2008R0273 — EN — 01.07.2013 — 001.001

This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents

Amended by:









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COMMISSION REGULATION (EC) No 273/2008

of 5 March 2008

laying down detailed rules for the application of Council Regulation (EC) No 1255/1999 as regards methods for the analysis and quality evaluation of milk and milk products







THE COMMISSION OF THE EUROPEAN COMMUNITIES, Having regard to the Treaty establishing the European Community, Having regard to Council Regulation (EC) No 1255/1999 of 17 May 1999 on the common organisation of the market in milk and milk products ( 1 ), and in particular Articles 10 and 15 and Articles 26(3), 29(1) and 31(4) thereof, Whereas: (1) Commission Regulation (EC) No 213/2001 ( 2 ) lays down detailed rules for the application of Council Regulation (EC) No 1255/1999 as regards methods for the analysis and quality evaluation of milk and milk products. In the light of technical developments in the field of analytical methodology further substantial changes need to be made. In the interest of clarity and efficiency and given the number and technical nature of the amendments, Regulation (EC) No 213/2001 should be repealed and replaced by a new regulation. (2) The composition and quality requirements for milk and milk products laid down under the arrangements provided for in Regulation (EC) No 1255/1999 must be verified to ensure that they are strictly complied with. (3) The reference methods for such verifications are often methods published by international organisations such as the European Committee for Standardization (CEN), the International Dairy Federation (IDF), the International Organisation for Standardisation (ISO) and the Scientific Association dedicated to the analytical Excellence (AOAC International), which are regularly updated by those organisations. In some cases a Community reference method is laid down, while in other cases no reference method is specified in the Community rules. In order to ensure that reference methods are uniformly applied, a list of reference methods should be drawn up and provision should be made for the Commission to adapt the list where necessary. (4) The use of routine methods should not be ruled out. Minimal conditions for using them should therefore be specified. (5) Common procedures should also be established to ensure uniform practice in evaluating the results of analyses, in sensory evaluation of the products concerned and in re-examining results which have been disputed. (6) For some analyses, there are currently no internationally accepted reference methods which have been validated and thus no information is available on the between-laboratory-variation of analytical results. Community methods should therefore be laid down, which have been validated according to internationally established rules and should be applied as reference methods. (7) Commission Regulation (EC) No 1898/2005 ( 3 ) lays down detailed rules for implementing Council Regulation (EC) No 1255/1999 as regards measures for the disposal of cream, butter and concentrated butter on the Community market and provides for the tracing of cream, butter and concentrated butter in certain circumstances in order to ensure the correct end use of these products. Tracing is important for the proper functioning of the scheme. In order to ensure that operators participating in it receive equal treatment, common methods should be established for determining some of these tracers. (8) Under Article 9 of Regulation (EC) No 1255/1999, private storage aid may be granted for cheeses made from ewes’ milk. A special refund for the same products can be granted under Article 31 of that Regulation. Cheeses made from ewes’ milk, goats’ milk, buffalos’ milk and mixtures of ewes’, goats’ and buffalos’ milk may be imported into the Community under preferential arrangements from certain third countries. In view of the above, appropriate checks are needed to ensure that no cow’s milk has been incorporated in the products concerned. A Community reference method should therefore be established for detecting cow’s milk, without prejudice to the use of routine methods, provided they comply with certain criteria. (9) Under Commission Regulation (EEC) No 2921/90 of 10 October 1990 on aid for the production of casein and caseinates from skimmed milk ( 4 ), the absence of coliforms must be detected. The internationally accepted reference methods for detecting coliforms in milk and milk products is ISO 4831. A Community reference method for detecting coliforms has been established based on the abovementioned standard. (10) Council Regulation (EEC) No 2658/87 of 23 July 1987 on the tariff and statistical nomenclature and on the Common Customs Tariff ( 5 ) provides for different rates of customs duty for compound feedingstuffs falling within tariff heading No 2309, depending on their milk-product content. To ensure that the rules in question are uniformly applied, a generally recognised method for analysing lactose content should be laid down for compulsory use in all Member States. (11) Under Regulation (EC) No 1255/1999, butter and skimmed-milk powder intended for intervention or, in the case of skimmed-milk powder, for use as animal feed, must meet certain quality requirements. Reference methods should be laid down to verify that those requirements are met. (12) Some methods are introduced for the first time in this Regulation. A sufficient period from the time of entry into force of this Regulation should be provided in order to allow laboratories to correctly introduce and use these new methods. Whenever a reference method referred to in Annex I is revised and published by the Standards Developing Organisation, the laboratories should be allowed six months to update their analytical procedures to conform to the new standard. (13) The measures provided for in this Regulation are in accordance with the opinion of the Management Committee for Milk and Milk products, Whereas: HAS ADOPTED THIS REGULATION:







CHAPTER I

GENERAL PROVISIONS

Article 1

Subject matter and scope

1. This Regulation lays down certain reference methods for the chemical, physical and microbiological analysis and sensory evaluation of milk and milk products to be used under the arrangements provided for in the common organisation of the market in milk and milk products established by Regulation (EC) No 1255/1999 and the rules for applying those methods.

2. The list of the reference methods applicable to analyses as referred to in paragraph 1 is laid down in Annex I to this Regulation.

3. The Commission shall update the list in accordance with the procedure laid down in Article 42 of Regulation (EC) No 1255/1999.

Article 2

Routine methods

Routine methods may be used for analyses required by the Community rules provided that they are properly calibrated and regularly checked against the reference method. Results shall be compared taking into account the constant bias, the repeatability and the reproducibility.

In cases of dispute, the result obtained by the reference method shall be decisive.

Member States shall inform the Commission on the use of routine methods in the analysis referred to in Article 1.







CHAPTER II

METHODS OF ANALYSIS

Article 3

Evaluation of compliance of a consignment with a legal limit

Except for the analysis of tracers, Annex II to this Regulation shall apply in order to define compliance with legal compositional requirements.

Article 4

Sensory evaluation

1. For milk and milk products other than butter for public storage, the reference method to be used by the Member States for sensory evaluation shall be either IDF standard 99C:1997 or other comparable methods which they shall notify to the Commission.

The procedures described in Annex III shall be applied to check the performance of assessors and the reliability of results in sensory analyses.

2. For butter for public storage, the procedures described in Annex III shall be applied to check the performance of assessors and the reliability of results in sensory analyses.

The procedure laid down in Annex IV shall be applied as a reference method for sensory evaluation.

Article 5

Tracers

1. The method of analysis laid down in Annex V shall be used as the reference method for determining the content of enanthic acid triglyceride in butter, butter-oil and cream.

2. The method of analysis laid down in Annex VI shall be used as the reference method for determining vanillin in concentrated butter, butter and cream.

3. The method of analysis laid down in Annex VII shall be used as the reference method for determining the ethyl ester of beta-apo-8' carotenic acid content of concentrated butter and butter.

4. The analysis method laid down in Annex VIII shall be used as the reference method for determining the β-sitosterol or stigmasterol content of butter and concentrated butter.

5. Concentrated butter, butter and cream are considered to be traced in conformity with the relevant Community rules if the results obtained are in accordance with the specifications of points 10 and 11 of Annex V and point 8 of Annexes VI, VII and VIII.

Article 6

Detection of cows’ milk casein

1. The reference method of analysis laid down in Annex IX shall be used to ensure that cheese made exclusively from ewes’ milk, goats’ milk or buffalos’ milk or from a mixture of ewes’, goats’ and buffalos’ milk does not contain cows’ milk casein.

Cows’ milk casein is considered to be present if the cows’ milk casein content of the analysed sample is equal to or higher than the content of the reference sample containing 1 % cows’ milk as laid down in Annex IX.

2. Routine methods for detecting cows’ milk casein in cheeses referred to in paragraph 1 may be used provided that:

(a) the detection limit is maximum 0,5 %; and

(b) there are no false-positive results; and

(c) cows’ milk casein is detectable with the required sensitivity even after long ripening periods, as may occur in usual commercial conditions.

If any of the above mentioned requirements is not met, the reference methods laid down in Annex IX shall be used.

Article 7

Detection of coliforms

Coliforms in butter, skimmed-milk powder, casein and caseinates shall be detected in accordance with the reference method laid down in Annex X.

Article 8

Determination of the lactose content

The lactose content of products falling within CN code 2309 shall be determined in accordance with the reference method laid down in Annex XI.

Article 9

Detection of rennet whey

1. Rennet whey in skimmed-milk powder intended for public storage shall be detected in accordance with the reference method laid down in Annex XII.

2. Rennet whey in skimmed-milk powder and mixtures intended for use as animal feed shall be detected in accordance with the reference method laid down in Annex XII. In case of detection of rennet whey, Annex XIII should be implemented.

Article 10

Detection of buttermilk

Buttermilk in skimmed-milk powder shall be detected in accordance with the reference method laid down in Annex XIV.

Article 11

Detection of antimicrobiotic residues

Antimicrobial residues in skimmed-milk powder shall be detected in accordance with the reference method laid down in Annex XV.

Article 12

Determination of skimmed-milk powder content

Skimmed-milk powder content in compound feedingstuffs shall be determined in accordance with the reference method laid down in Annex XVI.

Article 13

Detection of starch

Starch in skimmed-milk powder, denatured milk powder and compound feedingstuffs shall be detected in accordance with the reference method laid down in Annex XVII.

Article 14

Determination of moisture content in dried cream

Moisture content in dried cream shall be determined in accordance with the reference method laid down in Annex XVIII.

Article 15

Determination of moisture content of acid buttermilk powder

Moisture content in acid buttermilk powder intended for use in feedingstuffs shall be determined in accordance with the reference method laid down in Annex XIX.

Article 16

Determination of milk fat purity

Milk fat purity shall be determined in accordance with the reference method laid down in Annex XX.







CHAPTER III

GENERAL AND FINAL PROVISIONS

Article 17

Quality assurance

Analyses shall be performed in laboratories having an analytical quality assurance system including internal quality control procedures. Not accredited laboratories shall participate in proficiency testing schemes at least once per year and their results shall not deviate by more than 2σR (reproducibility standard deviation of the reference method) from the consensus value. A detailed description of the systems used shall be available for consultation in the laboratory.

Laboratories that are accredited in accordance with the standards referred to in Article 12 of Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules ( 6 ) shall be exempted from the obligation to participate in proficiency testing.

Article 18

Sampling and disputes over the results of analysis

1. Sampling shall be carried out in accordance with the relevant regulation for the product under consideration. If no sampling provisions are presented, then the provision given in ISO 707 IDF 50, Milk and milk products — Guidance of sampling, shall be used.

2. Laboratory reports of the results of the analysis must contain sufficient information for an evaluation of the results to be made in accordance with Annex II and Annex XXI.

3. Duplicate samples must be taken for analyses required under Community rules.

4. The procedure described in Annex XXI shall be used in cases where the results of an analysis are not accepted by the operator.

5. If the manufacturer can prove, within five working days of sampling, that the sampling procedure was not carried out correctly, sampling must be repeated where possible. If sampling cannot be repeated, the consignment must be accepted.

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Article 19a

Notifications

The notifications provided for in Article 2, Article 4(1) and in Annex III C shall be made in accordance with Commission Regulation (EC) No 792/2009 ( 7 ).

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Article 20

Repeals

Regulation (EC) No 213/2001 is repealed.

References to the repealed Regulation shall be construed as references to this Regulation and shall be read in accordance with the correlation table in Annex XXII.

Article 21

Entry into force

This Regulation shall enter into force on the third day following that of its publication in the Official Journal of the European Union.

It shall apply from 31 March 2008.

This Regulation shall be binding in its entirety and directly applicable in all Member States.









ANNEX I

(Article 1)

LIST OF REFERENCE METHODS

Index Min. = minimum, Max. = maximum, Annex = Annex to quoted Regulation, SNF = solids non fat, PV = peroxide value, A = appearance, F = flavour, C = consistency, TBC = total bacterial count, Therm = thermophilic bacterial count, MS = Member State, IDF = International Dairy Federation, ISO = International Standards Organisation, IUPAC = International Union of Pure and Applied Chemistry, ADPI = American Dairy Products Institute, SCM = sweetened condensed milk, EMC = evaporated milk or cream.







