Animal feeding stuffs: Methods of sampling and analysis - Determination of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) with on-line HPLC-GC-FID analysis

This document specifies a method for the determination of saturated and aromatic hydrocarbons (from C10 to C50) in feed. The method has been interlaboratory validated with online-HPLC-GC-FID – see [1], [2] and [3]. This method is not intended to be applied to other matrices.
The method can be used for the analysis of mineral oil saturated hydrocarbons (MOSH) and/or mineral oil aromatic hydrocarbons (MOAH).
The method is applicable for feed materials, in particular vegetable oils and other fat rich feed materials, compound feeds and pre-mixtures. It is not applicable to additives or deodistillates.
The method has been tested in an interlaboratory study via the analysis of both naturally contaminated and spiked samples (pre-mixture, soybean meal, sunflower seeds, chicken feed, pig feed, vegetable oil) ranging from 3 mg/kg to 286 mg/kg for MOSH and from 1 mg/kg to 16 mg/kg for MOAH.
According to the results of the interlaboratory study, the method has been proven suitable for MOSH and MOAH mass concentrations, each above 10 mg/kg. However, the method was not fully validated during the collaborative study for the premixture sample due to too low concentrations of MOSH and MOAH. The method was also not fully validated during the collaborative study for the sunflower seeds sample due to a too low concentration of MOAH.
NOTE   The conclusions regarding MOAH are based on 4 analyte / matrix combinations while according to the IUPAC protocol [4] expects this to be a minimum of 5.
In case of suspected interferences from natural sources, the fossil origin of the MOSH and MOAH fraction can be verified by examination of the pattern by GC-MS.
For the determination of MOSH and MOAH in edible fats and oils, another CEN standard is also available: EN 16995. For more information see [5].
Annex C proposes a manual alternative method to online HPLC-GC-FID analysis that can be used as a screening method.

Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von mineralölgesättigten Kohlenwasserstoffen (MOSH) und mineralölaromatischen Kohlenwasserstoffen (MOAH) mit Online-Analyse durch HPLC-GC-FID

Dieses Dokument legt ein Verfahren zur Bestimmung von gesättigten und aromatischen Kohlenwasserstoffen (von C10 bis C50) in Futter fest. Das Verfahren wurde im Ringversuch mit Online HPLC GC FID validiert – siehe [1], [2] und [3]. Dieses Verfahren ist nicht für die Anwendung auf andere Matrices vorgesehen.
Das Verfahren kann zur Bestimmung von gesättigten Mineralöl Kohlenwasserstoffen (MOSH) und/oder aromatischen Mineralöl Kohlenwasserstoffen (MOAH) verwendet werden.
Das Verfahren ist anwendbar für Futtermittel, insbesondere Pflanzenöle und andere fettreiche Futtermittel, Mischfuttermittel und Vormischungen. Es ist nicht anwendbar auf Zusatzstoffe oder Deodestillate.
Das Verfahren wurde in einem Ringversuch sowohl an natürlich kontaminierten als auch an angereicherten Proben (Vormischung, Sojabohnenmehl, Sonnenblumenkerne, Hühnerfutter, Schweinefutter, Pflanzenöl) in Bereichen von 3 mg/kg bis 286 mg/kg für MOSH und von 1 mg/kg bis 16 mg/kg für MOAH getestet.
Nach den Ringversuchsergebnissen hat sich das Verfahren bei Massenkonzentrationen an MOSH und MOAH von jeweils über 10 mg/kg als geeignet erwiesen. Das Verfahren wurde jedoch während der gemeinsamen Studie für die Probe einer Vormischung wegen zu geringer MOSH  und MOAH Konzentrationen nicht vollständig validiert. Außerdem wurde das Verfahren während der gemeinsamen Studie für die Sonnenblumenkern-Probe wegen einer zu geringen MOAH Konzentration nicht vollständig validiert.
ANMERKUNG   Die Schlussfolgerungen in Bezug auf MOAH basieren auf 4 Analyt /Matrix Kombinationen, während nach dem IUPAC-Protokoll [4] davon ausgegangen wird, dass dies mindestens 5 sind.
Wenn Störungen durch natürliche Quellen vermutet werden, kann die fossile Herkunft der MOSH  und MOAH Fraktion durch Untersuchung des chromatographischen Profils mit GC MS verifiziert werden.
Für die Bestimmung von MOSH und MOAH in essbaren Fetten und Ölen steht auch eine andere Norm zur Verfügung: EN 16995. Weitere Informationen siehe [5].
In Anhang C wird ein manuelles Alternativverfahren zur Online HPLC GC FID Analyse vorgeschlagen, das als Screening-Verfahren verwendet werden kann.

Aliments pour animaux : Méthodes d’échantillonnage et d’analyse - Détermination des hydrocarbures saturés d’huile minérale (MOSH) et des hydrocarbures aromatiques d’huile minérale (MOAH) par analyse CLHP CG-FID en ligne

Le présent document décrit une méthode de détermination des hydrocarbures saturés et aromatiques (de C10 à C50) présents dans les aliments composés pour animaux. La méthode a fait l’objet d’une validation interlaboratoires par une analyse CLHP CG FID en ligne — voir [1], [2] et [3]. La présente méthode n’est pas destinée à être appliquée à d’autres matrices.
La méthode peut être employée pour l’analyse des hydrocarbures saturés d’huile minérale (MOSH) et/ou des hydrocarbures aromatiques d’huile minérale (MOAH).
La méthode est applicable aux matières premières pour aliments des animaux, notamment les huiles végétales et autres matières riches en matière grasse, aux aliments composés et aux prémélanges. Elle n’est pas applicable aux additifs et aux distillats de désodorisation.
Cette méthode a été soumise à essai lors d’une étude interlaboratoires en procédant à l’analyse d’échantillons naturellement contaminés et dopés (prémélange, farine de soja, graines de tournesol, aliments pour volailles, aliments pour porcs, huile végétale) à des teneurs comprises entre 3 mg/kg et 286 mg/kg pour les MOSH et entre 1 mg/kg et 16 mg/kg pour les MOAH.
D’après les résultats de l’étude interlaboratoires, il a été démontré que la méthode est adaptée pour les concentrations massiques des MOSH et des MOAH, au dessus de 10 mg/kg chacune. Toutefois, la méthode n’a pas été entièrement validée lors de l’étude interlaboratoires pour l’échantillon de prémélange en raison des concentrations trop faibles des MOSH et MOAH. La méthode n’a pas non plus été entièrement validée lors de l’étude interlaboratoires pour l’échantillon de graines de tournesol en raison de la concentration trop faible des MOAH.
NOTE   Les conclusions concernant les MOAH se fondent sur quatre combinaisons analytes/matrice alors que le protocole IUPAC [4] prévoit un minimum de cinq combinaisons.
En cas de suspicion d’interférences dues à des sources naturelles, l’origine fossile de la fraction des MOSH et des MOAH peut être vérifiée par un examen du profil par CG SM.
Une autre norme CEN est également disponible dans le cadre de la détermination des MOSH et MOAH dans les huiles et graisses alimentaires : EN 16995. Pour plus d’informations, voir [5].
L’Annexe C propose une méthode manuelle alternative pour l’analyse CLHP CG FID en ligne qui peut être utilisée en tant que méthode de criblage.

