CODA –CERVA

Belgian National Reference Laboratory

for Mycotoxins in Food and Feed

Report on the 2014 Proficiency Test for the determination of 19 mycotoxins and 4 sums of

mycotoxins in rye flour

final report

PART 1 : TEXT AND ANNEXES

February 2015

Ph. Debongnie, E. Tangni and A. Callebaut

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Table of Contents PART 1 : TEXT AND ANNEXES Summary Abbreviations Introduction Test materials Participants, instructions and schedule Calculation of assigned values and of z- and ζ-scores Results and discussion

Detailed example with assigned value : aflatoxin B1 Detailed example without assigned value : aflatoxin B2 Example with unsatisfactory left-censored results : aflatoxin G1 Example with a method-related trend : aflatoxin G2 Sum of aflatoxins Ochratoxin Deoxynivalenol 3- acetyl- deoxynivalenol and 5-acetyl- deoxynivalenol Zearalenone HT-2, T-2 and their sum Fumonisins Enniatins and beauvericin

Table : summary of statistics, assigned values and scores References Annexes :

A. List and addresses of the participating laboratories B. Instructions to participants, receipt form, report form C. Homogeneity tests

Rye flour material Fig. C.1 : (sanal. / σanal.recomm.) ratio Fig. C.2 : (between-samples RSD / σsampl.allow.), (ssampl. / σsampl.allow.) and (ssampl. /

√𝑐) ratios Table C.1 : Results of the homogeneity test

Vials containing evaporated-to-dryness solutions Table C.2 and Fig. C.3 : vial tests RSD Fig. C.4 : comparison of recoveries at lower (B) and higher (A) concentrations

D. Analytical details Fig. D.1 : distribution of clean-up and measurement methods for each group of molecules Fig. D.2 : number of different methods used, per set of results Fig. D.3 : number of analytes reported per method Fig. D.4 : subsample weight

PART 2 : RESULTS, STATISTICS AND SCORES, PER ANALYTE PART 3 : RESULTS AND SCORES, PER PARTICIPANT

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Summary A Proficiency Test (PT) on the determination of 19 mycotoxins and 4 sums of mycotoxins in rye flour was conducted with a total of 47 participants : 10 laboratories approved as Official Control Laboratories by the Belgian Competent Authority (FAVV-AFSCA) and 37 other laboratories from various countries. There were altogether 52 sets of results, because 3 participants sent 2 sets of independent results (i.e. obtained for the same toxin(s) but with different methods), and 1 participant sent 3 sets. Forty-six participants reported for all or part of the first group of 12 toxins (aflatoxins, OTA, ZEN, DON, T-2, HT-2 and fumonisins), 21 reported for the acetyl-DONs, and 11 reported for enniatins and beauvericin. The flour was a home-made material prepared by inoculation of wheat, maize and rice grains with selected Fusarium and Aspergillus strains, followed by grinding of these grains and mixing the flours obtained with non-contaminated rye flour. The concentrations in the final material were in the ranges of the Maximum Allowed Levels (MALs) for feed (all regulated analytes) and for food (all except aflatoxins and OTA). Two vials containing dry mycotoxins (known amounts of solution, evaporated to dryness) were also provided to those who expressed an interest in measuring and reporting the amounts of toxins in the vials, using their own solvent for reconstitution and their own reference solutions as calibrants. The flour material formally passed the critical value test for homogeneity for all toxins. However, for AfG2, OTA and ZEN the discriminating power of the test was clearly insufficient, and for OTA and ZEN closer examination of the results showed the presence of small “hot spots” (at 5-grams level, respectively 3/20 and 1/20 subsamples with concentrations >40% higher than average)

.............................. The standard deviations for proficiency assessment (σp) were calculated using the modified Horwitz equation. The assigned values and the standard uncertainties on these values were derived from the results of the participants, following the recommendations of the 2006 International Harmonized Protocol for the Proficiency Testing of Analytical Chemistry Laboratories. It was possible to assign reference values and score the results from the participants for 11 of the 19 toxins and for 3 of the 4 sums of toxins. The exceptions were :

- AfB2 and AfG2 (ca. 1.7 and 0.5 µg/kg), for which the HorRat values (σrob/σp) could have been considered acceptable (1.55 and 1.42) but the distribution of results was strongly bi- or trimodal. The lower mode was clearly linked with IAC clean-up, vs other types of clean-up or no clean-up. Two hypotheses may be considered : low bias due to partial saturation of columns in case of IAC clean-up, or high bias due to chromatographic interferences in the other cases.

- OTA and ZEN (ca. 190 and 560 µg/kg), with acceptable HorRat values (1.30 and 1.31) and consensus between participants, but for which it cannot be excluded that the few high outlier results are due to hot spots, as explained above

- 3-ac-DON, 15-ac-DON and their sum (MEDtot ca. 40, 110 and 150 µg/kg respectively) , for which the consensus was insufficient (HorRat 1.80, 2.20 and 1.77)

- FB3 and Enn-B, with clearly multimodal distributions (kernel density peaks at ca. 630 and 1300 µg/kg for FB3, ca. 14500 and 20000 µg/kg for Enn-B)

For those analytes or sums of analytes for which a reference value was assigned, the percentage ranges of satisfactory, questionable and unsatisfactory z-scores (corresponding to │z│<2, 2<│z│<3 and │z│>3) were 59%-92%, 0%-15% and 3%-27% respectively. The unsatisfactory z-scores correspond mainly to the outliers. On the whole the HorRat values and the distributions of z-scores are very comparable to those of the 2013 PT, which confirms the marked improvement relative to the 2011 and 2012 multi-mycotoxin PT.

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The ζ-scores are less satisfactory: the corresponding percentage ranges of satisfactory, questionable or unsatisfactory ζ-scores were 49%-75%, 0%-21% and 0%-21% (10%-25% of the results were

supplied without estimate of the MU and were assigned no ζ-scores). The participants still frequently

underestimate their MU. As a consequence, the risk of ‘false non-compliants’ remains much too high (e.g., for AfB1, ca. 22% instead of the targeted 5%).

.............................. In a few cases, low or high results for the flour were paralleled by equally low or high results for the vials, indicating a problem with the reference solutions of these participants. Forty-two of the participants, corresponding to 47 of the sets of results, completed the analytical questionnaire(s) :

The methods used were LC-MS (86% to 100%, depending on the analyte), LC coupled with fluorimetric or UV detection (0%-12%), or ELISA (0%-3%). Within the LC-MS results, 44% to 55% were obtained without any clean-up, 0% to 14% with IAC clean-up, and 39% to 50% with another type of clean-up. No clear method-related trend was observed, except for AfG2 and AfB2.

The weights of the subsamples analysed went from 0.25 g to 50 g, with 4 groups of approximately the same size : ≤4 g, >4 to 5 g, >5 to 10 g, and >10 to 50 g. No correlation was observed with the results, nor with the differences between duplicate results.

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Abbreviations

PT Proficiency Test MAL Maximum Allowed Level (for a toxin in a food or feed commodity) LRL Low Reporting Limit HRL High Reporting Limit

Analytes :

AfB1 aflatoxin B1 AfB2 aflatoxin B2 AfG1 aflatoxin G1 AfG2 aflatoxin G2 BEA beauvericin DON deoxynivalenol Enn-A enniatin A Enn-A1 enniatin A1 Enn-B enniatin B Enn-B1 enniatin B1 FB1 fumonisin B1 FB2 fumonisin B2 FB3 fumonisin B3 HT-2 HT-2 toxin OTA ochratoxin A T-2 T-2 toxin ZEN zearalenone 3-Ac-DON 3-acetyl- deoxynivalenol 15-Ac-DON 15-acetyl- deoxynivalenol

Homogeneity tests : σsampl. true sampling variance (= between bags) σsampl.allow. allowable sampling variance ssampl. observed sampling variance σanal.recomm. recommended limit for ‘analytical’ variance

(= between subsamples from the same bag) sanal. observed ‘analytical’ variance c critical value in the “sufficient homogeneity” test (ssampl.

2 must not be > c) MEDhom median of homogeneity tests results

Statistics on results from participants : Xlab (averaged) result reported by a participant ntot total number of results n”=” number of non-censored results n”<” or “>” number of left- or right-censored results MEDtot(=) median of all non-censored results MEDtot median of all results, including left-censored results with LRL<MEDtot and

right-censored results with HRL>MEDtot nref number of results in ‘reference’ dataset (i.e. after exclusion of outliers) MEDref median of reference dataset MADref median absolute difference from MEDref,, for the reference dataset σrob robust standard deviation for the reference dataset (= 1.4826 MADref)

uref standard (k=1) uncertainty on MEDref (=σrob / √𝑛𝑟𝑒𝑓 )

Vass ‘consensus’ or ‘reference’ value, concentration assigned to the material for the calculation of z- and ζ-scores

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σp target standard deviation for the PT (cfr Horwitz-Thompson equation) HorRat Horwitz Ratio = σrob / σp

Individual participant scoring :

z-score measure of the trueness of a result, defined as ((Xlab – Vass) / σp) MUlab extended (k=2) measurement uncertainty estimated and

reported by a participant ulab standard (k=1) measurement uncertainty of a participant (=MUlab/2)

ζ-score measure of the reliability of ulab, defined as ((Xlab – Vass) / √𝑢𝑟𝑒𝑓2 + 𝑢𝑙𝑎𝑏

2

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Introduction This was the fourth multi-mycotoxin PT organized by CODA-CERVA. In the first PT in 2011, 18 participants had contributed with their results for DON, ZEN, T-2 and T-2, and 10 participants contributed for enniatins (A, A1, B and B1) and beauvericin, The home-made material, intended primarily for animal experiments, had been obtained by inoculation of several grain batches with different selected mould strains, incubation, sterilization, grinding and blending with a non-contaminated wheat flour. The concentrations were relatively high (>250 µg/kg for all toxins except Enn-A). In the second PT in 2012, the range was broadened to 17 toxins, by adding aflatoxins (B1, B2, G1, G2), ochratoxin A and fumonisins (B1, B2, B3). Two maize flour materials were provided, at concentrations levels in the ranges of the MAL for feed and food respectively. The number of participants also increased, with up to 32 sets of results received for the regulated toxins and 11 sets for enniatins and beauvericin. In the third PT in 2013, the material was oats flour. The target concentrations were in the same range as the MAL for food and feed (except for OTA, which was in the range of the MAL for feed but over 10x the MAL for food). The number of participants further increased. In total 48 sets of results were received for the regulated toxins and again 11 sets for enniatins and beauvericin. The results and the consensus between the participants were markedly better than in the two previous PT. In this fourth PT in 2014, the material was rye flour. As in 2013, the target concentrations were broadly in the same ranges as the MAL for food and feed, except for OTA and aflatoxins (up to forty time the MAL for food). In total 51 sets of results were received for the regulated toxins and again 11 sets for enniatins and beauvericin. The quality of the results and of the consensus between the participants seem to confirm the over-all improvement observed in 2013.

