Animal feeding stuffs: Methods of sampling and analysis - Determination of dioxins and dioxin-like PCBs and of indicator PCBs by GC/HRMS

This document is applicable to the determination of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), (together termed ‘dioxins’ (PCDD/Fs)) and dioxin-like PCBs and non-dioxin-like PCBs (dl-PCBs and ndl-PCBs) in animal feeding stuffs. Collaborative studies have been carried out. The method is suitable for the determination of dioxins, dl-PCBs and ndl-PCBs at the appropriate MRL in compound feed and ingredients e.g. oil, mineral clay. The method is applicable to samples containing trace level amounts of one or more dioxins, dioxin-like PCBs and non-dioxin-likePCBs. The limit of quantification (LOQ) is
-   0,05 pg/g (OCDD/F = 0,1 pg/g) for the relevant individual congeners of dioxins/furans,
-   0,05 pg/g for non-ortho PCBs,
-   10 pg/g for mono-ortho PCBs, and
-   100 pg/g for non-dioxin-like-PCBs.
For determination of dioxins and dioxin-like PCBs, the procedure can be used as confirmatory method as defined by Commission Regulation (EC) No 152/2009 for dioxins and dl-PCB in feed [1]. Confirmatory methods as described in this standard are high-resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS) methods. If only the analysis of non-dioxin-like PCBs is required, a GC-LRMS method can be used (e.g. EN 15741 [2]) provided that appropriate analytical performance criteria are met in the relevant range for the matrix of interest.
This document is split into four modules. Each module describes a part of the whole procedure (see Figure 1 and Figure 2) to be followed:
a)   Module A:   Description of standards which might be used;
b)   Module B:   Description of extraction procedures;
c)   Module C:   Description of clean-up procedures;
d)   Module D:    GC/HRMS determination.
Each module describes a part of the whole method as well as, when applicable, alternatives which should be equivalent. Each module has to be regarded as an example. Combining modules and/or alternatives gives a highly flexible, "performance based" procedure. It is permitted to modify the method if all performance criteria laid down in Commission Regulation (EC) No 152/2009 [1] are met.
Any deviation of the described method, combination of modules needs to be recorded as part of the QA/QC procedures of accredited laboratories and should be available on request.
Figure 1 - Flow scheme for the determination of dioxins, dl-PCBs and non-dioxin-like-PCBs in feed
Figure 2 - Flow scheme for the determination of dioxins, dl-PCBs and non-dioxin-like-PCBs in oil and fat

Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von Dioxinen und dioxin-ähnlichen PCB mittels GC/HRMS und von Indikator-PCB mittels GC/HRMS

Dieses Dokument ist anwendbar für die Bestimmung von polychlorierten Dibenzo p dioxinen (PCDD), polychlorierten Dibenzofuranen (PCDF), (zusammen als „Dioxine“ bezeichnet [PCDD/F]) und dioxin-ähnlichen PCB und nicht dioxin ähnlichen PCB (dl PCB und ndl PCB) in Futtermitteln. Ringversuche sind durchgeführt worden. Das Verfahren ist für die Bestimmung von Dioxinen, dl PCB und ndl PCB bei der entsprechenden MRL in Mischfutter und Inhaltsstoffen, z. B. Öl, Tonmineral, geeignet. Das Verfahren ist auf Proben anwendbar, die Spurenkonzentrationen von einem Dioxin oder mehreren Dioxinen, dioxin-ähnlichen PCB und nicht dioxin ähnlichen PCB enthalten. Die Bestimmungsgrenze (LOQ) beträgt
- 0,05 pg/g (OCDD/F = 0,1 pg/g) für die relevanten einzelnen Kongeneren der Dioxine/Furane,
- 0,05 pg/g für non-ortho PCB,
- 10 pg/g für mono-ortho PCB und
- 100 pg/g für nicht dioxin ähnliche PCB.
Für die Bestimmung von Dioxinen und dioxin-ähnlichen PCB kann das Verfahren, wie in der Verordnung (EG) Nr. 152/2009 der Kommission [1] für Dioxine und dl PCB in Futtermitteln festgelegt, als Bestätigungsverfahren angewendet werden. Bestätigungsverfahren, wie in dieser Norm beschrieben, sind Verfahren mit hochauflösender Gaschromatographie/hochauflösender Massenspektrometrie (HRGC/HRMS). Falls nur die Analyse der nicht dioxin ähnlichen PCB erforderlich ist, kann ein GC LRMS Verfahren (z. B. EN 15741 [2]) angewendet werden, vorausgesetzt, dass für die zu untersuchende Matrix im relevanten Bereich die entsprechenden analytischen Leistungskriterien erfüllt werden.
Dieses Dokument ist in vier Module aufgeteilt. Jedes Modul beschreibt einen Teil des gesamten Verfahrens (siehe Bild 1 und Bild 2) wie folgt:
a) Modul A:   Beschreibung der Normen, die angewendet werden können;
b) Modul B:   Beschreibung der Extraktionsverfahren;
c) Modul C:   Beschreibung der Reinigungsverfahren;
d) Modul D:   GC/HRMS Bestimmung.
Jedes Modul beschreibt einen Teil des gesamten Verfahrens sowie, falls anwendbar, Alternativen, die gleich¬wertig sein sollten. Jedes Modul muss als ein Beispiel betrachtet werden. Das Kombinieren von Modulen und/oder Alternativen ermöglicht ein hochflexibles, „leistungsbezogenes“ Verfahren. Es ist zulässig, das Verfahren zu verändern, wenn sämtliche Leistungskriterien entsprechend der Verordnung (EG) Nr. 152/2009 der Kommission [1] erfüllt werden.
Jede Abweichung vom beschriebenen Verfahren und die Kombination der Module müssen als Teil der QA/QC Verfahren von anerkannten Laboratorien aufgezeichnet werden und sollten auf Anfrage verfügbar sein.

