SIST EN ISO 9167:2019

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

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.

Contents Page
European foreword . 3

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.

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
ISO 9167:2019(E)
© 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
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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

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 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

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.
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 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

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
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 9167:2019(E)
1)
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

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 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 prepared according to the HPLC elution mode and the assumed glucosinolate
content of the sample (5.5).
The temperature of the ethanol solution shall be as close as possible to the boiling point to ensure a
rapid denaturation of the enzyme myrosinase which generally occurs in the test portion.
NOTE Non-denatured myrosinase can break down the glucosinolates in a few minutes.
Continue heating at 75 °C for a further 10 min, shaking the tubes at regular intervals. Adjust the volume
in each tube A and B to approximately 4 ml with water, mix and then centrifuge at an acceleration of
5 000g for 3 min.
Transfer the supernatant liquid from each tube to two other tubes (6.6) labelled A′ and B′. Adjust the
volume in each tube A′ and B′ to approximately 5 ml with water and mix.
These extracts may be kept for two weeks if stored in the dark in a freezer at −18 °C.
[6].
NOTE With ethanol 50 % as solvent, a one-step extraction was found efficient enough
6 © ISO 2019 – All rights reserved

ISO 9167:2019(E)
9.3 Blank test
If required (5.5), carry out a blank test using the same procedure (see 9.2) on a test portion taken
from the same test sample, but omitting the internal standard solution (sinigrin or glucotropaeolin)
and replacing it by 200 µl of water, in order to detect and quantify any sinigrin or glucotropaeolin (or
coeluting compound) present in the test portion.
9.4 Preparation of ion-exchange columns
Cut the required number of Pasteur pipettes fitted with glass wood (6.8), i.e. one pipette per combined
extract, so as to leave a volume of 1,2 ml above the neck. Place the pipettes vertically on a stand.
Transfer 0,5 ml of a well-mixed suspension of ion-exchange resin (5.11) to each pipette and allow to settle.
Rinse the pipettes with 2 ml of the imidazole formiate (5.4) and then twice with 1 ml portions of water.
9.5 Purification and desulfatation
9.5.1 Carry out the operations given in 9.5.2 to 9.5.5 for each extract.
9.5.2 Transfer 1 ml or 0,5 ml of the extract (see 9.2) depending on the assumed glucosinolate content
of the sample (see Table 4) to a prepared column (see 9.4) without disturbing the resin surface and allow
to drain. Add two 1 ml portions of the 0,02 M, pH 4,0 sodium acetate buffer (5.2), allowing the buffer to
drain after each addition.
Table 4 — Volume of extract transferred to the ion-exchange column according to the expected
glucosinolate content of the sample
Glucosinolate content of the sample Volume of extract
µmol/g ml
< 50 1,0
> 50 0,5
9.5.3 Add to the column 75 μl of diluted purified sulfatase solution (see C.4.4). Leave to act
approximately 15 h at ambient temperature.
9.5.4 Place a tube (6.6) under the column to collect the eluate. Elute the desulfoglucosinolates obtained
with two 1 ml portions of water, allowing the water to drain after each addition.
NOTE The complete elution of the desulfoglucosinolates from the ion exchange column can be checked by
using an additional portion of water (1 ml) for elution. The chromatography of this additional portion will not
reveal any desulfoglucosinolate in significant amount.
9.5.5 Mix the eluate well. If not used immediately for chromatography, the eluate may be stored in the
dark in a freezer at −18 °C for up to one week.
9.6 Chromatography with gradient elution
9.6.1 General
The reference method uses a gradient elution with octyl or octadecyl stationary phase column (6.2) and
eluents (5.8, 5.9 and 5.10).
ISO 9167:2019(E)
9.6.2 Adjustment of the apparatus
9.6.2.1 General
Adjust the column temperature to 30 °C and the detector wavelength to 229 nm. Programme the HPLC
apparatus (6.1) so as to rinse the column by passing of eluent B at a flow rate of approximately 1 ml/
min. When the baseline is stable, assume the initial conditions of the elution gradient (eluent A) and
allow the system to establish equilibrium during 5 min.
If the baseline remains unstable after passing the eluent B during 20 min, use the rinsing eluent
(acetonitrile 70 %) to clean the column, then use again eluent B during 5 min and assume the initial
conditions (eluent A).
When the system is balanced with eluant A, the sample shall be injected without delay. If not, traces of
organic compounds occurring in water can be concentrated onto the column and be released during the
gradient elution. Coelution with desulfoglucosinolates may then occur.
9.6.2.2 Analysis
Inject into the chromatograph not more than 50 μl of the desulfoglucosinolate solution obtained in 9.5.5
and vary the concentration in acetonitrile of the mobile phase according to a linear gradient (from 0 %
to 25 % within approximately 20 min).
Use an elution gradient appropriate to the column employed.
The following elution gradients are given as examples and may be modified to give optimum separations
according to the columns used (see Annex D).
EXAMPLE 1 RP18 column, 5 μm (150 mm × 4,6 mm): Pass 100 % of eluent A (5.8) for 1 min. Apply a linear
elution gradient over 20 min until 0 % of eluent A and 100 % of eluent B are obtained. Apply a linear elution
gradient over 5 min until 100 % of eluent A and 0 % of eluent B are obtained. Pass 100 % of eluent A for 5 min to
establish equilibrium.
EXAMPLE 2 RP8 column, 5 μm (125 mm × 4 mm): Pass 100 % of eluent A for 2 min 30 s. Apply a linear elution
gradient over 18 min until 0 % of eluent A and 100 % of eluent B are obtained. Pass 100 % of eluent B for 5 min.
Apply a linear elution gradient over 2 min until 100 % of eluent A and 0 % of eluent B are obtained. Pass 100 % of
eluent A for 5 min to establish equilibrium.
9.6.2.3 Examination of chromatograms
9.6.2.3.1 Identification of the peaks
Using this chromatographic method, the desulfated derivatives of the rape glucosinolates can be
separated fairly easily and the order of elution is generally as shown in Figure 1. However, a few
problems, which can be solved after slight modification of the elution gradient or after change of the
column type, can exist for the separation of the following desulfoglucosinolates:
— desulfoepiprogoitrin and desulfoglucosinigrin;
— desulfogluconapoleiferin and desulfoglucoalyssin;
— desulfoglucobrassicanapin and desulfoglucotropaeolin;
— desulfogluconasturtiin and desulfo-4-methoxyglucobrassicin.
The identification of the peaks may be carried out by comparison with a chromatogram stemming from
a standard sample (6.2) or by UV detection at a specific wavelength (275 nm for indolic glucosinolates).
8 © ISO 2019 – All rights reserved

