italian national reference laboratory (nrl) proficiency test
TRANSCRIPT
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ITALIAN NATIONAL REFERENCE LABORATORY (NRL)
PROFICIENCY TEST
Fumonisins B1 and B2 in Maize Flour
December 2010 - April 2011
REPORT
Carlo Brera, Barbara De Santis, Francesca Debegnach, Elena Pannunzi,
Elisabetta Prantera
ISTITUTO SUPERIORE DI SANITA’ (ISS)
Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare
Reparto Ogm e Xenobiotici di Origine Fungina
Laboratorio Nazionale di Riferimento Micotossine
NATIONAL INSTITUTE OF HEALTH (ISS)
Department of Veterinary Public Health and Food Safety
GMO and Mycotoxins Unit
Italian National Reference Laboratory for Mycotoxins
Mycotoxins
2
TABLE OF CONTENTS
SUMMARY.........................................................................................................................................3
BACKGROUND .................................................................................................................................4
INTRODUCTION...............................................................................................................................4 Proficiency testing.......................................................................................................................................................4
FUMONISINS B1 AND B2 IN MAIZE...............................................................................................5
GENERAL OBSERVATIONS...........................................................................................................5
TEST MATERIAL .............................................................................................................................6 Preparation...................................................................................................................................................................6
Homogeneity ...............................................................................................................................................................6
Stability ........................................................................................................................................................................6
Distribution..................................................................................................................................................................6
INSTRUCTIONS TO PARTICIPANTS AND RESULTS ................................................................7
EVALUATION OF RESULTS...........................................................................................................7
ASSIGNED VALUES AND UNCERTAINTIES...............................................................................8
TARGET STANDARD DEVIATION FOR THE TEST, σP .............................................................8
INDIVIDUAL z-SCORE ....................................................................................................................8
INDIVIDUAL ZETA-SCORE............................................................................................................9
ASSESSMENT OF SCORES ...........................................................................................................10 Laboratories using ELISA kit...................................................................................................................................10
Laboratory with a z-score between -1 and 1............................................................................................................10
APPENDIX I .....................................................................................................................................12
APPENDIX II....................................................................................................................................14
APPENDIX III ..................................................................................................................................15
APPENDIX IV ..................................................................................................................................23
APPENDIX V....................................................................................................................................34
REFERENCES..................................................................................................................................42
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SUMMARY
1) The test material for the proficiency test was distributed in December 2010 to twenty-nine
laboratories, thirteen belonging to the network of the Italian official control service, fifteen
to the National Reference Laboratories (NRLs) in Europe and one to the European
Reference Laboratory (EU-RL). Each participant received a sample of maize flour test
material to be analyzed for fumonisins B1 (FB1) and B2 (FB2). Laboratories determined
fumonisins levels by either High-Performance Liquid-Chromatography (HPLC) followed by
fluorescence or mass selective detection (MS) or Enzyme Linked Immune-Sorbent Assay
(ELISA).
2) The assigned values ( ) were calculated as a robust measure of the central tendency of
participants’ results [1] [2] [3] [4].
3) The target standard deviation (σP) was derived from the Horwitz equation [4]. The assigned
value, the target standard deviation and uncertainties were used to calculate z-scores and
zeta-scores for participants’ results [1] [2] [3] [4].
4) The laboratory performance was expressed in terms of z-score and zeta-score.
5) Results are summarized as follows:
Analyte Assigned value,
µg/kg
Number of satisfactory
z-scores |z|≤2
Total number of z-scores
Satisfactory %
FB1 1257 23 26 88 FB2 425 23 26 88
FB1+FB2 1758 22 29 76
Analyte Assigned value,
µg/kg
Number of satisfactory zeta-scores
|zeta|≤2
Total number of zeta-scores
Satisfactory %
FB1 1257 16 24 66 FB2 425 18 24 75
4
BACKGROUND
In accordance with the Article 12 of EU Regulation (EC) 882/2004 [5] all laboratories involved in
official control analyses must give evidence of their analytical competence for a specific trial or
groups of trials in food and feed area. Paragraph 2 of the same article states that the laboratory
designated by the Competent Authority, must be assessed and accredited following the criteria
described from the international standard ISO 17025:2005 [6].
Among the various requirements provided by the ISO standard, the participation in a proficiency
test (PT), as a tool to check the performance of the analytical process used by the laboratory, must
be guaranteed at least for the trials for which accreditation has been requested or already received.
Within the quality control (QC) activities, any laboratory must implement and give evidence of both
internal and external QC. Internal QC implies that control measures, such as the use of control
charts, (certified) reference materials, recovery assessment, repeatability trials, are routinely put in
place by the laboratory. As for the external QC, the exchange of samples with another laboratory or
having own samples analyzed by another laboratory and the participation in inter-laboratory sample
exchange programs or PTs are key elements.
INTRODUCTION
Proficiency testing
Laboratory proficiency testing and the use of validated methods are crucial features elements of
laboratory quality assurance; their purpose is to assess the competence of the laboratories to
determine analytical performances. The laboratory proficiency testing allows to have an
independent assessment of the performances of laboratories in accordance with the requirements of
EU Regulation (EC) No. 882/2004 of the European Parliament and of the Council [5].
The present proficiency testing was designed following the International Harmonized Protocol for
the Proficiency Testing of Analytical Chemistry Laboratories [4]. Usually the results of a chemical
analysis are normally distributed, hence 95% of data points will lie between a z-score of -2 and +2.
Performances in proficiency testing are considered ‘satisfactory’ if a participant’s z-score lies
between the range of -2 and +2. However there is 1 in 20 chance that the result of a laboratory is
actually acceptable but is not included between -2 and +2. Acceptable results with │z-scores│>3
are expected at a rate of about 1 in 300.
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FUMONISINS B1 AND B2 IN MAIZE
Mycotoxins are natural food and feed contaminants, mainly produced by moulds of genera
Aspergillus, Penicillium and Fusarium. The number of mycotoxins known to exert toxic effect on
human and animal health is constantly increasing as well as the legislative provisions taken to
control their presence in food and feed. Fumonisins (FBs) belong to a quite wide spectrum of
mycotoxins commonly reported as Fusarium-toxins. FBs mainly occur in maize and maize based
products. The relatively recent setting of the legal limit for these toxins (Regulation EC/1126/2007)
[7], together with the occurrence rate and the high value of fumonisins contamination in maize and
maize based products, increased the attention on this issue.