Krma: metode vzorčenja in analize - Določevanje nasičenih ogljikovodikov iz mineralnih olj (MOSH) in aromatskih ogljikovodikov iz mineralnih olj (MOAH) z analizo on-line HPLC-GC-FID

General Information

Status
Published
Public Enquiry End Date
19-Jul-2020
Publication Date
12-Dec-2021
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
29-Nov-2021
Due Date
03-Feb-2022
Completion Date
13-Dec-2021

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Standards Content (sample)

SLOVENSKI STANDARD
SIST EN 17517:2022
01-januar-2022
Krma: metode vzorčenja in analize - Določevanje nasičenih ogljikovodikov iz
mineralnih olj (MOSH) in aromatskih ogljikovodikov iz mineralnih olj (MOAH) z
analizo on-line HPLC-GC-FID

Animal feeding stuffs: Methods of sampling and analysis - Determination of mineral oil

saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) with
on-line HPLC-GC-FID analysis
Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von
mineralölgesättigten Kohlenwasserstoffen (MOSH) und mineralölaromatischen
Kohlenwasserstoffen (MOAH) mit Online-Analyse durch HPLC-GC-FID

Aliments pour animaux : Méthodes d’échantillonnage et d’analyse - Détermination des

hydrocarbures saturés d’huile minérale (MOSH) et des hydrocarbures aromatiques
d’huile minérale (MOAH) par analyse CLHP CG-FID en ligne
Ta slovenski standard je istoveten z: EN 17517:2021
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN 17517:2022 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 17517:2022
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SIST EN 17517:2022
EN 17517
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2021
EUROPÄISCHE NORM
ICS 65.120
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of mineral oil saturated hydrocarbons
(MOSH) and mineral oil aromatic hydrocarbons (MOAH)
with on-line HPLC-GC-FID analysis

Aliments pour animaux : Méthodes d'échantillonnage Futtermittel: Probenahme und

et d'analyse - Détermination des hydrocarbures Untersuchungsverfahren - Bestimmung von

saturés d'huile minérale (MOSH) et des hydrocarbures mineralölgesättigten Kohlenwasserstoffen (MOSH) und

aromatiques d'huile minérale (MOAH) par analyse mineralölaromatischen Kohlenwasserstoffen (MOAH)

CLHP CG-FID en ligne mit Online Analyse durch HPLC GC FID
This European Standard was approved by CEN on 2 August 2021.

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, Turkey 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

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17517:2021 E

worldwide for CEN national Members.
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EN 17517:2021
Contents Page

European foreword ....................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Principle ............................................................................................................................................................. 6

5 Reagents ............................................................................................................................................................. 6

6 Apparatus ........................................................................................................................................................... 9

7 Sampling .......................................................................................................................................................... 10

8 Preparation of the test sample ................................................................................................................ 10

9 Preparation of the analytical sample .................................................................................................... 10

9.1 Fat extraction from feed sample ............................................................................................................. 10

9.2 Procedure for fats and fat extracts ........................................................................................................ 11

9.3 Blank ................................................................................................................................................................. 11

10 Liquid chromatography and gas chromatography .......................................................................... 12

10.1 Liquid chromatography setup ................................................................................................................. 12

10.2 Working conditions for HPLC .................................................................................................................. 12

10.3 HPLC-GC-interface ........................................................................................................................................ 13

10.4 Gas chromatography setup ....................................................................................................................... 14

10.5 Working conditions for GC ........................................................................................................................ 14

10.6 Solvent vapor exit ........................................................................................................................................ 15

10.7 Peak identification ....................................................................................................................................... 15

10.8 Performance of the HPLC-GC system..................................................................................................... 15

10.9 Quantitative determination of hydrocarbons attributed to mineral oil origin ..................... 16

11 Precision .......................................................................................................................................................... 17

11.1 Interlaboratory test ..................................................................................................................................... 17

11.2 Repeatability .................................................................................................................................................. 17

11.3 Reproducibility ............................................................................................................................................. 17

12 Test report ...................................................................................................................................................... 17

Annex A (informative) Examples of chromatograms ..................................................................................... 19

Annex B (informative) Precision data.................................................................................................................. 25

Annex C (informative) Determination of saturated hydrocarbons of mineral oil - Manual

alternative method to on line HPLC-GC-FID analysis ...................................................................... 28

Bibliography ................................................................................................................................................................. 40

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EN 17517:2021
European foreword

This document (EN 17517:2021) has been prepared by Technical Committee CEN/TC 327 “Animal

feeding stuffs - Methods of sampling and analysis”, the secretariat of which is held by NEN.

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 2022, and conflicting national standards shall be

withdrawn at the latest by April 2022.

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.

This document has been prepared under a standardization request given to CEN by the European

Commission and the European Free Trade Association.

Any feedback and questions on this document should be directed to the users’ national standards body.

A complete listing of these bodies can be found on the CEN website.

According to the CEN-CENELEC Internal Regulations, the national standards organisations 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, Turkey and the United

Kingdom.
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SIST EN 17517:2022
EN 17517:2021
Introduction

WARNING — The method described in this document implies the use of reagents that pose a hazard to

health. This document does not claim to address all associated safety problems. It is the responsibility of

the user of this document to take appropriate measures for the health and safety protection of the

personnel prior to use of the standard and to ensure that regulatory and legal requirements are complied

with.
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SIST EN 17517:2022
EN 17517:2021
1 Scope

This document specifies a method for the determination of saturated and aromatic hydrocarbons (from

C10 to C50) in feed. The method has been interlaboratory validated with on-line-HPLC-GC-FID – see [1],

[2] and [3]. This method is not intended to be applied to other matrices.

