SIST EN 17517:2022
(Main)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
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 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.
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 für die Analyse 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.
ANMERKUNG 1 Das Verfahren ist nicht für gekapselte Matrizen ausgelegt.
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 2 Die Schlussfolgerungen in Bezug auf MOAH basieren auf 4 Analyt /Matrix Kombinationen, während das IUPAC-Protokoll [4] mindestens 5 erwartet.
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 für die Bestimmung von MOSH 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.
NOTE 1 Cette méthode n’a pas été conçue pour les matrices encapsulées.
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 2 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 : l’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 pour la détermination des MOSH.
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
Ta dokument določa metodo za določevanje nasičenih in aromatskih ogljikovodikov (od C10 do C50) v krmi. Metoda je bila potrjena v medlaboratorijski študiji z analizo on-line HPLC-GC-FID – glej točke [1], [2] in [3]. Ta metoda ni namenjena za uporabo pri drugih matrikah.
Metodo je mogoče uporabiti za analizo nasičenih ogljikovodikov iz mineralnih olj (MOSH) in/ali aromatskih ogljikovodikov iz mineralnih olj (MOAH).
Metoda se uporablja za sestavine krme, zlasti rastlinska olja in druge sestavine krme, bogate z maščobami, krmne mešanice in premikse. Ne uporablja se za aditive ali deodestilate.
Ta metoda je bila preskušena v medlaboratorijski študiji z analizo naravno kontaminiranih in primešanih vzorcev (premiks, sojina moka, sončnična semena, krma za perutnino, krma za prašiče, rastlinsko olje) v razponu 3–286 mg/kg za nasičene ogljikovodike iz mineralnih olj in 1–16 mg/kg za aromatske ogljikovodike iz mineralnih olj.
Glede na rezultate medlaboratorijske študije je metoda dokazano primerna za masne koncentracije nasičenih ogljikovodikov iz mineralnih olj in aromatskih ogljikovodikov iz mineralnih olj nad 10 mg/kg. Vendar pa metoda med sodelovalno študijo za vzorec premiksa ni bila v celoti potrjena zaradi prenizkih koncentracij nasičenih ogljikovodikov iz mineralnih olj in aromatskih ogljikovodikov iz mineralnih olj. Metoda tudi ni bila v celoti potrjena med sodelovalno študijo za vzorec sončničnih semen zaradi prenizke koncentracije aromatskih ogljikovodikov iz mineralnih olj.
OPOMBA: Ugotovitve glede aromatskih ogljikovodikov iz mineralnih olj temeljijo na štirih kombinacijah analit/matrika, medtem ko se v skladu s protokolom UPAC [4] pričakuje najmanj pet kombinacij.
V primeru domnevnih motenj iz naravnih virov je mogoče fosilni izvor deleža nasičenih ogljikovodikov iz mineralnih olj in aromatskih ogljikovodikov iz mineralnih olj potrditi s pregledom vzorca z metodo GC-MS.
Za določevanje nasičenih ogljikovodikov iz mineralnih olj in aromatskih ogljikovodikov iz mineralnih olj v jedilnih maščobah in oljih je na voljo dodaten standard CEN: EN 16995. Za več informacij glej točko [5].
V dodatku C je predlagana alternativna metoda analizi on-line HPLC-GC-FID, ki jo je mogoče uporabiti kot presejalno metodo.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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.
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
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.
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.
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
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
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.
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.
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.
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).
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 obtained from the fat extraction step
(9.1) 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.
9.3 Blank
A procedural blank sample including the fat extraction step (from 9.1.2) should be analysed in order to
test the purity of the reagents but also other possible sources of contamination, such as the glassware
and the analytical instrument.
The mineral oil content of the procedural blank shall not exceed the level of 3 mg/kg of fat, considering a
test portion equal to 5 g of sample. If this level is exceeded, the source of contamination shall be identified
and eliminated.
Results shall not be corrected by deduction of the blank content.
