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Progetto Trieste 2014, 2nd

round Mycotoxins

FINAL REPORT

Laboratory Proficiency Testing for Food analysis

2014

Mycotoxins 2

nd round


round – Mycotoxins

Proficiency Testing Service by Test Veritas S.r.l.

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CONTENTS

1 INTRODUCTION 1

2 PREPARATION OF TEST MATERIALS, HOMOGENEITY, DISTRIBUTION 3

3 PERFORMANCE EVALUATION CRITERIA 4

4 CALCULATION OF THE ASSIGNED VALUE 6

5 PARTICIPANTS 8

6 ABSTRACT 11

7 REFERENCES 12

8 RESULTS 13

MI1460, lyophilized bovine milk 15

CE1461, hard cheese 22

SP1462, paprika 25

WH1463, common wheat 36

L1475, lyophilized swine liver 45

ANNEX 49




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About this proficiency test, participant should contact:

Test Veritas S.r.l.

Tel. +39 040 3755560

Fax +39 049 8256292

[email protected]

[email protected]

Report authorised by:

Bacer Vanessa

Round coordinator

20/01/2015 rev.0


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1. INTRODUCTION

The quality management system of Test Veritas S.r.l. has been assessed and certified by SGS Italia as meeting the requirements of

ISO 9001 / UNI EN ISO 9001:2008 for development and production of materials for agri-food analyses and proficiency supply.

84 laboratories asked to participate to “Progetto Trieste 2014, 2nd

round - Laboratory Proficiency Testing – Analytical field: mycotoxins”.

The test materials were dispatched on October 20th and 21

st, 2014. The deadline for sending the results was November 21

st, 2014.

80 laboratories returned some test results.

Unless 5 out of the 80 laboratories (participants n. 29, 55, 79, 80 and 84) sent back the test results after the deadline, all partecipans

data are included.

Participants were from 21 countries (Austria, Belgium, Bosnia-Herzegovina, Brazil, Denmark, France, Germany, Japan, Italy, Namibia,

Poland, Rumania, Serbia, Spain, Sud Africa, Sweden, Switzerland, Turkey, Uganda, United Republic of Tanzania and Uruguay).

This Proficiency Testing Scheme offered 5 different sets of test materials (biological matrices, see Table a).

Test materials “A” were provided for performance evaluation of confirmatory techniques, while test materials “B” were provided for

performance evaluation of screening methods. Same materials are composed by two different subsamples.

Table a: Test materials

Test material code matrices

MI1460: subsample “-1” and “-2” lyophilized bovine milk 110 ml A x 2 – 20 ml B x 2

CE1461: only one sample hard cheese 50 g A x 1 – 50 g B x 1

SP1462: only one sample paprika 55 g A x 1 – 55 g B x 1

WH1463: subsample “-1” and “-2” common wheat 55 g A x 2 – 55 g B x 2

L1475: subsample “-1” and “-2” lyophilized swine liver 20 g A x 2 – 10 g B x 2


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The molecules that could be present in the test materials of Progetto Trieste 2014, 2

nd round Mycotoxins are shown in Tables b and c.

The analytes that were actually present are in bold.

Table b: Class of substances and molecules that had to be examined for screening and confirmatory methods (test material “1”).

Test Materials Class Molecules Incurred

MI1460-1 Aflatoxins M1 Aflatoxin M1 yes

CE1461 Aflatoxins M1 Aflatoxin M1 yes

SP1462 Aflatoxins B/G Aflatoxins B1; Aflatoxins B2; Aflatoxins G1; Aflatoxins G2 yes

WH1463-1 DON and other

trichothecenes deoxynivalenol, 3-acetyl-DON, 15-acetyl-DON, T2, HT-2, nivalenol yes

L1475-1 Ochratoxin A Ochratoxin A no

Table c: Class of substances and molecules that had to be examined for screening and confirmatory methods (test material “2”).