The method can be used for the analysis of mineral oil saturated hydrocarbons (MOSH) and/or mineral

oil aromatic hydrocarbons (MOAH).

The method is applicable for feed materials, in particular vegetable oils and other fat rich feed materials,

compound feeds and pre-mixtures. It is not applicable to additives or deodistillates.

NOTE 1 The method was not designed for encapsulated matrices.

The method has been tested in an interlaboratory study via the analysis of both naturally contaminated

and spiked samples (pre-mixture, soybean meal, sunflower seeds, chicken feed, pig feed, vegetable oil)

ranging from 3 mg/kg to 286 mg/kg for MOSH and from 1 mg/kg to 16 mg/kg for MOAH.

According to the results of the interlaboratory study, the method has been proven suitable for MOSH and

MOAH mass concentrations, each above 10 mg/kg. However, the method was not fully validated during

the collaborative study for the premixture sample due to too low concentrations of MOSH and MOAH. The

method was also not fully validated during the collaborative study for the sunflower seeds sample due to

a too low concentration of MOAH.

NOTE 2 The conclusions regarding MOAH are based on 4 analyte / matrix combinations while the IUPAC

protocol [4] expects this to be a minimum of 5.

In case of suspected interferences from natural sources, the fossil origin of the MOSH and MOAH fraction

can be verified by examination of the pattern by GC-MS.

For the determination of MOSH and MOAH in edible fats and oils, another CEN standard is also available:

EN 16995. For more information see [5].

Annex C proposes a manual alternative method to on-line HPLC-GC-FID analysis that can be used as a

screening method for the determination of MOSH.
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.

EN ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological 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/
3.1
mineral oil saturated hydrocarbons
MOSH

paraffinic (open-chain, usually branched) and naphthenic (cyclic, alkylated) hydrocarbons

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SIST EN 17517:2022
EN 17517:2021
3.2
mineral oil aromatic hydrocarbons
MOAH
aromatic mainly alkylated hydrocarbons
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
4 Principle

The fatty material is extracted from the commodity using organic solvent. After concentration of part of

the solvent, the extract is submitted to an epoxidation step. The fractions of MOSH and MOAH are isolated

and separated by an HPLC-GC-FID system. MOSH and MOAH fractions are separated on a silica gel column

using an n-hexane/dichloromethane gradient and each transferred as 450 µl fractions to GC using the Y-

interface [6], while triglycerides are kept on the HPLC column. Solvent vapours are discharged via a

solvent vapour exit located between the uncoated pre-column and the GC separation column. Volatile

components are retained by solvent trapping applying partially concurrent eluent evaporation. High

boiling components are spread over the entire length of the flooded zone and refocused by the retention

gap technique [2].

The area attributed to mineral oil is calculated by subtraction of peaks due to n-alkanes (naturally

occurring hydrocarbons), terpenes, squalene and its isomerization products, sterenes and olefins with

the structure of carotenoids. MOSH and MOAH are quantitated by internal standard added before

analysis. Verification standards are added for monitoring proper HPLC fractionation and GC transfer

conditions.

Epoxidation is a purification step that is necessary for the quantification of MOAH. This purification step

allows the elimination of olefins like squalene, which elute within the MOAH fraction and interfere with

quantification (e.g. olive oil, palm oil). Epoxidation also removes certain olefins co-eluting with the MOSH

fraction, therefore epoxidation also may be used as a purification step for the MOSH fraction. The

epoxidation step is the best compromise to remove olefins, even though it is not fully quantitative and

the efficiency may be sample dependent. Depending on the sample, this reaction may induce the

epoxidation of a part of the MOAH or incomplete removal of the interfering olefins.

5 Reagents

WARNING — The method described in this document implies the use of reagents that pose a hazard to

health. This document does not claim to address all associated safety problems. It is the responsibility of

the user of this document to take appropriate measures for the health and safety protection of the

personnel prior to use of the standard and to ensure that regulatory and legal requirements are complied

with.
Unless otherwise specified, use only reagents of recognized analytical grade.
5.1 Demineralized water, stored in a glass bottle.
5.2 n-Hexane, trace organic analysis grade, for pesticide residue analysis.

n-Hexane purity can be checked by concentrating 30 ml of n-hexane mixed with 25 µl of internal standard

solution (5.16) and 2 drops of keeper (5.27) using a rotary evaporator, dissolving the residue in 0,2 ml of

n-hexane and the analysis of 50 µl by on-line-HPLC-GC-FID (6.10). Take care that in the evaporation step

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SIST EN 17517:2022
EN 17517:2021

the residue is not evaporated to dryness to avoid loss of volatile hydrocarbons. The signal abundance of

the residue after evaporation should not exceed a tenth of the signal abundance obtained at the

quantification limit.
5.3 Toluene.
5.4 1,1,2-Trichloroethane.
5.5 Perylene (Per), purity ≥ 99 %.
5.6 α-Cholestane (Cho), purity ≥ 97 %.
5.7 n-Undecane (n-C11), purity ≥ 98 %.
5.8 n-Tridecane (n-C13), purity ≥ 97 %.
5.9 Tri-tert-butylbenzene (TBB).
5.10 Bicyclohexyl (CyCy), purity ≥ 99 %.
5.11 1-Methylnaphthalene (1-MN), purity ≥ 95 %.
5.12 2-Methylnaphthalene (2-MN), purity ≥ 97 %.
5.13 Pentylbenzene (5-PB), purity ≥ 96 %.
5.14 Stock solutions, mass concentration ρ = 10 mg/ml.

Prepare individual stock solutions by weighing, to the nearest 1 mg, 100 mg of n-C11 (5.7), n-C13 (5.8),

TBB (5.9), CyCy (5.10), 1-MN (5.11), 2-MN (5.12) and 5-PB (5.13) into a 10 ml volumetric flask and dilute

to the mark with 1,1,2-trichloroethane (5.4) or toluene (5.3). Store the solutions at room temperature. If

crystals precipitate during storage, warm the solution until everything has dissolved.

5.15 Internal standard solution 1 (ISTD1).

Weigh, to the nearest 0,5 mg, 12 mg of Per (5.5) and Cho (5.6) in a volumetric flask of 20 ml (6.21), to

which 600 µl of each stock solution (5.14) is added with the exception of n-C13, of which 300 µl is added.