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Test materials The preparation of the rye flour material was comparable to the previous PT. The material was produced by inoculating cereal grains with Fusarium spp. and Aspergillus spp strains selected from the UCL Mycotheque for their ability to produce high amounts of mycotoxins. These materials were sterilized, dried, ground, homogenized, and mixed with a non-contaminated rye flour in several steps, with careful homogenization after each step. The final material was ca. 99% rye. As already stated in the introduction, the target concentrations were broadly in the same ranges as the MAL for food and feed, except for OTA and aflatoxins (up to forty time the MAL for food). This final material was tested for homogeneity according to the IUPAC International Harmonized Protocol (see annex C). Briefly, 10 samples were selected at random, and from each sample two 5 g subsamples were extracted and analyzed. Four dilutions (5, 50, 500 and 5000 mL/g) were prepared from each extract and analyzed by LC-MS/MS. For each toxin the most appropriate dilution was chosen and the 10 pairs of averaged results were subjected to the tests for sufficient homogeneity as described in appendix 1 of the IUPAC protocol. The material passed the tests, at least formally, for the 19 toxins investigated. However in 4 cases the analytical repeatability (sanal) was too high (> σp/2) for the test to be considered as sufficiently discriminating (see fig. C1 and C2) :

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for DON, sufficient homogeneity may be safely assumed, because the recommended σanal. (7.1%) was only slightly exceeded (7.5%) and furthermore even the ‘raw’ between-samples RSD (4.8%) almost complied with the criterion for allowable variability (4.3%)

for AfG2, sufficient homogeneity may be safely assumed insofar as the other aflatoxins may be considered as tracers

for OTA and ZEN, the presence of a few residual “hot spots” cannot be excluded In plots of part 2 of this report, for each analyte the ‘kernel density plot’ of the homogeneity tests results is superimposed to that of the participants’ results, which allows to check visually whether any multi-modality in the distributions of results from the participants could possibly be due to insufficient homogeneity. In addition to the flour sample, the participants who manifested their interest received 2 vials containing mycotoxins (evaporated-to-dryness mixtures) in different amounts. These 2 solutions were prepared by dilutions of mono-analyte stock solutions, themselves prepared by dissolving commercially available reference compounds in acetonitrile. The participants were asked to re-dissolve the toxins in the solvent of their choice and to measure the amounts, using their own reference solutions. The objective was to check whether strongly deviating results could be due to differences between the reference solutions used rather than to analytical method biases.

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Participants, instructions and schedule A total of 50 laboratories registered for the PT. Among these, 10 were laboratories approved by the Belgian Food Safety Agency (FAVV-AFSCA) and 40 were laboratories from various countries. Three participants did not send any results, so the final number of effective participants, listed by alphabetical order in annex A, is 47. Each participant received one or more container(s) with approximately 55 g of the rye flour material. Thirty-eight participants also asked for the vials described above. The instructions, receipt form and report form are given in annex B. The participants were invited to extract and analyse separately two sub-samples, preferably ≥ 20 g each, but, apart from this, to use the method of their choice. They were to report the duplicate and averaged results in µg/kg and an estimate of their uncertainty of measurement (MU(k=2)) either in µg/kg or in %. In case their result was outside their normal reporting range, they were asked to specify their reporting limit(s) but nevertheless to report also their measured results, unless they had reason to believe that these results were totally meaningless, e.g. because of a strong chromatographic interference. The participants who requested vials were invited to reconstitute the toxins in the solvent of their choice, and report the contents in ng (equivalent to µg/L if reconstituted in 1 mL solvent). The participants were also asked to answer a few questions about the method(s) used. The results and answers to the questions were to be reported on the protected Excel form file provided. The majority of samples were sent on Sept. 16 and 17, and the deadline for reporting was Oct. 30. For a few participants who registered late or encountered technical problems, the deadline for reporting was postponed for a few days. The last set of results was received on Nov. 13

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Three laboratories sent 2 separate sets of results (i.e. obtained with different methods, for the same analyte(s)), and 1 sent 3 separate sets, which brings the total number of sets of results to 52. The randomly assigned code numbers range are E1 to E11 for enniatins and beauvericin, and L1 to L51 for the other toxins. Per analyte, the number of results received for the flour ranged from 10 (Enn-B and -B1) to 48 (DON), and for the vials from 4 (enniatins) to 31 (DON).

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Calculation of assigned values and of z- and ζ-scores The assigned values were derived from the results of the participants in the manner described below and based on the IUPAC 2006 recommendations. The principles of the calculations are summarized in this paragraph, and detailed examples are given in the discussions in the following paragraphs. In a first step :

the median of all the (averaged) non-censored results (MEDtot(=)) is calculated

the median of all results (MEDtot) is then calculated by including : the LRL of any left-censored result which can be considered as potentially adding useful

information on the true concentration, i.e. if this LRL is lower than MEDtot inversely, the HRL of any right-censored result if this HRL is higher than MEDtot

In a second step :

results outside the 50% to 150% range around the MEDtot are removed as ‘outliers’

the medians of the remaining sets of results (‘reference’ sets) are calculated (MEDref)

the median absolute differences of the remaining sets of non-censored results are calculated (MADref)

the robust standard deviations are calculated as σrob = 1.4826 MADref

the uncertainties on the MEDref (with coverage factor k = 1) are calculated as

uref = σrob / √𝑛𝑟𝑒𝑓

the “standard deviations for proficiency assessment" σp are calculated from the MEDref, using the Horwitz-Thompson equation

the ‘HorRat’ values (=σrob / σp) are calculated and compared with 1.2, the maximum value recommended by IUPAC for deriving a consensus value.

the ‘kernel density plots’ are drawn, for: o the full datasets o the ‘reference’ datasets o the results from the homogeneity tests, “re-scaled” around MEDref :

Xi’ = Xi . (MEDref / average(Xi)) The ‘bandwidths’ (h) used for the calculation of these curves are 0.75 σp, as recommended by IUPAC, for the participants’ results, and 0.30 σp (weaker “smoothing” effect than 0.75 σp) for the homogeneity tests results For these visual comparisons all the kernel density curves are re-scaled along the Y-axis (min = 0, max = 1)

In a third step, for the analytes for which the kernel density plots showed a convincing central tendency and the HorRat value was satisfactory :

the MEDref are assigned as ‘consensus’ or ‘ reference’ values :

Vass = MEDref

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individual laboratory performance was expressed in terms of z- and ζ-scores in accordance with ISO 13528 and the International Harmonised Protocol.

𝑧 =(𝑋𝑙𝑎𝑏 − 𝑉𝑎𝑠𝑠)

𝜎𝑝

𝜁 =(𝑋𝑙𝑎𝑏 − 𝑉𝑎𝑠𝑠)

√𝑢𝑙𝑎𝑏2 + 𝑢𝑟𝑒𝑓

2

where

Xlab is the measurement result reported by a participant

p is the "standard deviation for proficiency assessment"

uref is the standard uncertainty on Vass ulab is the standard uncertainty reported by the participant

(all in µg/kg)

(Note that in the previous PT reports ulab was set to 0 if not reported, which usually led to high absolute values of the ζ-scores. In the present PT, since several participants did provide the information that the MU of the method used had not yet been evaluated, it was considered more appropriate to calculate ζ-scores only when the MU was given)

The z- and ζ-scores are considered :

satisfactory if between -2 and 2

questionable between -3 and -2 or between 2 and 3

unsatisfactory if <-3 or >3 For left- or right-censored results, z- and ζ-scores are calculated :

for the corresponding LRL or HRL : these are written with the appropriate sign, “<” or “>”, and considered :

o unsatisfactory if “< -3” or “> 3” o questionable if “< -2” or “> 2” o “no conclusion” in the other cases (e.g. “<4”), because correct but uninformative

for the actual measurement results, when given : these scores are written between parentheses, and have only an indicative or “informal” value

For the analytes for which there was no satisfactory central tendency and/or the HorRat value was too high, no reliable consensus values can be derived ; z- and ζ-scores are calculated on the basis of MEDref, but these scores have only an indicative value and are given between parentheses in the results tables.

