SIST EN 17853:2023

SLOVENSKI STANDARD
01-september-2023
Krma: metode vzorčenja in analize - Ugotavljanje nepoškodovanih glukozinolatov
v sestavinah krme in krmni mešanici z 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
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von intakten
Glucosinolaten in Futtermittel-Ausgangserzeugnissen und Mischfuttermitteln mittels LC-
MS/MS
Alimentation animale : Méthodes d’échantillonnage et d’analyse - Dosage des
glucosinolates intacts dans les matières premières pour l’alimentation animale et les
aliments composés par CLHP MS/MS
Ta slovenski standard je istoveten z: EN 17853:2023
ICS:
65.120 Krmila Animal feeding stuffs
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 17853
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2023
EUROPÄISCHE NORM
ICS 65.120
English Version
Animal feeding stuff: Methods of sampling and analysis -
Determination of intact glucosinolates in feed materials
and compound feed by LC-MS/MS
Alimentation animale : Méthodes d'échantillonnage et Futtermittel - Probenahme- und
d'analyse - Dosage des glucosinolates intacts dans les Untersuchungsverfahren - Bestimmung von intakten
matières premières pour l'alimentation animale et les Glucosinolaten in Futtermittel-Ausgangserzeugnissen
aliments composés par CLHP MS/MS und Mischfuttermitteln mittels LC-MS/MS
This European Standard was approved by CEN on 3 March 2023.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17853:2023 E
worldwide for CEN national Members.

Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 7
3 Terms and definitions . 7
4 Principle . 7
5 Reagents . 7
5.1 Analytical standards . 7
5.2 Chemicals . 8
5.3 Standard solutions . 8
5.4 Reagents . 11
5.5 Quality control material . 11
6 Apparatus . 12
7 Procedure . 13
7.1 Sample pre-treatment . 13
7.2 Test portion . 13
7.2.1 Oilseeds and oilseed products . 13
7.2.2 Compound feed . 13
7.3 Extraction . 13
7.3.1 Oilseeds and oilseed products . 13
7.3.2 Compound feed . 13
7.3.3 Recovery sample for compound feed . 14
7.3.4 Preparation of calibration standards in blank feed extract . 14
8 LC-MS/MS analysis . 15
8.1 General. 15
8.2 Analysis sequence . 16
8.2.1 Oilseeds and oilseed products . 16
8.2.2 Compound feed . 16
9 Evaluation of results . 17
9.1 Identification . 17
9.2 Quantification . 17
9.2.1 General. 17
9.2.2 Linearity of the calibration curve . 17
9.2.3 Calculation of the glucosinolate concentration in oilseed or oilseed product . 17
9.2.4 Calculation of the glucosinolate concentration in compound feed. 18
9.3 Expression of results . 19
10 Precision . 19
10.1 General. 19
10.2 Repeatability . 19
10.3 Reproducibility . 19
11 Test report . 20
Annex A (informative) Precision data . 21
Annex B (informative) Example of LC-MS/MS conditions . 45
B.1 General . 45
B.2 Chromatographic conditions . 45
B.3 MS conditions . 46
Annex C (informative) Examples of chromatograms . 48
Annex D (informative) Glucosinolate standards from commercial sources . 50
Bibliography . 52