Fill the volumetric flask up to 20 ml with 1,1,2-trichloroethane (5.4) or toluene (5.3). Resulting mass

concentrations are for n-C13: ρ = 150 µg/ml, for n-C11, TBB, CyCy, 1-MN, 2-MN and 5-PB: ρ = 300 µg/ml

and for Per, Cho: ρ = 600 µg/ml.

NOTE This mixture of internal standards is commercially available, ready to use product.

5.16 Internal standard solution 2 (ISTD2).

Dilute the ISTD1 solutions by a factor of 10, e.g. 1 ml filled up to 10 ml with n-hexane (5.2). Resulting mass

concentrations are for n-C13: ρ = 15 µg/ml, for n-C11, TBB, CyCy, 1-MN, 2-MN and 5-PB: ρ = 30 µg/ml and

for Per, Cho: ρ = 60 µg/ml.
5.17 Chloroperbenzoic acid (CPBA), purity 70 % to 75 %.

Restek Corp. ®, Cat.# 31070 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 CEN of this product. Other

products could be used, if the results are comparable.
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SIST EN 17517:2022
EN 17517:2021
5.18 CPBA solution, ρ = 0,2 g/ml in absolute ethanol.

For example 5 g of CPBA (5.17) in 25 ml of absolute ethanol (5.22). The solution can be used for up to one

week.
5.19 Carrier gas for gas chromatography, preferably hydrogen, purity ≥ 99,995 %.

5.20 Auxiliary gases for flame ionization detector, hydrogen, air, and nitrogen suitable for gas

chromatography.

5.21 Alkane standard mixture C10 to C40, solution of equal concentration in an apolar solvent,

ρ = 1 µg/ml.
5.22 Ethanol, absolute.

NOTE The ethanol purity can be checked by concentrating 50 ml of ethanol mixed with 25 µl of internal

standard solution (5.16) and 2 drops of keeper (5.27) using a rotary evaporator, dissolving the residue in 0,2 ml of

n-hexane and the analysis of 50 µl by on-line-HPLC-GC-FID (6.10).
5.23 n-Pentacontane (C50), purity ≥ 98 %.
5.24 n-Pentacontane (C50) solution in toluene, ρ approximately 10 µg/ml.

Weigh 2 mg of C50 (5.23) in a volumetric flask of 20 ml (6.21) and dilute to the mark with toluene (5.3).

Proceed to a second dilution of 1 ml in a 10 ml volumetric flask (6.21). Store the solutions at room

temperature.

NOTE 1 Solubility of pentacontane in toluene is limited at room temperature. However, the concentration of the

solution of pentacontane does not need to be accurate as it is used only to determine the limit of integration for

mineral oil peak.

NOTE 2 It is also possible to use a commercial mixture of n-alkanes from C12 to C60 that contains n-

pentacontane.
5.25 Sodium carbonate solution, ρ = 0,1 g/ml in water (5.1).
5.26 Dichloromethane (DCM), trace organic analysis grade, purity ≥ 99 %.

DCM purity can be checked by concentrating 50 ml of DCM mixed with 25 µl of internal standard solution

(5.16) and 2 drops of keeper (5.27) using a rotary evaporator, dissolving the residue in 0,2 ml of n-hexane

and the analysis of 50 µl by on-line-HPLC-GC-FID (6.10). Take care that in the evaporation step the

residue is not evaporated to dryness to avoid loss of volatile hydrocarbons. The signal abundance of the

residue after evaporation should not exceed a fifth of the signal abundance obtained at the quantification

limit.
5.27 Keeper solvent.

The keeper is a solvent that will not evaporate or evaporate to a lesser degree during the evaporation

step, e.g. bis(2-ethylhexyl) maleate. A keeper is used to enhance the recovery of volatile compounds.

ASTM® D5442 C12-C60 Qualitative Retention Time Mix available by e.g. Supelco® Cat.# 500623 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 CEN of this product. Other products could be used, if the results are

comparable.
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EN 17517:2021
6 Apparatus

IMPORTANT — The glassware used for the determination shall be thoroughly cleaned and rinsed with n-

hexane (5.2) before use so that it is free from impurities.

Usual laboratory apparatus and, in particular, the following. The glassware shall be thoroughly cleaned

and rinsed with n-hexane (5.2) or baked in an oven before use so that it is free from impurities.

6.1 Mill with stainless-steel rotor or ball mill, capable of reaching particles size ≤ 1 mm.

6.2 Magnetic stirrer.
6.3 Magnetic stir bars.
6.4 Analytical balance, reading accuracy 0,000 1 g.
6.5 Round-bottomed flasks, 250 ml capacity.
6.6 Glass vials with screw caps, 15 ml and 40 ml capacity.
6.7 Centrifuge and centrifuge tubes.
6.8 Automatic evaporator (optional) .
6.9 Glass sample vials, volume of 2 ml.

6.10 High performance liquid chromatograph, coupled with gas chromatograph and flame ionization

detector (HPLC-GC-FID).
6.11 Data acquisition system, with the possibility of manual integration.
6.12 LC column, 5 µm (250 mm x 2 mm inner diameter (i.d.)) or equivalent.
The silica gel column shall have a capacity to retain 20 mg fat.
6.13 Uncoated precolumn, 10 m x 0,53 mm or equivalent .
6.14 Capillary column 1, capable for temperatures up to 350 °C.

The column should have the following characteristics: 100 % dimethylpolysiloxane or 95 % dimethyl /

5 % phenyl methylpolysiloxane stationary phase, a length of 15 m, an internal diameter of 0,32 mm or

0,25 mm and a film thickness 0,10 µm to 0,15 µm or equivalent.

MicroDancer®, IR-Dancer (e.g. Zinser) or Syncore® Analyst (Büchi) are examples 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 CEN of these products. Other products could be used, if the results are comparable.

LiChrospher® Si 60 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 CEN of this product. Other

products could be used, if the results are comparable.

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 CEN of this product. Other

products could be used, if the results are comparable.
---------------------- Page: 11 ----------------------
SIST EN 17517:2022
EN 17517:2021

6.15 Capillary column 2, from transfer valve to first Y-piece, fused silica (FS) methyl silicone

deactivated (length 1 m, outside diameter (o.d.) 0,27 mm, inner diameter (i.d.) 0,1 mm).

