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.

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Publication Date
08-Oct-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
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SLOVENSKI STANDARD
SIST EN 17256:2019
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
SIST EN 17256:2019 en,fr,de

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

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SIST EN 17256:2019
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SIST EN 17256:2019
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.
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SIST EN 17256:2019
EN 17256:2019 (E)
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

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EN 17256:2019 (E)
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.
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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.
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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)

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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
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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 (tartrate)
5.1.12 Ergotaminine
5.1.13 Atropine or hyoscyamine
5.1.14 Scopolamine (hydrochloride)
5.2 Chemicals
5.2.1. Acetonitrile, LC-MS or HPLC quality
5.2.2. Methanol, LC-MS or HPLC quality
5.2.3. Formic acid, 98 to 100 %
5.2.4. Ammonium carbonate, anhydrous
5.2.5 Ammonia, 25 %
5.2.6 Water

Water of LC-MS grade, double-distilled or water of grade 1 as defined in EN ISO 3696:1995.

5.3 Standard solutions

Accurately weigh (6.1) between 5 mg and 6 mg of each standard (5.1.1 to 5.1.14) into a separate amber-

coloured glass bottle of 60 ml (6.12). Add a volume of acetonitrile (5.2.1) to produce a solution with a

concentration of 100 µg/ml. Take into account the weight, the purity and the appearance form of the

standard.

Many ergot alkaloid standards are commercially available only in small amounts (5 mg or less).

Preferably a standard should be prepared using a quantity of at least 5 mg. However, when the standard

is only available in a quantity of 5 mg or less, a smaller quantity can be weighed in, provided an accurate

weight measurement can be guaranteed. In principle this is preferred above flushing the contents of the

container with several volumes of solvent to dissolve and collect the material. Nevertheless, some ergot

standards may only be available as dried down standards that need to be reconstituted in solvent.

Stock solutions are stable for 6 months below -18 °C. However, ergot alkaloid standards are sensitive to

light and may epimerise rapidly in the presence of acid or base. Standard solutions should be kept in

amber coloured glass bottles and they should be stored at a temperature below -18 °C. Acetonitrile is the

preferred solvent because the rate of epimerisation is lowest in this solvent.
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Before use, stock solutions and mixed standard solutions that have been stored in the freezer, may need

to be vortexed (6.7) or sonicated (6.8) to ensure the complete dissolution of the analytes.

To further reduce the impact of epimerisation, mixed standard stock solutions containing the

corresponding epimeric pairs may be prepared.
5.3.1 Ergocornine stock solution ,100 µg/ml
5.3.2 Ergocorninine stock solution, 100 µg/ml
5.3.3 Ergocristine stock solution, 100 µg/ml
5.3.4 Ergocristinine stock solution, 100 µg/ml
5.3.5 α-Ergocryptine stock solution, 100 µg/ml
5.3.6 α-Ergocryptinine stock solution, 100 µg/ml
5.3.7 Ergometrine stock solution, 50 µg/ml

NOTE Ergometrine can be difficult to dissolve (particularly when present as the maleate salt form).

The solution can be sonicated (6.8) for up to 30 min or placed in a water bath of 60 °C for up to 30 min. Sonication

is preferred above warming in a water bath. Alternatively, a small amount of water (10 %) can be added to the

solvent to facilitate solvation of the salt form.
5.3.8 Ergometrinine stock solution, 100 µg/ml
5.3.9 Ergosine stock solution, 100 µg/ml
5.3.10 Ergosinine stock solution, 100 µg/ml
5.3.11 Ergotamine stock solution, 100 µg/ml
5.3.12 Ergotaminine stock solution, 100 µg/ml
5.3.13 Atropine stock solution, 100 µg/ml
NOTE Instead of atropine, hyoscyamine can be used.
5.3.14 Scopolamine stock solution, 100 µg/ml
5.3.15 Mixed standard solution, 5 µg/ml

Pipette (6.10) 1 ml of each stock solution 5.3.1 to 5.3.7 and 5.3.9 to 5.3.14 (100 µg/ml) and 2 ml of stock

solution 5.3.8 (50 µg/ml) in a calibrated volumetric flask of 20 ml (6.11) and make up the volume with

acetonitrile (5.2.1) and mix. Transfer the contents to an amber coloured glass bottle of 30 ml (6.12).

