SIST EN 17683:2023
(Main)Animal feeding stuffs - Methods of sampling and analysis - Determination of pyrrolizidine alkaloids in animal feeding stuff by LC-MS/MS
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 Pyrrolizidinalkaloiden 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.
Alimentation animale - Méthodes d’échantillonnage et d’analyse - Dosage des alcaloïdes pyrrolizidiniques dans l'alimentation animale 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
Ta dokument opisuje metodo za kvantitativno določevanje pirolizidinskih alkaloidov (PA) v polnovredni in dopolnilni krmi s tandemsko masno spektrometrijo s tekočinsko kromatografijo (LC-MS/MS) po izpiranju z ekstrakcijo na trdni fazi (SPE).
Ta metoda je bila uspešno potrjena v primerjalnem preskusu za matrice s polnovredno krmo za konje, dopolnilno krmo za konje, dopolnilno krmo za glodavce, seno, lucerno in travno silažo. Potrditev je bila izvedena za pirolizidinske alkaloide (PA) in vrednosti koncentracije, navedene v tabeli 1. Dokazano je bilo, da izomerna para pirolizidinskih alkaloidov sineciverin in senecionin ter senecivernin-N-oksid in senecionin-N-oksid, ni mogoče določiti posamezno zaradi neustreznega kromatografskega ločevanja. Vseeno pa so bile vsote posameznih pirolizidinskih alkaloidov izomernih parov kvantificirane z ustrezno stopnjo ponovljivosti. Upoštevati je treba sočasno eluacijo drugih izomerov pirolizidinskih alkaloidov, ki niso zajeti v metodo. Seznam izomerov s potencialno sočasno eluacijo je podan v dodatku E.
Čeprav je kalibracijsko območje protokola metode določeno od 10 μg/kg do 300 μg/kg, so rezultati primerjalne študije pokazali, da redčenje ekstraktov vzorcev z ekstrakti slepih vzorcev omogoča kvantitativno določanje koncentracij, ki presegajo kalibracijsko območje. Zadovoljiva ponovljivost je vila dosežena pri kvantificiranju do 1428 μg/kg za posamezen pirolizidinski alkaloid in do 887 μg/kg za vsoto izomernih parov.
OPOMBA 1: Druga metoda je bila del primerjalnega glavnega preskusa potrjevanja metode. Pri tej metodi so bili PA-N-oksidi zmanjšani z dodajanjem cinkovega prahu ekstraktu krmnega material. Spodnji koraki ustrezajo prvi in glavni metodi. Kvantitativni rezultati za vsak pirolizidinski alkaloid razen pirolizidinskega alkaloida senkirkin vrste otenocin predstavljajo vsoto proste baze pirolizidinskih alkaloidov in njen ustrezen N-oksid.
OPOMBA 2: Zaradi nezadostnega števila podatkov za nekatere kombinacije analita in matrike statistična ocena za standardizacijo ni bila veljavna. Pridobljeni rezultati so kazali uporabnost metod v izkušenih laboratorijih z ustrezno kakovostjo ukrepov za zagotavljanje kakovosti. Za to je opis metode vključen kot informativni dodatek (dodatek D).
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN 17683:2023
01-maj-2023
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 LC-MS/MS
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von
Pyrrolizidinalkaloiden in Futtermitteln mittels LC-MS/MS
Alimentation animale - Méthodes d’échantillonnage et d’analyse - Dosage des alcaloïdes
pyrrolizidiniques dans l'alimentation animale par CL-SM/SM
Ta slovenski standard je istoveten z: EN 17683:2023
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN 17683:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN 17683:2023
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SIST EN 17683:2023
EN 17683
EUROPEAN STANDARD
NORME EUROPÉENNE
March 2023
EUROPÄISCHE NORM
ICS 65.120
English Version
Animal feeding stuffs - Methods of sampling and analysis -
Determination of pyrrolizidine alkaloids in animal feeding
stuff by LC-MS/MS
Alimentation animale - Méthodes d'échantillonnage et Futtermittel - Probenahme- und
d'analyse - Dosage des alcaloïdes pyrrolizidiniques Untersuchungsverfahren - Bestimmung von
dans l'alimentation animale par CL-SM/SM Pyrrolizidinalkaloiden in Futtermitteln mittels LC-
MS/MS
This European Standard was approved by CEN on 17 January 2023.
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, Türkiye 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
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17683:2023 E
worldwide for CEN national Members.