FBs are generally considered as “field” mycotoxins as a result of poor implementation of
appropriate Good Agricultural Practices (GAP) such as variety selection, insects and pests control
(pyralid), soil tillage, crop rotation, use of fungicides, irrigation and sowing time.
As far as toxic effects of FBs, even if no updated information is currently available with regard to
the existing evidence of their involvement in encephalopathy in horses, cancer in rats, pulmonary
oedema in swine, oesophagus cancer and neural tube defects in humans, the level of contamination
of FBs in maize and maize based products should always be monitored in order to have available
updated data for handling emergency situations.
GENERAL OBSERVATIONS
Twenty-nine participants from seventeen countries participated to the PT (Table 1, Appendix I).
Twenty-six laboratories provided results for FB1 and FB2 and three laboratories provided results
only for the sum of FB1 and FB2 (FBs). Twenty-four laboratories provided the measurement
uncertainties for the results of FB1 and FB2 and only 16 laboratories returned the uncertainties for
FBs.
All laboratories were free to use the method of their choice.
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TEST MATERIAL
Preparation
Test material was prepared in ISS laboratory. The test material used was a naturally contaminated
fumonisins B1 and B2 maize flour.
Homogeneity
Sufficient homogeneity of the batch was assumed after that a double milling process was applied
(RETSCH - ZM 200 and Romer Labs - Ras Mill).
However, the homogeneity was tested according to the international harmonized protocol for the
proficiency testing of analytical chemistry laboratories [4]. Ten randomly selected test materials
were analyzed in duplicate for FB1 and FB2. The data showed satisfactory homogeneity and were
not included in calculation of the assigned value (APPENDIX II) (Table 2) [8].
Stability
All samples were packed in plastic bags and vacuum sealed. In order to monitor the stability of the
toxin, the packed samples were stored at room temperature and random analyses were performed at
a different timeline (the beginning of the study, during the study and after the receipt of
participant’s results). Among the obtained FB1 and FB2 results, statistically significant differences
were not found.
Distribution
The test material was dispatched on 17th December 2010 to twenty-nine laboratories of sixteen
countries.
Each participant received:
- One individually numbered maize test material containing approximately 50 g of test
material;
- A cover letter;
- A receipt form;
- A method form questionnaire;
- A results form;
- A laboratory code number.
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In APPENDIX V a copy of the cover letter, the receipt form, the method form and the results form
are reported.
Materials were shipped at room temperature and it was required to store them at -20°C until the
analysis.
INSTRUCTIONS TO PARTICIPANTS AND RESULTS
Participants were required i) to provide their data in µg/kg, ii) to correct for recovery, iii) to report
the percentage of recovery, iv) to provide the measurement uncertainty and the coverage factor.
All participants submitted the results before the closing date set on 21 February 2011.
The participants’ values were all reported on the results form. Laboratories also filled the method
form questionnaire where information on the analytical techniques was requested, however not all
fields were filled. The gathered information is reported in APPENDIX IV (Tables 10-35).
The reported FB1 and FB2 concentrations returned by laboratories are given in Table 3; z-scores
and zeta-scores outside the satisfactory range (i.e. |z|>2; |zeta|>2) are reported in bold in Table 3,
Table 4 and Table 5.
EVALUATION OF RESULTS
The submitted results were statistically analyzed in order to provide an assigned value for single
and grouped fumonisins. The assigned value, the target standard deviation and the measurement
uncertainties were used to calculate the z-scores and zeta-scores for every result. The performance
of laboratories was expressed in terms of z-score and zeta-score.
The evaluation procedure followed the FAPAS® protocol [1] and IUPAC International Harmonized
Protocol for the Proficiency Testing of Analytical Chemistry Laboratories [4].
The calculation of the assigned value was deduced throughout a robust central value of the results
provided by participants [2] [3].
Data was eliminated when:
1) results did not report the percentage of recovery;
2) results were 10/100/1000 times bigger than the majority of the results submitted;
3) results were reported in a semi-quantitative format.
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ASSIGNED VALUES AND UNCERTAINTIES
The laboratory results were winsorized for establishing a robust consensus value (assigned value,
) of the provided results. The winsoring process allowed to minimize the influence of outliers [2]
[3].
The uncertainty of the consensus value was also assessed:
Where = the robust standard deviation (not the target standard deviation for the test σP);
n=the number of data points used to calculate the robust mean.
Assigned values, the standard deviations and uncertainties for FB1, FB2 and their sum are reported
in Table 6.
TARGET STANDARD DEVIATION FOR THE TEST, σP
The target standard deviation was determined by the Horwitz equation as an indication of the best
practice [4] [9].
INDIVIDUAL z-SCORE
Participants’ z-scores were calculated as follows:
P
Xxz
!
)ˆ( "=
Where x = the participant’s result;
= the assigned value;
σP = the target value for standard deviation.
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The z-scores were interpreted as:
|z|≤2 satisfactory result;
2<|z|≤3 questionable result;
|z|>3 unsatisfactory result.
The z-score allows to compare the results provided by laboratories with the assigned value,
assumed to be very close to the true value.
Participants’ z-scores for FB1, FB2 and their sum are given in Table 3 and Table 5 and in Figures
1, 2 and 3 the corresponding histograms are shown.
INDIVIDUAL ZETA-SCORE
Participants’ zeta-scores were calculated as follows:
22
)ˆ(
alabuu
Xxzeta
+
!=
Where x = the participant’s result
= the assigned value;
ulab=uncertainty of the participant’s result;
ua=uncertainty of the assigned value.
The zeta-scores were interpreted as:
|zeta|≤2 satisfactory result;
2<|zeta|≤3 questionable result;
|zeta|>3 unsatisfactory result.