PART A Commission Regulation Product Parameter Limit (1) Reference method Remark Regulation (EC) No 2771/1999 — Public storage Unsalted butter Fat Min. 82 % m/m ISO 17189:2003IDF 194:2003 Water Up to 16 % m/m ISO 3727-1:2001IDF 80-1:2001 SNF Up to 2 % m/m ISO 3727-2:2001IDF 80-2:2001 Fat acidity 1,2 mmole/100 g of fat ISO 1740:2004IDF 6:2004 PV (max.) 0,3 meq. oxygen/1 000 g fat ISO 3976:2006IDF 74:2006 Note 1 Coliforms Not detectable in 1 g Annex X Note 3 Non-milk fat Not detectable by triglyceride analysis Annex XX Sterol tracers Not detectable, β-sitosterol ≤ 40 mg/kg Annex VIII Other tracers — vanillin Not detectable Annex VI — ethyl ester of carotenic acid ≤ 6 mg/kg Annex VII — triglycerides of enanthic acid Not detectable Annex V Sensory characteristics At least 4 out of 5 points for A, F and C Annex IV Water dispersion At least 4 points ISO 7586:1985 — IDF 112A:1989 Regulation (EC) No 2771/1999 — Private storage Unsalted butter Fat Min. 82 % m/m ISO 17189:2003IDF 194:2003 Water Up to 16 % m/m ISO 3727-1:2001IDF 80-1:2001 SNF Up to 2 % m/m ISO 3727-2:2001IDF 80-2:2001 Regulation (EC) No 2771/1999 — Private storage Salted butter Fat Min. 80 % m/m ISO 17189:2003IDF 194:2003 Water Up to 16 % m/m ISO 3727-1:2001IDF 80-1:2001 SNF (excluding salt) Up to 2 % m/m ISO 3727-2:2001IDF 80-2:2001 Salt Up to 2 % m/m ISO 15648:2004IDF 179:2004 Regulation (EC) No 1898/2005 chapter II Unsalted butter Fat Min. 82 % m/m ISO 17189:2003IDF 194:2003 Non-milk fat Annex XX Water Up to 16 % m/m ISO 3727-1 2001IDF 80-1:2001 SNF Up to 2 % m/m ISO 3727-2:2001IDF 80-2:2001 Tracers: — sterols See Annex VIII Annex VIII — vanillin See Annex VI Annex VI — ethyl ester of carotenic acid See Annex VII Annex VII — triglycerides of enanthic acid See Annex V Annex V Regulation (EC) No 1898/2005 chapter II Salted butter Fat Min. 80 % m/m ISO 17189:2003IDF 194:2003 Non-milk fat Annex XX Water Up to 16 % m/m ISO 3727-1:2001IDF 80-1:2001 SNF (excluding salt) Up to 2 % m/m ISO 3727-2:2001IDF 80-2:2001 Salt Up to 2 % m/m ISO 15648:2004IDF 179:2004 Tracers: — sterols See Annex VIII Annex VIII — vanillin See Annex VI Annex VI — ethyl ester of carotenic acid See Annex VII Annex VII — triglycerides of enanthic acid See Annex V Annex V Regulation (EC) No 1898/2005 chapter II Concentrated butter Fat Min. 99,8 % m/m IDF 24:1964 Water and SNF Up to 0,2 % m/m ISO 5536:2002IDF 23:2002 (moisture) IDF 24:1964 (SNF) Fat acidity 1,2 mmole/100 g of fat ISO 1740:2004IDF 6:2004 PV (max.) 0,5 meq. oxygen/1 000 g fat ISO 3976:2006IDF 74:2006 Note 1 Non-milk fat Absence Annex XX Flavour Clean Smell Absence of foreign odours Other Absence of neutralising agents, anti-oxidants and preservatives Tracers: — sterols See Annex VIII Annex VIII — vanillin See Annex VI Annex VI — ethyl ester of carotenic acid See Annex VII Annex VII — triglycerides of enanthic acid See Annex V Annex V Regulation (EC) No 1898/2005 chapter II Cream Fat Minimum 35 % m/m ISO 2450:1999IDF 16 C:1987 Non-milk fat Annex XX Tracers: — sterols See Annex VIII Note 2 — vanillin See Annex VI Annex VI — ethyl ester of carotenic acid See Annex VII Note 2 — triglycerides of enanthic acid See Annex V Annex V Regulation (EC) No 1898/2005 chapter III Concentrated butter Fat Min. 96 % m/m Note 2 Non-milk fat Annex XX SNF Up to 2 % m/m Note 2 Tracers: — stigmasterol (95 % m/m) 15 g/100 kg of concentrated butter Annex VIII — stigmasterol (85 % m/m) 17 g/100 kg of concentrated butter Annex VIII — triglycerides of enanthic acid 10,34 kg/t of concentrated butter Annex V — ethyl ester of butyric acid and stigmasterol — ethyl ester of butyric acid — stigmasterol: Annex VIII Note 2 — ethyl ester of butyric acid and triglycerides of enanthic acid — ethyl ester of butyric acid — triglycerides of enanthic acid: Annex V Note 2 lecithin (E 322) Up to 0,5 % m/m Note 2 NaC1 Up to 0,75 % m/m ISO 15648:2004IDF 179:2004 Fat acidity 1,2 mmole/100 g of fat ISO 1740:2004IDF 6:2004 PV (max.) Up to 0,5 meq. oxygen/1 000 g fat ISO 3976:2006IDF 74:2006 Note 1 Flavour Clean Smell Absence of foreign odours Other Absence of neutralising agents, anti-oxidants and preservatives Regulation (EC) No 1898/2005 chapter IV Unsalted butter Fat Min. 82 % m/m ISO 17189:2003IDF 194:2003 Water Up to 16 % m/m ISO 3727-1:2001IDF 80-1:2001 SNF Up to 2 % m/m ISO 3727-2:2001IDF 80-2:2001 Regulation (EC) No 1898/2005 chapter IV Salted butter Fat Min. 80 % m/m ISO 17189:2003IDF 194:2003 Water Up to 16 % m/m ISO 3727-1:2001IDF 80-1:2001 SNF (excluding salt) Up to 2 % m/m ISO 3727-2:2001IDF 80-2:2001 Salt Up to 2 % m/m ISO 15648:2004IDF 179:2004 Article 9 and Title II of Regulation (EC) No 1255/1999 Cheese made from ewes’ and/or goats’ milk Cows’ milk < 1 % m/m Annex IX Regulation (EEC) No 2921/90 Annex I — Acid casein Water Up to 12,00 % m/m ISO 5550:2006IDF 78:2006 Fat Up to 1,75 % m/m ISO 5543:2004IDF127:2004 Free acidity Up to 0,30 ml of 0,1 N NaOH solution/g ISO 5547:1978IDF 91:1979 Regulation (EEC) No 2921/90 Annex I — Rennet- casein Water Up to 12,00 % m/m ISO 5550:2006IDF 78:2006 Fat Up to 1,00 % m/m ISO 5543:2004IDF 127:2004 Ash Min. 7,50 % m/m ISO 5545:1978IDF 90:1979 Regulation (EEC) No 2921/90 Annex I — Caseinates Water Up to 6,00 % m/m ISO 5550:2006IDF 78:2006 Milk protein Min. 88,00 % m/m ISO 5549:1978IDF 92:1979 Fat and ash Up to 6,00 % m/m ISO 5543:2004IDF 127:2004 Fixed ash ISO 5544:1978IDF 89:1979 Ash ISO 5545:1978IDF 90:1979 Regulation (EEC) No 2921/90 Annex II — Acid casein Water Up to 10,00 % m/m ISO 5550:2006IDF 78:2006 Fat Up to 1,50 % m/m ISO 5543:2004IDF 127:2004 Free acidity Up to 0,20 ml of 0,1 N NaOH solution/g ISO 5547:1978IDF 91:1979 TBC (max.) 30 000/ g ISO 4833:2003 Note 3 Coliforms Absence in 0,1 g Annex X Note 3 Therm. (max.) 5 000/ g ISO 4833:2003 Notes 3 and 4 Regulation (EEC) No 2921/90 Annex II — Rennet-casein Water Up to 8,00 % m/m ISO 5550:2006IDF 78:2006 Fat Up to 1,00 % m/m ISO 5543:2004IDF 127:2004 Ash Min. 7,50 % m/m ISO 5545:1978IDF 90:1979 TBC (max.) 30 000/ g ISO 4833:2003 Note 3 Coliforms Absence in 0,1 g Annex X Note 3 Therm. (max.) 5 000/ g ISO 4833:2003 Notes 3 and 4 Regulation (EEC) No 2921/90 Annex II — Caseinates Water Up to 6,00 % m/m ISO 5550:2006IDF 78:2006 Milk protein Min. 88,00 % m/m ISO 5549:1978IDF 92:1979 Fat and ash Up to 6,00 % m/m ISO 5543:2004IDF 127:2004 ISO 5544:1978IDF 89:1979 or ISO 5545:1978IDF 90:1979 TBC (max.) 30 000/ g ISO 4833:2003 Note 3 Coliforms Absence in 0,1 g Annex X Note 3 Therm. (max.) 5 000/ g ISO 4833:2003 Notes 3 and 4 Regulation (EEC) No 2921/90 Annex III — Caseinates Water Up to 6,00 % m/m ISO 5550:2006IDF 78:2006 Milk protein Min. 85,00 % m/m ISO 5549:1978IDF 92:1979 Fat Up to 1,50 % m/m ISO 5543:2004IDF 127:2004 Lactose Up to 1,00 % m/m ISO 5548:2004IDF 106:2004 Ash Up to 6,50 % m/m ISO 5544:1978IDF 89:1979 or ISO 5545:1978IDF 90:1979 TBC (max.) 30 000/ g ISO 4833:2003 Note 3 Coliforms Absence in 0,1 g Annex X Note 3 Therm. (max.) 5 000/ g ISO 4833:2003 Notes 3 and 4 Regulation (EC) No 2799/1999 Compound feedingstuffs and skimmed-milk powder (SMP) (for use in feedingstuffs) Water (acid buttermilk powder) Up to 5 % m/m Annex XIX Protein 31,4 % m/m (min.) of the non-fat dry matter ISO 8968-123:2001IDF 20-123:2001 Water (SMP) Up to 5 % m/m ISO 5537:2004IDF 26:2004 Fats (SMP) Up to 11 % m/m ISO 1736:2000IDF 9C:1987 Rennet whey (SMP) Absence Annex XIII Note 6 Starch (SMP) Absence Annex XVII Water (mixtures) Up to 5 % m/m of the non-fat matter ISO 5537:2004IDF 26:2004 Fat (mixtures) Commission Directive 84/4/EEC (OJ L 15, 18.1.1984, p. 29) Rennet whey (mixtures) Absence Annex XIII SMP content (of end product) Min. 50 % m/m Annex XVI Fat (in end product) Min. 2,5 % m/m or 5 % m/m Commission Directive 84/4/EEC (OJ L 15, 18.1.1984, p. 29) Note 7 Starch (in end product) Min. 2 % m/m Annex XVII Note 8 Copper (in end product) 25 ppm Commission Directive 78/633/EEC (OJ L 206, 26.7.1987, p. 43) Regulation (EC) No 214/2001 SMP (spray) Fat Up to 1,0 % m/m ISO 1736:2000IDF 9C:1987 Protein 31,4 % (2) m/m (min.) of the non-fat dry matter ISO 8968-1/2:2001IDF 20-1/2:2001 Water Up to 3,5 % m/m ISO 5537:2004IDF 26:2004 Acidity Up to 19,5 ml, 0,1 N NaOH, 10 g solids non-fat ISO 6091:1980IDF 86:1981 Lactates Up to 150 mg/100 g solids non-fat ISO 8069:2005IDF 69:2005 Phosphatase Negative ISO 11816-1:2006IDF 155-1:2006 Insolubility index Up to 0,5 ml at 24 °C ISO 8156:2005IDF 129:2005 Scorched particles Disc A or B (15,0 mg) ADPI (1990) TBC 40 000/ g ISO 4833:2003 Note 3 Coliforms Negative/0,1 g Annex X Note 3 Buttermilk Negative Annex XIV Rennet whey Negative Annex XII Acid whey Negative Note 2 Anti-microbial agents Annex XV (1) Without prejudice to the requirements in the specific Regulation (2) The minimum protein content would be 34 % per 1 September 2009.







