SIST EN ISO 12228-1:2014

SLOVENSKI STANDARD
SIST EN ISO 12228-1:2014
01-oktober-2014
1DGRPHãþD
SIST EN ISO 12228:2000
'RORþHYDQMHSRVDPH]QLKLQFHORWQLKVWHURORY3OLQVNDNURPDWRJUDIVNDPHWRGD
GHO5DVWOLQVNHLQåLYDOVNHPDãþREHLQROMD,62
Determination of individual and total sterols contents - Gas chromatographic method -
Part 1: Animal and vegetable fats and oils (ISO 12228-1:2014)
Bestimmung der individuellen und der Gesamtsterine - Gaschromatographisches
Verfahren - Teil 1: Tierische und pflanzliche Fette und Öle (ISO 12228-1:2014)
Détermination de la teneur en stérols individuels et totaux - Méthode par
chromatographie en phase gazeuse - Partie 1: Corps gras d'origines animale et végétale
(ISO 12228-1:2014)
Ta slovenski standard je istoveten z: EN ISO 12228-1:2014
ICS:
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
SIST EN ISO 12228-1:2014 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 12228-1:2014

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SIST EN ISO 12228-1:2014

EUROPEAN STANDARD
EN ISO 12228-1

NORME EUROPÉENNE

EUROPÄISCHE NORM
July 2014
ICS 67.200.10 Supersedes EN ISO 12228:1999
English Version
Determination of individual and total sterols contents - Gas
chromatographic method - Part 1: Animal and vegetable fats and
oils (ISO 12228-1:2014)
Détermination de la teneur en stérols individuels et totaux - Bestimmung der individuellen und der Gesamtsterine -
Méthode par chromatographie en phase gazeuse - Partie 1: Gaschromatographisches Verfahren - Teil 1: Tierische und
Corps gras d'origines animale et végétale (ISO 12228- pflanzliche Fette und Öle (ISO 12228-1:2014)
1:2014)
This European Standard was approved by CEN on 14 June 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 12228-1:2014 E
worldwide for CEN national Members.

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SIST EN ISO 12228-1:2014
EN ISO 12228-1:2014 (E)
Contents Page
Foreword .3

2

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SIST EN ISO 12228-1:2014
EN ISO 12228-1:2014 (E)
Foreword
This document (EN ISO 12228-1:2014) 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 January 2015, and conflicting national standards shall be withdrawn at
the latest by January 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 12228-1:2014 has been approved by CEN as EN ISO 12228-1:2014 without any modification.

3

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SIST EN ISO 12228-1:2014

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SIST EN ISO 12228-1:2014
INTERNATIONAL ISO
STANDARD 12228-1
First edition
2014-07-15
Determination of individual
and total sterols contents — Gas
chromatographic method —
Part 1:
Animal and vegetable fats and oils
Détermination de la teneur en stérols individuels et totaux —
Méthode par chromatographie en phase gazeuse —
Partie 1: Corps gras d’origines animale et végétale
Reference number
ISO 12228-1:2014(E)
©
ISO 2014

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SIST EN ISO 12228-1:2014
ISO 12228-1:2014(E)

COPYRIGHT PROTECTED DOCUMENT
© 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

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SIST EN ISO 12228-1:2014
ISO 12228-1:2014(E)

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 2
6 Apparatus . 3
7 Sample . 3
7.1 Sampling . 3
7.2 Preparation of the test sample . 3
8 Procedure. 4
8.1 Preparation of the aluminium oxide column . 4
8.2 Test portion . 4
8.3 Extraction of the unsaponifiable matter . 4
8.4 Thin-layer chromatography . 4
8.5 Isolation of the sterols . 4
8.6 Preparation of sterol trimethylsilyl ethers . 5
8.7 Gas chromatography . 5
9 Expression of results . 5
9.1 Identification of sterols . 5
9.2 Composition of sterols . 5
9.3 Determination of the total sterol content . 6
10 Precision . 7
10.1 Interlaboratory test. 7
10.2 Repeatability limit, r . 7
10.3 Reproducibility limit, R . 7
11 Test report . 7
Annex A (informative) Figures . 8
Annex B (informative) Interlaboratory trial .15
Bibliography .23
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SIST EN ISO 12228-1:2014
ISO 12228-1:2014(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 12228-1, together with 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 content — Gas chromatographic method:
— Part 1: Animal and vegetable fats and oils
— Part 2: Olive oils and olive pomace oils
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SIST EN ISO 12228-1:2014
INTERNATIONAL STANDARD ISO 12228-1:2014(E)
Determination of individual and total sterols contents —
Gas chromatographic method —
Part 1:
Animal and vegetable fats and oils
1 Scope
This part of ISO 12228 specifies a procedure for the gas chromatographic determination of the content
and composition of sterols in animal and vegetable fats and oils. However, the determination of the
contents and composition of sterols in olive and olive pomace oils is to be carried out using ISO 12228-2.
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
ISO 3696, Water for analytical laboratory use — Specification and test methods
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 Δ7-
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 Δ7-avenasterol (see Table 1),
divided by the mass of the test portion
Note 1 to entry: The content is expressed in milligrams per kilogram.
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 cholestanol or betulin as the internal standard.
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SIST EN ISO 12228-1:2014
ISO 12228-1:2014(E)

