Rapeseed and rapeseed meals - Determination of glucosinolates content - Method using high-performance liquid chromatography (ISO 9167:2019)

This document specifies a method for the determination of the individual glucosinolates content in
rapeseeds and rapeseed meals using high-performance liquid chromatography with gradient elution.
This method was tested on rapeseeds and rapeseed meals (Brassica rapa, Brassica napus and Brassica
juncea) but is applicable to other plant materials, on the condition that the occurring glucosinolates
previously identified are described in this document. On the contrary, the quantitative analysis of the
concerned glucosinolate(s) is not carried out.
NOTE This method does not determine glucosinolates that are substituted on the glucose molecule, but
these compounds are of little importance in commercial rapeseed and rapeseed meal.
Annex A presents the results of the interlaboratory trials for the gradient elution HPLC method.
Annex B presents how to check the titre of the prepared internal standard solution. Annex C presents
how to prepare and test the purified sulfatase solution and how to check the desulphation step on the
ion exchange column. Annex D presents the HPLC and column performance criteria qualification.
The analysis of glucosinolates content in rapeseed can also be done using an isocratic elution mode.
This requires some modifications of the method (internal, standard, HPLC column and HPLC buffers),
as described in Annex E.

Rapssamen und Rapsschrot - Bestimmung des Glucosinolatgehaltes - Verfahren mittels Hochleistungsflüssigchromatographie (ISO 9167:2019)

Dieses Dokument legt ein Verfahren zur Bestimmung der einzelnen Glucosinolate in Rapssamen und Rapsschrot mittels Hochleistungsflüssigkeitschromatographie mit Gradientenelution fest.
Dieses Verfahren wurde an Rapssamen und Rapsschrot (Brassica rapa, Brassica napus und Brassica juncea) geprüft, ist jedoch auf andere Pflanzenmaterialien unter der Bedingung anwendbar, dass die vorkommenden Glucosinolate zuvor identifiziert wurden und in diesem Dokument beschrieben sind. Die quantitative Bestimmung des (der) betreffenden Glucosinolats (Glucosinolate) wird nicht durchgeführt.
ANMERKUNG   Glucosinolate, die am Glucosemolekül substituiert sind, werden mit diesem Verfahren nicht erfasst, sind für handelsübliche Rapssamen und Rapsschrot jedoch auch von untergeordneter Bedeutung.
Anhang A enthält die Ergebnisse der Ringversuche für das HPLC Verfahren mit Gradientenelution. Anhang B stellt dar, wie der Titer der hergestellten internen Standardlösung überprüft wird. Anhang C stellt dar, wie die gereinigte Sulfatase Lösung hergestellt und geprüft wird und wie der Desulfatierungsschritt an der Ionenaustauschersäule überprüft wird. Anhang D stellt die Qualifikation der HPLC- und Säulenleistungs¬kriterien dar.
Die Analyse des Glucosinolatgehalts in Rapssamen kann auch unter Anwendung eines isokratischen Elutionsmodus erfolgen. Das erfordert einige Modifikationen des Verfahrens (interner Standard, HPLC Säule und HPLC Pufferlösungen), wie in Anhang E beschrieben.

Graines et tourteaux de colza - Dosage des glucosinolates - Méthode par chromatographie liquide à haute performance (ISO 9167:2019)

Le présent document spécifie une méthode de dosage des glucosinolates individuels dans les graines et tourteaux de colza par chromatographie en phase liquide à haute performance avec élution par gradient.
Cette méthode a été soumise à essai sur les graines et tourteaux de colza (Brassica rapa, Brassica napus et Brassica juncea), mais elle est applicable à d'autres matériaux végétaux, à condition que les glucosinolates identifiés précédemment soient décrits dans le présent document. Sinon, l'analyse quantitative du ou des glucosinolates concernés n'est pas effectuée.
NOTE Cette méthode ne dose pas les glucosinolates ayant des substituants sur la partie glucose, mais ces composés sont peu importants dans les graines et tourteaux de colza commercialisés.
L'Annexe A présente les résultats des essais interlaboratoires pour la méthode de CLHP avec élution par gradient. L'Annexe B présente comment vérifier le titre de la solution préparée d'étalon interne. L'Annexe C présente comment préparer et analyser la solution purifiée de sulfatase et contrôler l'étape de désulfatation sur la colonne échangeuse d'ions. L'Annexe D présente la qualification du système de CLHP et des critères de performance de la colonne.
Les glucosinolates des graines de colza peuvent également être dosés en utilisant un mode d'élution isocratique. Cela nécessite un certain nombre de modifications de la méthode (étalon interne, colonne de CLHP et solutions tampons pour CLHP), comme décrit dans l'Annexe E.