PART B The reference methods listed in Part B may be used for analysing products covered by any of the Regulations listed in column 1. Commission Regulation Product CN code Parameter Limit Reference method Remark Regulation (EEC) No 2658/87 Regulation (EC) No 2535/2001 Regulation (EC) No 1282/2006 Milk and cream, not concentrated nor containing added sugar or other sweetening matter 0401 Fat (≤ 6 % m/m) The limits are those specified in the description of the CN code for the specific product, further specified, where applicable, in Commission Regulation (EEC) No 3846/87 (OJ L 366, 24.12.1987, p. 1), Part 9 of the export nomenclature or Regulation (EC) No 2535/2001 (OJ L 341, 22.12.2001, p. 29) ISO 1211:2001IDF 1D:1996 Fat (> 6 % m/m) ISO 2450:1999IDF 16C:1987 Milk and cream, concentrated or containing added sugar or other sweetening matter 0402 Fat (liquid form) ISO 1737:1999IDF 13C:1987 Fat (solid form) ISO 1736:2000IDF 9C:1987 Protein ISO 8968-123:2001IDF 20-123:2001 Sucrose (normal content) ISO 2911:2004IDF 35:2004 Sucrose (low content) Note 2 Solids (SCM) ISO 6734:1989IDF 15B:1991 Solids (EMC) ISO 6731:1989IDF 21B:1987 Water (milk powder) ISO 5537:2004IDF 26:2004 Water (cream powder) Annex XVIII Buttermilk, fermented or acidified milk and cream, concentrated or not concentrated, containing added sugar or other sweetening matter 0403 Fat ISO 1211:2001IDF 1D:1996 ISO 1736:2000IDF 9C:1987 ISO 2450:1999IDF 16 C:1987 ISO 7208:1999IDF 22B:1987 ISO 8262-3:2005IDF 124-3:2005 Protein ISO 8968-123:2001IDF 20-123:2001 Sucrose (normal content) ISO 2911:2004IDF 35:2004 Sucrose (low content) Note 2 Water (acid buttermilk powder) Annex XIX Water (sweet buttermilk powder) ISO 5537:2004IDF26:2004 Solids (other products) Methods approved by the competent authority Whey, whether or not concentrated or containing added sugar or other sweetening matter; products consisting of natural milk constituents 0404 Fat ISO 1736:2000IDF 9C:1987 ISO 2450:1999IDF 16C:1987 ISO 7208:1999IDF 22B:1987 Protein ISO 8968-123:2001IDF 20-123:2001 Sucrose (normal content) ISO 2911:2004IDF 35:2004 Sucrose (low content) Note 2 0404 90 Protein ISO 8968 1/2 2001IDF 20-1/2:2001 Water IDF 21B:1987 Solids ISO 6734:1989IDF 15B:1991 (Concentrated products) ISO 6731:1989IDF 21B:1987 Butter and other fats derived from milk; dairy spreads 0405 Fat (if ≤ 85 % m/m) ISO 17189:2003IDF 194:2003 Butter Water ISO 3727-1:2001IDF 80-1:2001 SNF ISO 3727-2:2001IDF 80-2:2001 NaCl ISO 15648:2004IDF 179:2004 Fat (if > 99 % m/m) IDF 24:1964 Butteroil Water (if fat < 99 % m/m) ISO 5536:2002IDF 23:2002 Cheese and curd 0406 Fat ISO 1735:2004IDF 5:2004 Solids ISO 5534:2004IDF 4:2004 Solids (Ricotta) ISO 2920:2004IDF 58:2004 NaCl ISO 5943:2006IDF 88:2006 Lactose ISO 5765-1/2:2002IDF 79-1/2:2002 Regulation (EEC) No 2658/87 Compound feedingstuffs 2309 Lactose Annex XI

Notes to list of European Union reference methods:

Note 1: Milk fat isolation as described in ISO 1740:1991 (protection from light).

Note 2: No reference method has been established. Methods approved by the competent authority.

Note 3: Sample to be prepared according to ISO 8261:2001IDF 122:2001.

Note 4: Incubation for 48 hours at a temperature of 55 °C, care should be taken to prevent the culture medium from drying out.

Note 5: % m/m SNF = % m/m solids — % m/m fat.

Note 6: Commission Directive 84/4/EEC.

Note 7: Commission Regulation (EC) No 2799/1999 (OJ L 340, 31.12.1999, p. 3-27).

Note 8: Commission Directive 78/633/EEC.









ANNEX II

(Article 3)

EVALUATION OF COMPLIANCE OF A CONSIGNMENT WITH THE LEGAL LIMIT

1. PRINCIPLE

In cases where detailed sampling procedures are given by the relevant legislation, these procedures are followed. In all other cases a sample of at least 3 sample units taken randomly from the consignment submitted to control is used. A composite sample may be prepared. The result obtained is compared with the legal limits by calculation of a 95 % confidence interval as 2 x standard deviation, where the relevant standard deviation depends on whether (1) the method is validated through international collaboration with values for σr and σR or (2) in the case of in-house validation, an internal reproducibility has been calculated. This confidence interval will then equate to the measurement uncertainty of the result.

2. THE METHOD IS VALIDATED THROUGH INTERNATIONAL COLLABORATION

In this case the repeatability standard deviation σr and the reproducibility standard deviation σR have been established and the laboratory can demonstrate compliance with the performance characteristics of the validated method.

Calculate the arithmetic mean

n

Calculate the expanded uncertainty (k = 2) of

If the final result x of measurement is calculated using a formula of the form x = y 1 + y 2, x = y 1 – y 2, x = y 1 · y 2 or x = y 1 / y 2 the usual procedures for combining standard deviations in such cases must be followed.

The consignment is judged to be not in compliance with the upper legal limit UL if

;

otherwise it is judged to be in compliance with UL.

The consignment is judged to be not in compliance with the lower legal limit LL if

;

otherwise it is judged to be in compliance with LL.

3. IN-HOUSE VALIDATION WITH CALCULATION OF INTERNAL REPRODUCIBILITY STANDARD DEVIATION

In cases where methods not specified in this Regulation are used and precision measures have not been established an in-house validation must be carried out. Internal repeatability standard deviation sir and the internal reproducibility standard deviation siR have to be used instead of σr and σ R , resp., in the formulae for the computation of the expanded uncertainty U.

The decision rules are as under (1). However, if the consignment is judged to be not in compliance with the legal limit the measurements must be repeated with the method specified in this Regulation and the decision reached according to (1).









ANNEX III

(Article 4)

EVALUATION OF ASSESSORS AND THE RELIABILITY OF RESULTS IN SENSORY ANALYSES

The following procedures are applicable if scoring methods are used (IDF Standard 99C:1997).

A. DETERMINATION OF THE ‘REPEATABILITY INDEX’

At least ten samples will be analysed as blind duplicates by an assessor within a period of 12 months. This will usually happen in several sessions. The results for individual product characteristics are evaluated using the following formula:

where:

w1 : repeatability index

xi1 : score for the first evaluation of sample xi

xi2 : score for the second evaluation of sample xi

n : number of samples

The samples to be evaluated should reflect a broad quality range. wI should not exceed 1,5 (5-point scales).

B. DETERMINING THE ‘DEVIATION INDEX’

This index should be used to check whether an assessor uses the same scale for quality evaluation as an experienced group of assessors. The scores obtained by the assessor are compared with the average of the scores obtained by the assessor group.

The following formula is used for the evaluation of results:

where:

xi1; xi2 : see section (A)

; : average score of the assessor group for the first and second evaluation respectively of sample xi

n : number of samples (at least 10 per 12 months).

The samples to be evaluated should reflect a broad quality range. DI should not exceed 1,5 (5-point scales).

Member States must notify any difficulties encountered when applying this procedure.

Where individual assessors are found to exceed the 1,5 limit for Deviation or Repeatability indices, the Official authority expert/s must perform one or more random ‘Re-performance’ checks on samples graded by them over the next few weeks, or perform one or more ‘Accompanied’ checks with those assessors. Close monitoring is necessary to decide whether to retain their services. Findings should be documented and retained as proof of follow up action.

C. COMPARISON OF THE RESULTS OBTAINED IN DIFFERENT REGIONS OF A MEMBER STATE AND IN DIFFERENT MEMBER STATES

Where applicable, a test must be organised at least once per year to compare the results obtained by assessors from different regions. If significant differences are observed, the necessary steps should be taken to identify the reasons and arrive at comparable results.

Member States may organise tests to compare the results obtained by their own assessors and by assessors from neighbouring Member States. Significant differences should lead to an in-depth investigation with the aim of arriving at comparable results.

Member States should notify the Commission of the results of these comparisons.









ANNEX IV

(Article 4)

SENSORY EVALUATION OF BUTTER

1. SCOPE

The purpose of this procedure for sensory evaluation of butter is to provide a uniform method applicable in all Member States.

Refer to the current IDF International Standard for Milk and Milk Products, IDF 99 — Parts 1, 2, 3 on Sensory Evaluation, for further detail.

2. DEFINITIONS

‘Sensory evaluation’ (assessment) means the examination of the attributes of a product by the sense organs.

‘Panel’ means a group of selected assessors working, during the assessment, without intercommunication, and without influencing one another.

‘Assessor’ is defined as someone chosen for his/her ability to perform a sensory test. This type of assessor may have limited experience.

‘Expert Assessor’ is defined as someone with a high degree of sensory sensitivity and experience of sensory methodology, who is able to make consistent and reliable sensory assessments of various products. This type of assessor will have a good long term sensory memory.

‘Scoring’ means sensory evaluation by a panel, using a numerical scale. A nomenclature of defects must be used.

‘Grading’ means a quality classification which is performed on the basis of scoring.

‘Control documents’: documents used to record the individual scores for each attribute and the final grade of the product. (This document may also be used to record chemical composition.)

3. TEST ROOM

Refer to ISO 8589 and ISO/DIS 22935-2 IDF 99-2 par 7 for more details.

Precautions must be taken in order that the assessors in the test room are not influenced by external factors.

The test room must be free from foreign odours and easy to clean. The walls must be of a light colour and non reflective.

The test room and its lighting must be such that the properties of the products to be scored are not affected.

The room must be equipped with appropriate thermostatic control to enable a constant temperature of butter to be maintained. Butter should have a temperature of 12 °C (±2 °C) at the time of grading.

4. SELECTION OF ASSESSORS

An assessor must be familiar with butter products and be competent to carry out sensory grading. His/her competence should be monitored on a regular basis (at least once a year) by the competent authority.

4.1. ISO/DIS 22935-1 IDF 99-1 par 4 (recruitment) and par 5.1 should be consulted for details on general requirements and screening tests which may be used prior to official use of a new assessor. It is essential that training is ongoing and general sessions should be held on a regular basis. Refer to ISO 8586-1 for information on panel training.

4.2. Initial training should cover the following: — general theory and practical importance of sensory evaluation, — methods, scales and description of sensory impressions, — detection and recognition of sensory attributes and specific sensory terms, — background training on the manufacture of butter, — validated references and samples to help the assessor to identify specific flavours and flavour intensity within the product.

5. REQUIREMENTS FOR THE PANEL

The number of assessors in the panel should be uneven, the minimum number being three. The majority must be employees of the competent authority or authorised persons not employed by the dairy industry.

A panel Leader shall be responsible for the entire procedure and may participate in the panel.

A number of factors must be taken into account before evaluation in order to obtain optimal performances from the subjects:

— subjects must not be suffering from an illness which could affect their performance. In such a case, the assessor concerned should be replaced on the panel by another,

— subjects must be on time to take part in the evaluation and make sure that they have enough time to make their evaluation,

— subjects must not use strong-smelling substances like perfume, after-shave lotion, deodorant, etc. and should avoid eating strong-flavoured (e.g. highly spiced) food, etc.,

— subjects may not smoke, eat or drink anything other than water during the half hour before the evaluation.

6. PERFORMANCE

All Assessors should participate in regular sensory evaluation panels to maintain their competence. The frequency will depend on the volume and throughput of butter, and where possible, should be at least one panel per month.

