kSIST FprEN 17683:2022

Animal feeding stuffs - Methods of sampling and analysis - Determination of pyrrolizidine alkaloids in animal feeding stuff by LC-MS/MS

This document describes a method for the quantitative determination of pyrrolizidine alkaloids (PA) in complete and supplementary feed and in forages by liquid chromatography tandem mass spectrometry (LC-MS/MS) after solid phase extraction (SPE) clean-up.
The method has been successfully validated in a collaborative trial for the matrices complete feed for horses, supplementary feed for horses, supplementary feed for rodents, hay, alfalfa and grass silage. Validation was carried out for the PA and concentrations ranges listed in Table 1. It was demonstrated that the PA isomeric pairs senecivernine and senecionine as well as senecivernine-N-oxide and senecionine-N-oxide cannot be determined individually due to insufficient chromatographic separation. However, the sums of the individual PA of the isomeric pairs were quantified with sufficient reproducibility. Co-elution of other PA-isomers not included in the scope of the method shall be taken into account. A list of potentially co-eluting isomers is presented in Annex E.
Although the calibration range of the method protocol is specified from 10 μg/kg to 300 μg/kg, the results of the collaborative study showed, that the dilution of sample extracts with blank sample extracts enables for the quantitation of concentrations exceeding the calibration range. Satisfactory reproducibility was achieved when quantifying up to 1428 μg/kg for individual PA and up to 887 μg/kg for the sum of isomeric pairs.
NOTE 1 A second method was part of the method validation collaborative main trial. For this method PA-N-Oxides are reduced by adding zinc powder to the extract of the feed material. The following steps correspond to the first and main method. Quantitative results for each PA except the otonecine type PA senkirkine represent the sum of the free PA base and its corresponding N-oxide.
NOTE 2 Due to insufficient numbers of data for some analyte-matrix combinations statistical evaluation was not valid for standardization. Received data indicated the methods applicability in experienced laboratories with appropriate quality assurance measures. Therefore, the method description is included as an informative annex (Annex D).

Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von Pyrrolizidinalkaloide in Futtermitteln mittels LC-MS/MS

Dieses Dokument beschreibt ein Verfahren zur quantitativen Bestimmung von Pyrrolizidinalkaloiden (PA) in Allein- und Ergänzungsfuttermitteln sowie in Futtermitteln mittels Flüssigchromatographie mit Massen-spektrometrie-Kopplung (LC-MS/MS) nach Aufreinigung durch Festphasenextraktion (SPE).
Das Verfahren wurde in einem Ringversuch für die Matrices Alleinfuttermittel für Pferde, Ergänzungs-futtermittel für Pferde, Ergänzungsfuttermittel für Nagetiere, Heu, Alfalfa und Grassilage erfolgreich validiert. Die Validierung wurde für die in Tabelle 1 aufgeführten PA- und Konzentrationsbereiche durchgeführt. Es wurde gezeigt, dass die PA-Isomerenpaare Senecivernin und Senecionin sowie Senecivernin-N-Oxid und Senecionin-N-Oxid aufgrund unzureichender chromatographischer Trennung nicht einzeln bestimmt werden können. Die Summen der einzelnen PA der Isomerenpaare wurden jedoch mit ausreichender Vergleichpräzision quantifiziert. Die Ko-Elution von anderen PA-Isomeren, die nicht in den Anwendungsbereich des Verfahrens fallen, muss berücksichtigt werden. Eine Liste der potenziell ko-eluierenden Isomere ist in Anhang E enthalten.
Obwohl der Kalibrierbereich des Verfahrensprotokolls von 10 μg/kg bis 300 μg/kg festgelegt ist, zeigten die Ergebnisse des Ringversuchs, dass die Verdünnung von Probenextrakten mit Blindprobenextrakten die Quantifizierung von Konzentrationen ermöglicht, die den Kalibrierbereich überschreiten. Eine zufriedenstellende Vergleichpräzision wurde bei der Quantifizierung von bis zu 1 428 μg/kg für einzelne PA und bis zu 887 μg/kg für die Summe der Isomerenpaare erreicht.
ANMERKUNG 1 Ein zweites Verfahren war Teil des Haupt-Ringversuchs zur Verfahrensvalidierung. Bei diesem Verfahren werden PA-N-Oxide durch Zugabe von Zinkpulver zum Extrakt des Futtermittels reduziert. Die folgenden Schritte entsprechen dem ersten und wichtigsten Verfahren. Die quantitativen Ergebnisse für jedes PA mit Ausnahme des PA vom Otonecin-Typ Senkirkin stellen die Summe der freien PA-Base und ihres entsprechenden N-Oxids dar.
ANMERKUNG 2 Aufgrund einer unzureichenden Anzahl von Daten für einige Analyt-Matrix-Kombinationen war die statistische Auswertung für die Normung nicht gültig. Die erhaltenen Daten weisen auf die Anwendbarkeit des Verfahrens in erfahrenen Laboratorien mit entsprechenden Qualitätssicherungsmaßnahmen hin. Daher ist die Verfahrensbeschreibung als informativer Anhang (Anhang D) beigefügt.

