EN 17256:2019
(Main)Animal feeding stuffs: Methods of sampling and analysis - Determination of ergot alkaloids and tropane alkaloids in feed materials and compound feeds by LC-MS/MS
Animal feeding stuffs: Methods of sampling and analysis - Determination of ergot alkaloids and tropane alkaloids in feed materials and compound feeds by LC-MS/MS
This document describes a method for the determination of individual ergot alkaloids and tropane alkaloids in unprocessed cereals and cereal-based compound feeds by high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS).
This method has been successfully validated by collaborative trial in the following matrices: rye, barley, wheat, complete feed for bovine, porcine and poultry. Validation in buckwheat produced acceptable results, but the relative standard reproducibility was higher for most analytes in comparison with the other matrices. This may be related to the matrix. The validated range of the method is approximately 10 to 250 µg/kg for individual alkaloids. Determination of concentrations above 250 µg/kg is possible by applying a higher spiking level and dilution of the sample extract, but this has not been validated in the collaborative trial.
The method is applicable for the determination, by means of one-point standard addition to the sample, of ergocornine in the tested range of 12 µg/kg to 221 µg/kg, ergocorninine in the tested range of 9 µg/kg to 196 µg/kg, ergocristine in the tested range of 14 µg/kg to 312 µg/kg, ergocristinine in the tested range of 12 µg/kg to 258 µg/kg, α-ergocryptine in the tested range of 10 µg/kg to 184 µg/kg, α-ergocryptinine in the tested range of 8 µg/kg to 171 µg/kg, ergometrine in the tested range of 12 µg/kg to 174 µg/kg, ergometrinine in the tested range of 3 µg/kg to 172 µg/kg, ergosine in the tested range of 12 µg/kg to 226 µg/kg, ergosinine in the tested range of 9 µg/kg to 273 µg/kg, ergotamine in the tested range of 11 µg/kg to 443 µg/kg, ergotaminine in the tested range of 10 µg/kg to 273 µg/kg, atropine in the tested range of 16 µg/kg to 252 µg/kg and scopolamine in the tested range of 15 µg/kg to 246 µg/kg.
Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung der Alkaloide des Mutterkorns und der Tropanalkaloiden in Einzelfuttermitteln und Mischfuttermitteln mittels LC-MS/MS
Dieses Dokument beschreibt ein Verfahren zur Bestimmung einzelner Mutterkornalkaloide und Tropanalkaloide in unverarbeitetem Getreide und getreidebasierten Mischfuttermitteln durch Hochleistungs Flüssigkeits¬chromatographie mit Tandem-Massenspektrometrie (LC MS/MS).
Dieses Dokument wurde im Rahmen eines Ringversuches in folgenden Matrices erfolgreich validiert: Roggen, Gerste, Weizen, Alleinfuttermittel für Rinder, Schweine und Geflügel. Die Validierung in Buchweizen ergab akzeptable Ergebnisse, die relative Standardvergleichpräzision war jedoch bei den meisten Analyten, verglichen mit anderen Matrices, höher. Dies kann mit der Matrix zusammenhängen. Der validierte Verfahrensbereich liegt etwa zwischen 10 µg/kg und 250 µg/kg für einzelne Alkaloide. Durch Anwendung eines höheren Dotierniveaus und der Verdünnung des Probenextrakts ist die Bestimmung von Konzentrationen über 250 µg/kg möglich, wurde jedoch im Ringversuch nicht validiert.
Dieses Dokument wird angewendet für die Bestimmung, mittels einer Ein-Punkt-Standardaddition der Probe, von:
— Ergocornin im geprüften Bereich von 12 µg/kg bis 221 µg/kg;
— Ergocorninin im geprüften Bereich von 9 µg/kg bis 196 µg/kg;
— Ergocristin im geprüften Bereich von 14 µg/kg bis 312 µg/kg;
— Ergocristinin im geprüften Bereich von 12 µg/kg bis 258 µg/kg;
— α Ergocryptin im geprüften Bereich von 10 µg/kg bis 184 µg/kg;
— der Summe von α und -Ergocryp¬tinin im geprüften Bereich von 8 µg/kg bis 171 µg/kg;
— Ergometrin im geprüften Bereich von 12 µg/kg bis 174 µg/kg;
— Ergometrinin im geprüften Bereich von 3 µg/kg bis 172 µg/kg;
— Ergosin im geprüften Bereich von 12 µg/kg bis 226 µg/kg;
— Ergosinin im geprüften Bereich von 9 µg/kg bis 273 µg/kg;
— Ergotamin im geprüften Bereich von 11 µg/kg bis 443 µg/kg;
— Ergotaminin im geprüften Bereich von 10 µg/kg bis 273 µg/kg;
— Atropin im geprüften Bereich von 16 µg/kg bis 252 µg/kg;
— Scopolamin im geprüften Bereich von 15 µg/kg bis 246 µg/kg.
