EN ISO 12228:1999

SLOVENSKI STANDARD
01-maj-2000
5DVWOLQVNHLQåLYDOVNHPDãþREHLQROMD'RORþHYDQMHSRVDPH]QLKLQFHORWQLK
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Animal and vegetable fats and oils - Determination of individual and total sterols contents
- Gas chromatographic method (ISO 12228:1999)
Tierische und pflanzliche Fette und Öle - Bestimmung der individuellen und der
Gesamtsterine - Gaschromatographisches Verfahren (ISO 12228:1999)
Corps gras d'origines animale et végétale - Détermination de la teneur en stérols
individuels et totaux - Méthode par chromatographie en phase gazeuse (ISO
12228:1999)
Ta slovenski standard je istoveten z: EN ISO 12228:1999
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.

INTERNATIONAL ISO
STANDARD 12228
First edition
1999-03-01
Animal and vegetable fats and oils —
Determination of individual and total sterols
contents — Gas chromatographic method
Corps gras d'origines animale et végétale — Détermination de la teneur en
stérols individuels et totaux — Méthode par chromatographie en phase
gazeuse
A
Reference number
ISO/FDIS 12228:1999(E)
ISO 12228:1999(E)
Contents
1 Scope .1
2 Normative references .1
3 Definitions .1
4 Principle.1
5 Reagents.2
6 Apparatus .2
7 Sampling.3
8 Preparation of test sample.3
9 Procedure .3
10 Expression of results .4
11 Precision.6
12 Test report .6
Annex A (informative) Interlaboratory test .9
Annex B (informative) Bibliography .16
©  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 iso@iso.ch
Printed in Switzerland
ii
© ISO
ISO 12228:1999(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.
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 12228 was prepared by ISO/TC 34, Agricultural food products, Subcommittee SC 11,
Animal and vegetable fats and oils.
Annexes A and B of this International Standard are for information only.
iii
INTERNATIONAL STANDARD  © ISO ISO 12228:1999(E)
Animal and vegetable fats and oils — Determination of individual
and total sterols contents — Gas chromatographic method
1 Scope
This International Standard specifies a method for the gas chromatographic determination of the contents and
compositions of sterols in animal and vegetable fats and oils.
2 Normative references
The following standards contain provisions which, through reference in this text, constitute provisions of this
International Standard. At the time of publication, the editions indicated were valid. All standards are subject to
revision, and parties to agreements based on this International Standard are encouraged to investigate the
possibility of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain
registers of currently valid International Standards.
ISO 661:1989, Animal and vegetable fats and oils — Preparation of test samples.
ISO 3696:1987, Water for analytical laboratory use — Specification and test methods.
3 Definitions
For the purposes of this International Standard, the following definitions apply.
3.1
composition of sterols
composition of individual sterols in the sample, beginning with cholesterol and ending with Δ7-avenasterol (see
table 1) under the conditions specified in this International Standard
NOTE The composition is expressed as peak area, in percent, and normalized to 100 %.
3.2
total sterol content
mass of the sum of all individual sterols, as determined in accordance with the method specified in this International
Standard, beginning with cholesterol and ending with Δ7-avenasterol (see table 1), divided by the mass of the test
portion
NOTE The content is expressed in milligrams per 100 g.
4 Principle
A test portion is saponified by boiling under reflux with an ethanolic potassium hydroxide solution. The
unsaponifiable matter is isolated by solid-phase extraction on an aluminium oxide column. The aluminium oxide
column is used to retain the fatty acid anions; sterols pass through the column. The sterol fraction from the
unsaponifiable matter is separated by thin-layer chromatography. The qualitative and quantitative compositions of
the sterol fraction are determined by gas chromatography using betulin as an internal standard.
© ISO
ISO 12228:1999(E)
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise stated, and water complying with grade 3 of
ISO 3696.
5.1  Potassium hydroxide, ethanolic solution, c(KOH) ≈ 0,5 mol/l.
Dissolve 3 g of potassium hydroxide in 5 ml of water and dilute to 100 ml with ethanol (5.3). The solution should be
colourless or straw-coloured.
5.2  Internal standard solution, betulin, 1,0 mg/ml solution in acetone (see note to 5.10).
NOTE In the case of olive pomace oils which may contain betulin, the use of 5α-cholestan-3β-ol (peak 2 in table 1) as
internal standard is recommended.
5.3  Ethanol, of minimum purity 95 % (V/V).
5.4  Aluminium oxide, neutral, 0,063 mm to 0,200 mm, activity step I.
5.5  Diethyl ether, freshly distilled, free from peroxides and residue.
WARNING: Diethyl ether is highly flammable and can form explosive peroxides. Explosive limits in air are
1,7 % (V/V) to 48 % (V/V). Take special precautions when using it.
5.6  Silica gel thin-layer chromatography (TLC) plates, commercially available, dimensions 20 cm x 20 cm,
thickness of layer 0,25 mm.
5.7  Developing solvent, hexane/diethyl ether [1/1 (V/V)].
5.8  Standard solution for thin-layer chromatography, 1,0 mg/ml cholesterol and 5,0 mg/ml betulin in acetone.
, methanol.