The solution can be used for 12 months when stored in the dark at below −18 °C.

NOTE The prepared mixed standard solution can be divided in 4 portions of 5 ml and stored in separate amber

coloured glass bottles of 10 ml (6.12).
5.3.16 Mixed standard solution, 1 000 ng/ml

Pipette (6.10) 4 ml of the mixed standard solution (5 µg/ml) (5.3.15) in a calibrated volumetric flask of

20 ml (6.11) and make up the volume with acetonitrile (5.2.1) and mix. Transfer the contents to an amber

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coloured glass bottle of 30 ml (6.12). The solution can be used for 12 months when stored in the dark at

below −18 °C.

NOTE A new mixed standard solution can be prepared every 3 months, by using a new, unused, portion of the

mixed standard solution 5.3.15.
5.3.17 Mixed standard solution, 200 ng/ml

Pipette (6.10) 4 ml of the mixed standard solution (1 000 ng/ml) (5.3.16) in a calibrated volumetric flask

of 20 ml (6.11) and make up the volume with acetonitrile (5.2.1) and mix. Transfer the contents to an

amber coloured glass bottle of 30 ml (6.12). The solution can be used for 12 months when stored in the

dark at below −18 °C.

NOTE A new mixed standard solution can be prepared every 3 months, from a freshly prepared mixed standard

solution 5.3.16.
5.3.18 Mixed standard solution, 50 ng/ml

Pipette (6.10) 5 ml of the mixed standard solution (200 ng/ml) (5.3.17) in a calibrated volumetric flask

of 20 ml (6.11) and make up the volume with acetonitrile (5.2.1) and mix. Transfer the contents to an

amber coloured glass bottle of 30 ml (6.12). The solution can be used for 12 months when stored in the

dark at below −18 °C.

NOTE A new mixed standard solution can be prepared every 3 months, from a freshly prepared mixed standard

solution 5.3.17.
5.3.19 Working standard solution, 10 ng/ml

Dilute the mixed standard solution 1 000 ng/ml (5.3.16) with extraction solvent (5.4.1) in a 1 to 100 ratio

by volume. Prepare a fresh solution every new day of analysis. Store at +4 °C.
5.4 Reagent solutions
5.4.1 Extraction solvent 0,4 % formic acid in methanol:water (60:40)

Mix 600 ml methanol (5.2.2), 400 ml water (5.2.6) and 4 ml formic acid (5.2.3) in a glass bottle of

1 000 ml. This solution is stored at room temperature and can be used for 3 months.

5.4.2 LC-MS/MS mobile phase A: 10 mM ammonium carbonate, pH 10,0

Weigh 0,96 g of ammonium carbonate (5.2.4) and dissolve in 1 000 ml water (5.2.6) in a glass bottle of

1 000 ml. Add with a positive displacement pipette (6.10) ammonia 25 % (5.2.5) to adjust the pH to

10,0 ± 0,1 using a pH meter (6.9). This solution is stored at room temperature and can be used for

1 month.

A mobile phase A consisting of 10 mM ammonium carbonate pH 9,0 has been shown to work equally well.

Weigh 0,96 g of ammonium carbonate and dissolve in 1 000 ml water (5.2.6) in a glass bottle of 1 000 ml.

If necessary, adjust the pH to 9,0 ± 0,1 with formic acid (5.2.3) or ammonia 25 % (5.2.5) using a pH meter

(6.9). This solution is stored at room temperature and can be used for 1 month.

