SIST EN ISO 20122:2024

SLOVENSKI STANDARD
01-junij-2024
Rastlinska olja - Določevanje nasičenih ogljikovodikov mineralnih olj (MOAH) in
aromatskih ogljikovodikov mineralnih olj (MOAH) z on-line sklopljeno analizo s
tekočinsko kromatografijo visoke ločljivosti in plinsko kromatografijo v povezavi s
plamenskim ionizacijskim detektorjem (HPLC-GC-FID) - Metoda za nizko mejo
določljivosti (ISO 20122:2024)
Vegetable oils - Determination of mineral oil saturated hydrocarbons (MOSH) and
mineral oil aromatic hydrocarbons (MOAH) with online-coupled high performance liquid
chromatography-gas chromatography-flame ionization detection (HPLC-GC-FID)
analysis - Method for low limit of quantification (ISO 20122:2024)
Pflanzliche Öle - Bestimmung von gesättigten Mineralölkohlenwasserstoffen (MOSH)
und aromatischen Kohlenwasserstoffen (MOAH) mit online gekoppelter HPLC-GC-FID-
Analyse - Verfahren für die niedrige Bestimmungsgrenze (ISO 20122:2024)
Huiles végétales - Dosage des hydrocarbures saturés d’huile minérale (MOSH) et des
hydrocarbures aromatiques d’huile minérale (MOAH) par analyse par chromatographie
en phase liquide haute performance et chromatographie en phase gazeuse couplées à
un détecteur à ionisation de flamme (CLHP-CG-FID) en ligne - Méthode pour une faible
limite de quantification (ISO 20122:2024)
Ta slovenski standard je istoveten z: EN ISO 20122:2024
ICS:
67.200.10 Rastlinske in živalske Animal and vegetable fats
maščobe in olja and oils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 20122
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2024
EUROPÄISCHE NORM
ICS 67.200.10
English Version
Vegetable oils - Determination of mineral oil saturated
hydrocarbons (MOSH) and mineral oil aromatic
hydrocarbons (MOAH) with online-coupled high
performance liquid chromatography-gas chromatography-
flame ionization detection (HPLC-GC-FID) analysis -
Method for low limit of quantification (ISO 20122:2024)
Huiles végétales - Dosage des hydrocarbures saturés Pflanzliche Öle - Bestimmung von gesättigten
d'huile minérale (MOSH) et des hydrocarbures Mineralölkohlenwasserstoffen (MOSH) und
aromatiques d'huile minérale (MOAH) par analyse par aromatischen Kohlenwasserstoffen (MOAH) mit online
chromatographie en phase liquide haute performance gekoppelter HPLC-GC-FID-Analyse - Verfahren für die
et chromatographie en phase gazeuse couplées à un niedrige Bestimmungsgrenze (ISO 20122:2024)
détecteur à ionisation de flamme (CLHP-CG-FID) en
ligne - Méthode pour une faible limite de quantification
(ISO 20122:2024)
This European Standard was approved by CEN on 12 January 2024.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20122:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 20122:2024) 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 2024, and conflicting national standards shall
be withdrawn at the latest by October 2024.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
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, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 20122:2024 has been approved by CEN as EN ISO 20122:2024 without any modification.