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Results and discussion The tables in part 2 give, for each toxin or sum of toxins:

- the duplicate and average results (in µg/kg) and the measurement uncertainties (in %) reported by the participants

- the z- and ζ-scores - the results (in ng) for the contents of vials A and B, as reported by the participants - the results of the statistical calculations - the numbers of satisfactory, questionable, unsatisfactory, and “no conclusion “ z- and ζ-scores

In these tables :

- the results (µg/kg or ng) are highlighted :

in yellow if < 0.5 MEDtot

in green if > 1.5 MEDtot. - the z- and ζ-scores are highlighted :

in orange if unsatisfactorily low

in yellow if questionably low

in green if questionably high

in blue if unsatisfactorily high - the σrob/σp ratio is highlighted :

in orange if above 1.5 (cases of AfB2, AfG2, ac-DONs, FB1 and Enn-B)

in yellow if between 1.2 and 1.5 (OTA, ZEN, T-2, HT-2, FB2, sum FB1+FB2, BEA) Each page comprises up to 7 plots :

- the kernel density plots of the participants’ results, before and after removal of outliers, and of the results of the homogeneity tests

- the distribution of these results among the following four groups :

IAC + LC-Fl or LC-UV

IAC + LC-MS

other clean-up + LC-MS

LC-MS without clean-up - the duplicate and averaged results in µg/kg, with error bars indicating the MU as reported - the z- and ζ-scores (on these plots any value <-5 or >5 is replaced by -5 or 5) - flour vs vial A (log-log plot of the ratios to the median) - vial B vs vial A (idem) - the numbers of satisfactory, questionable, unsatisfactory and “no conclusion “ z- and ζ-

scores, and (for the non-censored results only) the percentages These calculations and graphs are presented and discussed in more detail below, using as detailed examples the cases of AfB1 and AfB2, respectively with and without assigned values, AfG1 with unsatisfactory left-censored results, and AfG2 with a clear method-related trend..

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Detailed example with assigned value : aflatoxin B1 MEDtot(=), the median of the 45 non-censored averaged results is 22.32 µg/kg. The 2 right-censored results are “>6” and “>10” : since these HRL are lower than the median of non-censored results, they are not taken into account for the calculation of MEDtot, which remains at 22.32 µg/kg. MEDref, the median of the 40 results remaining after removal of 3 low outliers (5.8 to 11 µg/kg) and 2 high outliers (42 and 44 µg/kg), is 22.34 µg/kg. The ratio of the robust standard deviation (5.77 µg/kg = 25.8%) to the target standard deviation (22%, from the Horwitz-Thompson equation) is 1.17, below the recommended maximum (1.2). The 5 outliers are clearly visible on the µg/kg plot, and their removal is clearly visible on the kernel density plots. After this removal, the distribution is unimodal and symmetric. MEDref is therefore assigned as reference value Vass. The standard uncertainty (k=1) on this assigned

value is uref = 25.8% / √40 = 4.1%.

............. Among the 40 non-censored and non-outlier results, 39 results obtain satisfactory z-scores and 1 obtains a z-score slightly above 2 (2.14). Among the 5 outliers, 2 obtain questionable z-scores (-2.33 and -2.31) and 3 obtain unsatisfactory z-scores (-3.36, 4.01 and 4.44). The percentages of satisfactory, questionable and unsatisfactory z-scores are 87%, 7% and 7%. As often, the [satisfactory:questionable] ratio is close to [95:5] as expected, whereas the percentage of unsatisfactory results is higher than the 0.3% that would be expected from a Gaussian distribution without outliers. The 2 right-censored results would have obtained satisfactory z-scores (0.25 and -1.84), the first one being even among the best results of the whole set.

............. For the 45 non-censored results, 28 of the ζ-scores are satisfactory, 4 are questionable, 7 are unsatisfactory, and 6 were not calculated because the MU was unknown. Expressed in percentages, the [satisfactory : questionable : unsatisfactory : not calculated] ratio is [62% : 9% : 16% : 13%]. The right-censored results would have obtained ζ-scores 0.33 and -7.44. The ζ-scores calculated on the HRL (>-4.48 and >-10.2) are correct but uninformative.

............. In general the percentages of satisfactory z- and ζ-scores are quite comparable to those of the 2013 PT. As usual, the ζ-scores are thus in general less satisfactory than the z-scores, due to the number of participants underestimating their MU. Underestimation of MU has the undesirable consequence that the risk of wrongly declaring a feed lot as “non-compliant” largely exceeds the targeted ∝=5%:

According to Commission Decision 2002/657/EC, to respect this limit of 5% false non-compliants, a lot should be declared non-compliant, on the basis of a result from a single lab,

only if this result exceeds CC𝜶, i.e. the MAL plus a safety margin equal to 1.64 ulab(k=1) = 0.82 MUlab(k=2). For example, with a MU of 44%, corresponding to the σp of 22% derived from the Horwitz-Thompson equation below 120 µg/kg, the safety margin would be 36% of the MAL.

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The present PT sample is clearly non-compliant, because the median of the results of 40 different participants (22.34 µg/kg) is ca. 12% above the MAL for feed materials (20 µg/kg), and the uncertainty on this median is only ca. 4%.

If the participants had analysed this sample not for PT participation but for official control, without confirmation from other labs, each would have had to apply his own “single lab” safety margin (e.g. 36% = 7.2 µg/kg for ulab(k=1) = σp = 22%) to compare the MAL with his own result. Only 33% of the participants would have declared the sample as non-compliant. In other words the “β-error” (probability of “false compliant”), at 22 µg/kg real concentration, is ca. 67%.

This may seem high, but it is the price to pay for keeping down the risk of false non-compliant.

However, if the MAL was not 20 but 22.5 µg/kg and if the real concentration was indeed 22.34 µg/kg, then the sample would be compliant, but 22% of the participants would still have declared it non-compliant. This is over 4 times the risk of false non-compliant targeted by

2002/657/EC (∝ = 5%).

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The main objective of measuring the contents of vial A is to check, in case of strong outliers for the flour, whether this can be due to problems with the participants’ reference solutions. In such a case the points on the “flour vs vial A” plot should be close to the diagonal drawn across the plot. In the present set of data :

The 2 high outliers for the flour (L9 and L22, deviations +98% and +88%), which appear just before the upper red dotted line corresponding to z=3, have results close to the median for the vials (deviations -9% and +20%), so probably the causes for their deviation are elsewhere.

Among the 3 low outliers for the flour (L2, L4 and L39, dev. -74%, -51% and -51% resp.), only L2 and L4 have provided results for the vials : they are also lower than the median result, but less so than the flour results (dev. -38% and -20%), so the corresponding points (the 2 lowest of the plot) appear in the region of the diagonal but below it. It is possible that the reference solutions used by these labs are over-concentrated, but probably this would explain only part of the deviation observed for the flour.

(for examples of deviations for vial A close to those for the flour, see e.g. the results for FB1)

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The main objective of measuring the contents of vial B, ca. 10 times lower than those of vial A, is to provide a second check of the validity of these measurements and/or of the linearity within this range. However, as already observed and discussed after the 2013 PT, in the case of aflatoxins the correlation is poor, because of the highly variable recovery of low levels of aflatoxins from vials B, also observed in the homogeneity tests (see annex C). In contrast, examples of very good linearity can be seen for e.g. OTA, T-2 or the fumonisins.

............. The type of clean-up and detection used has no significant impact on the distribution of the results, as can be seen on the plot on the upper right of the page. Also the size of the subsamples for analysis (from 0.25 to 50 grams) has no significant impact (results not shown) on the distribution of results, nor on the differences between duplicate results.

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Detailed example without assigned value : aflatoxin B2

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The concentration is around 1 or 2 µg/kg, much lower than for AfB1. Out of the 43 results received, 8 are left-censored, with LRL from 0.015 to 5 µg/kg. MEDtot(=), the median of the non-censored results, is 1.727 µg/kg. Since the LRL of 4 of the left-censored results are lower than this value, these results are considered as potentially relevant for the determination of the reference value. MEDtot, taking these 4 results into account, is 1.708 µg/kg. For the determination of MEDref, among the non-censored results 4 are eliminated as high outliers and 3 as low outliers. Among the 4 left-censored results included in the calculation of MEDtot, 2 (<0.015 and <0.5) are eliminated as low outliers, the other 2 (<1 and <1.5) are included in the reference dataset. The final value of MEDref is 1.714. However, the consensus between participants is not sufficient to consider this MEDref as “assigned value” Vass, because :

- The kernel density plot shows a distinctly bi-modal distribution - The HorRat value is 1.55

Therefore, the z- and ζ-scores have an indicative value only, and are given between parentheses.

The same type of bi-modal distribution had already been observed in 2013, with maxima at ca. 0.75 and 1 µg/kg. In contrast to AfB1, there is a marked influence of the clean-up on the distribution of the results : Clearly the results obtained after IAC are around 1 µg/kg, while those obtained after other types of clean-up are mostly between 1.5 and 3 µg/kg, and those obtained without clean-up are divided between these two modes. An even stronger method-related trend is observed for AfG2 (see below).

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Example with unsatisfactory left-censored results : aflatoxin G1 The case of AfG1 is as straightforward as that of AfB1 :

- MEDtot(=) : 10.535 µg/kg (for 40 non-censored results) - MEDtot : 10.435 µg/kg (after inclusion of the 2 left-censored results) - MEDref : 10.48 µg/kg (after removal of 7 outliers,

including the 2 left-censored results) - MADref : 1.54 µg/kg (nref = 35) - σrob : 2.38 µg/kg

- The HorRat (σrob/ σp) is 0.99, well below the recommended limit 1.2, and the kernel density plot

is unimodal after removal of the outliers MEDref is therefore assigned as Vass. As in the case of AfB1, the z-score calculated from the HRL of the right-censored result (“>6 µgkg”) is correct but uninformative (z > -1.94). In contrast, those calculated from the LRL of the 2 left-censored results (“<0.089 µg/kg” and “<0.5 µg/kg”) are unsatisfactory (z < -4.51 and < -4.33).

