Table 1: test materials. MA1140 maize 55 gr A x 2 55 gr B x 2. MA1141 maize 55 gr A x 2 55 gr B x 2. MI1142 lyophilized milk 110 ml A x 2 20 ml B x 2

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1 1. REFERENCES Progetto Trieste 2011, Mycotoxins was managed in agreement with the principles of ISO GUIDE 17043:2010 and also in agreement with the procedure described in The International Harmonised Protocol for The Proficiency Testing of (Chemical) Analytical Laboratories, Thompson and Wood, Journal of Association of Analytical Chemists International, 1993, Vol. 76, No 4, INTRODUCTION 104 laboratories asked to participate to Progetto Trieste 2011, - Laboratory Proficiency Testing Analytical field: Mycotoxins. The test materials were dispatched on June 13 rd and 14 th, The deadline for sending the results was July 15 th, laboratories returned some test results. 101 out of the 101 laboratories sent back the test results before the deadline. Participants were from 12 countries (Austria, Brazil, Chile, Croatia, Germany, Greece, Italy, Montenegro, Republic of San Marino, Serbia, Spain and Thailand). This Proficiency Testing Scheme offered 6 different sets of test materials (biological matrices, see Table 1). Test materials A were provided for performance evaluation of confirmatory techniques while test materials B were provided for performance evaluation of screening methods. All materials A and B are composed of two different subsample (A1 and A2, B1 and B2) Table 1: test materials Test material code matrices MA1140 maize 55 gr A x 2 55 gr B x 2 MA1141 maize 55 gr A x 2 55 gr B x 2 MI1142 lyophilized milk 110 ml A x 2 20 ml B x 2 WH1143 common wheat 55 gr A x 2 55 gr B x 2 WH1144 durum wheat 55 gr A x 2 55 gr B x 2 W1145 wine 55 ml A x 2 25 ml B x 2 Page 1 of 28

2 The molecules that could be present in the test materials of Progetto Trieste 2011, Mycotoxins are shown in Table 2a and 2b. The analytes that were actually present are in bold. Incurred test materials are in grey. Table 2a: groups of substances and relative compounds that had to be examinated. Class Test materials 1 MA1140 MA1141 MI1142 WH1143 WH1144 W1145 Fumonisins B 1 Fumonisins B 2 Fumonisins and Fumonisins B 3 B 1 B 2 G 1 G 2 Deoxynivalenol and Zearalenone Deoxynivalenol 3-acetyl-DON 15-acetyl-DON Zearalenone M Aflatoxin M 1 Deoxynivalenol and T2 Toxin Deoxynivalenol Ochratoxin A Deoxynivalenol 3-acetyl-DON 15-acetyl-DON T2 toxin HT2 toxin Deoxynivalenol 3-acetyl-DON 15-acetyl-DON Ochratoxin A Confirmatory test materials A-1 and screening test materials B-1 were the same test materials. Page 2 of 28

3 Table 2b: groups of substances and relative compounds that had to be examinated. Class Test materials 2 MA1140 MA1141 MI1142 WH1143 WH1144 W1145 Fumonisins B 1 Fumonisins B 2 Fumonisins and Fumonisins B 3 B 1 B 2 G 1 G 2 Deoxynivalenol and Zearalenone Deoxynivalenol 3-acetyl-DON 15-acetyl-DON Zearalenone M Aflatoxin M 1 Deoxynivalenol and T2 Toxin Deoxynivalenol Ochratoxin A Deoxynivalenol 3-acetyl-DON 15-acetyl-DON T2 toxin HT2 toxin Deoxynivalenol 3-acetyl-DON 15-acetyl-DON Ochratoxin A Confirmatory test materials A-2 and screening test materials B-2 were the same test materials. Page 3 of 28

