Report on the 2011 Proficiency Test for the determination of T-2 and HT-2 toxins in wheat flour

CODA CERVA Belgian National Reference Laboratory for Mycotoxins in Food and Feed Report on the 2011 Proficiency Test for the determination of T-2 and HT-2 toxins in wheat flour May 2012 Ph. Debongnie

Table of Contents Summary Test materials Participants, instructions and schedule Assigned values and z- and ζ-scores Results and discussion Conclusions References Tables : Reference concentrations T-2 results, flours T-2 results, solutions HT-2 results, flours HT-2 results, solutions Figures, from top to bottom (T-2 and HT-2 in the left and right columns respectively) : Results, as % of expected values Results and MU for flour B z- and ζ-scores for flour B Results and MU for flour A z- and ζ-scores for flour A Annexes : A. List and addresses of the participating laboratories B. instructions to participants C. Receipt form D. Results form p. 2/14

Summary A Proficiency Test (PT) on the determination of T-2 and HT-2 toxins in wheat flour was conducted with 6 laboratories in Belgium The samples were a blank wheat flour supplemented at medium and low levels (respectively flour B, ca. 115 µg/kg, and flour A, ca. 40 µg/kg). These flours were accompanied by 2 solutions. The participants were asked to measure and report the concentrations of these 2 flours and 2 solutions, using their own reference solutions as calibrants. The standard deviations for proficiency assessment (σ p ) were calculated using the modified Horwitz equation. One participant reported only less than values, out of which 3 were actually below the reference values. For the other 5 participants, 50% of the 20 z-scores were satisfactory, 20% borderline (z -2), 20% questionably low, and 10% unacceptably high (due to HT-2 calibrants problems and/or calculation errors). Only 35% of the ζ-scores were satisfactory, while 5% were questionably low, 50% unacceptably low and 10% (HT-2 calibrants problems) unacceptably high. These results are not so good on the whole. Fortunately, the results of the participants to the present PT were much better in the multi-mycotoxin international PT conducted at the same time and reported elsewhere : all of them obtained more than satisfactory z- and ζ-scores for T-2 and HT-2, except in the few cases where calibrants problems and/or calculation errors again compromised the trueness of the results. The difference with this PT might be explained by the higher concentration levels (274 and 577 µg/kg). p. 3/14

Test materials The test materials were produced by supplementing 2 series of subsamples (20 g each) of a commercial blank wheat flour with mixed T-2/HT-2 solutions. The original mono-toxin stock solutions had been prepared by dissolving crystalline reference compounds (purchased from Biopure) in acetonitrile. One series of subsamples was supplemented at medium concentrations ( flour B, ca. 100 µg/kg), the other at low concentrations ( flour A, ca. 40 µg/kg). The solvent was allowed to evaporate overnight. The containers were then agitated on an over-head shaker. Each participant received 2 samples of each of the 2 materials. Participants, instructions and schedule A total of 6 laboratories approved by the Belgian Food Safety Agency (FAVV-AFSCA) participated in the PT. Each participant received 2 containers with 20 g flour A, 2 containers with 20 g flour B, and 2 vials containing the solutions described above. In addition to these 4 flour samples, each participant received 2 vials containing T-2/HT-2 mixtures at different concentrations in ACN:HAc:water 16:1:83. They were asked to measure the concentrations of these mixtures against 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 The participants were invited to extract the whole content of each of the 4 flour containers, follow their routine procedures in all other respects, and report (see annexes) : - the results of the 2 independent measurements of each flour, corrected for recovery - the recovery factor used for correction - the results of 3 replicate measurement of each of the 2 solutions - an estimate of their uncertainty of measurement (MU = U k=2 ) The results were to be reported on a form (protected Excel file) provided in attachment. The samples were dispatched in June 2011 and the deadline for reporting was Sept. 1. Upon receiving the draft report, a participant pointed out a mismatch in lab numbers in the table of results for the HT-2 solutions and communicated that they themselves had mismatched their recoveries and measurement uncertainties for HT-2 and T-2. Before issuing this final report, a further check also revealed a slight calculation error on the ζ-scores in flour B, which fortunately did not affect any of the previously reported qualitative conclusions in terms of satisfactory, questionable or unsatisfactory ζ-scores. The present report is corrected for these three errors. p. 4/14