Senior Assessors should also participate in a number of panels each year, and where possible, at least once per quarter.

7. SAMPLING AND PREPARATION OF THE SAMPLE

It is essential that the identity of the samples is concealed during the assessment so that any possible bias is avoided. The samples should be coded.

This should be organized prior to the evaluation. Requirement for temperature of butter during its transportation to the test room should be set (6 °C ± 2 °C).

When the sensory evaluation is carried out at a cold store, the sample is taken using a butter trier. If the sensory evaluation is carried out at another location other than the cold store, then at least a 500 g sample should be taken. During the evaluation, the butter should have the temperature of 12 °C (±2 °C) (refer: in ISO/DIS 22935-2 IDF 99-2 the evaluation temperature of butter is 14 °C ± 2 °C). Large deviations should be avoided at all cost.

8. ASSESSMENT OF THE VALUE OF EACH ATTRIBUTE

8.1. The sensory evaluation is to be carried out in relation to the following three attributes: appearance, consistency and flavour: ‘Appearance’ involves the following features: colour, visible purity, absence of physical contamination, absence of mould growth and uniformity of water dispersion. Water dispersion is tested according to IDF-Standard 112A/1989. ‘Consistency’ involves the following features: Body, texture and firmness. Spreadability may be monitored using physical means should an Individual Member State so wish in order to satisfy customer requirements. The Commission may decide to harmonise methodology in the future. ‘Body’ is the term which refers to the cohesiveness of the product as it is being consumed. It is normally associated with firmness and spreadability, and should be uniform throughout the product. It is closely related to texture and is the ability of the product to stand up under its own weight. It is indicated by resistance during cutting and can be measured mechanically and by mouthfeel and fingerfeel. ‘Flavour’ is the characteristic as perceived in the mouth, predominantly by the taste buds of the tongue. ‘Aroma’ is the characteristic as perceived by the nose and sense of smell. A significant deviation from the recommended temperature prevents a reliable evaluation of consistency and flavour. The temperature is of paramount importance. Grading of butter must be deferred if the temperature is outside the recommended band.

8.2. Each attribute has to be sensory evaluated separately. The scoring has to be done according to table 1.

8.3. It may be desirable for the assessors to score together, before starting the assessment, one or more reference samples for appearance, consistency and flavour, in order to achieve uniformity.

8.4. Scoring for acceptance is as follows: Refer to part 7 — Nomenclature, and description of criteria applicable to points, when scoring.



Maximum Required Appearance 5 4 Consistency 5 4 Flavour/aroma 5 4 — Where the required score is not obtained, a description of the defect has to be given. — The score given by each assessor for each attribute must be recorded in the control document. — The product is accepted or rejected on the basis of a majority decision. — Cases where differences between the individual scoring for each attribute are wider than adjacent points should not occur frequently (not more than once per 20 samples). Otherwise the competence of the panel should be checked by the panel leader.

9. SUPERVISION

A panel leader who must be an official employee of the competent authority and may be a member of the panel must be generally responsible for the entire procedure. He/she must record the individual scores for each attribute in the control document and certify whether the product is accepted or rejected.

10. NOMENCLATURE

Refer to the appended table 2.

11. REFERENCE

FIL-IDF 99C:1997 Sensory evaluation of dairy products by scoring — Reference method

ISO/DIS 22935 IDF 99 International Standard for Milk and Milk Products — Sensory analysis — Parts 1-3

ISO 8586-1 Sensory analysis — General guidance for selection, training and monitoring of assessors — Part 1

ISO 8589 Sensory analysis — General guidance for the design of test rooms

FIL-IDF 112A:1989 Butter — Determination of water dispersion value







Table 1 Butter scoring Appearance Consistency Flavour + aroma Points No (1) Remarks Points (quality class) No (1) Remarks Points (quality class) No (1) Remarks 5 Very good ideal type highest quality (equal dry) 5 Very good ideal type highest quality (well spreadable) 5 Very good ideal type highest quality (absolutely pure finest aroma) 4 Good (2) no evident defects 4 17 18 Good (2) hard soft 4 Good (2) no evident defects 3 1 2 3 4 5 6 7 8 Fair (slight defects) loose (free), moisture not uniform, two coloured streaky mottled, marbled speckled oil separation overcoloured weak, open texture 3 14 15 16 17 18 Fair (slight defects) short, brittle, crumbly pasty, doughy, greasy sticky hard soft 3 21 22 25 27 33 34 35 Fair (slight defects) unclean foreign flavour acid cooked flavour, scorched flavour feed flavour coarse, bitter oversalted 2 1 3 4 5 6 10 11 12 Poor (evident defects) loose (free) moisture streaky mottled, marbled speckled oil separation foreign matter mouldy undissolved salt 2 14 15 16 17 18 Poor (evident defects) short, brittle, crumbly pasty, doughy, greasy sticky hard soft 2 21 22 23 25 32 33 34 35 36 38 Poor (evident defects) unclean foreign flavour stale acid oxidized flavour, metallic flavour feed flavour coarse, bitter oversalted musty-flat, putrid chemical flavour 1 1 3 4 5 6 7 9 10 11 12 Very poor (strong defects) loose (free) moisture streaky mottled, marbled speckled oil separation overcoloured granular foreign matter mouldy undissolved salt 1 14 15 16 17 18 Very poor (strong defects) short, brittle, crumbly pasty, doughy, greasy sticky hard soft 1 22 24 25 26 28 29 30 31 32 34 35 36 37 38 Very poor (strong defects) foreign flavour cheesy, lactic cheese flavour acid yeasty mould flavour rancid oily, fishy tallowy oxidized flavour, metallic flavour coarse, bitter oversalted musty-flat, putrid malty chemical flavour (1) Table 2. (2) The defects mentioned under ‘good’ are only very small deviations from the ideal type.







Table 2 Table of butter defects I. Appearance 1. loose (free), moisture 2. not uniform, two coloured 3. streaky 4. mottled, marbled 5. speckled 6. oil separation 7. overcoloured 8. weak (open texture) 9. granular 10. foreign matter 11. mouldy 12. undissolved salt II. Consistency 14. short, brittle, crumbly 15. pasty, doughy, greasy 16. sticky 17. hard 18. soft III. Flavour and aroma 20. without flavour 21. unclean (1) 22. foreign flavour 23. stale 24. cheesy, lactic cheese flavour 25. acid 26. yeasty 27. (a) cooked flavour (b) scorched flavour 28. mouldy flavour 29. rancid 30. oily, fishy 31. tallowy 32. (a) oxidized flavour (b) metallic flavour 33. feed flavour 34. coarse, bitter 35. oversalted 36. musty-flat, putrid 37. malty 38. chemical flavour (1) This designation should be used as seldom as possible and only when the defect cannot be described more accurately.









ANNEX V

(Article 5)

DETERMINATION OF THE CONTENT OF ENANTHIC ACID TRIGLYCERIDE IN BUTTER, BUTTER-OIL AND CREAM BY GAS CHROMATOGRAPHIC ANALYSIS OF TRIGLYCERIDES

1. SCOPE

This method lays down a method for the determination of the content of the triglyceride of enanthic acid in butter-oil, butter and cream.

2. TERMS AND DEFINITION

Enanthic acid content: content of the triglyceride of enanthic acid determined by the procedure specified in this method.

Note: The enanthic acid content is expressed in kg per ton of product for butter-oil and butter, and it is expressed in kg per ton of milk fat for cream.

3. PRINCIPLE

Milk fat is extracted from the different products according to ISO 14156 IDF 172:2001. The quantitative determination of the content of the triglyceride of enanthic acid in the extracted fat is determined by capillary gas chromatography (GC). The result obtained for the sample is evaluated by reference to the triglyceride of caproic acid as internal standard.

Note: Tributyrin has also been found to be a satisfactory internal standard.

4. REAGENTS

Use only reagents of recognized analytical grade.

4.1. n-Hexane

4.2. Standard triglyceride of caproic acid, at least 99 % pure

4.3. Standard triglyceride of enanthic acid, at least 99 % pure

4.4. Anhydrous sodium sulfate (Na2SO4).

5. APPARATUS

Usual laboratory equipment and particularly the following:

5.1. Analytical balance precise at 1 mg

5.2. Volumetric flasks, of capacities 10 ml and 20 ml

5.3. Tubes for centrifuge, of capacity 30 ml

5.4. Rotary evaporator

5.5. Oven, capable of being maintained at a temperature of 50 °C ± 5 °C

5.6. Filter paper, medium porosity, of diameter about 15 cm

5.7. Gas chromatography equipment 5.7.1. Gas chromatograph equipped with a split/splitless or on-column injector and a flame ionization detector (FID) 5.7.2. GC column, with a stationary phase which has successfully employed to perform triglyceride separation (100 % dimethylpolysiloxane or 5 % phenyl-95 % methylpolysiloxane). Select the stationary phase, the column length (between 4 m and 15 m), the internal diameter (between 0,22 mm and 0,50 mm) and the film thickness (0,12 μm or more) taking into account the laboratory experience and the injection system applied. In any case the selected column shall produce both a complete separation between the solvent peak and the triglyceride of caproic acid and a baseline resolution between triglyceride of caproic and enanthic acid peaks. Examples of applicable conditions are listed below. 5.7.2.1. Example of applicable conditions using a split injector: — Carrier gas: helium — Column head pressure: 100 KPa — Column: 12 m length, 0,5 mm internal diameter, 0,1 μm film thickness fused silica column — Stationary phase: 100 % dimethylpolysiloxane or 5 % phenyl-95 % dimethylpolysiloxane (for ex. HT5) — Column temperature: initial temperature of 130 °C, maintained for 1 min, raised at a rate of 20 °C/min up to 260 °C and then raised at a rate of 30 °C/min up to 360 °C; maintain 10 mn at 360 °C — Detector temperature: 370 °C — Injector temperature: 350 °C — Split ratio 1:30 — Amount of sample injected: 1 μl. 5.7.2.2. Example of applicable conditions using an on-column injector: — Carrier gas: hydrogen (constant flow system) — Column head pressure: 89 kPa — Column: 4 m length, 0,32 mm internal diameter, 0,25 μm film thickness, fused silica column — Stationary phase: 5 % phenyl, 95 % dimethylpolysiloxane — Column temperature: initial temperature of 60 °C, maintained for 2 min, raised at a rate of 35 °C/min up to 340 °C, maintained at this temperature for 5 min — Detector temperature: 350 °C — Amount of sample injected: 1 μl

5.8. Injection syringe, of capacity 5 μl.

6. SAMPLING

It is important that the laboratory receives a sample which is truly representative and has not been damaged or changed during transport or storage.

Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in IDF: standard 50C:1995 or ISO 707-1997 — Milk and milk products — Methods of sampling.

7. PROCEDURE

7.1. Preparation of the test sample and test portion

Proceed according to ISO 14156 IDF 172:2001

7.1.1. Butter-oil, Butter

7.1.1.1. Melt 50 g to 100 g of test sample in the oven (5.5)

7.1.1.2. Place 0,5 g to 1,0 g of anhydrous sodium sulfate (5.4) in a folded filter paper

7.1.1.3. Filter the fat through the filter paper containing anhydrous sodium sulfate collecting the filtrate in a beaker maintained in the oven (5.5). When decanting the melted butter onto the filter paper, take care that no serum is transferred

7.1.2. Cream

7.1.2.1. Bring the test sample to a temperature of 20 °C ± 2 °C

7.1.2.2. Mix or stir the sample thoroughly

7.1.2.3. Dilute a suitable amount of test sample so as to obtain 100 ml of test portion with a mass fraction of fat of approximately 4 %

7.1.2.4. Proceed as with raw milk and homogenized milk (see ISO 14156 IDF 172:2001, §8.3) to extract the fat from the cream

7.1.2.5. Weigh in a 10 ml volumetric flask (5.2), to the nearest 1 mg, 1 g of the extracted fat. Add 1 ml of the solution 7.2.2. Complete to 10 ml with n-hexane (4.1) and homogenise

7.1.2.6. Introduce 1 ml of the solution 7.1.1.2 in a 10 ml volumetric flask (5.2) and dilute to 10 ml with n-hexane (4.1)

7.2. Preparation of the calibration standards

7.2.1. Dissolve 100 mg of the triglyceride of enanthic acid (4.3) in 10 ml of n-hexane (4.1)

7.2.2. Dissolve 100 mg of the triglyceride of caproic acid (4.2) in 10 ml of n-hexane (4.1)

7.2.3. Introduce 1 ml of the solution 7.2.2 in a 10 ml volumetric flask (5.2). Complete to 10ml with n-hexane (4.1)

7.2.4. Introduce 1 ml of the solution 7.2.1 and 1 ml of the solution 7.2.2 in a 10 ml volumetric flask (5.2). Complete to 10 ml with n-hexane (4.1)

7.2.5. Introduce 1 ml of the solution 7.2.4 in a 10 ml volumetric flask (5.2) and complete to 10 ml with n-hexane (4.1)

7.3. Chromatographic determination

7.3.1. Inject 1 μl of the standard solution 7.2.5 twice

7.3.2. Inject 1 μl of each sample solution Note: If the on column injector system is adopted an increased dilution should be applied both to the standard and sample solutions.