International
Standard
ISO 20122
First edition
Vegetable oils — Determination of
2024-04
mineral oil saturated hydrocarbons
(MOSH) and mineral oil aromatic
hydrocarbons (MOAH) with
online-coupled high performance
liquid chromatography-gas
chromatography-flame ionization
detection (HPLC-GC-FID) analysis
— Method for low limit of
quantification
Reference number
ISO 20122:2024(en) © ISO 2024
ISO 20122:2024(en)
© ISO 2024
All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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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CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 20122:2024(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents . 3
6 Apparatus . 6
7 Sample . 7
7.1 Sampling .7
7.2 Preparation of the final sample for liquid and solid fats .7
8 Procedures . 8
8.1 General .8
8.2 Hexane/ethanol distribution for removal of interfering substances .8
8.3 Saponification . .8
8.4 Removal of biogenic n-alkanes with aluminium oxide for determination of the MOSH
fraction .9
8.5 Clean-up before epoxidation to separate polar substances .9
8.6 Ethanolic epoxidation of the MOAH fraction to oxidize unsaturated non-aromatic
compounds .9
8.7 HPLC-GC separation .10
8.7.1 HPLC conditions .10
8.7.2 GC configuration .10
8.7.3 Solvent vapour exit configuration .11
8.7.4 Peak identification .11
8.7.5 System suitability test . 12
8.8 Blank run . 13
8.9 Quality control . 13
9 Result of the determination .13
9.1 Testing the chromatograms for sufficient epoxidation and other relevant parameters. 13
9.2 Calculation .14
10 Precision of the method .15
10.1 Repeatability limit . 15
10.2 Reproducibility limit . 15
11 Test report .15
Annex A (informative) Graphics and chromatograms. 17
Annex B (informative) Precision data .28
Annex C (informative) Alternative method for the epoxidation of the MOAH fraction (performic
acid epoxidation) . 41
Bibliography .42

iii
ISO 20122:2024(en)
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO’s adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34 Food products, Subcommittee SC 11,
Animal and vegetable fats and oils, in collaboration with the European Committee for Standardization
(CEN) Technical Committee CEN/TC 307, Oilseeds, vegetable and animal fats and oils and their by-products —
Methods of sampling and analysis, in accordance with the Agreement on technical cooperation between ISO
and CEN (Vienna Agreement).
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
ISO 20122:2024(en)
Introduction
In order to achieve a low limit of quantification (LOQ), the method contains additional and partially modified
processing steps, specifications for the uniform processing of defined product groups and additional
requirements for system suitability compared to EN 16995:2017.
The method has been tested in an interlaboratory study via the analysis of both naturally contaminated and
spiked vegetable oil samples, ranging from 1 mg/kg to 75 mg/kg for MOSH, and from 1 mg/kg to 7 mg/kg
for MOAH.
v
International Standard ISO 20122:2024(en)
Vegetable oils — Determination of mineral oil saturated
hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons
(MOAH) with online-coupled high performance liquid
chromatography-gas chromatography-flame ionization
detection (HPLC-GC-FID) analysis — Method for low limit of
quantification
1 Scope
This document specifies a procedure for the determination of saturated and aromatic hydrocarbons (from
C10 to C50) in vegetable fats and oils using the online-coupled high performance liquid chromatography-gas
[4][5][6]
chromatography-flame ionization detection (HPLC-GC-FID). This document does not apply to other
matrices.
The method is applicable for the analysis of mineral oil saturated hydrocarbons (MOSH) and/or mineral oil
aromatic hydrocarbons (MOAH).
According to the results of the interlaboratory studies, the method has been proven suitable for MOSH mass
concentrations above 3 mg/kg and MOAH mass concentrations above 2 mg/kg.
In case of suspected interferences, the fossil origin of the MOSH and MOAH fraction can be verified by
examination by GC⨯GC-MS.
An alternative method for the epoxidation of the MOAH fraction (performic acid epoxidation) is proposed
in Annex C. This alternative method provides comparable results to the ethanolic epoxidation of the MOAH
fraction described in 8.6. This alternative method for epoxidation has proven to be efficient for samples with
[14]
a high amount of interferences in the MOAH fraction (e.g. tropical oils).
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. 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.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/

ISO 20122:2024(en)
3.1
mineral oil saturated hydrocarbons
MOSH
paraffinic (open-chain, usually branched) and naphthenic (cyclic, alkylated) hydrocarbons in the boiling
range of n-alkanes with a chain length of 10 to 50 carbon atoms, which are obtained from mineral oil by this
method by means of online-coupled high performance liquid chromatography-gas chromatography-flame
ionization detection (HPLC-GC-FID)
3.2
mineral oil aromatic hydrocarbons
MOAH
aromatic mainly alkylated hydrocarbons from mineral oil in the boiling range of n-alkanes with a chain
length of 10 to 50 carbon atoms, determined by means of online-coupled high performance liquid
chromatography-gas chromatography-flame ionization detection (HPLC-GC-FID)
3.3
unresolved complex mixture
UCM
complex mixture of saturated or aromatic hydrocarbons not resolved by gas chromatography such as
branched paraffins, alkylated naphthenes and alkylated aromatics, that produces a hump when analysed by
gas chromatography-flame ionization detection (GC-FID)
3.4
polyolefin oligomeric saturated hydrocarbons
POSH
synthetic hydrocarbons from oligomers of polyolefins, such as polyethylene, polypropylene and
polybutylenes
Note 1 to entry: Food contact uses comprise plastic bags, containers or films, heat sealable layers and other lamination
as well as adhesives and plasticizers.