Aliments des animaux : Méthodes d'échantillonnage et d'analyse - Détermination de la teneur en alcaloïdes de l'ergot et en alcaloïdes tropaniques dans les matières premières et les aliments composés par CL-SM/SM
Le présent document décrit une méthode de détermination de la teneur en alcaloïdes de l’ergot et en alcaloïdes tropaniques dans les céréales non transformées et les aliments composés à base de céréales par chromatographie liquide à haute performance couplée à une spectrométrie de masse en tandem (CL-SM/SM).
Le présent document a été validé lors d’un essai interlaboratoires dans les matrices suivantes : seigle, orge, blé, aliments complets pour bovins, porcins et volailles. La validation dans le sarrasin a donné des résultats acceptables, mais le coefficient de variation de la reproductibilité était plus élevé pour la plupart des analytes par rapport aux autres matrices. Ceci peut être dû à la matrice. La gamme validée de la méthode est d’environ 10 µg/kg à 250 µg/kg pour les alcaloïdes. Il est possible de déterminer des concentrations supérieures à 250 µg/kg en appliquant un niveau de dopage et une dilution plus élevés de l’extrait d’échantillon, mais ceci n’a pas été validé lors de l’essai interlaboratoires.
Le présent document est applicable à la détermination, par ajout dosé en un point dans l’échantillon :
• de la teneur en ergocornine dans la gamme d’essai de 12 µg/kg à 221 µg/kg ;
• de la teneur en ergocorninine dans la gamme d’essai de 9 µg/kg à 196 µg/kg ;
• de la teneur en ergocristine dans la gamme d’essai de 14 µg/kg à 312 µg/kg ;
• de la teneur en ergocristinine dans la gamme d’essai de 12 µg/kg à 258 µg/kg ;
• de la teneur en α-ergocryptine dans la gamme d’essai de 10 µg/kg à 184 µg/kg ;
• de la teneur totale en α-ergocryptinine et en β-ergocryptinine dans la gamme d’essai de 8 µg/kg à 171 µg/kg ;
• de la teneur en ergométrine dans la gamme d’essai de 12 µg/kg à 174 µg/kg ;
• de la teneur en ergométrinine dans la gamme d’essai de 3 µg/kg à 172 µg/kg ;
• de la teneur en ergosine dans la gamme d’essai de 12 µg/kg à 226 µg/kg ;
• de la teneur en ergosinine dans la gamme d’essai de 9 µg/kg à 273 µg/kg ;
• de la teneur en ergotamine dans la gamme d’essai de 11 µg/kg à 443 µg/kg ;
• de la teneur en ergotaminine dans la gamme d’essai de 10 µg/kg à 273 µg/kg ;
• de la teneur en atropine dans la gamme d’essai de 16 µg/kg à 252 µg/kg ;
• de la teneur en scopolamine dans la gamme d’essai de 15 µg/kg à 246 µg/kg.
