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Comparison of Data from a Single and a Multi-analyte Method for Determination of Urinary Total Deoxynivalenol in Human Samples Paul Craig Turner, Michele Solfrizzo, Allison Gost, Lucia Gambacorta, Monica Olsen, Stina Wallin, and Natalie Kotova J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04755 • Publication Date (Web): 16 Nov 2016 Downloaded from http://pubs.acs.org on November 19, 2016
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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Agricultural and Food Chemistry
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Comparison of Data from a Single and a Multi-Analyte Method for Determination of Urinary
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Total Deoxynivalenol in Human Samples
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Paul C Turner*†, Michele Solfrizzo*‡, Allison Gost†, Lucia Gambacorta‡, Monica Olsen§, Stina
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Wallin§ and Natalia Kotova§
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†
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College Park, MD, US; ‡ Institute of Sciences of Food Production (ISPA) of the National Research Council
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(CNR), Bari, Italy. § The National Food Agency, Box 622, SE 751 26 Uppsala, Sweden.
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* Corresponding author (Tel: +39-080-592-9367 ; Fax: +39-080-592-9374; E-mail:
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Maryland Institute for Applied Environmental Health, School of Public Health, University of Maryland,
[email protected])
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Abstract
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Deoxynivalenol (DON) exposure is estimated by the combined measures of urinary DON and
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DON-glucuronides. In this study, data from a single mycotoxin (SM) and a multi-mycotoxin
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(MM) method were compared for 256 Swedish adult urine samples. Both methods included β-
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glucuronidase pre-digestion, immunoaffinity enrichment and LC-MS/MS. However, the specific
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reagents, apparatus and conditions were not identical in part because the MM method
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measures additional mycotoxins. DON was detected in 88% and 63% of samples using the SM
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and MM, methods respectively, with mean and median concentrations (SM: mean 5.0ng/mL,
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SD 7.4, range of positives 0.5-60.2ng/mL, median 2.5ng/mL, IQR 1.0-5.5ng/mL; MM: mean
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4.4ng/mL, SD 12.9, range of positives 0.5-135.2ng/mL, median 0.8ng/mL, IQR 0.3-3.5). Linear
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regression showed a significant, albeit modest, correlation between the two measures
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(p=0.0001, r = 0.591). The differences observed may reflect subtle handling differences in DON
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extraction and quantitation between the methods.
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Keywords: deoxynivalenol, multi-mycotoxin method, single-mycotoxin method, exposure,
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Introduction
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Deoxynivalenol (DON), 1 (Figure 1) also known as vomitoxin, is a type B-trichothecene
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mycotoxin produced by various Fusarium species.1 DON is one of the more frequently detected
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mycotoxins in cereal crops, including wheat, maize and barley, and tends to occur more
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frequently in temperate regions of the world.2 In animals DON and other trichothecenes
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adversely affect the immune system via binding to ribosomes. Additionally, they suppress
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appetite, reduce food utilization and interfere with the concentration of serum insulin-like
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growth factor.3-6 There are numerous incidences of human toxicosis linked to Fusarium
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mycotoxin contamination of cereal crops, and DON contamination of these cereals are
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associated in some of the larger incidences, involving tens of thousands of individuals from
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countries including India, China, Japan and Korea. Typical symptoms include abdominal pain
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and fullness, nausea, diarrhea, vomiting, fatigue and fever,7-10 though causal relationships are
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not always clearly defined. In part this reflects the acute nature of the events, limited possibility
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to confirm levels in contaminated food consumed at the time of poisoning, and inherent
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difficulties in accurate exposure assessment for mycotoxins based on food sampling.