Assigned values and z- and ζ-scores The concentrations of the flours and the solution are given in the following table : HT-2 T-2 flours (µg/kg) low (A) 42.5 37.5 high (B) 105 125 solutions (µg/l) low (C) 5 5 high (D) 200 175 These are nominal concentrations, i.e. as calculated from the theoretical amount of toxin according to the supplier and the successive dilution factors applied, without correction for purity of the original compounds of for dilution errors. It was assumed that the differences between these nominal concentrations and the true concentrations would be negligible compared to the target standard deviation σ p ( 22%) and to the dispersion of results from the participants. This assumption was confirmed by the very good results of one participant (not the PT provider) for flour B as well as solution D and by the generally good results of the other participants at least for solution D. Individual laboratory performance is expressed in terms of z- and ζ-scores in accordance with ISO 13528 and the International Harmonised Protocol. ( ) ( ) where X lab is the measurement result reported by a participant X ref is the assigned value p is the "standard deviation for proficiency assessment" u ref is the standard uncertainty on the reference value u lab is the standard uncertainty reported by a participant (all in µg/kg) As a conservative worst-case, the uncertainties u ref were set at 7% ( σ p /3) of the assigned values X ref. p. 5/14

Results and discussion The results as reported by the participants and the deviations, z- and ζ-scores are given in the tables and figures below. A few results were reported as less than : Lab 1 reported < 25 µg/kg T-2 and < 75 µg/kg HT-2 for both flours. This is correct for HT-2 in flour A but not in the 3 other cases. Therefore biases, z- and ζ-scores were calculated, also as less than, on the basis of the reporting limits 25 and 75 µg/kg. The symbols in the corresponding plots were left empty to emphasize the difference in meaning with the scores of the other participants. Lab 5 reported 29.6 µg/kg and <20 µg/kg for HT-2 in flour A, and 29.6 µg/kg as average. Lab 4 used 25 µg/kg as reporting limit for both T-2 and HT-2, but nevertheless provided their full quantitative results (e.g. <25 (estimated 15,6) ), a useful and commendable practice that allowed to check that the RSD were acceptable (<12% in general, 22% for T-2 in flour A). The histograms at the top of the page summarize all results, expressed as percentage of the expected values. For solution D (175-200 µg/l), the deviations were within ±10% in 5 cases out of 6 for T-2, but in only 3 cases for HT-2. For the more dilute solution C (5 µg/l), these numbers fall to 3 cases out of 4 for T-2 and 1 case out of 4 for HT-2. These histograms show probable links between the deviations for the flours and those for the solutions in 1 case for T-2 (lab 4), 2 cases for HT-2 (labs 2 and 4). The results of lab 6 are consistently very close to the expected values for the medium concentrations (deviations between -2% and 1% in general, 6% for T-2 in flour B). The z- scores (0.29 and 0.04 for T-2 and HT-2 respectively) and ζ-scores (0.30 and 0.03) are very good. They are, expectedly, not quite so good at low concentrations, but still quite acceptable even though the ζ-score for T-2 in flour A (-2.06) is just slightly below -2. Lab 5 also obtains satisfactory z-scores (-0.29 to -1.59). Its ζ-scores are satisfactory for T-2 (- 0.33 and -1.88) but not for HT-2 (-3.98 and -3.36), which suggests an underestimation of their MU (only 16.4% for HT-2, vs 37.6 % for T-2). Lab 4 obtained satisfactory z-scores for T-2 (-1.77 and -1.07) but questionable z-scores for HT-2 (-2.94 and -2.72). In both cases the deviations for the flours were in the same ranges as those for the solutions (ca. -40% for T-2 and -60% for HT-2), which suggests a problem with their reference solutions rather than with their recovery or analytical method. Lab 3 obtained good results for the solutions (deviations between -8% and -1%) but systematically low results for the flours (-43% to -45%). Their z-scores were all between - 1.96 and -2.03, i.e. at the limit between satisfactory and questionable, and their ζ-scores were unsatisfactory (-3.23 to -3.41). In the case of lab 2 there seems to be a problem with the HT-2 reference solution, leading to very high positive deviations both for the solutions (3500% and 1690%) and the flours (365% p. 6/14