Seme in obroki oljne repice - Določevanje glukozinolatov - Metoda s tekočinsko kromatografijo visoke ločljivosti (ISO 9167:2019)

Ta dokument določa metodo za določevanje vsebnosti posameznih glukozinolatov v semenu in obrokih oljne repice s pomočjo tekočinske kromatografije visoke ločljivosti z gradientnim eluiranjem. Ta metoda je bila preskušena na semenu in obrokih oljne repice (Brassica rapa, Brassica napus in Brassica juncea), vendar se uporablja tudi za druge rastlinske materiale pod pogojem, da so v tem dokumentu opisani obstoječi glukozinolati, ki so bili predhodno prepoznani. Nasprotno se ne opravi kvantitativna analiza zadevnega glukozinolata oziroma glukozinolatov.
OPOMBA: Ta metoda ne določa glukozinolatov, ki so v molekuli glukoze nadomeščeni, vendar te spojine v okviru obroka komercialne oljne repice in oljne repice nimajo velikega pomena. V dodatku A so predstavljeni rezultati medlaboratorijskih preskusov za gradientno eluiranje po metodi HPLC. V dodatku B je predstavljeno, kako je mogoče preveriti porabo pripravljene raztopine iz internega standarda. V dodatku C je predstavljeno, kako pripraviti in preskusiti prečiščeno raztopino sulfataze ter kako preveriti korak desulfatizacije v koloni za ionsko izmenjavo. V dodatku D sta predstavljeni metoda HPLC in uvrstitev merila uspešnosti kolone. Analiza vsebnosti glukozinolatov v oljni repici je mogoča tudi z načinom izokratskega eluiranja. Za to je potrebnih nekaj prilagoditev metode (notranje, standardne, kolona HPLC in puferske raztopine za HPLC), kot je opisano v dodatku E.

General Information

Status
Published
Public Enquiry End Date
04-Mar-2018
Publication Date
18-Jul-2019
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
11-Jul-2019
Due Date
15-Sep-2019
Completion Date
19-Jul-2019

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SLOVENSKI STANDARD
SIST EN ISO 9167:2019
01-september-2019
Nadomešča:
SIST EN ISO 9167-1:1998
SIST EN ISO 9167-1:1998/A1:2013
Seme in obroki oljne repice - Določevanje glukozinolatov - Metoda s tekočinsko
kromatografijo visoke ločljivosti (ISO 9167:2019)

Rapeseed and rapeseed meals - Determination of glucosinolates content - Method using

high-performance liquid chromatography (ISO 9167:2019)

Rapssamen und Rapsschrot - Bestimmung des Glucosinolatgehaltes - Verfahren mittels

Hochleistungsflüssigchromatographie (ISO 9167:2019)
Graines et tourteaux de colza - Dosage des glucosinolates - Méthode par
chromatographie liquide à haute performance (ISO 9167:2019)
Ta slovenski standard je istoveten z: EN ISO 9167:2019
ICS:
67.200.20 Oljnice Oilseeds
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
SIST EN ISO 9167:2019 en

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

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SIST EN ISO 9167:2019
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SIST EN ISO 9167:2019
EN ISO 9167
EUROPEAN STANDARD
NORME EUROPÉENNE
June 2019
EUROPÄISCHE NORM
ICS 67.200.20
English Version
Rapeseed and rapeseed meals - Determination of
glucosinolates content - Method using high-performance
liquid chromatography (ISO 9167:2019)

Graines et tourteaux de colza - Dosage des Rapssamen und Rapsschrot - Bestimmung des

glucosinolates - Méthode par chromatographie liquide Glucosinolatgehaltes - Verfahren mittels

à haute performance (ISO 9167:2019) Hochleistungsflüssigchromatographie (ISO 9167:2019)

This European Standard was approved by CEN on 4 May 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 ISO 9167:2019 E

worldwide for CEN national Members.
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SIST EN ISO 9167:2019
EN ISO 9167:2019 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 9167:2019
EN ISO 9167:2019 (E)
European foreword

This document (EN ISO 9167:2019) has been prepared by Technical Committee ISO/TC 34 "Food

products" in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats

and oils and their by-products - Methods of sampling and analysis” the secretariat of which is held by

AFNOR.