4 3. PREPARATION OF TEST MATERIALS, HOMOGENEITY, DISTRIBUTION 3.1 Preparation Corn and wheat: A number of samples were provided by mills, with various contamination levels of mycotoxins. After grinding by a centrifugal mill fatted with a 0.5 µm scem, each sample was tested in order to determinate the contamination level. Sample contaminated at different levels were mixed at different ratio to make two materials for each toxin. Mixing was going on until the sample passed a preliminary homogeneity check (1 to 4 days). Individual sub-sample were dispensed into foil-laminated pouches, heat sealed, subjected to irradiation with beta rays and kept refrigerated until distribution. Milk: Milk sample were provided by farms. By a preliminary test, two samples were chosen, one essentially blank (aflatoxin M1 content less than 5 ppt) and the other heavily contaminated (about 600 ppt). Two test materials were produced mixing the blank and the highly contaminated sample at different ratio in order to obtain two different concentration of aflatoxin M1 in milk. After homogenization the samples were lyophilized. Individual sub-sample, after carefully mixing, were dispensed into a vials and kept refrigerated until distribution. Wine: The wine sample was purchased in a supermarket and spiked with Ochratoxin A. After preparation, the sample were distributed in containers and kept at -20 C until distribution. 3.2 Homogeneity Five randomly selected pouches for each corn test materials and 5 vials of lyophilized milk were sampled. From each pouche or vial 2 laboratory samples were taken. The 10 samples for each test material were analysed and results submitted to a statistical evaluation in order to stop any eventually unsuitable test material lot (see Annex II). 3.3 Distribution Each participant received the Test Material in a proper container (refrigerated, in case of milk powder, with dry ice in case of wine), together a covering letter with the necessary instruction. Page 4 of 28

5 4. CALCULATION OF THE ASSIGNED VALUE The Assigned Value X is the best possible estimate of the True Value x. For the spiked and incurred test materials X represents the mean of the concentration values as determined by the participants (only confirmatory analysis) without considering the outliers (robust mean). In some cases, may happen that only a few participants confirm the presence of some analytes in the test materials. In order to keep low wrong evaluations, we choose to consider the presence of an analyte: unconfirmed, when less then 25 % of participants detect the compound; confirmed, when 25% of participants, or more, detect the compund (the minimum number of positive results is anyway three). When the analytical uncertainty is too high, because few participants sent results, few detected the analyte, and / or because of an high variability of measurements, the assigned value could be anyway far from the true value. In such cases the assigned value is reported in italics, as well as the z-scores, that are just given for information. The analytical uncertainty (u) is calculated as: u = σ rm / n (where σ rm = the standard deviation of the robust mean) u is considered too high when its value was above 50% of the target standard deviation of the round or when n < 8 where n is the number of data after outlier rejection. Page 5 of 28

6 5. PERFORMANCE EVALUATION CRITERIA 5.1 Confirmatory analyses The identity of the laboratories is confidential. Each laboratory was given a code number Result (concentration value) Concentration value is the result obtained by the laboratory, corrected for recovery. The concentration data were used for a statistical analysis (see further). NS means Not Searched, when the laboratory did not perform the analysis. < of or ND means Not Detected z-score Each laboratory was assigned a z-score calculated from: z-score = (x X) / σ where: x is the analyte concentration value reported by the laboratory; X is the assigned value, as defined in chapter 4; σ is the target value for standard deviation calculated from bx; b=%rsd / 100, (RSD = Relative Standard Deviation). %RSD is defined taking into account Commission Regulation (EC) n. 401/2006 of February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs, which indicates that the precision values are calculated from the Horwitz equation (Horwitz, W., 1988, Pure Appl. Chem. 60, ): (1-0.5 log X) %RSD = 2 where X is expressed as a dimensionless concentration. (EC) n. 401/2006 provides several maximum values for RSD depending on the analyte and its concentration. The lowest of these maximum values is 25%. Therefore, we applied this criteria (b=0.25) for all mycotoxins at any level of concentration. This criteria has been extended to aflatoxin M1 and M2. The laboratory performance evaluation was established taking in to account the following criteria for z-score: satisfactory when IzI < 2 questionable when 2 < IzI < 3 unsatisfactory when IzI > Limit of Detection (LOD) The limit of detection for the relative compounds were claimed by the laboratories. - means Not Reported Limit of Quantification (LOQ) The limit of quantification for the relative compounds were claimed by the laboratories. - means Not Reported Not searched compunds Some laboratory did not look for all the compounds potentially present in the test material; such missed analyses are listed Not Searched, NS. Page 6 of 28

