kSIST FprEN 17853:2023
(Main)Animal feeding stuff: Methods of sampling and analysis - Determination of intact glucosinolates in feed materials and compound feed by LC-MS/MS
Animal feeding stuff: Methods of sampling and analysis - Determination of intact glucosinolates in feed materials and compound feed by LC-MS/MS
This document describes a method for the determination of individual intact glucosinolates in rapeseed
by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry
(MS/MS). Progoitrin, gluconapin, glucobrassicanapin, glucobrassicin, nasturtiin, neoglucobrassicin and
4-methoxyglucobrassicin are quantitatively determined. Other glucosinolates such as
4-hydroxyglucobrassicin, glucnapoliferin, glucoalysin, can only be qualitatively detected when
analytical standards become commercially available.
The method has been in-house validated for rapeseeds in the range 0.05-60 mmol/kg for individual
glucosinolates.
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von intakten Glucosinolaten in Futtermittel-Ausgangserzeugnissen und Mischfuttermitteln mittels LC‑MS/MS
Dieses Dokument legt ein Verfahren zur Bestimmung der einzelnen Glucosinolate in Ölsaaten, Ölsaaten- erzeugnissen und Alleinfuttermitteln durch Hochleistungsflüssigkeitschromatographie(HPLC) in Verbindung mit Tandem-Massenspektrometrie(MS/MS) fest.
Das in diesem Dokument beschriebene Verfahren wurde im Rahmen eines Ringversuchs mit den folgenden Matrices erfolgreich validiert: Rapssamen, Leindottersamen, Brassica-oleracea-Samen, gemischte Ölsaaten, Rapssamenflocken, Mischfuttermittel für Rinder, Schweine und Geflügel.
Das Verfahren ist anwendbar für die quantitative Bestimmung von Epiprogroitrin, Glucoalyssin, Glu
coarabin, Glucobrassicanapin, Glucobrassicin, Glucocamelinin, Glucoerucin, Glucoiberin, Gluconapin, Gluconapoleiferin, Gluconasturtiin, Glucoraphanin, Glucoraphenin, Glucotropaeolin, Homoglucocamelinin, 4-Hydroxyglucobrassicin, 4-Methoxyglucobrassicin, Neoglucobrassicin, Progoitrin, Sinalbin und Sinigrin.
Die im Ringversuch geprüften Konzentrationsbereiche für jedes einzelne Glucosinolat und für den Gesamt- gehalt an Glucosinolaten sind in Tabelle1 zusammengefasst.
Aliments pour animaux : Méthodes d’échantillonnage et d’analyse - Dosage des glucosinolates intacts dans les matières premières pour aliments des animaux et les aliments composés par CLHP MS/MS
Le présent document spécifie une méthode pour le dosage de différents glucosinolates intacts dans les graines oléagineuses, les produits oléagineux et les aliments composés par chromatographie liquide haute performance (CLHP) couplée à la spectrométrie de masse en tandem (SM/SM).
La méthode décrite dans le présent document a été validée par un essai interlaboratoires avec les matrices suivantes : graines de colza, graines de cameline, graines de Brassica oleracea, mélange de graines oléagineuses, flocons de graines de colza, aliments composés pour bovins, porcins et volaille.
La méthode est applicable pour la détermination quantitative de l’épiprogroitrine, la glucoalyssine, la glucoarabine, la glucobrassicanapine, la glucobrassicine, la glucocamélinine, la glucoérucine, la glucoibérine, la gluconapine, la gluconapoléiférine, la gluconasturtiine, la glucoraphanine, la glucoraphénine, la glucotropaéoline, l’homoglucocamélinine, la 4-hydroxyglucobrassicine, la 4-méthoxyglucobrassicine, la néoglucobrassicine, la progoitrine, la sinalbine et la sinigrine.
Les plages de concentration soumises à essai dans le cadre de l’essai interlaboratoires pour déterminer la teneur des différents glucosinolates et des glucosinolates totaux sont présentées dans le Tableau 1.