European foreword
This document (EN 17853:2023) has been prepared by Technical Committee CEN/TC 327 “Animal
feeding stuffs: Methods of sampling and analysis”, the secretariat of which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by October 2023, and conflicting national standards shall
be withdrawn at the latest by October 2023.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a Standardization Request given to CEN by the European
Commission and the European Free Trade Association.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the United
Kingdom.
Introduction
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
measures and to ensure that regulatory and legal requirements are complied with.
1 Scope
This document specifies a method for the determination of individual intact glucosinolates in feed
materials including oilseeds and oilseed products and in compound feeds by high performance liquid
chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS).
The method specified 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
Glucosinolate Number of samples Tested concentration range
with 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
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)
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminology 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/ui
4 Principle
Glucosinolates are extracted from the homogenized sample with methanol:water (70:30) (V:V). 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 (see Annex D). 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 (CAS 19041-10-2)
5.1.5 Glucobrassicin (3-indolylmethyl 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
(CAS 19764-03-5)
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-methylsulfinylundecyl glucosinolate) or its potassium salt
(CAS 186037-18-3)
5.1.16 4-Hydroxyglucobrassicin (4-hydroxy-3-indolylmethyl glucosinolate) or its potassium salt
(CAS 83327-20-2)
5.1.17 4-Methoxyglucobrassicin (4-methoxy-3-indolylmethyl glucosinolate) or its potassium salt
(CAS 83327-21-3)
5.1.18 Neoglucobrassicin (1-methoxy-3-indolylmethyl glucosinolate) or its potassium salt
(CAS 5187-84-8)
5.1.19 Progoitrin (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 Chemicals
5.2.1 Methanol, LC-MS grade
5.2.2 Acetic acid, 98 % to 100 %, HPLC grade
5.2.3 Water
Water of LC-MS grade, double-distilled or water of grade 1 as specified in EN ISO 3696.
5.3 Standard solutions
5.3.1 General
Accurately weigh (6.1) between 5 mg 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 mg and 10 mg
quantities. Since glucosinolates are highly hygroscopic compounds, the solid standard is only used 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 < −18 °C. Methanol:water (70:30)
(V:V) is the preferred solvent because it provides a better stability of glucosinolates than water.
Table 2 — Preparation of stock standard solutions
Glucosinolate Molecular weight Weight standard for 1 ml
potassium form standard solution of 10 µmol/ml
g/mol mg
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)
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 (6.12) in a 10 ml volumetric flask (6.11) 100 µl of each stock solution 5.3.2-5.3.22 (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 < −18 °C the solution is stable for 1 year. Extraction solvent (methanol:water (70:30) (V:V))
is the preferred solvent because it provides a better stability of glucosinolates than water.
5.3.24 Mixed standard solution (10 nmol/ml)
Pipette (6.12) 2 ml of mixed standard solution 100 nmol/ml (5.3.23) in a 20-ml volumetric flask (6.11)
and fill to the mark with extraction solvent (5.4.1).
NOTE When stored < −18 °C the solution is stable for 1 year. Extraction solvent (methanol:water (70:30) (V:V))
is the preferred solvent because it provides a better stability of glucosinolates than water.
5.3.25 Mixed standard solution (1 nmol/ml)
Pipette (6.12) 500 µl of mixed standard solution 100 nmol/ml (5.3.23) in a 50 ml volumetric flask (6.11)
and fill to the mark with extraction solvent (5.4.1).
NOTE When stored < −18 °C the solution is stable for 1 year. Extraction solvent (methanol:water (70:30) (V:V))
is the preferred solvent because it provides a better stability of glucosinolates than water.
5.3.26 Calibration solutions in aqueous solution
Prepare calibration solutions according to Table 3. Pipette (6.12) directly in HPLC vials (see NOTE).
Table 3 — Preparation of calibration standards in aqueous solution
Code Concentration Mixed standard Mixed standard Mixed standard Water
solution solution solution (5.2.3)
(nmol/ml)
1 nmol/ml 10 nmol/ml 100 nmol/ml µl
(5.3.25) (5.3.24) (5.3.23)
µl 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
Cal 8 1,0 0 0 10 990
Cal 9 2,5 0 0 25 975
Cal 10 5,0 0 0 50 950
Calibration standards should be prepared each new day of analysis. It is advised to use all calibration
standards, but at least calibration standards Cal 1,3-8.
NOTE The calibration points required depend on the concentrations expected in the samples (see 7.3), the
dilution factor used (see 7.3) and the dynamic range of the mass spectrometer (see Clause 8).
5.4 Reagents
5.4.1 Extraction solvent: methanol:water (70:30) (V:V)
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 (6.12) 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 is included in each series and used for quality purposes. This material can be a
reference material , or a material with known natural contamination, or a blank material fortified with
known amounts of the glucosinolates.