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 December 2019, and conflicting national standards

shall be withdrawn at the latest by December 2019.

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 supersedes EN ISO 9167-1:1995.

According to the CEN-CENELEC Internal Regulations, the national standards organizations 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.
Endorsement notice

The text of ISO 9167:2019 has been approved by CEN as EN ISO 9167:2019 without any modification.

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SIST EN ISO 9167:2019
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SIST EN ISO 9167:2019
INTERNATIONAL ISO
STANDARD 9167
First edition
2019-05
Rapeseed and rapeseed meals —
Determination of glucosinolates
content — Method using high-
performance liquid chromatography
Graines et tourteaux de colza — Dosage des glucosinolates —
Méthode par chromatographie liquide à haute performance
Reference number
ISO 9167:2019(E)
ISO 2019
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SIST EN ISO 9167:2019
ISO 9167:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
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SIST EN ISO 9167:2019
ISO 9167:2019(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Reagents ........................................................................................................................................................................................................................ 2

6 Apparatus ..................................................................................................................................................................................................................... 4

7 Sampling ........................................................................................................................................................................................................................ 5

8 Preparation of the test sample ............................................................................................................................................................... 6

9 Procedure..................................................................................................................................................................................................................... 6

9.1 Test portion ................................................................................................................................................................................................ 6

9.2 Extraction of glucosinolates ........................................................................................................................................................ 6

9.3 Blank test ..................................................................................................................................................................................................... 7

9.4 Preparation of ion-exchange columns ................................................................................................................................ 7

9.5 Purification and desulfatation ................................................................................................................................................... 7

9.6 Chromatography with gradient elution ............................................................................................................................. 7

9.6.1 General...................................................................................................................................................................................... 7

9.6.2 Adjustment of the apparatus ................................................................................................................................. 8

10 Expression of results ........................................................................................................................................................................................ 9

10.1 Calculation of the content of each glucosinolate ....................................................................................................... 9

10.2 Relative proportionality factors ............................................................................................................................................... 9

10.3 Calculation of the total glucosinolate content ...........................................................................................................10

11 Precision ....................................................................................................................................................................................................................10

11.1 Interlaboratory test..........................................................................................................................................................................10

11.2 Repeatability ..........................................................................................................................................................................................10

11.3 Reproducibility ....................................................................................................................................................................................10

12 Test report ................................................................................................................................................................................................................11

Annex A (informative) Results of interlaboratory trials — Gradient elution HPLC method ...................12

Annex B (normative) Checking of the titre of the prepared internal standard solution.............................14

Annex C (normative) Preparation and test of purified sulfatase solution and checking of the

desulphation step on ion-exchange columns .......................................................................................................................15

Annex D (informative) HPLC system and column performance criteria qualification ..................................20

Annex E (informative) Elution in the isocratic mode .......................................................................................................................22

Bibliography .............................................................................................................................................................................................................................28

© ISO 2019 – All rights reserved iii
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SIST EN ISO 9167:2019
ISO 9167:2019(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso

.org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 2,

Oleaginous seeds and fruits and oilseed meals.

This first edition cancels and replaces ISO 9167-1:1992, which has been technically revised. It also

incorporates the amendment ISO 9167-1:1992/Amd.1:2013. The main changes are as follows:

— rapeseed meals have been added to the scope with the addition of a new collaborative trial;

[6]
— in 9.2, methanol 70 % has been replaced by ethanol 50 % for lower toxicity ;
— in 9.2, only one extraction is carried out instead of two;

— in 10.2 and E.5.1, the term “relative proportionality factor” has been used instead of “response

factor”;
— the isocratic mode has been added in Annex E.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2019 – All rights reserved
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SIST EN ISO 9167:2019
ISO 9167:2019(E)
Introduction

The glucosinolates in rapeseed can be analysed by chromatographic, enzymatic or spectroscopic

methods. This document describes a chromatographic method with two conditions (gradient and

isocratic) of elution for qualitative and quantitative analysis of individual glucosinolates in rapeseed

and rapeseed meals. The method with gradient elution is considered as the reference method whereas

the method with isocratic elution is considered as a simplified method and is presented in Annex E as

information.

