SIST EN 16995:2017
(Main)Foodstuffs - Vegetable oils and foodstuff on basis of vegetable oils - Determination of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) with on-line HPLC-GC-FID analysis
Foodstuffs - Vegetable oils and foodstuff on basis of vegetable oils - Determination of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) with on-line HPLC-GC-FID analysis
This European Standard specifies a method for the determination of saturated and aromatic hydrocarbons (from C10 to C50) in vegetable fats and oils and foodstuff on basis of vegetable oils with online-HPLC-GC-FID [1], [2]. HPLC-GC-FID provides a highly efficient method for the determination of mineral oils in different foodstuffs [3].
The method can be used for the analysis of MOSH and/or MOAH. Mineral oil saturated hydrocarbons (MOSH) are paraffinic (open-chain, usually branched) and naphthenic (cyclic, alkylated) hydrocarbons, mineral oil aromatic hydrocarbons (MOAH) are aromatic mainly alkylated hydrocarbons.
The method has been tested in an interlaboratory study via the analysis of both naturally contaminated and spiked vegetable oil samples and mayonnaise and margarine samples, ranging from 4 mg/kg to 197 mg/kg for MOSH, and from 2 mg/kg to 51 mg/kg for MOAH.
The method has been proved suitable above 10 mg/kg on basis on the results of the interlaboratory tests.
Lebensmittel - Pflanzliche Öle und Lebensmittel auf Basis pflanzlicher Öle - Bestimmung von Mineralölen aus gesättigten Kohlenwasserstoffen (MOSH) und aus aromatischen Kohlenwasserstoffen (MOAH) mit on-line HPLC-GC-FID
Diese Europäische Norm legt ein Verfahren zur Bestimmung von gesättigten und aromatischen Kohlenwasserstoffen (von C10 bis C50) in pflanzlichen Fetten und Ölen und Nahrungsmitteln auf der Basis von Pflanzenölen mit Hilfe einer Online-HPLC-GC-FID [1], [2] (HPLC: Hochleistungsflüssig-chromatographie, en: high performance liquid chromatography; GC: Gaschromatographie, en: gas chromatographie; FID: Flammenionisationsdetektor, en: flame ionization detector) fest. HPLC-GC-FID ist ein hochwirksames Verfahren für die Bestimmung von Mineralölen in verschiedenen Lebensmitteln [3].
Das Verfahren kann zur Bestimmung von MOSH und/oder MOAH herangezogen werden. Gesättigte Mineralöl-Kohlenwasserstoffe (MOSH) sind paraffinisch (offenkettig, für gewöhnlich verzweigt) und naphthenisch (ringförmig alkyliert). Aromatische Mineralöl-Kohlenwasserstoffe (MOAH) sind aromatische hauptsächlich alkylierte Kohlenwasserstoffe.
Das Verfahren wurde in einem Ringversuch anhand der Analyse von sowohl natürlich als auch künstlich kontaminierten Proben von Pflanzenölen, Mayonnaise und Margarine in Mengen 4 mg/kg bis 197 mg/kg für MOSH und von 2 mg/kg bis 51 mg/kg für MOAH getestet.
Die Ergebnisse des Ringversuchs ergaben, dass sich das Verfahren bei Mengen über 10 mg/kg eignet.
Produits alimentaires - Huiles végétales et denrées à base d'huiles végétales - Dosage des hydrocarbures saturés d’huile minérale (MOSH) et des hydrocarbures aromatiques d’huile minérale (MOAH) par analyse par CLHP-CG-FID en ligne
La présente Norme européenne spécifie une méthode applicable pour le dosage des hydrocarbures saturés et aromatiques (de C10 à C50) présents dans les corps gras végétaux et les denrées alimentaires à base d’huiles végétales par CLHP-CG-FID en ligne [1], [2]. La méthode CLHP-CG-FID est hautement efficace pour le dosage des huiles minérales dans différentes denrées alimentaires [3].