Test Materials Class Molecules Incurred

MI1460-2 Aflatoxins M1 Aflatoxin M1 yes

WH1463-2 DON and other

trichothecenes deoxynivalenol, 3-acetyl-DON, 15-acetyl-DON, T2, HT-2, nivalenol yes

L1475-2 Ochratoxin A Ochratoxin A blank


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2. PREPARATION OF TEST MATERIALS, HOMOGENEITY, DISTRIBUTION

Preparation

Milk (incurred)

A number of blank and incurred samples were provided by farms. Milk was centrifuged and fat was removed. Incurred matrices were

mixed with blank matrices. 0.09% sodium azide was added as preservative. The obtained materials were homogenized with a magnetic

stirrer and lyophilized. After mixing thoroughly, test materials were dispensed into vials, checked for homogeneity and kept refrigerated

until dispatch.

Spices and wheat (incurred)

Contamined matrices are obtained from a competent laboratory. Contaminated matrices were mixed with a worm shaft mixer.

The obtained test materials were dispensed into foil-laminate pouches, heat-sealed, irradiated with beta rays for preventing fungal

growth and checked for homogeneity. The test materials were kept refrigerated until dispatch.

Cheese (incurred)

Blank matrices were purchased in a supermarket, incurred samples were provided by supplier. Blank matrices and contaminated

matrices were mixed with a worm shaft mixer.

The obtained test materials were dispensed into foil-laminate pouches, heat-sealed and checked for homogeneity. The test materials

were kept refrigerated until dispatch.

Liver (spiked)

Spiking solutions were prepared. Blank matrices were spiked. The obtained samples were homogenized with a cutter mixer, dispensed

into amber bottles and lyophilized. Homogeneity was assessed, then test materials were were kept refrigerated until dispatch.

Subcontractor

In order to prevent fungal growth, incurred test materials such as spices and wheat are beta-irradiated. Irradiation is performed by a

competent subcontractor.

Homogeneity and stability

Appropriate homogeneity studies assessed that test materials are sufficiently homogeneous for the measurands under study.

Appropriate stability studies assessed that test materials are stable for the time that elapsed between the dispatch of the test material

and the deadline for submission of results.

Labels

To avoid labeling errors, the Test Material “-1” and “-2” are always labeled on different days.


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Distribution

In accordance with the stability established, the test material were shipped in proper containers and conditions.

Dispatch was followed by the courier's parcel tracking system on the internet.

Shipments outside the EU follows different procedures.

The parcel contains:

• A number of units of test material (see Table a)

• A covering letter with the necessary instructions for use and storage of the materials, together with indications for reporting

results in the on-line form.

An e-mail informed participants that the materials were shipped. The e-mail contains an attached copy of the covering letter and MSDS.

3. PERFORMANCE EVALUATION CRITERIA

Laboratory code and confidentiality

To each participant a web restricted area was given. Therefore, confidentiality of results is guaranteed. Participants are identified in the

final report by a randomly assigned code.

The laboratory codes were communicated to each participant trough his web restricted area .

Confidentiality is guaranteed.

Confirmatory and screening result: concentration value

Participants were asked to report quantitative or semiquantitative results corrected for recovery. In case of quantitative results, if

correction is not applied, the value is not included in the calculation of the assigned value.

Semiquantitative results can be given as:

- “< of…” means that the analyte was not detected.

- “> of…” means that the analyte was detected but not quantified.

- “NS” (Not Searched) means that the laboratory did not perform the analysis.

Depending of the reported method, screening results can be expressed for a single analyte or for an analyte group.

Limit of detection (LOD), limit of quantification (LOQ) and uncertainty

Limit of detection, limit of quantification and uncertainty for the relative compounds were claimed by the laboratories.

“-” means Not Reported.


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False positive results

If it is possible, false positive results obtained by the laboratories are shown.

- "None" means that no false positive results were reported. This corresponds to a good performance for both screening as well

as confirmatory methods.

- “-” means that evaluation was not applicable (e.g. the laboratory found a compound that was present but did not searched for

other analytes that were absent in the test material)

It is not possible to classify as false positives concentrations lower than the assigned value of an intended blank test material. In this

case concentrations are reported just for information.

False negative results

In case of false negative confirmatory results, no evaluation is given.

In case of false negative screening results, evaluation is given as described (see screening assessment).


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4. CALCULATION OF THE ASSIGNED VALUE

The Assigned Value X

The assigned value X is the best possible estimate of the True Value X.