6.16 Capillary column 3, for hydrogen carrier gas, FS methyl silicone (length 1 m, o.d. 360 µm, i.d.

25 µm).

6.17 Capillary column 4, for solvent vapour exit, FS methyl silicone (length 1 m, o.d. 0,68 mm, i.d.

0,53 mm).

The columns given in 6.15, 6.16 and 6.17 have proven to be suitable for the analysis. However these

columns can be adjusted in accordance with the characteristics of the HPLC-GC apparatus and the

analytical conditions.

6.18 Restriction capillary column, transfer valve and solvent vapor exit, FS uncoated (length 1 m, o.d.

360 µm, i.d. 50 µm).

6.19 Microsyringe, 5 µl to 100 µl capacity, suitable for injection in liquid chromatography.

6.20 Pasteur pipette, glass.
The use of plastic pasteur pipettes and polyethylene film shall be avoided.
6.21 Volumetric flasks, various sizes.
7 Sampling

The sample should be truly representative and not damaged or changed during transport or storage.

Samples should be packed in glass bottles or aluminium foil in order to prevent additional contamination.

Plastic and paper packaging are unsuitable.

Sampling is not part of the method specified in this document. A recommended sampling method is given

in EN ISO 6497 [7].
8 Preparation of the test sample
Prepare the test sample in accordance with EN ISO 6498.

Grind the laboratory sample (typically 50 g) to a particle size of at least 1 mm in the mill (6.1) in order to

ensure representative data. Mix the sample thoroughly.
9 Preparation of the analytical sample
9.1 Fat extraction from feed sample
9.1.1 Fatty material extraction from samples with fat content lower than 30 %

Weigh 5 g of milled sample in a 250 ml round-bottomed flask (6.5), add a magnetic stir bar (6.3). Add

500 µl ISTD2 (5.16) and 100 ml of n-hexane (5.2) to the sample and mix for 1 h with a magnetic stirrer

(6.2).

Transfer 20 ml of the solvent phase into a 40 ml glass vial (6.6) and wash with 5 ml of demineralized

water (5.1). Centrifuge for 2 min at a speed of 2 500 rpm and transfer 10 ml of sample extract to a 15 ml

glass vial (6.6) and concentrate the solvent down to 1 ml (triglycerides from the sample act as a keeper)

under a stream of nitrogen, using either a water bath at 35 °C or an automatic evaporator (6.8).

---------------------- Page: 12 ----------------------
SIST EN 17517:2022
EN 17517:2021
9.1.2 Fatty material extraction from sample with fat content higher than 30 %

Weigh 2 g of milled sample in a 250 ml round-bottomed flask (6.5), add a magnetic stir bar (6.3). Add

250 µl ISTD2 (5.16) and 100 ml of n-hexane (5.2) to the sample and mix for 1 h with a magnetic stirrer

(6.2). Transfer 30 ml of the solvent phase into a 40 ml glass vial (6.6) and wash with 5 ml of demineralized

water (5.1). Centrifuge for 2 min at a speed of 2 500 rpm and transfer 20 ml of sample extract to a 40 ml

glass vial (6.6) and concentrate the solvent down to 1 ml (triglycerides from the sample act as a keeper)

under a stream of nitrogen, using either a water bath at 35 °C or an automatic evaporator (6.8).

9.2 Procedure for fats and fat extracts
9.2.1 Procedure for liquid and solid fats

Weigh, to the nearest 1 mg, 300 mg sample into a 10 ml vial, and fill it up with 600 µl n-hexane and add

50 µl ISTD2 (5.16). Shake the vial.

The amount of the added internal standards may be increased, in order to lower the impact of the matrix

interferences, if necessary.

The amount of the added internal standards may be decreased, in case low concentrations shall be

measured.

Add 500 µl of CPBA ethanolic solution (5.18) and place the vial into an agitator to be shaken for 15 min

at a speed of 1 800 rpm at room temperature. Immediately, add 3 ml of sodium carbonate solution (5.25),

shake the mixture for 2 min and then centrifuge for 2 min at a speed of 2 500 rpm.

NOTE The collaborative study was performed in 2018. Therefore the epoxidation procedure does not

correspond to the one proposed during the Roundtable Workshop on the Determination of MOAH in Infant

Formula [11].

Transfer 500 µl of the hexane phase to an autosampler vial. At a maximum, inject 50 µl into the HPLC.

Depending on the level of contamination, the injected volume may be adapted in order to avoid the

overloading of the chromatograms.
9.2.2 Procedure for extracted fats
Add 500 µl of CPBA ethanolic solution (5.18) to the sample extract obtain
...

SLOVENSKI STANDARD
oSIST prEN 17517:2020
01-julij-2020
Krma: metode vzorčenja in analize - Določevanje mineralnih olj nasičenih
ogljikovodikov (MOSH) in mineralnih olj aromatskih ogljikovodikov (MOAH) z
analizo on-line HPLC-GC-FID

Animal feeding stuffs: Methods of sampling and analysis - Determination of mineral oil

saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) with
on-line HPLC-GC-FID analysis
Futtermittel: Probenahme- und Untersuchungsverfahren - Bestimmung von
mineralölgesättigten Kohlenwasserstoffen (MOSH) und mineralölaromatischen
Kohlenwasserstoffen (MOAH) mit Online-Analyse durch HPLC-GC-FID

Aliments pour animaux - Méthodes d’échantillonnage et d’analyse - Détermination des

hydrocarbures saturés d’huile minérale (MOSH) et des hydrocarbures aromatiques
d’huile minérale (MOAH) par analyse CLHP CG FID en ligne
Ta slovenski standard je istoveten z: prEN 17517
ICS:
65.120 Krmila Animal feeding stuffs
oSIST prEN 17517:2020 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN 17517:2020
---------------------- Page: 2 ----------------------
oSIST prEN 17517:2020
DRAFT
EUROPEAN STANDARD
prEN 17517
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2020
ICS 65.120
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Determination of mineral oil saturated hydrocarbons
(MOSH) and mineral oil aromatic hydrocarbons (MOAH)
with on-line HPLC-GC-FID analysis

Aliments pour animaux - Méthodes d'échantillonnage Futtermittel: Probenahme- und

et d'analyse - Détermination des hydrocarbures Untersuchungsverfahren - Bestimmung von

saturés d'huile minérale (MOSH) et des hydrocarbures mineralölgesättigten Kohlenwasserstoffen (MOSH) und

aromatiques d'huile minérale (MOAH) par analyse mineralölaromatischen Kohlenwasserstoffen (MOAH)