..............................

Example with a method-related trend : aflatoxin G2 The case of aflatoxin G2 is interesting.

p. 16/40

The real concentration is very low (<1 µg/kg). Nearly half of the results received (21 out of 43) are left-censored, with LRL ranging from 0.046 to 2.5 µg/kg. MEDtot(=) is 0.822 µg/kg, The LRL of 6 of the left-censored results are below this MEDtot(=) and are therefore included for the calculation of MEDtot, which as a consequence is significantly lower (0.643 µg/kg) than MEDtot(=). After removal of 7 high outliers and the 2 lowest LRL (“<0.046” and “<0.2” µg/kg), MEDref is down to 0.5 µg/kg. MADref is 0.105 µg/kg, which gives 1.42 as HorRat value. This is higher than the recommended value 1.2, but could still have been considered acceptable (< 1.5) if the kernel density plot did not show clearly a multimodal distribution of the results. Therefore no Vass is assigned, and the scores have an indicative value only. The interesting part is the comparison with the method details, for the 19 non-censored results for which this information was supplied by the participants. It is quite clear that :

the 1st group (n=6), centred around 0.45 µg/kg, comprises the 5 results obtained after IAC clean-up (by either LC-Fl or LC-MS), plus one result from those obtained by LC-MS after another type of clean-up

the 2d and 3d groups, centred around 0.83 and 1.28 µg/kg, comprise 10 results obtained by LC-MS, either without clean-up (n = 6) or with a non-IAC type of clean-up (n = 4)

the 3 high outlier results were obtained by LC-MS, again either without clean-up (n = 2, with >8 µg/kg, not visible on the kernel plots) or with a non-IAC type of clean-up (n = 1, with 2.15 µg/kg)

Two hypotheses among others might explain the method-related trends observed for AfB2 and AfG2. On the one hand, the IAC results could be too low because of incomplete recovery (partial saturation of the columns ?). On the other hand it is also possible that, without IAC, some of the results are too high because of the superposition of AfB2 and some interfering substance(s) in the same chromatographic peak. The further surprising observation is that the AfG2 results from the homogeneity tests (which are below the usual LRL of the “dilute-and-shoot” LC-MS method used for these tests, and for which the ‘qualifier’ peaks was too small to confirm the purity of the observed peaks) give a bimodal profile which fits the 2d and 3d groups described above (for this reason, for AfG2 the homogeneity test results have not been rescaled along the X-axis through multiplication by the MEDref/MEDhom ratio as usual, but by the MEDtot(=)/MEDhom ratio, which superimposed them on these two groups of results).

..............................

Sum of aflatoxins Fortunately, the uncertainties about the true concentrations of AfB2 and AfG2 have a negligible impact on the consensus about the sum of aflatoxins, and the results are on the whole very good. The HorRat value is 1.15 and the distribution is unimodal. MEDref (33.84 µg/kg) is therefore assigned as Vass. Among the 36 non-censored results, 33 are satisfactory, 2 are questionable and only one is far too high. The only right-censored result is correct as statement (>24 µg/kg, z(HRL)>-1.32), and the actual measurement result (43.97 µg/kg) would have obtained a satisfactory z-score (1.36). ,

..............................

p. 17/40

Ochratoxin The MEDref is 190.8 µg/kg. The HorRat (1.34) is only slightly higher than the recommended value and the distribution seems reasonably unimodal. However, the kernel density plot and the detailed results (annex C) shows that during the homogeneity tests 3 out of the 10 samples (in fact, 3 out of the 20 subsamples) seemed to have concentrations >20% higher than the others. Therefore it cannot be excluded that some OTA “hot spots” remained in the material. For this reason, and despite the generally good consensus and the relatively low number of outliers among the participants, only indicative z-scores are given because the “questionable” or “unsatisfactory” status of the high z-scores would in itself be questionable.

.............................. Deoxynivalenol The results are straightforward, with MEDref = 2139 µg/kg, HorRat = 1.11, and a unimodal distribution. The MEDref is assigned as Vass, Forty z-scores are satisfactory, 2 are questionably low, 5 are <-3, and 1 is >3.

.............................. 3- acetyl- deoxynivalenol and 5-acetyl- deoxynivalenol There is no satisfying consensus for either of these analytes (multimodal distributions, HorRat 1.53 and 2.22). There is no reason to believe this could come from insufficient homogeneity, since the material passed the tests with a comfortable margin, and both of these toxins were natural companions of DON, for which the consensus is quite satisfying. There was no acetyl-DON in the vials, so it is unfortunately impossible to check the role of reference solution in this dispersion. For the sum, surprisingly the distribution seems much more unimodal. However the HorRat value (1.77) is much higher than recommended.

.............................. Zearalenone The case of zearalenone is similar to that of OTA :

- The MEDref is 563.2 µg/kg. The HorRat (1.32) is only slightly higher than the recommended value and the distribution seems reasonably unimodal.

- However, even though ZEN came from the same starting material as DON, the kernel density plot and table C.1 show that during the homogeneity tests 1 out of the 20 subsamples seemed to have a concentration >40% higher than the others.

Therefore it cannot be excluded that some ZEN hot spots remained in the material, and only indicative z-scores are given.

..............................

p. 18/40

HT-2, T-2 and their sum For HT-2 and T-2, the homogeneity tests were passed with a comfortable margin, and the distribution is unimodal after exclusion of the few outliers. The HorRat values (1.17 and 1.27) are acceptable. Interestingly, the HorRat value for the sum is significantly lower (1.02), which suggests that the analytical methods applied by some participants might de-acetylate part of the T-2 into HT-2.

.............................. Fumonisins For the 3 fumonisins, the material passed the homogeneity tests with more than comfortable margins: even the “raw” observed between-bags RSD was below the allowed ssampl. For FB1, FB2 and their sum, the kernel density plots show unimodal central tendencies, despite the presence of a relatively large number of outliers for FB1. The HorRat values are 1.51, 1.34 and 1.37 respectively, higher than 1.2 but still acceptable. On the flour vs vial A plot for FB1, two points lie close to the diagonal :

- L26 is one of the 2 lowest outliers for the flour (653 µg/kg = 0.2 MEDref) and the lowest outlier for both vials (0.3 MEDtot)

- Inversely, L50 is the highest outlier both for the flour (7108 µg/kg = 2.15 MEDref) and the vials (3.5 to 3.8 MEDtot)

In these 2 cases, the deviation is probably linked with the concentration of the reference solutions used (or possibly the solvents, since fumonisins are poorly soluble in low or high percentages of organic solvent). Apart from the obvious outliers, the only unsatisfactory z-score is the L44 result for the sum of FB1 and FB 2 : the FB1 result was questionably low (2302 µg/kg, z = -2.26), the FB2 result was left-censored (<1000, z < 4.76, actual measurement value not provided), and the sum was reported as 2032 µg/kg (z = -3.06), equal to the FB1 result. In this case at least, using the actual FB2 measurement value, even though <LRL, would probably have been a safer choice than the “lower-bound approach” (setting the FB2 concentration to 0 for calculating and reporting the sum) There is no significant trend linked with the type of clean-up used. The results obtained after IAC clean-up are generally as high as the others, so potential saturation of IAC columns is not a possible explanation for the low outliers.

.............................. Enniatins and beauvericin Despite the lower number of participants, a satisfying consensus was obtained for enniatins A (Vass = 63 µg/kg), A1 (Vass = 968 µg/kg) and B1 (Vass = 5850 µg/kg), and for beauvericin (Vass = 1562 µg/kg). The concentration of enniatin B was very high (MEDref = 14680 µg/kg), and no consensus was obtained (besides 1 low and 2 high outliers, there were 4 results at ca. 14000 µg/kg and 3 results at ca. 20000 µg/kg).

..............................

p. 19/40

Summary of statistics, assigned values and scores

aflatoxin B1

(AfB1)

aflatoxin B2

(AfB2)

aflatoxin G1

(AfG1)

aflatoxin G2

(AfG2)

sum aflatoxins

ochratoxin A

(OTA)

deoxynivaleno

l (DON)

3-acetyl-DON

15-acetyl-DON

sum 3- and 15-

acetyl-DON

zearalenone

(ZEN)

HT-2 toxin

T-2 toxin

sum HT-2 + T-2

toxins

Fumonisin B1

(FB1)

Fumonisin B2

(FB2)

sum

fumonisins B1

+ B2

Fumonisin B3

(FB3)

Enniatin A

(Enn-A)

Enniatin A1

(Enn-A1)

Enniatin B

(Enn-B)

Enniatin B1

(Enn-B1)

Beauvericin

(BEA)

Nto

t(=)

4535

4022

3640

4813

1614

4541

4435

3934

3116

1110

88

10

N(<

LRL)

04

09

00

05

12

00

00

02

00

00

00

0

N(x

>HR

L)2

01

01

50

00

02

11

01

01

10

12

21

MED

tot(

=)22

.32

1.72

710

.54

0.82

234

.49

187.

121

2741

.599

.17

156.

255

4.9

176.

932

9.1

546.

632

5053

9.7

3779

1935

67.1

194

2.2

1739

361

2814

36

MED

tot(

=,x<

,x>)

22.3

21.

727

10.5

90.

815

35.0

918

4.5

2126

.539

.22

109

156.

1555

2.5

177.

732

6.3

546.

632

02.5

531.

8537

9218

4367

.11

967.

717

094

6128

1368

MED

tot

22.3

21.

708

10.4

550.

6425

34.4

8518

9.7

2126

.541

.589

.275

153.

855

4.9

176.