X derives from the consensus of quantitative data results obtained from confirmatory analysis given from participants.

After excluding non valid results, the data population was checked for normality and for the presence of outliers by applying appropriate

statistics and visual presentations.

For both spiked and incurred test materials, X represents the mean (robust mean) of the concentration values without considering the

outliers. In some cases, the median is considered as the best possible estimate of the True Value.

In case of “blank” test materials, the value is assigned on the basis of the capability to detect the analyte. Currently the 8th value reported

by participants in increasing order is taken.

Sometimes very low concentrations are quantified. When it occurs, the concentration value is assigned only if proper statistic elaboration

is available.

The standard uncertainty u

The standard uncertainty (u) is calculated as:

u = !rm / !n (where !rm is the standard deviation of the robust mean).

In case of median as estimator, the standard deviation is calculated as !rm=1.4826*MAD (where MAD is the Median Absolute Deviation).

u is considered too high its value divided by the standard deviation for proficiency assessment (!P ) of the round was above 0.51 or

when n < 8, where “n” is the number of data after outlier rejection.

When the analytical uncertainty is too large (it occurs when few participants report results, when few participants detect the analyte or

when !rm is high), the assigned value could be anyway far from the true value.

In case of high uncertainty, the assigned value X is not the best possible estimate of the true value x.

- In case u /!P is higher than 0.51, the consensus value is not determined and individual laboratory performance scores are not

reported. Summary statistics are provided only for information.

- In case u /!P is between 0.31 and 0.50 or “n” is lower than 8, the assigned value and the z-score will be given in italics

(provided only for information). Uncertainty is not negligible.

It may happen that only few participants confirm the presence of some analytes in the test materials, In such cases, the presence of an

analyte is considered:

• “unconfirmed”, when less then 25 % of participants detect the compound;

• “confirmed”, when 25% of participants, or more, detect the compound (the minimum number of positive results is anyway three).


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z-score

For quantitative data, a participant!s result was converted into a z-score according to the equation:

z-score = (x – X) / !P

where:

• x is the analyte concentration value reported by the laboratory;

• X is the assigned value;

• !P is the standard deviation for proficiency assessment calculated from bX.

• b = %RSD / 100, (RSD = Relative Standard Deviation)

the %RSD value comes from the Horwitz equation (Horwitz, W., 1988, Pure Appl. Chem. 60, 855-864) unless otherwise specified.

%RSD = 2

(1-0.5 log X)

where X is expressed as a dimensionless concentration.

In some cases, the Thompson modified Horwitz equation (Thompson, M., 2000, Analyst 125, 385-386) or an expert judgment can be

applied. Chosen criteria is reported on the specific test material table.

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 > 3

Screening assessment

The semi-quantitative results are classified as satisfactory in the following cases:

• The laboratory detects the analyte or the group of analytes that are effectively present in the test material.

• The laboratory does not detect the analyte or the group of analytes that are not effectively present in the sample.

• The laboratory does not detect the analyte that is effectively present, because his method does not allow this.

The results are classified as unsatisfactory in the following case:

• The laboratory does not detect the analyte or the group of analytes that is/are effectively present in the sample, but according

to the method specifications the analyte/analytes is/are detectable. It means that a false negative has been reported.

The results are classified as questionable in the following case:

• The laboratory detects an analyte or a group of analytes that were not effectively present in the sample. It means that false

positive has been detected.

Because of the different country legislations, the regulatory limits are not included in the results evaluation.


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5. PARTICIPANTS

AUSTRIA:

- AGES GmbH, Linz

BELGIUM:

- Federal Agency for the Safety of Food Chain (FLVVT) – VAR–CODA–CERVA, Tervuren

BOSNIA:

- Veterinary Institute Bihac, Bihac

BRAZIL:

- LANAGRO – MG, National Laboratory of the Ministry of Agriculture, Livestock and Food Supply of Brasil, Pedro Leopoldo, Minas

Gerais

DENMARK:

- Eurofins Steins Laboratorium A/S, Vejen

- Fodevarestyrelsen – Danish Veterinary & Food Administration, Ringsted

EGYPT:

