Milk and milk products - Determination of milk fat purity by gas chromatographic analysis of triglycerides (ISO 17678:2019)

This document specifies a reference method for the determination of milk fat purity using gas chromatographic analysis of triglycerides. The method utilizes the differences in triglyceride fingerprint of milk fat from the individual triglyceride fingerprints of other fats and oils to determine samples which are outside the range normally observed for milk fat. This is achieved by using the defined triglyceride formulae based on the normalized weighted sum of individual triglyceride peaks which are sensitive to the integrity of the milk[6][7]. The integrity of the milk fat can be determined by comparing the result of these formulae with those previously observed for a range of pure milk fat samples[12]. Both vegetable fats and animal fats such as beef tallow and lard can be detected.
The method is applicable to bulk milk, or products made thereof, irrespective of the variation in common feeding practices, breed or lactation conditions. In particular, the method is applicable to fat extracted from milk products purporting to contain pure milk fat with unchanged composition, such as butter, cream, milk and milk powder.
Because a false-positive result can occur, the method does not apply to milk fat related to these circumstances:
a) obtained from bovine milk other than cow's milk;
b) obtained from single cows;
c) obtained from cows whose diet contained a particularly high proportion of vegetable oils such as rapeseed, cotton or palm oil, etc.;
d) obtained from cows suffering from serious underfeeding (strong energy deficit);
e) obtained from colostrum;
f) subjected to technological treatment such as removal of cholesterol or fractionation;
g) obtained from skim milk, buttermilk or whey;
h) obtained from cheeses showing increased lipolysis;
i) extracted using the Gerber, Weibull?Berntrop or Schmid?Bondzynski?Ratzlaff methods, or that has been isolated using detergents (e.g. the Bureau of Dairy Industries method).
With the extraction methods specified in i), substantial quantities of partial glycerides or phospholipids can pass into the fat phase.
NOTE 1 In nature, butyric (n-butanoic) acid (C4) occurs exclusively in milk fat and enables quantitative estimations of low to moderate amounts of milk fat in vegetable and animal fats to be made. Due to the large variation of C4, for which the approximate content ranges from 3,1 % fat mass fraction to 3,8 % fat mass fraction, it is difficult to provide qualitative and quantitative information for foreign fat to pure milk fat ratios of up to 20 % mass fraction[11].
NOTE 2 In practice, quantitative results cannot be derived from the sterol content of vegetable fats, because they depend on production and processing conditions. Furthermore, the qualitative determination of foreign fat using sterols is ambiguous.
NOTE 3 Due to special feeding practices such as those related to c) and d), false-positive results have sometimes been reported for milk from certain Asian regions[15]. Moreover, grass-only diets such as mountain and, in particular, highland pasture feeding sometimes cause false-positive results, which can be substantiated by a content of conjugated linoleic acid (C18:2 c9t11) of ≥ 1,3 % fatty acid mass fraction[16][17]. Nevertheless, results conforming to the criteria of milk fat purity specified in this document are accepted, even if samples were undoubtedly produced under conditions reported in this note, including those described in h).
NOTE 4 In cases where a positive resu

Milch und Milcherzeugnisse - Bestimmung der Reinheit des Milchfetts durch gaschromatographische Triglyceridanalyse (Referenzverfahren) (ISO 17678:2019)