A mobile phase A consisting of a solution of 6 mM ammonia in water has been shown to work equally

well. Mix 500 µl ammonia 25 % (5.2.5) with 1 000 ml water (5.2.6) in a glass bottle of 1 000 ml. This

solution is stored at room temperature and can be used for 1 week.
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EN 17256:2019 (E)
6 Apparatus
Usual laboratory equipment and, in particular, the following items.
6.1 Analytical balance, with a mass resolution of 0,1 mg or better
6.2 Laboratory balance, with a mass resolution of 0,1 g or better
6.3 Mill

Single or multiple mills capable of grinding test materials to particle sizes of ≤ 500 µm.

6.4 Mixer
Capable of sufficiently homogenizing the ground test materials.
6.5 Vertical or horizontal shaker, adjustable
6.6 Centrifuge

Suitable for 50 ml centrifuge tubes (6.13) and ultra-filters (6.14) and capable of generating a relative

centrifugal force (rcf) of 3 000 g.
6.7 Minishaker or vortex mixer
6.8 Ultrasonic bath
6.9 pH meter
6.10 Pipettors

Adjustable, suitable for organic solvents, properly calibrated, with appropriate tips.

6.11 Volumetric flasks, calibrated, 20 ml
6.12 Amber coloured glass bottle of 10, 30 and 60 ml size with screw cap
6.13 Centrifuge tube, polypropylene, 50 ml with screw cap
6.14 Ultrafilter (centrifugal filter unit) with a cut off of 30 kDa

The centrifugal filter unit shall be compatible with 60 % methanol and 0,4 % formic acid solution. The

membrane should be made of regenerated cellulose. The ultrafilter should have a capacity of 4 ml and the

membrane should have an active surface of ≥ 300 mm .
6.15 HPLC vial, glass or polypropylene, 2 ml
6.16 HPLC system consisting of
6.16.1 Autosampler, thermostated
Capable of maintaining a temperature of 10 °C± 1 °C.
6.16.2 Binary pump system

Capable of delivering a binary gradient at flow rates appropriate for the analytical column in use with

sufficient accuracy.
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EN 17256:2019 (E)
6.16.3 Column oven, thermostated
Capable of maintaining a temperature of least 50 °C ± 1 °C.
6.16.4 Analytical column

Containing high pH-resistant cross-linked C18 reversed phase packing material, capable of the base-line

separation of analytes with identical molecular mass.
6.16.5 Pre-column, optional

With the same stationary phase material as the analytical column and with appropriate dimensions.

6.17 Tandem mass spectrometer

Capable of performing multiple selected reaction monitoring in positive 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.
7 Procedure
7.1 General

Animal feed is a complex matrix containing a wide range of ingredients in varying amounts. For this

reason it is not possible, due to variable and sample-dependent matrix effects

(suppression/enhancement), to conduct quantification of the analytes in a sample by a direct comparison

with a set of calibration samples. Each feed sample is quantified by means of standard addition to the

sample prior to extraction. It is not necessary to correct the results for recovery.

Depending on the sensitivity and linear range of the mass spectrometric instrument it may be necessary,

to dilute the sample extracts (7.4, 7.5, 7.6) by an appropriate factor with extraction solvent (5.4.1), taking

into account the sum of the expected concentration of the analyte in the sample and the added

concentration, to keep the response of the detector within the dynamic range of the mass spectrometer.

Alternatively, the injection volume of the sample can be reduced, within the calibrated range of the

injection system.
7.2 Sample pre-treatment

Laboratory samples should be taken and prepared in accordance with European legislation [2] where

applicable or, in any other case with EN ISO 6498.

In order to obtain a homogeneous sample it is essential that the sample is finely ground (≤0,5 mm) and

homogenized with a grinding mill (6.3). Depending on the starting material (ground or unground

material), it may advisable to first grind the sample through a sieve of 1 mm to prevent excessive heat

formation during milling, which could lead to partial decomposition of the analytes. Next, the sample is

ground through a sieve of 0,5 mm. The samples are stored at room temperature.
7.3 Test sample amount
The amount of homogenized test sample examined is 4,0 g ± 0,1 g.