and 446%). In contrast, with T-2 the deviations are small for the solutions (2% and -8%) but large for the flours (-55% and -47%), leading to questionable z-scores (-2.53 and -2.13). Finally, the less than z-scores of lab 1, calculated using their less than results (when this reporting limit was actually lower than the expected value), range from <-3.66 to <-1.30, and the corresponding ζ-scores from <-2.08 to <-11.21. It is worth mentioning that all 6 participants also took part in the international multi-mycotoxin PT conducted at the same time with 19 participants and reported elsewhere, and that 9 out of their 12 z-scores for T-2 and HT-2 (X ref = 263 and 562 µg/kg) were quite good (between -0.5 and +1.2), with corresponding ζ-scores between -0.7 and +0.7. The 3 exceptions (1 for T-2 and 2 for HT-2) were again clearly due to calibrants problems and/or calculation errors. The poor results obtained by labs 3, 1 and 2 in the present PT might be due to the lower concentration levels. Conclusions Out of the 20 z-scores (not counting the less than cases ) in the present PT, only 10 were really satisfactory, 4 were borderline (z=-1.96 to -2.03), 4 were questionably low (z=-2.13 to - 2.96), and 2 were unacceptably high (z>16, due to HT-2 calibrants problems). Fortunately the good results of the same laboratories (except again a few cases due to calibrants problems and/or calculation errors) in the multi-mycotoxin PT shows that there is no fundamental problem with the analytical methods used, but possibly only at low concentrations. It would be advisable : - For labs 2 and 4 to check the validity of their reference solutions - For labs 3, 2 and 1 to investigate the causes of their questionably low z-scores, and in function of the results of these investigations, possibly to revise their reporting limits and/or their MU - For lab 5 to consider revising its MU for HT-2. References THE INTERNATIONAL HARMONIZED PROTOCOL FOR THE PROFICIENCY TESTING OF ANALYTICAL CHEMISTRY LABORATORIES (IUPAC Technical Report), Pure Appl. Chem., Vol. 78, No. 1, pp. 145 196, 2006. p. 7/14