Aliments des animaux : Méthodes d’échantillonnage et d’analyse - Dosage des dioxines, des PCB de type dioxine et des PCB indicateurs par GC/HRMS

Le présent document concerne le dosage des dibenzo-p-dioxines polychlorés (PCDD), des dibenzofuranes polychlorés (PCDF) (conjointement appelés « dioxines » (PCDD/F)) ainsi que des PCB de type dioxine et des PCB autres que ceux de type dioxine (dl-PCB et ndl-PCB) dans les aliments des animaux. Des études comparatives ont été menées. La méthode convient au dosage des dioxines, des dl PCB et des ndl-PCB à la LMR appropriée dans les aliments et les ingrédients composés tels que l’huile et l’argile minérale. La méthode est applicable aux échantillons contenant, à l’état de traces, un(e) ou plusieurs dioxines, PCB de type dioxine et PCB autres que ceux de type dioxine. La limite de quantification (LQ) est de :
-   0,05 pg/g (OCDD/F = 0,1 pg/g) pour les congénères individuels concernés des dioxines/furanes ;
-   0,05 pg/g pour les PCB non-ortho ;
-   10 pg/g pour les PCB mono-ortho ; et
-   100 pg/g pour les PCB autres que ceux de type dioxine.
Pour le dosage des dioxines et des PCB de type dioxine, le mode opératoire peut être utilisé comme méthode de confirmation, conformément aux définitions du Règlement (CE) no 152/2009 de la Commission relatif aux dioxines et aux dl-PCB dans les aliments [1]. Les méthodes de confirmation décrites dans la présente norme sont les méthodes de chromatographie en phase gazeuse haute résolution/spectrométrie de masse haute résolution (HRGC/HRMS). Si seule l’analyse des PCB autres que ceux de type dioxine est requise, une méthode GC-LRMS peut être utilisée (par exemple, EN 15741 [2]), à condition que les critères de performance analytique appropriés soient atteints dans la gamme de concentration correspondant aux aliments concernés.
Le présent document se divise en quatre modules. Chaque module décrit une partie de l’ensemble du mode opératoire (voir la Figure 1 et la Figure 2) à respecter :
a)   Module A :   Description des normes susceptibles d’être utilisées ;
b)   Module B :   Description des modes opératoires d’extraction ;
c)   Module C :   Description des modes opératoires de purification ;
d)   Module D :    Dosage par GC/HRMS.
Chaque module décrit une partie de l’ensemble de la méthode ainsi que, le cas échéant, des alternatives équivalentes. Chaque module doit être considéré comme un exemple. La combinaison des modules et/ou alternatives donne un mode opératoire très souple qui est « basé sur les performances ». Il est possible de modifier la méthode si tous les critères de performance énoncés dans le Règlement (CE) no 152/2009 de la Commission [1] sont remplis.
En cas d’écart par rapport à la méthode décrite, il est nécessaire d’enregistrer la combinaison de modules dans le cadre des procédures AQ/CQ des laboratoires accrédités et il convient que cette combinaison soit disponible sur demande.
[sans figure]
Figure 1 - Logigramme relatif au dosage des dioxines, des dl-PCB et des PCB autres que ceux de type dioxine dans les aliments
[sans figure]
Figure 2 - Logigramme relatif au dosage des dioxines, des dl-PCB et des PCB autres que ceux de type dioxine dans l’huile/la graisse