Note 2 to entry: POSH can be distinguished from mineral oil saturated hydrocarbons (MOSH) by their chromatographic
[5]
pattern, but it is difficult to differentiate and chromatographically separate them from the MOSH if both are present.
3.5
resin oligomeric saturated hydrocarbons
ROSH
synthetic saturated hydrocarbons (oligomers from monoterpenes, cyclopentadienes and other C5- or C9-
monomeres) that are ingredients of hot-melt adhesives and can migrate into the sample mostly via gas phase
transfer or via direct contact
3.6
resin oligomeric aromatic hydrocarbons
ROAH
synthetic aromatic hydrocarbons that are ingredients of hot-melt adhesives and can migrate into the sample
mostly via gas phase transfer or by direct contact
3.7
poly-alpha-olefins
PAO
synthetic iso-paraffins with short and long side chains, used as lubricants or in adhesives and hotmelts
Note 1 to entry: When analysed by gas chromatography-flame ionization detection (GC-FID), they are recognized by
[5]
series of rather narrow humps of unresolved branched hydrocarbons with regular distance between them.
4 Principle
The sample is saponified and from the unsaponifiable residue, purified fractions are obtained following
additional steps. These fractions are separated on a silica gel column of the HPLC-GC-FID system into MOSH

ISO 20122:2024(en)
and MOAH fractions; each is transferred separately to the GC by online coupling. Most of the solvent is
removed via a solvent vapour exit between the uncoated pre-column and the GC separation column.
In order to meet the requirements of the various interfering accompanying substances occurring in the
samples, specific sample preparation procedures are described for different product groups. However,
epoxidation is a purification step that is necessary for the quantification of MOAH for all vegetable oil
samples. This purification step allows the elimination of olefins such as squalene, which elute within the
MOAH fraction and interfere with quantification. Depending on the sample, this reaction can induce the
epoxidation of a part of the MOAH or incomplete removal of the interfering olefins.
The signal area for mineral oil is calculated by subtracting riding peaks from the total area. The riding peaks
can be caused by n-alkanes (naturally occurring hydrocarbons), terpenes, sterenes, squalene and their
isomerization products as well as other substances. MOSH and MOAH are quantitated by internal standard
added before analysis. Verification standards are added for monitoring proper HPLC fractionation and GC
transfer conditions.
NOTE Epoxidation step can induce degradation of MOAH with three or more aromatic rings.
5 Reagents
WARNING — Reference is drawn to regulations that specify the handling of hazardous substances.
Technical, organizational and personal safety measures shall be followed.
All materials shall be tested for their influence in a blank run. It is recommended to heat all glassware in an
oven according to the instructions. All other materials that come into direct contact with the sample should
also be heated and should not be made of polyethylene or polypropylene.
Unless otherwise stated:
— analytical pure reagents shall be used;
— water shall be either distilled or of corresponding purity;
— a solution is understood to be an aqueous solution.
1)
5.1 Silica gel 60 , extra pure, for column chromatography, with a particle size between 60 μm and
200 μm (70 to 230 mesh) stored in a glass bottle (protection against contamination). The silica gel is heated
in an oven at 400 °C for at least 16 h and cooled in a clean desiccator (without ground grease).
5.2 Sodium sulfate, anhydrous, analytical grade, purity ≥ 99 %.
In case of contamination, heat the sodium sulfate in an oven at 400 °C for at least 16 h and allow to cool down
in a clean desiccator (without ground grease).
5.3 n-Hexane, free of hydrocarbons in the boiling range of the n-alkanes C10 to C50 and other impurities
such as hexane oxidation products.
Check the purity of the n-hexane as follows:
— mix 30 ml n-hexane with 25 μl of the internal standard solution (5.17) and two drops of bis(2-ethylhexyl)
maleate (5.26);
— evaporate using an evaporator unit;
— dissolve the residue in 0,2 ml n-hexane;
— inject 50 μl into the HPLC-GC-FID system for analysis.
