Animal and vegetable fats and oils - Determination of tocopherol and tocotrienol contents by high-performance liquid chromatography (ISO 9936:2016)

ISO 9936:2016 specifies a method for the determination of the contents of free α-, β-, γ-, and δ-tocopherols and tocotrienols (referred to jointly as tocols) in animal and vegetable fats and oils (referred to hereinafter as fats) by high-performance liquid chromatography (HPLC).
For products containing tocopherol or tocotrienol esters, it is necessary to carry out a preliminary saponification.
Milk and milk products (or fat coming from milk and milk products) are excluded from the Scope of this International Standard.
NOTE          A suitable method involving a cold saponification procedure is described in Annex B for information only.

Tierische und pflanzliche Fette und Öle - Bestimmung des Tocopherol- und Tocotrienol-Gehaltes mittels Hochleistungsflüssigchromatographie (ISO 9936:2016)

Diese Internationale Norm legt ein Verfahren zur Bestimmung des Gehalts an freien α-, β-, γ- und δ Tocopherolen und Tocotrienolen (nachfolgend insgesamt als Tocole bezeichnet) in tierischen und pflanzlichen Fetten und Ölen (nachfolgend als Fette bezeichnet) durch Hochleistungsflüssigchromatographie (HPLC, en: high-performance liquid chromatography) fest.
Bei Produkten, die Ester von Tocopherolen oder Tocotrienolen enthalten, ist es erforderlich, eine voraus-gehende Verseifung durchzuführen.
Milch und Milcherzeugnisse (oder Fett aus Milch und Milcherzeugnissen) sind vom Anwendungsbereich dieser Internationalen Norm ausgenommen.
ANMERKUNG   Ein geeignetes Verfahren mit einer Kaltverseifung wird in Anhang B nur zur Information beschrieben.

Corps gras d'origines animale et végétale - Détermination des teneurs en tocophérols et en tocotriénols par chromatographie en phase liquide à haute performance (ISO 9936:2016)

ISO 9936:2016 spécifie une méthode pour la détermination des teneurs en α-, β-, γ- et δ-tocophérols et tocotriénols libres (appelés globalement tocols) des graisses et des huiles (appelés corps gras dans la suite du texte) d'origines animale et végétale, par chromatographie en phase liquide haute performance (CLHP).
Pour les produits contenant des esters de tocophérols ou de tocotriénols, il est nécessaire qu'ils subissent une saponification préalable.
Le lait et les produits laitiers (ou les corps gras issus du lait et des produits laitiers) sont exclus du domaine d'application de la présente Norme internationale.
NOTE          Une méthode appropriée incluant un protocole de saponification à froid est décrite, pour information uniquement, dans l'Annexe B.

Rastlinske in živalske maščobe in olja - Določevanje tokoferola in tokotrienola s tekočinsko kromatografijo visoke ločljivosti (ISO 9936:2016)

Ta mednarodni standard določa metodo za določevanje vsebnosti prostih α-, β-, γ- in δ-tokoferolov ter tokotrienolov (s skupnim imenom tokoli) v živalskih in rastlinskih maščobah ter oljih (v nadaljevanju »maščobe«) s tekočinsko kromatografijo visoke ločljivosti (HPLC).
Za proizvode, ki vsebujejo tokoferolne ali tokotrienolne estre, je treba izvesti predhodno
umiljenje. Mleko in mlečni izdelki (ali maščoba iz mleka in mlečnih izdelkov) niso zajeti v tem mednarodnem standardu.
OPOMBA: Ustrezna metoda, ki vključuje postopek hladnega umiljenja, je v dodatku B opisana
samo informativno.