CLHP CG FID en ligne mit Online-Analyse durch HPLC-GC-FID

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

CEN/TC 327.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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, Turkey and

United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without

notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17517:2020 E

worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
oSIST prEN 17517:2020
TC 327 WI 00327116:2020 (E)
Contents Page

European foreword ....................................................................................................................................................... 4

Introduction .................................................................................................................................................................... 5

1 Scope .................................................................................................................................................................... 6

2 Normative references .................................................................................................................................... 6

3 Terms and definitions ................................................................................................................................... 6

4 Principle ............................................................................................................................................................. 7

5 Reagents ............................................................................................................................................................. 7

6 Apparatus ........................................................................................................................................................ 10

7 Sampling .......................................................................................................................................................... 11

8 Preparation of the test sample ................................................................................................................ 11

9 Preparation of the analytical sample .................................................................................................... 11

9.1 Fat extraction from feed sample ............................................................................................................. 11

9.1.1 Fatty material extraction for samples with fat content lower than 30 %................................ 11

9.1.2 Fatty material extraction for sample with fat content higher than 30 % ................................ 12

9.2 Procedure for fats and fat extracts ........................................................................................................ 12

9.2.1 Procedure for liquid and solid fats ........................................................................................................ 12

9.2.2 Procedure for extracted fats .................................................................................................................... 12

9.3 Blank ................................................................................................................................................................. 12

10 Liquid chromatography and gas chromatography .......................................................................... 13

10.1 Liquid chromatography setup ................................................................................................................. 13

10.2 Working conditions for HPLC .................................................................................................................. 13

10.3 HPLC-GC-interface ........................................................................................................................................ 14

10.4 Gas chromatography setup ....................................................................................................................... 15

10.5 Working conditions for GC ........................................................................................................................ 15

10.6 Solvent vapor exit ........................................................................................................................................ 16

10.7 Peak identification ....................................................................................................................................... 16

10.8 Performance of the HPLC-GC system..................................................................................................... 16

10.9 Quantitative determination of hydrocarbons attributed to mineral oil origin ..................... 17

11 Precision .......................................................................................................................................................... 18

11.1 Interlaboratory test ..................................................................................................................................... 18

11.2 Repeatability .................................................................................................................................................. 18

11.3 Reproducibility ............................................................................................................................................. 18

12 Test report ...................................................................................................................................................... 18

Annex A (informative) Examples of chromatograms ................................................................................... 20

Annex B (informative) Precision data ............................................................................................................... 26

Annex C (informative) Determination of saturated hydrocarbons of mineral oil - Manual

alternative method to on line HPLC-GC-FID analysis ...................................................................... 29

C.1 General ............................................................................................................................................................. 29

C.2 Principle .......................................................................................................................................................... 29

C.3 Reagents .......................................................................................................................................................... 29

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oSIST prEN 17517:2020
TC 327 WI 00327116:2020 (E)

C.4 Apparatus ........................................................................................................................................................ 31

C.5 Sampling .......................................................................................................................................................... 32

C.6 Preparation of the test sample ................................................................................................................ 32

C.7 Procedure ........................................................................................................................................................ 32

C.7.1 Fat extraction from feed sample ............................................................................................................. 32

C.7.1.1 General ............................................................................................................................................................. 32

C.7.1.2 Fatty material extraction for animal feed (fat content lower than 15 %) ............................... 32

C.7.1.3 Fatty material extraction for oilseeds (fat content higher than 30 %) ..................................... 32

C.7.1.4 Fatty material extraction for oilseeds (fat content between 15 % and 30 %) ....................... 32

C.7.1.5 Evaporation of the solvent ........................................................................................................................ 33

C.7.2 Chromatography column preparation .................................................................................................. 33

C.7.2.1 Preparation of AgNO3 impregnated silica gel .................................................................................... 33

C.7.2.2 Column packing ............................................................................................................................................. 33

C.7.3 Isolation of the hydrocarbon fraction from the fatty extract (or the oil sample) ................. 33

C.7.3.1 Oil sample preparation ............................................................................................................................... 33

C.7.3.2 Elution of the hydrocarbon fraction ...................................................................................................... 34

C.7.4 Gas Chromatography ................................................................................................................................... 34

C.7.4.1 Gas chromatography setup ....................................................................................................................... 34

C.7.4.2 Working conditions for gas chromatography analysis ................................................................... 34

C.7.4.3 Peak identification ....................................................................................................................................... 34

C.7.4.4 Performance of the gas chromatography system ............................................................................. 35

C.7.5 Procedural blank .......................................................................................................................................... 35

C.7.6 Quantitative determination ...................................................................................................................... 35

C.8 Determination of hydrocarbons attributed to mineral origin ..................................................... 38

C.9 Precision data ................................................................................................................................................ 38

Bibliography ................................................................................................................................................................. 41

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oSIST prEN 17517:2020
TC 327 WI 00327116:2020 (E)
European foreword

This document (prEN 17517:2020) has been prepared by Technical Committee CEN/TC 327 “Animal

feeding stuffs - Methods of sampling and analysis”, the secretariat of which is held by NEN.

This document is currently submitted to the CEN Enquiry.

This document has been prepared under a standardization request given to CEN by the European

Commission and the European Free Trade Association.
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oSIST prEN 17517:2020
TC 327 WI 00327116:2020 (E)
Introduction

WARNING — The method described in this document implies the use of reagents that pose a hazard to

health. The standard does not claim to address all associated safety problems. It is the responsibility of

the user of this document to take appropriate measures for the health and safety protection of the

personnel prior to use of the standard and to ensure that regulatory and legal requirements are complied

with.
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oSIST prEN 17517:2020
TC 327 WI 00327116:2020 (E)
1 Scope

This document specifies a method for the determination of saturated and aromatic hydrocarbons (from

C10 to C50) in feed. The method has been interlaboratory validated with online-HPLC-GC-FID – see [1],

[2] and [3]. This method is not intended to be applied to other matrices.

The method can be used for the analysis of mineral oil saturated hydrocarbons (MOSH) and/or mineral

oil aromatic hydrocarbons (MOAH).

The method is applicable for feed materials, in particular vegetable oils and other fat rich feed materials,

compound feeds and pre-mixtures. It is not applicable to additives or deodistillates.