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In 2003, Meky et al.11 suggested that urinary measurement of DON and a DON-glucuronide
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(now termed total DON (t-DON)) may improve exposure assessment. Subsequently urinary t-
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DON was validated as a reliable tool to assess recent DON exposure,12-14 based on analytical
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precision using immuno-affinity enrichment and LC-MS; and demonstration of both dose-
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response relationships between exposure and the measure, combined with stability surveys
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through collection and long term cryo-storage of urine. Novel approaches to analytical
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detection are now reported e.g. GC-MS and direct analysis of DON and DON-conjugates in urine
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by LC-MS;15-17 and some approaches report quantitative data for several mycotoxins, and
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metabolites thereof, in a single analysis.18-22 It is important to understand and be able to
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compare biomarker levels and frequencies from different laboratories, especially when subtle
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methodological differences exist; yet to date such data are mostly absent for mycotoxin
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biomarkers. The aim of this study was to compare the urinary t-DON concentrations in a single
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set of samples, initially reported using the single mycotoxin (SM) approach,
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independently obtained using the multi-mycotoxin (MM) method used to measure six distinct
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mycotoxins, including total DON.24 One smaller study of a similar nature was conducted using
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samples from South Africa,25 though here we have five times the number of samples. The
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parent toxin, 1 (Figure 1) and the two major DON-glucuronide conjugates 2, 3 (Figure 1) can be
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observed in urine of individuals exposed to DON,11-16 though determinants of their relative
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contribution to t-DON in urine has not been reported.
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Materials and Methods
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The study population was previously described.23 In brief, the majority of the participants took
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part in Riksmaten, a Swedish national survey investigating dietary habits among adults (18-80
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years), conducted from May 2010 - July 2011.26 In addition, 32 participants from a pilot study
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for Riksmaten, from September and October 2009 were included. The designs of the studies
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were very similar and the sampling procedure was identical. The urine samples were stored at -
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20 oC before shipment. Urine of DON exposed individuals contains a mix of free DON and one or
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more DON-glucuronides.27-28 Both analytical methods in this comparison used an enzymic
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with data
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digest step to convert any DON-glucuronide to DON prior to quantitation of t-DON. Data
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collection for each approach and the analytical differences are described in brief below, though
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data from each study has already separately been published.23, 24 The two published studies had
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different objectives but included subsets of samples from the main study involving 299
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278
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method in the UK was conducted approximately one year before those in Italy using their MM
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method. Separate aliquots were sent to each laboratory, so while no freeze/thaw issues are
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relevant, the samples for MM had an additional 12 months of storage at -20 oC. Two
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independent surveys reported negligible loss of urinary total DON following either, two months
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at -20 oC21 or after both a one-year and three-year storage at -40 oC.29
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Single mycotoxin (SM) method performed at University of Leeds
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This laboratory analyzed 299 urine samples using the single mycotoxin (SM) method described
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by Wallin et al.23 Briefly, frozen samples were thawed and centrifuged at 2,000 x g for 15 min at
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4 °C. The supernatant was collected and 1 mL (adjusted to pH 6.8) was treated with 250 µL of
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75 mM phosphate buffer (pH 6.8) containing 7,000 units of β-glucuronidase type IX-A from
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Escherichia coli (Sigma, Poole, UK) and 20 ng
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Samples were digested overnight at 37 °C, then centrifuged and the supernatants were diluted
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with 3 mL phosphate buffered saline (pH 7.4). Diluted supernatants were passed through wide-
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bore DON immunoaffinity columns under gravity, according to the manufacturer’s instructions
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(Vicam, Milford, MA). DON was eluted with 4 mL of MeOH and dried in vacuo. The residue was
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reconstituted in 250 µL 10% (v/v) ethanol and analyzed by LC-MS by injecting 5 µL, equivalent
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samples each, and with 256 samples common to both surveys. The analysis by the SM
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C15 DON internal standard (Sigma, Poole, UK).
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to 20 µL urine. Two quality control (QC) samples (urine spiked with 10 ng/mL DON) and two PBS
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blanks were run with each batch of twenty samples. DON was analyzed using a Waters 2795
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HPLC separation module and MS detection with a Micromass Quattro Micro (Waters, Milford,
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USA). The column used was a 150 mm × 4.6 mm, 5.0 µm Luna C18 column (Phenomonex,
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Macclesfield, UK); all conditions, values and concentrations were as documented by Turner et
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al.12 The range of the standard curve was equivalent to 0.5-62.5 ng DON /mL urine, additionally
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containing the
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C15 DON as an internal standard; standard curve R2 was always >0.990. The
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limit of detection (LOD) was 0.1ng/mL and limit of quantitation (LOQ) for this analysis was 0.5
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ng DON/mL urine. The mean value for the in house QC was 9.9 ng/mL with a CV of 2.5% (n=32).