Krma: metode vzorčenja in analize - Določevanje alkaloidov rožička in tropanskih alkaloidov v sestavinah krme in krmni mešanici z LC-MS/MS
Ta dokument opisuje metodo za določanje posameznih alkaloidov rožička in tropanskih alkaloidov v nepredelanih žitih ter žitnih krmnih mešanicah z visokozmogljivo tekočinsko kromatografijo v povezavi s tandemsko masno spektrometrijo (LC-MS/MS). Alkaloidi rožička, zajeti v tej metodi, so: ergokorin, ergokorninin, ergokristin, ergokristinin, α-ergokriptin, α-ergokriptinin, ergometrin, ergometrinin, ergosin, ergosinin, ergotamin in ergotaminin. Tropanski alkaloidi, zajeti v tej metodi, so: atropin (hiosciamin) in skopolamin. Mejna vrednost kvantifikacije za vse zmesi naj bi bila vsaj 10 µg/kg. Ta metoda je bila interno potrjena v območju od 10 do 500 µg/kg za posamezne alkaloide. Zaznavanje koncentracij nad 500 µg/kg je mogoče z redčenjem ekstrakta vzorca, vendar ni bilo potrjeno.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-december-2019
Krma: metode vzorčenja in analize - Določevanje alkaloidov rožička in tropanskih
alkaloidov v sestavinah krme in krmni mešanici z LC-MS/MS
Animal feeding stuffs: Methods of sampling and analysis - Determination of ergot
alkaloids and tropane alkaloids in feed materials and compound feeds by LC-MS/MS
Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung der Alkaloide des
Mutterkorns und der Tropanalkaloiden in Einzelfuttermitteln und Mischfuttermitteln
mittels LC-MS/MS
Aliments des animaux: Méthodes d'échantillonnage et d'analyse - Détermination de la
teneur en alcaloïdes de l'ergot et en alcaloïdes tropaniques dans les matières premières
et les aliments composés par CL-SM/SM
Ta slovenski standard je istoveten z: EN 17256:2019
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 17256
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2019
EUROPÄISCHE NORM
ICS 65.120; 71.040.40
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of ergot alkaloids and tropane alkaloids in
feed materials and compound feeds by LC-MS/MS
Aliments des animaux : Méthodes d'échantillonnage et Futtermittel: Probenahme- und
d'analyse - Détermination de la teneur en alcaloïdes de Untersuchungsverfahren - Bestimmung der Alkaloide
l'ergot et en alcaloïdes tropaniques dans les matières des Mutterkorns und der Tropanalkaloiden in
premières et les aliments composés par CL-SM/SM Einzelfuttermitteln und Mischfuttermitteln mittels LC-
MS/MS
This European Standard was approved by CEN on 28 July 2019.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17256:2019 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 6
4 Principle . 6
5 Reagents . 6
6 Apparatus . 10
7 Procedure . 11
8 Analysis . 13
9 Results . 14
10 Precision . 15
11 Test report . 17
Annex A (informative) Precision data . 18
Annex B (informative) Example of LC-MS/MS conditions . 34
Annex C (informative) Example LC-MS/MS chromatograms of ergot alkaloids and tropane
alkaloids in a sample of complementary bovine feed . 36
Bibliography . 38
European foreword
This document (EN 17256:2019) has been prepared by Technical Committee CEN/TC 327 “Animal
feeding stuffs: Methods of sampling and analysis”, the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by March 2020, and conflicting national standards shall be
withdrawn at the latest by March 2020.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United
Kingdom.
Introduction
Ergot alkaloids are mycotoxins produced by species of the genus Claviceps. In Europe, Claviceps purpurea
is the most widespread fungal species. The fungus may infest plant species of the Poaceae family (true
grasses), producing dark coloured bodies, called sclerotia or (rye) ergot. Economically important cereal
grains that may be infected by C. purpurea are rye, wheat, triticale, barley, millet and oats. The sclerotia
contain a suit of ergot alkaloids, of which twelve have been recognized as major components:
ergocornine, ergocorninine, ergocristine, ergocristinine, ergocryptine, ergocryptinine, ergometrine,
ergometrinine, ergosine, ergosinine, ergotamine and ergotaminine. Ergocryptine and ergocryptinine
occur as a mixture of α- and β-isomers.
Tropane alkaloids are plant toxins produced by several species within the family of Solanaceae
(nightshades). The most relevant are Datura (thornapple), Hyoscyamus (henbane) and Atropa
(belladonna, deadly nightshade) species. Seeds and other plant parts contain substantial amounts of
atropine (hyoscyamine) and scopolamine, which are the most important toxic principles. Datura,
Hyoscyamus and Atropa species can be present as weeds in arable fields and may be co-harvested,
resulting in contaminated feed grains and feed products.
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 safety and health protection
measures and to ensure that regulatory and legal requirements are complied with.
1 Scope
This document describes a method for the determination of individual ergot alkaloids and tropane
alkaloids in unprocessed cereals and cereal-based compound feeds by high performance liquid
chromatography with tandem mass spectrometry (LC-MS/MS).
