EN ISO 15788-1:2001

Animal and vegetable fats and oils - Determination of stigmastadienes in vegetable oils - Part 1: Method using capillary-column gas chromatography (Reference method) (ISO 15788-1:1999)

Tierische und pflanzliche Fette und Öle - Bestimmung der Stigmastadiene in Pflanzenölen - Teil 1: Verfahren mit Kapillarsäulen-Gaschromatographie (Referenzverfahren) (ISO 15788-1:1999)

Dieser Teil von ISO 15788 legt ein Verfahren zur Bestimmung von Stigmastadienen in nativen pflanzlichen Ölen und insbesondere in nativen Olivenölen fest, die diese Kohlenwasserstoffe in geringen Konzentrationen enthalten.
Dieses Verfahren ist auf alle pflanzlichen Öle anwendbar, obgleich die Messungen nur zuverlässig sind, wenn der Gehalt an diesen Kohlenwasserstoffen zwischen 0,01 mg/kg und 4,0 mg/kg liegt. Das Verfahren ist zum Nachweis raffinierter pflanzlicher Öle (Olivenöl, Oliventresteröl, Sonnenblumenöl, Palmöl usw.) in nativen Olivenölen geeignet, da raffinierte Öle im Gegensatz zu kaltgepressten Ölen Stigmastadiene enthalten.

Corps gras d'origines animale et végétale - Dosage des stigmastadiènes dans les huiles végétales - Partie 1: Méthode par chromatographie en phase gazeuse sur colonne capillaire (Méthode de référence) (ISO 15788-1:1999)

La présente partie de l'ISO 15788 spécifie une méthode de dosage des stigmastadiènes dans les huiles végétales vierges contenant de faibles concentrations de ces hydrocarbures, et notamment dans l'huile d'olive vierge.  Cette méthode est applicable à toutes les huiles végétales bien que les mesurages ne soient fiables que lorsque la teneur en hydrocarbures se situe entre 0,01 mg/kg et 4,0 mg/kg. Elle permet de détecter la présence d'huiles végétales raffinées (d'olive, de grignon d'olive, de tournesol, de palme, etc.) dans les huiles d'olive vierges, dans la mesure où les huiles raffinées renferment des stigmastadiènes, alors que les huiles vierges obtenues par pression à  froid n'en renferment pas.

Rastlinske in živalske maščobe in olja - Določevanje stigmastadienov v rastlinskih oljih - 1. del: Metoda uporabe kapilarno-kolonske plinske kromatografije (Referenčna metoda) (ISO 15788-1:1999)

General Information

• Status: Published
• Publication Date: 19-Jun-2001
• ICS: 67.200.10 - Animal and vegetable fats and oils
• Technical Committee: CEN/TC 307 - Oilseeds, vegetables and animal fats and oils and their by-products - Methods of sampling and analys
• Drafting Committee: CEN/TC 307 - Oilseeds, vegetables and animal fats and oils and their by-products - Methods of sampling and analys
• Current Stage: 9093 - Decision to confirm - Review Enquiry
• Start Date: 01-Jun-2007
• Completion Date: 01-Jun-2007
• Ref Project: SIST EN ISO 15788-1:2001 - Animal and vegetable fats and oils - Determination of stigmastadienes in vegetable oils - Part 1: Method using capillary-column gas chromatography (Reference method) (ISO 15788-1:1999)

Overview

EN ISO 15788-1:2001 is a European standard adopted from the international ISO 15788-1:1999 standard, focusing on the determination of stigmastadienes in vegetable oils, primarily virgin olive oil. Stigmastadienes, steroidal hydrocarbons formed during thermal processing of oils, serve as crucial chemical markers for assessing the quality and refining status of vegetable oils. This standard prescribes a precise reference method using capillary-column gas chromatography (GC) for the quantitative analysis of these compounds.

Developed under ISO Technical Committee 34, which deals with agricultural food products, the method is essential for detecting minor amounts of refined oils in virgin oil batches, thereby ensuring product authenticity and quality control.