General Information

Status
Published
Publication Date
12-Apr-2016
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
13-Apr-2016
Completion Date
13-Apr-2016

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SLOVENSKI STANDARD
SIST EN ISO 9936:2016
01-junij-2016
1DGRPHãþD
SIST EN ISO 9936:2006
SIST EN ISO 9936:2006/A1:2011
SIST EN ISO 9936:2006/AC:2009

5DVWOLQVNHLQåLYDOVNHPDãþREHLQROMD'RORþHYDQMHWRNRIHURODLQWRNRWULHQRODV

WHNRþLQVNRNURPDWRJUDILMRYLVRNHORþOMLYRVWL ,62

Animal and vegetable fats and oils - Determination of tocopherol and tocotrienol contents

by high-performance liquid chromatography (ISO 9936:2016)

Tierische und pflanzliche Fette und Öle - Bestimmung des Tocopherol- und Tocotrienol-

Gehaltes mittels Hochleistungsflüssigchromatographie (ISO 9936:2016)

Corps gras d'origines animale et végétale -- Détermination des teneurs en tocophérols et

en tocotriénols par chromatographie en phase liquide à haute performance (ISO
9936:2016)
Ta slovenski standard je istoveten z: EN ISO 9936:2016
ICS:
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
SIST EN ISO 9936:2016 en

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

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SIST EN ISO 9936:2016
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SIST EN ISO 9936:2016
EN ISO 9936
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2016
EUROPÄISCHE NORM
ICS 67.200.10 Supersedes EN ISO 9936:2006
English Version
Animal and vegetable fats and oils - Determination of
tocopherol and tocotrienol contents by high-performance
liquid chromatography (ISO 9936:2016)

Corps gras d'origines animale et végétale - Tierische und pflanzliche Fette und Öle - Bestimmung

Détermination des teneurs en tocophérols et en des Tocopherol- und Tocotrienol-Gehaltes mittels

tocotriénols par chromatographie en phase liquide à Hochleistungsflüssigchromatographie (ISO 9936:2016)

haute performance (ISO 9936:2016)
This European Standard was approved by CEN on 4 March 2016.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, 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: Avenue Marnix 17, B-1000 Brussels

© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 9936:2016 E

worldwide for CEN national Members.
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SIST EN ISO 9936:2016
EN ISO 9936:2016 (E)
Contents Page

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

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SIST EN ISO 9936:2016
EN ISO 9936:2016 (E)
European foreword

This document (EN ISO 9936:2016) 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 October 2016, and conflicting national standards shall

be withdrawn at the latest by October 2016.

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

patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent

rights.
This document supersedes EN ISO 9936:2006.

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, Former Yugoslav Republic of Macedonia,

France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,

Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
Endorsement notice

The text of ISO 9936:2016 has been approved by CEN as EN ISO 9936:2016 without any modification.

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SIST EN ISO 9936:2016
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SIST EN ISO 9936:2016
INTERNATIONAL ISO
STANDARD 9936
Third edition
2016-04-01
Animal and vegetable fats and
oils — Determination of tocopherol
and tocotrienol contents by high-
performance liquid chromatography
Corps gras d’origines animale et végétale — Détermination des
teneurs en tocophérols et en tocotriénols par chromatographie en
phase liquide à haute performance
Reference number
ISO 9936:2016(E)
ISO 2016
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SIST EN ISO 9936:2016
ISO 9936:2016(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, Published in Switzerland

All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2016 – All rights reserved
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SIST EN ISO 9936:2016
ISO 9936:2016(E)
Contents Page

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

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

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

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

4 Principle ........................................................................................................................................................................................................................ 1

5 Reagents ........................................................................................................................................................................................................................ 1

6 Apparatus ..................................................................................................................................................................................................................... 2

7 Sampling ........................................................................................................................................................................................................................ 3

8 Preparation of test sample ......................................................................................................................................................................... 3

9 Procedure..................................................................................................................................................................................................................... 3

9.1 Preparation of calibration solutions ..................................................................................................................................... 4

9.1.1 Stock calibration solutions ...................................................................................................................................... 4

9.1.2 Standard solution ............................................................................................................................................................ 4

9.2 Optimization of working parameters .................................................................................................................................. 4

9.3 Preparation of test solution ......................................................................................................................................................... 5