Krma: metode vzorčenja in analize - Ugotavljanje nepoškodovanih glukozinolatov v sestavinah krme in krmni mešanici z LC-MS/MS
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 17853:2022
01-julij-2022
Krma: metode vzorčenja in analize - Ugotavljanje nepoškodovanih glukozinolatov
v oljni ogrščici z LC-MS/MS
Animal feeding stuffs: Methods of sampling and analysis - Determination of intact
glucosinolates in rapeseed by LC-MS/MSFuttermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von intakten
Glucosinolaten in Rapssaat mittels LC-MS/MS
Aliments pour animaux - Méthodes d’échantillonnage et d’analyse - Dosage des
glucosinolates intacts dans les matières premières pour aliments des animaux et les
aliments composés par CL-SM/SMTa slovenski standard je istoveten z: prEN 17853
ICS:
65.120 Krmila Animal feeding stuffs
oSIST prEN 17853:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 17853:2022
DRAFT
EUROPEAN STANDARD
prEN 17853
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2022
ICS 65.120
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of intact glucosinolates in rapeseed by LC-
MS/MS
Aliments pour animaux - Méthodes d'échantillonnage Futtermittel - Probenahme- und
et d'analyse - Dosage des glucosinolates intacts dans Untersuchungsverfahren - Bestimmung von intakten
les matières premières pour aliments des animaux et Glucosinolaten in Rapssaat mittels LC-MS/MS
les aliments composés par CL-SM/SMThis draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 327.If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17853:2022 E
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Contents Page
European foreword ............................................................................................................................................ 3
Introduction .......................................................................................................................................................... 4
1 Scope .......................................................................................................................................................... 5
2 Normative references .......................................................................................................................... 6
3 Terms and definitions ......................................................................................................................... 6
4 Principle ................................................................................................................................................... 6
5 Reagents ................................................................................................................................................... 6
6 Apparatus .............................................................................................................................................. 11
7 Procedure .............................................................................................................................................. 12
8 LC-MS/MS analysis .............................................................................................................................. 14
9 Evaluation of results .......................................................................................................................... 16
10 Accuracy ................................................................................................................................................. 18
11 Test report ............................................................................................................................................. 19
Annex A (informative) Precision data ...................................................................................................... 20
Annex B (informative) Example of LC-MS/MS conditions ................................................................. 44
Annex C (informative) Examples of chromatograms .......................................................................... 47
Annex D (informative) Glucosinolate standards from commercial sources .............................. 49
Bibliography ....................................................................................................................................................... 51
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European foreword
This document (prEN 17853:2022) 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 document is currently submitted to the CEN Enquiry.This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of EU Directive(s).
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Introduction
Glucosinolates are a group of plant produced secondary metabolites predominantly found in the family
Brassicaceae (mustards and cabbages) ([1], [2], [3]). Many common vegetables such as broccoli, Brussels
sprouts, cabbage and cauliflower, belong to this plant family. At the same time species from the genera
Brassica, Camelina, Crambe, Rhaphanus and Sinapis are agricultural crops used for the production of plant
oils, such as rapeseed oils. The press cake is used as animal feed material. Glucosinolates are considered
undesirable substances in feed [4]. Glucosinolates in rapeseed and rapeseed products can also be
measured after enzymatic desulfation by high-performance liquid chromatography (HPLC) coupled with
UV detection. This method is described in standard EN ISO 9167 [5].WARNING — 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
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1 Scope
This document specifies a method for the determination of individual intact glucosinolates in oilseeds,
oilseed products and complete feeds by high performance liquid chromatography (HPLC) coupled with
tandem mass spectrometry (MS/MS).The method described in this document has been successfully validated by collaborative trial in the
following matrices: rape seed, camelina seed, Brassica oleracea seeds, mixed oilseeds, rape seed flakes,
compound feed for bovine, porcine and poultry.The method is applicable for the quantitative determination of epiprogroitrin, glucoalyssin, glucoarabin,
glucobrassicanapin, glucobrassicin, glucocamelinin, glucoerucin, glucoiberin, gluconapin,
gluconapoleiferin, gluconasturtiin, glucoraphanin, glucoraphenin, glucotropaeolin, homoglucocamelinin,
4-hydroxyglucobrassicin, 4-methoxyglucobrassicin, neoglucobrassicin, progoitrin, sinalbin and sinigrin.