La méthode peut être employée pour l’analyse des MOSH et/ou des MOAH. Les hydrocarbures saturés d’huile minérale (MOSH) sont des hydrocarbures paraffiniques (chaîne ouverte, généralement ramifiée) et naphténiques (cycliques, alkylés) ; les hydrocarbures aromatiques d’huile minérale (MOAH) sont des hydrocarbures aromatiques essentiellement alkylés.
Cette méthode a été soumise à essai lors d’une étude interlaboratoires en procédant à l’analyse d’échantillons d’huile végétale naturellement contaminés et dopés et d’échantillons de mayonnaise et de margarine à des teneurs comprises entre 4 mg/kg et 197 mg/kg pour les MOSH et entre 2 mg/kg et 51 mg/kg pour les MOAH.
Il a été démontré que la méthode est adaptée au-dessus de 10 mg/kg sur la base des résultats des essais interlaboratoires.
Živila - Rastlinska olja in živila na osnovi rastlinskih olj - Določevanje mineralnih olj nasičenih ogljikovodikov (MOSH) in mineralnih olj aromatskih ogljikovodikov (MOAH) z analizo on-line HPLC-GC-FID
Ta evropski standard opredeljuje metodo za določanje nasičenih in aromatskih ogljikovodikov (od C10 do C50) v rastlinskih maščobah, oljih in živilih na osnovi rastlinskih olj z analizo on-line HPLC-GC-FID [1], [2]. Analiza HPLC-GC-FID je zelo učinkovita metoda za določanje mineralnih olj v različnih živilih [3].
To metodo je mogoče uporabiti za analizo mineralnih olj nasičenih ogljikovodikov (MOSH) in/ali mineralnih olj aromatskih ogljikovodikov (MOAH). Mineralna olja nasičenih ogljikovodikov (MOSH) so parafinski (odprtoverižni, običajno razvejani) in naftenski (ciklični, alkilirani) ogljikovodiki; mineralna olja aromatskih ogljikovodikov (MOAH) so aromatski in v večji meri alkilirani ogljikovodiki.
Ta metoda je bila preskušena v medlaboratorijski študiji z analizo naravno kontaminiranih in primešanih vzorcev rastlinskih olj, majoneze ter margarine v razponu 4–197 mg/kg za mineralna olja nasičenih ogljikovodikov in 2–51 mg/kg za mineralna olja aromatskih ogljikovodikov.
Ta metoda je dokazano primerna za vsebnosti nad 10 mg/kg na podlagi rezultatov medlaboratorijskih preskusov.
General Information
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.RJOMLNRYRGLNRYLebensmittel - Pflanzliche Öle und Lebensmittel auf Basis pflanzlicher Öle - Bestimmung von Mineralölen aus gesättigten Kohlenwasserstoffen (MOSH) und aus aromatischen Kohlenwasserstoffen (MOAH) mit on-line HPLC-GC-FIDProduits alimentaires - Huiles végétales et denrées à base d'huiles végétales - Dosage des hydrocarbures saturés d’huile minérale (MOSH) et des hydrocarbures aromatiques d’huile minérale (MOAH) par analyse par CLHP-CG-FID en ligneFoodstuffs - Vegetable oils and foodstuff on basis of vegetable oils - Determination of mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH) with on-line HPLC-GC-FID analysis67.200.10Animal and vegetable fats and oilsICS:Ta slovenski standard je istoveten z:EN 16995:2017SIST EN 16995:2017en,fr,de01-september-2017SIST EN 16995:2017SLOVENSKI
STANDARD
SIST EN 16995:2017
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16995
June
t r s y ICS
x yä t r rä s r English Version
Foodstuffs æ Vegetable oils and foodstuff on basis of vegetable oils æ Determination of mineral oil saturated Produits alimentaires æ Huiles végétales et produits alimentaires à base d 5huiles végétales æ Dosage des par analyse par CLHPæCGæFID en ligne
Lebensmittel æ Pflanzliche Öle und Lebensmittel auf Basis pflanzlicher Öle æ Bestimmung von gesättigten mit onæline HPLCæGCæFID This European Standard was approved by CEN on
s r March
t r s yä
egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alterationä Upætoædate lists and bibliographical references concerning such national standards may be obtained on application to the CENæCENELEC Management Centre or to any CEN memberä
translation under the responsibility of a CEN member into its own language and notified to the CENæCENELEC Management Centre has the same status as the official versionsä
CEN members are the national standards bodies of Austriaá Belgiumá Bulgariaá Croatiaá Cyprusá Czech Republicá Denmarká Estoniaá Finlandá Former Yugoslav Republic of Macedoniaá Franceá Germanyá Greeceá Hungaryá Icelandá Irelandá Italyá Latviaá Lithuaniaá Luxembourgá Maltaá Netherlandsá Norwayá Polandá Portugalá Romaniaá Serbiaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey and United Kingdomä
EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Avenue Marnix 17,
B-1000 Brussels
9
t r s y CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN
s x { { wã t r s y ESIST EN 16995:2017
EN 16995:2017 (E) 2 Contents
Page European foreword . 3 1 Scope . 4 2 Normative references . 4 3 Terms and definitions . 4 4 Principle . 4 5 Reagents . 5 6 Apparatus . 8 7 Sample storage . 9 8 Preparation of the test sample . 10 9 Preparation of the analytical sample . 10 10 Liquid chromatography and gas chromatography . 12 11 Precision . 17 12 Test report . 17 Annex A (informative)
Examples of chromatograms . 18 Annex B (informative)
Precision data . 32 Bibliography . 35
SIST EN 16995:2017
EN 16995:2017 (E) 3 European foreword This document (EN 16995:2017) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. 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 December 2017, and conflicting national standards shall be withdrawn at the latest by December 2017. 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 mandate given to CEN by the European Commission and the European Free Trade Association. WARNING — The use of this standard can involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this document to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 16995:2017
EN 16995:2017 (E) 4 1 Scope This European Standard specifies a highly efficient method for the determination of saturated and aromatic hydrocarbons (from C10 to C50) in vegetable fats and oils and foodstuff on basis of vegetable oils for which it has been interlaboratory validated, with online-HPLC-GC-FID [1], [2] and [3]. This standard 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 has been tested in an interlaboratory study via the analysis of both naturally contaminated and spiked vegetable oil samples and mayonnaise and margarine samples, ranging from 4 mg/kg to 197 mg/kg for MOSH, and from 2 mg/kg to 51 mg/kg for MOAH. According to the results of the interlaboratory studies, the method has been proven suitable for MOSH and MOAH mass concentrations each above 10 mg/kg. 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. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 661, Animal and vegetable fats and oils - Preparation of test sample (ISO 661) 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 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 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 [4], 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 SIST EN 16995:2017
EN 16995:2017 (E) 5 applying partially concurrent eluent evaporation. High boiling components spread over the entire length of the flooded zone and are refocused by the retention gap technique [2]. The area attributed to mineral oil is calculated by subtraction of sharp 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. Some vegetable oils contain odd-numbered n-alkanes in the range of C21-C33 in such quantities that the chromatograms of the MOSH fraction are severely overloaded and that they might overlap with the mineral oil hump. In this case, it is recommended to use an additional clean-up technique. Activated aluminium oxide strongly retains long chain n-alkanes. Mineral oil which contaminates edible oil almost exclusively consists of branched and cyclic components which are not retained by activated aluminium oxide. Therefore, the use of activated aluminium oxide enables the removal of plant paraffins. 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. Since now, 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 Unless otherwise specified, use only reagents of recognized analytical grade. 5.1 Silica Gel 601), extra pure for column chromatography with particle size from 60 µm to 200 µm (70 mesh to 230 mesh) in glass bottle to prevent contamination. 5.2 Silica Gel 60, activated. Condition silica gel (5.1) in an oven for at least 16 h at 400 °C. 5.3 Demineralized water. 5.4 Anhydrous sodium sulfate, analytical gradeá purity
·
{ { %. 5.5 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 (5.21) and 2 drops of bis(2-ethylhexyl) maleate (5.29) 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 (6.8). 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.