7.3.3. Repeat the operation 7.3.1 every 3 samples in order to bracket samples between duplicate standard injections. Results are based upon the mean average response factors from the standard chromatograms.

8. CALCULATION OF RESULTS

For each chromatogram, integrate the area of the peaks associated with the triglycerides of enanthic acid and caproic acid.

Follow those instructions for each bracketed sequence i.e. for a set of bracketed samples, the standard injected twice immediately before them is STD1 and the standard injected twice immediately after them is STD2.

8.1. Calibration

8.1.1. Calculate the response factor for each duplicate of STD1, Rf1(a) and Rf1(b) Rf1 (a) or (b) = (Peak area for caproic acid triglyceride/Peak area for enanthic acid triglyceride) × 100 Calculate the mean average response factor, Rf1 Rf1 = (Rf1(a) + Rf1(b)) / 2

8.1.2. Similarly, calculate the mean average response factor STD2, Rf2

8.1.3. Calculate the mean average response factor, Rf Rf = (Rf1 + Rf2) /2

8.2. Test samples

For each sample chromatogram obtained between STD1 and STD2, calculate the enanthic acid content, C (kg/t):

C = (Peak area for enanthic acid triglyceride × Rf × 100)/(Peak area for caproic acid triglyceride × Wt × 1 000)

where:

— Wt = weight of fat taken (g),

— 100 = dilution volume for sample,

— 1 000 = conversion factor (for μg/g to kg/t)

For butter samples, take the fat content of butter into account and calculate a corrected concentration value, Cbutter (kg/t of butter)

Cbutter = Cfat × F

where F is the fat content of butter.

9. PRECISION

Details of an interlaboratory test on butter in accordance with ISO 5725-1 and ISO 5725-2 on the precision method are shown in (12.).

The values for repeatability and reproducibility limit are expressed for the 95 % probability level and may not be applicable to concentration ranges and matrices other than those given.

9.1. Repeatability

The absolute differences between two individual single test results, obtained with the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in not more than 5 % of cases be greater than 0,35 kg/t.

9.2. Reproducibility

The absolute differences between two individual single test results, obtained with the same method on identical test material in different laboratories with different operators using different equipment will in not more than 5 % of cases be greater than 0,66 kg/t.

10. TOLERANCE LIMITS: LOWER LIMITS (CASE OF INSUFFICIENT QUANTITIES)

10.1. Three samples must be taken from the traced product in order to check on the correct tracing of the product

10.2. Butter and concentrated butter 10.2.1. The incorporation rate is 11 kg of at least 95 % pure enanthic acid triglyceride per tonne of butter, i.e. 10,45 kg/t 10.2.2. The results of three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits: — 9,51 kg/t (95 % of the minimum incorporation rate of 95 % pure enanthic acid triglyceride, single determination), — 6,89 kg/t (70 % of the minimum incorporation rate of 95 % pure enanthic acid triglyceride, single determination), — The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation respectively between 9,51 kg/t and 6,89 kg/t.

10.3. Cream 10.3.1. The incorporation is 10 kg of at least 95 % pure enanthic acid triglyceride per tonne of milk fat, i.e. 9,50 kg/t traced milk fat 10.3.2. The results of the three samples obtained from the analysis of the product are used to check the rate of the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits: — 8,60 kg/t (95 % of the minimum incorporation rate of 95 % pure enanthic acid triglyceride, single determination), — 6,23 kg/t (70 % of the minimum incorporation rate of 95 % pure enanthic acid triglyceride, single determination), — The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation respectively between 8,60 kg/t and 6,23 kg/t.

11. TOLERANCE LIMITS: UPPER LIMITS (CASE OF EXCEEDING QUANTITY BY MORE THAN 20 %)

11.1. Three samples must be taken from the traced product in order to check on the correct tracing of the product

11.2. Butter and concentrated butter 11.2.1. The results of three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the mean of these results is compared with the following limits: — Upper limit is 12,96 kg/t

11.3. Cream 11.3.1. The results of three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the mean of these results is compared with the following limits: — Upper limit is 11,82 kg/t.

12. ADDITIONAL INFORMATION: STATISTICAL ANALYSIS OF RESULTS ON THE DETERMINATION OF TRIENANTOATE IN BUTTERFAT BY TRIGLYCERIDE ANALYSIS

Four collaborative trials have been carried out to determine the trienantoate content in traced butter.

Nine laboratories participated to the 1st ring test and no specifications were provided about the analytical methods to use:

10 laboratories participated to the 2nd ring test and 4 different methods were applied:

— Quantification of methylheptanoate by using n-nonane or methylnonanoate as internal standard

— Quantification of trienantoate by using tricaproate as internal standard

— Quantification of methylheptanoate by using a calibration sample/mixture

— Quantification of methylheptanoate by using a calibration mixture.

Moreover, if FAME were analysed, two different methylation procedures were used (De Francesco and Christopherson & Glass).

Due to the results obtained, two methods were chosen to perform the 3rd ring test:

— Quantification of methylheptanoate by using n-nonane or methylnonanoate as internal standard

— Quantification of trienantoate by using tricaproate as internal standard.

The results of 7 labs showed that the FAME method produced a higher variability and consequently it was decided to use only the determination of trienantoate as triglyceride following the procedure of the q Quantification of trienantoate by using tricaproate as internal standard. Moreover the triglyceride analysis has to be carried out by capillary column.

In the 4th ring test four samples (A, B, C, D) were circulated and nine laboratories provided results (Tables 1-2).

Two laboratories (DE and UE) analysed the samples by using FAME method.

Due to the reduced number of laboratories, the Statistical calculation has been performed both on the complete set (Figures 1-2) of data including FAME results and on the data obtained from TG analysis.

Tests for outliers:

— sample A. Dixon, Cochran and Grubbs tests at levels 1 and 5 %, showed one laboratory outlier.

— sample B. Grubbs test at level 5 % showed one laboratory outlier.

— sample C. Dixon and Grubbs tests at levels 1 and 5 %, showed one laboratory outlier.

— sample D. Dixon and Grubbs tests at levels 1 end 5 %, showed one laboratory outlier.

The outlier has been excluded from the calculation.

It is worth noting that the results obtained by FAME method were never considered as outliers by the tests applied.

Precision parameters

Tables 1 and 2 report the results of all the laboratories and the precision parameters calculated on an acceptable number (8) of labs but, unfortunately not deriving from the same analytical method.

Tables 3 and 4 report the results deriving only from TG method and the corresponding precision parameters. The acceptance of these parameters is subjected to the acceptance of the low number of laboratories (6).

Figures 2 and 3 show the trend of Sr and SR calculated on the 4 samples of the 2 data set described above.

Table 5 reports the Sr and SR values together with the corresponding pooled values and overall r and R parameters.

Finally the Critical Difference at 95 % of probability level has been calculated.







Table 1 Statistical Results of TG + FAME* methods Sample A R1 R2 Mean N. of labs retained after eliminating outliers 8 RENNES FR1 11,0 11,1 11,1 N. of outliers 1 RIKILT NL 11,2 11,2 11,2 Outliers DК ZPLA DE* 11,6 11,8 11,7 Mean value 11,3 ADAS GB 11,4 11,2 11,3 True value 11,0 CNEVA FR2 11,4 11,4 11,4 Repeatability standard deviation (Sr) 0,09 LODI IT 11,1 11,3 11,2 Repeatability relative sd (RSDr%) 0,80 EELA FI 11,3 11,2 11,3 Repeatability r (95 %) 0,26 ISPRA UE* 11,0 11,0 11,0 Relative Repeatability r % 2,24 D.V.F.A. DK 13,3 11,8 12,6 Reproducibility standard deviation (SR) 0,23 Reproducibility relative sd (RSDR%) 2,04 Reproducibility R (95 %) 0,84 Relative Reproducibility R % 5,71 Sample B R1 R2 Mean N. of labs retained after eliminating outliers 8 RENNES FR1 12,7 12,8 12,8 N, of outliers 1 RIKILT NL 13,5 13,3 13,4 Outliers DK ZPLA DE* 14,0 13,8 13,9 Mean value 13,4 ADAS GB 13,4 13,5 13,5 True value 13,5 CNEVA FR2 13,3 13,4 13,4 Repeatability standard deviation (Sr) 0,14 LODI IT 13,9 13,5 13,7 Repeatability relative sd (RSDr%) 1,04 EELA FI 13,4 13,2 13,3 Repeatability r (95 %) 0,40 ISPRA UE* 13,2 13,3 13,3 Relative Repeatability r % 2,91 D.V.F.A. DK 14,1 14,8 14,5 Reproducibility standard deviation (SR) 0,35 Reproducibility relative sd (RSDR%) 2,61 Reproducibility R (95 %) 0,99 Relative reproducibility R % 7,31







Table 2 Statistical Results of TG + FAME* methods Sample C R1 R2 Mean N. of labs retained after eliminating outliers 8 RENNES FR1 8,9 9,2 9,1 N. of outliers 1 RIKILT NL 9,2 9,3 9,3 Outliers DK ZPLA DE* 9,2 9,4 9,3 Mean value 9,3 ADAS GB 9,5 9,3 9,4 True value 9,3 CNEVA FR2 9,4 9,4 9,4 Repeatability standard deviation (Sr) 0,14 LODI IT 9,2 9,5 9,4 Repeatability relative sd (RSDr%) 1,50 EELA FI 9,4 9,6 9,5 Repeatability r (95 %) 0,40 ISPRA UE* 9,4 9,3 9,4 Relative Repeatability r % 4,20 D.V.F.A. DK 10,7 10,9 10,8 Reproducibility standard deviation (SR) 0,17 Reproducibility relative sd (RSDR%) 1,82 Reproducibility R (95 %) 0,47 Relative Reproducibility R % 5,10 Sample D R1 R2 Mean N. of labs retained after eliminating outliers 8 RENNES R1 1,6 1,6 1,6 N. of outliers 1 RIKILT NL 2,1 2,1 2,1 Outliers DK ZPLA DE* 2,3 2,3 2,3 Mean value 2,1 ADAS GB 2,1 2,2 2,2 True value 2,1 CNEVA FR2 2,1 2,1 2,1 Repeatability standard deviation (Sr) 0,08 LODI IT 2,2 1,9 2,1 Repeatability relative sd (RSDr%) 3,81 EELA FI 2,3 2,3 2,3 Repeatability r (95 %) 0,22 ISPRA UE* 2,3 2,3 2,3 Relative Repeatability r % 10,67 D.V.F.A. DK 3,4 2,9 3,2 Reproducibility standard deviation (SR) 0,24 Reproducibility relative sd (RSDR%) 11,43 Reproducibility R (95 %) 0,67 Relative Reproducibility R % 32,00







