SIST EN 17194:2020
(Main)Animal feeding stuffs: Methods of sampling and analysis - Determination of Deoxynivalenol, Aflatoxin B1, Fumonisin B1 & B2, T-2 & HT-2 toxins, Zearalenone and Ochratoxin A in feed materials and compound feed by LC-MS/MS
Animal feeding stuffs: Methods of sampling and analysis - Determination of Deoxynivalenol, Aflatoxin B1, Fumonisin B1 & B2, T-2 & HT-2 toxins, Zearalenone and Ochratoxin A in feed materials and compound feed by LC-MS/MS
This European Standard method of analysis is applicable for the determination of Deoxynivalenol (DON) in the tested range of 96,2 µg/kg to 3 269 µg/kg, Aflatoxin B1 (AfB1) in the tested range of 2,62 µg/kg to 444 µg/kg, Fumonisin B1 (FB1) in the tested range of 693 µg/kg to 7 529 µg/kg, Fumonisin B2 (FB2) in the tested range of 203 µg/kg to 2 465 µg/kg, T-2 toxin in the tested range of 7,47 µg/kg to 360 µg/kg and HT-2 toxin in the tested range of 13,9 µg/kg to 1 758 µg/kg, Zearalenone (ZON) in the tested range of 34,3 µg/kg to 593 µg/kg and Ochratoxin A (OTA) in the tested range of 10,8 µg/kg to 228 µg/kg in cereals and cereal-based compound feed by liquid-chromatography tandem mass spectrometry (LC-MS/MS). The actual working ranges may extend beyond the tested ranges.
Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von Deoxynivalenol, Aflatoxin B1, Fumonisin B1 und B2, T-2- und HT-2-Toxine, Zearalenon und Ochratoxin A in Einzelfuttermitteln und Mischfuttermitteln mittels LC-MS/MS
Dieses Europäische Normanalyseverfahren ist gültig für die Bestimmung von Deoxynivalenol (DON) im untersuchten Bereich von 96,2 µg/kg bis 3 269 µg/kg, Aflatoxin B1 (AfB1) im untersuchten Bereich von 2,62 µg/kg bis 444 µg/kg, Fumonisin B1 (FB1) im untersuchten Bereich von 693 µg/kg bis 7 529 µg/kg, Fumonisin B2 (FB2) im untersuchten Bereich von 203 µg/kg bis 2 465 µg/kg, T 2 Toxin im untersuchten Bereich von 7,47 µg/kg bis 360 µg/kg und HT 2 Toxin im untersuchten Bereich von 13,9 µg/kg bis 1 758 µg/kg, Zearalenon (ZON) im unter¬suchten Bereich von 34,3 µg/kg bis 593 µg/kg und Ochratoxin A (OTA) im untersuchten Bereich von 10,8 µg/kg bis 228 µg/kg in Getreide und Getreidemisch-futtermittel durch Flüssig¬chromatographie mit Tandem Massenspektrometrie (LC MS/MS). Die tatsächlichen Arbeitsbereiche dürfen über die geprüften Bereiche hinausgehen.
Aliments des animaux : Méthodes d’échantillonnage et d’analyse — Détermination du déoxynivalénol, de l’aflatoxine B1, de la fumonisine B1 et B2, des toxines T-2 et HT-2, de la zéaralénone et de l’ochratoxine A dans les matières premières pour aliments et les aliments composés pour animaux par CL-SM/SM
La méthode d’analyse de la présente Norme européenne s’applique à la détermination du déoxynivalénol (DON) dans la plage soumise à essai allant de 96,2 µg/kg à 3 269 µg/kg, de l’aflatoxine B1 (AfB1) dans la plage soumise à essai allant de 2,62 µg/kg à 444 µg/kg, de la fumonisine B1 (FB1) dans la plage soumise à essai allant de 693 µg/kg à 7 529 µg/kg, de la fumonisine B2 (FB2) dans la plage soumise à essai allant de 203 µg/kg à 2 465 µg/kg, de la toxine T-2 dans la plage soumise à essai allant de 7,47 µg/kg à 360 µg/kg et de la toxine HT-2 dans la plage soumise à essai allant de 13,9 µg/kg à 1 758 µg/kg, de la zéaralénone (ZON) dans la plage soumise à essai allant de 34,3 µg/kg à 593 µg/kg et de l’ochratoxine A (OTA) dans la plage soumise à essai allant de 10,8 µg/kg à 228 µg/kg dans les céréales et les aliments composés à base de céréales, par chromatographie en phase liquide couplée à de la spectrométrie de masse (CL-SM/SM). Les plages de travail réelles peuvent s’étendre au-delà des plages soumises à essai.