Results and calculations, tables T-2 in flours results from participants : calculations : flour B flour A flour B flour A res. 1 res. 2 aver. res. 1 res. 2 aver. recov. MU dev. dev. z-score ζ-score RSD dev. (µg/kg) (µg/kg) (µg/kg) (µg/kg) (µg/kg) (µg/kg) (%) (%, k=2) (µg/kg) (%) (µg/kg) dev. (%) z-score ζ-score RSD Lab 1 <25 <25 <25 <25 <25 <25 100 14 (-100) (-80.0%) (<-3.66) (<-11.21) (<-12.5) (<-33.3%)(<-1.52) (<-3.96) Lab 2 65.8 45.7 55.75 21 18.9 19.95 102 23-69.25-55% -2.53-6.38 25% -17.55-47% -2.13-5.03 7% Lab 3 84.5 55.8 70.15 22.1 19.4 20.75 ca. 100 40-54.85-44% -2.01-3.32 29% -16.75-45% -2.03-0.91 9% Lab 4 83 70 76.5 33.2 24.1 28.7 71 65-48.5-39% -1.77-1.84 12% -8.8-23% -1.07-1.15 22% Lab 5 99.5 134.9 117.2 23.2 30.3 26.8 37.6-7.8-6% -0.29-0.33 21% -10.7-29% -1.30-1.88 19% Lab 6 137 129 133 25 27 26 101 38 8 6% 0.29 0.30 4% -11.5-31% -1.39-2.06 5% T-2 in solutions results from participants : calculations : sol. D sol. C sol. D sol. C res. 1 res. 2 res. 2 average res. 1 res. 2 res. 2 average dev. dev. RSD dev. (µg/l) (µg/l) (µg/l) (µg/l) (µg/l) (µg/l) (µg/l) (µg/l) (µg/l) (%) (%) (µg/l) µg/kg % µg/kg % X ref 125 X ref 37.5 σ p 27.4 21.9% σ p 8.3 22.0% u ref 8.8 7% u ref 2.6 7% Lab 1 188 198 191 192.3 <25 <25 <25 <25 17.3 10% 2.7% Lab 2 174 179.3 180 177.8 4.6 4.7 4.6 4.6 2.8 2% 1.8% -0.4-8% 1.3% Lab 3 166.9 163.4 166.7 165.7 5 4.8 4.7 4.9-9.3-5% 1.2% -0.1-2% 3.1% Lab 4 114.1 nd nd 2.6 nd nd -60.9-35% -2.4-48% Lab 5 160.4 162.6-161.5 < 10 < 10 < 10 < 10-13.5-8% 1.0% Lab 6 168 173 175 172 4.8 5 4.9 4.9-3.0-2% 2.1% -0.1-2% 2.0% dev. (%) µg/l µg/l X ref 175 X ref 5 RSD (%) HT-2 in flours results from participants : calculations : flour B flour A flour B flour A res. 1 res. 2 aver. res. 1 res. 2 aver. recov. MU dev. dev. z-score ζ-score RSD dev. (µg/kg) (µg/kg) (µg/kg) (µg/kg) (µg/kg) (µg/kg) (%) (%, k=2) (µg/kg) (%) (µg/kg) dev. (%) z-score ζ-score RSD Lab 1 <75 <75 <75 <75 <75 <75 98 33 (<-30) (<-29%) (<-1.30) (<-2.08) Lab 2 564.1 411.4 487.8 241.8 222.4 232.1 102.4 33.6 382.8 365% 16.57 4.65 22% 189.6 446% 20.28 4.85 6% Lab 3 77.4 42 59.7 27 20.9 23.95 ca. 100 40-45.3-43% -1.96-3.23 42% -18.55-44% -1.98-3.29 18% Lab 4 39 35 37 18.5 15.6 17.05 124 50-68 -65% -2.94-5.76 8% -25.45-60% -2.72-4.90 12% Lab 5 67.4 68.9 68.2 29.6 < 20 29.6 (*) 16.4-36.8-35% -1.59-3.98 2% -12.9-30% -1.38-3.36 Lab 6 111 101 106 38 38 38 98 72 1 1% 0.04 0.03 7% -4.5-11% -0.48-0.32 0% µg/kg % µg/kg % X ref 105 X ref 42.5 σ p 23.1 22.0% σ p 9.4 22.0% u ref 7.35 7% u ref 3.0 7% HT-2 in solutions results from participants : calculations : sol. D sol. C sol. D sol. C res. 1 res. 2 res. 2 average res. 1 res. 2 res. 2 average dev. dev. RSD dev. (µg/l) (µg/l) (µg/l) (µg/l) (µg/l) (µg/l) (µg/l) (µg/l) (µg/l) (%) (%) (µg/l) Lab 1 218 232 200 216.667 <25 <25 <25 <25 16.7 8% 7.4% Lab 2 6844 6951 7800 7198 76.9 86.4 102 89.5 6998.0 3499% 7.3% 84.5 1690% 14.2% Lab 3 196.2 203.6 194.8 198.2 4.3 4.9 4.6 4.6-1.8-1% 2.4% -0.4-8% 6.5% Lab 4 102.6 nd nd 102.6 1.4 nd nd 1.4-97.4-49% -3.6-72% Lab 5 227.1 239.4-233.3 < 10 < 10 < 10 < 10 33.3 17% 3.7% Lab 6 197 198 205 200 4.4 4.4 4.5 4.4 0.0 0% 2.2% -0.6-12% 1.3% dev. (%) µg/l µg/l X ref 200 X ref 5 RSD (%) p. 8/14

Results and calculations, figures p. 9/14

Annexes Annex A. List and addresses of the participating laboratories (by alphabetical order) Organisation Address CER Groupe rue du Point du Jour, 8 B-6900 Marloie (Marche-en-Famenne) CODA-CERVA Leuvensesteenweg, 17 B-3080 Tervuren ECCA Ambachtsweg, 3 B-9820 Merelbeke FLVV-T Leuvensesteenweg, 17 B-3080 Tervuren Fytolab Technologiepark, 2/3 B-9052 Zwijnaarde SGS Belgium N.V Division IAC Polderdijkweg, 16 B-2030 antwerpen p. 10/14

Annex B. Instructions to the participants p. 11/14

Annex C. Receipt form p. 12/14

Annex D. Results form (p. 1 to 2) p. 13/14

p. 14/14