Key Topics

• Stigmastadienes in Vegetable Oils
  Stigmastadienes (primarily 3,5-stigmastadiene) arise during high-temperature refining steps such as bleaching and deodorizing. Virgin oils contain negligible amounts (<0.01 mg/kg), whereas refined oils have much higher concentrations (1–120 mg/kg). Measuring stigmastadienes helps distinguish between virgin and refined oils.

• Scope and Applicability
  The method applies to all vegetable oils but is particularly reliable for those with stigmastadiene concentrations between 0.01 mg/kg and 4.0 mg/kg, supporting quality control in virgin olive oil and related products.

• Analytical Principle
  The procedure involves isolating the unsaponifiable fraction from the oil, extracting steroidal hydrocarbons using silica gel column chromatography, and analyzing by capillary-column gas chromatography with flame ionization detection. Quantification uses internal standards and specific chromatographic parameters.

• Reagents and Equipment
  Essential reagents include hexane, ethanol, potassium hydroxide, activated silica gel, and standard solutions of 3,5-cholestadiene and 3,5-stigmastadiene. Equipment includes rotary evaporators, separating funnels, silica gel chromatography columns, and gas chromatographs with fused silica capillary columns.

• Chromatographic Conditions
  The method specifies injector and detector temperatures (300 °C and 320 °C, respectively), carrier gases (helium or hydrogen), oven temperature programming, split ratios, and integration modes. These ensure reproducible and well-resolved chromatograms for accurate peak identification and quantification.

Applications

• Quality Control of Virgin Olive Oil
  EN ISO 15788-1:2001 allows producers and laboratories to verify the purity of virgin olive oils by detecting adulteration with refined vegetable oils.

• Authentication of Vegetable Oils
  By measuring stigmastadiene levels, this standard supports certification and compliance testing to international quality requirements.

• Refining Process Monitoring
  Refiners can utilize this method to monitor the formation of stigmastadienes during heating steps, optimizing processes to limit thermal degradation.

• Research and Development
  The method provides a reliable tool for scientific investigations into the chemical changes during oil processing and refining, supporting innovation in vegetable oil products.

Related Standards

• EN ISO 15788-2
  This is the complementary part of the series that specifies an alternative method for identifying stigmastadienes using high-performance liquid chromatography (HPLC).

• Codex Alimentarius Standards
  Harmonized international food standards sometimes reference stigmastadiene determination for establishing vegetable oil authenticity.

• IUPAC Methods on Oils and Fats Analysis
  The standardized method reflects recommendations and collaborative studies approved by the IUPAC Commission on Oils, Fats, and Derivatives.

• Other CEN Standards on Fats and Oils
  Integrated quality testing of vegetable oils often includes methods for determining fatty acid profiles, sterols, and unsaponifiable matter alongside stigmastadienes.

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By adhering to the robust and validated procedures in EN ISO 15788-1:2001, laboratories and industry players ensure accurate, reproducible, and internationally recognized testing for stigmastadienes. This supports regulatory compliance, product quality assurance, and consumer protection in the global vegetable oil market.

Standards Content (Sample)

SLOVENSKI STANDARD
01-december-2001
5DVWOLQVNHLQåLYDOVNHPDãþREHLQROMD'RORþHYDQMHVWLJPDVWDGLHQRYYUDVWOLQVNLK
ROMLKGHO0HWRGDXSRUDEHNDSLODUQRNRORQVNHSOLQVNHNURPDWRJUDILMH
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Animal and vegetable fats and oils - Determination of stigmastadienes in vegetable oils -
Part 1: Method using capillary-column gas chromatography (Reference method) (ISO
15788-1:1999)
Tierische und pflanzliche Fette und Öle - Bestimmung der Stigmastadiene in
Pflanzenölen - Teil 1: Verfahren mit Kapillarsäulen-Gaschromatographie
(Referenzverfahren) (ISO 15788-1:1999)
Corps gras d'origines animale et végétale - Dosage des stigmastadienes dans les huiles
végétales - Partie 1: Méthode par chromatographie en phase gazeuse sur colonne
capillaire (Méthode de référence) (ISO 15788-1:1999)
Ta slovenski standard je istoveten z: EN ISO 15788-1:2001
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 15788-1
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2001
ICS 67.200.10
English version
Animal and vegetable fats and oils - Determination of
stigmastadienes in vegetable oils - Part 1: Method using
capillary-column gas chromatography (Reference method) (ISO
15788-1:1999)
Corps gras d'origines animale et végétale - Dosage des Tierische und pflanzliche Fette und Öle - Bestimmung der
stigmastadiènes dans les huiles végétales - Partie 1: Stigmastadiene in Pflanzenölen - Teil 1: Verfahren mit
Méthode par chromatographie en phase gazeuse sur Kapillarsäulen-Gaschromatographie (Referenzverfahren)
colonne capillaire (Méthode de référence) (ISO 15788- (ISO 15788-1:1999)
1:1999)
This European Standard was approved by CEN on 13 May 2001.
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 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 Management Centre has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2001 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 15788-1:2001 E
worldwide for CEN national Members.