Krma: metode vzorčenja in analize - Določevanje deoksinivalenola, aflatoksina B1, fumonizina B1 in B2, toksinov T-2 in HT-2, zearalenona in ohratoksina A v sestavinah krme in krmni mešanici z LC-MS/MS
Ta analitska metoda evropskega standarda se uporablja za določanje deoksinivalenola (DON) v preskusnem obsegu od 96,2 µg/kg do 3269 µg/kg, aflatoksina B1 (AfB1) v preskusnem obsegu od 2,62 µg/kg do 444 µg/kg, fumonisina B1 (FB1) v preskusnem obsegu od 693 µg/kg do 7529 µg/kg, fumonisina B2 (FB2) v preskusnem obsegu od 203 µg/kg do 2465 µg/kg, T- 2 toksina v preskusnem obsegu od 7,47 µg/kg do 360 µg/kg in HT-2 toksina v preskusnem obsegu od 13,9 µg/kg do 1758 µg/kg, zeralenona (ZON) v preskusnem obsegu od 34,3 µg/kg do 593 µg/kg ter ohratoksina A (OTA) v preskusnem obsegu od 10,8 µg/kg do 228 µg/kg pri žitih in krmnih mešanicah na osnovi žit s tandemsko masno spektrometrijo s tekočinsko kromatografijo (LC-MS/MS). Dejanski delovni obsegi lahko presegajo presušene obsege.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN 17194:2020
01-januar-2020
Krma: metode vzorčenja in analize - Določevanje deoksinivalenola, aflatoksina B1,
fumonizina B1 in B2, toksinov T-2 in HT-2, zearalenona in ohratoksina A v
sestavinah krme in krmni mešanici z LC-MS/MS
Animal feeding stuffs: Methods of sampling and analysis - Determination of
Deoxynivalenol, Aflatoxin B1, Fumonisin B1 & B2, T-2 & HT-2 toxins, Zearalenone and
Ochratoxin A in feed materials and compound feed by LC-MS/MS
Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von
Deoxynivalenol, Aflatoxin B1, Fumonisin B1 und B2, T-2- und HT-2-Toxine, Zearalenon
und Ochratoxin A in Einzelfuttermitteln und Mischfuttermitteln mittels LC-MS/MS
Aliments des animaux : Méthodes d’échantillonnage et d’analyse — Détermination du
déoxynivalénol, de l’aflatoxine B1, de la fumonisine B1 et B2, des toxines T-2 et HT-2, de
la zéaralénone et de l’ochratoxine A dans les matières premières pour aliments et les
aliments composés pour animaux par CL-SM/SM
Ta slovenski standard je istoveten z: EN 17194:2019
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN 17194:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN 17194:2020
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SIST EN 17194:2020
EN 17194
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2019
EUROPÄISCHE NORM
ICS 65.120
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of Deoxynivalenol, Aflatoxin B1, Fumonisin
B1 & B2, T-2 & HT-2 toxins, Zearalenone and Ochratoxin A
in feed materials and compound feed by LC-MS/MS
Aliments des animaux : Méthodes d'échantillonnage et Futtermittel: Probenahme- und
d'analyse - Détermination du déoxynivalénol, de Untersuchungsverfahren - Bestimmung von
l'aflatoxine B1, de la fumonisine B1 et B2, des toxines Deoxynivalenol, Aflatoxin B1, Fumonisin B1 und B2, T-
T-2 et HT-2, de la zéaralénone et de l'ochratoxine A 2- und HT-2-Toxine, Zearalenon und Ochratoxin A in
dans les matières premières pour aliments et les Einzelfuttermitteln und Mischfuttermitteln mittels LC-
aliments composés pour animaux par CL-SM/SM MS/MS
This European Standard was approved by CEN on 9 September 2019.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17194:2019 E
worldwide for CEN national Members.