932

9.05

543.

4532

5053

4.7

3779

1934

.567

.11

967.

717

392.

561

2814

35.5

Nto

t(=,

x<,x

>,<,

>,?)

)47

4343

4337

4548

2121

1747

4245

3640

3732

1711

1110

1011

Nre

f40

3035

1934

3644

812

1340

3634

3430

3128

98

95

78

Nto

t -

Nre

f0

00

00

00

00

00

00

00

00

00

00

00

MED

ref

22.3

351.

714

10.4

80.

533

.835

190.

821

3939

.22

82.1

8515

3.8

563.

218

6.2

329.

0554

9.5

3300

.554

538

3422

8563

.005

967.

714

680

5850

1562

σ(H

orw

-Th

)22

.0%

22.0

%22

.0%

22.0

%22

.0%

20.5

%14

.3%

22.0

%22

.0%

21.2

%17

.4%

20.6

%18

.9%

17.5

%13

.4%

17.5

%13

.1%

14.1

%22

.0%

16.1

%10

.7%

12.3

%15

.0%

σ(H

orw

-Th

)4.

914

0.37

72.

306

0.11

7.44

439

.17

305.

28.

628

18.0

832

.62

98.2

538

.37

62.2

496

.21

441.

295

.54

501.

132

2.8

13.8

615

5.6

1568

717.

523

3.7

crit

eri

um

ou

tlie

rs50

.0%

50.0

%50

.0%

50.0

%50

.0%

50.0

%50

.0%

50.0

%50

.0%

50.0

%50

.0%

50.0

%50

.0%

50.0

%50

.0%

50.0

%50

.0%

50.0

%50

.0%

50.0

%50

.0%

50.0

%50

.0%

MA

D/M

EDre

f0.

170.

230.

150.

210.

170.

190.

110.

230.

330.

250.

160.

160.

160.

120.

140.

160.

120.

100.

070.

090.

110.

100.

15

σ(r

ob

ust

)25

.8%

34.1

%21

.8%

31.1

%25

.3%

27.5

%15

.8%

33.7

%48

.8%

37.6

%23

.0%

24.1

%24

.1%

17.9

%20

.2%

23.4

%17

.8%

14.6

%9.

7%12

.6%

16.2

%14

.1%

21.5

%

Ho

rRat

1.17

1.55

0.99

1.42

1.15

1.34

1.11

1.53

2.22

1.77

1.32

1.17

1.27

1.02

1.51

1.34

1.37

1.03

0.44

0.78

1.52

1.15

1.44

Ho

rRat

/re

com

m0.

981.

290.

831.

180.

961.

110.

921.

281.

851.

481.

100.

981.

060.

851.

261.

111.

140.

860.

370.

651.

270.

961.

20

Vas

s (=

MED

ref)

22.3

35-

10.4

8-

33.8

35-

2139

--

--

186.

232

9.05

549.

533

00.5

545

3834

-63

.005

967.

7-

5850

1562

u(M

EDre

f)4.

1%6.

2%3.

7%7.

1%4.

3%4.

6%2.

4%11

.9%

14.1

%10

.4%

3.6%

4.0%

4.1%

3.1%

3.7%

4.2%

3.4%

4.9%

3.4%

4.2%

7.3%

5.3%

7.6%

0.91

20.

107

0.38

60.

036

1.46

68.

735

51.0

724.

678

11.5

8416

.04

20.5

237.

487

13.6

0316

.870

121.

808

22.9

5412

9.30

511

1.19

52.

152

40.6

7310

65.5

0331

1.56

611

8.72

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p. 20/40

References Université catholique de Louvain (UCL), Earth and Life Institute (ELI), Applied Microbiology, Mycothèque de l'Université catholique de Louvain (BCCMTM /MUCL), Croix du Sud 3/6, 1348 Louvain-la-Neuve, Belgium THE INTERNATIONAL HARMONIZED PROTOCOL FOR THE PROFICIENCY TESTING OF ANALYTICAL CHEMISTRY LABORATORIES (IUPAC Technical Report), Pure Appl. Chem., 78 (1), pp. 145–196, 2006. Manfred Metzler, “Proposal for a uniform designation of zearalenone and its metabolites”, Mycotox Res, 27 (1), pp. 1-3, 2011 Solfrizzo et al., “Results of a proficiency test for multi-mycotoxin determination in maize by using methods based on LC-MS/(MS)”, Quality Assurance and Safety of crops & foods, March 2013, 5 (1), pp. 15-48, 2013. CODA-CERVA, Report on the 2011 Proficiency Test for the determination of deoxynivalenol, HT-2 and T-2 toxins, zearalenone, beauvericin and enniatins A, A1 B and B1 in cereal flour CODA-CERVA, Report on the 2012 Proficiency Test for the determination of 17 mycotoxins in cereal flour CODA-CERVA, Report on the 2013 Proficiency Test for the determination of 17 mycotoxins in oats flour

……………………..

p. 21/40

Annexes Annex A. List and addresses of the participating laboratories (by alphabetical order)

AGES GmbH Wieningerstrasse 8, 4020 Linz Austria

Agriculture and Agri-Food Canada 1391 Sandford Street, N5V4T3-London Canada

Agrolab S.C.R.L Route de Herve, 104, 4651-Battice Belgium

ANALIZA Calidad Madrid, S.L. Avenida Castilla, 32. nave 56. Polígono

Industrial San Fernando de Henares,

28830-San Fernando de Henares (Madrid)

Spain

Australian Superintendence Company 467 Vulture Street, 4169-East Brisbane,

Queensland

Australia

Azienda Sanitaria Locale di Milano -

Laboratorio di Prevenzione

Corso Italia, 19, 20122-MILANO Italy

Canadian Food Inspection Agency 3155 Willingdon Green, V5G 4P2-Burnaby Canada

Canadian Grain Commission 1404-303 Main St, R3C3G8-Winnipeg Canada

Center for Analytical Chemistry,

Department IFA-Tulln, University of

Natural Resources and Life Science

Vienna

Konrad Lorenzstr. 20, A-3430-Tulln Austria

Central Institute for Supervising and

Testing in Agriculture (UKZUZ)

Hroznova 2, 65606-Brno Czech

Republic

CER Groupe rue du point du jour, 8, 6900-Marloie Belgium

Chinese Academy of Agricultural

Sciences, Oil Crops research institute

No.2 Xudong Second Road, Hubei

Province, 430062-Wuhan

P. R. China

CNTA (Centro Nacional de Tecnología

y Seguridad Alimentaria)

Ctra. NA-134- KM.53, 31570-SAN

ADRIÁN

Spain

CODA-CERVA Leuvensesteenweg 17, 3080-Tervuren Belgium

Eurofins WEJ Contaminants GmbH Neulaender Kamp 1, 21079-Hamburg Germany

FLVVT Leuvensesteenweg 17, 3080-Tervuren Belgium

Fytolab Belgium cvba Technologiepark 2/3, 9052-Zwijnaarde Belgium

Ghent University, Laboratory of Food

Analysis

Ottergemsesteenweg 460, 9000-Ghent Belgium

Glasgow Scientific Services 64 Everard Drive, Glasgow, G21 1XG-

Glasgow

United

Kingdom

Handelslab Dr A Verwey Agrogruppe Coolhaven 32, 3024AC-Rotterdam Netherlands

p. 22/40

Institute for Global Food Security,

Queen''s University Belfast

Room 0G.326, David Keir Building, 18-30

Malone Road, BT9 5BN-Belfast

United

Kingdom

Institute of Food Safety, Animal Health

and Environment BIOR

Lejupes Street 3, LV-1076-Riga Latvia

Institute of quality standards and testing

technology for agricultural products,

Chinese Academy of Agricultural

Science

No.12 Southern Street of Zhong Guan

Cun, Hai Dian District, 100086-Beijing

P. R. China

Laboratorium ECCA NV Ambachtsweg 3, 9820-Merelbeke Belgium

LAVES- Futtermittelinstitut Stade Heckenweg 6, 21680-Stade Germany

LOVAP NV KLAUS-MICHAEL KHUENELAAN 11, 2440-

GEEL

Belgium

MTA TKI Nádor street 7., H-1051-Budapest Hungary

National Food Agency Box 622, SE-751 26-Uppsala Sweden

National Institute of Health Dr. Ricardo

Jorge

Av., Av. Padre Cruz, 1649-016-LISBOA Portugal

National Veterinary Institute (SVA) SVA, SE-75189-Uppsala Sweden

National Veterinary Research Institute

Department of Pharmacology and

Toxicology

Partyzantow 57, 24-100-Pulawy Poland

Nestle R&D Center SG (NQAC) 29, Quality Road, 618802-Singapore Singapore

NOFALAB Jan van Galenstraat 51, 3115JG-

Schiedam

Netherlands

Nutreco Nederland BV - Masterlab Veerstraat 38, 5831 JN-Boxmeer Netherlands

OLEOTEST LAGE WEG 427, B-2660-ANTWERPEN Belgium

Public Analyst''s Laboratory Dublin Sir Patrick Dun''s, Lower Grand Canal

Street, 2-Dublin

Ireland

Qualtech 7 rue du bois de la champelle, 54500-

Vandoeuvre-lès-Nancy

France

R-Biopharm AG An der neuen Bergstrasse 17, 64297-

Darmstadt

Germany

R-Biopharm Rhone Ltd Block 10 Todd Campus, Acre Road, G20

0XA-GLASGOW

United

Kingdom

RIKILT Wageningen UR Akkermaalsbos 2, 6708 WB-Wageningen Netherlands

Romer Labs Diagnostic GmbH Technopark 1, 3430-Tulln Austria

SGS Belgium - Division IAC Polderdijkweg 16, Haven 407, 2030-

Antwerpen

Belgium

p. 23/40

SGS Nederland B.V. Malledijk 18, Puntweg 6, 3208 LD-

Spijkenisse

Netherlands

The Southern African Grain Laboratory

NPC

SAGL, Grain Building, 477 Witherite Road,

The Willows, 0040-Pretoria

South Africa

The State Laboratory Young''s Cross, Celbridge, 1-Celbridge,

Co. kildare

Ireland

Trilogy Analytical Laboratory 870 Vossbrink Dr, 63090-Washington United States

Zhejiang Provincial Center for Disease

Control and Provention

630 Xincheng Road, Binjiang District,

Hangzhou City, China, 310051-Hangzhou

P. R. China

p. 24/40

C O D A - C E R V A

Centrum voor Onderzoek in Diergeneeskunde en Agrochemie

Centre d’Etude et de Recherches Vétérinaires et Agrochimiques

Annex B. Instructions to the participants, receipt form and report form

p. 25/40

p. 26/40

p. 27/40

p. 28/40

p. 29/40

p. 30/40

p. 31/40

Annex C. Homogeneity tests Rye flour material Introduction : description of test, calculations, and tables of results