Table 3 Statistical Results of TG + FAME* methods Sample A R1 R2 Mean N. of labs retained after eliminating outliers 6 RENNES FR1 11,0 11,1 11,1 N. of outliers 1 RIKILT NL 11,2 11,2 11,2 Outliers DК ADAS GB 11,4 11,2 11,3 Mean value 11,2 CNEVA FR2 11,4 11,4 11,4 True value 11,0 LODI IT 11,1 11,3 11,2 Repeatability standard deviation (Sr) 0,09 EELA FI 11,3 11,2 11,3 Repeatability relative sd (RSDr%) 0,80 D.V.F.A. DK 13,3 11,8 12,6 Repeatability r (95 %) 0,25 Relative Repeatability r % 2,24 Reproducibility standard deviation (SR) 0,13 Reproducibility relative sd (RSDR%) 1,16 Reproducibility R (95 %) 0,36 Relative Reproducibility R % 3,25 Sample B R1 R2 Mean N. of labs retained after eliminating outliers 6 RENNES FR1 12,7 12,8 12,8 N. of outliers 1 RIKILT NL 13,5 13,3 13,4 Outliers DК ADAS GB 13,4 13,5 13,5 Mean value 13,3 CNEVA FR2 13,3 13,4 13,4 True value 13,5 LODI IT 13,9 13,5 13,7 Repeatability standard deviation (Sr) 0,15 EELA FI 13,4 13,2 13,3 Repeatability relative sd (RSDr%) 1,13 D.V.F.A. DK 14,1 14,8 14,5 Repeatability r (95 %) 0,42 Relative Repeatability r % 3,16 Reproducibility standard deviation (SR) 0,33 Reproducibility relative sd (RSDR%) 2,48 Reproducibility R (95 %) 0,93 Relative Reproducibility R % 6,94







Table 4 Statistical Results of TG method Sample C R1 R2 Mean N. of labs retained after eliminating outliers 6 RENNES FR1 8,9 9,2 9,1 N. of outliers 1 RIKILT NL 9,2 9,3 9,3 Outliers DК ADAS GB 9,5 9,3 9,4 Mean value 9,3 CNEVA FR2 9,4 9,4 9,4 True value 9,3 LODI IT 9,2 9,5 9,4 Repeatability standard deviation (Sr) 0,15 EELA FI 9,4 9,6 9,5 Repeatability relative sd (RSDr%) 1,61 D.V.F.A. DK 10,7 10,9 10,8 Repeatability r (95 %) 0,42 Relative Repeatability r % 4,51 Reproducibility standard deviation (SR) 0,19 Reproducibility relative sd (RSDR%) 2,04 Reproducibility R (95 %) 0,53 Relative Reproducibility R % 5,71 Sample D R1 R2 Mean N, of labs retained after eliminating outliers 6 RENNES FR1 1,6 1,6 1,6 N. of outliers 1 RIKILT NL 2,1 2,1 2,1 Outliers DK Mean Value 2,1 ADAS GB 2,1 2,2 2,2 True value 2,1 CNEVA FR2 2,1 2,1 2,1 Repeatability standard deviation (Sr) 0,09 LODI IT 2,2 1,9 2,1 Repeatability relative sd (RSDr%) 4,29 EELA FI 2,3 2,3 2,3 Repeatability r (95 %) 0,26 D.V.F.A. DK 3,4 2,9 3,2 Relative Repeatability r % 12,01 Reproducibility standard deviation (SR) 0,25 Reproducibility relative sd (RSDR%] 11,90 Reproducibility R (95 %) 0,69 Relative Reproducibility R % 33,32







Table 5 Repeatability and reproducibility (with FAME) No of labs Outlier Repeatability Sr (95 %) Reproducibility SR (95 %) Sample A 8 1 0,09 0,23 Sample Β 8 1 0,14 0,35 Sample C 8 1 0,14 0,17 Sample D 8 1 0,08 0,24 Pooled value 0,116 0,256 r R Pooled value* 2,8 0,324 0,716 CrD95 =0,40 Minimum purity stated for trienantoate = 95 % Minimum limit stated for trienantoate in butterfat = 11 kg/t Taking the Critical Difference for a 95 % probability level into consideration, the mean of the two results shall not be less than: in the case of incorporation of 95 % pure trienantoate 10,05 kg/t







Repeatability and reproducibility (without FAME) No of labs Outlier Repeatability Sr (95 %) Reproducibility SR (95 %) Sample A 6 1 0,09 0,13 Sample B 6 1 0,15 0,33 Sample C 6 1 0,15 0,19 Sample D 6 1 0,09 0,25 Pooled value 0,124 0,237 r R Pooled value * 2,8 0,347 0,663 CrD95 = 0,36 Minimum purity stated for trienantoate = 95 % Minimum limit stated for trienantoate in butterfat = 11 kg/t Taking the Critical Difference for a 95 % probability level into consideration, the mean of the two results shall not be less than: in the case of incorporation of 95 % pure trienantoate 10,09 kg/t.

Figure 1 ( 8 )

Experimental results: Sample A

Experimental results: Sample B

Experimental results: Sample C

Experimental results: Sample D

Figure 2

Repeatability and Reproducibility standard deviation at different levels (TG+FAME)

Figure 3

Repeatability and Reproducibility standard deviation at different levels (TG)

Figure 4

Example using an on-column injector









ANNEX VI

(Article 5)

DETERMINING THE VANILLIN CONTENT IN CONCENTRATED BUTTER, BUTTER OR CREAM BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

1. SCOPE AND FIELD OF APPLICATION

The method describes a procedure for the quantitative determination of vanillin in concentrated butter, butter or cream.

2. PRINCIPLE

Extraction of a known quantity of sample with a mixture of isopropanol/ethanol/acetonitrile (1:1:2). Precipitation of the majority of fat by cooling between –15 °C and –20 °C, followed by centrifugation.

After dilution with water, determination of the vanillin content by high-performance liquid chromatography (HPLC).

3. APPARATUS

Usual laboratory apparatus and, in particular, the following:

3.1. Freezer, operating in the temperature range –15 °C to –20 °C

3.2. Syringes, disposable of 2 ml capacity

3.3. Membrane microfilters of 0,45 μm pore size, resistant to a solution containing 5 % extraction solution (4.4)

3.4. Liquid chromatography system consisting of a pump (flow of 1,0 ml/min), an injector (20 μl injection, automatic or manual), an UV detector (operated at 306 nm, 0,01 Å full scale), a recorder or integrator and a column thermostat operating at 25 °C

3.5. Analytical column (250 mm × 4,6 mm ID) packed with LiChrospher RP 18 (Merck, 5 μm) or equivalent

3.6. Guard column (ca. 20 mm × 3 mm ID) dry-packed with LiChrospher RP 18 (5 to 10 μm) or equivalent

3.7. Centrifuge operating at 2 000 rpm.

4. REAGENTS

All reagents used must be of recognised analytical quality.

4.1. Isopropanol

4.2. Ethanol 96 % (v/v)

4.3. Acetonitrile

4.4. Extraction solution Mix isopropanol (4.1), ethanol (4.2) and acetonitrile (4.3) in the ratio of 1:1:2 (v/v).

4.5. Vanillin (4-hydroxy-3-methoxybenzaldehyde) ≥ 98 % 4.5.1. Vanillin stock solution (= 500 μg/ml) Weigh accurately to 0,1 mg about 50 mg (CM mg) vanillin (4.5) in a 100 ml volumetric flask, add 25 ml extraction solution (4.4) and make up with water. 4.5.2. Vanillin standard solution (= 10 μg/ml) Pipette 5 ml of the vanillin stock solution (4.5.1) into a volumetric flask of 250 ml and make up with water. 4.5.3. Methanol, HPLC quality 4.5.4. Acetic acid, glacial 4.5.5. Water, HPLC quality 4.5.6. HPLC mobile phase Mix 300 ml methanol (4.5.3) with about 500 ml water (4.5.5) and 20 ml acetic acid (4.5.4) in a volumetric flask of 1 000 ml and make up with water (4.5.5). Filter through 0,45 μm filter (3.3).

5. PROCEDURE

5.1. Preparation of the test sample

5.1.1. Butter

Heat the sample until melting starts. Avoid local overheating at about 30 °C. The butter may not separate in two phases, in any case. When the sample becomes sufficiently plastic, homogenise it by shaking. Stir the butter for 15 s before taking a sample. Weigh, to the nearest 1 mg, about 5 g (SM g) of butter into a 100 ml volumetric flask.

5.1.2. Concentrated butter

Immediately before sampling place the container, with concentrated butter, into an oven at 40 to 50 °C until it is melted completely. Mix the sample by swirling or stirring, avoiding formation of air bubbles by too vigorous stirring. Weigh, to the nearest 1 mg, about 4 g (SM g) of concentrated butter into a 100 ml volumetric flask.

5.1.3. Cream

Heat the sample in a waterbath or incubator at a temperature of 35 to 40 °C. Distribute the fat homogeneously by swirling and, if necessary, by stirring. Cool the sample quickly to 20 ± 2 °C. The sample should look homogenous; otherwise the procedure should be repeated. Weigh, to the nearest 1 mg, about 10 g (SM g) of cream into a 100 ml volumetric flask.

5.2. Preparation of the test solution

Add about 75 ml extraction solution (4.4) to the test portion (5.1.1, 5.1.2 or 5.1.3), stir, or shake vigorously, for about 15 minutes and make up with extraction solution (4.4). Transfer about 10 ml of this extract to a test tube fitted with stopper. Place the test tube in the freezer (3.1) and allow it to stand for about 30 minutes. Centrifuge the cold extract for 5 minutes at approximately 2 000 rpm and decant immediately. Allow the decanted solution to adjust to room temperature. Pipette 5 ml of the decanted solution into a 100 ml volumetric flask and make up with water. Filter an aliquot through a membrane microfilter (3.3) using a syringue (3.2). The filtrate is ready for determination by HPLC.

5.3. Calibration

Pipette 5 ml of the vanillin standard solution (4.5.2) into a 100 ml volumetric flask. Add 5 ml extraction solution (4.4) and make up to the mark with water. This solution contains 0,5 μg/ml of vanillin.

5.4. Determination by HPLC

Allow the chromatographic system to stabilise for about 30 minutes. Inject the standard solution (5.3). Repeat this until the difference in peak area or peak height between two successive injections is less than 2 %. Under the conditions described the retention time of vanillin is about 9 minutes. Analyse the standard solution (5.3) in duplicate by injecting 20 μl. Inject 20 μl of the test solutions (5.2). Determine the area or height of the vanillin peak obtained. Repeat the duplicate injection of the standard solution (5.3) after 10 injections of test samples (5.2).

6. CALCULATION OF THE RESULTS

Calculate the average peak area (or height) (AC), of the vanillin peaks associated with the bracketing duplicate injections for each batch of test solutions (four areas or heights in total).

Calculate the response factor (R):

where CM is the mass of vanillin in mg (4.5.1).

The content (mg/kg) of vanillin I in the test sample is given by:

where:

AS = peak area or height of the vanillin peak of the test sample

SM = mass of test sample in g (5.1.1, 5.1.2 or 5.1.3).

Note: Where cream is analysed for vanillin, the tracer concentration has to be expressed as mg tracer/kg milk fat. This is done by multiplying C by 100/f. f is the fat content of the cream in percent (m/m).

20 = factor which takes into account the dilutions of the standard and the test sample

0,96 = correction factor for the fat content in first dilution of the test sample

Note: Instead of peak area, peak heights can be used (see 8.3).

7. ACCURACY OF THE METHOD

7.1. Repeatability (r)

The difference between the results of two determinations carried out within the shortest feasible time interval, by one operator using the same apparatus on identical test material, may not exceed 16 mg/kg.

7.2. Reproducibility (R)

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 27 mg/kg.

8. TOLERANCE LIMITS

8.1. Three samples must be taken from the traced product in order to check homogeneity

8.2. Tracer obtained either from vanilla or from synthetic vanillin 8.2.1. The incorporation rate for 4-hydroxy-3-methoxybenzaldehyde is 250 g per tonne of concentrated butter or butter. Where cream is traced, the incorporation rate is 250 g per tonne of milk fat 8.2.2. The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits: — 220,8 mg/kg (95 % of the minimum incorporation rate), — 158,3 mg/kg (70 % of the minimum incorporation rate). The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation between 220,8 mg/kg and 158,3 mg/kg.

8.3. Tracer obtained exclusively from vanilla beans or integral extracts thereof: 8.3.1. The incorporation rate for 4-hydroxy-3-methoxybenzaldehyde is 100 g per tonne of concentrated butter or butter. Where cream is traced, the incorporation rate is 100 g per tonne of milk fat. 8.3.2. The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits: — 78,3 mg/kg (95 % of the minimum incorporation rate), — 53,3 mg/kg (70 % of the minimum incorporation rate). The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation between 78,3 mg/kg and 53,3 mg/kg.