9.4 Determination ......................................................................................................................................................................................... 5

10 Expression of results ........................................................................................................................................................................................ 6

11 Precision ....................................................................................................................................................................................................................... 6

11.1 Interlaboratory test............................................................................................................................................................................. 6

11.2 Repeatability ............................................................................................................................................................................................. 6

11.3 Reproducibility ....................................................................................................................................................................................... 7

12 Test report ................................................................................................................................................................................................................... 7

Annex A (informative) Examples of chromatograms ........................................................................................................................... 8

Annex B (informative) Saponification ..............................................................................................................................................................10

Annex C (informative) Results of interlaboratory tests .................................................................................................................12

Bibliography .............................................................................................................................................................................................................................16

© ISO 2016 – All rights reserved iii
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SIST EN ISO 9936:2016
ISO 9936:2016(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 on the meaning of ISO specific terms and expressions related to conformity

assessment, as well as information about ISO’s adherence to the WTO principles in the Technical

Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information

The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 11, Animal

and vegetable fats and oils.

This third edition cancels and replaces the second edition (ISO 9936:2006), which has been technically

revised. It also incorporates the Amendment ISO 9936:2006/Amd.1:2011 and the Technical Corrigendum

ISO 9936:2006/Cor.1:2008. A non-applicability statement for milk and milk products has been added to

the Scope.
iv © ISO 2016 – All rights reserved
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SIST EN ISO 9936:2016
INTERNATIONAL STANDARD ISO 9936:2016(E)
Animal and vegetable fats and oils — Determination of
tocopherol and tocotrienol contents by high-performance
liquid chromatography
1 Scope

This International Standard specifies a method for the determination of the contents of free α-, β-, γ-,

and δ-tocopherols and tocotrienols (referred to jointly as tocols) in animal and vegetable fats and oils

(referred to hereinafter as fats) by high-performance liquid chromatography (HPLC).

For products containing tocopherol or tocotrienol esters, it is necessary to carry out a preliminary

saponification.

Milk and milk products (or fat coming from milk and milk products) are excluded from the Scope of this

International Standard.

NOTE A suitable method involving a cold saponification procedure is described in Annex B for

information only.
2 Normative references

The following documents, in whole or in part, are normatively referenced in this document and are

indispensable for its application. For dated references, only the edition cited applies. For undated

references, the latest edition of the referenced document (including any amendments) applies.

ISO 661, Animal and vegetable fats and oils — Preparation of test sample
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
tocol content

mass fraction of the individual tocols, determined using the method specified in this International

Standard

Note 1 to entry: The content is expressed in milligrams per kilogram as a whole number.

4 Principle

A test portion is dissolved in n-heptane and the individual tocols are separated by high-performance

liquid chromatography (HPLC). The content of each tocol is calculated using calibration factors

determined from calibration solutions.
5 Reagents
Use only reagents of HPLC grade or equivalent.
5.1 α-, β-, γ- and δ-tocopherol and tocotrienol standards.

If tocopherol standards are not available, a blend of wheat germ and soya bean oil may be used to

identify α-, β-, γ- and δ-tocopherols.
© ISO 2016 – All rights reserved 1
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SIST EN ISO 9936:2016
ISO 9936:2016(E)

If tocotrienol standards are not available, palm oil may be used to identify α- and γ-tocotrienols. The

chromatograms obtained can be used to assist peak identification in test sample chromatograms, in

which case the calibration factors for the corresponding tocopherols should be used.

NOTE α-, β-, γ- and δ-tocopherol and tocotrienol standards can be obtained from Merck ; α-tocopherol

can be obtained from various suppliers. Tocotrienol standards are available from Sigma Aldrich . It has been

reported that the purity of some commercially available tocopherol standards may vary between 85 % and

100 %. Thus, it is important to determine the concentration of prepared calibration solutions by UV spectrometry

(see 9.1.1).
5.2 Tetrahydrofuran, filtered through an HPLC nylon filter (0,45 µm).
5.3 n-Heptane, filtered through an HPLC nylon filter (0,45 µm).
5.4 HPLC mobile phase.

