SIST EN ISO 12228-2:2015

SLOVENSKI STANDARD
01-januar-2015
1DGRPHãþD
SIST EN ISO 12228:2000
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GHO2OMþQDROMDLQROMDL]ROMþQLKWURSLQ,62
Determination of individual and total sterols contents - Gas chromatographic method -
Part 2: Olive and olive pomace oils (ISO 12228:2014)
Bestimmung der individuellen und der Gesamtsterine - Gaschromatographisches
Verfahren - Teil 2: Oliven- und Oliventresteröle (ISO 12228:2014)
Détermination de la teneur en stérols individuels et totaux - Méthode par
chromatographie en phase gazeuse - Partie 2: Huile d'olive et huile de grignons d'olive
(ISO 12228:2014)
Ta slovenski standard je istoveten z: EN ISO 12228-2:2014
ICS:
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 12228-2
NORME EUROPÉENNE
EUROPÄISCHE NORM
October 2014
ICS 67.200.10 Supersedes EN ISO 12228:1999
English Version
Determination of individual and total sterols contents - Gas
chromatographic method - Part 2: Olive and olive pomace oils
(ISO 12228:2014)
Détermination de la teneur en stérols individuels et totaux - Bestimmung der individuellen und der Gesamtsterine -
Méthode par chromatographie en phase gazeuse - Partie 2: Gaschromatographisches Verfahren - Teil 2: Oliven- und
Huile d'olive et huile de grignons d'olive (ISO 12228-2:2014) Oliventresteröle (ISO 12228-2:2014)
This European Standard was approved by CEN on 6 September 2014.

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
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 12228-2:2014 E
worldwide for CEN national Members.

Contents Page
Foreword .3

Foreword
This document (EN ISO 12228-2:2014) 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 April 2015, and conflicting national standards shall be withdrawn at the
latest by April 2015.
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 12228:1999.
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 12228-2:2014 has been approved by CEN as EN ISO 12228-2:2014 without any modification.
INTERNATIONAL ISO
STANDARD 12228-2
First edition
2014-10-01
Determination of individual
and total sterols contents — Gas
chromatographic method —
Part 2:
Olive oils and olive pomace oils
Détermination de la teneur en stérols individuels et totaux —
Méthode par chromatographie en phase gazeuse —
Partie 2: Huile d’olive et huile de grignons d’olive
Reference number
ISO 12228-2:2014(E)
©
ISO 2014
ISO 12228-2:2014(E)
© ISO 2014
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
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Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2014 – All rights reserved

ISO 12228-2:2014(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents . 2
6 Apparatus . 3
7 Sample . 4
7.1 Sampling . 4
7.2 Preparation of the test sample . 4
8 Procedure. 4
8.1 Test portion . 4
8.2 Preparation of unsaponifiable matter. 4
8.3 Separation of the sterol and triterpene dialcohols (erythrodiol, uvaol) fractions by TLC . 5
8.4 Preparation of the trimethylsilyl ethers . 6
8.5 Gas chromatographic analysis . 6
9 Expression of results . 7
9.1 Quantitative evaluation . 7
9.2 Determination of the total sterol content . 8
9.3 Composition of sterols . 8
9.4 Composition of triterpene dialcohols . 8
10 Precision . 9
10.1 Interlaboratory test. 9
10.2 Repeatability limit, r . 9
10.3 Reproducibility limit, R . 9
11 Test report . 9
Annex A (informative) Figures .10
Annex B (informative) Interlaborative trial .13
Bibliography .16
ISO 12228-2:2014(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 first edition of ISO 12228-2 cancels and replaces ISO 12228:1999, which has been technically
revised.
ISO 12228 consists of the following parts, under the general title Determination of individual and total
sterols contents — Gas chromatographic method:
— Part 1: Animal and vegetable fats and oils
— Part 2: Olive oils and olive pomace oils
iv © ISO 2014 – All rights reserved