1) Silica gel is available from Merck, reference 7754 or 7734. It is an example of a suitable product available commercially.
This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of
this product. Equivalent products may be used if they can be shown to lead to the same results.

ISO 20122:2024(en)
The hump signal (excluding any sharp single peaks of the solvent blank) should not exceed one-tenth of the LOQ.
NOTE Hydrocarbons in the boiling range under investigation interfere with the specific detection of mineral
oil constituents in gas chromatography of the MOSH and MOAH fractions, while polar compounds such as hexane
oxidation products interfere with the separation of long-chain n-alkanes in column chromatography on alumina.
5.4 Dichloromethane (DCM), purity ≥ 99 %.
Test the purity as for n-hexane (5.3) with 30 ml DCM.
5.5 Toluene.
5.6 Perylene (PER), purity > 99 %.
5.7 5-alpha-cholestane (CHO), purity ≥ 97 %.
5.8 n-Undecane (C11), purity ≥ 99 %.
5.9 n-Tridecane (C13), purity ≥ 99 %.
5.10 Tri-tert-butylbenzene (TBB), purity ≥ 97 %.
5.11 Bicyclohexyl (CYCY), purity ≥ 99 %.
5.12 1-Methyl naphthalene (1-MN), purity ≥ 95 %.
5.13 2-Methyl naphthalene (2-MN), purity ≥ 97 %.
5.14 Pentyl benzene (PB), purity ≥ 99 %.
5.15 Stock solution, mass concentrations, ρ = 5 mg/ml, 10 mg/ml or 20 mg/ml. Weigh, for example, 50 mg
of C13 (5.9), 100 mg each of C11 (5.8), TBB (5.10), CYCY (5.11), 1-MN (5.12), 2-MN (5.13) and PB (5.14) as well
as 200 mg CHO (5.7) and PER (5.6) to the nearest 1 mg and fill up to the mark in a 10 ml volumetric flask
with toluene (5.5). Store at room temperature to keep the solutions stable. Dissolve any crystals formed
during storage by gentle heating.
The verification of the start of the MOAH fraction, based on TBB, can result in losses of higher alkylated
benzenes and naphthalenes, if present in some samples (i.e. cosmetics) and when the chromatographic
performance of the column is limited. In such cases, di(2-ethylhexyl) benzene (DEHB) can be used in addition
[10]
as verification standard and the fractionation shall be adapted.
2)
5.16 Internal standard solution 1 (ISTD1) , mass concentrations ρ = 150 μg/ml (C13), 300 μg/ml (C11,
CYCY, PB, 1-MN, 2-MN and TBB) and 600 μg/ml (CHO and PER). Transfer 300 μl stock solution (5.15) into a
10 ml volumetric flask and fill up to the mark with toluene (5.5).
5.17 Internal standard solution 2 (ISTD2), mass concentrations ρ = 30 μg/ml (C13), 60 μg/ml (C11, TBB,
CYCY, 1-MN, 2-MN and PB) and 120 μg/ml (PER and CHO) Dilute ISTD1 solution (5.16) by a factor of 5, e.g. fill
up 1 000 μl ISTD1 solution (5.16) to 5 ml with n-hexane.
5.18 Aluminium oxide 90, alkaline, for column chromatography, 0,063 mm to 0,2 mm, activated. Heat the
alumina before use for at least 16 h at 500 °C in an oven and cool down to room temperature in a cleaned
desiccator (without ground grease).
2) This standard mixture is available from, for example, Restek Corp., Cat.# 31070. It is an example of a suitable product
commercially available. This information is given for the convenience of users of this document and does not constitute
an endorsement by ISO of this product. Equivalent products may be used if they can be shown to lead to the same results.

ISO 20122:2024(en)
5.19 meta-chloroperbenzoic acid (mCPBA), stated quantities based on a purity of about ≤ 77 %;
Commercially available mCPBA contains varying amounts of mCPBA, meta-chlorobenzoic acid and water.