The concentration ranges tested in the collaborative trial for each individual glucosinolate and for the
total glucosinolate content are summarized in Table 1.Table 1 — Summary of glucosinolate concentration ranges tested in the collaborative trial
Number of samples Tested concentration rangewith acceptable
mmol/kg
results
Min Max
Epiprogoitrin 7 0,01 0,93
Glucoalyssin 6 0,02 2,10
Glucoarabin 3 0,31 6,15
Glucobrassicanapin 5 0,01 0,38
Glucobrassicin 5 0,02 0,31
Glucocamelinin 3 0,82 16,1
Glucoerucin 3 1,07 15,6
Glucoiberin 3 1,51 18,5
Gluconapin 6 0,23 1,68
Gluconapoleiferin 5 0,01 0,33
Gluconasturtiin 7 0,01 11,0
Glucoraphanin 5 0,01 3,11
Glucoraphenin 1 15,6
Glucotropaeolin 2 0,03 18,3
Homoglucocamelinin 3 0,17 3,23
4-Hydroxyglucobrassicin 6 0,23 7,33
4-Methoxyglucobrassicin 1 0,16
Neoglucobrassicin 5 0,01 0,13
Progoitrin 6 0,62 14,8
Sinalbin 4 0,01 41,1
Sinigrin 3 0,25 23,7
Total content 8 1,48 117,3
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2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO
3696:1987)EN ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498:2012)
3 Terms and definitionsNo terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at https://www.electropedia.org/• ISO Online browsing platform: available at https://www.iso.org/obp
4 Principle
Glucosinolates are extracted from the homogenized sample with methanol:water (70:30). After
centrifugation, the extracts are diluted, filtered and measured by liquid chromatography coupled to
tandem mass spectrometry (HPLC-MS/MS). In oilseeds and oilseed products individual glucosinolates
are quantified by multi-level calibration using standards in aqueous solution. In compound feeds
individual glucosinolates are quantified by multi-level calibration using standards in blank feed matrix
extract.5 Reagents
5.1 Analytical standards
Analytical standards should have a demonstrated purity of at least 90 %, preferably of 95 % or higher.
NOTE In this section glucosinolate standards are listed that are currently available from at least one
commercial supplier. Depending on the intended application a selection of the listed glucosinolate standards can be
used.5.1.1 Epiprogoitrin (2-(S)-hydroxy-3-butenyl glucosinolate) or its potassium salt (CAS 19237-18-4)
5.1.2 Glucoalyssin (5-(methylsulfinyl)pentyl glucosinolate) or its potassium salt (CAS 499-37-6)
5.1.3 Glucoarabin (9-(methylsulfinyl)nonyl glucosinolate) or its potassium salt (CAS 67920-64-3)
5.1.4 Glucobrassicanapin (4-pentenyl glucosinolate) or its potassium salt (19041-10-2)
5.1.5 Glucobrassicin (3-indoylmethyl glucosinolate) or its potassium salt (CAS 4356-52-9)
5.1.6 Glucocamelinin (10-(methylsulfinyl)decyl glucosinolate) or its potassium salt (CAS 67884-10-0)
5.1.7 Glucoerucin (4-methoxythiobutyl glucosinolate) or its potassium salt (CAS 21973-56-8)
5.1.8 Glucoiberin (3-methylsulfinylpropyl glucosinolate) or its potassium salt (CAS 554-88-1)
5.1.9 Gluconapin (3-butenyl glucosinolate) or its potassium salt (CAS 19041-09-9)
5.1.10 Gluconapoleiferin (2-(S)-hydroxy-4-pentenyl glucosinolate) or its potassium salt
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Gluconapoleiferin is only available as a 1:1 mixture of 2-S and 2-R isomers. A purity of 50 % is taken for
2S-gluconapoleiferin.5.1.11 Gluconasturtiin (2-phenylethyl glucosinolate) or its potassium salt (CAS 499-30-9)
5.1.12 Glucoraphanin (4-methylsulfinylbutyl glucosinolate) or its potassium salt (CAS 21414-41-5)
5.1.13 Glucoraphenin (4-methylsulfinylbutenyl glucosinolate) or its potassium salt (CAS 28463-24-3)
5.1.14 Glucotropaeolin (benzyl glucosinolate) or its potassium salt (CAS 499-26-3)
5.1.15 Homoglucocamelinin (11-(methylsulfinyl)undecyl glucosinolate) or its potassium salt
(CAS 186037-18-3)5.1.16 4-Hydroxyglucobrassicin (4-hydroxy-3-indoylmethyl glucosinolate) or its potassium salt
(CAS 83327-20-2)5.1.17 4-Methoxyglucobrassicin (4-methoxy-3-indoylmethyl glucosinolate) or its potassium salt
(CAS 83327-21-3)5.1.18 Neoglucobrassicin (1-methoxy-3-indoylmethyl glucosinolate) or its potassium salt
(CAS 5187-84-8)5.1.19Progoitrin (2-(R)-hydroxy-3-butenyl glucosinolate) or its potassium salt (CAS 585-95-5)
5.1.20 Sinalbin (4-hydroxybenzyl glucosinolate) or its potassium salt (CAS 16411-05-5)
5.1.21 Sinigrin (2-propenyl glucosinolate), monohydrate or its potassium salt (CAS 3952-98-5)
5.2 Chemicals5.2.1 Methanol (HPLC grade)
5.2.2 Acetic acid (98-100 %)
5.2.3 Water
Water of LC-MS grade, double-distilled or water of grade1 as defined in EN ISO 3696.