This document has been successfully validated by collaborative trial in the following matrices: rye, barley,
wheat, complete feed for bovine, porcine and poultry. Validation in buckwheat produced acceptable
results, but the relative standard reproducibility was higher for most analytes in comparison with the
other matrices. This may be related to the matrix. The validated range of the method is approximately
10 µg/kg to 250 µg/kg for individual alkaloids. Determination of concentrations above 250 µg/kg is
possible by applying a higher spiking level and dilution of the sample extract, but this has not been
validated in the collaborative trial.
This document is applicable for the determination, by means of one-point standard addition to the
sample, of:
• ergocornine in the tested range of 12 µg/kg to 221 µg/kg;
• ergocorninine in the tested range of 9 µg/kg to 196 µg/kg;
• ergocristine in the tested range of 14 µg/kg to 312 µg/kg;
• ergocristinine in the tested range of 12 µg/kg to 258 µg/kg;
• α-ergocryptine in the tested range of 10 µg/kg to 184 µg/kg;
• the sum of α- and β-ergocryptinine in the tested range of 8 µg/kg to 171 µg/kg;
• ergometrine in the tested range of 12 µg/kg to 174 µg/kg;
• ergometrinine in the tested range of 3 µg/kg to 172 µg/kg;
• ergosine in the tested range of 12 µg/kg to 226 µg/kg;
• ergosinine in the tested range of 9 µg/kg to 273 µg/kg;
• ergotamine in the tested range of 11 µg/kg to 443 µg/kg;
• ergotaminine in the tested range of 10 µg/kg to 273 µg/kg;
• atropine in the tested range of 16 µg/kg to 252 µg/kg;
• scopolamine in the tested range of 15 µg/kg to 246 µg/kg.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN ISO 3696:1995, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987)
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
4 Principle
The alkaloids are extracted by mixing 4 g of a homogenized, finely ground, sample with 40 ml of 0,4 %
formic acid in methanol:water (60:40). The mixture is shaken for 30 min. After centrifugation, a portion
of the supernatant is further purified by passing it through a 30 kDa ultrafilter. The filtrate is transferred
to a vial and it is analysed with a liquid chromatography – tandem mass spectrometry (LC-MS/MS)
system. A reverse-phase column in combination with an aqueous mobile phase with a pH > 7 and an
organic modifier is used to separate the analytes. Quantification is performed by one-point standard
addition to the sample.
5 Reagents
WARNING: Mycotoxins may be highly hazardous to health. Certain mycotoxins have carcinogenic,
mutagenic, toxic, teratogenic and immunotoxic effects. Inhalation or dermal exposure to mycotoxins may
occur at workplaces. Depending on the level of exposure, both acute and chronic effects are possible.
The tropane alkaloids atropine and scopolamine are acutely toxic, may be fatal if swallowed or if inhaled.
In addition, scopolamine may be fatal if in contact with skin.
5.1 Analytical standards
Analytical standards should have a demonstrated purity of at least 90 %, preferably of 95 % or higher.
NOTE 1 Ergotamine and ergometrine are listed as category I drug precursors and for these compounds an official
licence would be required and special procedures for storage and management would need to be followed
(EC 273/2004) [1].Atropine is the racemic mixture of L-(-)-hyoscyamine and D-(+)-hyoscyamine. In this method
the enantiomers of hyoscyamine are not separated. Both enantiomers produce identical fragmentation spectra.
In this method either a standard of atropine or hyoscyamine can be used.
NOTE 2 β-Ergocryptine and β-ergocryptinine are currently not available from commercial providers as
analytical standards of sufficient purity and quality. In this document α-ergocryptinine is used for the determination
of the sum of α- and β-ergocryptinine. For determination of β-ergocryptine, α-ergocryptine should be used, but this
has not been validated in the interlaboratory study. See under Clause 8 for more information on the analysis of these
alkaloids.
NOTE 3 Isotopically labelled analogues of ergometrine, ergometrinine, atropine and scopolamine are available
from commercial providers. Optionally, these isotopically labelled analogues can be used as internal standards,
provided that the mass increment in the molecule by the isotope labels is at least 3.
5.1.1 Ergocornine
5.1.2 Ergocorninine
5.1.3 Ergocristine
5.1.4 Ergocristinine
5.1.5. α-Ergocryptine
5.1.6. α-Ergocryptinine
5.1.7 Ergometrine (maleate)
5.1.8 Ergometrinine
5.1.9 Ergosine
5.1.10 Ergosinine
5.1.11 Ergotamine (tartra
...
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