7 5.2 Screening analyses The identity of the laboratories is confidential. Each laboratory was given a code number Results (concentration value) and qualitative results (if any) Concentration value is the result obteined by laboratoty, corrected for recovery. When the concentration is not reported, qualitative result is indicate ( compliance or not-compliance ) Quantitative results: The laboratory performance evaluation was established taking in to account the following criteria for z-score: satisfactory when IzI < 2 questionable when 2 < IzI < 3 unsatisfactory when IzI > Qualitative results: By this evaluation the results are classified "satisfactory" when the laboratory is able to detect the analyte, in respect to his limit of decision, while "unsatisfactory" when the result is a false compliant. False not-compliant results are reported as questionable. Page 7 of 28

8 6. PARTECIPANTS AUSTRIA : - AGES GmbH, Linz - Institute Dr. Wagner, Technisches Buro fur Lebensmittel- und Biotechnologie Chemisches Laboratorium, Lebring BRAZIL: - National Laboratory of the Ministry of Agriculture, Livestock and Food Supply of Brasil, LANAGRO-MG, Pedro Leopoldo, Minas Gerais CHILE: - Servicio Agricola y Ganadero, Laboratorio Quimica Ambiental y Alimentaria, Santiago - Viamed Technical Laboratory S.A., Nunoa, Santiago CROATIA: - Croatian Veterinary Institute, Zagreb GERMANY: - BFEL Max Rubner Institut, Inst. Fur Sicherheit und Qualitat bei Milch und Fisch, Kiel - Eurofins WEJ Contaminants GmbH, Hamburg - LADR GmbH, Geesthacht - LUFA - ITL Gmbh, Kiel - LUFA Speyer, Speyer GREECE - Mevgal S.A., Thessaloniki ITALY: - AAFVG - Associazione Allevatori del Friuli Venezia Giulia, Lab. Analisi, Codroipo, Udine - Agenzia delle Dogane, Laboratori e servizi chimici per la Liguria, Savona - Agricola Tre Valli S.c.a.r.l., Quinto di Valpantena, Verona - Agricola Tre Valli S.c.a.r.l., Putignano, Bari - Agricola Tre Valli S.c.a.r.l., Fossano, Cuneo - Agricola Tre Valli S.c.a.r.l., Ospedaletto Euganeo, Padova - Agricola Tre Valli S.c.a.r.l., San Pietro in Giù, Padova - Agricola Tre Valli S.c.a.r.l., San Polo di Torrile, Parma - Agricola Tre Valli S.c.a.r.l., Acquanegra, Cremona - Ambiente S.c., Loc. Nazzano Carrara, Massa Carrara - Artest S.r.l., Modena - A.S.L. della Provincia di Cremona, Lab. sanità pubblica, Cremona - A.S.L. Laboratorio di sanità pubblica, Milano - Associazione Granaria di Milano, Rozzano, Milano - Azienda Sanitaria di Firenze, Laboratorio di Sanità Pubbica, Firenze - Bioaesis S.r.l., Jesi, Ancona - Bioqualità, Guarene, Cuneo - CAIP - Consorzio Agrario di Bologna e Modena Soc. Coop. Laboratorio di Analisi c/o Sementificio, San Giorgio di Piano, Bologna - Cargill Animal Nutrition S.r.l., Laboratorio Regionale Europeo, Spessa Po, Pavia - Caseificio Pugliese F.lli Radicci S.p.A, Lauriano, Torino - CCIAA Laboratorio Chimico Merceologico - Azienda Speciale per la Formazione, Albenga, Savona - CCIAA - Laboratorio Agroalimentare, Verona - Cereal Docks S.p.A., Summaga di Portoguaro, Venezia - Chelab S.r.l., Resana, Treviso - Chemical Control S.r.l., Madonna dell Olmo, Cuneo - Colussi S.p.A. Laboratorio Analisi, Imperia Page 8 of 28