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 (KOH), ethanolic solution, molar concentration c(KOH) approximately
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, cholestanol (5α-cholestan-3β-ol) or betulin, volume fraction of
1,0 mg/ml solution in ethanol (see note to 5.10).
NOTE In case of hydrogenated oils, which may contain cholestanol, the use of betulin (peak 17 in Table 1) is
recommended.
5.3 Ethanol, of minimum volume fraction φ = 95 %.
5.4 Aluminium oxide, neutral, particle size 0,063 mm to 0,200 mm, activity grade I (water
content = 0 %).
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 % to 48 % (volume fraction). Take special precautions when using it. Keep
away from heat sources and sunlight.
5.6 Silica gel thin-layer chromatography (TLC) plates, commercially available, dimensions
20 cm × 20 cm, thickness of layer 0,25 mm.
5.7 Developing solvent, hexane/diethyl ether.
Volume fraction of each solvent is 50 ml/100 ml.
5.8 Standard solution for thin-layer chromatography, volume fraction of 1,0 mg/ml
cholesterol/cholestanol in acetone or 5,0 mg/ml betulin in acetone.
NOTE 1 Cholesterol and cholestanol have the same Rf value (0,35) in TLC while the Rf value for betulin is 0,30
(see Figure A.1).
NOTE 2 In case of hydrogenated oils, which may contain cholestanol, the use of betulin (peak 17 in Table 1) is
recommended.
5.9 Spraying reagent, methanol.
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 Ready-to-use solutions are commercially available. Other silylation reagents, e.g. bis trimethylsilyl
trifluoroacetamide with 1 % trimethylchlorosilane, are also available and can be used when cholestanol is used
as internal standard. However for betulin special precautions are taken to ensure that both hydroxyl groups of
betulin are silylated. If not, betulin may show two peaks in the chromatogram.
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SIST EN ISO 12228-1:2014
ISO 12228-1:2014(E)

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.
6.2 Reflux condenser, with ground glass joint to fit a flask (6.1).
6.3 Glass column, with polytetrafluoroethylene (PTFE) stopper, sintered glass filter 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 × 20 cm.
6.6 Microsyringe, to deliver 100 µl.
6.7 Oven, maintained at 105 °C ± 3 °C.
6.8 Desiccator, containing an efficient desiccant, for storing the plates.
6.9 Reaction vials, of 0,3 ml to (1,0 to 1,5) 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.
NOTE A better resolution of the peaks is obtained with a film thickness of 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 0,000 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
[1]
is given in ISO 5555 .
It is important that the laboratory receives a sample which is truly representative and has not been
damaged or changed during transport or storage.
7.2 Preparation of the test sample
Prepare the test sample in accordance with ISO 661.
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SIST EN ISO 12228-1:2014
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8 Procedure
8.1 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.
8.2 Test portion
Weigh, to the nearest 1 mg, about 250 mg of the test sample into a 25 ml flask (6.1), proceed with 8.3.
For fats and oils with a low sterol content (for example less than 2 000 mg per kilogram) or for other
reasons, proceed using a threefold amount of the test sample. Adjust reagents and apparatus accordingly.
8.3 Extraction of the unsaponifiable matter
Add exactly 1,00 ml of internal standard solution (5.2) to the test portion (8.2). 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 (8.1). 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 flow rate 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.
8.4 Thin-layer chromatography
Dissolve the unsaponifiable matter obtained in 8.3 in a small amount (approximately 0,5 ml) 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 (8.3) to the TLC plate is not necessary in this step. Automatic
apparatus for applying streaks may be used. Saturation of the chamber is not required.
8.5 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 cholestanol is part of the Δ5-sterol zone (see Figure A.1). Mark the zones at
the height of the standard spots 2 mm above and 4 mm below the visible zones (see Figure A.1). Scratch
off this part of the layer completely using a spatula and quantitatively collect the silica in a small beaker.
NOTE 1 The wider margin at the lower edge of the visible zones (4 mm vs. 2 mm at the upper edge) is a precaution
to avoid partial loss of betulin in this step. Sunflower seed oil may show three bands (∆5-sterols, ∆7-sterols and
betulin).
NOTE 2 Betulin, if used as internal standard, appears slightly below the sterol zone (see Figure A.1).
Add 0,5 ml of ethanol to the collected silica gel. Extract the silica gel in the beaker three times with 5 ml
of diethyl ether (5.5) and filter into a flask (6.1). Reduce the combined ether extracts to about 1 ml in
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SIST EN ISO 12228-1:2014
ISO 12228-1:2014(E)