Aliments pour animaux : Méthodes d’échantillonnage et d’analyse — Dosage des alcaloïdes pyrrolizidiniques dans les aliments pour animaux par CL-SM/SM

Le présent document décrit une méthode de détermination quantitative des alcaloïdes pyrrolizidiniques (AP) dans les aliments pour animaux complets et complémentaires ainsi que dans les fourrages, par chromatographie liquide couplée à une spectrométrie de masse en tandem (CL-SM/SM) après purification par extraction en phase solide (SPE).
La méthode a été validée avec succès lors d’un essai interlaboratoires portant sur les matrices suivantes : aliments complets pour chevaux, aliments complémentaires pour chevaux, aliments complémentaires pour rongeurs, foin, luzerne et ensilage. La validation a été effectuée pour les AP à des gammes de concentrations indiqué dans le Tableau 1. Il a été démontré que les paires d’isomères d’AP (sénécivernine et sénécionine ainsi que sénécivernine N-oxyde et sénécionine N-oxyde) ne peuvent pas être déterminées individuellement en raison d’une séparation chromatographique insuffisante. Cependant, les sommes des AP individuels des paires d’isomères ont été quantifiées avec une reproductibilité acceptable. La co-élution des autres isomères d’AP non inclus dans le domaine d’application de la méthode doit être prise en compte. Une liste des isomères potentiellement co-éluants est donnée à l’Annexe E.
Bien que la gamme d’étalonnage du protocole de la méthode se situe entre 10 μg/kg et 300 μg/kg, les résultats de l’essai interlaboratoires ont montré que la dilution des extraits d’échantillon avec des extraits d’échantillon blanc permet de quantifier des concentrations supérieures à la gamme d’étalonnage. Une reproductibilité satisfaisante a été atteinte lors de la quantification jusqu’à 1 428 μg/kg pour les AP individuels et jusqu’à 887 μg/kg pour la somme des paires d’isomères.
NOTE 1 Une seconde méthode faisait partie de l’essai interlaboratoires principal de validation de la méthode. Pour cette méthode, les N-oxydes d‘AP sont réduits en ajoutant de la poudre de zinc à l’extrait d’aliment pour animaux. Les étapes suivantes correspondent à la première et principale méthode. Les résultats quantitatifs pour chaque AP, excepté l’AP senkirkine de type otonécine, représentent la somme de la base libre d’AP et de son N-oxyde correspondant.
NOTE 2 En raison du nombre insuffisant de données pour certaines combinaisons analyte-matrice, l’évaluation statistique n’était pas valide pour la normalisation. Les données reçues ont indiqué l’applicabilité des méthodes au sein de laboratoires expérimentés avec des mesures d’assurance qualité appropriées. Par conséquent, la description de la méthode est incluse à titre d’annexe informative (Annexe D).