Note : this section is almost identical to the equivalent section in the 2013 report, except for: - the number of samples (10 instead of 12) - the number of dilutions of each extract (4 instead of 2) and the highest dilution factor

(5000 instead of 50) - the consequence of the number of samples on the values of F1 and F2 (1.88 and

1.01 instead of 1.79 and 0.86) and therefore on the minimum possible for the critical

value c (√𝑐 𝜎sampl.allow.⁄ = √1.79 = 1.37 𝑖𝑛𝑠𝑡𝑒𝑎𝑑 𝑜𝑓 √1.88 = 1.34)

The material was tested for homogeneity according to the IUPAC International Harmonized Protocol. Briefly, 10 samples were selected at random, and from each sample two 5 g subsamples were extracted and analyzed with a “dilute-and-shoot” LC-MS/MS method. From each extract 4 dilutions, with final dilution factors 5-50-500-5000 mL/g, were prepared and injected into the LC-MS/MS for analysis. The target standard deviations σp used were calculated by applying the Horwitz-Thompson function to the MEDref (medians after exclusion of outliers) of the results from the PT.

The calculations performed and the acceptance criteria are based on the IUPAC 2006 protocol :

the condition for “sufficient homogeneity” is that the true between-samples variability (σsampl.) does not exceed σsampl.allow. = 0.3 σp

the SD of the 12 average results includes a contribution from the between-samples variability and a contribution from the analytical variability sanal ; therefore, if this SD is:

o < 0.3 σp : then the material is certainly sufficiently homogenous o > 0.3 σp : then the critical value test described below should be applied

the observed between-samples variability ssampl. is calculated by ANOVA substraction of the observed analytical variability sanal. from the variability of the 12 averages :

(ssampl.)2 = (s(12 averages))

2 – (sanal.)2 / 2

for this calculation, the analytical variability is assimilated to the between-subsamples variability (which implies that each sample has been perfectly homogenized before sub-sampling) :

(sanal.)2 = average of the 12 (s(2 subsamples))

2

it is not uncommon in actual practice that (sanal.2) / 2 > (s(12 averages))

2, so that the result of the ANOVA substraction is negative. In this case ssampl. is set to 0. This can happen when the real between-samples variability σsampl. is indeed small, but also when σsampl. is relatively large but

sanal. /√2 is even larger (see next point)

because of the ANOVA substraction, if sanal. is not small enough, important sampling variation may be obscured by analytical variation. We may get a non-significant result when testing for heterogeneity, not because it is not present, but because the test has no power to detect it. Therefore it is recommended that σanal. < 0.5 σp.

Finally the hypothesis of sufficient homogeneity

H:(σsampl.)2≤(σsampl.allow).

2

is tested, and rejected if the 95% confidence interval around σsampl. does not include σsampl.allow., i.e. when the observed sampling variability exceeds a certain critical value :

(ssampl.)

2 > c = F1 (σsampl.allow.)2 + F2 (sanal.)

2

p. 32/40

where F1 and F2 are constants that may be derived from standard statistical tables. For 10

samples, F1 = 1.88 and F2 = 1.01, so that √𝑐 ≥ 1.37 σsampl.allow.. In other words this test, being based on a 95% confidence interval, is less stringent than direct comparison between ssampl. and σsampl.allow. (a test which, in case a material had ssampl. = σsampl.allow., would actually give 50% probability of wrongly rejecting it)

Table C1 below gives, for each toxin :

- the results for each subsample and sample, expressed as ratios to the average of the 12 samples

- the assigned values : o MEDref, the median of the participants’ results after exclusion of outliers o σp, the target standard deviation derived from MEDref using the Horwitz-Thompson

equation o the recommended maximum for sanal., equal to 0.5 σp, o σsampl.allow., the maximum between-samples variability, equal to 0.3 σp

- the between-samples RSD, with the comment “PASS” if this RSD is already smaller than σsampl.allow.

- the ANOVA calculations : o sanal., with the comment “PASS”/“(FAIL)” when this sanal. is below/above the

recommended value o ssampl., with the comment “PASS” if ssampl. is already smaller than σsampl.allow.

- the details of the critical value test : o σsampl.allow., sanal. and ssampl., in µg/kg o the critical value c o the square root of this critical value, expressed as % of MEDref o the ratio of this square root to σsampl.allow. o the ratio of ssampl. to the square root of c o ssampl.

2 and the result of the test : “PASS” if (ssampl.

2 < c) and (sanal. < 0.5 σp) “(PASS)” if (ssampl.

2 < c) but (sanal. > 0.5 σp) “FAIL” if (ssampl.

2 > c) Fig. C1 and C2 below summarize the four ratios :

Fig. C1 : (sanal. / σanal.recomm.)

Fig. C2 : (between-samples RSD / σsampl.allow.), (ssampl. / σsampl.allow.) and (ssampl. / √𝑐).

p. 33/40

Discussion The results are on the whole very similar to those of the 2013 PT:

all molecules pass the critical value test.

However, for 3 of them (AfG2, OTA and ZEN, the same as in 2013), although ‘sufficient homogeneity’ is not disproved, the relatively high analytical variability (within-bag differences) decreases the discriminating power of the test to the point that sufficient homogeneity may not be considered as adequately proved either.

In more detail, the results fall into 3 groups :

- For 11 toxins (AfB1, AfG1, 3-AcDON, fumonisins, enniatins and BEA), even the between-samples RSD were smaller than σsampl.allow. ; a fortiori the ANOVA test and the critical value test were passed.

- For 4 other toxins (AfB2, 15-AcDON, HT-2 and T-2), the sanal. were satisfactorily low ; the

ssampl. of 1 of them (15-AcDON) also passed the ANOVA test, and all 4 of them passed the critical value test

- For the last 4 toxins (AfG2, OTA, DON, ZEN), the ssampl. were higher than recommended ; the critical value tests were formally passed, but the discriminating power of the test was less than satisfactory. In this respect it is interesting to note that, for AfG2, OTA and ZEN,

the relatively high sanal. (1.3 to 1.8 times the recommended values) resulted in (√𝑐 / σsampl.allow.) ratios between 2.6 and 3.3, clearly above the 1.5-to-2.0 range obtained for the other toxins. In contrast, for DON the sanal. was only slightly above the recommended value

(7.5% instead of 7.1%), and therefore the (√𝑐 / σsampl.allow.) ratio also was only slightly higher (2.22) than for other molecules (e.g. 1.98 for FB1)

For the 4 toxins for which the discriminating power of the test is less than satisfactory, further discussion is needed :

- For DON, sufficient homogeneity seems a safe assumption, because :

o the recommended analytical variability (7.1%) is only marginally exceeded (7.5%), so the discriminating power of critical value test is close to satisfactory

o also the observed between-bag RSD (4.8%) is only marginally higher than σallowable, 4.3%), even though it includes a contribution from analytical variability

- for AfG2, the concentrations measured were below the LRL of the method, and the

“qualifier” peaks were too small to allow positive identification by the ion ratio. However, sufficient homogeneity seems a safe assumption, unless the distribution of AfG2 is different from that of AfB1 and AfG1

- for OTA and ZEN, sufficient homogeneity cannot be considered as either excluded or

adequately proved on the basis of the critical value test. Further examination of the results shows that respectively 3 and 1 out of the 20 subsamples (of 5 grams each) seem to have concentrations at leat 20% higher than the average. The presence of residual small-scale “hot spots” can therefore not be excluded, despite the careful multi-step homogenization.

Despite the very low incorporation levels, it is unlikely that these OTA and ZEN hot spots could be due to insufficient blending, because :

- the final material is homogenous for aflatoxins, even though the aflatoxin-rich material was incorporated at a lower level (0.03%) than the DON and ZEN-rich material (0.06%) and especially the OTA-rich material (0.5%).