5.3 Standard solutions5.3.1 General
Accurately weigh (6.1) between 5 and 10 mg of each standard (5.1.1-5.1.21) into a separate amber-
coloured glass bottle of 4 ml (6.8). Add a volume of extraction solvent (5.4.1) to produce a solution with
a concentration of 10 µmol/ml. Take into account the weight, the purity and the appearance form of the
standard (see NOTES 1-4). In Table 2 example calculations are given for the preparation of 1 ml stock
solution.NOTE 1 Glucosinolate standards are typically available as potassium salts. Some glucosinolate standards
additionally contain one molecule of water.NOTE 2 Most analytical standards of individual glucosinolates are typically obtained in 5-10 mg quantities. Since
glucosinolates are highly hygroscopic compounds it is preferable to use the solid standard only once.
NOTE 3 Depending on the intended application, a selection of the standard solutions listed below can be used.
NOTE 4 The stock standard solutions are stable for 24 months when stored below < −18 °C. Methanol:water
(70:30) is the preferred solvent because it provides a better stability of glucosinolates than water.
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Table 2 — Preparation of stock standard solutions
Molecular weight Weight standard for 1 ml
potassium form
standard solution of 10 µmol/ml
g/mol
Epiprogoitrin 427,48 4,2748
Glucoalyssin 489,63 4,8963
Glucoarabin 545,73 5,4573
Glucobrassicanapin 425,51 4,2551
Glucobrassicin 486,26 4,8626
Glucocamelinin 559,76 5,5976
Glucoerucin 459,61 4,5961
Glucoiberin 461,56 4,6156
Gluconapin 411,49 4,1149
Gluconapoleiferin 441,51 4,4151
Gluconasturtiin 461,16 4,6116
Glucoraphanin 475,66 4,7566
Glucoraphenin 473,58 4,7358
Glucotropaeolin 447,52 4,4752
Homoglucocamelinin 573,79 5,7379
4-Hydroxyglucobrassicin 502,56 5,0256
4-Methoxyglucobrassicin 516,59 5,1659
Neoglucobrassicin 516,59 5,1659
Progoitrin 427,49 4,2749
Sinalbin 463,52 4,6352
Sinigrin 397,47 3,9747
5.3.2 Epiprogoitrin (10 µmol/ml)
5.3.3 Glucoalyssin (10 µmol/ml)
5.3.4 Glucoarabin (10 µmol/ml)
5.3.5 Glucobrassicanapin (10 µmol/ml)
5.3.6 Glucobrassicin (10 µmol/ml)
5.3.7 Glucocamelinin (10 µmol/ml)
5.3.8 Glucoerucin (10 µmol/ml)
5.3.9 Glucoiberin (10 µmol/ml)
5.3.10 Gluconapin (10 µmol/ml)
5.3.11 Gluconapoleiferin (10 µmol/ml)
5.3.12 Gluconasturtiin (10 µmol/ml)
5.3.13 Glucoraphanin (10 µmol/ml)
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5.3.14 Glucoraphenin (10 µmol/ml)
5.3.15 Glucotropaeolin (10 µmol/ml)
5.3.16 Homoglucocamelinin (10 µmol/ml)
5.3.17 4-Hydroxyglucobrassicin (10 µmol/ml)
5.3.18 4-Methoxyglucobrassicin (10 µmol/ml)
5.3.19 Neoglucobrassicin (10 µmol/ml)
5.3.20 Progoitrin (10 µmol/ml)
5.3.21 Sinalbin (10 µmol/ml)
5.3.22 Sinigrin (10 µmol/ml)
5.3.23 Mixed standard solution (100 nmol/ml)
Pipette in a 10 ml volumetric flask 100 µl of each stock solution 5.3.1-5.3.22 (10 µmol/ml) and fill to the
mark with extraction solvent (5.4.1).NOTE 1 Depending on the intended application, a selection of the standard solutions can be used.