Krma - Metode vzorčenja in analize - Določanje pirolizidinskih alkaloidov v krmi z LCMS/MS

General Information

• Status: Not Published
• Public Enquiry End Date: 14-Sep-2021
• ICS: 65.120 - Animal feeding stuffs
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 5020 - Formal vote (FV) (Adopted Project)
• Start Date: 30-Nov-2022
• Due Date: 18-Jan-2023
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Mandate: M/523 - Mandate for standardisation addressed to CEN for methods of analysis in the field of animal nutrition - Part III
• Ref Project: FprEN 17683 - Animal feeding stuffs - Methods of sampling and analysis - Determination of pyrrolizidine alkaloids in animal feeding stuff by LC-MS/MS

Standards Content (sample)

SLOVENSKI STANDARD
oSIST prEN 17683:2021
01-september-2021

Krma - Metode vzorčenja in analize - Določanje pirolizidinskih alkaloidov v krmi z

LCMS/MS

Animal feeding stuffs- Methods of sampling and analysis - Determination of pyrrolizidine

alkaloids in animal feeding stuff by LCMS/MS
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von
Pyrrolizidinalkaloide in Futtermitteln mittels LC-MS/MS
Aliments pour animaux : Méthodes d’échantillonnage et d’analyse — Dosage des
alcaloïdes pyrrolizidiniques dans les aliments pour animaux par CL-SM/SM
Ta slovenski standard je istoveten z: prEN 17683
ICS:
65.120 Krmila Animal feeding stuffs
oSIST prEN 17683:2021 en,fr,de

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

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oSIST prEN 17683:2021
DRAFT
EUROPEAN STANDARD
prEN 17683
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2021
ICS 65.120
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of pyrrolizidine alkaloids in animal feeding
stuff by LCMS/MS
Futtermittel - Probenahme- und
Untersuchungsverfahren - Bestimmung von
Pyrrolizidinalkaloide in Futtermitteln mittels LC-
MS/MS; Deutsche und Englische Fassung prEN WI
00327113:2018

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

CEN/TC 327.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without

notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17683:2021 E

worldwide for CEN national Members.
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Contents Page

European foreword ............................................................................................................................................ 3

Introduction .......................................................................................................................................................... 4

1 Scope .......................................................................................................................................................... 5

2 Normative references .......................................................................................................................... 7

3 Terms and definitions ......................................................................................................................... 7

4 Principle ................................................................................................................................................... 7

4.1 General...................................................................................................................................................... 7

4.2 Reagents ................................................................................................................................................... 7

4.3 Analytical standards ............................................................................................................................ 7

4.4 Chemicals ................................................................................................................................................. 8

4.5 Solutions ................................................................................................................................................... 9

5 Apparatus .............................................................................................................................................. 10

6 Procedure .............................................................................................................................................. 11

6.1 General.................................................................................................................................................... 11

6.2 Sample preparation ........................................................................................................................... 11

6.3 Extraction .............................................................................................................................................. 12

6.4 SPE procedure ...................................................................................................................................... 12

6.5 Reconstitution of the sample .......................................................................................................... 13

6.6 Quality control samples .................................................................................................................... 13

6.7 Calibration by means of matrix matched standards (MMS) ................................................ 13

7 HPLC-MS/MS analysis ........................................................................................................................ 14

7.1 Liquid chromatographic separation ............................................................................................ 14

7.2 Mass spectrometric operating conditions ................................................................................. 15

7.3 Analysis sequence ............................................................................................................................... 15

8 Results ..................................................................................................................................................... 15

8.1 Peak identification ............................................................................................................................. 15

8.2 Calibration function ........................................................................................................................... 16

8.3 Quantification ...................................................................................................................................... 16

8.4 Reporting of results ........................................................................................................................... 16

8.5 Quality control – Performance criteria ....................................................................................... 17

9 Precision ................................................................................................................................................ 17

9.1 General.................................................................................................................................................... 17