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Contents Page
European foreword . 3
1 Scope . 4
2 Normative references . 4
3 Terms and definitions . 4
4 Principle . 4
5 Reagents . 5
6 Apparatus . 7
7 Procedure. 8
7.1 Sample preparation . 8
7.2 Extraction . 9
7.3 Test solution . 9
7.4 Spiking procedure . 10
8 Measurements . 10
8.1 General . 10
8.2 LC conditions. 10
8.3 MS conditions . 10
8.4 Batch composition . 11
8.5 Peak identification . 11
8.6 Determination of mycotoxins in calibration and test solutions . 11
8.7 Calibration . 11
9 Determination of mass fraction . 12
10 Precision . 13
10.1 Interlaboratory study . 13
10.2 Repeatability . 13
10.3 Reproducibility . 14
11 Test report . 14
Annex A (informative) Precision data . 15
Annex B (informative) Examples . 25
Annex C (informative) Example chromatograms . 30
Bibliography . 32
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European foreword
This document (EN 17194:2019) has been prepared by Technical Committee CEN/TC 327 “Animal
feeding stuffs - Methods of sampling and analysis”, the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2020, and conflicting national standards shall be
withdrawn at the latest by May 2020.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a standardization request given to CEN by the European
Commission and the European Free Trade Association.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United
Kingdom.
3
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1 Scope
This document's method of analysis is applicable for the determination of:
— deoxynivalenol (DON) in the tested range of 100 µg/kg to 3 300 µg/kg,
— aflatoxin B1 (AfB1) in the tested range of 2,5 µg/kg to 440 µg/kg,
— fumonisin B1 (FB1) in the tested range of 690 µg/kg to 7 500 µg/kg,
— fumonisin B2 (FB2) in the tested range of 200 µg/kg to 2 500 µg/kg,
— T-2 toxin in the tested range of 7,5 µg/kg to 360 µg/kg,
— HT-2 toxin in the tested range of 14 µg/kg to 1 800 µg/kg,
— zearalenone (ZEN) in the tested range of 30 µg/kg to 600 µg/kg, and
— ochratoxin A (OTA) in the tested range of 10 µg/kg to 230 µg/kg
in cereals and cereal-based compound feed by liquid-chromatography tandem mass spectrometry (LC-
MS/MS). The actual working ranges could extend beyond the tested ranges.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN ISO 3696:1995, Water for analytical laboratory use — Specification and test methods (ISO 3696:1987)
EN ISO 6498, Animal feeding stuffs — Guidelines for sample preparation (ISO 6498)
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at https://www.iso.org/obp.
4 Principle
A test portion of finely ground and homogeneous material is extracted by shaking with a mixture of
acetonitrile and aqueous formic acid solution. The extract is centrifuged and an aliquot of the supernatant
extract is transferred to a deactivated glass vial, mixed with an appropriate amount of stable-isotope
labelled analogues and evaporated to dryness. The reconstituted sample is filtered and quantified with a
Liquid Chromatography - Mass Spectrometry (LC-MS) system. Laboratories using this method shall
demonstrate the following limits of quantitation (LOQs) in order to be able to apply this method over the
whole validation range: for DON ≤ 100 µg/kg, for AfB1 ≤ 2 µg/kg, for FB1 and FB2 ≤ 500 µg/kg
(FB1 ≤ 375 µg/kg and FB2 ≤ 125 µg/kg), for T-2 and HT-2 toxin ≤ 10 µg/kg, for ZEN ≤ 20 µg/kg and for
OTA ≤ 10 µg/kg.
4
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NOTE The performance data collected in this study came from laboratories that verified to be able to achieve
the above mentioned LOQs. Applicants that could not verify this, were excluded in order to ensure that validation
data could be obtained over the targeted concentration range.
5 Reagents
Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696:1995,
unless otherwise specified. Commercially available solutions with equivalent properties to those listed
may be used.
WARNING — Environmental regulations and rules apply when disposing of waste solvents.
Decontamination procedures for laboratory wastes have been reported by the International Agency for
Research on Cancer (IARC), see [1, 2].