9. NOTES

9.1. Recovery of added vanillin at a level of 250 mg/kg butteroil varies from 97,0 to 103,8. The average content found was 99,9 % with a standard deviation of 2,7 %.

9.2. The standard solution contains 5 % extraction solution to compensate for peak broadening caused by the presence of 5 % of the extraction solution of the test samples. This enables quantification by peak height.

9.3. The analysis is based on a linear calibration line with a zero intercept.

9.4. By using appropriate dilutions of the standard solution (4.5.2), the linearity should be checked the first time the analysis is carried out and then at regular intervals and after changes in or repair of the HPLC equipment. Vanillin may be degraded to vanillin acid, divanillin and other compounds by activity of intrinsic enzymes in unpasteurised cream or products thereof.









ANNEX VII

(Article 5)

DETERMINING THE ETHYL ESTER OF BETA-APO-8'-CAROTENIC ACID IN CONCENTRATED BUTTER AND BUTTER BY SPECTROMETRY

1. SCOPE AND FIELD OF APPLICATION

The method describes a procedure for the quantitative determination of the ethyl ester of beta-apo-8'-carotenic acid (apo-carotenic ester) in concentrated butter and butter. The apo-carotenic ester is the sum of all substances present in an extract of samples obtained under the conditions described in the method which absorb light at 440 nm.

2. PRINCIPLE

The butterfat is dissolved in light petroleum and the absorbance measured at 440 nm. The apo-carotenic ester content is determined by reference to an external standard.

3. APPARATUS

3.1. Pipettes — graduated, of capacity 0,25, 0,50, 0,75 and 1,0 ml

3.2. Spectrophotometer — suitable for use at 440 nm (and 447-449 nm) and equipped with cells of optical path length 1 cm

3.3. Volumetric flasks, 20 ml and 100 ml

3.4. Analytical balance, sensitivity 0,1 mg capable of weighing to the nearest 1 mg, with a readability of 0,1 mg

3.5. Oven, 45 °C ± 1 °C

3.6. Fast-filtering ashless filters.

4. REAGENTS

All reagents must be of recognised analytical grade.

4.1. Apo-carotenic ester suspension (approximately 20 %) 4.1.1. Establish the content of the suspension as follows: Warm the suspension between 45 °C and 50 °C and homogenize in the unopened original container. Weigh about 400 mg in a volumetric flask (100 ml), dissolve in 20 ml chloroform (4.4) and make up the volume with cyclohexane (4.5). Dilute 5 ml of this solution to 100 ml with cyclohexane (solution A). Dilute 5 ml of solution A to 100 ml with cyclohexane. Measure the absorbance at 447-449 nm (measure the maximum against cyclohexane as a blank using cells with 1 cm optical path length). Apo-carotenic ester content P (%) = (Absmax × 40 000) / (Msusp × 2 550) or develop: (Absmax / 2 550) × (100 / 5) × (100 / 5) × (100 / Msusp) Absmax = absorbance of the measuring solution at the maximum Msusp = mass of suspension (g) 2 550 = reference Abs (1 %, 1 cm) value P = Purity (content) of the suspension (%) Note: Apo-carotenic ester suspension is sensitive to air, heat and light. In the unopened, original container (sealed under nitrogen) and in a cool place it can be stored for about 12 months. After opening the contents should be used within a short period. 4.1.2. Apo-carotenic ester standard solution, approx. 0,2 mg/ml Weigh to the nearest 1 mg about 0,100 g of apo-carotenic ester suspension (4.1.1) (W), dissolve in petroleum spirit (4.2), transfer quantitatively into a volumetric flask of capacity 100 ml, and make up to the mark with petroleum spirit. This solution contains (W × P) / 10 mg/ml of apo-carotenic ester. Note: The solution must be stored in a cool place in the dark. Discard unused solution after one month.

4.2. Petroleum spirit (40-60 °C)

4.3. Sodium sulphate, anhydrous, granular, previously dried at 102 °C for two hours

4.4. Chloroform

4.5. Cyclohexane

5. PROCEDURE

5.1. Preparation of the test sample

5.1.1. Concentrated butter

Melt the sample in an oven at approximately 45 °C.

5.1.2. Butter

Melt the sample in an oven at approximately 45 °C and filter a representative portion through a filter containing about 10 g of anhydrous sodium sulphate (4.3) in an environment shielded from strong natural and artificial light and maintained at 45 °C. Collect a suitable amount of butterfat.

5.2. Determination

Weigh, to the nearest 1 mg approximately 1 g of concentrated butter (or extracted butterfat (5.1.2)), (M). Transfer quantitatively to a 20 ml (V) volumetric flask using petroleum spirit (4.2), make up to the mark and mix thoroughly.

Transfer an aliquot to a 1 cm cell and measure the absorbance at 440 nm, against a petroleum spirit blank. Obtain the concentration of apo-carotenic ester in the solution by referring to the obtained standard curve (C μ/ml).

5.3. Calibration

Pipette 0, 0,25, 0,5, 0,75 and 1,0 ml of apo-carotenic ester standard solution (4.1.2) into five 100 ml volumetric flasks. Dilute to volume with petroleum spirit (4.2) and mix.

The approximate concentrations of the solutions range from 0 to 2 μg/ml and are calculated accurately by reference to the concentration of the standard solution (4.1.2) (W × P) / 10 mg/ml. Measure the absorbances at 440 nm against a petroleum spirit (4.2) blank.

Plot the values of absorbance on the y axis against apo-carotenic ester concentration on the x axis. Calculate the equation of the standard curve.

6. CALCULATION OF THE RESULTS

6.1. Apo-carotenic ester content, expressed as mg/kg product, is given by: Concentrated butter: (C × V)/M Butter: 0,82 (C × V)M where: C = apo-carotenic ester content, μg/ml, read from the calibration graph (5.3) V = volume (ml) of the test solution (5.2) M = mass (g) of the test portion (5.2) 0,82 = a correction factor for the butterfat content of butter.

7. ACCURACY OF THE METHOD

7.1. Repeatability

7.1.1. Butter analysis

The difference between the results of two determinations carried out within the shortest feasible time interval, by one operator using the same apparatus on identical test material, may not exceed 1,4 mg/kg.

7.1.2. Concentrated butter analysis

The difference between the results of two determinations carried out within the shortest feasible time interval, by one operator using the same apparatus on identical test material, may not exceed 1,6 mg/kg.

7.2. Reproducibility

7.2.1. Butter analysis

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 4,7 mg/kg.

7.3. Concentrated butter analysis

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 5,3 mg/kg.

7.4. Source of precision data

The precision data were determined from an experiment conducted in 1995 involving 11 laboratories and 12 traced samples (six blind duplicates) for butter and 12 traced (six blind duplicates) for concentrated butter.

8. TOLERANCE LIMITS

8.1. Three samples must be taken from the traced product in order to check on the correct tracing of the product.

8.2. Butter 8.2.1. The incorporation rate for butter, taking into account background absorbance, is 22 mg/kg. 8.2.2. The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits: — 17,7 mg/kg (95 % of the minimum incorporation rate), — 12,2 mg/kg (70 % of the minimum incorporation rate). The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation between 17,7 mg/kg and 12,2 mg/kg.

8.3. Concentrated butter 8.3.1. The incorporation rate for concentrated butter, taking into account background absorbance, is 24 mg/kg The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits: — 19,2 mg/kg (95 % of the minimum incorporation rate), — 13,2 mg/kg (70 % of the minimum incorporation rate). The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation between 19,2 mg/kg and 13,2 mg/kg.









ANNEX VIII

(Article 5)

DETERMINING SITOSTEROL OR STIGMASTEROL IN BUTTER OR CONCENTRATED BUTTER BY CAPILLARY-COLUMN GAS CHROMATOGRAPHY

1. SCOPE AND FIELD OF APPLICATION

The method describes a procedure for the quantitative determination of sitosterol or stigmasterol in butter and concentrated butter. Sitosterol is taken to be the sum of β-sitosterol and 22 dihydro-β-sitosterol, other sitosterols are assumed to be insignificant.

2. PRINCIPLE

The butter or concentrated butter is saponified with potassium hydroxide in ethanolic solution and the unsaponifiables are extracted with diethyl ether.

The sterols are transformed into trimethyl-silyl ethers and are analysed by capillary-column gas chromatography with reference to an internal standard/betulin.

3. APPARATUS

3.1. 150 ml saponification flask fitted with a reflux condenser having ground-glass joints

3.2. 500 ml separating funnels

3.3. 250 ml flasks

3.4. Pressure equalising funnels, 250 ml or similar, to collect waste diethyl ether

3.5. Glass column, 350 mm × 20 mm fitted with sintered glass plug

3.6. Waterbath or isomantle

3.7. Reaction vials, 2 ml

3.8. Gas chromatograph suitable for use with a capillary column, provided with a splitting system consisting of: 3.8.1. a thermostatic chamber for columns capable of maintaining the desired temperature with an accuracy of ±1 °C 3.8.2. a temperature-adjustable vaporisation unit 3.8.3. a flame ionisation detector and converter-amplifier 3.8.4. an integrator-recorder suitable for use with the converter-amplifier (3.8.3)

3.9. A fused-silica capillary column entirely coated with BP1 or equivalent (or any other column of at least equal resolution) in a uniform thickness 0,25 μm; the column must be capable of resolving trimethyl-silyl derivatives of lanosterol and sitosterol. A column, length 12 m, internal diameter 0,2 mm, is suitable

3.10. A 1 μl gas chromatography microsyringe with hardened needle.

4. REAGENTS

All reagents must be of recognised analytical grade. The water used must be distilled water or water of at least equivalent purity.

4.1. Ethanol, at least 95 % pure

4.2. Potassium hydroxide, 60 % solution, dissolve 600 g potassium hydroxide (minimum 85 %) in water and make up to one litre with water

4.3. Betulin of at least 99 % purity 4.3.1. Solutions of betulin in diethyl ether (4.4) 4.3.1.1. The concentration of betulin solution used for sitosterol determination should be 1,0 mg/ml 4.3.1.2. The concentration of betulin solution used for stigmasterol determination should be 0,4 mg/ml

4.4. Diethyl ether, analytical purity (free from peroxides or residue)

4.5. Sodium sulphate, anhydrous, granular, previously dried at 102 °C for two hours

4.6. Silylating reagent, for example TRI-SIL (available from Pierce Chemical Co, Cat No 49001) or equivalent (Important: TRI-SIL is inflammable, toxic, corrosive and possibly carcinogenic. Laboratory personnel must be familiar with TRI-SIL safety data and take the appropriate precautions.)

4.7. Lanosterol

4.8. Sitosterol, of known purity not less than 90 % pure (P) Note 1: The purity of standard materials used for calibration must be determined using the method of normalisation. Assume that all sterols present in the sample are represented on the chromatogram, the total area of the peaks represents 100 % of the sterol constituents and that the sterols give the same detector response. Linearity of the system must be validated over the concentration ranges of interest. 4.8.1. Sitosterol standard solution — prepare a solution containing, to the nearest 0,001 mg/ml, approximately 0,5 mg/ml (W1) sitosterol (4.8) in diethyl ether (4.4)

4.9. Stigmasterol, of known purity not less than 90 % pure (P) 4.9.1. Stigmasterol standard solution — prepare a solution containing, to the nearest 0,001 mg/m, approximately 0,2 mg/ml (W1) stigmasterol (4.9) in diethyl ether (4.4)

4.10. Resolution test mixture. Prepare a solution containing 0,05 mg/ml lanosterol (4.7) and 0,5 mg/ml sitosterol (4.8) in diethyl ether (4.4).