Any suitable mixture of solvents that has been proven to reach a chromatographic resolution of peaks

as good as the one presented in Table 2 (relative retention time of tocopherols and tocotrienols) and in

Annex A (chromatograms of a mixture of vegetable oils), should be used (see Table C.3).

A good separation of γ-tocopherol and β-tocotrienol can be achieved by using a mixture of 5 % volume

fraction t-butyl methyl ether + 95 % volume fraction n-heptane and a diol-column.

The preparation of a suitable mobile phase, 3,85 % (volume fraction) tetrahydrofuran solution in

n-heptane, is as follows. Using a 1 000 ml graduated cylinder (6.5), introduce 1 000 ml of n-heptane

(5.3) in a 2 litre bottle. Add twice 20 ml of tetrahydrofuran (5.2) using a 20 ml volumetric pipette (6.6).

Homogenize the mobile phase by means of an ultrasonic bath (6.8) for 15 min.
5.5 Methanol.
6 Apparatus
Usual laboratory apparatus and, in particular, the following.

6.1 HPLC system, consisting of a high-pressure pump, a sample injection device, column thermostat

adjusted to 25 °C (optional), a fluorescence detector with the excitation wavelength set at 295 nm and

emission wavelength at 330 nm, and a recording integrator.

An ultraviolet (UV) detector may be used if a fluorescence detector is not available but it is not

recommended. However, if a UV detector is used, the wavelength should be set at 292 nm.

6.2 HPLC analytical column, two types are possible:

— 250 mm × 4 mm, packed with microparticulate diol having a mean particle size of about 5 µm, or

— 250 mm × 4,6 mm, packed with microparticulate silica having a mean particle size of about 5 µm.

1) Merck Tocopherol set 613424 is available from Calbiochem (www.calbiochem.com). It contains one 50 mg

vial each of dl-α-tocopherol, d-β-tocopherol, d-γ-tocopherol, and d-δ-tocopherol with a purity of 95 % by HPLC (for

each component). Merck Tocotrienol set 613432 is available from Calbiochem also. It contains one 50 mg vial each of

α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol with a purity of 95 % by HPLC (75 % for γ-tocotrienol).

This information is given for the convenience of users of this document and does not constitute an endorsement by

ISO of the products named. Equivalent products may be used if they can be shown to lead to the same results.

2) Tocotrienols are available from Sigma Aldrich (www.sigmaaldrich.com) and from Chromadex (www.chromadex.

com) with purities between 65 % and 98 %. This information is given for the convenience of users of this document

and does not constitute an endorsement by ISO of the products named. Equivalent products may be used if they can

be shown to lead to the same results.
2 © ISO 2016 – All rights reserved
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SIST EN ISO 9936:2016
ISO 9936:2016(E)

NOTE 1 Suitable diol silica column packing material available commercially is 5 µm LiChrospher 100 Diol;

suitable silica column packing materials available commercially are 5 µm LiChrosob SI 60 and Kromasil 100 .

When β-tocotrienol is expected in the sample, the diol silica column is preferred as γ-tocopherol and β-tocotrienol

are co-eluted when using the silica column.

NOTE 2 The length and the diameter of the column can be varied according to the HPLC technique used.

NOTE 3 Both types of columns have been used for the evaluation of the precision data (Annex C).

6.3 UV spectrometer, capable of absolute measurement of absorbance at precisely defined

wavelengths, with a 10-mm path length cell.
6.4 Rotary evaporator.
6.5 Graduated cylinder, of 1 000 ml capacity.
6.6 Volumetric pipettes, of 5 ml, 10 ml and 20 ml capacities.
6.7 Volumetric flasks, 50 ml and 25 ml capacities.
6.8 Ultrasonic bath.
7 Sampling

A representative sample should be sent to the laboratory. It is important that the sample has not been

damaged or changed during transport or storage.