Dieses Dokument legt ein Referenzverfahren zur Bestimmung der Reinheit des Milchfetts durch gaschro¬mato¬graphische Triglyceridanalyse fest. Bei diesem Verfahren werden die Unterschiede zwischen dem Triglycerid Fingerprint von Milchfett und den individuellen Triglycerid Fingerprints anderer Fette und Öle zur Bestimmung von Proben analysiert, die außerhalb des bei Milchfett üblicherweise festgestellten Bereichs liegen. Dies wird anhand von festgelegten Triglycerid-gleichungen erreicht, die auf der normalisierten gewichteten Summe der einzelnen Triglycerid¬spitzen beruht, die auf die Integrität der Milch [6] [7] reagieren. Die Integrität des Milchfetts kann durch Vergleiche der Ergebnisse dieser Gleichungen mit den zuvor bei einer Reihe reiner Milchfettproben [12] beobachteten Ergebnissen bestimmt werden. Es können sowohl pflanzliche als auch tierische Fette wie Rindertalg und Schweinefett nachgewiesen werden.
Grundsätzlich gilt das Verfahren für Sammelmilch oder daraus hergestellte Produkte, unabhängig von Abweichungen bei den allgemeinen Fütterungs , Zucht  oder Laktationsbedingungen. Insbesondere ist das Verfahren auf Fett anwendbar, das aus Milchprodukten extrahiert wurde, die reines Milchfett in unver¬änderter Zusammensetzung enthalten sollen, wie z. B. Butter, Sahne, Milch und Milchpulver.
Jedoch kann in den nachstehend erwähnten Fällen ein falsch positives Ergebnis ermittelt werden. Deshalb ist das Verfahren nicht anwendbar auf Milchfett, das:
a)   aus anderer Milch als Kuhmilch gewonnen wurde;
b)   von einzelnen Kühen gewonnen wurde;
c)   von Kühen gewonnen wurde, deren Futter einen besonders hohen Pflanzenölanteil aufwies, wie Raps-, Baumwollsamen  oder Palmöl usw.;
d)   von Kühen gewonnen wurde, die an erheblicher Unterernährung leiden (starkes Energiedefizit);
e)   aus Kolostrum (Vormilch) gewonnen wurde;
f)   einer technologischen Behandlung unterzogen wurde, wie z. B. dem Entfernen von Cholesterol oder einer Fraktionierung;
g)   aus Magermilch, Buttermilch oder Molke gewonnen wurde;
h)   aus Käse mit gesteigerter Lipolyse gewonnen wurde;
i)   mit Verfahren nach Gerber, Weibull Berntrop oder Schmid Bondzynski Ratzlaff extrahiert oder mittels Detergenzien isoliert wurde [z. B. mit dem Bureau of Dairy Industries (BDI) Verfahren].
Mit den in Punkt i) festgelegten Extraktionsverfahren können beträchtliche Mengen von Partialglyceriden oder Phospholipiden in die Fettphase übertreten.

Lait et produits laitiers - Détermination de la pureté des matières grasses laitières par analyse chromatographique en phase gazeuse des triglycérides (ISO 17678:2019)