The zeta-score was used as an indication of the consistency of the uncertainty provided by the
participant with the observed deviation from the assigned value. Unsatisfactory zeta-scores may
indicate an underestimation of uncertainty or a large deviation of the result from the assigned value;
it indicates an uncertainty value not consistent with the uncertainty of the reference value. High
zeta-scores and z-scores are usually expected when uncertainties values are not in agreement with
the observed deviation from the assigned value, so it is useful to consider z and zeta scores together.
Participants’ zeta-scores for FB1 and FB2 are given in Table 4 and Table 5 and the corresponding
histograms are shown in Figures 4 and 5.
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ASSESSMENT OF SCORES
Not all participants provided all values of measurement uncertainties, hence for those laboratories
zeta-scores were not calculated. Moreover, zeta-scores of the sum of FBs were not computed due to
the scarce number of uncertainties values collected.
From the results provided by participants, the percentage of z-scores in the satisfactory range, |z|≤2,
was 88% for FB1, 88% for FB2 and 76% for their sum (Table 8).
The percentage of zeta-scores in the satisfactory range, |zeta|≤2, was 66% for FB1 and 75% for FB2
(Table 9).
Laboratories 18 and 32 failed z-scores and zeta-scores both for FB1 and FB2 underlining a large
deviation between the results and the assigned value and possibly an underestimation of the
uncertainties (Table 5). Laboratory number 11 had unsatisfactory scores only for FB1, while
laboratory number 16 failed zeta-scores both for FB1 and FB2, and z-score only for FB2 .
Z-score and zeta-scores were unsatisfactory more frequently for FB1 than for FB2.
Laboratory 1, 16, 20, 25 and 33 for FB1 and 20, 25 and 33 for FB2 failed only zeta-score,
evidencing possible underestimation of uncertainty values.
Laboratories using ELISA kit
Three participants (laboratory number 13, 24 and 28) used an ELISA kit, providing only results for
the sum of FB1 and FB2.
The z-scores of the participants using ELISA KIT were respectively 1.8, -0.2 and 2.6; the
uncertainties of measurements for the ELISA method were not provided, hence zeta-scores were not
computed.
Tables 36-45 describe the features related to ELISA technique, derived by the information collected
by the questionnaire answers returned by participants.
Laboratory with a z-score between -1 and 1
Fourteen laboratories (Labs 2-4-5-6-7-9-10-12-14-15-17-22-26-27) resulted to have a satisfactory z-
score included in the range from -1 to 1 for FB1, FB2 and their sum, all using HPLC determination.
Tables 46-69 describe the features of the analytical methods of participants with a z-score between
-1 and 1 for FB1, FB2 and their sum.
The analytical methods information reported in Tables 46-71 summarize the questionnaire answers
returned by participants.
11
In most cases, laboratories with the best performance used an accredited (8 laboratories out of 14-
Table 46) and validated method (11 laboratories out of 11-Table 47). The most frequently used
method was the JRC method (3 laboratories out of 7-Table 48).
Acetonitrile, methanol and water mixture was the most widely extracting solvent used by
laboratories (6 laboratories out of 13-Table 50). The homogenization with solvent (10 laboratories
out of 14-Table 51) and a single extraction type (11 laboratories out of 14-Table 52) were the most
common extraction procedures used by laboratories.
The most used immunoaffinity column brand was R-Biopharm (9 laboratories out of 12-Table 54).
The following operating HPLC conditions were mostly used:
Injection volume : ≤25 µL (8 laboratories out of 13-Table 57)
Column packing: C18 (9 laboratories out of 13-Table 58)
HPLC column temperature: ≥ 30 and ≤40 °C (7 laboratories out of 13-Table 59)
Mobile phase flow rate: >0.5 and ≤1.0 mL/min (8 laboratories out of 14-Table 61)
HPLC derivatisation: pre-column with OPA (8 laboratories out of 10-Table 63).
HPLC type of detector: FLD (10 laboratories out of 14-Table 66),
Ion source for LC/MS-MS : ESI (4 laboratories out of 4-Table 67).
The most frequently primary reference standards were certified (10 laboratories out of 10-Table 64)
and the brand was Biopure (8 laboratories out of 12-Table 64).
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APPENDIX I
Laboratories registered to the proficiency test
Table 1 Participant Laboratories.
Country Name of the organization
Luxembourg, Luxembourg
Laboratoire national de Santé- Contrôle alimentaire Contact person: Gilbert Moris
Hungary, Budapest
Central Agricultural Office Food and Feed Safety Directorate Contact person: József Dömsödi
Denmark, Copenhagen
Danish Plant Directorate Laboratorium for Foder og Gødning Contact person: Yvonne Simonsen
Romania, Bucarest
Sanitary Veterinary and Food Safety Directorate- Feed Investigation NRL Contact person: Catalin Negreanu
Belgium, Geel
Institute for Reference Materials and Measurement Food Safety and Quality Unit- (EU-RL) Contact person:Donata Lerda
Sweden, Uppsala
National Veterinary Institute Department of Chemistry, Environment and Feed Hygiene Contact person: Alexey Solyakov
France, Rennes
Service Commun des Laboratories du MINEFI Laboratorie de RENNES, NRL for Mycotoxins Contact person: Marie Paule Herry
Finland, Espoo
Finnish Customs Laboratory Customs Laboratory Tekniikantie Contact person: Kirsti Nuotio
Greece, Athens
General Chemical State Laboratory of Athens- Division of Environment Contact person: Panagiota Giannikopoulou
Spain, Majadahonda