For purification of the reagent, remove contaminating hydrocarbons, e.g. finely suspend 5 g mCPBA with
200 ml n-hexane in a polyethylene terephthalate (PET) beaker in an ultrasonic bath and filter by using a
vacuum frit. Let the purified mCPBA dry in a fume cupboard. Do not store in glass containers, as mCPBA
decomposes on glass surfaces. Commercially available mCPBA still contains meta-chlorobenzoic acid and
residual moisture to make handling in the laboratory safe. Pure mCPBA, on the other hand, is explosive, so
isolation of mCPBA as a pure substance, which goes beyond the cleaning described here, is not recommended.
Washing with a solvent mixture of 200 ml n-hexane and 20 ml DCM can remove further impurities, but also
leads to significant higher losses (yields only 75 % of the initial mCPBA with a content of about 74 g to 84 g
mCPBA per 100 g starting material in the purified product).
To determine the mCPBA content of the reagent, weigh about 0,2 g mCPBA into a PET beaker, add 50 ml
distilled water and mix thoroughly. Add 5 ml concentrated acetic acid and 10 ml sodium iodide solution (10 g
sodium iodide in 100 ml water). Then pre-titrate with 0,1 N sodium thiosulfate solution from dark red to
light yellow. Add a few drops of starch indicator solution and titrate from dark blue to colourless at the end
point (consumption usually below 20 ml).
Calculate the content w (mCPBA) in per cent by mass as shown by Formula (1):
NV××86,29×100
w= (1)
E
where
N is the normality of the sodium thiosulfate solution;
V is the total volume of consumed sodium thiosulfate solution in l;
E is the mass of the reagent in g.
5.20 mCPBA solution in ethanol, ρ = 100 mg/ml, e.g. dissolve 1 g mCPBA (5.19) in 10 ml ethanol (5.28).
Prepare the solution fresh every working day.
5.21 Sodium thiosulfate, anhydrous, purity > 90,0 %.
5.22 Sodium hydrogen carbonate (or sodium carbonate), anhydrous, purity > 90,0 %.
5.23 Solution for deactivation of the excess of mCPBA: Sodium thiosulfate and sodium carbonate
solution, ρ = 50 g/l, e.g. dissolve 5 g of sodium thiosulfate and 5 g of sodium hydrogen carbonate (or sodium
carbonate) in 100 ml distilled water and mix thoroughly.
5.24 Alumina column with silica gel cover. Place a filter (6.4) in a glass column (6.3). Add and compress
10 g of alumina (5.18), 3 g of silica gel (5.1) and 1 g of sodium sulfate (5.2).
5.25 Clean-up column. Place a filter (6.4) in an empty SPE glass cartridge (volume 6 ml), add 3 g of silica
gel (5.1), compress and cover with 1 g of sodium sulfate (5.2).
5.26 bis (2-ethylhexyl) maleate, purity 90 %. Check the purity in a blank run.
Bis (2-ethylhexyl) maleate may be replaced by bis(2-ethylhexyl) sebacate in order to limit the risk of
epoxidation process disturbance.
5.27 Standard solution of the n-alkanes with chain lengths of 10 to 50 in the same mass concentration
for checking for discrimination against low- or high-boiling substances, ρ = 1 μg/ml. Store this solution
at room temperature, otherwise C50 can crystallize.

ISO 20122:2024(en)
5.28 Ethanol, absolute.
Test the purity as for n-hexane (5.3) with 30 ml ethanol.
5.29 Mixture of ethanol and n-hexane, volume fraction φ = 50 %, e.g. mix 50 ml of ethanol (5.28) with
50 ml of n-hexane (5.3).
5.30 Elution mixture of n-hexane and DCM, e.g. mix 30 ml DCM (5.4) with 70 ml n-hexane (5.3). Due to
the volatility of DCM, the solution shall be freshly prepared.
5.31 Potassium hydroxide solution, e.g. 50 g potassium hydroxide in 100 ml distilled water, w = 33 g/100 g.
6 Apparatus
In order to achieve a sufficiently low blank level, the following process has proven to be effective: glassware
(except volumetric flasks) should be heated in an oven at 430 °C for 4 h or overnight at 400 °C and kept in
desiccators or other containers for use. In addition, it is recommended to:
— perform multiple determinations in different series and not directly one after another;
— not use grease for ground joints;
— not use hand cream;
— handle samples with gloves, only;
— use glassware where possible;
— check nitrogen for purity, when drying in a stream of nitrogen;
— rinse volumetric flasks, glass pipettes and other required glassware with n-hexane before use.
