kSIST FprEN 17517:2021

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

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

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

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)

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

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

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

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

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

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

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

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

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

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

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],

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

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

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

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

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

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

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)

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

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

complex mixture of saturated or aromatic hydrocarbons not resolved by gas chromatography such as

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

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

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.

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

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

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

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.

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

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

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.

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

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

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

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

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

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

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

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.

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

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

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.

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

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

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

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.

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.

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

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

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