NOTE 2 When stored below < −18 °C the solution is stable for 1 year. Extraction solvent (methanol:water
(70:30)) is the preferred solvent because it provides a better stability of glucosinolates than water.
5.3.24 Mixed standard solution (10 nmol/ml)Pipette 2 ml of mixed standard solution 100 nmol/ml (5.3.23) in a 20-ml volumetric flask and fill to the
mark with extraction solvent (5.4.1).NOTE When stored below < −18 °C the solution is stable for 1 year Extraction solvent (methanol:water
(70:30)) is the preferred solvent because it provides a better stability of glucosinolates than water.
5.3.25 Mixed standard solution (1 nmol/ml)Pipette 500 µl of mixed standard solution 100 nmol/ml (5.3.23) in a 50 ml volumetric flask and fill to the
mark with extraction solvent (5.4.1).NOTE When stored below < −18 °C the solution is stable for 1 year. Extraction solvent (methanol:water
(70:30)) is the preferred solvent because it provides a better stability of glucosinolates than water.
5.3.26 Calibration solutions in aqueous solutionPrepare calibration solutions according to Table 3. Pipette directly in HPLC vials (see NOTE).
Table 3 — Preparation of calibration standards in aqueous solutionConcentration Mixed standard Mixed standard Mixed standard solution Water (5.2.3)
solution solution(nmol/ml) 100 nmol/ml (5.3.23) µl
1 nmol/ml 10 nmol/ml
(5.3.25) (5.3.24)
µl µl
Cal 0 0 0 0 1 000
Cal 0,01 10 0 0 990
Cal 0,02 20 0 0 980
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Concentration Mixed standard Mixed standard Mixed standard solution Water (5.2.3)
solution solution(nmol/ml) 100 nmol/ml (5.3.23) µl
1 nmol/ml 10 nmol/ml
(5.3.25) (5.3.24)
µl µl
Cal 0,05 50 0 0 950
Cal 0,10 0 10 0 990
Cal 0,25 0 25 0 975
Cal 0,50 0 50 0 950
Cal 1,0 0 0 10 990
Cal 2,5 0 0 25 975
Cal 5,0 0 0 50 950
Calibration standards should be prepared each new day of analysis.
NOTE The calibration points required depend on the concentrations expected in the samples (see Clause 7.3),
the dilution factor used (see Clause 7.3) and the dynamic range of the mass spectrometer (see Clause 8). It is advised
to use all calibration standards, but at least calibration standards Cal 1,3-8.5.4 Reagents
5.4.1 Extraction solvent: methanol:water (70:30)
Mix 700 ml methanol (5.2.1) with 300 ml water (5.2.3). The solvent is stored at room temperature and
can be used for one month.5.4.2 Mobile phase A: 0,1 % acetic acid in water
Pipette 1 ml of acetic acid (5.2.2) in 1 000 ml water (5.2.3). The solvent is stored at room temperature
and can be used for one month.NOTE The acetic acid concentration can be adjusted in the range of 0,01 % to 0,1 % to optimize the retention
of the analytes on the analytical column.5.4.3 Mobile phase B: methanol (5.2.1)
5.5 Quality control material
An appropriate material: a reference material (5.6), or a material with known natural contamination, or
a blank material fortified with known amounts of the glucosinolates, is included in each series and used
for quality purposes.5.6 Reference materials
Reference materials can be used for internal quality control purposes. Example reference materials are:
5.6.1 ERM®-BC366R Rapeseed5.6.2 ERM®-BC190R Rapeseed
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5.6.3 ERM®-BC367R Rapeseed
6 Apparatus
Usual laboratory equipment and, in particular, the following items
6.1 Analytical balance with a precision of 0,02 mg
6.2 Analytical balance with a precision of 1 mg
6.3 (Micro)grinder
6.4 Polypropylene centrifuge tubes 50 ml with screw cap
6.5 Water bath
Capable of maintaining a temperature of 75 °C ± 1 °C.