9.2 Repeatability ........................................................................................................................................ 17

9.3 Reproducibility .................................................................................................................................... 17

10 Test report ............................................................................................................................................. 18

Annex A (informative) Precision data ...................................................................................................... 19

Annex B (informative) Example of LC-MS/MS conditions ................................................................. 52

Annex C (informative) Example LC-MS/MS chromatogram of a pyrrolizidine alkaloid

mixture ................................................................................................................................................... 55

Annex D (informative) Method protocol for the determination of pyrrolizidine alkaloids in

animal feeding stuff by LC-MS/MS after reduction of N-oxides using metallic zinc ... 56

Annex E (informative) List of potentially co-eluting pyrrolizidine alkaloid (PA) isomers ... 65

Bibliography ....................................................................................................................................................... 66

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European foreword

This document (prEN 17683:2021) has been prepared by Technical Committee CEN/TC 327 “Animal

feeding stuffs”, the secretariat of which is held by NEN.
This document is currently submitted to the CEN Enquiry.
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Introduction

Pyrrolizidine alkaloids (PA) are secondary metabolites of flowering plants. Ingestion of high doses results

in acute liver damage. In animal studies some PA have proven to be genotoxic carcinogens. Therefore, PA

are undesired substances in food and feed [1], [2]. Poisoning in animals has been reported with known

outbreaks attributed to Heliotropium, Trichodesma, Senecio, and Crotalaria species. In general, grazing

animals will avoid PA-bearing plants. However, if weedy crops are used for the production of hay, silage

or other plant derived feed materials the animals can no longer exercise discrimination when feeding

because the toxins survive storage processes and are completely intermixed with the feed. Therefore,

analytical methods for the control of PA levels in animal feed are needed [1], [2].

WARNING — The use of this protocol involves hazardous materials, operations and equipment. This

protocol does not purport to address all the safety problems associated with its use. It is the responsibility

of the user of this protocol to establish appropriate health and safety practices and determine the

compatibility with regulatory limitations prior to use.
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1 Scope

This document describes a method for the quantitative determination of pyrrolizidine alkaloids (PA) in

complete and supplementary feed and in forages by liquid chromatography tandem mass spectrometry

(LC-MS/MS) after solid phase extraction (SPE) clean-up.

The method has been successfully validated in a collaborative trial for the matrices complete feed for

horses, supplementary feed for horses, supplementary feed for rodents, hay, alfalfa and grass silage.

Validation was carried out for the PA and concentrations ranges listed in Table 1. It was demonstrated

that the PA isomeric pairs senecivernine and senecionine as well as senecivernine-N-oxide and

senecionine-N-oxide cannot be determined individually due to insufficient chromatographic separation.

However, the sums of the individual PA of the isomeric pairs were quantified with sufficient

reproducibility. Co-elution of other PA-isomers not included in the scope of the method shall be taken

into account. A list of potentially co-eluting isomers is presented in Annex E.

Although the calibration range of the method protocol is specified from 10 µg/kg to 300 µg/kg, the results

of the collaborative study showed, that the dilution of sample extracts with blank sample extracts enables

for the quantitation of concentrations exceeding the calibration range. Satisfactory reproducibility was

achieved when quantifying up to 1428 µg/kg for individual PA and up to 887 µg/kg for the sum of

isomeric pairs.
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Table 1 — Summary of concentration ranges per PA tested in the collaborative trial