Centro Nacional de Alimentaciòn Contact person: Pedro A. Burdaspal
Slovenia, Ljubljana
University in Ljubljana National veterinary institute Contact person: Gabrijela Tavcar-Kalcher
Ireland, Dublin
Public Analyst’s Laboratory - Sir Patrick Dun’s Contact person: John Keegan
Estonia, Saku
Laboratory for Residues and Contaminants Agricultural Research Centre Contact person: Merike Toome
Poland, Pulawy
National Veterinary Research Institute- Department of Pharmacology and Toxicology Contact person: Jan Zmudzki
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The Netherland, Wageningen
RIKILT Institute of Food Safety Contact person: Hans Mol
Belgium, Tervuren
Veterinary and Agrochemical Research Centre Unit Toxins and Natural Substances Contact person: Philippe Debongnie
Italy, Roma
Istituto Zooprofilattico Sperimentale Lazio e Toscana Contact person: Alessandro Ubaldi
Italy, Palermo
Istituto Zooprofilattico Sperimentale della Sicilia Area Chimica e Tecnologie Alimentari Contact person: Francesca Grippi
Italy, Asti
Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Sezione di Asti Contact person: Adriano Rosso
Italy, Portici
Istituto Zooprofilattico Sperimentale del Mezzogiorno Contact person: Pasquale Gallo
Italy, Bologna
Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna Reparto Chimico degli Alimenti Bologna Contact person:Simonetta Menotta
Italy, Legnano
Istituto Zooprofilattico Sperimentale delle Venezie S.C. Chimica - Laboratorio Residui e Farmaci Contact person: Giancarlo Biancotto
Italy, Foggia
Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata Micotossine e Tecniche Immunoenzimatiche Contact person: Rita De Pace
Italy, Brescia
Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna Dipartimento Alimenti e Sicurezza Alimentare Laboratorio Micotossine e Tossicologia Contact person: Alberto Biancardi
Italy, Foggia
Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata Unita’ Operativa: Farmaci e Tossicologia Contact person: Marilena Muscarella
Italy, Perugia
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche Laboratorio Contaminanti Ambientali Area Sicurezza Alimentare Contact person: Ivan Pecorelli
Italy, Teramo
Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise Reparto Bromatologia e Residui negli alimenti per l’uomo e gli animali Contact person: Giampiero Scortichini
Italy, Torino
ARPA Piemonte Polo Alimenti - Arpa Piemonte Contact person: Sara Coluccia
Italy, Genova
ARPA Liguria Dipartimento di Genova U.O. Laboratorio Contact person: Marta Ferro
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APPENDIX II
Homogeneity data for maize flour test material
Table 2 Homogeneity data for maize flour test material. FB1
µg/kg FB2 µg/kg
Target standard deviation σP 191.6 74.2 S2
an 10946.1 3426.2 S2
sam 3709.8 1297.8 σ2
all 3300.5 495.5 critical 18553.1 4764.3 S2
sam<critical ACCEPT ACCEPT
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APPENDIX III
Results tables
Table 3 Results and z-scores for FB1, FB2 and FB1+FB2 in maize flour test material. Analyte
Laboratory number
FB1 Assigned value
1257 µg/kg
FB2 Assigned value
425 µg/kg
FB1+FB2 Assigned value
1758 µg/kg Results
µg/kg Rec %
z-score
Results µg/kg
Rec %
z-score
Results µg/kg
Rec %
z-score
1 1040.6 100.0 -1.1 439.7 100.0 0.2 1480.3 - -1.1 2 1310 80 0.3 434.5 77 0.1 1744 - -0.1 4 1380 100 0.6 470 90 0.6 1850 - 0.4 5 1267 88 0.1 448 75 0.3 1715 - -0.2 6 1240 96 -0.1 468 77 0.6 1708 - -0.2 7 1458 100 1.0 425 100 0.0 1883 100 0.5 8 1586.6 96.4 1.7 435.5 86.3 0.1 2022.1 94.3 1.0 9 1127.2 102.9 -0.7 361.2 89.1 -0.8 1488.4 - -1.0 10 1283.8 96.5 0.1 454.4 79.0 0.4 1738.2 - -0.1 11 2103.3 68.9 4.4 478.2 64.7 0.7 2581.5 67.9 3.2 12 1301 73.7 0.2 453 76.8 0.4 1754 75.2 0.0 13 * * 2640* 104.6 3.4 14 1245 122.1 -0.1 416.3 120.9 -0.1 1661.3 122.2 -0.4 15 1430 90 0.9 392 90 -0.4 1822 - 0.2 16 960 100 -1.5 200 100 -2.9 1160 100 -2.3 17 1237 98 -0.1 481 105 0.7 1718 - -0.2 18 2060 80 4.1 1560 79 14.7 3620 - 7.2 20 1070 92.3 -1.0 540 94 1.5 1610 - -0.6 21 1463 99 1.1 402 129 -0.3 1865 - 0.4 22 1258 89 0.0 376 86 -0.6 1634 - -0.5 23 1178 90 -0.4 532 75 1.4 1710 - -0.2 24 * * 1650* 104 -0.4 25 978 64 -1.4 335 63 -1.2 1313 - -1.7 26 1187 83 -0.4 408 71 -0.2 1595 - -0.6 27 1423 81 0.9 408 90 -0.2 1831 - 0.3 28 * * 3000* 160 4.8 30 1079 81 -0.9 330 80 -1.2 1409 - -1.4 32 564.6 85.8 -3.6 247.4 82.2 -2.3 811.9 - -3.7 33 874 82 -2.0 297 71 -1.7 1171 - -2.3 *=participant reported only total fumonisins value.
16
Table 4 Results, extended uncertainty and zeta-scores for FB1, FB2 in maize flour test material. Analyte
Laboratory
number
FB1 Assigned value
1257 µg/kg
FB2 Assigned value
425 µg/kg Results
µg/kg Extended
uncertainty zeta-Score Results
µg/kg Extended
uncertainty zeta-Score
1 1040.6 193 -3.1 439.7 53.7 0.5 2 1310 236 0.4 434.5 73.8 0.2 4 1380 300 0.8 470 120 0.7 5 1267 139 0.1 448 54 0.6 6 1240 496 -0.1 468 187 0.4 7 1458 816 0.5 425 55 0.0 8 1586.6 317.4 2.0 435.5 304.8 0.1 9 1127.2 116.1 -1.6 361.2 75.9 -1.4 10 1283.8 320.95 0.2 454.4 113.6 0.5 11 2103.3 582.6 2.9 478.2 130.1 0.8 12 1301 195 0.4 453 68 0.7 13 * * 14 1245 381 -0.1 416.3 34.9 -0.3 15 1430 245 1.3 392 65 -0.8 16 960 240 -2.3 200 50 -6.4 17 1237 186 -0.2 481 72 1.3 18 2060 494.4 3.1 1560 327.6 6.7 20 1070 77.04 -2.8 540 41.04 3.6 21 1463 402 22 1258 276.76 0.0 376 106.8 -0.8 23 1178 294.5 -0.5 532 133 1.5 24 * * 25 978 147 -3.0 335 60 -2.3 26 1187 188 -0.6 408 53.04 -0.5 27 1423♥ 408♥ 28 * * 30 1079 239 -1.4 330 84 -1.9 32 564.6 196.9 -6.1 247.4 97.7 -3.2 33 874 220 -3.1 297 101 -2.3 *=participant reported only total fumonisins value. ♥=participant didn’t provide an uncertainty value.