Le présent document spécifie une méthode de référence pour la détermination de la pureté des matières grasses laitières par analyse chromatographique en phase gazeuse des triglycérides. La méthode utilise les différences d'empreintes des triglycérides présents dans les matières grasses laitières par rapport aux empreintes des triglycérides individuels des autres matières grasses et huiles pour déterminer des échantillons se situant en dehors de la plage normalement observée pour les matières grasses laitières. Cela est réalisé en utilisant les formules de triglycérides définies basées sur la somme pondérée normalisée des pics de triglycérides individuels, qui sont sensibles à l'intégrité du lait[6][7]. L'intégrité des matières grasses laitières peut être déterminée en comparant les résultats de ces formules à ceux précédemment observés pour une gamme d'échantillons de matières grasses laitières pures[12]. Il est possible de détecter des matières grasses d'origines végétale et animale telles que la graisse de bœuf et le saindoux.
La méthode s'applique au lait en vrac, ou aux produits laitiers dérivés, indépendamment de la variation des pratiques d'alimentation courantes et des conditions d'élevage ou de lactation. Elle s'applique en particulier aux matières grasses extraites de produits laitiers supposés contenir des matières grasses laitières pures, présentant une composition non modifiée, comme le beurre, la crème, le lait et le lait en poudre.
Un résultat faux positif pouvant être obtenu, la méthode n'est pas applicable à la matière grasse laitière liée aux circonstances listées ci-après:
a) issue de lait de bovin autre que le lait de vache;
b) issue de vaches individuelles;
c) issue de vaches dont l'alimentation contenait une proportion particulièrement élevée d'huiles végétales telles que l'huile de colza, l'huile de coton ou de palme, etc.;
d) issue de vaches souffrant d'une grave sous-alimentation (puissant déficit énergétique);
e) issue du colostrum;
f) soumise à un traitement technologique tel que l'élimination du cholestérol ou le fractionnement;
g) issue du lait écrémé, du lait battu (babeurre) ou du lactosérum;
h) issue de fromages présentant une lipolyse importante;
i) extraite selon les méthodes Gerber, Weibull?Berntrop ou Schmid?Bondzynski?Ratzlaff, ou isolée au moyen de détergents [par exemple méthode BDI (Bureau of Dairy Industries)].
Avec les méthodes d'extraction spécifiées en i), d'importantes quantités de glycérides ou phospholipides partiels peuvent passer dans la phase grasse.
NOTE 1 Dans la nature, l'acide butyrique (n-butanoïque) (C4) se trouve exclusivement dans les matières grasses laitières et permet d'effectuer des estimations quantitatives de petites et moyennes quantités de matières grasses laitières dans les graisses végétales et animales. En raison de la grande variation de C4, pour lequel la fraction massique est comprise approximativement entre 3,1 % et 3,8 % de la matière grasse, des informations qualitatives et quantitatives peuvent difficilement être fournies lorsque le rapport des matières grasses étrangères aux matières grasses laitières pures atteint jusqu'à 20 % en fraction massique[11].
NOTE 2 Dans la pratique, des résultats quantitatifs ne peuvent pas être déduits de la teneur en stérols des matières grasses végétales, étant donné que celles-ci sont fonction des conditions de production et de traitement. En outre, la détermination qualitative de matières grasses étrangères au moye

Mleko in mlečni proizvodi - Določanje čistosti mlečne maščobe s plinsko kromatografsko analizo trigliceridov (referenčna metoda) (ISO 17678:2019)