Krma: metode vzorčenja in analize - Določevanje dioksinov in dioksinu podobnih PCB z GC/HRMS in indikatorjev PCB z GC/HRMS

Ta dokument se uporablja za določevanje polikloriranih dibenzo-p-dioksinov (PCDD), polikloriranih dibenzofuranov (PCDF) (s skupnim imenom »dioksini« (PCDD/F)) ter dioksinom podobnih polikloriranih bifenilov in dioksinom nepodobnih polikloriranih bifenilov (dl-PCB in ndl-PCB) v krmi. Izvedene so bile medlaboratorijske študije. Metoda je primerna za določevanje dioksinov, dioksinom podobnih polikloriranih bifenilov in dioksinom nepodobnih polikloriranih bifenilov v krmi in sestavinah, na primer olju ter mineralni glini, pri ustrezni mejni vrednosti ostankov. Metoda se uporablja za vzorce, ki vsebujejo enega ali več dioksinov, dioksinom podobne poliklorirane bifenile in dioksinom nepodobne poliklorirane bifenile v sledovih. Mejne vrednosti kvantifikacije (LOQ) so:
– 0,05 pg/g (OCDD/F = 0,1 pg/g) za ustrezne posamezne analoge dioksinov/furanov,
– 0,05 pg/g za neorto poliklorirane bifenile,
– 10 pg/g za za monoorto poliklorirane bifenile in
– 100 pg/g za dioksinom nepodobne poliklorirane bifenile.
Pri določevanju dioksinov in dioksinom podobnih polikloriranih bifenilov je postopke mogoče uporabiti kot potrditveno metodo v skladu z Uredbo Komisije (ES) št. 152/2009 za dioksine in dioksinom podobne poliklorirane bifenile v krmi [1]. Potrditvene metode, kot je opisano v tem standardu, so metode visokoločljivostne plinske kromatografije/visokoločljivostne masne spektrometrije (HRGC/HRMS). Če je potrebna samo analiza dioksinom nepodobnih polikloriranih bifenilov, je mogoče uporabiti metodo plinske kromatografije/nizkoločljivostne masne spektrometrije (GC-LRMS) (npr. EN 15741 [2]) pod pogojem, da so za zadevno matriko v ustreznem območju izpolnjena ustrezna merila analitskega izvajanja.
Ta dokument je razdeljen v štiri module. Vsak modul opisuje del celotnega postopka (glej sliki 1 in 2), ki ga je treba upoštevati:
a) Modul A: opis standardov, ki se lahko uporabljajo;
b) Modul B: opis postopkov za ekstrakcijo;
c) Modul C: opis postopkov za očiščenje;
d) Modul D: določitev s plinsko kromatografijo/visokoločljivostno masno spektrometrijo.
Vsak modul opisuje del celotne metode in, kadar je to primerno, alternative, ki naj bodo enakovredne. Posamezni modul je treba obravnavati kot primer. S kombinacijo modulov in/ali alternativ se oblikuje izredno prilagodljiv postopek, ki temelji na učinkovitosti. Metodo je mogoče spremeniti, če so izpolnjena vsa merila učinkovitosti, ki so navedena v Uredbi Komisije (ES) št. 152/2009 [1]. V primeru odstopanj od opisane metode je treba kombinacijo modulov zabeležiti kot del postopkov za zagotavljanje in nadzor kakovosti (QA/QC) akreditiranih laboratorijev, ki naj bo na voljo za zahtevo.
Slika 1 – Shema poteka za določevanje dioksinov, dioksinom podobnih polikloriranih bifenilov in dioksinom nepodobnih polikloriranih bifenilov v krmi
Slika 2 – Shema poteka za določevanje dioksinov, dioksinom podobnih polikloriranih bifenilov in dioksinom nepodobnih polikloriranih bifenilov v olju in maščobi