17
Table 5 Results, z-score and zeta-scores for FB1 and FB2 in maize flour test material.
Laboratory
number
FB1 Assigned value
1257 µg/kg
FB2 Assigned value
425 µg/kg Results
µg/kg z-Score zeta-Score Results
µg/kg z-Score zeta-Score
1 1040.6 -1.1 -3.1 439.7 0.2 0.5 2 1310 0.3 0.4 434.5 0.1 0.2 4 1380 0.6 0.8 470 0.6 0.7 5 1267 0.1 0.1 448 0.3 0.6 6 1240 -0.1 -0.1 468 0.6 0.4 7 1458 1.0 0.5 425 0.0 0.0 8 1586.6 1.7 2.0 435.5 0.1 0.1 9 1127.2 -0.7 -1.6 361.2 -0.8 -1.4 10 1283.8 0.1 0.2 454.4 0.4 0.5 11 2103.3 4.4 2.9 478.2 -0.1 0.8 12 1301 0.2 0.4 453 -0.4 0.7 13 * * 14 1245 -0.1 -0.1 416.3 -0.1 -0.3 15 1430 0.9 1.3 392 -0.4 -0.8 16 960 -1.5 -2.3 200 -2.9 -6.4 17 1237 -0.1 -0.2 481 0.7 1.3 18 2060 4.1 3.1 1560 14.7 6.7 20 1070 -1.0 -2.8 540 1.5 3.6 21 1463♥ 1.1 402♥ -0.3 22 1258 0.0 0.0 376 -0.6 -0.8 23 1178 -0.4 -0.5 532 1.4 1.5 24 * * 25 978 -1.4 -3.0 335 -1.2 -2.3 26 1187 -0.4 -0.6 408 -0.2 -0.5 27 1423♥ 0.9 408♥ -0.2 28 * * 30 1079 -0.9 -1.4 330 -1.2 -1.9 32 564.6 -3.6 -6.1 247.4 -2.3 -3.2 33 874 -2.0 -3.1 297 -1.7 -2.3 *= participant reported only FB1+FB2 value. ♥=participant didn’t provide any uncertainty value.
18
Table 6 Assigned values, target standard deviations and uncertainties.
Assigned value X̂ , µg/kg Target standard deviation
Analyte Data points, n
Winsorized mean X̂
Robust standard deviation
Standard uncertainty
of X̂
Derived from
σP
FB1 26 1257 271.4 53.2 Horwitz 194.2 FB2 26 425 126.6 24.8 Horwitz 77.3 FBS 29 1758 479.1 89.0 Horwitz 258.3
Table 7 Results, extended uncertainties, z-score and zeta-scores for FB1 and FB2 in maize flour test material. Laboratory number
FB1 Assigned value
1257 µg/kg
FB2 Assigned value
425 µg/kg Results
µg/kg Extended
uncertainty z-
Score zeta-Score
Results µg/kg
Extended uncertainty
z- Score
zeta-Score
1 1040.6 193 -1.1 -3.1 439.7 53.7 0.2 0.5 2 1310 236 0.3 0.4 434.5 73.8 0.1 0.2 4 1380 300 0.6 0.8 470 120 0.6 0.7 5 1267 139 0.1 0.1 448 54 0.3 0.6 6 1240 496 -0.1 -0.1 468 187 0.6 0.4 7 1458 816 1.0 0.5 425 55 0.0 0.0 8 1586.6 317.4 1.7 2.0 435.5 304.8 0.1 0.1 9 1127.2 116.1 -0.7 -1.6 361.2 75.9 -0.8 -1.4 10 1283.8 320.95 0.1 0.2 454.4 113.6 0.4 0.5 11 2103.3 582.6 4.4 2.9 478.2 130.1 -0.1 0.8 12 1301 195 0.2 0.4 453 68 -0.4 0.7 13 * * 14 1245 381 -0.1 -0.1 416.3 34.9 -0.1 -0.3 15 1430 245 0.9 1.3 392 65 -0.4 -0.8 16 960 240 -1.5 -2.3 200 50 -2.9 -6.4 17 1237 186 -0.1 -0.2 481 72 0.7 1.3 18 2060 494.4 4.1 3.1 1560 327.6 14.7 6.7 20 1070 77.04 -1.0 -2.8 540 41.04 1.5 3.6 21 1463♥ 1.1 402♥ -0.3 22 1258 276.76 0.0 0.0 376 106.8 -0.6 -0.8 23 1178 294.5 -0.4 -0.5 532 133 1.4 1.5 24 * * 25 978 147 -1.4 -3.0 335 60 -1.2 -2.3 26 1187 188 -0.4 -0.6 408 53.04 -0.2 -0.5 27 1423♥ 0.9 408♥ -0.2 28 * * 30 1079 239 -0.9 -1.4 330 84 -1.2 -1.9 32 564.6 196.9 -3.6 -6.1 247.4 97.7 -2.3 -3.2 33 874 220 -2.0 -3.1 297 101 -1.7 -2.3 *= participant reported only FB1+FB2 value. ♥=participant didn’t provide any uncertainty value.
19
Table 8 Number and percentage of satisfactory z-scores. Analyte Number of
satisfactory scores |z|≤2
Total number of scores
Satisfactory %
FB1 23 26 88 FB2 23 26 88
FB1 + FB2 22 29 76 Table 9 Number and percentage of satisfactory zeta-scores.
Analyte Number of satisfactory scores
|zeta|≤2
Total number of scores
Satisfactory %
FB1 16 24 67 FB2 18 24 75
20
Figure 1 z-Scores for FB1 (Assigned Value=1257 µg/kg).