5. METHOD

5.1. Preparation of standard solutions for chromatography

The internal standard solution (4.3.1) must be added to the appropriate sterol standard solution at the same time as it is added to the saponified sample (see 5.2.2)

5.1.1. Sitosterol standard chromatographic solution: transfer 1 ml of sitosterol standard solution (4.8.1) to each of two reaction vials (3.7) and remove the diethyl ether with a stream of nitrogen. Add 1 ml of internal solution (4.3.1.1) and remove the diethyl ether with a stream of nitrogen

5.1.2. Stigmasterol standard chromatographic solution: transfer 1 ml of stigmasterol standard solution (4.9.1) to each of two reaction vials (3.7) and remove the diethyl ether with a stream of nitrogen. Add 1 ml of internal standard solution (4.3.1.2) and remove the diethyl ether with a stream of nitrogen

5.2. Preparation of the unsaponifiables

5.2.1. Melt the butter sample at a temperature not exceeding 35 °C, mix the sample thoroughly by stirring Weigh, to the nearest 1 mg, approximately 1 g of butter (W2) or concentrated butter (W2) into a 150 ml flask (3.1). Add 50 ml ethanol (4.1) and 10 ml potassium hydroxide solution (4.2). Fit the reflux condenser and heat at approximately 75 °C for 30 minutes. Detach the condenser and cool the flask to approximately ambient temperature.

5.2.2. Add 1,0 ml of internal standard solution (4.3.1.1) to the flask if sitosterol is to be determined, or (4.3.1.2) if stigmasterol is to be determined. Mix thoroughly. Transfer the contents of the flask quantitatively into a 500 ml separating funnel (3.2), washing the flask in turn with 50 ml water and 250 ml diethyl ether (4.4). Shake the separating funnel vigorously for two minutes and allow the phases to separate. Run off the lower aqueous layer and wash the ether layer by shaking with four successive 100 ml aliquots of water Note 2: To avoid formation of an emulsion, it is essential that the first two water washes are carried out gently (10 inversions). The third wash can be shaken vigorously for 30 seconds. If an emulsion is formed it can be destroyed by the addition of 5-10 ml of ethanol. If ethanol is added it is essential to carry out a further two vigorous water washes.

5.2.3. Pass the clear, soap-free ether layer through a glass column (3.5) containing 30 g anhydrous sodium sulphate (4.5). Collect the ether in a 250 ml flask (3.3). Add one anti-bumping granule and evaporate to near dryness in a waterbath or isomantle, taking care to collect the waste solvents. Note 3: If sample extracts are taken to complete dryness at too high temperature sterol losses may occur.

5.3. Preparation of trimethyl silyl ethers

5.3.1. Transfer the ether solution remaining in the flask to a 2 ml reaction vial (3.7) with 2 ml diethyl ether and remove the ether using a stream of nitrogen. Wash the flask with two further 2 ml aliquots of diethyl ether, transferring to the vial and removing the ether with nitrogen each time

5.3.2. Silylate the sample by addition of 1 ml TRI-SIL (4.6). Close the vial and shake vigorously to dissolve. If dissolution is incomplete, warm to 65-70 °C. Allow to stand for at least five minutes before injecting into the gas-chromatograph. Silylate standards in the same way as samples. Silylate the resolution test mixture (4.10) in the same way as samples Note 4: Silylation must be effected in a water-free environment. Incomplete silylation of betulin is indicated by a second peak close to that of betulin. The presence of ethanol at the silylation stage will interfere with silylation. This may result from inadequate washing at the extraction stage. If this problem persists, a fifth wash may be introduced at the extraction stage, shaking vigorously for 30 seconds.

5.4. Gas-chromatographic analysis

5.4.1. Choice of operating conditions

Set up the gas-chromatograph according to the manufacturer’s instructions.

The guideline operating conditions are as follows:

— column temperature: 265 °C

— injector temperature: 265 °C

— detector temperature: 300 °C

— carrier gas flow rate: 0,6 ml/min.

— hydrogen pressure: 84 kPa

— air pressure: 155 kPa

— sample split: 10:1 to 50:1; the split ratio must be optimised in accordance with the manufacturer’s instructions and linearity of detector response, then validated over the concentration range of interest. — Note 5: It is especially important that the injection liner is regularly cleared.

— amount of substance injected: 1 μl of TMSE solution.

Allow the system to equilibrate and obtain a satisfactory stable response before commencing any analysis.

These conditions must be varied in the light of column and gas-chromatograph characteristics so as to obtain chromatograms which meet the following requirements:

— the sitosterol peak must be adequately resolved from lanosterol. Figure 1 shows a typical chromatogram which should be obtained from a silylated resolution test mixture (4.10),

— the relative retention times of the following sterols should be approximately: — cholesterol: 1,0 stigmasterol: 1,3 sitosterol: 1,5 betulin: 2,5

— the retention time for betulin should be approximately 24 minutes.

5.4.2. Analytical procedure

Inject 1 μl of silylated standard solution (stigmasterol or sitosterol) and adjust the integrator calibration parameters.

Inject a further 1 μl of silylated standard solution to determine the response factors with reference to betulin.

Inject 1 μl of silylated sample solution and measure peak areas. Each chromatographic run must be bracketed by an injection of standards.

As a guide, six injections of sample should be included in each bracketed run.

Note 6: Integration of the stigmasterol peak should include any tailing as defined by points 1, 2 and 3 in Figure 2b.

Integration of the sitosterol peak should include the area of the 22 dihydro-β-sitosterol (stigmastanol) peak which elutes immediately after sitosterol (see Figure 3b) when evaluating total sitosterol.

6. CALCULATION OF RESULTS

6.1. Determine the area of the sterol peaks and betulin peaks in both standards bracketing a batch, and calculate R1: R1 = (average area of the sterol peak in the standard)/(average area of the betulin peak in the standard) Determine the area of the sterol peak (stigmasterol and sitosterol) and betulin peak in the sample and calculate R2: R2 = (area of the sterol peak in the sample)/(area of the betulin peak in the sample) W1 = sterol content of the standard (mg) contained in 1 ml of standard solution (4.8.1 or 4.9.1) W2 = weight of sample (g) (5.2.1) P = purity of standard sterol (4.8 or 4.9) Sterol content of the sample (mg/kg) = ((R 2) / (R 1)) × ((W 1) / (W 2)) × P × 10.

7. ACCURACY OF THE METHOD

7.1. Butter

7.1.1. Repeatability

7.1.1.1. Stigmasterol

The difference between the results of two determinations carried out within the shortest feasible time interval, by one operator using the same apparatus on identical test material, may not exceed 19,3 mg/kg.

7.1.1.2. Sitosterol

The difference between the results of two determinations carried out within the shortest feasible time interval, by one operator using the same apparatus on identical test material, may not exceed 23,0 mg/kg.

7.1.2. Reproducibility

7.1.2.1. Stigmasterol

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 31,9 mg/kg.

7.1.2.2. Sitosterol

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 8,7 % relative to the mean of the determination.

7.1.3. Source of precision data

The precision data were determined from an experiment conducted in 1992 involving eight laboratories and six samples (three blind duplicates) for stigmasterol and six samples (three blind duplicates) for sitosterol.

7.2. Concentrated butter

7.2.1. Repeatability

7.2.1.1. Stigmasterol

The difference between the results of two determinations carried out within the shortest feasible time interval, by one operator using the same apparatus on identical test material, may not exceed 10,2 mg/kg.

7.2.1.2. Sitosterol

The difference between the results of two determinations carried out within the shortest feasible time interval, by one operator using the same apparatus on identical test material, may not exceed 3,6 % relative to the mean of the determinations.

7.2.2. Reproducibility

7.2.2.1. Stigmasterol

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 25,3 mg/kg.

7.2.2.2. Sitosterol

The difference between the results of two determinations carried out by operators in different laboratories, using different apparatus on identical test material, may not exceed 8,9 % relative to the mean of the determinations.

7.2.3. Source of precision data

The precision data were determined from an experiment conducted in 1991 involving nine laboratories and six samples (three blind duplicates) for stigmasterol and six samples (three blind duplicates) for sitosterol.

8. TOLERANCE LIMITS

8.1. Three samples must be taken from the traced product in order to check on the correct tracing of the product.

8.2. Butter 8.2.1. Stigmasterol 8.2.1.1. The incorporation rate for stigmasterol is 150 g of at least 95 % pure stigmasterol per tonne of butter, i.e. 142,5 mg/kg, or 170 g of at least 85 % pure stigmasterol per tonne of butter, i.e. 144,5 mg/kg. 8.2.1.2. The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits: — 115,8 mg/kg (95 % of the minimum incorporation rate for 95 % pure stigmasterol), — 117,7 mg/kg (95 % of the minimum incorporation rate for 85 % pure stigmasterol), — 80,1 mg/kg (70 % of the minimum incorporation rate for 95 % pure stigmasterol), — 81,5 mg/kg (70 % of the minimum incorporation rate for 85 % pure stigmasterol). The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation respectively between 115,8 mg/kg and 80,1 mg/kg or 117,7 mg/kg and 81,5 mg/kg. 8.2.2. Sitosterol 8.2.2.1. The incorporation rate for sitosterol is 600 g of at least 90 % pure sitosterol per tonne of butter, i.e. 540 mg/kg 8.2.2.2. The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits: — 482,6 mg/kg (95 % of the minimum incorporation rate for 90 % pure sitosterol), — 347,6 mg/kg (70 % of the minimum incorporation rate for 90 % pure sitosterol). The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation between 482,6 mg/kg and 347,6 mg/kg.

8.3. Concentrated butter 8.3.1. Stigmasterol 8.3.1.1. The incorporation rate for stigmasterol is 150 g of at least 95 % pure stigmasterol per tonne of concentrated butter, i.e. 142,5 mg/kg; or 170 g of at least 85 % pure stigmasterol per tonne of concentrated butter, i.e. 144,5 mg/kg 8.3.1.2. The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits: — 118,5 mg/kg (95 % of the minimum incorporation rate for 95 % pure stigmasterol), — 120,4 mg/kg (95 % of the minimum incorporation rate for 85 % pure stigmasterol), — 82,9 mg/kg (70 % of the minimum incorporation rate for 95 % pure stigmasterol), — 84,3 mg/kg (70 % of the minimum incorporation rate for 85 % pure stigmasterol). The tracer concentration of the sample giving the lowest result is used in conjunction with interpolation respectively between 118,5 mg/kg and 82,9 mg/kg or 120,4 mg/kg and 84,3 mg/kg. 8.3.2. Sitosterol 8.3.2.1. The incorporation rate for sitosterol is 600 g of at least 90 % pure sitosterol per tonne of concentrated butter, i.e. 540 mg/kg 8.3.2.2. The results for the three samples obtained from the analysis of the product are used to check the rate and the homogeneity of tracer incorporation and the lowest of these results is compared with the following limits: — 480,9 mg/kg (95 % of the minimum incorporation rate for 90 % pure sitosterol), — 345,9 mg/kg (70 % of the minimum incorporation rate for 90 % pure sitosterol). The tracer concentration in the sample giving the lowest result is used in conjunction with interpolation between 480,9 mg/kg and 345,9 mg/kg. Complete resolution is preferable, i.e. the peak trace for lanosterol should return to baseline before leaving for the sitosterol peak although incomplete resolution is tolerable.



Figure 2a Figure 2b Stigmasterol standard Butter sample denatured with stigmasterol Note: Integration of the stigmasterol peak should include any tailing as defined by points 1, 2 and 3.



Figure 3a Figure 3b Sitosterol standard Butter sample denatured with β-Sitosterol Note: β-sitosterol often contains an impurity (identified as stigmastanol) which elutes immediately after β-sitosterol. The areas of these two peaks should be summed when evaluating the total β-sitosterol present.









ANNEX IX

(Article 6)

REFERENCE METHOD FOR THE DETECTION OF COWS’ MILK AND CASEINATE IN CHEESES FROM EWES’ MILK, GOATS’ MILK OR BUFFALOS’ MILK OR MIXTURES OF EWES’, GOATS’ AND BUFFALOS’ MILK

1. SCOPE

Detection of cows’ milk and caseinate in cheeses made from ewes’ milk, goats’ milk, buffalos’ milk or mixtures of ewes’, goats’ and buffalos’ milk by isoelectric focusing of γ-caseins after plasminolysis.

2. FIELD OF APPLICATION

The method is suitable for sensitive and specific detection of native and heat-treated cows’ milk and caseinate in fresh and ripened cheeses made from ewes’ milk, goats’ milk, buffalos’ milk or mixtures of ewes’, goats