Ta dokument določa referenčno metodo za določanje čistosti mlečne maščobe z uporabo analize trigliceridov s plinsko kromatografijo. Pri tej metodi se uporabljajo razlike med identifikacijo trigliceridov v mlečni maščobi in identifikacijo posameznih trigliceridov v drugih masteh in oljih za določanje vzorcev, ki so zunaj razpona takih, ki so običajno opazovani pri mlečni maščobi. To se doseže z uporabo določenih formul za trigliceride, ki temeljijo na normalizirani ponderirani vsoti posameznih najvišjih vrednosti trigliceridov, ki so občutljivi na integriteto mleka [6][7]. Integriteto mlečne maščobe je mogoče ugotoviti s primerjavo rezultata teh formul s predhodno opazovanimi za različne vzorce čiste mlečne maščobe [12]. Mogoče je odkriti tako rastlinske kot tudi živalske maščobe, kot sta goveji loj in mast. Ta metoda se uporablja za velike količine mleka ali iz njega narejene izdelke, ne glede na razlike pri običajnih praksah krmljenja, pasmah ali laktacijskih pogojih. Metoda se uporablja zlasti za maščobo, ekstrahirano iz mlečnih izdelkov, ki domnevno vsebujejo čisto mlečno maščobo z nespremenjeno sestavo, na primer maslo, smetana, mleko in mleko v prahu.
Zaradi možnosti lažnega pozitivnega rezultata se metoda ne uporablja za mlečno maščobo, kadar
je ta:
a) pridobljena iz drugega mleka kot kravjega;
b) pridobljena od posameznih krav;
c) pridobljena od krav, ki so imele v prehrani še posebej visok delež rastlinskih olj, na primer
repično, bombažno ali palmovo olje ipd.;
d) pridobljena od resno podhranjenih krav (velik primanjkljaj energije);
e) pridobljena iz mleziva;
f) tehnološko obdelana, na primer z odstranitvijo holesterola ali frakcioniranjem;
g) pridobljena iz posnetega mleka, pinjenca ali sirotke;
h) pridobljena iz sirov, ki izkazujejo povečano lipolizo;
i) ekstrahirana z metodami po Gerberju, Weibull–Berntropu ali Schmid–Bondzynski–Ratzlaffu ali izolirana z detergenti (npr. z metodo ameriškega urada za mlečno industrijo).
Pri metodi ekstrakcije, določeni pod točko i), lahko znatne količine delnih gliceridov ali fosfolipidov preidejo v maščobno fazo.
OPOMBA 1: V naravi se butirična (n-butanojska) kislina (C4) pojavlja izključno v mlečni maščobi in omogoča kvantitativno ocenjevanje nizkih do srednjih količin mlečne maščobe v rastlinskih in živalskih maščobah. Zaradi velikih razlik pri C4, pri čemer je približna vsebnost med 3,1 % masnega deleža maščobe in 3,8 % masnega deleža maščobe, je težko podati kvalitativne in kvantitativne informacije za razmerje med tujo maščobo in čisto mlečno maščobo z do 20 % masnega deleža [11].
OPOMBA 2: Kvantitativnih rezultatov v praksi ni mogoče pridobiti iz vsebnosti sterolov v rastlinskih maščobah, ker so odvisni od proizvodnje in pogojev predelave. Poleg tega je kvalitativno določevanje tuje maščobe z uporabo sterolov dvoumno.
OPOMBA 3: Zaradi posebnih praks krmljenja, podobnih tistim v zvezi s točkama c) in d),
včasih poročajo o lažnih pozitivnih rezultatih iz določenih regij v Aziji [15]. Poleg tega lahko krmljenje izključno s travo, na primer pri pašnji na planinskih pašnikih in zlasti na visokogorskih pašnikih, povzroči lažne pozitivne rezultate, ki jih je mogoče potrditi s konjugirano linolno kislino (C18:2 c9t11) z vsebnostjo ≥ 1,3 % masnega deleža maščobnih kislin [16][17]. Kljub temu so rezultati sprejemljivi, če so skladni s kriteriji čistosti mlečne maščobe, ki so določeni v tem dokumentu, čeprav so bili vzorci nedvomno odvzeti v pogojih, poročanih v teh opombi, vključno z opisanimi pod točko h).
OPOMBA 4: V primerih, ko pozitiven rezultat domnevno nastane zaradi okoliščin, povezanih s točkama c) in d), je za potrditev izsledkov dovoljeno uporabiti drugo analitično metodo, na primer analizo maščobnih kislin ali analizo sterolov. Zaradi podobnih ali povečanih omejitev (npr. opisanih v OPOMBI 1 in OPOMBI 2) z drugo metodo pridobljen negativni rezultat ni primeren za nasprotno potrditev čistosti mlečne maščobe.

General Information

Status
Published
Publication Date
25-Jun-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
26-Jun-2019
Completion Date
26-Jun-2019

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SLOVENSKI STANDARD
SIST EN ISO 17678:2019
01-september-2019
Nadomešča:
SIST EN ISO 17678:2010
Mleko in mlečni proizvodi - Določanje čistosti mlečne maščobe s plinsko
kromatografsko analizo trigliceridov (referenčna metoda) (ISO 17678:2019)

Milk and milk products - Determination of milk fat purity by gas chromatographic analysis

of triglycerides (ISO 17678:2019)
Milch und Milcherzeugnisse - Bestimmung der Reinheit des Milchfetts durch
gaschromatographische Triglyceridanalyse (Referenzverfahren) (ISO 17678:2019)

Lait et produits laitiers - Détermination de la pureté des matières grasses laitières par

analyse chromatographique en phase gazeuse des triglycérides (ISO 17678:2019)
Ta slovenski standard je istoveten z: EN ISO 17678:2019
ICS:
67.100.10 Mleko in predelani mlečni Milk and processed milk
proizvodi products
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
SIST EN ISO 17678:2019 en

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

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SIST EN ISO 17678:2019
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SIST EN ISO 17678:2019
EN ISO 17678
EUROPEAN STANDARD
NORME EUROPÉENNE
June 2019
EUROPÄISCHE NORM
ICS 67.100.10 Supersedes EN ISO 17678:2010
English Version
Milk and milk products - Determination of milk fat purity
by gas chromatographic analysis of triglycerides (ISO
17678:2019)