- Central Laboratory at Giza – Central Laboratory of Residue Analysis of Pesticides and Heavy Metals in Food, Giza

FRANCE:

- Eurofins Analytics France, Nantes

- Laboratoire phytocontrol, Nimes

GERMANY:

- Aokin Ag, Berlin

- Becit GmbH, Bitterfeld-Wolfen

- BFEL – Max Rubner Institut – Institut für Sicherheit und Qualitat bei Milch unf Fisch, Kiel

- BioCheck GmbH, Liepzig

- Chemisches und Veterinaruntersuchungsamt Sigmaringen – CVUA, Sigmaringen

- Lufa Speyer, Speyer

ITALY:

- AAFVG – Associazone Allevatorio del Friuli Venezia Giulia, Codroipo, Udine

- Agenzia Regionale Protezione Ambiente Ligure – ARPAL – Dipartimento di Genova, Genova

- Agricola Tre Valli S.c.a.r.l., Quinto di Valpantena, Verona

- ASL della provincia di Varese – PMIP Chimico, Varese

- Azienda Sanitaria di Firenze – Laboratorio di Sanità Pubblica, Firenze

- Azienda Speciale per la Formazzione – CCIAA – Laboratorio chimico merceologico, Albenga, Savona

- Calabrialatte S.p.A. – Stab. Prod. Caseari Castrovillari, Castrovillari, Cosenza

- Casearia Podda Srl, Sestu, Cagliari

- Caseificio Pugliese F.lli Radicci S.p.A., Lauriano, Torino

- Colussi S.p.A., Petrignano d!Assisi, Assisi, Perugia

- ERSA – Agenza regionale per lo Sviluppo Rurale, Pozzuolo del Friuli, Udine

- Eurofins – Centro Caseario Pivetti S.r.l., Castenovo di Sotto, Reggio Emilia

- Favero Antonio S.r.l., Camin, Padova

- Gesco Consorzio Cooperativo, Società Cooperativa Agricola - Gruppo Amadori, San Vittore di Cesena, Cesena

- Granarolo S.p.A., Bologna

- Granarolo S.p.A. Gioia del Colle, Bari

- Granarolo S.p.A., Usmate Velate, Monza Brianza

- Granarolo S.p.A., Filiare di Anzio, Anzio, Roma

- Granarolo S.p.A., Laboratorio Pastorizzato, Bologna


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- Granarolo S.p. A, Stabilimento Yogurt Pasturago, Pasturago di Vernate, Milano

- Granarolo S.p.A., Stabilimento di Soliera, Soliera, Modena

- Istituto Polidiagnostico S. Chiara Dr.ssa Rossetta di Buono Srl, Agropoli, Salerno

- Istituto Zooprofilattico Sperimentale dell!Abruzzo e del Molise “G. Caporale”, Teramo

- Istituto Zooprofilattico Sperimentale del Mezzogiorno, della Campania e della Calabria, Portici, Napoli

- Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D!Aosta “I. Altara”, Torino

- Istituto Zooprofilattico Sperimentale della Lombardia e dell!Emilia Romagna “Bruno Ubertini”, Brescia

- Istituto Zooprofilattico Sperimentale della Lombardia e dell!Emilia Romagna “Bruno Ubertini”, Sezione Bologna, Bologna

- Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata – Laboratorio Micotossine e tecniche immunoenzimatiche, Foggia

- Istituto Zooprofilattico Sperimentale della Sicilia “A. Mirri”, Palermo

- Istituto Zooprofilattico Sperimentale della Sardegna “G.Pegreffi”, Sassari

- Istituto Zooprofilattico Sperimentale dell!Umbria e delle Marche, Perugia

- Istituto Zooprofilattico Sperimentale dell!Umbria e delle Marche – sede di Fermo, Fermo

- Istituto Zooprofilattico Sperimentale delle Venezie, Basaldella di Campoformido, Udine

- Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padova

- I.R.S.A.Q. S.r.l., Potenza

- La Chimica S.r.l., Cerese di Virgilio, Mantova

- Laboratorio Centrale Parmalat, Castellaro di Sala Baganza, Parma

- Laboratorio Gamma di Fornaciari – Pittano, Cavriago, Reggio Emilia

- Metralab S.r.l., Cadorago, Como

- NDF Azteca Milling Europe S.r.l., Ceggia, Venezia

- Pastificio De Cecco – Laboratorio CQ, Fara San Martino, Chieti

- Pastificio Mediterranea Srl, Silvano d!Orba, Alessandria

- Pastificio Rana SpA, San Giovanni Lupatoto, Verona

- Progeo S.c.a., Masone, Reggio Emilia

- R&C Lab S.r.l., Altavilla Vicentina, Vicenza

- Salchim S.c.r.l., Cavriago, Reggio Emilia

- Stazione sperimentale per l!industria delle conserve alimentari in Parma, S.S.I.C.A, Parma

- Tecna S.r.l., Trieste

- Tecnal S.r.l., Bazzano, Bologna

- Trentingrana CON.CA.S.T. s.c.a., Trento

- Università Cattolica del Sacro Cuore, ISAN Agraria Piacenza, Piacenza

- Univerisità di Parma – Dipartimento Chimica Organica, Parma

- Voltan S.p.A., Marcon, Venezia

JAPAN:

- Hokuren Federation of Agricultural Cooperatives, Sapporo

NAMIBIA:

- Central Veterinary Laboratory, Windhoek

POLAND:

- Lajkonik Snacks Sp. Zo. O. Laboratorium, Skawina

ROMANIA:

- Institutul de Bioresurse Alimentarie, Bucharest

SERBIA:

- Veterinarski Specijalisticki Institut Sabac, Sabac

SPAIN:

- Agencia de Gestion Agraria y Pesquera de Andalucia, Junta de Andalucia, Alcolea, Cordoba

- Laboratorio Agroalimentario de Valencia, Burjassot, Valencia


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- Laboratorio de Salud Publica Murcia – Conserelia De Sanidad, Murcia

- Paprimur S.L., Fortuna, Murcia

SUD AFRICA:

- Agricultural Research Council – Onderstepoort Veterinary Institute, Pretoria

SWEDEN:

- SVA, Uppsala

SWITZERLAND:

- Provimi Kliba SA, Kaiseraugst

TURKEY:

- Bornova Veteriner Kontrol Enstitüsü Müdürlügü, Bornova, Izmir

UNITED REPUBLIC OF TANZANIA:

- Tanzania Food and Drug Authoruty (TFDA), Dar es Salaam

UGANDA:

- Ministry of Agriculture, Animal Industry and Fisheries, Kampala

URUGUAY:

- Division de Laboratorios Veterinarios – MGAP, Montevideo


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6. ABSTRACT

Table e: Overall evaluation

Test Material code analyte number of

labs

z-score < 2 or

satisfactory (n)

z-score < 2 or

satisfactory (%)

A Aflatoxin M1 17 13 76.4 MI1460-1

B Aflatoxin M1 28 27 96.4

A Aflatoxin M1 17 14 82.3 MI1460-2

B Aflatoxin M1 28 28 100

aflatoxin B1 11 11 100

aflatoxin B2 11 10 90.9

aflatoxin G1 11 10 90.9

aflatoxin G2 10 7 70

A

aflatoxin B1 + B2 + G1 + G2 10 10 100

SP1462

B aflatoxin B1+ B2 + G1 + G2 1 0 0

A deoxynivalenol 12 10 83.3 WH1463-2

B deoxynivalenol 12 10 83.3

L1475-2 B blank 3 3 100


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7. REFERENCES

“Progetto Trieste” is managed in agreement to:

• UNI CEI EN ISO/IEC 17043:2011 Conformity assessment – General requirements for proficiency testing

• EURACHEM Selection, Use and Interpretation of Proficiency Testing (PT) Schemes, 2nd edition,

2011

• ISO 13528:2005 Statistical method for use in proficiency testing by interlaboratory comparisons

• ISO GUIDE 35 Reference materials – General ans statistical principles for certification, 2006

• IUPAC Technical Report The International Harmonized Protocol for the Proficiency Testing of Analytical

Chemistry Laboratories, 2006

• EURACHEM/CITAC Guide CG4 Quantifying Uncertainty in Analytical Measurement, 3rd edition, 2012