Page 2
Foreword
The text of the International Standard from Technical Committee ISO/TC 34 "Agricultural food
products" of the International Organization for Standardization (ISO) has been taken over as
an European Standard by 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 2001, and conflicting
national standards shall be withdrawn at the latest by December 2001.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy,
Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United
Kingdom.
Endorsement notice
The text of the International Standard ISO 15788-1:1999 has been approved by CEN as a
European Standard without any modification.

INTERNATIONAL ISO
STANDARD 15788-1
First edition
1999-10-15
Animal and vegetable fats and oils —
Determination of stigmastadienes in
vegetable oils —
Part 1:
Method using capillary-column gas
chromatography (Reference method)
Corps gras d'origines animale et végétale — Dosage des stigmastadiènes
dans les huiles végétales —
Partie 1: Méthode par chromatographie en phase gazeuse sur colonne
capillaire (Méthode de référence)
A
Reference number
ISO 15788-1:1999(E)
© ISO
ISO 15788-1:1999(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3.
Draft International Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote.
International Standard ISO 15788-1 was prepared by Technical Committee ISO/TC 34, Agricultural food products,
Subcommittee SC 11, Animal and vegetable fats and oils.
ISO 15788 consists of the following parts, under the general title Animal and vegetable fats and oils —
Determination of stigmastadienes in vegetable oils:
 Part 1: Method using capillary-column gas chromatography
 Part 2: Method using high-performance liquid chromatography
Annex A of this part of ISO 15788 is for information only.
©  ISO 1999
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic
or mechanical, including photocopying and microfilm, without permission in writing from the publisher.
International Organization for Standardization
Case postale 56 • CH-1211 Genève 20 • Switzerland
Internet [e-mail]
Printed in Switzerland
ii
© ISO
ISO 15788-1:1999(E)
Introduction
Significant amounts of hydrocarbons are formed in vegetable oils as a consequence of thermal treatments during
the refining processes [1]. Among these hydrocarbons, 3,5-stigmastadiene, a steroidal compound, is the most
abundant in all refined vegetable oils since it is derived from b-sitosterol by dehydration [2]. The 3,5-stigmastadiene
is produced together with minor amounts of the 2,4-isomer and both substances give a single well-defined gas-
chromatographic peak when the hydrocarbon fraction is analysed on a low polar column [3]. Therefore, the sum of
both isomers can be easily quantified by gas-chomatographic analysis of the steroidal hydrocarbon fraction [2], [4].
For instance, for virgin olive oils, the usual oil production processes (pressure or centrifuging) do not produce
measurable amounts of stigmastadienes (less than 0,01 mg/kg). In crude olive residue oil, small concentrations of
stigmastadienes are found (ranging between 0,2 mg/kg and 3 mg/kg) due to the high temperatures applied during
the drying of the raw olive residue.
In the refining processes, stigmastadienes are formed in all the steps involving high temperatures, such as
bleaching and deodorizing, but more amounts are formed in the first step using acid bleaching earth than in the
second [2]. Depending on the conditions applied during the refining process, commercial refined vegetable oils
show stigmastadiene concentrations ranging between 1 mg/kg and 120 mg/kg and, therefore, the assessment of
stigmastadienes allows not only the identification of thermally treated oils but also the detection of minor amounts of
refined vegetable oils in virgin oils.
A method for the determination of stigmastadienes and the results of a collaborative study carried out under the
auspices of the International Olive Oil Council were presented to the International Union of Pure and Applied