General Information

Status
Published
Publication Date
07-Jan-2020
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
08-Jan-2020
Completion Date
08-Jan-2020

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SLOVENSKI STANDARD
SIST EN 16215:2020
01-marec-2020
Nadomešča:
SIST EN 16215:2012
Krma: metode vzorčenja in analize - Določevanje dioksinov in dioksinu podobnih
PCB z GC/HRMS in indikatorjev PCB z GC/HRMS

Animal feeding stuffs: Methods of sampling and analysis - Determination of dioxins and

dioxin-like PCBs by GC/HRMS and of indicator PCBs by GC/HRMS
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von Dioxinen
und dioxin-ähnlichen PCBs mittels GC/HRMS und von Indikator-PCBs mittels GC/HRMS

Aliments des animaux : Méthodes d’échantillonnage et d’analyse - Dosage des dioxines,

des PCB de type dioxine et des PCB indicateurs par GC/HRMS
Ta slovenski standard je istoveten z: EN 16215:2020
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN 16215:2020 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 16215:2020
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SIST EN 16215:2020
EN 16215
EUROPEAN STANDARD
NORME EUROPÉENNE
January 2020
EUROPÄISCHE NORM
ICS 65.120 Supersedes EN 16215:2012
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of dioxins and dioxin-like PCBs by
GC/HRMS and of indicator PCBs by GC/HRMS

Aliments des animaux : Méthodes d'échantillonnage et Futtermittel - Probenahme- und

d'analyse - Dosage des dioxines, des PCB de type Untersuchungsverfahren - Bestimmung von Dioxinen

dioxine et des PCB indicateurs par GC/HRMS und dioxin-ähnlichen PCBs mittels GC/HRMS und von

Indikator-PCBs mittels GC/HRMS
This European Standard was approved by CEN on 11 November 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

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 16215:2020 E

worldwide for CEN national Members.
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SIST EN 16215:2020
EN 16215:2020 (E)
Contents Page

European foreword ....................................................................................................................................................... 4

Introduction .................................................................................................................................................................... 5

1 Scope .................................................................................................................................................................... 6

2 Normative references .................................................................................................................................... 8

3 Terms and definitions ................................................................................................................................... 8

4 Principle ............................................................................................................................................................. 9

5 Reagents .......................................................................................................................................................... 10

5.1 General ............................................................................................................................................................. 10

5.2 Dioxins, furans, non-ortho PCBs, mono-ortho PCBs and non-dioxin-like PCBs and

their labelled analogues ............................................................................................................................ 10

6 Apparatus ........................................................................................................................................................ 10

7 Sampling .......................................................................................................................................................... 10

8 Preparation of test sample ....................................................................................................................... 11

9 Procedure........................................................................................................................................................ 11

9.1 General ............................................................................................................................................................. 11

9.2 Animal feed stuff sample and oil/fat sample ..................................................................................... 11

10 Extraction ........................................................................................................................................................ 11

10.1 General ............................................................................................................................................................. 11

10.2 Module BI Extraction using automated Pressurized Fluid Extraction (PFE) system .......... 12

10.3 Module BII: Manual extraction procedure .......................................................................................... 13

11 Clean-up ........................................................................................................................................................... 14

11.1 General ............................................................................................................................................................. 14

11.2 Module CI: Automated clean-up .............................................................................................................. 16

11.3 Module CII: Manual sample clean-up, removal of fat and group separation .......................... 19

11.4 Additional clean-up-steps for fraction A and fraction B of module CII ..................................... 22

11.5 Module CIII: Removal of matrix, manual sample clean-up and group separation ............... 24

12 Module D: Gas chromatograph-high resolution mass spectrometer (GC-HRMS) ................. 29

12.1 General ............................................................................................................................................................. 29

12.2 Reagents and materials .............................................................................................................................. 29

12.3 Procedure........................................................................................................................................................ 29

13 Calculation and expression of results ................................................................................................... 31

13.1 General ............................................................................................................................................................. 31

13.2 Calibration by Isotope Dilution ............................................................................................................... 31

13.3 Linearity .......................................................................................................................................................... 31

