and processing was performed with Analyst version 1.5.1 software (Applied Biosystems 2011, Foster City, CA, USA). Detailed chromatographic and mass spectrometric operating conditions are reported elsewhere [24,25]. Crea concentrations in urine samples were analyzed according to an enzymatic method described by [26].
4.4. Calibration Curves
Quantification of mycotoxin biomarkers in the 195 purified pig urine sample extracts was performed using matrix-matched calibration curves. For each set of samples (one for each day), matrix-matched calibration solutions were prepared for 6 purified urinary extracts: one aliquot was analyzed as a control and 5 aliquots were used to prepare calibration samples. In particular, aliquots of urine from 5 pigs were pooled and mixed, then 6 aliquots (5 mL each) were purified according to the protocol reported above. After purification, adequate and increasing amounts of standard solutions of DON, DOM-1, AFM1, FB1, ZEA,α-ZOL,β-ZOL and OTA were added to the 5 purified extracts, dried down, reconstituted in 200μL of LC–MS/MS mobile phase and filtered. The calibration ranges in the matrix ranged between: 0.1–100 ng/mL for DON, 0.75–24.85 ng/mL for DOM-1, 0.01–7 ng/mL for AFM1, 0.10–101.6 ng/mL for FB1, 0.03–20.6 ng/mL forβ-ZOL andα-ZOL, 0.01–100 ng/mL for ZEA, 0.01–5.01 ng/mL for OTA.
4.5. Recovery Experiment
A mixture of 3 blank pig urine samples was used for the recovery experiment of DON, DOM-1, AFM1, FB1, ZEA,α-ZOL,β-ZOL and OTA. Triplicate experiments were performed. The spiking concentration of each analyte was reported in Table1. They ranged from 0.9 ng/mL for OTA to 90 ng/mL for DON. Matrix matched calibration curves were used to quantify each analyte. LOD and LOQ were calculated as 3 times and 10 times the noise, respectively.
4.6. Censoring
Left-censored analytical results, i.e.; values below the limit of detection (LOD) and the limit of quantification (LOQ), were evaluated with the substitution method as suggested by the European Food Safety Authority (EFSA 2010) [27]. Within the three scenarios proposed by EFSA (lower, middle and upper bound), we used the middle bound approach, i.e.; results below the LOD (or the LOQ), were given the value LOD/2 (or LOQ/2). To calculate the mean concentration in positive samples (Table3), the values between the LOD and the LOQ were assigned a fixed value of LOQ/2. LOD and LOQ values for all mycotoxins and metabolites were reported in Table1.
4.7. Estimation of Mycotoxin Intake
Calculation of intake estimation was performed for DON, ZEA, FB1, OTA and AFB1in pigs using urinary biomarker concentrations measured in this study according Formula (1), reported by [25]:
PDI=(C×V×100)/(W×E) (1)
where:
PDI: probable daily intake of each mycotoxin (μg/kg body weight);
C: pig urinary biomarker concentration (μg/L);
V: mean 24 h pig urine volume (2.5 L);
W: mean pig body weight (110 kg);
E: mean urinary excretion rate of each mycotoxin in 24 h post dose in piglets (36.8% for total ZEA, 27.9% for total DON, 2.6% for FB1, 2.6% for OTA and 2.5% for AFB1excreted as AFM1[6]).
4.8. Estimation of Feed Contamination
The DON, ZEA, FB1, OTA and AFB1contamination in feeds consumed by the pigs monitored in this study was calculated using the calculated PDI of each mycotoxin and Formula (2).
ML=PDI×W/V (2)
where:
ML: mycotoxin level in the feed (μg/kg);
PDI: probable daily intake of the mycotoxin (μg/kg body weight);
W: mean pig body weight (110 kg) V: mean 24 h pig urine volume (2.5 L)
4.9. Statistical Analyses of Urinary Biomarker Concentrations and Geographical Areas
Mean, median and standard deviation of the results were calculated using Microsoft Excel 2013 software (Microsoft Corporation, Redmond, WA, USA). Statistical analyses were performed using GraphPad Instat software version 3.00 (Instat 1997, San Diego, CA, USA). Data were subjected to the unpaired t-test (one-tailpvalue). Values were judged to be significantly different ifpvalues were<0.05.
The Mann–Whitney test and the Kruskal–Wallis equality of population rank test were performed in STATA version 12.1 (STATA Corp. 2017, College Station, TX, USA). Ap-value < 0.05 was considered significant.
Author Contributions:Funding acquisition, M.O.; Investigation, L.G. and M.O.; Methodology, L.G. and M.S.;
Project administration, M.O.; Resources, M.O.; Validation, M.S.; Writing – original draft, M.S.; Writing – review and editing, M.O.
Funding: This research was funded by the Swedish National Food Agency and the Swedish Civil Contingencies Agency.
Conflicts of Interest:The authors declare no conflict of interest.
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