INTERNATIONAL STANDARD ISO 12228-2:2014(E)
Determination of individual and total sterols contents —
Gas chromatographic method —
Part 2:
Olive oils and olive pomace oils
1 Scope
This part of ISO 12228 specifies a procedure for the gas chromatographic determination of the contents
and composition of sterols and triterpene dialcohols in olive and olive pomace oils. For the determination
of the contents and composition of sterols in all other animal and vegetable fats and oils, ISO 12228-1 is
to be used.
NOTE This part of ISO 12228 is technically identical to IOC Standard COI/T.20/Doc. No. 30 (November 2011).
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
composition of sterols
composition of individual sterols in the sample, beginning with cholesterol and ending with ∆7-avenasterol
(see Table 1) under the conditions specified in this part of ISO 12228
Note 1 to entry: The composition is expressed as a percentage of all peak areas, normalized to 100 %.
3.2
total sterol content
mass fraction of the sum of all individual sterols, as determined in accordance with the method specified
in this part of ISO 12228, beginning with cholesterol and ending with ∆7-avenasterol (see Table 1),
divided by the mass of the test portion
Note 1 to entry: The content is expressed in milligrams per kilogram.
3.3
composition of triterpene dialcohols
composition of erythrodiol and uvaol in the sample under the conditions specified in this part of
ISO 12228
Note 1 to entry: The composition is expressed as a percentage of all peak areas, beginning with cholesterol and
ending with uvaol (see Table 1) under the conditions specified in this part of ISO 12228, normalized to 100 %.
ISO 12228-2:2014(E)
4 Principle
A test portion is saponified by boiling under reflux with an ethanolic potassium hydroxide solution. The
unsaponifiable matter is extracted with diethyl ether. The sterol and triterpene dialcohol fractions are
separated from the unsaponifiable matter by thin-layer chromatography on a basic silica gel plate. The
qualitative and quantitative compositions of the sterol and triterpene dialcohol fractions are determined
by gas chromatography of the trimethylsilyl ethers using cholestanol as internal standard.
5 Reagents
WARNING — Attention is drawn to the regulations which specify the handling of hazardous
substances. Technical, organizational and personal safety measures shall be followed.
Use only reagents of recognized analytical grade, unless otherwise stated, and water complying with
[1]
grade 3 of ISO 3696.
5.1 Potassium hydroxide, minimum mass fraction w = 85 g/100 g.
5.2 Potassium hydroxide, ethanolic solution, amount concentration, c, approximately 2 mol/l.
While cooling, dissolve 130 g of potassium hydroxide (5.1) in 200 ml of distilled water and then make up
to 1 l with ethanol (5.9). Keep the solution in well-stoppered dark glass bottles and store for a maximum
of 2 d.
5.3 Potassium hydroxide, ethanolic solution, amount concentration, c, approximately 0,2 mol/l.
Dissolve 13 g of potassium hydroxide (5.1) in 20 ml of distilled water and make up to 1 l with ethanol
(5.9).
5.4 Diethyl ether, for chromatography.
WARNING — Diethyl ether is highly flammable and can form explosive peroxides. Explosive
limits in air are 1,7 % to 48 % (volume fraction). Take special precautions when using it.
5.5 Anhydrous sodium sulfate.
5.6 Silica gel thin-layer chromatography (TLC) plates, commercially available, dimensions
20 cm × 20 cm, thickness of layer 0,25 mm, without fluorescence indicator.
5.7 Acetone, for chromatography.
5.8 n-Hexane, for chromatography.
5.9 Ethanol 96 %, minimum volume fraction φ = 95 %.
5.10 Ethyl acetate.
5.11 Reference solution for thin-layer chromatography, cholesterol or mixture of phytosterols, and
erythrodiol solution in ethyl acetate (5.10), mass concentration, ρ = 5 %.
5.12 2,7-dichlorofluorescein, ethanolic solution, mass concentration, ρ = 0,2 %.
Make slightly basic by adding a few drops of 2 mol/l alcoholic potassium hydroxide solution (5.2). Store
for a maximum of 1 year.
2 © ISO 2014 – All rights reserved