the rotary evaporator (6.4) and transfer the remaining solution into the reaction vial (6.9). Blow off the
solvent in the reaction vial with a stream of nitrogen.
8.6 Preparation of sterol trimethylsilyl ethers
Add 100 µl of the silylation reagent (5.10) to the reaction vial (6.9) containing the isolated sterols. Seal
and heat the vial for 15 min in the oven set at (105 ± 3) °C. Allow the reaction vial to cool to room
temperature and inject the solution directly into the gas chromatograph (6.10).
8.7 Gas chromatography
Optimize the temperature programme and the carrier gas flow rate so that chromatograms similar to
Figures A.2 to A.7 are obtained. Test the separation with silylated sterol fractions obtained from known
oils, as shown in Figures A.2 to A.7.
NOTE 1 The following parameters were tested and found useful (see chromatograms in Annex A): GC column:
SE-54, 50 m length, 0,25 mm internal diameter, 0,10 µm film thickness; carrier gas H , carrier gas flow rate 36 cm/s,
2
split 1:20, detector/injector 320° C, temperature programme 245 °C to 265 °C at 5 °C/min, 40 min isothermal at
265 °C; injection volume 1 µl. Capillary columns of equivalent quality can be used.
NOTE 2 A standard solution containing cholesterol, campesterol, stigmasterol, and sitosterol may be used to
check the retention times. Use a blank run to test for possible contamination (e.g. cholesterol) from solvents, glass
walls, filter, fingerprints, etc.
9 Expression of results
9.1 Identification of sterols
To identify the sterols present in the test sample, determine the relative retention times (RRT) by
dividing the retention time (RT) of the sterol in question by the RT of cholesterol and/or betulin. Table 1
shows the RRT of the various sterols corresponding to cholesterol (RRT ) and betulin (RRT ), with SE-
C B
54 stationary phase.
NOTE The RRT in Table 1 (determined under the conditions of note 1 in 8.7) are mentioned as an aid for the
identification of the individual sterols only, and to illustrate the elution sequence (cf. also Figure A.1). The actual
RRT found may deviate slightly from the RRT given in Table 1 because the RRT depends on the experimental
conditions (type and length of GLC column, temperature programme, and quality of stationary phase).
9.2 Composition of sterols
Calculate the mass fraction w , of the individual sterol i, in g/100 g (percent), according to the following
i
equation:
A
i
w =⋅100 (1)
i
A
∑
where
A is the area of the peak of sterol i;
i
∑A is the sum of the peak areas of all sterols (peaks 1, 3 to 16 or 1 to 16, if betulin is used).
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SIST EN ISO 12228-1:2014
ISO 12228-1:2014(E)

Table 1 — Gas chromatograhic peak identification of individual sterols and betulin by RRT (SE-
54 stationary phase)
Peak
Common names of sterols Systematic names of sterols RRT RRT
C B
No
1 Cholesterol Cholest-5-en-3β-ol 1,00 0,44
2 Cholestanol 5α-Cholestan-3β-ol 1,02 0,45
3 Brassicasterol [24S]-24-Methyl cholesta-5,22-dien-3β-ol 1,09 0,48
4 24-Methylene cholesterol 24-Methylene cholesta-5,24-dien-3β-ol 1,21 0,53
5 Campesterol [24R]-24-Methyl cholest-5-en-3β-ol 1,23 0,54
6 Campestanol [24R]-24-Methyl cholestan-3β-ol 1,25 0,55
7 Stigmasterol [24S]-24-Ethyl cholesta-5,22-dien-3β-ol 1,31 0,57
8 ∆7-Campesterol [24R]-24-Methyl cholest-7-en-3β-ol 1,38 0,59
9 ∆5,23-Stigmastadienol [24R,S]-24-Ethyl cholesta-5,23-dien-3β-ol 1,40 0,60
10 Clerosterol [24S]-24-Ethyl cholesta-5,25-dien-3β-ol 1,42 0,62
11 Sitosterol [24R]-24-Ethyl cholest-5-en-3β-ol 1,47 0,64
12 Sitostanol [24R]-24-Ethyl cholestan-3β-ol 1,50 0,65
13 ∆5-Avenasterol [24Z]-24(28)-Ethylidene cholest-5-en-3β
...