This document specifies a method using high-performance liquid chromatography (HPLC) with

gradient elution as reference method. For the isocratic mode, the choice of the internal standard, the

chromatographic conditions and the separation results are different from the reference method. These

aspects are discussed in Annex E.
© ISO 2019 – All rights reserved v
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SIST EN ISO 9167:2019
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SIST EN ISO 9167:2019
INTERNATIONAL STANDARD ISO 9167:2019(E)
Rapeseed and rapeseed meals — Determination of
glucosinolates content — Method using high-performance
liquid chromatography
1 Scope

This document specifies a method for the determination of the individual glucosinolates content in

rapeseeds and rapeseed meals using high-performance liquid chromatography with gradient elution.

This method was tested on rapeseeds and rapeseed meals (Brassica rapa, Brassica napus and Brassica

juncea) but is applicable to other plant materials, on the condition that the occurring glucosinolates

previously identified are described in this document. On the contrary, the quantitative analysis of the

concerned glucosinolate(s) is not carried out.

NOTE This method does not determine glucosinolates that are substituted on the glucose molecule, but

these compounds are of little importance in commercial rapeseed and rapeseed meal.

Annex A presents the results of the interlaboratory trials for the gradient elution HPLC method.

Annex B presents how to check the titre of the prepared internal standard solution. Annex C presents

how to prepare and test the purified sulfatase solution and how to check the desulphation step on the

ion exchange column. Annex D presents the HPLC and column performance criteria qualification.

The analysis of glucosinolates content in rapeseed can also be done using an isocratic elution mode.

This requires some modifications of the method (internal, standard, HPLC column and HPLC buffers),

as described in Annex E.
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.

ISO 664, Oilseeds — Reduction of laboratory sample to test sample
ISO 665, Oilseeds — Determination of moisture and volatile matter content

ISO 771, Oilseed residues — Determination of moisture and volatile matter content

ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 5502, Oilseed residues — Preparation of test samples
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:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
© ISO 2019 – All rights reserved 1
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SIST EN ISO 9167:2019
ISO 9167:2019(E)
4 Principle

Extraction of glucosinolates by a water-ethanol mixture, then purification and enzymatic desulfatation

on ion-exchange columns. Determination using reverse phase liquid chromatography with gradient

elution (reference method) or isocratic elution (rapid method) and detection by ultraviolet

absorptiometry.
5 Reagents

Use only reagents of recognized analytical grade, unless otherwise specified, and water conforming to

grade 2 of ISO 3696.
5.1 Ethanol, volume fraction = 50 %.

5.2 Sodium acetate, c = 0,02 mol/l at pH 4,0, prepared by mixing sodium acetate, c = 0,02 mol/l and

acetic acid, c = 0,02 mol/l as to obtain a solution having a pH = 4,0.

5.3 Sulfatase, Helix pomatia, type H1 purified and diluted as described in Annex C.

5.4 Imidazole formiate, c = 6 mol/l.

Dissolve 204 g of imidazole in 113 ml of formic acid in a 500 ml beaker. Transfer the mixture in a 500 ml

cylinder and make up to 500 ml with water.
5.5 Internal standard.

Use either sinigrin (potassium allyglucosinolate monohydrate, M = 415,5 g/mol) (5.6) or glucotropaeolin

(potassium benzylglucosinolate, M = 447,5 g/mol or tetramethylammonium benzylglucosinolate,

M = 482,6 g/mol) (5.7). The glucotropaeolin may be used in a hydrated form, then the molar mass and

the purity shall be known and taken into consideration for the preparation of the solution.

The choice of the internal standard will be conditioned by its perfect chromatographic separation from

the other glucosinolates of the sample. The natural absence in the sample of the internal standard or of

glucosinolates unseparated from the latter may be checked with a blank test (see 9.3).

