SIST EN 17256:2019

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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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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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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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
2
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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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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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
...