ISO 12228-2:2014(E)
5.13 α-cholestanol internal standard solution, mass concentration, ρ = 0,2 g/100 ml, in ethyl acetate
(5.10).
5.14 Phenolphthalein solution, mass concentration, ρ = 10 g/l, in ethanol (5.9).
5.15 Carrier gas for gas chromatography, helium or preferably hydrogen.
5.16 Auxiliary gases for gas chromatography, hydrogen, helium, nitrogen, and air.
5.17 Developing solvent, mixture of n-hexane and diethyl ether, volume concentrations are: σ(n-
hexane) = 65 ml/100 ml, σ(diethyl ether) = 35 ml/100 ml.
5.18 Hexamethyldisilazane.
5.19 Trimethylchlorosilane.
5.20 Silylation reagent, mixture of pyridine, hexamethyldisilazane, and trimethylchlorosilane.
Volume concentration σ(pyridine) = 9 ml/13 ml, σ(hexamethyl disilazane) = 3 ml/13 ml,
σ(trimethylchlorosilane) = 1 ml/13 ml. Prepare the mixture fresh daily.
NOTE Other silylation reagents can be used, e. g., mixture of N,O-bis-trimethylsilyl-trifluoroacetamide
(BSTFA) and trimethylchlorosilane (TMCS), σ(BSTFA) = 99 ml/100 ml and σ(TMSC) = 1 ml/100ml.
6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1 Round-bottomed flasks, of 250 ml, with ground neck.
6.2 Reflux condenser, with ground glass joint to fit the flask (6.1).
6.3 Separating funnel, of 500 ml capacity.
6.4 Developing tank, made of glass, with a ground glass lid, suitable for use with plates of dimensions
20 cm × 20 cm.
6.5 Ultraviolet lamp, wavelength of 366 nm or 254 nm.
6.6 Microsyringe, to deliver 100 µl, 500 µl, and 1000 µl.
6.7 Cylindrical filter funnel, with sintered-glass filter (G3, porosity 15 µm to 40 µm), diameter
approximately 2 cm, depth 5 cm, suitable for filtration under vacuum with male ground glass joint.
6.8 Conical flask, for operation under a vacuum, 50 ml with ground glass female joint to fit to the filter
funnel (6.7).
6.9 Test tube, 10 ml with a tapering bottom and a sealing glass stopper.
6.10 Gas chromatograph, for capillary columns, with split injector, consisting of:
— column oven, capable of maintaining the temperature with an accuracy of ±1°C;
ISO 12228-2:2014(E)
— temperature-controlled injection unit, with persilanised glass vaporising element and split
system;
— flame ionization detector (FID);
— data acquisition system.
6.11 Capillary column, made of fused silica, length 20 m to 30 m, internal diameter 0,25 mm to 0,32 mm,
coated with 5 % Diphenyl, 95 % Dimethyl polysiloxane (SE-52 or SE-54 or equivalent stationary phase),
with a temperature limit of at least 280 °C to 300 °C, film thickness between 0,1 µm and 0,30 µm.
6.12 Microsyringe for gas chromatography, of 1 µl and 10 µl capacity, for gas chromatography, with
cemented needle suitable for split injection.
6.13 Desiccator, containing an efficient desiccant, for storing the plates, e.g. calcium dichloride.
6.14 Oven, maintained at 103 °C ± 2 °C.
6.15 Rotary evaporator, attached to a vacuum pump and water bath maintained at 40 °C.
6.16 Analytical balance, capable of weighing to the nearest 0,001 g and displaying 0,000 1 g.
7 Sample
7.1 Sampling
Sampling is not part of the method specified in this part of ISO 12228. A recommended sampling method
[2]
is given in ISO 5555.
It is
...