ISO 9167:2019(E)
9.6.2.3.2 Quantification
Only take into account the peaks which correspond to desulfoglucosinolates and where the area exceeds
1 % of the total surface of the areas of these peaks.
Make certain that the integration measurement device operates in a suitable manner for badly
separated peaks and for high intensity peaks. If necessary, reinject the desulfoglucosinolate solution
(see 9.5.5) after diluting with water.
NOTE In spite of the choice of two internal standards, the chromatogram of the blank analysis (tube B,
see 9.2) can show a peak with the same retention time than the internal standard used in the tube A (see 9.2).
Nevertheless, the calculation of the glucosinolates content can be possible by elimination of the influence of the
coeluting peak on the internal standard peak area. If the coeluting peak area is less than the half of the internal
standard area, the former can be subtracted from the latter with a correction made in comparison with the areas
of another major peak present in both chromatograms. The test report specifies this calculation mode.
10 Expression of results
10.1 Calculation of the content of each glucosinolate
The content C of each glucosinolate, expressed in micromoles per gram of dry matter of the product is
g
equal to Formula (1):
A K
g n g 100
C =× ×× (1)
g
A m Kw()100−
s s
where
A is the numeric value of the peak area, in integrator units, corresponding to the
g
desulfoglucosinolate;
A is the numeric value of the peak area, in integrator units, corresponding to the
s
desulfoglucosinolate used as internal standard;
K is the numeric value of the relative proportionality factor of the desulfoglucosinolate;
g
K is the numeric value of the relative proportionality factor of the desulfoglucosinolate used as
s
internal standard;
m is the numeric value of the mass, in grams, of the test portion;
n is the numeric value of the quantity, in micromoles, of internal standard added to the tube in 9.2;
w is the numeric value of the moisture and volatile matter content, expressed in percentage by
mass, of the test sample.
If it is desired to express the result relative to a specific moisture and volatile matter content, w (e.g.
s
w = 9 %), multiply the result C obtained for dry matter (as above) by Formula (2):
s g
100− w
s
(2)
10.2 Relative proportionality factors
The relative proportionality factors (Ki) given in Table 5 shall be adopted.
NOTE These relative proportionality factors have been determined experimentally and have been fixed by
consensus between the various laboratories who took part in the test; they may need to be revised in due course.
ISO 9167:2019(E)
Table 5 — Relative proportionality factors (Ki) to adopt
Desulfo-glucosinolate Ki
1 Desulfoglucoiberin 1,07
2 Desulfoprogoitrin 1,09
3 Desulfoepiprogoitrin 1,09
4 Desulfosinigrin 1,00
5 Desulfoglucoraphanin 1,07
6 Desulfogluconapoleiferin 1,00
7 Desulfoglucoalyssin 1,07
8 Desulfosinalbin 0,50
9 Desulfogluconapin 1,11
10 Desulfo-4-hydroxyglucobrassicin 0,28
11 Desulfoglucobrassicanapin 1,15
12 Desulfoglucotropaeolin 0,95
13 Desulfoglucobrassicin 0,29
14 Desulfogluconasturtiin 0,95
15 Desulfo-4-methoxyglucobrassicin 0,25
16 Desulfoneoglucobrassicin 0,20
17 Other desulfoglucosinolates 1,00
10.3 Calculation of the total glucosinolate content
The total glucosinolate content, expressed in micromoles per gram of dry matter of the product, is equal
to the sum of the content of each glucosinolate (the corresponding peak area of which is greater than
1 % of the sum total of the peak areas).
11 Precision
11.1 Interlaboratory test
Details of an interlaboratory test on the precision of the method are summarized in Annex A. The values
derived from this interlaboratory test may not be applicable to concentration ranges and matrices other
than those given.
11.2 Repeatability
The absolute difference between two independent single test results, obtained using the same method
on identical test materials in the same laboratory by the same operator using the same equipment
within a short interval of time, should not be greater than 2 µmol/g for glucosinolates contents lower
than 20 µmol/g and 4 µmol/g for glucosinolates contents ranging from 20 µmol/g to 35 µmol/g.
11.3 Reproducibility
The absolute difference between two single test results, obtained using the same method on identical
test materials in different equipment, should not be greater than 4 µmol/g for glucosinolate contents
lower than 20 µmol/g and 8 µmol/g for glucosinolate contents ranging from 20 µmol/g to 35 µmol/g.
10 © ISO 2019 – All rights reserved