ERM®-BC366R Rapeseed, ERM®-BC190R Rapeseed and ERM®-BC367R Rapeseed are examples of suitable
products available commercially. This information is given for the convenience of users of this document and does
not constitute an endorsement by CEN of these products.
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 Analytical balance with a mass resolution of 1 mg or better
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 ml, 20 ml, 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 °C ± 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 °C ± 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 spectrometer
Capable 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.
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 1,00 g ± 0,02 g.
The amount of homogenized oilseed or oilseed product sample may be reduced to 0,50 g ± 0,01 g. The
amount of extraction solvent (7.3.1) shall in that case be reduced accordingly.
7.2.2 Compound feed
The amount of homogenized compound feed sample (7.1) examined is 1,00 g ± 0,02 g.
7.3 Extraction
7.3.1 Oilseeds and oilseed products
Weigh (6.2) a test portion of 1,00 g ± 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 (6.6) 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 % (volume fraction) 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 % (volume fraction) 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 (6.2) a test portion of 1,00 g ± 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 (6.6) 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.
7.3.3 Recovery sample for compound feed
For preparation of the recovery sample take a representative blank feed sample (see NOTE), preferably
the same that is used for the preparation of the calibration range in blank extract (7.3.4).
Weigh (6.2) a test portion of 1,00 g ± 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 (6.6).
Add 25 ml extraction solvent (5.4.1), vortex for 10 s (6.6) 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 blank sample is 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 (see NOTE).
Weigh (6.2) a test portion of 1,00 g ± 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 (6.6) 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 (6.12) directly in HPLC vials.
NOTE A blank sample is 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
Code Concentration Mixed standard Mixed standard Mixed standard Blank feed
solution solution solution extract
nmol/ml
1 nmol/ml 10 nmol/ml 100 nmol/ml (7.3.4)
(5.3.25) (5.3.24) (5.3.23) µl
µl 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
Cal 8 1,0 0 0 10 990
Cal 9 2,5 0 0 25 975
Cal 10 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. The chosen column dimensions and chromatographic conditions should be appropriate to
obtain base line separation of epimers or isomers with the same molecular mass-to-charge ratio.
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 analyzed in negative ionization mode. At least two transitions
should be selected and included in the multiple reaction monitoring (MRM) method. In general, the most
intense product ion is the formation of hydrogen sulphate anion (m/z 97). Additional product ions that
are present in the spectra of most glucosinolates are the C H OS anion (m/z 75) and glucose-sulphate
2 3
anion (m/z 259). For several glucosinolates structure specific anions are also formed. See Table B.3 for
detailed information on which other product ions are formed. Preferably, product ions formed by the loss
of water from the de protonated molecular ion should not be selected.
Glucosinolates containing an indole substituent (glucobrassicin and its 4-hydroxy, 4-methoxy and N-
methoxy derivatives) can produce 7-hydroxy and 7-methoxy adducts in the mass spectrometer. The
amount of adducts formed depends on the design of the mass spectrometer and therefore varies
substantially between type or brand of mass spectrometer. The tendency to produce these adducts
diminishes in the order: 4-hydroxyglucobrassicin > 4-
methoxyglucobrassicin > glucobrassicin > > neoglucobrassicin. Adduct formation can result in a
(strongly) reduced sensitivity of the parent molecular ion. It is therefore advised to include the 7-hydroxy
and 7-methoxy adducts in the analytical method. The product ions that are the most sensitive and stable
during the analytical run should be used for quantification.
The instrument should have sufficient sensitivity to detect the analytes at a level of 0,1 mmol/kg in the
oilseed or oilseed product at a level of 0,02 mmol/kg in compound feed with a signal-to-noise ratio of 3
for the weakest selected product ion and signal-to-noise ratio of 10 for the most intense product ion.
The linearity of the mass detector should be verified over the full calibration range before the analysis of
the samples is conducted. Depending on the sensitivity and linear dynamic range of the mass
spectrometric instrument it can be necessary to reduce the injection volume. When this is not possible, it
is advised to dilute the calibration standards and sample extracts by an additional factor with extraction
solvent (5.4.1).
An example of suitable measuring conditions is provided in Annex B and example chromatograms are
given in Annex C.
8.2 Analysis sequence
8.2.1 Oilseeds and oilseed products
An appropriate analysis sequence for oilseeds and oilseed products:
— calibration standard 8 of 1 nmol/ml in water, injected at least three times or until stable
measurement conditions are obtained;
— water;
— calibration standard range in aqueous solution (5.3.26);
— water;
— quality control sample extract (5.5);
— sample extracts (7.3.1);
— water;
— calibration standard range in aqueous solution (5.3.26).
Depending on the number of samples, additional calibration standards may be included in between the
samples.
8.2.2 Compound feed
An appropriate analysis sequence for compound feeds:
— calibration standard 8 of 1 nmol/ml in blank matrix extract, injected at least three times or until
stable measurement conditions are obtained;
— water;
— calibration standard range in blank matrix extract (7.3.4);
— water;
— recovery sample (7.3.3);
— sample extracts (7.3.2);
— water;
— calibration standard range in blank matrix extract (7.3.4).
Depending on the number of samples, additional calibration standards may be included in between the
samples.
9 Evaluation of results
9.1 Identification
The detection and identification of individual glucosinolates in the sample is based on the presence of co-
eluting chromatographic peaks for the transitions measured. Retention time and relative abundance of
the transitions should match with those in the calibration standards. The deviation of the retention time
of the analyte in the sample should not exceed 0,2 min in comparison to the average retention time in the
calibration extracts. The relative intensities of the transitions in the sample should not deviate more than
30 % in comparison to the average of the relative intensities of the transitions in the calibration extracts.
9.2 Quantification
9.2.1 General
For oilseeds and oilseed products quantification is based on multi-level calibration using calibration
standard in aqueous solution (5.3.26). For compound feeds quantification is based on multi-level
calibration using calibration standards in blank matrix extract (7.3.4) (see NOTE 1). The sum area of both
product ions is used for all calculations (see NOTE 2).
NOTE 1 At the conditions described in this document, matrix effects (ion suppression or enhancement) were not
significant for oilseed samples. For compound feed samples matrix effects can be observed.
NOTE 2 Optionally, the area of the quantifier ion only can be used in the calculations of analyte concentration
and recovery.
9.2.2 Linearity of the calibration curve
Plot the sum of the product ion areas for the analyte in the calibration standards in aqueous solution
(5.3.26) or for the calibration standards in matrix extract (7.3.4) as function of the added concentration.
Use weighed least-square linear regression over all data points to estimate slope and intercept of the
calibration lines. Test for linearity of the calibration curves using a suitable test for linearity (e.g. residual
versus fitted-values plot; calculation of correlation coefficient). If the test indicates nonlinearity, identify
its cause and take appropriate measures (e.g. by reduction of the injection volume, by reduction of the
working range of the calibration curve, by using weighted calibration or by dilution of the extracts).
In the residuals versus fitted-values plot, calculated concentrations for individual calibration points
should deviate less than 20 %. The correlation coefficient of the calibration curve should be at least 0,990.
9.2.3 Calculation of the glucosinolate concentration in oilseed or oilseed product
The concentration of the analyte in the oilseed sample is calculated according to Formula (1).