- ZEN seems much less homogeneously distributed than DON (between-bags RSD 12.1% vs 4.8%) even though these two toxins come from the same material, wherein they were produced at the same time by the same fungi

p. 34/40

Fig. C1 : (sanal. / σanal.recomm.) ratio

Fig. C2 : (between-samples RSD / σsampl.allow.), (ssampl. / σsampl.allow.) and (ssampl. / √𝑐) ratios

p. 35/40

Table C.1 : Results of the homogeneity test

AfB

1A

fB2

AfG

1A

fG2

OTA

DO

N3-A

cD

ON

15-A

cD

ON

ZE

N

resu

lts

(re

lati

ve

to

ave

rag

e)

av

a-b

av

a-b

av

a-b

av

a-b

av

a-b

av

a-b

av

a-b

av

a-b

av

a-b

1a

1.0

27

1.2

13

1.0

82

1.0

15

0.9

02

0.9

47

1.0

21

1.0

37

1.0

12

1b

0.9

77

1.0

02

1.0

29

1.1

21

1.0

04

1.0

43

0.7

93

0.9

04

1.1

65

1.0

34

0.9

46

0.9

46

0.9

17

0.9

69

1.0

06

1.0

22

0.8

51

0.9

31

2a

1.1

26

0.9

86

1.1

28

1.3

76

0.9

61

1.0

15

1.0

35

1.1

31

0.9

94

2b

0.9

06

1.0

16

0.9

76

0.9

81

0.9

23

1.0

26

1.2

10

1.2

93

0.9

79

0.9

70

1.1

11

1.0

63

1.0

67

1.0

51

1.0

66

1.0

99

0.8

22

0.9

08

3a

1.0

67

1.0

69

1.0

18

1.2

60

1.0

12

1.0

20

0.9

46

0.9

65

1.0

18

3b

0.9

76

1.0

21

1.1

07

1.0

88

0.9

53

0.9

85

1.0

26

1.1

43

0.8

45

0.9

28

1.1

24

1.0

72

1.1

17

1.0

32

1.2

28

1.0

96

1.1

64

1.0

91

4a

0.8

57

0.7

87

0.9

01

0.8

42

0.7

96

0.9

87

0.9

05

0.9

94

0.9

35

4b

0.9

84

0.9

21

0.8

66

0.8

26

1.0

23

0.9

62

0.9

41

0.8

92

0.8

92

0.8

44

0.9

56

0.9

71

0.8

92

0.8

98

1.0

34

1.0

14

1.1

13

1.0

24

5a

1.0

69

0.9

80

1.0

92

0.8

76

0.7

81

0.9

47

0.9

71

0.9

67

0.9

31

5b

0.9

45

1.0

07

0.9

68

0.9

74

0.9

45

1.0

18

0.7

20

0.7

98

0.8

42

0.8

12

1.0

36

0.9

91

1.0

32

1.0

02

0.9

73

0.9

70

1.0

44

0.9

88

6a

1.0

12

1.0

41

1.0

22

1.1

96

0.9

28

0.9

61

1.0

25

0.9

70

1.0

72

6b

1.0

94

1.0

53

1.1

33

1.0

87

0.9

96

1.0

09

1.0

26

1.1

11

0.7

51

0.8

40

0.9

48

0.9

55

0.9

82

1.0

04

0.9

11

0.9

40

0.9

82

1.0

27

7a

1.0

45

1.0

32

1.0

24

0.8

54

1.4

51

0.8

77

0.8

80

0.9

80

0.8

85

7b

1.0

98

1.0

71

1.1

49

1.0

90

1.0

60

1.0

42

1.2

01

1.0

28

0.9

81

1.2

16

1.1

13

0.9

95

1.0

59

0.9

69

0.9

98

0.9

89

0.8

02

0.8

43

8a

1.0

30

0.9

58

1.0

13

0.7

10

1.2

82

1.0

66

1.0

67

0.9

76

1.0

94

8b

0.9

21

0.9

75

0.9

32

0.9

45

0.8

64

0.9

39

1.0

57

0.8

84

1.0

62

1.1

72

1.0

07

1.0

36

1.0

14

1.0

40

0.9

28

0.9

52

1.4

61

1.2

77

9a

0.9

40

0.8

74

1.0

29

0.9

59

1.4

83

1.0

77

0.9

25

0.9

50

0.8

91

9b

0.9

42

0.9

41

0.8

56

0.8

65

1.0

01

1.0

15

1.2

86

1.1

23

1.0

35

1.2

59

0.9

81

1.0

29

1.1

07

1.0

16

1.1

59

1.0

54

0.9

28

0.9

09

10a

0.9

51

1.0

74

0.9

13

0.8

80

0.7

88

0.8

82

0.9

49

0.8

71

0.9

95

10b

1.0

33

0.9

92

0.9

70

1.0

22

1.0

12

0.9

62

0.7

69

0.8

25

1.0

62

0.9

25

0.9

98

0.9

40

1.0

89

1.0

19

0.8

54

0.8

63

1.0

05

1.0

00

ass

ign

ed

va

lue

s

ME

Dre

f (µ

g/k

g)

22.3

51.

714

10.4

80.

519

1.9

2130

41.5

82.1

8556

1.15

σp (

Horw

-Th)

22.0

%22.0

%22.0

%22.0

%20.5

%14.3

%22.0

%22.0

%17.5

%

σa

na

l.re

co

mm

. = 0

.5 σ

p11.0

%11.0

%11.0

%11.0

%10.3

%7.1

%11.0

%11.0

%8.7

%

σsa

mp

l.a

llow

.

= 0

.3 σ

p6.6

%6.6

%6.6

%6.6

%6.2

%4.3

%6.6

%6.6

%5.2

%

RS

D(b

etw

ee

n b

ag

s)

4.6%

PASS

10.0

%3.

6%PASS

16.3

%16

.4%

4.8%

4.5%

PASS

7.3%

12.1

%A

NO

VA

te

st

san

al. :

PA

SS

if <

0.5

σp

7.7%

PASS

6.2%

PASS

7.9%

PASS

16.7

%(FAIL)

18.5

%(FAIL)

7.5%

(FAIL)

8.2%

PASS

7.9%

PASS

11.7

%(FAIL)

ssa

mp

l. :

PA

SS

if <

0.3

σp

0.0%

PASS

9.1%

0.0%

PASS

11.3

%9.

9%0.

0%0.

0%PASS

4.7%

PASS

8.9%

cri

tica

l va

lue

te

st

σsa

mp

l.a

llow

. (µ

g/k

g)

1.48

0.11

312

0.69

168

0.03

311

.809

391

.242

2.73

95.

4242

129

.382

4

san

al. (µ

g/k

g)

1.72

0.10

552

0.82

307

0.08

331

35.5

388

159.

069

3.40

133

6.52

816

65.4

012

ssa

mp

l. (

µg/k

g)

00.

1552

30

0.05

666

18.9

295

00

3.86

954

50.0

18

c (

= F

1 (

σsa

mp

l.allo

w.)

2 +

F2 (

san

al)2

)7.

090.

0353

11.

5836

50.

0090

615

37.8

241

207.

125

.788

798

.356

559

43.1

5

11.9

%11.0

%12.0

%19.0

%20.4

%9.5

%12.2

%12.1

%13.7

%

1.80

568

1.66

098

1.81

938

2.88

395

3.32

069

2.22

481.

8540

61.

8283

72.

6237

4

00.

8261

50

0.59

534

0.48

271

00

0.39

017

0.64

881

(ssa

mp

l.)2

: P

AS

S if <

c0

PASS

0.02

41PASS

0PASS

0.00

321

(PA

SS)

358.

324

(PA

SS)

0(P

ASS

)0

PASS

14.9

733

PASS

2501

.8(P

ASS

)

𝑐(a

s %

of M

EDre

f)

𝑐/

σsa

mpl.

allo

w.

s sam

pl.

/ 𝑐

𝑐(a

s %

of M

EDre

f)

𝑐/

σsa

mpl.

allo

w.

s sam

pl.

/ 𝑐

p. 36/40

Table C.1 (continued) : Results of the homogeneity test

HT2

T2

FB

1F

B2

FB

3E

nn-A

Enn-A

1E

nn-B

Enn-B

1B

EA

resu

lts

(re

lati

ve

to

ave

rag

e)