The method has been tested in an interlaboratory study via the analysis of both naturally contaminated

and spiked samples (pre-mixture, soybean meal, sunflower seeds, chicken feed, pig feed, vegetable oil)

ranging from 3 mg/kg to 286 mg/kg for MOSH and from 1 mg/kg to 16 mg/kg for MOAH.

According to the results of the interlaboratory study, the method has been proven suitable for MOSH and

MOAH mass concentrations, each above 10 mg/kg. However, the method was not fully validated during

the collaborative study for the premixture sample due to too low concentrations of MOSH and MOAH. The

method was also not fully validated during the collaborative study for the sunflower seeds sample due to

a too low concentration of MOAH.

NOTE The conclusions regarding MOAH are based on 4 analyte / matrix combinations while according to the

IUPAC protocol [4] expects this to be a minimum of 5.

In case of suspected interferences from natural sources, the fossil origin of the MOSH and MOAH fraction

can be verified by examination of the pattern by GC-MS.

For the determination of MOSH and MOAH in edible fats and oils, another CEN standard is also available:

EN 16995. For more information see [5].

Annex C proposes a manual alternative method to online HPLC-GC-FID analysis that can be used as a

screening method.
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.

EN ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498)

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at http://www.electropedia.org/
3.1
mineral oil saturated hydrocarbons
MOSH

paraffinic (open-chain, usually branched) and naphthenic (cyclic, alkylated) hydrocarbons

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oSIST prEN 17517:2020
TC 327 WI 00327116:2020 (E)
3.2
mineral oil aromatic hydrocarbons
MOAH
aromatic mainly alkylated hydrocarbons
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
4 Principle

The fatty material is extracted from the commodity using organic solvent. After concentration of part of

the solvent, the extract is submitted to an epoxidation step. The fractions of MOSH and MOAH are isolated

and separated by an HPLC-GC-FID system. MOSH and MOAH fractions are separated on a silica gel column

using a n-hexane/dichloromethane gradient and each transferred as 450 µl fractions to GC using the Y-

interface [6], while triglycerides are kept on the HPLC column. Solvent vapours are discharged via a

solvent vapour exit located between the uncoated pre-column and the GC separation column. Volatile

components are retained by solvent trapping applying partially concurrent eluent evaporation. High

boiling components are spread over the entire length of the flooded zone and refocused by the retention

gap technique [2].

The area attributed to mineral oil is calculated by subtraction of peaks due to n-alkanes (naturally

occurring hydrocarbons), terpenes, squalene and its isomerization products, sterenes and olefins with

the structure of carotenoids. MOSH and MOAH are quantitated by internal standard added before

analysis. Verification standards are added for monitoring proper HPLC fractionation and GC transfer

conditions.

Epoxidation is a purification step that is necessary for the quantification of MOAH. This purification step

allows the elimination of olefins like squalene, which elute within the MOAH fraction and interfere with

quantification (e.g. olive oil, palm oil). Epoxidation also removes certain olefins co-eluting with the MOSH

fraction, therefore epoxidation also may be used as a purification step for the MOSH fraction. The

epoxidation step is the best compromise to remove olefins, even though it is not fully quantitative and

the efficiency may be sample dependent. Depending on the sample, this reaction may induce the

epoxidation of a part of the MOAH or incomplete removal of the interfering olefins.

5 Reagents

WARNING — The method described in this document implies the use of reagents that pose a hazard to

health. The standard does not claim to address all associated safety problems. It is the responsibility of

the user of this document to take appropriate measures for the health and safety protection of the

personnel prior to use of the standard and to ensure that regulatory and legal requirements are complied

with.
Unless otherwise specified, use only reagents of recognized analytical grade.
5.1 Demineralized water, stored in a glass bottle
5.2 n-Hexane, trace organic analysis grade, for pesticide residue analysis

n-Hexane purity can be checked by concentrating 30 ml of n-hexane mixed with 25 µl of internal standard

solution (5.16) and 2 drops of keeper (5.27) using a rotary evaporator, dissolving the residue in 0,2 ml of

n-hexane and the analysis of 50 µl by online-HPLC-GC-FID (6.10). Take care that in the evaporation step

the residue is not evaporated to dryness to avoid loss of volatile hydrocarbons. The signal abundance of

---------------------- Page: 9 ----------------------
oSIST prEN 17517:2020
TC 327 WI 00327116:2020 (E)

the residue after evaporation should not exceed a tenth of the signal abundance obtained at the

quantification limit.
5.3 Toluene
5.4 1,1,2-Trichloroethane
5.5 Perylene (Per), purity ≥ 99 %
5.6 α-Cholestane (Cho), purity ≥ 97 %
5.7 n-Undecane (n-C11), purity ≥ 98 %
5.8 n-Tridecane (n-C13), purity ≥ 97 %
5.9 Tri-tert-butylbenzene (TBB)
5.10 Bicyclohexyl (CyCy), purity ≥ 99 %
5.11 1-Methylnaphthalene (1-MN), purity ≥ 95 %
5.12 2-Methylnaphthalene (2-MN), purity ≥ 97 %
5.13 Pentylbenzene (5-PB), purity ≥ 96 %
5.14 Stock solutions, mass concentration ρ = 10 mg/ml

Prepare individual stock solutions by weighing, to the nearest 1 mg, 100 mg of n-C11 (5.7), n-C13 (5.8),

TBB (5.9), CyCy (5.10), 1-MN (5.11), 2-MN (5.12) and 5-PB (5.13) into a 10 ml volumetric flask and dilute

to the mark with 1,1,2-trichloroethane (5.4) or toluene (5.3). Store the solutions at room temperature. If

crystals precipitate during storage, warm the solution until everything has dissolved.

5.15 Internal standard solution 1 (ISTD1)

Weigh, to the nearest 0,5 mg, 12 mg of Per (5.5) and Cho (5.6) in a volumetric flask of 20 ml (6.21), to

which 600 µl of each stock solution (5.14) is added with the exception of n-C13, of which 300 µl is added.