Figure 2 z-Scores for FB2 (Assigned Value=425 µg/kg). The maximum z-score (laboratory N°18, z-score=14.7) is not shown.
21
Figure 3 z-Scores for FBs (Assigned Value=1758 µg/kg).
Figure 4 zeta-Scores for FB1 (Assigned Value=1257 µg/kg).
22
Figure 5 zeta-Scores for FB2 (Assigned Value=425 µg/kg).
23
APPENDIX IV
Analytical methods used by participants
Table 10. Accredited method Laboratory number Yes 2-4-5-7-8-9-12-13-14-22-24-30-32 No 1-6-10-11-15-16-17-18-20-21-23-25-26-27-28-
33
Table 11. Validated method Laboratory number Yes 2-4-5-6-7-8-9-10-12-13-14-15-16-17-18 No 1-11
Table 12. Reference Laboratory number R-Biopharm Manual 1 In house validated method 2-13 DIN EN 14352 4-6-8-15-32-33 EC, JRC, IRMM 10 DIN EN 16006 17 Dossier di validazione 15CH 030/0/0-RAC C3 576/2006-REG CE 882/2004
18
Italian National Institute for Health (ISS) 21 Journal of chromatography A, 1203 (2008) pp.88-93; Journal of food science Vol 76 N1 (2011)
25
AOAC OFFICIAL METHOD 995.15 26 Journal of AOAC 84(6) (2001) 1828-1837 30
Table 13. Sample weight, g Laboratory number ≤10 4-5-11-13-20-24-25-26-28-32 >10-≤20 2-6-15-16-17-21-22-33 >20-≤30 1-7-8-9-12-14-18-27-30
24
Table 14. Extraction solvent Laboratory number AcCN:MeOH:H2O 1-2-4-6-8-9-11-14-18-21-25-27-32-33 AcCN:H2O 5-12 AcCN:H2O:HCOOH 7 MeOH:PBS 10-17 MeOH:H2O 13-20-22-24-26-28-30 AcCN:CH3COOH:H2O 16
Table 15. Extraction procedure Laboratory number +NaCl 1-8-9-18-27-28 Blender 5-8-10-11-14-18-22-23-26-28-33 Homogenisation with solvent 1-2-4-6-7-8-9-12-13-14-15-16-17-20-23-24-25-
27-32 Magnetic agitator 21
Table 16. Extraction type Laboratory number Single 2-4-5-7-9-10-12-13-16-17-18-20-21-22-23-24-
26-27-30-33 Multiple 6-8-11-14-15-25-28-32
Table 17. Sample work up Laboratory number Dilution 2-5-8-9-10-12-13-16-17-18-21-22-23-24-25-30-
32-33 Filtration 1-2-4-5-6-7-8-9-10-11-12-13-14-17-18-20-21-
22-24-25-26-27-28-30-32-33 Centrifugation 2-6-7-9-11-12-13-14-15-16-20-22-25-26-28-33
Table 18. Sample clean-up by IAC (brand) Laboratory number R-Biopharm 1-2-4-6-8-9-11-14-15-17-18-21-23-25-27 Romer Labs 12-32 VICAM 5-10-30-33
Table 19. Sample clean-up by SPE Laboratory number Multisep Romer 12 Sax isolute 20 Quaternary Amine Baker 26
25
Table 20. Mycotoxin determination Laboratory number ELISA 13-24-28 HPLC 1-2-4-5-6-7-8-9-10-11-12-14-15-16-17-18-20-
21-22-23-25-26-27-30-32-33
Table 21. ELISA brand Laboratory number R-Biopharm 13 Euroclone 24 Tecna 28
Table 22. ELISA Laboratory number Automatic Manual 13-24-28
Table 23. HPLC injection volume (µL) Laboratory number ≤25 1-5-6-7-11-12-14-16-18-21-22-23-26-27-30-32-
33 >25 ≤50 4-8-17-20 >50 2-9-15-25
Table 24. HPLC column packing Laboratory number C18 1-4-5-6-11-12-14-15-16-17-20-21-22-23-27-30-
33 WATER NOVA-PAK C18 2 Ultra aqueous C18 7 INTERSIL ODS-2 8-9 RP-18 18-26 RP-ODS 32
Table 25. HPLC column temperature (°C) Laboratory number Ambient 1-4-5-6-20-23-32 ≤30 8-11-14-21-26-30-33 >30 ≤40 2-7-9-12-15-16-18-22-25-27 >40 17
26
Table 26. Mobile phase components Laboratory number AcCN:H20:HCOOH 1-4-12-16-17-21 MeOH:H2O 2 MeOH:phosphate buffer 5-25 MeOH:NaH2PO4 6-8-9-10-14-26-30-32 H2O:MeOH:NH4HCO2:HCOOH 7 MeOH:NaH2PO42H2O 11 H2O:MeOH:HCOOH 15 AcCN:HCOOH 18-22 H2O:MeOH:CH3COOH 20 MeOH:CH3COONH4 :CH3COOH:H2O 27 MeOH :Na3PO4 33
Table 27. Mobile phase flow rate (mL/min) Laboratory number ≤0.3 1-12-18-21-22-23 >0.3-≤0.5 7-16-20-27 >0.5-≤1.0 2-4-5-6-8-9-11-14-15-25-26-30-32-33 >1.0 10-17
Table 28. Post-column mobile phase flow rate (mL/min)
Laboratory number
≤0.5 4-17-21-25 >0.5 11
Table 29. HPLC derivatisation Laboratory number OPA pre-column 2-5-6-8-9-10-11-14-15-26-30-32-33 OPA post-column 4-17-25
Table 30. Source of standard Laboratory number Biopure 1-2-4-5-6-7-12-15-16-17-21-26 Sigma-Aldrich 8-10-14-23-32-33 LGC 9 Panreac 22 Riedel-de-Haen 25
Table 31. Is the standard certified? Laboratory number Yes 1-2-4-5-6-7-8-9-10-12-14-16-17 No
27
Table 32. HPLC Detector type Laboratory number FLD 2-4-5-6-8-9-10-11-14-15-17-25-26-30-32-33 MS 21-22 MS/MS 1-7-12-16-18-20-23-27
Table 33. Ion source Laboratory number ESI 1-7-12-16-18-20-21-22-23-27 APCI APPI
Table 34. Acquisition mode Laboratory number Full scan Limited m/z range Selected ion mode (SIM) 12-21-22 Selected/multiple reaction mode (SRM/MRM) 1-7-16-18-20-23-27 Full scan product ion spectra
Table 35. Detector Laboratory number Single quadrupole 12-21-22 Orbitrap Time of flight Triple quadrupole 1-7-16-23 Ion trap 18-20 Hybrid MS (e.g. Q-TOF, Q-trap, LTQ-Orbitrap) 27