With gradient elution on octyl or octadecyl stationary phases, sinigrin or glucotropaeolin can be used.

However, glucotropaeolin is sometimes difficult to separate from other natural minor glucosinolates.

With isocratic elution on cyano propyl stationary phase (see Annex E), sinigrin cannot be used for

rapeseed analysis because of the non-separation from the other glucosinolates. Glucotropaeolin shall

be used instead.

In the most frequent cases (i.e. when rapeseeds have an assumed glucosinolates content between

10 μmol/g and 50 μmol/g inclusive) the internal standard is used in solution form at 20 mmol/l. For

rapeseeds where the assumed glucosinolates content is less than 10 μmol/g or greater than 50 μmol/g,

the concentrations of the internal standard solutions used per sample are given in Table 1.

Check the titre of the prepared internal standard solution as described in Annex B.

2 © ISO 2019 – All rights reserved
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SIST EN ISO 9167:2019
ISO 9167:2019(E)

Table 1 — Concentration of the internal standard solution to use according to the assumed

glucosinolates content of the sample

Glucosinolates content of the sample Concentrations of the internal standard solutions

μmol/g mmol/l
< 10 5
> 10 and < 50 20
> 50 40
5.6 Sinigrin solution.

The sinigrin solution with the required concentration (see Table 1) is prepared according to Table 2.

Weigh the sinigrin to the nearest 0,5 mg and dissolve it in water in a 100 ml volumetric flask. Make up

to the mark with water. The solution thus prepared may be stored in a refrigerator at approximately

4 °C up to a week or in a freezer at −18 °C for a longer period.

Table 2 — Weight of sinigrin in 100 ml water for preparation of 5 mmol/l, 20 mmol/l and

40 mmol/l solutions
Molecular weight Sinigrin weight
Sinigrin form g
g/mol
5 mmol/l 20 mmol/l 40 mmol/l
Potassium monohydrate 415,5 0,207 7 0,831 0 1,662 0
5.7 Glucotropaeolin solution.

The glucotropaeolin solution with the required concentration (see Table 1) is prepared according to

Table 3. Weigh the glucotropaeolin to the nearest 0,5 mg and dissolve it in water in a 100 ml volumetric

flask. Make up to the mark with water. The solution thus prepared may be stored in a refrigerator at

approximately 4 °C up to a week or in a freezer at –18 °C for a longer period.

Table 3 — Weight of glucotropaeolin in 100 ml water for preparation of 5 mmol/l, 20 mmol/l

and 40 mmol/l solutions
Glucotropaeolin weight
Molecular weight
Glucotropaeolin form
g/mol
5 mmol/l 20 mmol/l 40 mmol/l
Potassium 447,5 0,223 7 0,895 0 1,790 1
Tetramethylammonium 482,6 0,241 3 0,965 2 1,930 3

5.8 Eluent A: water, purified by passing it through an activated charcoal cartridge or water of

equivalent purity.

NOTE The use of insufficiently purified water can lead to ghost peaks during the analysis due to impurities

eluted when the proportion of acetonitrile in the eluent increases.

5.9 Eluent B: acetonitrile, HPLC gradient grade, solution in purified water, volumic fraction = 20 %.

The concentration may be modified in relation to the column used.

5.10 Rinsing solvent: acetonitrile, HPLC grade, solution in water, volumic fraction = 70 %.

© ISO 2019 – All rights reserved 3
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SIST EN ISO 9167:2019
ISO 9167:2019(E)
5.11 Ion-exchange resin: DEAE Sephadex A25 suspension, prepared as follows.

Mix 10 g of DEAE Sephadex A25 resin (or an equivalent resin) in excess 2 mol/l acetic acid solution. Leave

to settle. Add 2 mol/l acetic acid until the total volume is equal to twice the volume of the sediment.

6 Apparatus
Usual laboratory apparatus and, in particular, the following.

6.1 HPLC apparatus with gradient or isocratic elution, column temperature adjustment at 30 °C and

detection by ultraviolet absorptiometry at wavelength of 229 nm and, if possible, at wavelength of 275 nm.

An efficient column temperature regulation at 30 °C can be impossible when the ambient temperature

is above 25 °C. An oven with a cooling-heating device is recommended in this case.