• ILAC-P10:2002 ILAC Policy on Traceability of Measurement Results

• ILAC-G8:03/2009 Guidelines on the Reporting of Compliance with Specification




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RESULTS




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Proficiency Test MI1460, result contents

Test material MI1460-1, lyophilized bovine milk

! Result of confirmatory “A”

! Result of screening “B”

Test material MI1460-2, lyophilized bovine milk

! Result of confirmatory “A”

! Result of screening “B”




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Test material MI1460, lyophilized bovine milk, confirmatory and

screening

Table 1: results of confirmatory and screening analyses performed on test material MI1460-1 (incurred sample):

aflatoxins M1 detection.

MI1460-1

aflatoxin M1, assigned value: 40.67 ng/kg

satisfactory range: 22.78 – 58.56 ng/kg

Lab. code Result

(ng/kg)

Uncertainty

(ng/kg)

Recovery

%

z-score /

evaluation

LOD

(ng/kg)

LOQ

(ng/kg)

ISO

17025

02 27 12 88 - 1.53 10 - y

04 39 - - - 0.19 5 5 -

07 38.69 17 - - 0.22 - - y

09 43 - - 0.26 - - -

10 40 - - - 0.07 - 5 n

11 37 - - - 0.41 - 5 -

12 35 - - - 0.63 - - -

13 38 7 76 - 0.30 3 10 y

13 41 12 100 0.04 5 5 n

14 39.24 0.71 - - 0.16 - 5 -

17 44 3 96 0.37 2 5 n

18 44.97 5 92 0.48 1 2 y

18 41 8 - 0.04 3 5 y

19 43 - - 0.26 - - -

20 40 - - - 0.07 5 5 n

21 270 23 100 > 10 15 25 y

22 53.71 0.01 74 1.46 0.01 0.01 y

22 > 0.01 - - satisfactory 0.01 - y

24 54.00 - 109 1.49 5 - y

27 36.32 - - - 0.49 - - y

29 65 - - 2.72 - - -

32 39.1 7.4 99.2 - 0.18 5 10 y

32 49.52 22.78 - 0.99 - 5 y

35 41.74* - - 0.12 - - -

* no correct for recovery




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screening

MI1460-1

aflatoxin M1, assigned value: 40.67 ng/kg

satisfactory range: 22.78 – 58.56 ng/kg

Lab. code Result

(ng/kg)

Uncertainty

(ng/kg)

Recovery

%

z-score /

evaluation

LOD

(ng/kg)

LOQ

(ng/kg)

ISO

17025

38 72.86 - - 3.60 20 20 n

39 47.6 9.52 90 0.77 3.0 10.0 n

41 38.94 3.9 - - 0.19 < 5 < 10 y

42 42.7 4.3 100 0.23 2 10 y

42 35.6 - - - 0.57 - 5 -

43 46.73 - 94 0.68 5 - y

44 39 - - - 0.19 5 5 -

46 35 5 104 - 0.63 - 10 y

48 35.23 9.17 - - 0.61 - 5.00 y

52 38.1 2.8 94 - 0.29 5.0 10.0 y

55 10 - 3.43

61 64 - 68 2.61 10 20 -

62 40.11 8.02 - - 0.06 5 20 y

64 50.98 28 114 1.15 25 125 -

66 44.49 7.1 114 0.43 5 20 y

70 36.56 9.07 - - 0.46 - 12.5 n

73 49 6 - 0.93 3 5 y

80 41.22 17.43 83.39 0.06 20.00 50.00 y

81 41.97 - 99.81 0.15 5 - y

82 46.2 - 62.8 0.62 36 36 y

84 37 - - - 0.41 5 5 n

screening method

Confirmatory methods: Screening methods:

Concentration mean (n = 17) 55.12 ng/kg Concentration mean (n = 27) 42.83 ng/kg

Standard deviation (n = 17) 56.80 ng/kg Standard deviation (n = 27) 7.81 ng/kg

Coefficient of variation (n = 17) 103.05% Coefficient of variation (n = 27) 18.23%




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screening

Table 2: assigned value and standard deviations.