9 - Consorzio Vino Chianti Classico, San Casciano, Firenze - CRA-MAC Unità di ricerca per la maiscoltura, Bergamo - ERSA, Servizio Fitosanitario e Chimico, Pozzuolo del Friuli, Udine - Favero Antonio S.r.l., Padova - Ferrero Mangimi S.p.A., Fossano, Cuneo - G.I.Ma. Gruppo Italiano Mangimi S.p.A., Rubiera, Reggio Emilia - Galeno RP S.r.l., Ortona, Chieti - Gesco Consorzio Cooperativo - Soc. Coop. Agricola Gruppo Amadori, San Vittore di Cesena, Forlì Cesena - Granarolo S.p.A., Anzio, Roma - Granarolo S.p.A., Bologna - Granarolo S.p.A. Laboratorio Centrale, Bologna - Granarolo S.p.A. Gioia del Colle, Bari - Granarolo S.p.A., Pasturago di Vernate, Milano - Granarolo S.p.A., Soliera Modena - Gruppo C.S.A. S.p.A., Rimini - I.R.S.A.Q. S.r.l., Potenza - Istituto Sperimentale per l Enologia di Asti, Asti - Istituto Superiore di Sanità, Reparto OGM e Xenobiotici di origine fungina, Roma - Istituto Polidiagnostico Santa Chiara, lab. Analisi Chimiche Dr. Di Buono, Agropoli, Salerno - Istituto Zooprofilattico Sperimentale dell Abruzzo e del Molise G. Caporale, Teramo - Istituto Zooprofilattico Sperimentale del Lazio e della Toscana Laboratorio chimico, Roma - Istituto Zooprofilattico Sperimentale del Lazio e della Toscana - sede di Grosseto, Grosseto - Istituto Zooprofilattico Sperimentale del Lazio e della Toscana - sezione Firenze, San Martino alla Palma, Firenze - Istituto Zooprofilattico Sperimentale della Lombardia e dell Emilia, sede di Forlì - Istituto Zooprofilattico Sperimentale della Lombardia e dell Emilia Romagna "Bruno Ubertini - sezione di Bologna, Bologna - Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, Napoli - Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D aosta I.Altara sezione di Asti, Asti - Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata Micotossine e tecniche immunoenzimatiche, Foggia - Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata - sede di Potenza, Tito Potenza, Potenza - Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata - sede di Putignano, Bari - Istituto Zooprofilattico Sperimentale della Sardegna G. Pegreffi - Sassari - Istituto Zooprofilattico Sperimentale della Sicilia A. Mirri - Area di chimica, Palermo - Istituto Zooprofilattico Sperimentale dell'umbria e delle Marche, Perugia - Istituto Zooprofilattico Sperimentale dell'umbria e delle Marche, magazzino Istituzionale, Perugia - Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padova - Laboratorio Bonazzi S.a.s. di Bonazzi Vanessa & C., Novellara, Reggio Emilia - La Chimica S.r.l., Cerese di Virgilio, Mantova - MAIA Agroindustriale S.r.l., Pieve di Soligo, Treviso - MB Mangimi S.p.A., Longiano, Forlì-Cesena - Mediterranea S.r.l. Specialità Alimentare, Silvano d Orba, Alessandria - Microlab S.n.c., Analisi microbiologiche e consulenza alimenti e ambiente, Amaro, Udine - Molino Rossetto S.p.A., Pontelongo, Padova - Molini Lario S.p.A., Alzate Brianza, Como - Mondeco S.r.l., Piobesi d Alba, Cuneo - Monviso S.p.A., Andezeno, Torino - NDF Azteca Milling Europe S.r.l., Ceggia, Venezia - Panalisi S.r.l., Bari - Pastifici Rana S.p.A., San Giovanni Lupatoto, Verona Page 9 of 28