10 Liquid chromatography and gas chromatography
10.1 Liquid chromatography setup
Install the column (6.12) in the liquid chromatography system (6.10) and check the working conditions
by injecting the solvent. Use n-hexane (5.2) and DCM (5.26) as eluent. Backflush the HPLC column after
every run primarily in order to remove the lipids. For this, backflush the column 6 min after injection
with 500 µl/min DCM for 9 min, then recondition with 500 µl/min n-hexane.
10.2 Working conditions for HPLC
The following working conditions have proven to be suitable to the analysis (see Table 1). However, the
conditions shall be adjusted and checked by the verification standards (5.16).
Table 1 — Working conditions for HPLC
Time n-hexane (5.2) DCM (5.26) Flow
(min) (%) (%) (µl/min)
Initial 100,0 0,0 300
0,10 100,0 0,0 300
1,50 65,0 35,0 300
5,90 65,0 35,0 300
6,00 0,0 100 500
15,00 0,0 100 500
15,30 100,0 0,0 500
25,00 100,0 0,0 500
25,20 100,0 0,0 300
30,00 100,0 0,0 300
The gradient is checked in the column effluent by UV-detection at 230 nm. Breakthrough of DCM is
observed after 4,5 min as a steep increase of absorption to a plateau level.
The elution of perylene is detected with a retention time of around 5,6 min (see Figure 1). The MOSH is
eluted from 2 min to 3,5 min, the fraction comprising the MOAH ranges from 4,7 min to 6,2 min.
The described retention times may vary depending on the system and the columns used for analysis.
Key
X time, in min 2 MOAH 5 reconditioning
Y1, Y2 abundance 3 perylene
1 MOSH 4 backflush (6 min after injection)
Figure 1 — HPLC-UV chromatogram
10.3 HPLC-GC-interface
The carrier gas and the line from the transfer valve are joined in a press-fit Y-piece. For convenience, the
Y-piece is positioned above the GC oven. The carrier gas, e.g. from an injector, is fed into the Y-piece using
a fused silica capillary. The inlet of the precolumn passes through the top of the oven into the leg of the
Y-piece, e.g. through an unused detector base block. The transfer line is backflushed via the transfer valve
(see Figure 2).
Key
1 HPLC pump 5 transfer line 9 reconditioning
2 HPLC column 6 backflush of transfer line 10 inlet of precolumn
3 HPLC waste 7 carrier gas 11 GC oven
4 transfer valve 8 Y-piece
NOTE Available commercial equipment can replace the HPLC-GC interface described above.
Figure 2 — Schematic drawing of HPLC–GC interface [2]
10.4 Gas chromatography setup
Install the columns (6.14 and 6.15) in the gas chromatograph (6.10) and check the working conditions by
injecting the solvent, n-hexane (5.2). The baseline should be straight with a small positive drift. If the drift
is high, proceed to condition the column. In case of a negative drift, check the connections of the column.
If the column is used for the first time, it is necessary to condition the column by heating it in the column
oven using a temperature gradient up to 350 °C (depending on the oven temperature chosen for the
analysis) in 4 h, maintain the temperature for 0,5 h.
10.5 Working conditions for GC
The following working conditions have proven to be suitable to the analysis, however they may be
adjusted in accordance with the characteristics of the HPLC-GC-apparatus and the column. Typical
chromatograms are presented in Annex A.
— Pre-column: uncoated, deactivated, 7 m to 10 m x 0,53 mm;
— Column: 100 % dimethyl polysiloxane, low bleed (15 m long, 0,25 mm i.d., 0,1 µm to 0,15 µm film
thickness);
— Oven temperature: initial temperature 55 °C for 7 min, programmed at 20 °C/min to 350 °C, hold for
5 min;
— Carrier gas: hydrogen. For MOSH an inlet pressure of 70 kPa, after closing solvent vapor exit 60 kPa.
For MOAH an inlet pressure of 65 kPa, after closing solvent vapor exit 60 kPa;
— Detector temperature: 380 °C;
— Transfer volume: 450 µl.
10.6 Solvent vapor exit
Solvent evaporation forms large volumes of vapor diluted with carrier gas which need to be discarded.
Therefore a solvent vapor exit is used, located between the uncoated pre-column and the GC separation
column.