Chemistry (IUPAC) Commission on Oils, Fats and Derivatives. The stigmastadiene content has been adopted as an
identity criterion for virgin olive oils and the resulting analytical method has been adopted as an international
standard method.
iii
INTERNATIONAL STANDARD  © ISO ISO 15788-1:1999(E)
Animal and vegetable fats and oils — Determination of
stigmastadienes in vegetable oils —
Part 1:
Method using capillary-column gas chromatography (Reference
method)
1 Scope
This part of ISO 15788 specifies a method for the determination of stigmastadienes in virgin vegetable oils
containing low concentrations of these hydrocarbons, particularly in virgin olive oil.
This method is applicable to all vegetable oils although measurements are reliable only where the content of these
hydrocarbons lies between 0,01 mg/kg and 4,0 mg/kg. The method is suited to detecting the presence of refined
vegetable oils (olive, olive pomace, sunflower, palm, etc.) in virgin olive oils, since refined oils contain stigmasta-
dienes and cold-extracted oils do not.
2 Principle
Unsaponifiable matter is isolated. The steroidal hydrocarbon fraction is separated by column chromatography on
silica gel and analysed by capillary gas chromatography.
3 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or
water of equivalent purity.
3.1  Hexane, or mixture of alkanes of boiling point from 65 °C to 70 °C, distilled with a rectifying column.
The hexane shall be redistilled to remove impurities.
3.2  Ethanol, 96 % (volume fraction).
3.3  Anhydrous sodium sulfate.
3.4  Alcoholic potassium hydroxide solution, of concentration 50 g per 500 ml.
Add 10 ml of water to 50 g potassium hydroxide, stir, and then dilute the mixture with ethanol to 500 ml.
Alcoholic potassium hydroxide solution turns brown on standing. It should be prepared freshly each day and kept in
well-stoppered dark glass bottles.
© ISO
ISO 15788-1:1999(E)
1)
, for column chromatography, 70 to 230 mesh.
3.5 Silica gel 60
Usually, silica gel can be used directly from the container without any treatment. However, some batches of silica
may show low activity resulting in poor chromatographic separations. Under these circumstances, the silica gel
should be treated in the following way.
Activate the silica gel by heating for at least 4 h at 550 °C. After heating, place the silica gel in a desiccator while the
gel is cooling and then transfer the silica gel to a stoppered flask. Add 2 % of water and shake until no lumps can be
seen and the powder flows freely.
If batches of silica gel result in chromatograms with interfering peaks, the silica gel should be treated as above. An
1)
alternative would be the use of extra pure silica gel 60 .
2)
3.6  3,5-Cholestadiene , 99 % purity (mass fraction), stock solution in hexane, of concentration 10 mg per 50 ml.
3.7  3,5-Cholestadiene, standard solution in hexane, of concentration of 1 mg per 50 ml, obtained by dilution of the
stock solution (3.6).
NOTE If kept at a temperature of below 4 °C, solutions (3.6) and (3.7) will not deteriorate over a period of at least
4 months.
3.8  n-Nonacosane, solution in hexane, of concentration approximately 10 mg per 100 ml.
3)
(24-ethylcholesta-3,5-diene), solution in hexane, of concentration approximately 10 mg
3.9 3,5-Stigmastadiene
per 100 ml.
3.10  Carrier gas, for chromatography: helium or hydrogen of 99,9990 % purity (mass fraction).
3.11  Auxiliary gases, for flame ionization detector: hydrogen of 99,9990 % purity (mass fraction), and purified air.
4 Apparatus
Usual laboratory apparatus and, in particular, the following.
4.1  Flasks, of 250 ml capacity, suitable for use with a reflux condenser.
4.2  Separating funnel, of 500 ml capacity.
4.3  Round-bottomed flasks, of 100 ml capacity.
4.4  Rotary evaporator.
4.5  Glass chromatography column of 1,5 cm i.d. and 50 cm length, with Teflon tap and a plug of glass wool fibre
or sintered glass disc at the bottom.
...