1) Silica gel is available from Merck, reference 7754 or 7734 (www.merck-chemicals.com). It is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 16995:2017
EN 16995:2017 (E) 6 5.6 Dichloromethane (DCM)á trace organic analysis gradeá purity
·
{ {
¨ä DCM purity can be checked by concentrating 50 ml of DCM mixed with 25 µl of internal standard solution (5.21) and 2 drops of bis(2-ethylhexyl) maleate (5.29) 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 (6.8). 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. 5.7 Dichloromethane solution. Mix 30 ml DCM (5.6) with n-hexane (5.5) up to a volume of 100 ml. The solution should be freshly prepared daily. 5.8 Toluene. 5.9 1,1,2-Trichloroethane. 5.10 Perylene (Per %. 5.11 5-ƒ-Cholestane (Cho), purity
·
{ y %. 5.12 n-Undecane (n-C11), purity
·
{ z %. 5.13 n-Tridecane (n-C13), purity
·
{ y %. 5.14 Tri-tert-butylbenzene (TBB). 5.15 Bicyclohexyl (CyCy)á purity
·
{ { %. 5.16 1-Methylnaphthalene (1-MN)á purity
·
{ w %. 5.17 2-Methylnaphthalene (2-MN)á purity
·
{ y %. 5.18 Pentylbenzene (PB)á purity
·
{6 %. 5.19 Stock solutions, mass concentration
= 10 mg/ml. Prepare individual stock solutions by weighing, to the nearest 1 mg, 100 mg of n-C11 (5.12), n-C13 (5.13), TBB (5.14), CyCy (5.15), 1-MN (5.16), 2-MN (5.17) and PB (5.18) into a 10 ml volumetric flask and dilute to the mark with 1,1,2-trichloroethane (5.9) or toluene (5.8). Store the solutions at room temperature. If crystals precipitate during storage, warm the solution until everything has dissolved. 5.20 Internal standard solution 1 (ISTD1)2). Weigh, to the nearest 0,5 mg, 12 mg of Per (5.10) and Cho (5.11) in a volumetric flask of 20 ml (6.22), to which 600 µl of each stock solution (5.19) 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 (5.9) or toluene (5.8). Resulting mass concentrations are for n-C13:
= 150 µg/ml, for n-C11, TBB, CyCy, 1-MN, 2-MN and PB:
= 300 µg/ml and for Per, Cho:
= 600 µg/ml.
2) This standard mixture is available by e.g. Restek Corp., Cat.# 31070. It is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 16995:2017
EN 16995:2017 (E) 7 5.21 Internal standard solution 2 (ISTD2). Dilute the ISTD1 solutions by a factor of 30, e.g. 333 µl filled up to 10 ml with n-hexane (5.5). Resulting mass concentrations are for n-C13:
= 5 µg/ml, for n-C11, TBB, CyCy, 1-MN, 2-MN and PB:
= 10 µg/ml and for Per, Cho:
= 20 µg/ml. 5.22 Aluminium oxide 90, alkaline, for column chromatography 0,063 mm to 0,2 mm. 5.23 Aluminium oxide, activated (ALOX). Condition aluminium oxide 90 (5.22) for at least 16 h at 500 °C in an oven before using. 5.24 Chloroperbenzoic acid (CPBA), purity 70 % to 75 %. 5.25 CPBA solution,
= 0,1 g/ml in dichloromethane. For example 1 g of CPBA (5.24) in 10 ml of DCM (5.6). Clouding of solution does not disturb the reaction. The solution can be used for up to one week. 5.26 Ascorbic acid. 5.27 Silica-ALOX column. Insert a filter (6.3) in each glass column (6.2). Then, fill in 10 g of ALOX (5.23) and 3 g of silica gel (5.2) and compress. 5.28 Cleanup column. Insert a filter (6.3) in a glass SPE tube (6.20). Then, fill in 3 g of silica gel (5.2), compress and overlay with 0,5 g of sodium sulfate (5.4). 5.29 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. 5.30 Carrier gas for gas chromatographyá preferably hydrogená purity
·
{ {á { { w %. 5.31 Auxiliary gases for flame ionization detector, hydrogen, air, and nitrogen suitable for gas chromatography. 5.32 Alkane standard mixture C10 to C40, solution of equal concentration in an apolar solvent,
