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

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
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 legt ein Verfahren zur quantitativen Bestimmung von Pyrrolizidinalkaloiden (PA) in den in Tabelle 1 angegebenen Konzentrationsbereichen in Allein- und Ergänzungsfuttermitteln sowie in Grünfutter mittels Flüssigchromatographie mit Massenspektrometrie-Kopplung (LC MS/MS) nach Aufreinigung durch Festphasenextraktion (SPE, en: solid phase extraction) fest.
Tabelle 1 - Zusammenfassung der im Ringversuch geprüften Konzentrationsbereiche je PA
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 Laboren 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 spécifie une méthode de dosage des alcaloïdes pyrrolizidiniques (AP) dans les gammes de concentrations indiquées dans le Tableau 1 dans les aliments composés 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).
Tableau 1 - Résumé des gammes de concentrations par AP soumis à essai lors de l’essai interlaboratoires
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 composé 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

Status
Published
Publication Date
28-Feb-2023
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
01-Mar-2023
Due Date
02-Dec-2020
Completion Date
01-Mar-2023

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

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.

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
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
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.
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.

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 spe
...

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