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Data presented was automatically fully adjusted for recovery and that is presented. To allow
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method comparison to the MM we additionally report “recoveries” without such adjustment,
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thus the mean recovery through the entire extraction process was as calculated as 65% (range
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61-73%).
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Multi-mycotoxin (MM) method performed at ISPA
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This laboratory analyzed 278 urine samples using the multi-mycotoxin (MM) method described
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by Solfrizzo et al.19 Briefly, frozen urine samples were thawed and centrifuged at 3000 × g for 5
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min at 4°C to remove particulate matter. Enzymatic deconjugation of DON glucuronides was
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performed by adding 300 μL of β-glucuronidase/sulfatase solution containing 11,700 units of β-
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glucuronidase and 64 units of sulfatase type H-2 from Helix pomatia (Sigma-Aldrich, Milan,
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Italy) to 6 mL urine which was then incubated at 37 °C overnight. The digested urine samples
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were then diluted with 6 mL ultrapure water and purified on Myco6in1 IAC (Vicam, Watertown,
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MA) and Oasis HLB column (Waters, Milford, MA) connected in cascade. After sample elution
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the two columns were separated and treated separately as follows. The Myco6in1 IAC was
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washed with 4 mL water, then vacuum-dried for 15 s. DON was eluted with 3 mL MeOH and 2
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mL water in a vial. The Oasis HLB column was washed with 1 mL MeOH/water (20:80), then
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vacuum-dried for 15 s. DON was eluted from the column by gravity with 1 mL of MeOH/water
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(40:60) and collected in the vial containing the eluates from Myco6in1 IAC. The combined final
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eluates were dried under an air stream at 55 °C, reconstituted in 200 μl MeOH/water (20:80)
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containing 0.5% acetic acid, filtered with regenerated cellulose filter (0.20 µm) and analysed by
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UPLC and triple quadrupole API 5000 system MS/MS (Applied Biosystems, Foster City, CA) by
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injecting 10 µl, equivalent to 300 µl urine. The column used was a 150 mm x 2.1 mm i.d., 1.7 μm
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Acquity UPLC BEH phenyl column (Waters, Milan, Italy). Quantitation was performed by using a
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5-point matrix matched calibration curve prepared by using five purified urine extracts
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obtained from a pool of urine samples. The range of the calibration curve was 0.5-120 ng/mL
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for DON and results were reported as ng/mL after correction for method recovery (77%). The
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LOD was 0.45ng/mL and LOQ was 1.5ng/mL.
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Results & Discussion
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All data below the LOQ were assigned a value of half the LOQ, and if below the LOD a value of
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half the LOD. The data obtained with the multi-mycotoxin (MM) method were corrected for the
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method recovery, whereas the data obtained with the single mycotoxin (SM) method were
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generated using an internal standard and thus reported data are individually adjusted.
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Recoveries and repeatability of results (RSDr) for both methods were within the criteria for
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methods established by EU Regulation n. 401/200630 (RSDr < 20%, recovery range 60 – 110%). 7 ACS Paragon Plus Environment
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In addition to DON the MM method analyzed fumonisin B1 and B2, zearalenone, α- and β-
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zearalenols, ochratoxin A and nivalenol, published previously.24
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Table 1 reports the summary of results obtained in the two laboratories. For all samples
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analyzed t- DON was detected in 89% of 299 samples analyzed by using the SM method (overall
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mean 5.3 ng/ml; range 0.5 – 60.2 ng/mL; median 2.8 ng/mL, inter quartile range (IQR) 1.2 – 5.9
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ng/mL;), whereas, it was detected in 62% of 278 samples analyzed by using the MM method
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(mean 4.3 ng/mL, range 0.5 – 135.2 ng/mL, median 0.7 ng/mL; IQR 0.3 – 3.4 ng/mL).
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Within the 256 common samples comparable mean values but not median values were
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obtained for the two methods, with the median value significantly lower using the MM method
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compared to the SM method (Table 1). The t-DON concentration was below the LOD for 30
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samples (12%) and 97 samples (38%) by the SM and MM methods, respectively; while 146
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(57%) of samples were above the LOD and 25 (10%) were below the LOD, by both methods. For
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the 97 samples below the LOD using the MM method 78 (80%) were above the LOD by the SM
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method; 60 (77%) of which were < 5 ng/mL and 18 (23%) were