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

method is given in ISO 5555.
8 Preparation of test sample

In the case of liquid laboratory samples, prepare the test sample by homogenization as described in

ISO 661, except that filtration should be avoided.

In the case of solid samples, transfer a representative portion (i.e. not less than 10 % by mass of the

laboratory sample) to a glass beaker and carefully homogenize by melting, with gentle mixing, in a

water bath at a temperature not exceeding 40 °C.

Preparation of the test samples should be carried out, as far as is practicable, in subdued light and in all

cases out of direct sunlight.
9 Procedure

IMPORTANT — In general, the oxidation of tocols during the analysis may lead to low results.

The rate of oxidation is increased in the presence of alkalis, or under the influence of heat or

light, and measures should be taken to guard against these influences.

3) These types of columns 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 ISO of these products.

© ISO 2016 – All rights reserved 3
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SIST EN ISO 9936:2016
ISO 9936:2016(E)
9.1 Preparation of calibration solutions
9.1.1 Stock calibration solutions

Prepare a stock solution of each tocol by weighing 10 mg ± 1 mg of the standard (5.1) into a 50 ml

volumetric flask (6.7) and diluting to the mark with n-heptane (5.3).

Pipette 5 ml (6.6) of this solution into an amber glass round-bottomed flask and remove all n-heptane on

a rotary evaporator (6.4) under vacuum at a temperature not greater than 40 °C. Restore atmospheric

pressure with nitrogen and remove the flask from the evaporator as soon as all the solvent has been

removed. Pipette into the flask 10 ml (6.6) of methanol (5.5) and swirl to dissolve the residue. Measure

the maximum absorbance of this solution in a wavelength range between 270 nm and 310 nm (see

appropriate wavelength in Table 1) using the UV spectrometer (6.3) with a 10-mm path length cell. The

measured absorbance should be between 0,2 and 0,8. Calculate the concentration (in micrograms per

millilitre) by dividing the absorbance value by the appropriate factor given in Table 1.

Table 1 — Division factors
Wavelength
Tocopherol Division factor
292 α-tocopherol 0,007 6
296 β-tocopherol 0,008 9
298 γ-tocopherol 0,009 1
298 δ-tocopherol 0,008 7

NOTE The factors quoted are derived from the E values (1 %/1 cm) of the tocopherols.

For example, the E value (1 %/1 cm) of α-tocopherol is 76 at 292 nm (in methanol);

therefore a 1 µg/ml solution of α-tocopherol will have an absorbance of 0,007 6 at 292 nm.

9.1.2 Standard solution

A suitable standard solution should be prepared, according to the sensitivity of the fluorescence

detector used.

The following preparation of working solution is given as an example: mix appropriate volumes, for

example 1 ml, of the stock calibration solutions (see 9.1.1) to obtain a mixed tocol standard solution,

and dilute with n-heptane to give a solution containing between 1 µg and 5 µg of each standard per

millilitre.
The standard solution shall be freshly prepared each working day.
Protect all solutions from light and store them at between 0 °C and 4 °C.

Stock standard solutions can be satisfactorily stored in amber glassware for up to 1 week if refrigerated.

Flasks may be wrapped in aluminium foil.
NOTE If a UV detector is used, a more concentrated solution might be needed.
9.2 Optimization of working parameters

9.2.1 If the column (6.2) is new or of unknown history, or if for any other reason it is necessary to

condition it, wash and condition it for about 10 min with methanol, then dichloromethane, followed by

n-heptane at a flow rate of about 1 ml/min.

Pump the HPLC mobile phase (5.4) through the column at a flow rate of 1 ml/min for at least 30 min.

WARNING — Methanol and dichloromethane are hazardous to humans and to the environment.