Lait et produits laitiers - Détermination de la pureté Milch und Milcherzeugnisse - Bestimmung der Reinheit

des matières grasses laitières par analyse des Milchfetts durch gaschromatographische

chromatographique en phase gazeuse des triglycérides Triglyceridanalyse (Referenzverfahren) (ISO

(ISO 17678:2019) 17678:2019)
This European Standard was approved by CEN on 6 March 2018.

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

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 17678:2019 E

worldwide for CEN national Members.
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SIST EN ISO 17678:2019
EN ISO 17678:2019 (E)
Contents Page

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

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SIST EN ISO 17678:2019
EN ISO 17678:2019 (E)
European foreword

This document (EN ISO 17678:2019) has been prepared by Technical Committee ISO/TC 34 "Food

products" in collaboration with Technical Committee CEN/TC 302 “Milk and milk products - 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 December 2019, and conflicting national standards

shall be withdrawn at the latest by December 2019.

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 supersedes EN ISO 17678:2010.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the

United Kingdom.
Endorsement notice

The text of ISO 17678:2019 has been approved by CEN as EN ISO 17678:2019 without any modification.

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SIST EN ISO 17678:2019
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SIST EN ISO 17678:2019
INTERNATIONAL ISO
STANDARD 17678
IDF 202
Second edition
2019-06
Milk and milk products —
Determination of milk fat purity
by gas chromatographic analysis of
triglycerides
Lait et produits laitiers — Détermination de la pureté des matières
grasses laitières par analyse chromatographique en phase gazeuse
des triglycérides
Reference numbers
ISO 17678:2019(E)
IDF 202:2019(E)
ISO and IDF 2019
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SIST EN ISO 17678:2019
ISO 17678:2019(E)
IDF 202:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO and IDF 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office International Dairy Federation
CP 401 • Ch. de Blandonnet 8 Silver Building • Bd Auguste Reyers 70/B
CH-1214 Vernier, Geneva B-1030 Brussels
Phone: +41 22 749 01 11 Phone: +32 2 325 67 40
Fax: +41 22 749 09 47 Fax: +32 2 325 67 41
Email: copyright@iso.org Email: info@fil-idf.org
Website: www.iso.org Website: www.fil-idf.org
Published in Switzerland
ii © ISO and IDF 2019 – All rights reserved
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SIST EN ISO 17678:2019
ISO 17678:2019(E)
IDF 202:2019(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

1 Scope ................................................................................................................................................................................................................................. 1

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

3 Terms and definitions ..................................................................................................................................................................................... 2

4 Principle ........................................................................................................................................................................................................................ 2

5 Reagents ........................................................................................................................................................................................................................ 3

6 Apparatus ..................................................................................................................................................................................................................... 3

7 Sampling ........................................................................................................................................................................................................................ 4

8 Procedure..................................................................................................................................................................................................................... 5

8.1 Preparation of test samples ......................................................................................................................................................... 5

8.1.1 General...................................................................................................................................................................................... 5

8.1.2 Isolation from butter or butteroil...................................................................................................................... 5

8.1.3 Extraction according to the Röse–Gottlieb gravimetric method ............................................ 5

8.1.4 Extraction from milk using silica gel columns ....................................................................................... 5

8.1.5 Extraction from cheese ............................................................................................................................................... 6

8.2 Preparation of fat sample solution ........................................................................................................................................ 6

8.3 Chromatographic triglyceride determination .............................................................................................................. 6

8.3.1 Baseline drift ....................................................................................................................................................................... 6

8.3.2 Injection technique ........................................................................................................................................................ 6

8.3.3 Calibration ............................................................................................................................................................................. 6

8.3.4 Chromatographic conditions ................................................................................................................................. 7

9 Integration, evaluation and control of the analytical performance ................................................................8

10 Calculation and expression of results ..........................................................................................................................................10