Tested pyrrolizidine alkaloid (PA) Abbreviation Tested concentration range
(µg/kg)
From To
Echimidine Em 20 435
Echimidine-N-oxide EmN 5 30
Erucifoline Er 20 245
Erucifoline-N-oxide ErN 20 370
Europine Eu 15 330
Europine-N-oxide EuN 25 285
Heliotrine Hn 25 280
Heliotrine-N-oxide HnN 25 245
Jacobine Jb 20 230
Jacobine-N-oxide JbN 20 215
Lasiocarpine Lc 20 350
Lasiocarpine-N-oxide LcN 5 250
Intermedine Im 25 560
Intermedine-N-oxide ImN 5 395
Lycopsamine La 25 500
Lycopsamine-N-oxide LaN 20 280
Monocrotaline Mc 20 360
Monocrotaline-N-oxide McN 20 365
Retrorsine Re 250 375
Retrorsine-N-oxide ReN 5 285
Senecionine Sc 25 205
Senecionine-N-oxide ScN 5 300
Senecivernine Sv 20 205
Senecivernine-N-oxide SvN 5 165
Senkirkine Sk 20 275
Seneciphylline Sp 25 225
Seneciphylline-N-oxide SpN 5 225
Trichodesmine Td 5 250
Intermedine + Lycopsamine Im+La 50 890
Intermedine-N-oxide + Lycopsamine-N- ImN+LaN 5 645
Senecivernine + Senecionine Sv+Sc 30 280
Senecivernine-N-oxide + Senecionine-N- SvN+ScN 10 380
Rounded figures

Individual PA of the isomeric pairs Sv+Sc and SvN+ScN were not evaluated statistically due to insufficient

chromatographic separation
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NOTE 1 A second method was part of the method validation collaborative main trial. For this method PA-N-

Oxides are reduced by adding zinc powder to the extract of the feed material. The following steps correspond to the

first and main method. Quantitative results for each PA except the otonecine type PA senkirkine represent the sum

of the free PA base and its corresponding N-oxide.

NOTE 2 Due to insufficient numbers of data for some analyte-matrix combinations statistical evaluation was not

valid for standardization. Received data indicated the methods applicability in experienced laboratories with

appropriate quality assurance measures. Therefore, the method description is included as an informative annex

(Annex D).
2 Normative references
There are no normative references in this document.
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 https://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
4 Principle
4.1 General

A test portion of 2 g feed material is sonicated twice in aqueous sulphuric acid solution for PA extraction.

After centrifugation, an aliquot of the supernatant is purified by solid phase extraction (SPE) using

reversed phase (C18) material. PA are released from the cartridge using methanol. Subsequently, the

eluate is evaporated to dryness and reconstituted in methanol/water (initial HPLC conditions).

For chromatographic separation, an RP-HPLC column is used with a binary gradient. Analytes are

detected by triple stage quadrupole mass spectrometry. Quantification of PA is accomplished by means

of matrix matched calibration.
4.2 Reagents

All chemicals should at least be of pro-analysis quality or higher. References to products or vendors are

just for general information and do not imply that other products or producers with the same or similar

characteristics are ruled out.

If not specified elsewise, use only reagents of analytical grade and solvents suitable for HPLC-MS/MS.

Water shall be distilled in glass vessels or demineralized before use, or shall be of equivalent purity.

4.3 Analytical standards
4.3.1 Senecionine (Sc)
4.3.2 Senecionine-N-oxide (ScN)
4.3.3 Seneciphylline (Sp)
4.3.4 Seneciphylline-N-oxide (SpN)
4.3.5 Monocrotaline (Mc)
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4.3.6 Monocrotatline-N-oxide (McN)
4.3.7 Retrorsine (Re)
4.3.8 Heliotrine (Hn)
4.3.9 Heliotrine-N-oxide (HnN)
4.3.10 Trichodesmine (Td)
4.3.11 Retrorsine-N-oxide (ReN)
4.3.12 Echimidine (Em)
4.3.13 Echimidine-N-oxide (EmN)
4.3.14 Intermedine (Im)
4.3.15 Intermedin-N-oxide (ImN)
4.3.16 Lycopsamine (La)
4.3.17 Lycopsamine-N-oxide (LaN)
4.3.18 Erucifoline (Er)
4.3.19 Erucifoline-N-oxide (ErN)
4.3.20 Senecivernine (Sv)
4.3.21 Senecivernine-N-oxide (SvN)
4.3.22 Jacobine (Jb)
4.3.23 Jacobine-N-oxide (JbN)
4.3.24 Lasiocarpine (Lc)
4.3.25 Lasiocarpine-N-oxide (LcN)
4.3.26 Europine (Eu)
4.3.27 Europine-N-oxide (EuN)
4.3.28 Senkirkine (Sk)
4.4 Chemicals
4.4.1 Formic acid 98 – 100 %
4.4.2 Methanol (MeOH) in LC-MS quality
4.4.3 Sulphuric acid 98 %
4.4.4 Ammonia 32 %
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4.4.5 Ammonium formate in LC-MS quality
4.4.6 Acetonitrile
4.5 Solutions
4.5.1 Extraction solution