Analyte Assigned value (ng/kg) Standard deviation for proficiency assessment

aflatoxin M1

Data points, n

16

Median

40.67

sMAD

6.23

Standard uncertainty u

1.56

b

0.22*

!P = bX

8.95

* Thompson correction

The median has been considered as the best possible estimate of the True Value

Horrat value

CV (RDS), n = 16: 31.36%

Expected CV: 22% Horrat (= CV / Expected CV): 1.43

u / !P = 0.17

The assigned value has been calculated by confirmatory methods only.

|z|<2=satisfactory 2<|z|<3=questionable

|z|>3=unsatisfactory *=outliers

confirmatory z-score

screening z-score

22.78 ng/kg

58.56 ng/kg

Test material MI1460-1, aflatoxin M1, 40.67 ng/kg

z-score

laboratory code


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ANNEX


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ANALYTICAL METHOD USED

AFLATOXIN M1 IN LYOPHILIZED BOVINE MILK, MI1460A Analytical methods used by participants

Aflatoxin M1

Methods Laboratory code Methods Laboratory code

HPLC/FLD 02-13-17-18-21-22-32-

39-42-46-61-62-80 HPLC-MS/MS 70

HPLC/UV 52 LC-MS/MS 29

Aflatoxin M1

Is the analytical method a routine one?

Yes 02-13-17-18-21-22-29-32-39-42-46-52-61-62-70-80

Action level

Aflatoxin M1

Action level µg/kg Laboratory code

0.015 21

0.025 70

0.05 22-42-70

0.061 13

0.5 80

50 18-39-46

Number of replicates (1 replicates = 1 extracion)

Aflatoxin M1

Number of replicates Laboratory code Number of replicates Laboratory code

1 02-22-32-42-46 3 29-70

2 18-21-39-62-80

Recovery

Aflatoxin M1

Recovery calculated by Laboratory code Recovery calculated by Laboratory code

Internal standard 22-32-70 Matrix matched calibration 18-21

Spiked sample 02-13-39-46-61-62-80 Certified Reference Material 42

In-house Reference Material (RM) 29

Quality

Aflatoxin M1

Quality control

implemented Laboratory code

Quality control

implemented Laboratory code

Control chart 22-42 Spiked sample 21-46

Control chart with in-house

Reference Material (RM) 32-80

Regular partecipation to

proficency test (PT) 02-13-18-39-70

Measurement uncertainty

Aflatoxin M1

Laboratory code Laboratory code

Type of uncertainty

expanded 02-13-18-21-22-32-39-42-46-62-70-80

Coverage factor

2 02-18-21-39-32-42-46-62-70-80 2.2 13


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Reference

Aflatoxin M1

Reference Laboratory code

Internal method 02-13-18-21-22-29-42-46-70-80

Source of standards for chromatographyc analysis

Aflatoxin M1

Laboratory code

Sigma, Aldrich, Fluka 02-18-22-29-39-70-80

Biopure 32-42-62

Supelco 13-21-46

Sample preparation

Amount of test sample for the extraction

Aflatoxin M1

Sample volume (ml) Laboratory code Sample volume (g) Laboratory code

10 70 1 21

25 18 5.2 62

50 02-22-32-39-42 50 13-46-80

Sample treatment

Aflatoxin M1


Additives for Extraction

NaCl 39

Solvent extraction

water 21-22-29-62

Extraction procedure

shaking 62 defatting 13

vortex 22

Extraction time (minutes)

30 21 120 29

60 62

Sample clean up

IAC

Vicam 02-39-80 r-Biopharm Rhone 13-32-42-46-62-70

SPE

Vicam hplc 22 C18 21

Sample work up

centrifugation 13-21-22-70-80 defatting 18

filtration 46-62

HPLC methodology and conditions

Aflatoxin M1


gradient 29-70 isocratic 02-13-18-21-22-29-32-39-42-46-62-80

Column paking

C18 02-13-18-21-22-29-32-

39-42-46-62-70-80

Injection volume (µ l)