A larger test sample size may be used by the laboratory in order to improve the representativeness of the

sample. The amount of extraction solvent shall in that case be adjusted accordingly (7.4). The use of a

larger test sample size should not be used as a solution for grinding the sample through a sieve larger

than 0,5 mm.
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EN 17256:2019 (E)
7.4 Sample extraction

Weigh a test portion of 4,0 g ± 0,1 g in a centrifuge tube of 50 ml (6.13). Add 40 ml extraction solvent

(5.4.1) to the centrifuge tube, close and shake vigorously by hand or vortex (6.7). Place the tube for 30 min

in a shaker moving at moderate speed (6.5).

Centrifuge the tube for 15 min at 3 000 g at room temperature (6.6). Transfer 2 ml of the supernatant to

an ultrafilter tube (6.14). Centrifuge the tube for 15 min at 3 000 g at room temperature (6.6) (see NOTE).

Transfer 1 ml of the ultrafiltrate to a HPLC vial (6.15) and close the vial.

NOTE The required time will depend on the matrix type and the minimum volume needed for preparation of

the sample extracts.
7.5 Standard addition sample

For preparation of the standard addition sample weigh a test portion of 4,0 g ± 0,1 g in a centrifuge tube

of 50 ml (6.13). Spike (6.10) the test portion with 200 µl of mixed standard solution 5 µg/ml (5.3.15).

This is equivalent to 250 µg/kg. Vortex (6.7) the spiked sample for 15 s and let soak for 30 min.

Add 40 ml extraction solvent (5.4.1) to the centrifuge tube, close and shake vigorously by hand or vortex

(6.7). Place the tubes for 30 min in a shaker rotating at moderate speed (6.5).

Centrifuge the tube for 15 min at 3 000 g at room temperature (6.6). Transfer 2 ml of the supernatant to

an ultrafilter tube (6.14). Centrifuge the tube for 15 min at 3 000 g at room temperature (6.6). (see NOTE).

Transfer 1 ml of the ultrafiltrate to a HPLC vial (6.15) and close the vial.

NOTE The required time will depend on the matrix type and the minimum volume needed for preparation of

the sample extracts.
7.6 Preparation of calibration range in blank extract

A calibration curve in blank extract is prepared for identification of the analytes (9.1) and to check the

linearity of the mass spectrometric detector (9.2.2). It is not to be used for quantification of samples. A

blank sample is used.

NOTE 1 A blank sample is a sample shown by a preceding analysis to contain none of the ergot alkaloids and

tropane alkaloids above the limit of detection (LOD). The matrix should be of similar composition as the samples to

be investigated, i.e. an unprocessed cereal or a cereal-based compound feed.

Weigh a test portion of 4,0 g ± 0,1 g in a centrifuge tube of 50 ml (6.13). Add 40 ml extraction solvent

(5.4.1) to the centrifuge tube, close and shake vigorously by hand or vortex (6.7). Place the centrifuge

tube for 30 min in a shaker rotating at moderate speed (6.5).

Centrifuge the tube for 15 min at 3 000 g at room temperature (6.6). Transfer up to 9 aliquots of 2 ml of

the supernatant to ultrafilter tubes (6.14).

NOTE 2 The number of calibration extracts required depends on the concentrations expected in the samples

(7.4 and 7.5) and the dynamic range of the mass spectrometer. It is advised to include at least calibration extracts

1 and 3 to 8.
Centrifuge the tubes for 15 min at 3 000 g at room temperature (6.6).

NOTE 3 The required time will depend on the matrix type and the minimum volume needed for preparation of

the sample extracts.

Aliquots of the ultrafiltrate are used to prepare calibration extracts according to Ta

...

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