10.1 Triglyceride composition ............................................................................................................................................................10

10.1.1 Calculation ..........................................................................................................................................................................10

10.1.2 Expression of test results ......................................................................................................................................10

10.2 S-values .......................................................................................................................................................................................................11

10.2.1 Calculation ..........................................................................................................................................................................11

10.2.2 Expression of test results ......................................................................................................................................11

10.3 Detection of foreign fat..................................................................................................................................................................11

11 Precision ....................................................................................................................................................................................................................12

11.1 Interlaboratory test..........................................................................................................................................................................12

11.2 Repeatability ..........................................................................................................................................................................................12

11.3 Reproducibility ....................................................................................................................................................................................12

12 Test report ................................................................................................................................................................................................................13

Annex A (normative) Preparation of the packed column ............................................................................................................14

Annex B (informative) Quantification of the foreign fat content .........................................................................................18

Annex C (informative) Uncertainty of measurement .......................................................................................................................20

Annex D (informative) Interlaboratory test ...............................................................................................................................................21

Bibliography .............................................................................................................................................................................................................................23

© ISO and IDF 2019 – All rights reserved iii
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SIST EN ISO 17678:2019
ISO 17678:2019(E)
IDF 202:2019(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national

standards bodies (ISO member bodies). The work of preparing International Standards is normally

carried out through ISO technical committees. Each member body interested in a subject for which

a technical committee has been established has the right to be represented on that committee.

International organizations, governmental and non-governmental, in liaison with ISO, also take part

in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all

matters of electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso

.org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5,

Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO

and IDF.

This second edition cancels and replaces the first edition (ISO 17678 | IDF 202:2010), which has been

technically revised. The following changes have been made:

— the Scope has been restricted to exclude milk fat obtained from special feeding practices and

from whey;

— the Scope has been extended to include milk fat obtained from cheese showing low lipolysis;

— the Normative references have been updated to reflect the modified scope;
— a method has been added for the fat extraction from cheese;
— the Bibliography has been expanded.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO and IDF 2019 – All rights reserved
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SIST EN ISO 17678:2019
ISO 17678:2019(E)
IDF 202:2019(E)

IDF (the International Dairy Federation) is a non-profit private sector organization representing the

interests of various stakeholders in dairying at the global level. IDF members are organized in National

Committees, which are national associations composed of representatives of dairy-related national

interest groups including dairy farmers, dairy processing industry, dairy suppliers, academics and

governments/food control authorities.

ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis

and sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International

Standards using the logos and reference numbers of both organizations.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. IDF shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

This document was prepared by the IDF Standing Committee on Analytical Methods for Composition and

ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is

being published jointly by ISO and IDF.

The work was carried out by the Joint ISO/IDF Action Team C23 of the Standing Committee on Analytical

Methods for Composition under the aegis of its project leader, Mr J. Molkentin (DE).

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SIST EN ISO 17678:2019
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SIST EN ISO 17678:2019
ISO 17678:2019(E)
INTERNATIONAL STANDARD
IDF 202:2019(E)
Milk and milk products — Determination of milk fat purity
by gas chromatographic analysis of triglycerides
1 Scope

This document specifies a reference method for the determination of milk fat purity using gas

chromatographic analysis of triglycerides. The method utilizes the differences in triglyceride

fingerprint of milk fat from the individual triglyceride fingerprints of other fats and oils to determine

samples which are outside the range normally observed for milk fat. This is achieved by using the

defined triglyceride formulae based on the normalized weighted sum of individual triglyceride peaks

[6][7]

which are sensitive to the integrity of the milk . The integrity of the milk fat can be determined

by comparing the result of these formulae with those previously observed for a range of pure milk fat

[12]

samples . Both vegetable fats and animal fats such as beef tallow and lard can be detected.

The method is applicable to bulk milk, or products made thereof, irrespective of the variation in

common feeding practices, breed or lactation conditions. In particular, the method is applicable to fat

extracted from milk products purporting to contain pure milk fat with unchanged composition, such as

butter, cream, milk and milk powder.