= 1 µg/ml. 5.33 Ethanol, absolute. NOTE The ethanol purity can be checked by concentrating 50 ml of ethanol mixed with 25 µl of internal standard solution (5.21) 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 (6.8). 5.34 Mixture of ethanol and n-hexane %. Mix 50 ml of ethanol (5.33) with 50 ml of n-hexane (5.5). 5.35 n-Pentacontane (n-C50), purity
·
{ z %. SIST EN 16995:2017
EN 16995:2017 (E) 8 5.36 n-Pentacontane (n-C50) solution in toluene,
³ 10 µg/ml. Weigh 2 mg of C50 (5.35) in a volumetric flask of 20 ml (6.22) and dilute to the mark with toluene (5.8). Proceed to a second dilution of 1 ml in a 10 ml volumetric flask (6.22). 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-pentacontane3). 5.37 Sodium carbonate solution,
= 0,1 g/ml in water (5.3). 5.38 Mixture of DCM and n-hexane. Mix 20 ml DCM (5.6) with n-hexane (5.5) up to a volume of 100 ml. The solution should be freshly prepared daily. 5.39 Blank refined sunflower oil. 6 Apparatus Usual laboratory apparatus and, in particular, the following. The glassware shall be thoroughly cleaned and rinsed with n-hexane (5.5) or baked in an oven before use so that it is free from impurities. 6.1 Centrifuge and centrifuge tubes. 6.2 Glass column for cleanup, 15 cm to 20 cm length and 15 mm to 20 mm internal diameter. 6.3 Filter for glass column. 6.4 Glass vials with screw caps, volume of 40 ml. 6.5 Rotary evaporator, with vacuum and a water bath at 35 °C (recommended). Care should be taken to prevent cross contamination. Clean the system thoroughly between determinations. 6.6 Automatic evaporator.4) 6.7 Glass sample vials, volume of 2 ml. 6.8 High performance liquid chromatograph, coupled with gas chromatograph and flame ionization detector (HPLC-GC-FID).
3) ASTM D5442 C12-C60 Qualitative Retention Time Mix is available by e.g. Supelco Cat.# 500623. It is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. Equivalent products may be used if they can be shown to lead to the same results. 4) For example, MicroDancer, IR-Dancer (e.g. Zinser) or Syncore Analyst (Büchi). These are examples of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 16995:2017
EN 16995:2017 (E) 9 6.9 Data acquisition system, with the possibility of manual integration. 6.10 LC column, e.g. LiChrospher Si 605), 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. 6.11 Uncoated precolumn, e.g. Hydroguard® MXT®5), 10 m x 0,53 mm or equivalent. 6.12 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. 6.13 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). 6.14 Capillary column 3, for hydrogen carrier gas, FS methyl silicone (length 1 m, o.d. 360 µm, i.d. 25 µm). 6.15 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 6.13, 6.14 and 6.15 have proven to be suitable for the analysis, however they can be adjusted in accordance with the characteristics of the HPLC-GC apparatus and the analytical conditions. 6.16 Restriction capillary column, transfer valve and solvent vapor exit, FS uncoated (length 1 m, o.d. 360 µm, i.d. 50 µm). 6.17 Microsyringe, 5 µl to 100 µl capacity, suitable for injection in liquid chromatography. 6.18 Analytical balance, reading accuracy 0,000 1 g. 6.19 Pasteur pipette, glass. The use of plastic Pasteur pipettes and polyethylene film shall be avoided. 6.20 Empty glass column for solid phase extraction (SPE), with glass fibre frits, 6 ml volume. 6.21 Conical flask, volume of 100 ml. 6.22 Volumetric flasks, various sizes. 7 Sample storage Analyse only samples packed in glass bottles or aluminium foil in order to prevent additional contamination. Plastic and paper packaging are unsuitable.