Handle them with care.
4 © ISO 2016 – All rights reserved
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SIST EN ISO 9936:2016
ISO 9936:2016(E)

9.2.2 Inject 10 µl or 20 µl (according to detector sensitivity) of the standard solution (see 9.1.2) into

the column and, if necessary, adjust the tetrahydrofuran content of the mobile phase and the flow rate to

achieve the following conditions:
a) α-tocopherol retention time between 8 min and 12 min;

b) resolution factor, RF, for the separation of β- and γ-tocopherols of not less than 1,0; i.e. almost

baseline separation, where RF is calculated using Formula (1):
ddII− I
() ()
RF = (1)
 
05, ⋅ bbII+ I
() ()
 
 
where
d (I) is the retention distance of γ-tocopherol;
d (II) is the retention distance of β-tocopherol;
b(I) is the width at the base of the γ-tocopherol peak;
b(II) is the width at the base of the β-tocopherol peak.

9.2.3 Select the optimum settings for the detection and integration system. Inject 10 µl or 20 µl of

the standard solution (see 9.1.2). Repeat the injection and check that reproducible chromatograms are

obtained.
9.3 Preparation of test solution

Depending on the tocol concentration (see 9.1.2), weigh, to the nearest 1 mg, 0,25 g ± 0,1 g of the test

sample (see Clause 8) into a 25 ml one-mark volumetric flask. Add a quantity of n-heptane (5.3), swirling

to dissolve the test portion, and dilute to the mark with the same solvent. Filter the solution with an

HPLC nylon filter 0,45 µm if not clear.

It is important that the test solutions be protected from light prior to analysis, and analysed on the day

of preparation.

NOTE It may be necessary to prepare a more concentrated solution or to dilute the solution further prior to

chromatography.
9.4 Determination

9.4.1 Inject 10 µl or 20 µl (according to detector sensitivity) of the standard solution (see 9.1.2) into

the column and record the areas of the peaks.

9.4.2 Inject 10 µl or 20 µl (according to detector sensitivity) of the test solution (see 9.3) into the

column and identify the tocols present by reference to the calibration chromatograms. Record the areas

of the peaks. Repeat the injection of the test solution and the measurement. Use the mean values of the

two measurements as the result of one determination.

Inject a further 10 µl or 20 µl (according to detector sensitivity) of the standard solution (see 9.1.2) and

record the areas of the peaks.
The relative retention times shown in Table 2 have been found to be typical.
© ISO 2016 – All rights reserved 5
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SIST EN ISO 9936:2016
ISO 9936:2016(E)
Table 2 — Example of relative retention time of tocopherols and tocotrienols
Silica column Diol column
(α-tocopherol as reference substance) (α-tocopherol as reference substance)

α-tocopherol = 1,00 α-tocotrienol = 1,19 α-tocopherol = 1,00 α-tocotrienol = 1,24

β-tocopherol = 1,34 β-tocotrienol = 1,63 β-tocopherol = 1,59 β-tocotrienol = 2,03

γ-tocopherol = 1,63 γ-tocotrienol = 2,00 γ-tocopherol = 1,74 γ-tocotrienol = 2,22

δ-tocopherol = 2,24 δ-tocotrienol = 2,79 δ-tocopherol = 2,46 δ-tocotrienol = 3,19

10 Expression of results

The α-tocopherol content, w, of the sample, expressed in milligrams per kilogram (mg/kg), is given by

Formula (2):
ρ××AV
w= (2)
Am×
where

ρ is the concentration, in micrograms per millilitre, of α-tocopherol in the standard solution

(9.1.2);
is the mean of the peak areas obtained for the α-tocopherol standard;
is the mean of the peak areas obtained for the α-tocopherol in the test sample;
m is the mass, in grams, of the test sample (9.3);
V is the volume of test solution prepared (= 25 ml).

Calculate the remaining tocol contents of the test sample in the same way using the data obtained from

the corresponding standard.

If the only standard available is α-tocopherol, relate all tocopherols to this standard, but make this clear

when reporting the results. If UV detection is used, again relate all tocopherols to the α-tocopherol

standard, but normalize the peak areas to α-tocopherol using the division factors given in 9.1.1.

NOTE The fluorescence intensity of tocotri
...

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