Fill the volumetric flask up to 20 ml with 1,1,2-trichloroethane (5.4) or toluene (5.3). Resulting mass

concentrations are for n-C13: ρ = 150 µg/ml, for n-C11, TBB, CyCy, 1-MN, 2-MN and 5-PB: ρ = 300 µg/ml

and for Per, Cho: ρ = 600 µg/ml.
NOTE 1 This document mixture is available.
5.16 Internal standard solution 2 (ISTD2)

Dilute the ISTD1 solutions by a factor of 10, e.g. 1 ml filled up to 10 ml with n-hexane (5.2). Resulting mass

concentrations are for n-C13: ρ = 15 µg/ml, for n-C11, TBB, CyCy, 1-MN, 2-MN and 5-PB: ρ = 30 µg/ml and

for Per, Cho: ρ = 60 µg/ml.
5.17 Chloroperbenzoic acid (CPBA), purity 70 % to 75 %
5.18 CPBA solution, ρ = 0,2 g/ml in absolute ethanol

Restek Corp., Cat.# 31070 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 CEN of this product.

---------------------- Page: 10 ----------------------
oSIST prEN 17517:2020
TC 327 WI 00327116:2020 (E)

For example 5 g of CPBA (5.17) in 25 ml of absolute ethanol (5.22). The solution can be used for up to one

week.
5.19 Carrier gas for gas chromatography, preferably hydrogen, purity ≥ 99,995 %

5.20 Auxiliary gases for flame ionization detector, hydrogen, air, and nitrogen suitable for gas

chromatography

5.21 Alkane standard mixture C10 to C40, solution of equal concentration in an apolar solvent,

ρ = 1 µg/ml
5.22 Ethanol, absolute

NOTE 2 The ethanol purity can be checked by concentrating 50 ml of ethanol mixed with 25 µl of internal

standard solution (5.16) and 2 drops of keeper (5.27) using a rotary evaporator, dissolving the residue in 0,2 ml of

n-hexane and the analysis of 50 µl by online-HPLC-GC-FID (6.10).
5.23 n-Pentacontane (C50), purity ≥ 98 %
5.24 n-Pentacontane (C50) solution in toluene, ρ approximately 10 µg/ml

Weigh 2 mg of C50 (5.23) in a volumetric flask of 20 ml (6.21) and dilute to the mark with toluene (5.3).

Proceed to a second dilution of 1 ml in a 10 ml volumetric flask (6.21). Store the solutions at room

temperature.

NOTE 3 Solubility of pentacontane in toluene is limited at room temperature. However, the concentration of the

solution of pentacontane does not need to be accurate as it is used only to determine the limit of integration for

mineral oil peak.

NOTE 4 It is also possible to use a commercial mixture of n-alkanes from C12 to C60 that contains n-

pentacontane.
5.25 Sodium carbonate solution, ρ = 0,1 g/ml in water (5.1)
5.26 Dichloromethane (DCM), trace organic analysis grade, purity ≥ 99 %

DCM purity can be checked by concentrating 50 ml of DCM mixed with 25 µl of internal standard solution

(5.16) and 2 drops of keeper (5.27) using a rotary evaporator, dissolving the residue in 0,2 ml of n-hexane

and the analysis of 50 µl by online-HPLC-GC-FID (6.10). Take care that in the evaporation step the residue

is not evaporated to dryness to avoid loss of volatile hydrocarbons. The signal abundance of the residue

after evaporation should not exceed a fifth of the signal abundance obtained at the quantification limit.

5.27 Keeper solvent

The keeper is a solvent that will not evaporate or evaporate to a lesser degree during the evaporation

step, e.g. bis(2-ethylhexyl) maleate. A keeper is used to enhance the recovery of volatile compounds.

ASTM D5442 C12-C60 Qualitative Retention Time Mix available by e.g. Supelco Cat.# 500623 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 CEN of this product.
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oSIST prEN 17517:2020
TC 327 WI 00327116:2020 (E)
6 Apparatus

IMPORTANT — The glassware used for the determination shall be thoroughly cleaned and rinsed with n-

hexane (5.2) before use so that it is free from impurities.

Usual laboratory apparatus and, in particular, the following. The glassware shall be thoroughly cleaned

and rinsed with n-hexane (5.2) or baked in an oven before use so that it is free from impurities.

6.1 Mill with stainless-steel rotor, for grinding to at least 1 mm
6.2 Magnetic stirrer
6.3 Magnetic stir bars
6.4 Analytical balance, reading accuracy 0,000 1 g
6.5 Round-bottomed flasks, 250 ml capacity
6.6 Glass vials with screw caps, 15 ml and 40 ml capacity
6.7 Centrifuge and centrifuge tubes
6.8 Automatic evaporator (optional)
6.9 Glass sample vials, volume of 2 ml

6.10 High performance liquid chromatograph, coupled with gas chromatograph and flame ionization

detector (HPLC-GC-FID)
6.11 Data acquisition system, with the possibility of manual integration
6.12 LC column, 5 µm (250 mm x 2 mm inner diameter (i.d.)) or equivalent
The silica gel column shall have a capacity to retain 20 mg fat.
6.13 Uncoated precolumn, 10 m x 0,53 mm or equivalent
6.14 Capillary column 1, capable for temperatures up to 350 °C

The column should have the following characteristics: 100 % dimethylpolysiloxane or 95 % dimethyl /

5 % phenyl methylpolysiloxane stationary phase, a length of 15 m, an internal diameter of 0,32 mm or

0,25 mm and a film thickness 0,10 µm to 0,15 µm or equivalent.

MicroDancer, IR-Dancer (e.g. Zinser) or Syncore Analyst (Büchi) are examples 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 CEN of these products.

LiChrospher Si 60 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 CEN of this product.

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 CEN of this product.

---------------------- Page: 12 ----------------------
oSIST prEN 17517:2020
TC 327 WI 00327116:2020 (E)

6.15 Capillary column 2, from transfer valve to first Y-piece, fused silica (FS) methyl silicone

deactivated (length 1 m, outside diameter (o.d.) 0,27 mm, inner diameter (i.d.) 0,1 mm)

6.16 Capillary column 3, for hydrogen carrier gas, FS methyl silicone (length 1 m, o.d. 360 µm, i.d.

Twenty-five µm)

6.17 Capillary column 4, for solvent vapour exit, FS methyl silicone (length 1 m, o.d. 0,68 mm, i.d.

0,53 mm)

The columns given in 6.15, 6.16 and 6.17 have proven to be suitable for the analysis. However these

columns can be adjusted in accordance with the characteristics of the HPLC-GC apparatus and the

analytical conditions.

6.18 Restriction capillary column, transfer valve and solvent vapor exit, FS uncoated (length 1 m, o.d.

360 µm, i.d. 50 µm)
6.19 Micros
...

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