The time for closing the vapor exit is determined by the transfer of the intended fraction with a
permanently open solvent vapor exit. The effluent of the vapor exit is lit and the duration up to the
extinction of the yellow flame measured. This duration minus 3 s is entered for closure of the vapor exit,
in order to direct the last portion of solvent containing the reconcentrated volatile solutes into the
separation column. The time varies depending on the length of the retention gap.
10.7 Peak identification
Identify the internal standards by injecting 50 µl of a 1/10-dilution of the standard solutions (5.16 and
5.24) (Figure A.1 and Figure A.2, Annex A). Check the resolution of n-undecane (n-C11) separated of the
solvent peak for the standard solution (5.16). Use the retention time of pentacontane (C50) to determine
the limit of integration for mineral oil peak (Figure A.6, Annex A).
The dilution of the standard solutions (5.16 and 5.24) can be adjusted in order to allow to use the
concentrations which are the best for each system. The absolute injection amount of standard substances
is each in the range of 25 ng up to 60 ng.
If necessary, inject 50 µl of the alkane standard mixture C10 to C40 (5.22) in order to identify the areas
to take into consideration for the calculation of mass concentrations of C10 to C50 fractions of mineral
oil hydrocarbons (MOSH and MOAH) (Figure A.3, Annex A).
In sunflower oils the major peaks correspond to saturated aliphatic hydrocarbons C27, C29 and C31. A
broad peak (of about 5 min to 15 min width, depending on the GC conditions) represents a complex
mixture of hydrocarbons (UCM) that the chromatographic system cannot resolve and it is attributed to
mineral oil hydrocarbons (Figure A.4 to A.9, Annex A).
10.8 Performance of the HPLC-GC system
Inject an aliquot of a dilution of the standard solution 2 (5.16) with an absolute amount of standard
substances each in the range of 10 ng up to 50 ng (e.g. 20 µl of a 1/150-dilution or 50 µl of a 1/500-
dilution). Use bicyclohexyl (CyCy) (5.10) as internal standard for the MOSH fraction, 1-
methylnaphthalene (1-MN) as an internal standard for the MOAH fraction. All substances of the internal
standard serve as marker substances. The MOSH/MOAH separation is checked by these verification
standards. After a successful separation of the fractions CyCy, n-C11, n-C13 and Cho shall be detected in
the MOSH fraction, while 5-PB, 1-MN, 2-MN, TBB and Per elute in the MOAH fraction (see Figure 3). If
there is a marker in the wrong fraction, it shall be checked whether the HPLC column is functioning
properly. If necessary, replace the HPLC column.
Key
1 injection 6 highly alkylated polycyclic aromatics 11 cholestane
2 high mass paraffins 7 little alkylated polycyclic aromatics 12 tri-tert-butylbenzene
3 low mass paraffins 8 saturated wax esters 13 perylene
4 naphthenes 9 MOSH 14 wax ester
5 alkylated benzenes 10 MOAH t retention time
Figure 3 — Elution sequence of MOSH and MOAH compounds (upper part) and the verification
standards used for establishing and checking the fraction window (lower part) [2]
The gas chromatography system is considered to be optimized when the ratio of C40 to C20 is equal or
higher than 80 %.
10.9 Quantitative determination of hydrocarbons attributed to mineral oil origin
Determine the area of total hydrocarbons (A ) by integrating manually the total signal composed of the
UCM and the sharp peaks above the UCM (Figure A.6 and Figure A.8, Annex A), from the point that the
baseline starts to increase until the retention time of pentacontane (C50).
Subtract all sharp peaks above the UCM. To determine the area (A ) to be subtracted, re-integrate the
chromatogram by drawing manually the valley-to-valley baseline over the UCM profile for all the sharp
peaks including the small ones (Figure A.7 and Figure A.9, Annex A).
The resultant area for quantitative determination is A (A = A – A ).
i i 1 2
If the bicyclohexyl (CyCy) peak or the 1-methylnaphthalene (1-MN) peak lies on the UCM, determine the
area of the standard by integrating the area of the internal standard valley-to-valley.