av

a-b

av

a-b

av

a-b

av

a-b

av

a-b

av

a-b

av

a-b

av

a-b

av

a-b

av

a-b

1a

1.0

28

0.9

54

1.0

65

1.0

66

1.0

70

1.1

78

1.0

73

1.0

19

1.0

68

0.9

83

1b

1.1

67

1.0

98

1.0

03

0.9

78

1.0

13

1.0

39

1.0

06

1.0

36

0.9

93

1.0

32

0.9

72

1.0

75

0.9

51

1.0

12

0.9

56

0.9

88

0.9

38

1.0

03

0.9

80

0.9

82

2a

1.1

05

1.1

10

1.0

09

0.9

80

1.0

01

0.9

43

0.9

54

0.9

92

0.9

56

1.0

33

2b

1.1

16

1.1

11

1.0

26

1.0

68

1.0

13

1.0

11

0.9

69

0.9

75

0.9

78

0.9

89

0.9

24

0.9

34

0.9

56

0.9

55

0.9

76

0.9

84

0.9

76

0.9

66

1.0

22

1.0

27

3a

0.9

86

1.1

20

0.9

63

0.9

64

0.9

80

0.9

67

0.9

82

0.9

96

0.9

84

0.9

91

3b

0.9

68

0.9

77

1.1

15

1.1

17

1.0

50

1.0

06

1.0

21

0.9

92

1.0

44

1.0

12

1.1

10

1.0

38

1.0

75

1.0

29

1.0

34

1.0

15

1.0

42

1.0

13

1.0

37

1.0

14

4a

1.0

87

0.9

21

1.0

11

1.0

15

1.0

00

0.9

95

1.0

21

1.0

24

1.0

11

0.9

73

4b

1.0

28

1.0

58

1.0

16

0.9

68

1.0

23

1.0

17

0.9

98

1.0

07

1.0

08

1.0

04

1.0

26

1.0

10

1.0

04

1.0

12

1.0

07

1.0

16

1.0

34

1.0

22

1.0

23

0.9

98

5a

1.0

31

1.1

15

1.0

06

0.9

99

1.0

09

1.0

08

1.0

23

1.0

08

1.0

08

1.0

26

5b

0.9

22

0.9

76

0.9

98

1.0

57

0.9

90

0.9

98

0.9

73

0.9

86

0.9

66

0.9

88

1.0

19

1.0

13

1.0

50

1.0

37

1.0

39

1.0

24

1.0

59

1.0

33

1.0

14

1.0

20

6a

0.9

46

0.8

82

1.0

13

0.9

92

1.0

19

1.0

18

1.0

37

1.0

13

1.0

22

1.0

22

6b

0.9

55

0.9

51

0.9

36

0.9

09

1.0

01

1.0

07

0.9

95

0.9

94

0.9

96

1.0

08

1.0

25

1.0

21

1.0

69

1.0

53

1.0

16

1.0

15

1.0

33

1.0

27

1.0

23

1.0

22

7a

0.8

63

0.9

80

1.0

53

1.0

71

1.0

41

0.9

88

0.9

64

0.9

56

0.9

56

0.9

91

7b

0.9

04

0.8

84

0.9

46

0.9

63

1.0

04

1.0

28

1.0

28

1.0

50

1.0

28

1.0

34

1.0

69

1.0

29

1.0

00

0.9

82

1.0

40

0.9

98

1.0

63

1.0

10

0.9

82

0.9

87

8a

1.0

01

1.0

15

0.9

72

1.0

24

1.0

06

0.9

45

1.0

22

1.0

20

1.0

19

1.0

25

8b

0.9

98

1.0

00

1.1

34

1.0

74

1.0

02

0.9

87

0.9

95

1.0

09

0.9

73

0.9

89

0.9

47

0.9

46

0.9

33

0.9

78

0.9

58

0.9

89

0.9

45

0.9

82

0.9

68

0.9

97

9a

1.1

05

0.8

94

0.9

70

0.9

83

0.9

83

0.9

96

1.0

03

1.0

21

1.0

20

0.9

86

9b

0.9

88

1.0

46

0.9

19

0.9

07

0.9

77

0.9

73

1.0

16

1.0

00

0.9

76

0.9

79

0.9

62

0.9

79

0.9

99

1.0

01

0.9

96

1.0

08

0.9

91

1.0

06

0.9

69

0.9

77

10a

0.8

09

0.9

58

0.8

20

0.8

35

0.8

51

0.8

98

0.9

18

0.9

53

0.9

20

0.9

40

10b

0.9

92

0.9

01

0.9

58

0.9

58

1.0

47

0.9

33

1.0

69

0.9

52

1.0

79

0.9

65

1.0

11

0.9

54

0.9

64

0.9

41

0.9

74

0.9

64

0.9

56

0.9

38

1.0

13

0.9

77

ass

ign

ed

va

lue

s

ME

Dre

f (µ

g/k

g)

178.

533

7.1

3270

.554

4.85

2856

.25

63.0

0596

7.7

1468

058

5015

62

σp

(Horw

-Th)

20.7

%18.8

%13.4

%17.5

%13.7

%22.0

%16.1

%10.7

%12.3

%15.0

%

σa

na

l.re

co

mm

. = 0

.5 σ

p10.4

%9.4

%6.7

%8.8

%6.8

%11.0

%8.0

%5.3

%6.1

%7.5

%

σsa

mp

l.a

llow

.

= 0

.3 σ

p6.2

%5.7

%4.0

%5.3

%4.1

%6.6

%4.8

%3.2

%3.7

%4.5

%

RS

D(b

etw

ee

n b

ag

s)

7.8%

7.4%

3.0%

PASS

2.8%

PASS

2.2%

PASS

4.5%

PASS

3.6%

PASS

1.9%

PASS

3.0%

PASS

1.9%

PASS

AN

OV

A t

est

san

al. :

PA

SS

if <

0.5

σp

6.5%

PASS

5.1%

PASS

5.7%

PASS

5.8%

PASS

5.8%

PASS

6.5%

PASS

4.3%

PASS

3.1%

PASS

4.6%

PASS

2.6%

PASS

ssa

mp

l. :

PA

SS

if <

0.3

σp

6.3%

6.4%

0.0%

PASS

0.0%

PASS

0.0%

PASS

0.0%

PASS

1.9%

PASS

0.0%

PASS

0.0%

PASS

0.6%

PASS

cri

tica

l va

lue

te

st

σsa

mp

l.a

llow

. (µ

g/k

g)

11.1

0519

.058

113

1.34

28.6

558

117.

067

4.15

833

46.6

797

470.

281

215.

243

70.1

085

san

al. (µ

g/k

g)

11.5

502

17.0

472

187.

377

31.4

424

164.

492

4.10

509

41.5

142

451.

308

271.

381

41.2

015

ssa

mp

l. (

µg/k

g)

11.1

807

21.6

689

00

00

18.6

615

00

8.75

913

c (

= F

1 (σ

sam

pl.a

llow

.)2 +

F2

(san

al)2 )

366.

586

976.

349

6789

1.9

2542

.28

5309

2.9

49.5

287

5837

.17

6215

0516

1484

1095

5.1

10.7

%9.3

%8.0

%9.3

%8.1

%11.2

%7.9

%5.4

%6.9

%6.7

%

1.72

412

1.63

954

1.98

386

1.75

954

1.96

827

1.69

242

1.63

672

1.67

635

1.86

696

1.49

292

0.58

396

0.69

348

00

00

0.24

426

00

0.08

369

(ssa

mp

l.)2

: P

AS

S if <

c12

5.00

9PASS

469.

542

PASS

0PASS

0PASS

0PASS

0PASS

348.

251

PASS

0PASS

0PASS

76.7

223

PASS

𝑐(a

s %

of M

EDre

f)

𝑐/

σsa

mp

l.al

low

.

s sam

pl.

/ 𝑐

p. 37/40

Vials containing evaporated-to-dryness solutions Three vials of each type were selected at random and their contents reconstituted in 1 mL ACN:HAc:H20 (80:2:18). From each solution 40 µL were drawn and mixed with 10 µL internal standard solution (13C analogs of each toxin except acetyl-DONs, enniatins and beauvericin) in an HPLC vial with a small volume insert. Each of these mixtures was injected 3 times into the LC-MS/MS system. Table C.2 below gives, for each level (A and B) :

the analytical variability (square root of the average variance of the 3 sets of 3 injections)

the RSD of the 3 averages

the between-vials difference, obtained by ANOVA subtraction of the analytical repeatability from the RSD of the averages

The values above 5% are highlighted in yellow, those above 10% in pink. At the higher concentration level (vials A), all values are below 5%. At the lower concentration level (vials B) also, all values are below 5% except for :

zearalenone: the between-vials RSD is 7%, but the estimated between-vials difference is back to 4.6% after ANOVA subtraction of the analytical repeatability (4.3%)

aflatoxins, with RSD up to 23%: as in 2013, the poor and variable recoveries of low levels of aflatoxins after drying suggest chemical degradation and/or irreversible adsorption. (see also figures C.3 and C.4)

In conclusion, the vials are fit for the primary purpose intended, i.e. detecting whether >50% deviations for the flour might be explained by >50% deviations in reference solutions. Except for aflatoxins, they are also fit for the secondary purpose, i.e. checking linearity and LOQ. Table C.2 : vial tests

vials A vials B vials A vials B vials A vials B

AfB1 2.0% 2.4% 1.8% 21.4% 0.7% 20.1%

AfB2 3.4% 2.6% 2.0% 3.8% 0.0% 2.3%

AfG1 1.5% 1.8% 1.8% 23.3% 1.0% 22.2%

AfG2 4.1% 2.2% 0.4% 5.9% 0.0% 4.6%

OTA 2.9% 5.0% 2.5% 1.2% 0.8% 0.0%

DON 1.3% 1.9% 3.3% 4.3% 2.5% 3.2%

ZEN 1.5% 4.3% 1.7% 7.0% 0.9% 4.6%

HT2 2.3% 2.8% 2.1% 2.0% 0.8% 0.4%

T2 1.6% 2.7% 0.6% 0.5% 0.0% 0.0%

FB1 1.5% 4.0% 1.4% 4.4% 0.5% 2.1%

FB2 3.1% 1.8% 1.0% 3.7% 0.0% 2.7%

FB3 1.8% 3.3% 1.1% 3.8% 0.1% 1.9%

Enn-A 2.6% 3.6% 0.9% 1.7% 0.0% 0.0%

Enn-A1 2.8% 4.8% 1.3% 2.6% 0.0% 0.0%

Enn-B 2.7% 3.1% 0.0% 1.7% 0.0% 0.0%

Enn-B1 3.6% 3.9% 0.4% 0.9% 0.0% 0.0%

BEA 2.4% 3.1% 0.3% 0.6% 0.0% 0.0%

analytical

repeatability RSD of vial averages

between vials

difference (ANOVA)

p. 38/40

Fig. C.3 : vial tests

Fig. C.4 : comparison of recoveries at lower (B) and higher (A) concentrations

..........................

p. 39/40

Annex D. Analytical details The analytical questionnaire was completed by 42 of the 47 participants. Most of the results were obtained using LC-MS (see fig. D.1) in multi-mycotoxin methods (see fig. D.2 and D.3). The “box-and-whiskers” plots in part 2 allow the visual detection of any method-related trends. The subsamples weights ranged from 0.25 to 50 grams and fell into 4 classes (see fig. D.4). No correlation was observed with the results, nor with the differences between duplicate results.

Fig. D.1 : distribution of clean-up and measurement methods for each group of molecules.

p. 40/40

Fig. D.2 : number of different methods used, per set of results

Fig. D.3 : number of analytes reported per method

Fig. D.4 : subsample weight

..............................