6.6 Vortex mixer or minishaker
6.7 Vertical or horizontal shaker adjustable
6.8 Amber coloured glass bottle 4 ml size with screw cap
6.9 HPLC autosampler vial glass or polypropylene 1,5 ml
6.10 Centrifuge suitable for use with the 50 ml centrifuge tubes (6.4)
6.11 Volumetric flasks calibrated 10, 20, 50 ml
6.12 Calibrated micrometric pipette(s)
6.13 HPLC system consisting of:
6.13.1 Autosampler thermostated
Capable of maintaining a temperature of 10 ± 1 °C.
6.13.2 Binary pump system
Capable of delivering a binary gradient at flow rates appropriate for the analytical column in use with
sufficient accuracy.6.13.3 Column oven thermostated
Capable of maintaining a temperature of 50 ± 1 °C.
6.13.4 Analytical column
Containing C18 reversed phase packing material, capable of the base-line separation of analytes with
identical molecular mass. A C18 stationary phase with charged surface hybrid chemistry has shown to
work well.6.13.5 Pre-column, optional
Containing the same stationary phase material as the analytical column and with appropriate dimensions.
6.14 Tandem mass spectrometerCapable of performing multiple selected reaction monitoring in negative mode, with a sufficiently wide
dynamic range, sufficient scan speed and capable of unit mass separation and equipped with a computer-
based data processing system. Any ionization source giving sufficient yield may be employed.
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7 Procedure
7.1 Sample pre-treatment
Laboratory samples should be taken and prepared in accordance with European legislation [6] where
applicable or, in any other case with EN ISO 6498.Homogenize samples in a grinder (6.3) to < 1 mm.
7.2 Test portion
7.2.1 Oilseeds and oilseed products
The amount of homogenized oilseed or oilseed product sample (7.1) examined is 1,0 ± 0,02 g.
The amount of homogenized oilseed or oilseed product sample may be reduced to 0,5 ± 0,01 g. The
amount of extraction solvent (Clause 7.3.1) shall in that case be reduced accordingly.
7.2.2 Compound feedThe amount of homogenized compound feed sample (7.1) examined is 1,0 ± 0,02 g.
7.3 Extraction
7.3.1 Oilseeds and oilseed products
Weigh a test portion of 1,0 ± 0,02 g homogenized sample (7.2.1) into a centrifuge tube of 50 ml (6.4). Add
25 ml extraction solvent (5.4.1), vortex for 10 s and place the tube in a water bath at 75 °C (6.5) (see
NOTE 1). Heat the sample for 15 min and then let cool down to room temperature. Place the tube for 60
min in a shaker rotating at moderate speed (6.7).Centrifuge the tube for 5 min at 2 000 g at room temperature (6.10). Transfer 5 µl of the supernatant to
a HPLC vial (6.9) and add 995 µl of water and close the vial (see NOTE 2).NOTE 1 An extraction solvent containing 70 % methanol gives the best compromise between analyte stability
and extraction efficiency. An extraction solvent with a higher organic content (e.g. 80 % methanol) increases the
stability of the analytes (slow degradation by myrosinase), but also results in a lower extraction efficiency. An
extraction solvent with a lower organic content (e.g. 60 % methanol or 50 % ethanol) results in a comparable
extraction efficiency, but also in a strongly increased myrosinase activity. This can result in partial degradation of
the analytes and in a reduced reproducibility. ISO 9167:2019 describes an alternative extraction procedure for
rapeseed and rapeseed meals using 50 % ethanol as extraction solvent. This alternative extraction procedure was
not tested in this collaborative trial.NOTE 2 In specific products concentrations can be present that exceed the working range of the calibration
curve. For these products the extract is further diluted with water to obtain a concentration that falls within the
working range of the calibration curve.7.3.2 Compound feed
Weigh a test portion of 1,0 ± 0,02 g homogenized sample (7.2.2) into a centrifuge tube of 50 ml (6.4). Add
25 ml extraction solvent (5.4.1), vortex for 10 s and place the tube in a water bath at 75 °C (6.5). Heat the
sample for 15 min and then let cool down to room temperature. Place the tube for 60 min in a shaker
rotating at moderate speed (6.7)Centrifuge the tube for 5 min at 2 000 g at room temperature (6.10). Transfer 25 µl of the supernatant to
a HPLC vial (6.9) and add 975 µl of water and close the vial.---------------------- Page: 14 ----------------------
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7.3.3 Recovery sample for compound feed
For preparation of the recovery sample take a representative blank feed sample (5.5) (see NOTE),
preferably the same that is used for the preparation of the calibration range in blank extract (7.3.4).