Prepare a 0,05 °M solution of aqueous sulphuric acid. Make up 2,665 ml of sulphuric acid (H2SO4) (see

4.4.3) to 1 l with water. The extraction solution can be used for 1 month when stored at room

temperature.
4.5.2 Aqueous ammoniacal solution (for neutralization of extracts before SPE)

Dilute ammonia 32 % (see 4.4.4) with water in a 1 to 5 ratio by volume (V:V), e.g. mix 5 ml of ammonia

32 % with 20 ml of water.
The solution shall be prepared every working day.
4.5.3 Examples of HPLC mobile phase
• Eluent A:

Weigh 315 mg ammonium formate (see 4.4.5) and dissolve in 5 ml of water in a 1000 ml volumetric

flask, add 1 ml of formic acid (see 4.4.1) and make up to 1 l with water. The solution can be used for

1 month when stored at room temperature.
• Eluent B:

Weigh 315 mg ammonium formate (see 4.4.5) and dissolve in 5 ml of water in a 1000 ml volumetric

flask, add 1 ml of formic acid (see 4.4.1) and make up to 1 l with methanol (see 4.4.2). The solution

can be used for 3 months when stored at room temperature.
4.5.4 PA stock solutions

To create a stock solution, weigh 1 mg to 10 mg (depending on the amount available per unit purchased)

of a pyrrolizidine alkaloid standard in a 10 ml volumetric flask using an analytical balance (see 5.22) and

make up to 10 ml with a suitable organic solvent like methanol or acetonitrile. The resulting

concentration of the stock solution is 0,1 mg/ml to 1°mg/ml. Stock solutions are stable for at least 2 years

when stored < −18 °C.

If the available analytical balance does not provide sufficient accuracy, higher quantities shall be weighed.

NOTE Instructions of standard providers can indicate suitable solvents for the preparation of stock solutions

but are not mandatory to be used.
4.5.5 Standard working solution (PA mixture), 1 µg/ml

Transfer respective volumes each PA stock solution (between 0,1 mg/ml to 10 mg/ml) into a volumetric

flask and make up with acetonitrile (see 4.4.6), to achieve a concentration of 1 µg/ml for each PA

contained in the mixture. Long term stability tests proved acetonitrile to be the most suitable solvent, but

is not mandatory to be used. The PA mixture is stable for at least 2 years when stored < −18 °C.

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If no baseline separation can be achieved for an isomeric pair (intermedine(-N-oxide) and lycopsamine

(-N-oxide); senecivernine(-N-oxide) and senecionine(-N-oxide)), the sum concentration of both PA can

be calculated via the calibration of one of them. The composition of the standard mix shall be prepared

accordingly by including only one isomer of the pair.
5 Apparatus
Use laboratory equipment and, in particular, the following elements.

NOTE References to products, instruments or vendors are just for general information and do not imply that

other products or producers with the same or similar characteristics are ruled out.