28
Analytical methods used by participants using kit ELISA
Table 36. Is the method used accredited Laboratory number Yes 13-24 No 28
Table 37. Is the method used validated Laboratory number Yes 13
Table 38. Reference Laboratory number In house validated method 13
Table 39. Sample weight Laboratory number ≤10 13-24-28
Table 40. Extraction solvent Laboratory number MeOH: H2O 13-24-28
Table 41. Extraction procedure Laboratory number +NaCl 28 Homogenisation with solvent 13-24
Table 42. Extraction type Laboratory number Single 13-24 Multiple 28
Table 43. Sample work up Laboratory number Dilution 13-24 Filtration 13-24-28 Centrifugation 13-28
29
Table 44. ELISA brand Laboratory number R-Biopharm 13 Euroclone 24 Tecna 28
Table 45. ELISA Laboratory number Manual 13-24-28
30
Analytical methods by participants with a z-score between -1 and 1
Table 46. Is the method used accredited Laboratory number Yes 2-4-5-7-9-12-14-22 No 6-10-15-17-26-27
Table 47. Is the method used validated Laboratory number Yes 2-4-5-6-7-9-10-12-14-15-17 No
Table 48. Reference Laboratory number In house validated method 2 DIN EN 14352 4-6-15 EC JRC 10 DIN EN 16006 17 AOAC OFFICIAL METHOD 995.15 26
Table 49. Sample weight Laboratory number ≤10 4-5-26 >10-≤20 2-6-15-17-22 >20-≤30 7-9-12-14-27
Table 50. Extraction solvent Laboratory number AcCN:MeOH:H2O 2-4-6-9-14-27 AcCN:H2O 5-12 AcCN:H2O:HCOOH 7 MeOH:PBS 10-17 MeOH: H2O 22-26
Table 51. Extraction procedure Laboratory number +NaCl 9-27 Blender 5-10-14-22-26 Homogenisation with solvent 2-4-6-7-9-12-14-15-17-27
31
Table 52. Extraction type Laboratory number Single 2-4-5-7-9-10-12-17-22-26-27 Multiple 6-14
Table 53. Sample work up Laboratory number Dilution 2-5-9-10-12-17-22 Filtration 2-4-5-6-7-9-10-12-14-17-22-26-27 Centrifugation 2-6-7-9-12-14-15-22-26
Table 54. Sample clean-up by IAC (brand) Laboratory number R-Biopharm 2-4-6-9-14-15-17-23-27 Romer Labs 12 VICAM 5-10
Table 55. Sample clean-up by SPE Laboratory number Multistep Romer 12 Quaternary Amine Baker 26
Table 56. Mycotoxin determination Laboratory number HPLC 2-4-5-6-7-9-10-12-14-15-17-22-26-27
Table 57. HPLC injection volume (µL) Laboratory number ≤25 5-6-7-12-14-22-26-27 >25 ≤50 4-17 >50 2-9-15
Table 58. HPLC column packing Laboratory number C18 4-5-6-12-14-15-17-22-27 WATER NOVA-PAK 2 Ultra aqueons C18 7 INTERSIL ODS-2 9 RP-18 26
32
Table 59. HPLC column temperature (°C) Laboratory number ambient 4-5-6 ≤30 14-26 >30 ≤40 2-7-9-12-15-22-27 >40 17
Table 60. Mobile phase components Laboratory number AcCN:H2O:HCOOH 4-12-17 MeOH:H2O 2 MeOH:phosphate buffer 5 MeOH:NaH2PO4 6-9-10-14-26 H20:MeOH:NH4HCO2:HCOOH 7 H2O:MeOH:HCOOH 15 AcCN:HCOOH 22 MeOH:CH3COONH4 :CH3COOH:H2O 27
Table 61. Mobile phase flow rate (mL/min) Laboratory number ≤0.3 12-22 >0.3-≤0.5 7-27 >0.5-≤1.0 2-4-5-6-9-14-15-26 >1.0 10-17
Table 62. Post column mobile phase flow rate (mL/min)
Laboratory number
≤0.5 4-17
Table 63. HPLC derivatisation Laboratory number OPA pre-column 2-5-6-9-10-14-15-26 OPA post-column 4-17
Table 64. Source of standard Laboratory number Biopure 4-5-6-7-12-15-17-26 Sigma-Aldrich 10-14 LGC 9 Panreac 22
Table 65. Is the standard certified? Laboratory number Yes 2-4-5-6-7-9-10-12-14-17
33
Table 66. HPLC Detector type Laboratory number FLD 2-4-5-6-9-10-14-15-17-26 MS 22 MS/MS 7-12-27
Table 67. Ion source Laboratory number ESI 7-12-22-27
Table 68. Acquisition mode Laboratory number Selected ion mode (SIM) 12-22 Selected/multiple reaction mode (SRM/MRM) 7-27
Table 69. Detector Laboratory number Single quadrupole 12-22 Triple quadrupole 7 Hybrid MS (e.g. Q-TOF, Q-trap, LTQ-Orbitrap 27
34
APPENDIX V
Cover letter
Rome, 18 December 2010
Ref: 2010 Proficiency Testing on fumonisins B1 and B2 in maize flour
Dear Participant, As previously communicated, I am sending you the material necessary for the performance of the
proficiency test on corn test material. Please read the following information carefully before starting any analysis. In case of
questions, do not hesitate to contact us by either phone or email (see details below). You are kindly requested to check the content of the parcel and fill in the annexed "Receipt form" to
send back as confirmation of the receipt of the material in proper conditions. If any material has been received damaged, immediately request a new material.