5.1 Water (deionized).
5.2 Water, LC-MS grade or of comparable purity (e.g. resistance of 18,2 MΩcm or respectively
conductivity of 55 nS/cm at 20 °C).
5.3 Methanol, LC-MS grade.
5.4 Formic acid (FA), 98-100 %.
5.5 Acetonitrile (ACN), LC grade.
5.6 Extraction solvent composed of 20 parts water (5.1), 79 parts acetonitrile (5.5) and 1 part formic
acid (5.4) (v/v/v).
5.7 20 % acetic acid solution, for washing glassware.
5.8 Aflatoxin B1 (AfB1), purity ≥ 95 %.
5.9 Deoxynivalenol (DON), purity ≥ 95 %.
5.10 Fumonisin B1 (FB1), purity ≥ 95 %.
5.11 Fumonisin B2 (FB2), purity ≥ 95 %.
5.12 HT-2 toxin (HT-2), purity ≥ 95 %.
5.13 T-2 toxin (T-2), purity ≥ 95 %.
5.14 Zearalenone (ZEN), purity ≥ 95 %.
5.15 Ochratoxin A (OTA), purity ≥ 95 %.
13 13
5.16 C -Aflatoxin B1 ( C -AfB1).
17 17
13 13
5.17 C -Deoxynivalenol ( C -DON).
15 15
13 13
5.18 C -Fumonisin B1 ( C -FB1).
34 34
13 13
5.19 C -Fumonisin B2 ( C -FB2).
34 34
13 13
5.20 C -HT-2 toxin ( C -HT2).
22 22
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13 13
5.21 C -T-2 toxin ( C -T2).
24 24
13 13
5.22 C -Zearalenone ( C -ZEN).
18 18
13 13
5.23 C -Ochratoxin A ( C -OTA).
20 20
5.24 Stock standard solution
A mixture containing Deoxynivalenol (5.9), Aflatoxin B1 (5.8), Fumonisin B1 (5.10) and Fumonisin B2
(5.11), T-2 toxin (5.13) and HT-2 toxin (5.12), Zearalenone (5.14) and Ochratoxin A (5.15) in
acetonitrile/water (80/20) with 0,1 % FA at relevant concentrations. When preparing this solution the
certified purities of the mycotoxin reference materials need to be properly accounted for.
NOTE 1 6,0 µg/ml DON, 0,040 µg/ml AfB1, 13,5 µg/ml FB1, 4,5 µg/ml FB2, 0,48 µg/ml T-2, 0,6 µg/ml HT-2,
0,9 µg/ml ZEN and 0,45 µg/ml OTA in ACN/H O/FA (80/20/0,1, v/v) has shown to work well. This solution is stable
2
up to half a year in the dark at −20 °C or at least three months in the dark at 2 °C to 8 °C.
Compare a new stock solution against the old one by adding 25 µl of each into separate deactivated vials
(6.9) and proceeding as described in 7.3.
NOTE 2 If 7.4 is executed at least 4 ml of the stock solution is required.
5.25 Working standard solution
Dilute stock standard solution (5.24) in amber glass acid-washed volumetric flask with
acetonitrile/water (80/20) with 0,1 % FA such that the resulting concentration in the working solution
is applicable to the calibration range of the different compounds. Prepare enough volume for only one
full calibration and use freshly prepared. This solution is stable up to one week when stored in dark at
−20 °C.
NOTE Adding 200 µl of the multimycotoxin stock standard solution as given in 5.24, NOTE 1, to a 3 ml
volumetric flask and making up to the mark with ACN/H O/FA (80/20/0,1, v/v) will result in a solution containing
2
0,4 µg/ml DON, 0,002 7 µg/ml AfB1, 0,9 µg/ml FB1, 0,3 µg/ml FB2, 0,032 µg/ml T-2, 0,04 µg/ml HT-2, 0,06 µg/ml
ZEN and 0,03 µg/ml OTA in ACN/H2O/FA (80/20/0,1, v/v). Alternatively, 333,3 µl of the multimycotoxin stock
standard solution can be diluted in a 5 ml volumetric flask. Pipetting 200 µl of the multimycotoxin stock standard
solution and adding 2 800 µl of the above mentioned solvent also gives comparable results.