Because a false-positive result can occur, the method does not apply to milk fat related to these

circumstances:
a) obtained from bovine milk other than cow’s milk;
b) obtained from single cows;

c) obtained from cows whose diet contained a particularly high proportion of vegetable oils such as

rapeseed, cotton or palm oil, etc.;

d) obtained from cows suffering from serious underfeeding (strong energy deficit);

e) obtained from colostrum;

f) subjected to technological treatment such as removal of cholesterol or fractionation;

g) obtained from skim milk, buttermilk or whey;
h) obtained from cheeses showing increased lipolysis;

i) extracted using the Gerber, Weibull–Berntrop or Schmid–Bondzynski–Ratzlaff methods, or that

has been isolated using detergents (e.g. the Bureau of Dairy Industries method).

With the extraction methods specified in i), substantial quantities of partial glycerides or phospholipids

can pass into the fat phase.

NOTE 1 In nature, butyric (n-butanoic) acid (C4) occurs exclusively in milk fat and enables quantitative

estimations of low to moderate amounts of milk fat in vegetable and animal fats to be made. Due to the large

variation of C4, for which the approximate content ranges from 3,1 % fat mass fraction to 3,8 % fat mass fraction,

it is difficult to provide qualitative and quantitative information for foreign fat to pure milk fat ratios of up to

[11]
20 % mass fraction .

NOTE 2 In practice, quantitative results cannot be derived from the sterol content of vegetable fats, because

they depend on production and processing conditions. Furthermore, the qualitative determination of foreign fat

using sterols is ambiguous.
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SIST EN ISO 17678:2019
ISO 17678:2019(E)
IDF 202:2019(E)

NOTE 3 Due to special feeding practices such as those related to c) and d), false-positive results have

[15]

sometimes been reported for milk from certain Asian regions . Moreover, grass-only diets such as mountain

and, in particular, highland pasture feeding sometimes cause false-positive results, which can be substantiated

[16][17]

by a content of conjugated linoleic acid (C18:2 c9t11) of ≥ 1,3 % fatty acid mass fraction . Nevertheless,

results conforming to the criteria of milk fat purity specified in this document are accepted, even if samples were

undoubtedly produced under conditions reported in this note, including those described in h).

NOTE 4 In cases where a positive result is suspected to be caused by circumstances related to c) or d), another

analytical method, such as fatty acid or sterol analysis, can be applied to confirm the finding. Due to similar or

increased limitations (e.g. as described in NOTE 1 and NOTE 2), a negative result obtained by another method is

not appropriate to contrastingly confirm milk fat purity.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 1211 | IDF 1, Milk — Determination of fat content — Gravimetric method (Reference method)

ISO 1740 | IDF 6, Milkfat products and butter — Determination of fat acidity (Reference method)

ISO 1736 | IDF 9, Dried milk and dried milk products — Determination of fat content — Gravimetric method

(Reference method)

ISO 2450 | IDF 16, Cream — Determination of fat content — Gravimetric method (Reference method)

ISO 3696, Water for analytical laboratory use — Specification and test methods

ISO 7328 | IDF 116, Milk-based edible ices and ice mixes — Determination of fat content — Gravimetric

method (Reference method)

ISO 14156 | IDF 172, Milk and milk products — Extraction methods for lipids and liposoluble compounds

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:

— IEC Electropedia: available at http: //www .electropedia .org/
— ISO Online browsing platform: available at https: //www .iso .org/obp
3.1
milk fat purity

absence of vegetable and animal fats determined by the procedure specified in this document

Note 1 to entry: The purity is determined using S-values, which are calculated from the content of triglycerides.

Triglyceride mass fractions are expressed as percentages.
4 Principle

Fat extracted from milk or milk products is analysed by gas chromatography (GC) using a packed

or a short capillary column to determine triglycerides (TGs), separated by total carbon numbers. By

inserting the mass fraction, expressed as a percentage, of fat molecules of different sizes (C24 to C54,

using even C numbers only) into suitable TG formulae, S-values are calculated. If the S-values exceed the

limits established with pure milk fat, the presence of foreign fat is detected.