The materials are shipped at room temperature; however, storage should be at -18° C until the analysis is performed. A short period of 1-2 days without cooling is no harm for the material, but a longer period of storage above -18° C shall be avoided.
The ISS-2010 PT aims to assess the content of fumonisins in the contaminated test samples. You will be asked to report the recovery corrected value in μg/kg, including your recovery in % and measurement uncertainty plus coverage factor.
Participants are kindly requested to print out the “Results Forms” and return NOT later than 21st
January 2011, the signed sheet by fax (+3906 49902363) or by mail (and by regular mail to
Carlo Brera
Istituto Superiore di Sanità National Centre of Food Quality and Risk Assessment GMO and Mycotoxin Unit Viale Regina Elena, 299 00161 Rome - Italy
You will receive a statistical report with the results of the validation study.
Fumonisin B1 and B2 in maize flour
PROFICIENCY TESTING
35
The mentioned report will be confidential and each participating laboratory will be marked
only with a number.
For any clarification please do not hesitate to contact Carlo Brera or Barbara De Santis phone
+39 06 49902377 or +30 06 49902820, or by e-mail: [email protected];
Carlo Brera
(Operating Manager of the Italian Reference Laboratory for Mycotoxins)
Your laboratory has been assigned with
CODE:
36
Receipt form
Date and hour of receipt of the material:
Conservation status: Satisfying Unsatisfying
Remarks:
Signature:
Please return the form by fax (+39 06 49902363) or by e mail ([email protected])
37
Results Form
Laboratory Code:
Institute:
City:
Contact person:
Operator:
Date of analysis:
FB1 (µg/kg) FB2 (µg/kg) Sum
Sample code
Recovery (%)
Uncertainty
Coverage Factor:
Signature (Operator):
Signature (Contact person):
Please return the form by fax (+39 06 49902363) or by e mail ([email protected])
NO LATER THAN THE CLOSING DATE 21st JANUARY 2011
38
Method Form
Is the method used accredited?
Is the method validated?
Reference
Sample weight (g)
Extraction solvent
Extraction procedure ☐add NaCl
☐blender
☐homogenize with solvent
Extraction type ☐single ☐multiple
Sample work up ☐dilution ☐filtration ☐centrifugation
Sample clean-up ☐by IAC (brand)
☐SPE (column type and brand)
☐other (specify)
Mycotoxin determination ☐HPLC ☐ELISA)
ELISA – kit ELISA brand
ELISA ☐automatic ☐manual
HPLC – injection volume (µL)
HPLC – column packing
HPLC – column temperature (°C)
Mobile phase components
Mobile phase flow rate (mL/min)
Post column mobile phase flow rate (mL/min)
Derivatization ☐HPLC Pre column ☐HPLC Post column
Source of standards (brand)
Is the standard certified?
HPLC detector type ☐fluorimetry ☐MS/MS
39
MS detection mode
Ion source
☐ESI
☐APCI
☐APPI
☐Other (specify)
Acquisition mode
☐Full scan
☐Limited m/z range
☐Selected ion mode (SIM)
☐Selected/multiple reaction mode (SRM/MRM)
☐Full scan product ion spectra
� Other (please specify)
Detector
� Single quadrupole
� Orbitrap
� Time of flight
� Triple quadrupole
� Ion trap
� Hybrid MS (e.g. Q-TOF, Q-trap, LTQ-Orbitrap)
� Other (please specify)
40
Table 1. SRM/MRM or full scan product ion spectra parameters
Precursor ion (m/z) Adducta Product ion(s)
(m/z)b
FB1
FB2
a Please specify, e.g. [M+H] +, [M+NH4] +, [M-H]-, etc.
b Please mark with an asterisk the quantifier ion
Table 2. Low resolution MS parameters (full scan SIM, limited m/z range)
Diagnostic ion(s)a Adduct
FB1
FB2
41
a Please mark with an asterisk the quantifier ion
Table 3. High resolution MS (full scan SIM, limited m/z range)
Diagnostic ion (s)a Mass accuracy
(ppm) Adduct
FB1
FB2
a Please mark with an asterisk the quantifier ion
Please return the form by fax (+39 06 49902363) or by e-mail ([email protected]) NO LATER THAN THE CLOSING DATE 21st FEBRUARY 2011
42
REFERENCES
1) Food Analysis Performance Assessment Scheme (FAPAS®), 2002, Protocol for the
Organization and Analysis of Data, 6th Edition.
2) Analytical Methods Committee, 1989, Report on an experimental test of “recommendations
for the conduct and interpretation of co-operative trials”, Analyst, 114, 1489-1495.
3) Analytical Methods Committee, 1989, Robust Statistics-How not to reject outliers Part 1.
Basic Concepts, Analyst, 114, 1693-1697.
4) Thompson, M., Ellison, S., L., R. and Wood, R., 2006, The International Harmonized
Protocol for the Proficiency Testing of Analytical Chemistry Laboratories, Pure Appl.
Chem., 78 (1) 145-196.
5) Regulation (EC) 882/2004 of the European Parliament and of the Council of 29 April 2004
on official controls performed to ensure the verification of compliance with feed and food
law, animal health and animal welfare rules, Official Journal L191, 28/05/2004, 1-59.
6) International Organization for Standardization. ISO 17025:2005 General requirements for
the competence of testing and calibration laboratories, Geneva, Switzerland (2005).
7) Regulation (EC) 1126/2007 of 28 September 2007 amending regulation (EC) No 1181/2006
setting maximum levels for certain contaminants in foodstuffs as regards fusarium toxins in
maize and maize products, Official Journal L255, 14-17.
8) Fearn, T. and Thompson, M., 2002, A new test for ‘sufficient homogeneity’. Analyst, 126,
1414-1417.
9) Thompson, M., 2000, Recent trends in inter-laboratory precision at ppb and sub-ppb
concentrations in relation to fitness for purpose criteria in proficiency testing. Analyst, 125,
385-386.