13.4 Calibration criteria ...................................................................................................................................... 31

13.5 Identification and confirmation.............................................................................................................. 31

13.6 Calculation ...................................................................................................................................................... 32

13.7 Toxic Equivalent (TEQ) values ................................................................................................................ 33

13.8 Recovery .......................................................................................................................................................... 36

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EN 16215:2020 (E)

14 Precision .......................................................................................................................................................... 36

15 Test report ...................................................................................................................................................... 38

Annex A (informative) Description of PTV injection system ..................................................................... 39

A.1 Large volume injection (100 µl) – GC-HRMS Calibration mixtures dioxins and non-

ortho PCBs ....................................................................................................................................................... 39

A.2 Sample preparation procedure ............................................................................................................... 40

A.3 PTV injection conditions using 100 µl injection ................................................................................ 40

Annex B (informative) Description of standards and concentration of the standard

solutions ........................................................................................................................................................... 41

B.1 CAS Registry numbers for dioxins, furans, non-ortho PCBs, mono-ortho PCBs and

non-dioxin-like PCBs and their labelled analogues ......................................................................... 41

B.2 Standard solutions ....................................................................................................................................... 42

B.3 GC-standard solutions ................................................................................................................................. 45

Annex C (informative) Example of automated procedure .......................................................................... 48

Annex D (informative) Mass spectrometer (MS) ............................................................................................ 50

Annex E (informative) Use of additional clean-up after fractionation using a small multi-

layer silica column ....................................................................................................................................... 54

E.1 Preparation of the silica mixtures .......................................................................................................... 54

E.2 Preparation of the small multi-layer column ..................................................................................... 54

Bibliography ................................................................................................................................................................. 55

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SIST EN 16215:2020
EN 16215:2020 (E)
European foreword

This document (EN 16215:2020) 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 July 2020, and conflicting national standards shall be

withdrawn at the latest by July 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 supersedes EN 16215:2012.

In comparison with the previous edition, the following technical modifications have been made:

— incorrect technical details have been corrected,
— references have been updated,
— inconsistencies were removed, and
— editorial adaptations have been made.

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.
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SIST EN 16215:2020
EN 16215:2020 (E)
Introduction

The previous version of this document was developed in response to Directive 2002/32/EC of the

European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed. The

document provides analytical laboratories active in the field of feed analysis with guidance for the

analysis of dioxins and PCBs and meets criteria as set in Commission Regulation (EC) No 152/2009 of 27

January 2009 laying down the methods of sampling and analysis for the official control of feed.

In this updated version, obvious mistakes were corrected.

WARNING — The use of this document can involve hazardous materials, operations and

equipment. This standard does not purport to address all the safety problems associated with its

use. It is the responsibility of the user of this European Standard to establish appropriate safety

and health practices and determine the applicability of regulatory limitations prior to use.

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SIST EN 16215:2020
EN 16215:2020 (E)
1 Scope

This document is applicable to the determination of polychlorinated dibenzo-p-dioxins (PCDDs),

polychlorinated dibenzofurans (PCDFs), (together termed ‘dioxins’ (PCDD/Fs)) and dioxin-like PCBs and

non-dioxin-like PCBs (dl-PCBs and ndl-PCBs) in animal feeding stuffs. Collaborative studies have been

carried out. The method is suitable for the determination of dioxins, dl-PCBs and ndl-PCBs at the

appropriate MRL in compound feed and ingredients e.g. oil, mineral clay. The method is applicable to

samples containing trace level amounts of one or more dioxins, dioxin-like PCBs and non-dioxin-likePCBs.

The limit of quantification (LOQ) is

— 0,05 pg/g (OCDD/F = 0,1 pg/g) for the relevant individual congeners of dioxins/furans,

— 0,05 pg/g for non-ortho PCBs,
— 10 pg/g for mono-ortho PCBs, and
— 100 pg/g for non-dioxin-like-PCBs.

For determination of dioxins and dioxin-like PCBs, the procedure can be used as confirmatory method as

defined by Commission Regulation (EC) No 152/2009 for dioxins and dl-PCB in feed [1]. Confirmatory

methods as described in this standard are high-resolution gas chromatography/high resolution mass

spectrometry (HRGC/HRMS) methods. If only the analysis of non-dioxin-like PCBs is required, a GC-LRMS

method can be used (e.g. EN 15741 [2]) provided that appropriate analytical performance criteria are

met in the relevant range for the matrix of interest.