ISO 9167:2019(E)
12 Test report
The test report shall specify the following:
— all information necessary for the complete identification of the sample;
— the sampling method used, if known;
— the test method used, with reference to this document, i.e. ISO 9167;
— all operating details not specified in this document, or regarded as optional, together with details of
any incidents which may have influenced the test result(s);
— the test result(s) obtained;
— if the repeatability has been checked, the final quoted result obtained.
ISO 9167:2019(E)
Annex A
(informative)
Results of interlaboratory trials — Gradient elution HPLC method
An interlaboratory test, carried out at the international level in 1988, in which 11 laboratories
participated, each of which carried out two determinations on each sample A, B, C and D, gave the
[2]
statistical results (evaluated in accordance with ISO 5725:1986 ) shown in Table A.1. The gradient
elution method was used.
Table A.1 — Statistical results of the 1988 interlaboratory test — Gradient elution
Rapeseed Rapeseed Rapeseed Rapeseed
Sample
A B C D
Number of laboratories retained after 11 11 11 11
eliminating outliers
a
Mean glucosinolate content 20,6 14,1 4,9 25,6
a
Standard deviation of repeatability, s 1,7 0,6 0,3 0,8
r
Coefficient of variation of repeatability 8,5 4,4 6,7 3,3
(%)
a
Repeatability limit, r (2,83 × s ) 4,8 1,7 0,8 2,2
r
Standard deviation of reproducibility, 3,4 2,5 1,5 2,4
a
s
R
Coefficient of variation of reproduci- 17 18 31 9,4
bility (%)
a
Reproducibility limit, R (2,83 × s ) 9,5 7,0 4,2 6,7
R
a
Expressed in µmol/g dry matter.
An interlaboratory test, carried out at the international level in 2014, in which 18 laboratories
participated, each of which carried out two determinations on each sample A to F, gave the statistical
[2]
results (evaluated in accordance with ISO 5725:1986 ) shown in Table A.2. The gradient elution
method was used.
Table A.2 — Statistical results of the 2014 interlaboratory test
Sample A B C D E F
Sample type Brassica Brassica Brassica Brassica Brassica Brassica
napus meal napus seed juncea juncea napus (can- napus (can-
meal seed ola) seed ola) meal
Number of participating laborato- 18 18 15 18 18 18
ries
Number of laboratories retained 16 18 13 17 18 16
after eliminating outliers
a
Mean glucosinolates content 7,83 14,58 156,97 15,07 10,14 1,70
Standard deviation of repeatabil- 0,15 0,18 1,99 0,36 0,52 0,10
a
ity, s
r
Coefficient of variation of repeata- 1,9 1,2 1,3 2,4 5,2 6,1
bility (%)
a
Repeatability limit, r (2,83 × s ) 0,43 0,51 5,57 1,02 1,47 0,29
r
a
Expressed in µmol/g dry matter.
12 © ISO 2019 – All rights reserved

ISO 9167:2019(E)
Table A.2 (continued)
Sample A B C D E F
Standard deviation of reproducibil- 1,33 1,34 30,14 1,91 1,27 0,36
a
ity, s
R
Coefficient of variation of repro- 17,0 9,2 19,2 12,7 12,5 21,0
ducibility (%)
a
Reproducibility limit, R (2,83 × s ) 3,72 3,75 84,4 5,34 3,56 1,00
R
a
Expressed in µmol/g dry matter.
ISO 9167:2019(E)
Annex B
(normative)
Checking of the titre of
...