5.1 Various piston pipettes and multiple dispenser and respective tips
5.2 Analytical balance, capable of weighing to 0,1 mg
5.3 Compartment drier
5.4 Centrifugal mill with 0,5 mm sieve
5.5 Centrifuge for 50 ml centrifuge tubes, capable of at least 5 000 × g
5.6 Laboratory shaker
5.7 Overhead shaker
5.8 Evaporation station with or without vacuum support
5.9 Ultrasonic bath
5.10 Centrifuge tubes 50 ml
5.11 Test tubes 15 ml
5.12 Volumetric flasks, 10 ml and 20 ml
5.13 Folded filters
5.14 SPE cartridges: C18, 500 mg sorbent material
® 1

NOTE Supelco Discovery DSC18 500 mg/6 ml is an example of a suitable product available commercially .

5.15 SPE vacuum chamber
5.16 Membrane filter 0,2 µm or 0,45 µm

Centrifugal filters should have > 0,5 ml capacity and contain modified nylon membrane. Other filtering

tools can be used as well.
1 ®

Supelco Discovery is an example of a suitable product available commercially. This information is given for the

convenience of users of this document and does not constitute an endorsement by CEN of this product.

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5.17 HPLC vials 2 ml
5.18 Glass inserts, 250 µl conic for HPLC vials
5.19 Chromatographic column

C18 reversed phase packing material at acidic pH-conditions (pH 2-3), capable of baseline separation of

analytes with identical molecular mass, separation of isomeric pairs is desirable.

5.20 LC-MS/MS system

Capable of performing multiple selected reaction monitoring in positive ionization mode, with a

sufficiently wide dynamic range and capable of unit mass separation and equipped with a computer-

based data processing system. Any ionization source giving sufficient yield may be employed.

6 Procedure
6.1 General

Animal feed is a complex matrix containing a wide range of ingredients in varying amounts leading to

variable and sample dependent matrix effects (suppression/enhancement). For this reason, a

quantification of the analytes shall be accomplished by matrix-matched calibration.

Comparison of matrix effects of different feed materials has shown that there might be a difference in

signal suppression of around 50 % depending on the analyte-matrix combination. As usually a variety of

feed materials shall be analysed in one sequence, a representative mix of blank feed materials can be used

(e.g. 75 % different types of grass, like hay and silage, and legumes and 25 % cereals). If the sequence

contains only samples of one specific feed material, a blank matrix as similar as possible to the sample

matrix should be used for matrix-matched calibration.

Samples exceeding the calibration range can be diluted using blank sample extract. Recovery shall be

checked with every series of samples proving the required range of recovery. Recovery correction is

carried out if recovery is outside of 90 % - 110 % according to EURL-MP's Guidance document on

performance criteria for the analysis of mycotoxins and plant toxins in food and feed [3] (under

development).
6.2 Sample preparation

To determine the PA concentration that is representative for the entire sample, the sample material

should meet the following characteristics: uniform particle size and a homogenous distribution.

Therefore, an appropriate portion of the sample material is ground to a particle size of 0,5 mm (see 5.4)

and homogenized for example using an overhead shaker (see 5.7).

Prior to grinding fresh feed samples (for example silage or grass) are dried at 40 °C for 18 h in a drying

oven. Frozen samples are defrosted at room temperature before drying.

Dry samples are mixed with dry ice (ratio 2:1) and the mixture is allowed to stand for 10 minutes before

grinding to a particle size of 0,5 mm using an ultra-centrifugal mill (see 5.4). The ground material is

homogenized by shaking for example using an overhead shaker (see 5.7) for 1 h.

NOTE Grinding with ice has proven to obtain excellent grinding results due to shear forces and porosity of the

frozen sample material.

If the test material cannot be ground to a particle size of 0,5 mm or smaller without generation of heat,

increasing the weighed sample amount to at least 10 g is also possible to enhance the results

representativeness for the sample. In order to keep a constant ratio of sample amount to extraction

volume, the used volume of extraction solution (see 4.5.1) needs to be increased accordingly.

Laboratories shall prove that the performance of their homogenization procedure used is sufficient.

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6.3 Extraction
Weigh 2,0 g ± 0,1 g of the homogenized sample into a centrifuge tube (see 5.10).