10 - Progeo s.c.a., Masone, Reggio Emilia - R&C Lab S.r.l., Altavilla Vicentina, Vicenza - Tecna S.r.l., Trieste - Tecno Bios S.r.l., Apollosa, Benevento - Università Cattolica del Sacro Cuore, Piacenza - Università degli Studi di Bologna, Dipartimento di Scienze Mediche Veterinarie, Ozzano dell Emilia, Bologna - Water & Life Lab, Entratico, Bergamo MONTENEGRO - PI Center for Exotoxicological Researches of Montenegro, Podgorica REPUBLIC OF SAN MARINO - Istituto per la Sicurezza Sociale, Dipartimento Prevenzione Laboratorio di Sanità Pubblica, Borgo Maggiore SERBIA: - Enoloska Stanica Vrsac, Vrsac - Intitute for Food Technology Departement of Instrumental Chem. Analysis, Novi Sad SPAIN: - Capher Idi S.L., Zaragoza - Harivasa 2000 S.A., Noain, Navarra - Laboratori Agroalimentari DAR (Generalitat de Catalunya), Cabrils, Barcelona - Laboratorio De Analisis Dr. Echevarne, Barcellona - Servicio de Laboratorio y Control SeLyC, Santander THAILAND: - Central Laboratory Co., LTD., Bankok Page 10 of 28

11 7. ABSTRACT Table 3: Overall evaluation Test Material code MA A number of z-score < 2 or z-score < 2 or analyte labs satisfactory (n) satisfactory (%) Fumonisin B Fumonisin B total fumonisins Aflatoxin B Aflatoxin B Aflatoxin G MA B A total aflatoxins total fumonisins Aflatoxin B total aflatoxins Fumonisin B Fumonisin B total fumonisins Aflatoxin B Aflatoxin B Aflatoxin G MA MA MI MI total aflatoxins total fumonisins B Aflatoxin B total aflatoxins A deoxynivalenol zearalenone deoxynivalenol B zearalenone deoxynivalenol A zearalenone B deoxynivalenol zearalenone A aflatoxin M 1 B A aflatoxin M 1 B Page 11 of 28

12 number of z-score < 2 or z-score < 2 or Test Material code analyte labs satisfactory (n) satisfactory (%) deoxynivalenol A T2 toxin WH HT2 toxin deoxynivalenol B T2 toxin WH A B deoxynivalenol WH A deoxynivalenol B WH A deoxynivalenol B W A ochratoxin A B W A ochratoxin A B Page 12 of 28

13 Proficiency Test MI1142, result contents Test material MI1142-1, lyophilized milk Result of confirmatory A Result of screening B Test material MI1142-2, lyophilized milk Result of confirmatory A Result of screening B Page 13 of 28

14 Test material MI1142, lyophilized milk, confirmatory Table 60: results of confirmatory analyses performed on test material MI (incurred sample): aflatoxins detection. Lab. code Result (ng/kg) Uncertainty (ng/kg) MI aflatoxin M 1, assigned value: ng/kg satisfactory range: ng/kg Recovery % z-score LOD (ng/kg) LOQ (ng/kg) n n y n > y n y y y y y y y n * n y 102 < y milk density (1.03) *not correct for recovery ISO Mean concentration (n =16) ng/kg Standard deviation (n =16) ng/kg Coefficient of variation (n =16) % Page 14 of 28

15 Test material MI1142, lyophilized milk, confirmatory Table 61: assigned value and target standard deviations. Analyte Assigned value (ng/kg) Target standard deviation Data points, n Robust mean SD CV Analytical uncertainty u b σ = bx aflatoxin M Horrat value CV (RDS), n =14: % Horwitz expected CV: % Horrat (= CV / Horwitz CV): 0.25 u / σ = 18.84% ng/kg ng/kg z < 2 = satisfactory z > 3 = unsatisfactory 2 < z < 3 = questionable * = outliers confirmatory z-score screening z-score Page 15 of 28

16 Test material MI1142, lyophilized milk, screening Table 62: results of screening analyses performed on test material MI (incurred sample): aflatoxins detection. MI aflatoxins M 1, assigned value: ng/kg Lab code Result (ng/kg) Uncertainty (ng/kg) z-score Evaluation ISO y * n n n y n n n n n n y n y 58 non compliant - satisfactory y y y 71 non compliant - satisfactory n y 78 non compliant - satisfactory y y y n Page 16 of 28