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EN 17683:2023 (E)
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
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
4.5.1 Extraction solution 0,05 mol/l aqueous sulphuric acid . 9
4.5.2 Aqueous ammoniacal solution (20/80) (v/v) for neutralization of extracts before SPE
. 9
4.5.3 Examples of HPLC mobile phase . 9
4.5.4 PA stock solutions . 9
4.5.5 Standard working solution (PA mixture), 1 µg/ml . 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 . 18
10 Test report . 18
Annex A (informative) Precision data . 19
Annex B (informative) Example of LC-MS/MS conditions . 52
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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 . 66
Bibliography . 67
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European foreword
This document (EN 17683:2023) 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 September 2023, and conflicting national standards
shall be withdrawn at the latest by September 2023.
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.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
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, Türkiye and the United
Kingdom.
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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]. This document describes
methods of sampling and analysis for the determination of pyrrolizidine alkaloids in animal feeding stuff
by LC-MS/MS.
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 as described in Clause 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 need to be taken
into account. A list of potentially co-eluting isomers is presented in Commission Regulation (EU)
2020/2040 [3].
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 1 428 µg/kg for individual PA and up to 887 µg/kg for the sum of
isomeric pairs.
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.
5
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1 Scope
This document specifies a method for the quantitative determination of pyrrolizidine alkaloids (PA) in
the concentration ranges shown in Table 1 in complete and supplementary feed and in forages by liquid
chromatography tandem mass spectrometry (LC-MS/MS) after solid phase extraction (SPE) clean-up.
Table 1 — Summary of concentration ranges per PA tested in the collaborative trial
a
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
b
Senecionine Sc 25 205
b
Senecionine-N-oxide ScN 5 300
b
Senecivernine Sv 20 205
b
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-oxide ImN+LaN 5 645
Senecivernine + Senecionine Sv+Sc 30 280
Senecivernine-N-oxide + Senecionine-N-ide SvN+ScN 10 380
a
Rounded figures
b
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
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, Water for analytical laboratory use - Specification and test methods (ISO 3696)
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.
4.3 Analytical standards
4.3.1 Senecionine (Sc), CAS-Nr.: 130-01-8
4.3.2 Senecionine-N-oxide (ScN), CAS-Nr.: 13268-67-2
4.3.3 Seneciphylline (Sp), CAS-Nr.: 480-81-9
4.3.4 Seneciphylline-N-oxide (SpN), CAS-Nr.: 38710-26-8
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4.3.5 Monocrotaline (Mc), CAS-Nr.: 315-22-0
4.3.6 Monocrotaline-N-oxide (McN), CAS-Nr.: 35337-98-5
4.3.7 Retrorsine (Re), CAS-Nr.: 480-54-6
4.3.8 Heliotrine (Hn), CAS-Nr.: 303-33-3
4.3.9 Heliotrine-N-oxide (HnN), CAS-Nr.: 6209-65-0
4.3.10 Trichodesmine (Td), CAS-Nr.: 548-90-3
4.3.11 Retrorsine-N-oxide (ReN), CAS-Nr.: 15503-86-3
4.3.12 Echimidine (Em), CAS-Nr.: 520-68-3
4.3.13 Echimidine-N-oxide (EmN), CAS-Nr.: 41093-89-4
4.3.14 Intermedine (Im), CAS-Nr.: 10285-06-0
4.3.15 Intermedin-N-oxide (ImN), CAS-Nr.: 95462-14-9
4.3.16 Lycopsamine (La), CAS-Nr.: 10285-07-1
4.3.17 Lycopsamine-N-oxide (LaN), CAS-Nr.: 95462-15-0
4.3.18 Erucifoline (Er), CAS-Nr.: 40158-95-0
4.3.19 Erucifoline-N-oxide (ErN), CAS-Nr.: 123864-94-8
4.3.20 Senecivernine (Sv), CAS-Nr.: 72755-25-0
4.3.21 Senecivernine-N-oxide (SvN), CAS-Nr.: 101687-28-9
4.3.22 Jacobine (Jb), CAS-Nr.: 6870-67-3
4.3.23 Jacobine-N-oxide (JbN), CAS-Nr.: 38710-25-7
4.3.24 Lasiocarpine (Lc), CAS-Nr.: 303-34-4
4.3.25 Lasiocarpine-N-oxide (LcN), CAS-Nr.: 127-30-0
4.3.26 Europine (Eu) as europine hydrochloride, CAS-Nr.: 570-19-4
4.3.27 Europine-N-oxide (EuN), CAS-Nr.: 65582-53-8
4.3.28 Senkirkine (Sk), CAS-Nr.: 2318-18-5
4.4 Chemicals
4.4.1 Formic acid (HCOOH) 98 to 100 %
4.4.2 Methanol (MeOH) in LC-MS quality
4.4.3 Sulphuric acid (H SO ) 98 %
2 4
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4.4.4 Ammonia (NH ) 32 %
3
4.4.5 Ammonium formate (NH COOH) in LC-MS quality
4
4.4.6 Acetonitrile (CH CN) in LC-MS quality
3
4.4.7 Water (H O) in LC-MS quality, double-distilled or water of grade 1 as defined in EN ISO 3696
2
4.5 Solutions
4.5.1 Extraction solution 0,05 mol/l aqueous sulphuric acid
Prepare a 0,05 mol/l solution of aqueous sulphuric acid. Make up 2,665 ml of sulphuric acid (H SO )
2 4
(4.4.3) to 1 l with water (4.4.7). The extraction solution can be used for 1 month when stored at room
temperature.