5) These are examples of a suitable product available commercially. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of this product. Equivalent products may be used if they can be shown to lead to the same results. SIST EN 16995:2017
EN 16995:2017 (E) 10 8 Preparation of the test sample Prepare the test sample in accordance with EN ISO 661. 9 Preparation of the analytical sample 9.1 Procedure for liquid and solid fats (e.g. sunflower oil, olive oil) 9.1.1 Procedure for liquid and solid fats soluble in n-hexane Weigh, to the nearest 1 mg, 300 mg sample into a 1,5 ml autosampler vial, fill up with n-hexane (5.5) and add 30 µl ISTD2 (5.21). Shake the vial well and place it onto the autosampler. Afterwards, measure the solution by online HPLC-GC-FID. The injection volume is 100 µl. Another possibility is to weigh, to the nearest 1 mg, 300 mg sample into an autosampler vial, to fill it up with 600 µl n-hexane and to add 30 µl ISTD2. Shake the vial well and place it onto the autosampler. Afterwards measure the solution by online HPLC-GC-FID. The injection volume is 50 µl. Depending on the level of contamination, the injected volume may be adapted in order to avoid the overloading of the chromatograms. The amount of the added internal standards may be increased (e.g. by using 3 µl ISTD1 (5.20)) instead of 30 µl ISTD2, in order to lower the impact of the matrix interferences, if necessary. If there are strong interferences in the MOSH chromatogram observed by natural, primarily odd-numbered n-alkanes in the range of C23 to C33 (see example in Figure A.4 in Annex A), an additional purification of the sample with aluminium oxide is necessary (9.3) before HPLC-GC analysis. Vegetable oils often contain biogenic olefinic substances (e.g. squalene, terpenes, phytosterenes), which interfere with the chromatography of the MOAH. Remove these substances by epoxidation (9.4) before HPLC-GC analysis. 9.1.2 Procedure for solid fats not soluble in n-hexane If solid fats are not soluble in n-hexane, weigh, to the nearest 1 mg, 300 mg sample in a 40 ml glass vial (6.4). Add 30 µl ISTD2 (5.21) and dissolve in 2 ml DCM solution (5.7). Transfer the solution to the cleanup column (5.28). Wash the vial (6.4) first with 1 ml, then again with 1 ml, 2 ml and 10 ml DCM solution (5.7) and apply the solutions to the cleanup column. Elute the hydrocarbon fraction in another 40 ml glass vial. Evaporate the solvent under reduced pressure after addition of 2 drops of bis(2-ethylhexyl) maleate (5.29) at 40 °C. Take care that in the evaporation step the residue is not evaporated to dryness to avoid loss of volatile hydrocarbons. Dissolve the residue of the extract in n-hexane (5.5) and transfer it to a vial to a final volume of 1 ml. Centrifugation may be necessary if the solution is cloudy. Analyse the sample solution by online-HPLC-GC-FID. The injection volume is 50 µl. The amount of the added internal standards may be increased (e.g. by using 3 µl ISTD1 (5.20)) instead of 30 µl ISTD2 (5.21), in order to lower the impact of the matrix interferences, if necessary. If there are strong interferences in the MOSH chromatogram observed by natural, primarily odd-numbered n-alkanes in the range of C23 to C33 (see example in Figure A.4 in Annex A), an additional purification of the sample with aluminium oxide is necessary (9.3) before HPLC-GC analysis. Vegetable fats often contain biogenic olefinic substances (e.g. squalene, terpenes, phytosterenes), which interfere with the chromatography of the MOAH. Remove these substances by epoxidation (9.4) before HPLC-GC analysis. SIST EN 16995:2017