6.1 Analytical balance, readability 0,000 1 g, weighing accuracy 0,001 g.
6.2 Centrifuge and centrifuge tubes.
6.3 Glass column, without stopcock, 15 cm to 20 cm long and 15 mm to 20 mm inner diameter.
6.4 Filter for glass column, extracted or heated, filter made of quartz wool/glass fibre.
6.5 Glass vial, 40 ml, with polytetrafluoroethylene (PTFE) sealed screw cap.
6.6 Rotary evaporator, with vacuum and water bath at 35 °C.
Comparable devices can also be used. Take care to prevent contamination. If necessary, clean the system
thoroughly between determinations.
3)
6.7 HPLC column, e.g. LiChrospher Si 60 or Allure Silica , 5 µm material, 2 × 250 mm or comparable.
4)
6.8 Uncoated GC guard column, fused silica or metal capillary, e.g. HydroGuard® MXT® ,
10 m × 0,53 mm or comparable.
3) LiChrospher Si 60 and Allure Silica 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.
4) HydroGuard® MXT® is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by ISO of this product.

ISO 20122:2024(en)
NOTE The capillaries in 6.8 to 6.12 have proven to be suitable, but can be adapted to the system to meet the
requirements and yield comparable results.
6.9 GC separation column, fused silica or metal capillary, programmed temperature stable up to at
least 370 °C: 100 % dimethylpolysiloxane or 95 % dimethyl and 5 % phenyl methylpolysiloxane as stationary
phase, length 15 m, internal diameter (ID) 0,32 mm or 0,25 mm and film thickness 0,10 μm to 0,25 μm.
6.10 Fused silica or metal capillary, deactivated, for transfer the HPLC fractions from the valve to the
T-connector of the GC, 1 m long, 0,1 mm ID.
6.11 Capillary, deactivated, from the T-connector between pre- and separation column to the vapour exit.
6.12 Restriction capillary at the vapour exit, deactivated, 1 m long, ID 0,05 mm.
6.13 Syringe, 100 μl, suitable for injection of 5 to 100 µl in liquid chromatography.
6.14 Pasteur pipette made of glass.
NOTE The use of plastic pipette tips and polyethylene foil leads to increased blank levels.
6.15 Online-coupled HPLC-GC-FID system, consisting of an HPLC instrument capable of running a binary
gradient, injection valve, HPLC column (6.7), ultraviolet light (UV) detector (detection wavelength: 230 nm),
switching valves for column backflush and fraction transfer into GC, GC with solvent vapour exit (SVE),
pneumatic control and evaluation system. In addition, an automatic control system is recommended.
6.16 Test tube shaker with temperature control and agitation (e.g. 500 r/min or comparable).
7 Sample
7.1 Sampling
Sampling is not part of this method. The sample may only be stored in glass bottles, aluminium or other
materials that do not release hydrocarbons. Packaging made of paper, polyethylene or polypropylene is
5)
unsuitable. Containers made of PET or foil bags made of a high-performance polyamide such as RILSAN
may be used in some cases. Attention shall also be paid to the closure and sealing materials of the containers.
The use of hand cream should be avoided when handling samples. The sampling shall be checked by blank
runs using n-hexane instead of a sample.
A recommended sampling method is given in ISO 5555.
7.2 Preparation of the final sample for liquid and solid fats
Prepare the test sample in accordance with ISO 661.
Special treatments of the test sample (e.g. filtering, melting) shall be mentioned.
5) RILSAN is an example of a suitable product available commercially. This information is given for the convenience of
users of this document and does not constitute an endorsement by ISO of this product.

ISO 20122:2024(en)
8 Procedures
8.1 General
Depending on the type of fats and oils, the samples shall be prepared differently. The specific cases A to C
are as follows:
— A: Prepare oils and fats with unknown or high content of biogenic, long-chain alkanes and unsaturated
[7]
compounds such as olive oil, rapeseed oil, sunflower oil and comparable samples according to 8.2
(ethanol-hexane distribution). Using two separate 10 ml fractions of the extract, run 8.3 (saponification)
on the first fraction for the determination of the MOSH content according to 8.4 (Alox column), and run
8.3 (saponification) on the second fraction for the determination of the MOAH content according to 8.5
(clean-up) and 8.6 (epoxidation).