13 13
5.26 Multi Internal standard (IStd) solution, 3,6 µg/ml C -DON (5.17), 0,02 µg/ml C -AfB1
15 17
13 13 13
(5.16), 3,75 µg/ml C -FB1 (5.18), 1,25 µg/ml C -FB2 (5.19), 0,5 µg/ml C -T-2 toxin (5.21),
34 34 24
13 13 13
0,5 µg/ml C22-HT-2 toxin (5.20), 1,0 µg/ml C18-ZEN (5.22) and 0,4 µg/ml C20-OTA (5.23) in
acetonitrile/water (80/20) with 0,1 % FA has shown to work well. This solution is stable up to half a year
in the dark at −20 °C or at least three months at 2 °C to 8 °C.
5.27 Calibrations
Add different volumes of the working standard solution (5.25) to five deactivated glass vials (6.9) such
that five equidistant calibration levels across the calibration range are obtained. Continue the preparation
procedure as described in 7.3.
The solutions should be protected from light and can be stored in the freezer at ca. −20 °C. They are stable
up to one week.
NOTE Table 1 below shows example calibration levels using the solutions described in the notes above.
Once it has been shown that there is linearity, the number of levels may be adjusted to local needs and
requirements.
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The exact concentrations of the stock standard solution (5.24), the working standard solution (5.25) and
the calibration standard solutions shall be derived from the concentration of the reference standard
materials (5.8–5.15), taking into account the purity and the uncertainty declared on the certificate, and
the volumes used.
Table 1 — Calibration solutions
Calibration Volume of Total mass of analyte per vial [ng]
sample working
standard
DON AfB1 FB1 FB2 T-2 HT-2 ZEN OTA
solution
[µl]
Cal 1 20 8,0 0,053 18,0 6,0 0,64 0,8 1,2 0,6
Cal 2 230 92,0 0,613 207,0 69,0 7,36 9,2 13,8 6,9
Cal 3 440 176,0 1,173 396,0 132,0 14,08 17,6 26,4 13,2
Cal 4 650 260,0 1,733 585,0 195,0 20,8 26,0 39,0 19,5
Cal 5 860 344,0 2,293 774,0 258,0 27,52 34,4 51,6 25,8
5.28 Quality control material
An appropriate material with natural contamination or fortification of the tested mycotoxins that is
sufficiently stable.
6 Apparatus
6.1 Mill, single mill or multiple mills capable of comminuting test materials to particle sizes
of < 500 µm.
6.2 Mixer, capable of sufficiently homogenizing the comminuted test materials.
NOTE A tumble mixer that uses a folding action either through moving paddles or fins, or an end-over-end
movement has shown to work well.
6.3 Conical polypropylene (PP) screw-cap centrifuge tubes 50 ml with caps.
6.4 Balance, with a mass resolution of 0,001 g or better.
6.5 Adjustable vertical or horizontal shaker.
6.6 Centrifuge, capable of generating a relative centrifugal force (rcf) of 2 300 g.
6.7 Pipettors, adjustable 10 µl to 100 µl and adjustable 100 µl to 1000 µl, properly calibrated, with
appropriate tips.
6.8 Volumetric flasks, amber, deactivated (acid-washed or silanized) glass, or PP, 3 ml (optional), 5 ml
and 10 ml.
6.9 Deactivated glass vials (acid-washed or silanized) or PP vials, of appropriate size for the Auto
Liquid Sampler (ALS) in use (usually approximately 1,5 ml capacity).
In order to acid-wash glassware fill it with 20 % acetic acid (5.7) and leave overnight under a fume hood
(ca. 16 h to 24 h). Remove acid and rinse glassware with tap water, then deionized water and finally with
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ultrapure water to reach a pH of 5–7. Usually performing each rinse step three times enough. Dry in an
oven at 60 °C to 70 °C.
6.10 Sample concentrator, capable of maintaining a stable temperature in the range of 30 to 60 °C with
a constant flow of dry nitrogen.
6.11 Syringe filter, small internal volume, Nylon, pore size: 0,2 µm.
6.12 1 ml syringe with needle.