6.2 HPLC columns for gradient elution.

HPLC column containing an octyl (C8) or octadecyl (C18) stationary phase, fixed to silica column

packing, of particle size less than or equal to 5 μm.

The performance of the column should be checked regularly, preferably using a reference sample of

rapeseed. In particular, the column shall not degrade desulfo-4-hydroxyglucobrassicin, an important

but relatively unstable desulfoglucosinolate. Figure 1 shows an example of glucosinolates separations

using the HPLC gradient mode. New columns shall be subjected to preliminary conditioning in

accordance with the manufacturer’s instructions so that reproducible results can be obtained.

6.3 pH-meter.
6.4 Microgrinder, for example, a coffee mill.

6.5 Centrifuge, suitable for use with the tubes (6.6), capable of obtaining a centrifugal acceleration

of 5 000g.
6.6 Polypropylene tubes, of 6 ml capacity.

6.7 Water-bath or other heating apparatus, capable of maintaining a temperature of 75 °C ± 3 °C.

6.8 Pasteur pipettes fitted with glass wood, 150 mm long, and a suitable stand, or any other

appropriate apparatus.

1) DEAE Sephadex A25 is an example of a suitable product available commercially. This information is given for

the convenience of users of this document and does not constitute an endorsement by ISO of this product.

4 © ISO 2019 – All rights reserved
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SIST EN ISO 9167:2019
ISO 9167:2019(E)
Key
1 desulfoglucoiberin 9 desulfogluconapin
2 desulfoprogoitrin 10 desulfo-4-hydroxyglucobrassicin
3 desulfoepiprogoitrin 11 desulfoglucobrassicanapin
4 desulfosinigrin 12 desulfoglucotropaeolin
5 desulfoglucoraphanin 13 desulfoglucobrassicin
6 desulfogluconapoleiferin 14 desulfogluconasturtiin
7 desulfoglucoalyssin 15 desulfo-4-methoxyglucobrassicin
8 desulfosinalbin 16 desulfoneoglucobrassicin
Figure 1 — Example of a typical chromatogram of rape seeds with gradient elution
7 Sampling

It is important that the laboratory receive a sample that is truly representative and that has not been

damaged or changed during transport or storage.
© ISO 2019 – All rights reserved 5
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SIST EN ISO 9167:2019
ISO 9167:2019(E)

Sampling is not part of the method specified in this document. A recommended sampling method is

[4] [1]
given in ISO 21294 for oilseeds and ISO 5500 for oilseed meals.

If large non-oleaginous foreign bodies have been separated before the reduction of the laboratory

sample, allowance shall be made for this in the calculation.
8 Preparation of the test sample

Reduce the laboratory sample in accordance with ISO 664 for oilseeds and ISO 5502 for oilseed meals.

If the seeds have a moisture and volatile matter content in excess of w = 10 %, dry them beforehand

using a current of air at 45 °C ± 5 °C.

The impurities level is generally 2 % (mass fraction). If sinigrin is found in the sample (with the blank),

analyse the impurities separately, as the sinigrin may stem from seeds of adventitious cruciferae, which

are impurities in rapeseed.

If the seeds have been treated, wash them with dichloromethane and dry them in a current of air at

ambient temperature.
Reduce the sample to two sub-samples of 20 g each.

Determine the moisture and volatile matter content of a sub-sample in accordance with ISO 665 for

oilseeds and ISO 771 for oilseed meals or an adequate procedure.

Grind the seeds of other sub-sample in the microgrinder (6.4) for 20 s. Mix, then grind for a further 5 s.

Weigh the prepared sample (see 9.1) immediately to avoid modification of the moisture and volatile

matter content.
9 Procedure
9.1 Test portion

Label two tubes (6.6) as A and B and transfer 200 mg for oilseeds and 100 mg for oilseed meals, weighed

to the nearest 0,5 mg, of the prepared test sample (see Clause 8) to each tube. Use tube A for the test

sample and use the tube B as blank sample, if necessary.
9.2 Extraction of glucosinolates

Place the tubes in the water-bath or other heating apparatus (6.7), set at 75 °C and leave for 1 min. Add

3 ml of boiling ethanol solution (5.1) and then immediately add, to tube A, 200 μl to the nearest 3 μl of

internal standard solution
...

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