1) Extraction step 1: Add 20 ml of the extraction solution (see 4.5.1) to the sample and mix to wet the

sample material completely before extraction by ultra-sonication (see 5.9) for 15 min at ambient

temperature.

NOTE Extraction can be accomplished by other techniques than ultra-sonication (e.g. using an over-head

shaker), if sufficient extraction efficiency was shown during in-house validation.

2) Centrifugation: Centrifuge the tube for 10 min ± 2 min at 3800 × g (see 5.5). Transfer the

supernatant (extract 1) into a clean test tube and use the sediment for the second extraction step.

3) Extraction step 2: Add 20 ml of extraction solution (see 4.5.1) to the sediment. Shake the centrifuge

tube vigorously to distribute the sample (the sample can also be stirred if necessary) and extract

again by ultra-sonication* for 15 min at ambient temperature.

4) Centrifugation: Centrifuge the tube for 10 min ± 2 min at 3800 × g (see 5.5) and combine the

supernatant (extract 2) with extract 1.

5) Neutralization: Adjust the pH of the combined extracts to pH 7 using the neutralization solution

(see 4.5.2). Check the pH value using indicator strips. Usually, about 500 µl to 1000 µl of the solution

are required.

6) Filtration: Pass the complete neutralized extract through a filter (e.g. folded filters see 5.1.3) or

centrifuge alternatively. Use an aliquot of the filtered supernatant for SPE. Repeat the filtration step

in case of larger quantities of remaining particles in the solution. Thereby, blockage of SPE cartridges

can be avoided.
6.4 SPE procedure

1) Conditioning step 1: Load 5 ml of methanol (see 4.4.2) onto the SPE cartridge and let it pass through

under normal pressure.

2) Conditioning step 2: Load 5 ml of water onto the SPE cartridge and let it pass through under normal

pressure.

3) Sample load: Load 10 ml of the sample extract (combined supernatants from Clause 6.3) onto the

SPE cartridge and let it pass through without letting the cartridge run dry.
4) Washing step: Wash the SPE cartridge 2 × 5 ml of water.

5) Drying of cartridges: Dry the cartridges by applying vacuum for 5 - 10 min (use the vacuum

chamber (see 5.15).

6) Elution of PA: Elute the analytes from the cartridges using 2 × 5 ml methanol (see 4.4.2). The eluate

is dried under a nitrogen stream at 50 °C ± 5 °C, or according to the recommendations for the

evaporation equipment used.

NOTE If alternative SPE cartridges are used, solvent volumes for conditioning, sample loading, washing and

eluting can be adapted. Depending on the solid phase material, it might be necessary to protect the stationary from

running dry until the washing step.
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6.5 Reconstitution of the sample
Dissolve the residue in 1 ml of methanol/water (5/95, v/v) by shaking (see 5.7).

Filter the reconstituted sample extracts through a 0,2 to 0,45 µm membrane filter (see 5.16). When using

centrifugal filters, 500 µl of the sample are centrifuged at 20 000 × g for 10 min ± 3 min. Transfer 200 µl

of the filtrate into an HPLC vial (5.17) with a glass insert (see 5.18).
6.6 Quality control samples

In every sample batch, recovery as a sum of extraction efficiency, clean-up and LC-MS/MS detection shall

be checked. Preferably, a reference material or well characterized positive samples should be used. If no

reference material is available, recovery can be determined by spiking a representative feed material

(see 6.1) that is free from PA. Spiking procedure (example):

EXAMPLE Spike 2 g of the blank feed material with PA working solution (see 4.5.5), vortex or mix by hand

vigorously and leave open for 1 hour to allow the solvent to evaporate. Analyse the spiked sample alongside with

the unknown samples.

NOTE Addition of 50 µl of PA working solution (see 4.5.5) were used in the method validation study and

worked well.
6.7 Calibration by means of matrix matched standards (MMS)

For the correction of matrix effects, a matrix matched calibration is used. In order to obtain the same

matrix strength in the MMS and the samples, blank feed shall be processed as described in

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