10 13-21-29 50 02-80

20 42-46-70 100 18-22-32-39-62

Column brand

AccucoreTM

aQ 29 Lichrospher® 46

AcquityTM

70 Phenomenex Kinetex® 13

Discovery® 18 Supelcosil

TM 21

GeminiTM

02-39 Tracer Extrasil 32

LiChrosorb® 80 Zorbax

® 22-42-62


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AFLATOXIN M1 IN LYOPHILIZED BOVINE MILK, MI1460B

Analytical methods used by participants

Aflatoxin M1 Methods Laboratory code Methods Laboratory code

ELISA 04-07-13-14-18-22-24-27-32-35-

38-41-42-43-48-64-66-73-81-82 Lateral flow 10-12-20-44-84

Charm Test 09-11-19

Aflatoxin M1

Is the analytical method a routine one? Laboratory code

Yes 07-09-10-12-19-14-18-20-22-24-27-32-35-38-41-42-43-44-48-64-66-73-81-82-84

No 04

Action level

Aflatoxin M1

Action level µg/kg Laboratory code

0.01 22

0.035 38-42

0.046 82 0.050 13-14-24-35-48-81

40.8 43 50 10-12-18-44-66-84

200 27

Aflatoxin M1

In case of non-compliant resuslts by screening, do

you proceed with confirmatory analysis? Laboratory code

Yes 10-12-13-14-18-22-24-38-42-43-44-66-81-82-84

No 27-48

Number of replicates (1 replicates = 1 extracion)

Aflatoxin M1 Number of replicates Laboratory code Number of replicates Laboratory code

1 04-12-13-14-22-27-42-44-64-66 3 09-10-43-73

2 18-20-24-32-35-41-81-84 8 38

Recovery

Aflatoxin M1 Recovery calculated by Laboratory code Recovery calculated by Laboratory code

Spiked sample 22-24-64-66-81-82 In-house Reference Material (RM) 41-73

Reference Material 13 Validation data 43

Mobil phase components

water/ formic acid / ammonium acetate 29 acetonitrile / water / glacial acetic acid 62

acetonitrile / water 21-22 acetonitrile / water / formic acid 70

acetonitrile / methanol / water 02-13-18-39-

42-46-80

pH of mobile phase

7 02

Flow rate (ml/min)

0.5 70 0.9 18-22-32

0.6 42 1 02-39-46-62-80

0.8 13 1.2 21


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Quality

Aflatoxin M1 Quality control implemented Laboratory code Quality control implemented Laboratory code

Control chart 22-24-41-42-43-64-66 Regular partecipation to

proficency test (PT) 13-24-38

Control chart with in-house

Reference Material (RM)

04-10-12-14-20-35-

44-73-84

Control chart with Certified

Reference Material (CRM) 18-48

Blank sample 09 Spiked sample 81-82

Reference

Aflatoxin M1 Reference Laboratory code

Kit insert 04-07-10-12-13-14-18-20-22-27-32-35-38-41-42-44-48-66-81-82-84

Internal method 24-43-73

Source of test kits

Aflatoxin M1

Manufacturer kit code Laboratory code Manufacturer kit code Laboratory code

MA418 22-43 EuroClone AM11409 82

MA430 38 R1121 14-24-81

MA440 35 r-Biopharm

- 42-66

MA443 41 L013 10-11-20-44-84

MA777 04

Charm

L9935 12

Tecna

- 07-13-18-32-48-64

Sample preparation

Amount of test sample for the extraction

Aflatoxins

Sample volume (ml) Laboratory code Sample volume (ml) Laboratory code

0.1 13-18-32-42-66-73-81 5 04-82

0.3 10-11-12-20-44-84 10 24

1 22 1g 14

2 38

Sample treatment

Aflatoxin M1


Extraction procedure

shaking 81 healting to 55°C 10-11-20-44-84

Extraction time (minutes)

10 14 15 10-11-12-20-44-84

Sample work up

centrifugation 13-14-22-35-38-82 defatting 13-14-18-38

Calibration curve (for ELISA)

Aflatoxin M1 Calibration curve Laboratory code Calibration curve Laboratory code

4 parameters 04-14-18- Point to point 13-22-24-35-38-43-66-73-82

Spline 81- Cubic spline 48-

Logit / Log 41-42

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