Calculate the mass fraction, w , in milligrams per kilogram of the MOSH, MOAH content respectively,
hc
using Formula (1):
A × m × 1 000
i is
w =
(1)
hc
A × m
is
where
w is the mass fraction of the MOSH repectively MOAH content, in milligrams per kilogram;
hc
A is the peak area of the UCM after subtraction of all sharp peaks above the UCM (A – A );
i 1 2
A is the peak area of the internal standard peak;
is
m
is the mass, of the internal standard solution (5.16) added to the sample, in milligrams;
is
m is the mass of the test portion, in grams.
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.
The limit of quantification (LOQ) of the UCM peak is variable because it depends on the width and height
of the peak, which is analyte dependent.
To calculate the concentration of UCM that elutes from C10 to C35, it is necessary to superimpose the
chromatogram of the sample and the chromatogram of the alkane standard mixture C10 to C40 and to
integrate the part of the UCM which is included in the range of C10 to C35.
11 Precision
11.1 Interlaboratory test
Details of an interlaboratory test on the precision of the method are summarized in Annex B. It is possible
that the values derived from this interlaboratory test are not applicable to concentration ranges and
matrices other than those given.
11.2 Repeatability
The absolute difference between two single test results found on identical test material by one operator
using the same apparatus within the shortest feasible time interval will exceed the repeatability limit r,
given in Tables B.2 and B.3, in not more than 5 % of the cases.
11.3 Reproducibility
The absolute difference between two single test results found on identical test material reported by two
laboratories will exceed the reproducibility limit R, given in Tables B.2 and B.3, in not more than 5 % of
the cases.
12 Test report
The test report shall specify:
a) all information necessary for the complete identification of the sample;
b) the sampling method used, if known;
c) the test method used, with reference to this document (including its year of publication);
d) the date of receipt;
e) the date of test;
f) the mass(es) of the test portion(s);
g) the range of the hydrocarbon chain length analysed;
h) all operating details not specified in this document or regarded as optional, together with details of
any incidents that may have influenced the test result(s);
i) the test result(s) and the units in which they have been expressed, or, if the repeatability has been
checked, the final quoted result obtained.
Annex A
(informative)
Examples of chromatograms
Key
1 n-decane (n-C10) or n-undecane (n-C11): detection of losses of volatile 5 n-tetracontane (n-C40)
components: transfer or reconcentration of sample extracts
2 bicyclohexyl (CyCy): standard for quantification, not present in mineral oil t time, in min
products
3 n-tridecane (n-C13): proves no co-elution with bicyclohexyl Y abundance
4 cholestane (Cho): end of MOSH fraction
Figure A.1 — GC chromatogram of the saturated hydrocarbon standard: MOSH
Key
1 pentylbenzene (5-PB): detection of losses of volatile components
2 2-methylnaphthalene (2-MN): standard for quantification
3 1-methylnaphthalene (1-MN): peak pair for easy identification
4 tri-tert-butylbenzene (TBB): start of MOAH fraction
5 perylene (Per): end of MOAH fraction, determination also through HPLC-UV
t time, in min
Y abundance
Figure A.2 — GC chromatogram of the aromatic hydrocarbon standard: MOAH
Key
1 n-decane (n-C10) t time, in min
2 n-eicosane (n-C20) Y abundance
3 n-tetracontane (n-C40)
Figure A.3 — GC chromatogram of an alkane standard mixture C10 to C40
Key
1 n-decane (n-C10) 4 n-tetracontane (n-C40)
2 bicyclohexyl (CyCy) t time, in min
3 n-tridecane (n-C13) Y abundance
Figure A.4 — GC chromatogram of the saturated hydrocarbon fraction of a crude sunflower oil
Key
1 pentylbenzene (5-PB) 4 tri-tert-butylbenzene (TBB)
2 2-methylnaphthalene (2-MN) t time, in min
3 1-methylnaphthalene (1-MN) Y abundance
Figure A.5 — GC chromatogram of the aromatic hydrocarbon fraction of a crude sunflower oil
Key
1 n-decane (n-C10) 5 retention time of n-pentacontane (n-C50)
2 bicyclohexyl (CyCy) t time, in min
3 n-tridecane (n-C13) Y abundance
4 chole
...
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