— B: Prepare oils and fats with low biogenic, long-chain alkanes and disturbing unsaturated compounds
such as coconut fat, linseed oil, palm oil and comparable samples without aluminium oxide column
according to 8.2 (ethanol-hexane distribution), 8.3 (saponification), 8.5 (clean-up) and 8.6 (epoxidation).
Determine the MOSH and MOAH fractions from the solution obtained.
— C: For automated application of the alumina column, prepare oils and fats according to 8.2 (ethanol-
hexane distribution), 8.3 (saponification), 8.5 (clean-up) and 8.6 (epoxidation). Inject into the HPLC-GC-
FID system. After separation of the MOSH, which are passed to an integrated online Alox column, the
MOAH fraction can be determined in the same run using a two-channel system.
Every laboratory using automated procedures shall carry out tests to ensure that the results obtained with
the automated procedures do not deviate from results obtained with the manual procedure.
Unknown samples or mix oils samples may be analysed first without Alox cleanup. If the presence of
interfering long-chain n-alkanes significantly impact the hump and do not allow a proper integration, the
extract shall be re-injected using the alumina column to reduce interferences (e.g. for online Alox clean
up, the same sample extract used to determine MOAH is used for the subsequent separation of long-chain
n-alkanes with the aluminium oxide column for the determination of MOSH).
NOTE Only the manual purification method for MOSH fraction was validated during the collaborative study.
8.2 Hexane/ethanol distribution for removal of interfering substances
Weigh 3 g of the sample for oils and fats into a 40 ml centrifuge tube with screw cap. Add 30 ml of the
mixture of n-hexane (5.3) and ethanol (5.29) and 20 μl ISTD1 (5.16) or 100 μl ISTD2 (5.17), and homogenize.
Use 10 ml of this solution for the further procedure (see 8.3).
NOTE In case of oils and fats, no phase separation will be observed. Nevertheless, this step ensures a complete
saponification of oils and fats. If necessary, other quantities of internal standard can be added.
8.3 Saponification
Transfer an aliquot of 10 ml (see 8.2) into another sample tube and add 3 ml potassium hydroxide solution
(5.31). Saponify the solution for 30 min at 60 °C in a water bath while shaking until the solution becomes
clear. Cool down, add 5 ml n-hexane (5.3) and 5 ml mixture of ethanol and water (a volume fraction of 1:1),
shake the mixture again, transfer the lower phase after phase separation into a new vial and extract again
with an additional 5 ml of n-hexane. Combine both n-hexane extracts.
Depending on the sample preparation method, continue using the solution to separate biogenic, long-chain
n-alkanes with aluminium oxide (according to 8.4) or for epoxidation (according to 8.5 and 8.6).
NOTE The addition of ethanol after saponification enables a better phase separation and avoids foaming.

ISO 20122:2024(en)
8.4 Removal of biogenic n-alkanes with aluminium oxide for determination of the MOSH
fraction
Pre-clean the alumina column (5.24) with 20 ml of n-hexane in order to remove interfering substances (see
text on interfering substances below). Transfer the solution from 8.3 to the alumina column and elute the
hydrocarbons with 25 ml n-hexane (5.3). Collect the hydrocarbons beginning with the sample transfer.
Evaporate the solvent under vacuum (≥260 mbar) at 35 °C after adding two drops of bis (2-ethylhexyl)
maleate (5.26).
Dissolve the residue in about 1 ml n-hexane, centrifuge if necessary and fill into a vial. Inject 60 μl to 90 μl of
the solution for analysis with the HPLC-GC-FID system to determine the MOSH fraction.
The MOAH remain on the alumina column and shall not be determined from this eluate. It is recommended
to remove interfering substances from the alumina by pre-rinsing the column with 20 ml n-hexane. However,
clean alumina does not need to be pre-rinsed.
Removal of biogenic n-alkanes can also remove paraffinic waxes at the same time if present in the sample. In
these cases, analysis shall be carried out according to procedure B.
NOTE Paraffinic waxes are characterized by n-alkanes with n
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