4.5.2 Aqueous ammoniacal solution (20/80) (v/v) for neutralization of extracts before SPE
Dilute a volume fraction of 20 % ammonia 32 % (4.4.4) with a volume fraction of 80 % water (4.4.7). The
solution shall be prepared every working day.
EXAMPLE Mix 5 ml of ammonia 32 % with 20 ml of water (4.4.7).
4.5.3 Examples of HPLC mobile phase
• Eluent A 5 mmol/l ammonium formate in water with 0,1 % formic acid:
Weigh 315 mg ammonium formate (4.4.5) and dissolve in 5 ml of water (4.4.7) in a 1 000 ml
volumetric flask, add 1 ml of formic acid (4.4.1) and make up to 1 l with water (4.4.7). The solution
can be used for 1 month when stored at room temperature.
• Eluent B 5 mmol/l ammonium formate in methanol with 0,1 % formic acid::
Weigh 315 mg ammonium formate (4.4.5) and dissolve in 5 ml of water (4.4.7) in a 1 000 ml
volumetric flask, add 1 ml of formic acid (4.4.1) and make up to 1 l with methanol (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 (5.2) 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 below −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 1 mg/ml (4.5.4) using an
appropriate device (5.1) into a volumetric flask (5.12) and make up with acetonitrile (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 below −18 °C.
9
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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. In case of baseline separation of the isomeric pairs
all pa analytical standards including 4.3.20 and 4.3.21 should be included in the PA mixture.
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 accuracy
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 pH indicator strips
5.16 SPE vacuum chamber
5.17 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.
10
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EN 17683:2023 (E)
5.18 HPLC vials 2 ml
5.19 Glass inserts, 250 µl conic for HPLC vials
5.20 Chromatographic column
C18 reversed phase packing material applicable at acidic pH-conditions (pH 2-3), capable of baseline
separation of analytes with identical molecular mass, separation of isomeric pairs is desirable. Other
stationary phases (for example biphenyl phases) or mobile phases (for example with basic pH) can be
used if comparable or better baseline separation of isomers is achieved.
5.21 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. Exemplarily
instrument configuration and measuring conditions are shown in Annex B.
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 can 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 % to 110 % according to EURL-MP’s Guidance document on
performance criteria for the analysis of mycotoxins and plant toxins in food and feed [4]
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 (5.4) and
homogenized for example using an overhead shaker (5.7).
Prior to grinding fresh feed samples (for example silage or grass) are dried at 40 °C to 45 °C at
atmospheric pressure for approximately 18 h in a drying oven (5.3). 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 min before
grinding to a particle size of 0,5 mm using an ultra-centrifugal mill (5.4). The ground material is
homogenized by shaking for example using an overhead shaker (5.7) for 1 h.
NOTE Grinding with dry ice has proven to obtain excellent grinding results due to shear forces and porosity of
the frozen sample material.
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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 (4.5.1) needs to be increased accordingly.
Laboratories shall prove that the performance of their homogenization procedure used is sufficient.
6.3 Extraction
Weigh 2,0 g ± 0,1 g of the homogenized sample into a centrifuge tube (5.10).
1) Extraction step 1: Add 20 ml of the extraction solution (4.5.1) to the sample and mix (5.6) to wet
the sample material completely before extraction by ultra-sonication (5.9) for 15 min at ambient
temperature.
NOTE 1 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 appr
...
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
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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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prEN 17683:2021 (E)
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.
3
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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.
4
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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.
5
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Table 1 — Summary of concentration ranges per PA tested in the collaborative trial
a
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
b
Senecionine Sc 25 205
b
Senecionine-N-oxide ScN 5 300
b
Senecivernine Sv 20 205
b
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
a
Rounded figures
b
Individual PA of the isomeric pairs Sv+Sc and SvN+ScN were not evaluated statistically due to insufficient
chromatographic separation
6
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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)
7
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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 %
8
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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.
9
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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.
10
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prEN 17683:2021 (E)
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.
11
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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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oSIST prEN 17683:2021
prEN 17683:2021 (E)
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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