Weigh a test portion of 1,0 ± 0,02 g homogenized sample (7.2.2) into a centrifuge tube of 50 ml (6.4). Add
1,00 ml of mixed standard solution 100 nmol/ml (5.3.22) to the sample. This is equivalent to
0,1 mmol/kg. Vortex for 10 s.Add 25 ml extraction solvent (5.4.1), vortex for 10 s and place the tube in a water bath at 75 °C (6.5). Heat
the sample for 15 min and then let cool down to room temperature. Place the tube for 60 min in a shaker
rotating at moderate speed (6.7).Centrifuge the tube for 5 min at 2 000 g at room temperature (6.10). Transfer 25 µl of the supernatant to
a HPLC vial (6.9) and add 975 µl of water and close the vial.NOTE A sample shown by a preceding analysis not to contain the target analytes in a concentration above the
limit of detection.7.3.4 Preparation of calibration standards in blank feed extract
For preparation of the calibration curve in matrix take a representative blank feed sample (5.5) (see
NOTE).Weigh a test portion of 1,0 ± 0,02 g homogenized sample (7.2.2) into a centrifuge tube of 50 ml (6.4). Add
25 ml extraction solvent (5.4.1), vortex for 10 s and place the tube in a water bath at 75 °C (6.5). Heat the
sample for 15 min and then let cool down to room temperature. Place the tube for 60 min in a shaker
rotating at moderate speed (6.7)Centrifuge the tube for 5 min at 2 000 g at room temperature (6.10). Transfer an aliquot of 1 ml to a new
centrifuge tube of 50 ml (6.4), add 39 ml of water and mix well. This diluted extract is used to prepare
calibration solutions according to Table 4. Pipette directly in HPLC vials.NOTE A sample shown by a preceding analysis not to contain the target analytes in a concentration above the
limit of detection.Table 4 — Preparation of calibration standards in blank feed extract
Concentration Mixed standard Mixed standard Mixed standard Blank
solution solution solution feed
nmol/ml
extract
1 nmol/ml 10 nmol/ml 100 nmol/ml
(7.3.4)
(5.3.23)
(5.3.25) (5.3.24)
µl µl
Cal 1 0 0 0 0 1 000
Cal 2 0,01 10 0 0 990
Cal 3 0,02 20 0 0 980
Cal 4 0,05 50 0 0 950
Cal 5 0,10 0 10 0 990
Cal 6 0,25 0 25 0 975
Cal 7 0,50 0 50 0 950
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oSIST prEN 17853:2022
prEN 17853:2022 (E)
Concentration Mixed standard Mixed standard Mixed standard Blank
solution solution solution feed
nmol/ml
extract
1 nmol/ml 10 nmol/ml 100 nmol/ml
(7.3.4)
(5.3.23)
(5.3.25) (5.3.24)
µl µl
Cal 8 1,0 0 0 10 990
Cal 9 2,5 0 0 25 975
Cal 5,0 0 0 50 950
8 LC-MS/MS analysis
8.1 General
Chromatographic and mass spectrometric conditions can be chosen freely. The optimal measuring
conditions strongly depend on the instrumentation used. However, some important criteria and
parameters with respect to the chromatographic separation and detection of the analytes are discussed
below.Analytical columns containing C18 reversed phase packing material can be used for the separation of
analytes with identical molecular mass. Attention should be paid to the more polar glucosinolates, eluting
early in the chromatogram. These analytes are more sensitive to small differences in the composition of
the mobile phase resulting in unstable retention times and distorted peak shapes. A C18 stationary phase
with charged surface hybrid chemistry has shown to work well, providing improved retention and peak
shapes.The injection volume should be optimized for the column dimension and the sensitivity of the mass
spectrometric system. Depending on the sensitivity and linear dynamic range of the mass spectrometric
instrument it can be necessary to dilute the calibration standards and sample extracts by an additional
factor with water.Mass spectrometric conditions should be appropriate to measure the analytes with sufficient sensitivity
and specificity. Glucosinolates are best analysed in negative ionizat...
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