17 Test material MI1142, lyophilized milk, screening MI aflatoxins M 1, assigned value: ng/kg Lab code Result (ng/kg) Uncertainty (ng/kg) z-score Evaluation ISO milk density (1.03) * not corrected for recovery n n Mean concentration (n = 23) ng/kg Standard deviation (n = 23) ng/kg Coefficient of variation (n =23) % Page 17 of 28

18 Test material MI1142, lyophilized milk, confirmatory Table 63: results of confirmatory analyses performed on test material MI (incurred sample): aflatoxins detection. Lab. code Result (ng/kg) Uncertainty (ng/kg) MI aflatoxin M 1, assigned value: ng/kg satisfactory range: ng/kg Recovery % z-score LOD (ng/kg) LOQ (ng/kg) n n 20 < y n 27 < y n y y y 58 < y y y y n * n < y milk density (1.03) *not correct for recovery Mean concentration (n =12) ng/kg Standard deviation (n = 12) 3.13 ng/kg Coefficient of variation (n =12) 23.10% ISO Page 18 of 28

19 Test material MI1142, lyophilized milk, confirmatory Table 64: assigned value and target standard deviations. Analyte Assigned value (ng/kg) Target standard deviation Data points, n Robust mean SD CV Analytical uncertainty u b σ = bx aflatoxin M Horrat value CV (RDS), n = 12: 3.13% Horwitz expected CV: 86.47% Horrat (= CV / Horwitz CV): 0.27 u / σ = 26.68% ng/kg 6.77 ng/kg z < 2 = satisfactory z > 3 = unsatisfactory 2 < z < 3 = questionable * = outliers confirmatory z-score screening z-score Page 19 of 28

20 Test material MI1142, lyophilized milk, screening Table 65: results of screening analyses performed on test material MI (incurred sample): aflatoxins detection. MI aflatoxins M 1, assigned value: ng/kg Lab code Result (ng/kg) Uncertainty (ng/kg) z-score Evaluation ISO y * n n satisfactory n y n n n n n n y 53 < 20 - satisfactory n y 58 compliant - satisfactory y y y 71 compliant - satisfactory n y 78 compliant - satisfactory y y y n Page 20 of 28

21 Test material MI1142, lyophilized milk, screening MI aflatoxins M 1, assigned value: ng/kg Lab code Result (ng/kg) Uncertainty (ng/kg) z-score Evaluation ISO milk density (1.03) * not corrected for recovery > 10 n n Mean concentration (n = 21) ng/kg Standard deviation (n = 21) 13.78ng/kg Coefficient of variation (n =21) % Page 21 of 28

22 Test material MI1142, lyophilized milk, screening Table 66: limits of quantification in screening analyses of aflatoxins. MI1142 cut-off and LOQ (ng/kg) Lab. code cut-off LOQ > > Page 22 of 28

23 ANNEX I AFLATOXINS IN LYOPHILIZED MILK, MI1142A Analytical methods used by participants Methods Methods HPLC 45 HPLC-FLD HPLC/MS 40 HPLC/RF 87 HPLC/MS-MS 43 Is the analytical method a routine one? Yes No 97 Source of standards for chromatographyc analysis AFLA M 1 Sigma, Aldrich, Fluka Biopure 97 Supelco LGC Standards 45 Sample preparation Amount of test sample Sample volume (ml) Sample volume (ml) Sample treatment Extraction add NaCl add filter aid Solvent extraction water ethylacetate 43 methanol methanol / acetonitrile 101 acetonitrile Extraction procedure shaking vortex blending 101 centrifugation 45 Extraction time 5 minutes minutes minutes minutes 101 Sample clean up IAC Vicam Romer Labs 87 r-biopharm Rhone SPE C18 20 Isolute C18 EC 18 Sample work up centrifugation filtration dilution 18 defatting 22 Page 23 of 28