NOTE 1 The suitability and equivalence of both packed and capillary columns have been demonstrated

[8][9][10]
previously .
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SIST EN ISO 17678:2019
ISO 17678:2019(E)
IDF 202:2019(E)
NOTE 2 An S-value is the sum of weighted TG mass fractions.
5 Reagents

During the analysis, unless otherwise stated, use only reagents of recognized analytical grade.

5.1 Water, conforming to the requirements of ISO 3696, grade 2.

5.2 Carrier gas, nitrogen or, alternatively, helium or hydrogen, all with a purity of at least 99,995 %

volume fraction.
5.3 Fat standards, as described in 5.3.1 and 5.3.2.

5.3.1 Triglyceride standards, saturated, with a purity of at least 99 % mass fraction, for standardizing

the milk fat standard described in 8.3.3 (suitable products are available commercially).

5.3.2 Cholesterol standard, with a purity of at least 99 % mass fraction, for standardizing the milk fat

standard described in 8.3.3.
5.4 Methanol, with a water content of not more than 0,05 % mass fraction.
5.5 n-Hexane.
5.6 n-Heptane.

5.7 Other gases, hydrogen, purity at least 99,995 % volume fraction, free from organic impurities

(C H < 1 µl/l); synthetic air, free from organic impurities (C H < 1 µl/l).
n m n m
5.8 Anhydrous sodium sulfate.
6 Apparatus
Usual laboratory equipment and, in particular, the following.

6.1 High-temperature gas chromatograph, suitable for use at temperatures of at least 400 °C and

equipped with a flame ionization detector (FID). For capillary GC, an on-column or a programmed

temperature vaporization injector is indispensable while a split injector is unsuitable.

Septa used in the injector shall withstand high temperatures and exhibit a very low degree of “bleeding”.

Always use graphite seals to connect the column as well as injector and/or detector inserts (where

applicable).

6.2 Packed chromatography column, glass, of internal diameter 2 mm and length 500 mm, packed

with a stationary phase of 3 % OV-1 on 125 µm to 150 µm (100 to 120 mesh) Gas ChromQ .

The preparation, silanization, packing and conditioning of the packed column are described in Annex A.

Alternatively, a capillary column (6.3) may be used.

1) Example of a suitable product available commercially. This information is given for the convenience of users of

this document and does not constitute an endorsement by ISO or by IDF of this product.

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SIST EN ISO 17678:2019
ISO 17678:2019(E)
IDF 202:2019(E)

6.3 Capillary chromatography column, short, e.g. of length 5 m, with a non-polar stationary phase

that can withstand temperatures up to 400 °C or more .

Condition the column by performing 20 analyses of a milk fat solution (see 8.2) within no more than

two days using the settings given in 8.3.4.3. After that, ensure that the response factors (see 8.3.3) are

close to 1 and not higher than 1,250 0.

Because of the variable overlap between C24 and cholesterol, a higher response factor may be accepted

for C24.

Columns with different dimensions and a different non-polar, highly temperature-resistant phase may

be used as long as their performance is consistent with this document. However, the column length is

restricted by the indispensable limitation in resolution as shown in Figure 1. See also 8.3.4.3.

6.4 Extrelut column , capacity 1 ml to 3 ml, filled with silica gel, for the extraction of milk fat in

accordance with 8.1.4 only.

6.5 Graphite seals, capable of withstanding temperatures of at least 400 °C; for the connection of the

GC column as well as for the injector and/or detector inserts.
6.6 Water bath, capable of being maintained at 50 °C ± 2 °C.
6.7 Oven, capable of operating at 50 °C ± 2 °C and 100 °C ± 2 °C.
6.8 Micropipette.
[2]
6.9 Graduated pipette, capacity 5 ml, in accordance with ISO 835 , class A.
6.10 Round-bottomed flask, capacity 50 ml.
6.11 Erlenmeyer flask
...

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