5.9 Spraying reagent
5.10  Silylating reagent, prepared by adding 50 μl of 1-methyl imidazole to 1 ml of N-methyl-N-(trimethylsilyl)-
hepta-fluorobutyramide (MSHFBA).
NOTE Other silylating reagents should normally not be used unless special precautions are taken to ensure that both
hydroxyl groups of betulin are silylated. If not, betulin may show two peaks in the GLC.
6 Apparatus
Usual laboratory apparatus and, in particular, the following.
6.1  Round-bottomed flasks, of 25 ml and 50 ml capacity, with ground neck.
, with ground joint to fit a flask (6.1).
6.2 Reflux condenser
6.3  Glass column, with PTFE stopper, sintered glass frit and reservoir for 100 ml, length 25 cm, internal diameter
1,5 cm.
6.4  Rotary evaporator, attached to a vacuum pump and water bath maintained at 40 °C.
6.5  Developing tank, made of glass, with a ground glass lid, suitable for use with plates of dimensions 20 cm x
20 cm.
6.6  Microsyringe or micropipette, to deliver 100 μl.
6.7  Oven, maintained at 105 °C ± 3 °C.
© ISO
ISO 12228:1999(E)
6.8  Desiccator, containing an efficient desiccant, for storing the plates.
6.9  Reaction vials, of 0,3 ml capacity, with screw caps and PTFE-lined seals, for preparation of sterol derivatives.
6.10  Gas chromatograph, for capillary columns, with split injector, flame ionization detector and suitable recorder.
6.11  Capillary column, made of fused silica or glass, length 25 m to 60 m, internal diameter 0,2 mm to 0,25 mm,
stationary phase SE-54 (or equivalent non-polar phase with a temperature limit of at least 280 °C to 300 °C); film
thickness about 0,1 μm.
6.12  Microsyringe for gas chromatography, for injecting volumes of 1 μl.
6.13  Analytical balance, capable of weighing to the nearest 0,001 g and displaying 0,0001 g.
7 Sampling
It is important that the laboratory receive a sample which is truly representative and 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 [1].
8 Preparation of test sample
Prepare the test samples in accordance with ISO 661.
9 Procedure
9.1 Test portion
Weigh, to the nearest 1 mg, about 250 mg of the test sample into a 25 ml flask (6.1).
For fats and oils with a sterol content of less than 100 mg per 100 g, proceed using a three-fold amount of the test
sample. Adjust reagents and apparatus accordingly.
9.2 Determination
9.2.1 Preparation of the aluminium oxide column
Suspend 10 g of aluminium oxide (5.4) in 20 ml of ethanol (5.3) and pour the slurry into the glass column (6.3).
Allow the aluminium oxide to settle and let the solvent run out of the column until the level of the solvent reaches the
top level of the aluminium oxide layer.
9.2.2 Extraction of the unsaponifiable matter
Add exactly 1,00 ml of internal standard solution (5.2) to the test portion (9.1).
Add 5 ml of ethanolic potassium hydroxide solution (5.1) and a few anti-bumping granules. Attach the reflux
condenser (6.2) to the flask and keep the contents gently boiling for 15 min. Stop heating. Immediately dilute the
contents of the flask while still hot with 5 ml of ethanol (5.3) and swirl or shake to homogenize.
Pipette 5 ml of this solution onto the prepared aluminium oxide column (9.2.1). Collect the eluate in a 50 ml round-
bottom flask (6.1) and allow the column to run off until the solvent level has reached the top level of the aluminium
oxide layer. Elute the unsaponifiable matter first with 5 ml of ethanol (5.3) and then with 30 ml of diethyl ether (5.5),
with a flowrate of about 2 ml/min. Remove the solvents from the flask by means of the rotary evaporator (6.4).
WARNING: The aluminium oxide column is essential for this procedure. It shall not be replaced by silica or
other columns or by solvent extraction.
© ISO
ISO 12228:1999(E)
9.2.3 Thin-layer chromatography
Dissolve the unsaponifiable matter obtained in 9.2.2 in a small amount of diethyl ether (5.5). Apply the solution as a
line at a distance of 2 cm from the lower edge onto a TLC plate (5.6) using the microsyringe (6.6). Leave a gap of at
least 3 cm from each side edge of the plate. Apply a spot of 5 μl of the TLC standard solution (5.8) at 1,5 cm from
the edge. Fill the developing tank (6.5) with about 100 ml of developing solvent (5.7). Place the plate into the tank
and develop it until the solvent reaches the upper edge. Remove the plate from the tank and allow the solvent to
evaporate in a fume cupboard.
NOTE Quantitative transfer of the material (9.2.2) to the TLC plate is not necessary in this step. Automatic apparatus for
applying streaks may be used. No saturation chamber is used.
9.2.4 Isolation of the sterols
Spray the plates with methanol (5.9) until the sterol and betulin zones appear white on a translucent (darker)
background. The betulin zone appears slightly below the sterol zone. Mark the zones at the height of the standard
spots 2 mm above and 4 mm below the visible zones (see figure 1). Scratch off this part of the layer completely
using
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