6.13 Vortex mixer, optional.
6.14 LC-MS/MS:
6.14.1 Solvent delivery system, capable of delivering a binary gradient at flow rates appropriate for
the analytical column in use with sufficient accuracy.
6.14.2 Degasser, optional, for degassing LC mobile phases.
6.14.3 Auto Liquid Sampler, capable of injecting an appropriate volume of injection solution with
sufficient accuracy, cross-contamination below 0,1 %.
6.14.4 Column oven, optional, capable to operate in temperatures at least up to 40 °C ± 1 °C.
6.14.5 Analytical column, capable of separating analytes with the following performance:
— Peak asymmetry factor at 10 % height: 0,9 < A < 1,4;
s
— Minimum retention factor for the first eluting analyte: k' ≥ 1;
— Minimum plate number for any of the eight analytes: N ≥ 1 200, where
2
t
R
N= 5, 54 ;
w
1
2
— Minimum resolution between two adjacent peaks: R ≥ 1,7.
s
6.14.6 Pre-column, optional, with the same stationary phase material as the analytical column, and
corresponding dimensions.
6.14.7 Mass spectrometer, capable of performing selected reaction monitoring with a sufficiently wide
dynamic range. Any ionization source giving sufficient yield may be employed.
7 Procedure
7.1 Sample preparation
It is important that the laboratory receives a laboratory sample which is truly representative and has not
been damaged or altered during transport or storage. Laboratory samples shall be taken and prepared in
accordance with EN ISO 6498 unless European legislation [3] applies. The laboratory sample shall be
finely ground and thoroughly mixed using a mill (6.1) and a mixer (6.2) or another process for which
adequate homogenization has been demonstrated before a test portion is removed for analysis.
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The recommended way is to comminute the laboratory sample in several steps. Beginning with the
totality of the laboratory sample each step consists of taking a representative aliquot of the previous step
after sufficient homogenization. This aliquot is then comminuted to the next smaller particle size until
a subsample of ca. 50 g of the final particle size is obtained. It is of utmost importance that the test portion
is taken from a subsample which is sufficiently homogenous with a particle size of ≤ 500 µm. Care shall
be taken not to overheat the sample during this process.
In all instances everything should be at room temperature before any kind of manipulation takes place.
7.2 Extraction
7.2.1 Introduction
Some of the steps described below are more critical for the accuracy of the results than others. These
steps are marked as such and should be carried out with the necessary attention. A scale-up of the test
portion size is deemed to be acceptable if such a need is assumed. In that case the amounts of added
water, acetonitrile and formic acid need to be increased at the same rate, e.g. scale-up by a factor of 2:
10 g test portion, 50 ml extraction solvent (5.6). Regardless of the sample size the sample preparation
shall meet the requirements in EN ISO 6498.
7.2.2 Dry samples
Weigh 4,8 g to 5,2 g of the homogenous test portion into a polypropylene screw-cap tube (6.3), round and
record the weight to the second decimal. The accuracy of this weight is critical for the accuracy of the final
result.
Add 25,0 ml of acetonitrile/water/formic acid solution (79/20/1, v/v) (5.6), the accuracy of this volume
is critical for the accuracy of the final result. Vortex or mix by hand thoroughly until the test portion is
completely suspended and shake for 30 min in a shaker (6.5), that ensures thorough mixing of the sample.
Centrifuge sample at 3 000 g for at least 3 min (or 5 min at 2 300 g) to aid settlement of particulate matter.
If wanted for possible repeats: Transfer the extract into a clean polypropylene vial for storage of up to
7 days at 2 °C to 8 °C in the dark.
Take 500 µl aliquot of crude extract, transfer it to a deactivated glass vial (6.9) for further processing. The
accuracy of this volume is critical for the accuracy of the final result.
7.2.3 Slurry
Adjust the amount of slurry to 5 g of feed sample, e.g. for 1:1 slurry weigh in 10 g, for 1:2 slurry weigh in
15 g (equivalent to 5 g feed sample) of homogenized test portion into a polypropylene screw-cap tube
(6.3) or another appropriate container. The mass shall be recorded to 2 decimal places. If more than 5 ml
of water is in the slurry portion, remember to scale-up the amount of solvents to reach the final ratio of
extraction solvent composition acetonitrile/water/formic acid of 79/20/1 (v/v).