EN 16995:2017 (E) 11 9.2 Procedure for fatty food materials containing water (e.g. mayonnaise, margarine) Weigh, 5 g of sample in a 30 ml centrifuge tube with screw cap. Add 25 ml of ethanol (5.33) and 20 µl of ISTD1 (5.20), homogenize and allow to stand for at least 1 h at ambient temperature, centrifuge if the phases do not separate correctly. The ethanol extract is decanted into a 100 ml conical flask (6.21) and the solid residue is immersed in 20 ml of n-hexane (5.5). After extraction overnight at ambient temperature, shake the tube, centrifuge it and add the supernatant n-hexane to the previously decanted ethanol. Admix 50 ml water (5.3) and transfer 1,5 ml of the n-hexane phase to an autosampler vial of which 100 µl is injected into HPLC. If necessary, this 1,5 ml n-hexane solution may be submitted to clean-ups following
9.3 and/or 9.4. The amount of the added internal standards may be increased in order to lower the impact of the matrix interferences, if necessary. 9.3 Cleanup for MOSH fraction with aluminium oxide (optional) Weigh, to the nearest 1 mg, 300 mg of sample in a 40 ml glass vial (6.4). Add 30 µl of ISTD2 (5.21) and dissolve the mixture in 2 ml of n-hexane (5.5). Subsequently, transfer the sample solution to a silica-ALOX column (5.27) that was rinsed by using 20 ml of n-hexane prior to sample loading. Elute the aliphatic hydrocarbon fraction with 25 ml of n-hexane. Evaporate the solvent under reduced pressure after addition of 2 drops of bis(2-ethylhexyl) maleate (5.29) at 40 °C. Take care that in the evaporation step the residue is not evaporated to dryness to avoid loss of volatile hydrocarbons. Dissolve the extract in small portions of n-hexane and transfer it into a vial to a final volume of 1 ml. Centrifugation may be necessary. Analyse the sample solution by online-HPLC-GC-FID. The injection volume is 100 µl. NOTE After the cleanup with aluminium oxide, only the MOSH fraction can be analysed as the MOAH fraction is retained on the aluminium oxide bed. The amount of the added internal standards may be increased (e.g. by using 3 µl of ISTD1 (5.20)) instead of 30 µl of ISTD2 (5.21), in order to lower the impact of the matrix interferences, if necessary. 9.4 Cleanup for MOAH fraction with epoxidation Weigh, to the nearest 1 mg, 300 mg of sample in a 15 ml centrifuge glass tube and add 30 µl of ISTD2 (5.21). If the sample had to be extracted (see 9.2), transfer 1,5 ml of sample extract to the centrifuge tube and evaporate the solvent by a stream of nitrogen (triglycerides from the sample act as a keeper). Add 3 ml of DCM (5.6) and cool the solution in ice water (> 5 min). After addition of 1 ml of cooled CPBA solution (5.25), shake the sample and allow it to warm to ambient temperature for 5 min. After 15 min, add 3 ml of sodium carbonate solution (5.37), shake the mixture intensively for 15 s and then centrifuge. Discard the aqueous supernatant and wash the sample with an additional 3 ml of water (5.3). Transfer 1,5 ml of the dichloromethane phase to an autosampler vial and bring it to dryness by a stream of nitrogen (triglycerides from the sample act as a keeper). Redissolve the residue in 1 ml of n-hexane (5.5). At a maximum, inject 100 µl into the HPLC. If required, the detection limit may be lowered by enrichment prior to epoxidation. Pack 12 g of silica gel (5.2) into a glass column (6.2) and condition with 10 ml of a 20 % mixture of DCM and n-hexane (5.38). Dissolve 1 g of edible oil or fat in 2 ml of n-hexane, add 50 µl ISTD2 (5.21) and transfer the mixture to the prepared silica gel column. Elute the hydrocarbons with 45 ml of a 20 % mixture of DCM and n-hexane. Discard the first 10 ml of eluate, collect the subsequent 35 ml in a round flask containing 40 mg of clean sunflower oil (5.39). Evaporate the solvent using a rotary evaporator and transfer the residue to a 15 ml centrifuge tube. Epoxidation proceeds as described above using smaller volumes of solvents and solutions: 1 ml of DCM, 150 µl of CPBA solution, 1 ml of sodium carbonate solution and 1 ml of water. Transfer the dichloromethane phase to an autosampler vial and bring to dryness by a stream of nitrogen (triglycerides from the sunflower oil act as a keeper). Redissolve the residue
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.