This document is split into four modules. Each module describes a part of the whole procedure (see

Figure 1 and Figure 2) to be followed:
a) Module A: Description of standards which might be used;
b) Module B: Description of extraction procedures;
c) Module C: Description of clean-up procedures;
d) Module D: GC/HRMS determination.

Each module describes a part of the whole method as well as, when applicable, alternatives which should

be equivalent. Each module has to be regarded as an example. Combining modules and/or alternatives

gives a highly flexible, “performance based” procedure. It is permitted to modify the method if all

performance criteria laid down in Commission Regulation (EC) No 152/2009 [1] are met.

Any deviation of the described method, combination of modules needs to be recorded as part of the

QA/QC procedures of accredited laboratories and should be available on request.
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SIST EN 16215:2020
EN 16215:2020 (E)

Figure 1 — Flow scheme for the determination of dioxins, dl-PCBs and non-dioxin-like-PCBs in

feed
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SIST EN 16215:2020
EN 16215:2020 (E)

Figure 2 — Flow scheme for the determination of dioxins, dl-PCBs and non-dioxin-like-PCBs in

oil and fat
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 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

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 http://www.iso.org/obp
3.1
limit of detection

smallest measured content, from which it is possible to deduce the presence of the analyte with

reasonable statistical certainty

Note 1 to entry: The limit of detection is numerically equal to three times the standard deviation of the mean of

blank determinations (n > 10).
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SIST EN 16215:2020
EN 16215:2020 (E)
3.2
limit of quantification

lowest content of the analyte that can be measured with reasonable statistical certainty

Note 1 to entry: If both accuracy and precision are constant over a concentration range around the limit of

detection, then the limit of quantification is numerically equal to six times the standard deviation of the mean of

blank determinations (n > 10).

Note 2 to entry: For dl-PCBs and PCDD/F: Use correct definition for limit of quantification for each congener as

in Commission Regulation (EC) No 152/2009 [1].

Note 3 to entry: Limit of quantification should be in the range of about one fifth of the level of interest.

3.3
feed additives

substances that comply with the definition of feed additives given in the Commission Regulation (EC) No

1831/2003 [3]
3.4
upper, middle and lower bound concentrations for WHO-PCDD/F-TEQ and WHO-PCB-TEQ

concentrations calculated assuming that all values of the different congeners less than the limit of

quantification are equal to the limit of quantification

Note 1 to entry: For lower bound concentrations LOQ = 0 is used. For middle bound concentrations 1/2 LOQ is

used.
4 Principle

A test portion of animal feeding stuff or ingredient is fortified with C labelled internal standards

(dioxins, furans, dioxin-like PCBs and non-dioxin-like PCBs) and extracted using a manual or an

automated method.

After automated or manual clean-up an aliquot of the extract is concentrated and injected into a GC-HRMS

using a split less injector (an alternative here is PTV injection (Programmed Temperature Vaporizer

injection)), see the NOTE below.
Quantification is based on isotope dilution.
Preconditions of combining modules for extraction and clean-up are:

a) for each extraction module an equal sample intake of 10 g for feed or feed ingredients with a fat

content ≤ 25 % or 2,5 g fat or oil is required;

b) in order to achieve the required LOQ for dioxins a final volume of 10 μl in combination with an

injection volume of 2 μl is required. If a different injection volume is applied, the final volume has to

be adjusted directly proportional.

NOTE In case more sensitivity is necessary or less volume reduction is wanted, injection of a larger volume by

PTV (an example is described in Annex A) or higher sample intake is possible (see also 9.2.2 NOTE).

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5 Reagents
5.1 General

Use only reagents of recognized analytical grade and with purity suitable for dioxin and PCB residue

analysis. Check the purity of the reagents and reference materials (e.g. standard solutions) by performing

a blank test under the same conditions as used in the method. The chromatogram should not show any

interfering impurity at the retention time of compounds of interest.

5.2 Dioxins, furans, non-ortho PCBs, mono-ortho PCBs and non-dioxin-like PCBs and

their labelled analogues
— C-spiking solution for PCDD/F (internal standard);
— C-spiking solution for PCB (internal standard);
— calibration solutions PCDD/F;
— calibration solutions PCB;
— recovery standard PCDD/F;
— recovery standard PCB.