Ab− V
C ×× D
(1)

aW

where
C is the concentration of the analyte in oilseed or oilseed product, in mmol/kg;
A is the sum of the area of the product ions;
b is the intercept of the calibration line (5.3.26);
a is the slope of the calibration line (5.3.26);
V is the volume of extraction solvent added to the sample, in ml;
W is the amount of sample, in g;
D is the dilution factor of the initial extract.

9.2.4 Calculation of the glucosinolate concentration in compound feed
The concentration of the analyte in the compound feed sample is calculated according to Formula (2).

Ab− V 100
C ×××D
(2)

aW R

where
C is the concentration of the analyte in the compound feed, in mmol/kg;
A is the sum of the area of the product ions;
b is the intercept of the calibration line in matrix extract (7.3.4);
a is the slope of the calibration line in matrix extract (7.3.4);
V is the volume of extraction solvent added to the sample, in ml;
W is the amount of sample, in g;
D is the dilution factor of the initial extract;
R is the recovery, in %.
The recovery is calculated according to Formula (3).
 
Ab−
V
Rec
RD ×× (3)
 
a W
 
where
R is the recovery, in %;
A is the sum of the area of the product ions from the analyte in the recovery sample of
Rec
0,1 mmol/kg (7.3.3);
b is the intercept of the calibration line;
a is the slope of the calibration line;
V is the volume of extraction solvent added to the sample, in ml;
=
=
=
W is the amount of sample, in g;
D is the dilution factor of the initial extract;
R is the recovery, in %;
A is the sum of the area of the product ions from the analyte in the recovery sample of
Rec
0,1 mmol/kg (7.3.3);
b is the intercept of the calibration line.

9.3 Expression of results
The concentration obtained is in mmol/kg. Besides the concentration of the individual glucosinolates, the
total concentration of glucosinolates can be reported. The total glucosinolate concentration is the sum of
the quantified individual glucosinolates. Results obtained for oilseeds and oilseed products are not
corrected for recovery. Results obtained for compound feeds are corrected for recovery when the
recovery for the analyte is lower than 90 % or higher than 110 %.
In addition, for sample analysis in the frame of EU directive 2002/32, the final result for reporting needs
to be expressed on the basis of a 12 % moisture content and the concentration needs to be adjusted taking
the actual moisture content of the sample into account [6].
To express the total glucosinolate content as mg/kg allyl isothiocyanate, the concentration in mmol/kg is
multiplied with a factor 99,15.
10 Precision
10.1 General
Precision data were obtained in a collaborative study. Details of the precision of the method are provided
in Annex A. Besides repeatability and r
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