Add to the slurry an appropriate amount of formic acid (5.4), vortex or mix by hand for 5 s and then add
appropriate amount of acetonitrile (5.5), vortex or mix by hand thoroughly for 10 s and then shake for
30 min in a shaker (6.5).
From this point follow the procedure as for dry samples.
7.3 Test solution
Add 25 µl of labelled IStd solution (5.26) to the aliquot of extract and/or the calibration solutions and
evaporate to dryness at 50 °C under a gentle stream of nitrogen.
NOTE 1 To reduce the possibility of cross-contamination, labelled IStd solution can be added to the vial before
the aliquot of extract/calibration solution is added.
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NOTE 2 Leaving the solutions under a gentle stream of nitrogen for up to 20 min longer than just necessary for
complete evaporation did not show to have any negative effect on the final results.
Allow the vial to cool down to room temperature and add 250 µl of LC mobile phase B (e.g. see Annex B)
to the dry residue for reconstitution.
Cover the vial (with a cap) and vortex thoroughly for at least 15 s, making sure that also the upper part
of the vial is thoroughly rinsed by the solvent.
Add 250 µl of LC mobile phase A (e.g. see Annex B) to the reconstituted extract.
Vortex thoroughly for at least 5 s.
Transfer the test solution into an ALS vial (6.9) for analysis; if the solution is turbid it shall be filtered
through a syringe filter (6.11).
7.4 Spiking procedure
If recovery needs to be determined execute the following in duplicate.
Only for recovery determination, the sample size is reduced to 2 g in order to reduce the amount of
standard solution used.
To three 2 g portions of a material ideally free of all analytes (in case no blank sample can be identified,
use a sample with as little contamination as possible) add three different volumes of the Multi-mycotoxin
stock standard solution (5.24), such that three contamination levels across the calibration range are
obtained. Distribute the solutions evenly over the materials, mix to further distribute the spike and leave
containers open in a dark fume hood overnight (ca. 17 h to 24 h) with air flow on at room temperature to
allow for solvent evaporation. In case of centrifuge tubes, these can be placed either vertically (e.g. in a
rack) or on a clean surface in horizontal position. Both have been tested for sufficient evaporation of the
spiking solution solvent. Proceed to 7.2, but remember to use 10 ml of extraction solvent (5.6) to keep
the sample-to-solvent ratio constant. Calculate the recovered concentrations from the formula of
calibration (1).
NOTE Addition of 180 µl, 590 µl and 1000 µl of the Multitoxin stock standard solution (5.24) with the
concentrations described in 5.24, Note 1 gives satisfactory results. Volumes of 250 µl and 1000 µl were used in
method validation study and have shown to give satisfactory results.
8 Measurements
8.1 General
The LC-MS system shall meet the requirements laid out in 6.14 and subclauses.
8.2 LC conditions
A combination of analytical column, mobile phase, gradient settings and injection volume should be such
that it allows obtaining acceptable separation, as mentioned in 6.14.5 and reliable results at the required
levels (for examples see Annex B).
8.3 MS conditions
Choose an ion source with sufficient ionization yield for the eight analytes and ion source settings such
that a stable spray is achieved.
Choose for each analyte an appropriate precursor ion (adducts of the molecule with a proton, sodium,
ammonia, etc. in positive mode, or deprotonation, etc. in negative mode). If more than one precursor ion
per analyte is detectable then choosing the strongest is a good starting point. Be aware that the choice of
precursor ion will affect the repeatability and, by that, Limits of Detection and Quantification (LOD and
LOQ).
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For selected reaction monitoring (SRM) select two product ions in the MS/MS spectrum for each chosen
precursor ion of each analyte. Set up SRM transitions with these precursor/ product ion combinations
(for SRM example see Annex A).
The chosen MS settings shall be such that for a compound feed with a contamination of ca. 100 µg/kg
DON, 2 µg/kg AfB1, 375 µg/kg FB1, 125 µg/kg FB2, 10 µg/kg T-2, 10 µg/kg HT-2, 20 µg/kg ZEN and
10 µg/kg OTA prepared according to Clause 7, peak-to-peak signal-to-noi
...
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