See Annex B for a description of standards and concentrations of the standard solutions.

6 Apparatus

All technical descriptions are examples of possible system setups and parameters and should be scaled

or adopted to the user’s equipment.
6.1 Evaporator, suitable for volumes up to 200 ml and inlet for nitrogen gas.
6.2 Evaporator tubes, end point about 0,5 ml.
6.3 Homogenizer.
6.4 Pasteur pipette, borosilicate glass, approx. length 150 mm.
6.5 Vortex mixer.

6.6 Measuring cylinder, borosilicate glass, 100 ml, 2 ml graduations with a precision of ± 0,5 ml.

6.7 Measuring cylinder, borosilicate glass, glass-stoppered, 25 ml, 1 ml graduation with a precision

of ± 0,5 ml graduation and 50 ml, 2 ml graduation with a precision of ± 0,5 ml graduation.

7 Sampling

The sample should be truly representative and not damaged or changed during transport or storage.

Sampling is not part of the method specified in this document. A recommended sampling method is given

in EN ISO 6497 [4].
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8 Preparation of test sample
Prepare the test sample in accordance with EN ISO 6498.

Dry or low moisture products such as cereals and cereal products, mixed feeds, and hay should be ground

carefully so that it passes completely through a sieve with 1 mm apertures. Mix thoroughly.

High moisture products such as grasses and silages and liquid feed should be (freeze-)dried and after that

ground carefully so that it passes completely through a sieve with 1 mm apertures. Mix thoroughly.

Oil / fat are directly dissolved in n-hexane.
9 Procedure
9.1 General
Analyse the following samples in each series:
— procedure blank (n = 1);

— (certified) reference material at appropriate level or a home-made reference sample;

— all samples (maximum 20).

The procedure blank should be free of contaminants at or above the limits of quantification.

9.2 Animal feed stuff sample and oil/fat sample
9.2.1 10 g animal feed stuff sample (12 % moisture content)

Weigh an appropriate amount, e.g. 10,0 g (±0,10 g) of the prepared test sample into a 100 ml glass vial.

Sample amount is based on 12 % moisture content. If extracting by Pressurized Fluid Extraction (PFE),

add 3 g diatomaceous earth and mix thoroughly. Fortify the sample with 500 µl C-DIOXNOP-2 (Annex B,

B.2.31) and 500 µl C-MOPIP-2 (Annex B, B.2.33) and incubate until solvent has been evaporated and

continue at 10.2 (module BI) or 10.3 (module BII). For samples with more than 25 % fat, the sample

intake should be reduced proportionally.
9.2.2 2,5 g oil/fat sample

Weigh an appropriate amount, e.g. 2,5 g (±0,10 g) of the oil/fat sample into graduated cylinder of 25 ml

13 13

(6.7). Fortify the sample with 500 µl C-DIOXNOP-2 (Annex B, B.2.31) and 500 µl C-MOPIP-2 (Annex B,

B.2.33). Fill the graduated cylinder to 25 ml with n-hexane. Close the graduated cylinder with a glass

stopper and mix thoroughly. Continue sample clean-up procedure at paragraph 11.2 (module CI) or 11.3

(module CII) or 11.5 (module CIII).

NOTE The calculation in 13.6 is based on sample intake of 10 g for feed with fat content of ≤ 25 % and 2,5 g for

fat and oil. Deviations of sample intake can be considered in the formulas in 13.6 (M = sample intake in gram).

10 Extraction
10.1 General

The sample amount used for extraction may vary from 5 g to 50 g depending on the expected level of

contamination. However, the calculation in 13.6 is based on sample intake of 10 g for feed with fat content

of ≤ 25 % and 2,5 g for fat and oil. Deviations of sample intake should be considered in the formula’s in

13.6 (M = sample intake in gram).
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The internal standard consisting of C-labelled congeners listed in Table B.1 shall be added directly onto

the sample before extraction, or onto the oil sample before clean-up.

The extraction procedure is carried out using Pressurized Fluid Extraction (PFE) with consecutively

toluene and a mixture of toluene/ethanol (module BI) or Soxhlet extraction (module BII). Duration of

extraction should be adjusted according to kind and amount of sample used. The minimum requirement

for Soxhlet extraction is 50 extraction cycles.