24 HPLC methodology and conditions gradient isocratic Injection volume (µl) 5 < x < < x < < x < < x < < x < Column phase C Polar PR 45 Particle size (µm) Column brand Acquity TM 43 Lichrospher BetaBasic 40 Luna Gemini TM 58 Prodigy 79 Gemini TM NX 22 Symmetry 45 Hypersil 67 Supelcosil TM Kromasil Eternity 101 Zorbax 97 LiChrosorb RP 87 Mobil phase components methanol acetonitrile water acetic acid 97 ammonium formate 43 formic acid 40 ph of mobile phase x < < x < < x < x > Flow rate (ml/min) x < < x < < x < Post column derivatization... (specify) Number of replicates Number of extraction Number of extraction Quality Quality control implemented Control chart with in-house Reference Material (RM) Control chart with Certified Reference Material (CRM) Regular partecipation to proficency test (PT) Quality control implemented Duplicate analyses Spiked sample Recovery Recovery calculated by Recovery calculated by Internal standard 05 In-house Reference Material (RM) Spiked sample Certified Reference Material (CRM) 87 spiked curve Blank sample Page 24 of 28

25 Uncertainty Type of uncertainty expanded Coverage factor Reference Reference Reference Internal method UNI EN ISO 14501: IDF/IUPAC/IAEA Validation of an Analytical method for aflatoxin M1 in milk by immunoaffinity TLC (2001) utilizando como referência o método de Dragacci and Grosso (2001), para leite fluído e, para leite em pó no método Immunoaffinity column clean-up with liquid chromatography for determination of aflatoxin M1 in liquid milk: 102 Collaborative Study Journal of AOAC international vol., 84, No. 2, 2001 de Dragacci,S. and Grosso. AFLATOXINS IN LYOPHILIZED MILK, MI1142B Analytical methods used by participants Methods Methods ELISA lateral-flow Is the analytical method a routine one? Yes No cross-reactivity to aflatoxin M 2 cross-reactivity to aflatoxin M 2 3% 03 30% % Source of test kits Manufacturer kit code Manufacturer kit code MA418 / MA EuroProxima 5121AFM1p 33 Tecna MA440 / MA EuroClone EEM MA E R Charm Rosa MRL r-biopharm R In house kit 85 Ridascreen 83 Sample preparation Amount of test sample Sample volume (ml) Sample volume (ml) < > Page 25 of 28

26 Calibration curve (for ELISA) Calibration curve Calibration curve 4 parameters Spline 59 Linear regression Linear 6 point 33 Point to point Sample treatment Extraction water 33 Extraction procedure heating to 55 C 06 Extraction time 5 minutes minutes minutes minutes 05 Sample work up centrifugation filtration dilution 59 defatting Number of replicates (1 replicates = 1 extracion) Number of extraction Number of extraction Recovery Recovery calculated by Recovery calculated by Internal standard In-house Reference Material (RM) Spiked sample Quality Quality control Quality control implemented implemented Control chart with in-house Duplicate analyses Reference Material (RM) Control chart with Certified Spiked sample Reference Material (CRM) Regular partecipation to Reference Material 05 proficency test (PT) Blank sample Other Action level Action level (ng/kg) Action level (ng/kg) > < > In case of non-compliant resuslts by screening, do you proceed with confirmatory analysis? Yes No Page 26 of 28

27 Reference Reference Reference Internal method ISO / IDF 186: Kit insert ANNEX II LYOPHILIZED MILK M1142 Test material 1 Data from the homogeneity test on test material MI1142 are given. Sample id ng/l ng/l Analysis of variance Variance df Sum of squares Mean squares Between samples Analytical F < F critical: F= 0.17 F critical = 3.02 there are no significant differences among samples S s /σ 2 = 0.03 (S s /σ 2 )critical= 0.4 S s /σ 2 < ( S s /σ 2 ) critical: the material is sufficiently homogeneus for the proficiency test purposes Page 27 of 28

28 LYOPHILIZED MILK M1142 Test material 2 Data from the homogeneity test on test material MI1142 are given. Sample id µg/l µg/l Analysis of variance Variance df Sum of squares Mean squares Between samples Analytical F < F critical: F= 0.49 F critical = 3.02 there are no significant differences among samples S s /σ 2 = 0.03 (S s /σ 2 )critical= 0.4 S s /σ 2 < ( S s /σ 2 ) critical: the material is sufficiently homogeneus for the proficiency test purposes Page 28 of 28