Other extraction techniques like microwave assisted extraction can also be used but shall be of proven

equal performance.

10.2 Module BI Extraction using automated Pressurized Fluid Extraction (PFE) system

10.2.1 Reagents and materials
10.2.1.1 Diatomaceous earth.
10.2.1.2 n-Hexane, for dioxin and PCB analysis.
10.2.1.3 Toluene, for dioxin and PCB analysis.
10.2.1.4 Ethanol, for dioxin and PCB analysis.
10.2.1.5 Toluene/ethanol, in volume portions of 9/1.

Mix 900 ml toluene (10.2.1.3) with 100 ml ethanol (10.2.1.4) thoroughly. Store at room temperature in a

tightly closed bottle. The solution is tenable under these conditions for at least 1 year.

10.2.1.6 Anhydrous sodium sulphate, heated at 160 °C for at least 24 h.
10.2.1.7 Nitrogen.
10.2.1.8 Silanised glass wool.
10.2.1.9 Apparatus.
10.2.1.10 Pressurized Fluid Extraction apparatus.
The apparatus shall be able to extract the samples at 100 °C and 10 MPa.
10.2.1.11 Pressurized Fluid Extraction cell, e.g. 30 to 40 ml.

10.2.1.12 Measuring cylinder, borosilicate glass, 25 ml, 1 ml graduation with a precision of ± 0,5 ml.

10.2.1.13 Funnel.

10.2.1.14 Evaporator, suitable for volumes up to 200 ml and inlet for nitrogen gas.

10.2.1.15 Evaporator tubes, 0,5 ml end point.
10.2.2 Procedure

Put the sample (9.2.1) in a Pressurized Fluid Extraction cell (10.2.1.11) and fill with diatomaceous earth

(10.2.1.1) and place the extraction cell into the Pressurized Fluid Extraction apparatus (10.2.1.10). For

the extraction the following parameters could be used:
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— temperature 100 °C;
— pressure 10 MPa;
— preheat 0 min;
— heat 5 min;
— static 15 min;

— flush 40 vol. % of extraction cell, e.g. for a 33 ml extraction cell = 13,2 ml;

— purge 300 s;
— cycles 3;
— solvent cycle 1 toluene (10.2.1.3);
— solvent cycles 2 and 3 toluene/ethanol in volume portions of 9/1 (10.2.1.4).

Combine solvent obtained with each cycle and filter over a funnel (10.2.1.13) equipped with a glass wool

plug (10.2.1.8) and 5 g pre-dried sodium sulphate (10.2.1.6). Evaporate the filtrate using an evaporator

(10.2.1.14) until an end volume of 0,5 ml. Take the sample extract from the evaporator tube and place in

a glass-stoppered graduated 25 ml cylinder (6.7) and wash the evaporator tube 5 times with 4 ml n-

hexane each time (i.e. 20 ml total) (10.2.1.2). The n-hexane is added to graduated cylinder containing the

sample extract. Bring the volume to 25 ml with n-hexane (10.2.1.2), close the graduated cylinder with the

glass stopper and mix thoroughly. Continue sample clean-up procedure using the automated procedure

(module CI, 11.2) or at paragraph using the manual method (module CII, 11.3 or module CIII, 11.5).

NOTE Comparable techniques in combination with appropriate parameters can be used provided that

Commission Regulation (EC) No 152/2009 [1] is obeyed.
10.3 Module BII: Manual extraction procedure
10.3.1 Reagents and materials
10.3.1.1 DCM, dichloromethane, for dioxin and PCB analysis.
10.3.1.2 Toluene, for dioxin and PCB analysis.
10.3.1.3 Ethanol, for dioxin and PCB analysis.
10.3.1.4 n-Hexane, for dioxin and PCB analysis.
10.3.1.5 Toluene/ethanol, in volume portions of 9/1.

Mix 900 ml toluene (10.2.1.2) with 100 ml ethanol (10.2.1.3) thoroughly. Store at room temperature in a

tightly closed bottle. The solution is tenable under these conditions for at least 1 year.

10.3.1.6 Anhydrous sodium sulphate, heated at 160 °C for at least 24 h.

10.3.1.7 Measuring cylinder, borosilicate glass, 25 ml, 1 ml graduation with a precision of ± 0,5 ml.

10.3.1.8 Sila
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