Commission Regulation (EC) No 213/2001

of 9 January 2001

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 and amending Regulations (EC) No 2771/1999 and (EC) No 2799/1999

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(14) thereof,

Whereas:

(1) Commission Regulations (EEC) No 1216/68 and (EEC) No 3942/92 and (EC) No 86/94, (EC) No 2721/95, (EC) No 1080/96, (EC) No 1081/96, (EC) No 1082/96, (EC) No 1854/96, (EC) No 880/98 and (EC) No 1459/98, the full references of which are given in Annex XXVI to this Regulation, lay down reference and routine methods for the analysis and quality evaluation of milk and milk products and establish the scope and rules for applying those methods. In the interests of clarity and to provide those operating in the sector with a single set of methods and rules for applying them, the abovementioned Regulations should be recast and brought together in a single text. For the same reason, Commission Regulations (EC) No 2771/1999 of 16 December 1999 laying down detailed rules for the application of Council Regulation (EC) No 1255/1999 as regards intervention on the market in butter and cream(2) and (EC) No 2799/1999 of 17 December 1999 laying down detailed rules for applying Regulation (EC) No 1255/1999 as regards the grant of aid for skimmed milk and skimmed-milk powder intended for animal feed and the sale of such skimmed-milk powder(3) should be amended so that the annexes to those Regulations concerning analysis methods can be included in this 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 CEN, IDF, ISO and 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 each year and only methods included in the list may be applied.

(4) The use of routine methods should not be ruled out. The conditions for using them should therefore be specified.

(5) Common methods 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 are applied as reference methods.

(7) Commission Regulation (EC) No 2571/97 of 15 December 1997 on the sale of butter at reduced prices and the granting of aid for cream, butter and concentrated butter for use in the manufacture of pastry products, ice-cream and other foodstuffs(4), as last amended by Regulation (EC) No 635/2000(5), provides for the tracing of cream, butter and concentrated butter in certain circumstances in order to ensure the correct end use of these products. Since tracing is important if the scheme is to function properly, and 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 Commission Regulation (EEC) No 3143/85 of 11 November 1985 on the sale at reduced prices of intervention butter intended for direct consumption in the form of concentrated butter(6), as last amended by Regulation (EC) No 101/1999(7), Commission Regulation (EEC) No 429/90 of 20 February 1990 on the granting by invitation to tender of an aid for concentrated butter intended for direct consumption in the Community(8), as last amended by Regulation (EC) No 124/1999(9), and Regulation (EC) No 2571/97, concentrated butter must have tracers added to it under supervision. Compliance with the requirements for tracing concentrated butter must be strictly enforced to ensure that products are not diverted. A common method for detecting such tracers should therefore be laid down.

(9) 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 these 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 provisions, 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.

(10) Under Commission Regulation (EEC) No 2921/90 of 10 October 1990 on the granting of an aid for skimmed milk used for the production of casein and caseinates(10), as last amended by Regulation (EC) No 2654/1999(11), the absence of coliforms must be detected. The internationally accepted reference method for detecting coliforms in milk and milk products is International Standard IDF 73A: 1985. However, that standard is applicable only in a modified form for detecting coliforms in a certain quantity of product. A Community reference method for detecting coliforms has therefore been established based on the abovementioned standard.

(11) Council Regulation (EEC) No 2658/87 of 23 July 1987 on the tariff and statistical nomenclature and on the Common Customs Tariff(12), as last amended by Regulation (EC) No 254/2000(13), 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.

(12) 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.

(13) Implementation of some of the methods introduced for the first time in this Regulation will require a period of adaptation. Application of those methods should therefore be deferred.

(14) The Management Committee for Milk and Milk Products has not delivered an opinion within the time limit set by its chairman,

HAS ADOPTED THIS REGULATION:

CHAPTER I

GENERAL PROVISIONS

Article 1

Scope and field of application

This Regulation lays down the rules for applying the 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. It also lays down some of those methods.

Article 2

List of methods

1. Annex I to this Regulation contains the list of reference methods applicable to analyses as referred to in Article 1.

2. The Commission shall update the list at least once a year in accordance with the procedure laid down in Article 42 of Regulation (EC) No 1255/1999.

Article 3

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.

The procedure described in Annex II may be applied for checking results obtained by routine methods which are close to the limits specified in the Regulations concerned.

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

Article 4

Validation of reference methods

1. Reference methods shall be validated if they meet pre-determined precision criteria concerning the repeatability and reproducibility limit.

2. If a reference method laid down in the Regulations concerned has not been validated, Member States shall set a provisional reproducibility limit.

That limit shall be obtained according to the procedure described in Annex III(b). However, for the first 18 months following the entry into force of this Regulation, the Member States may use an equivalent procedure.

Compliance with the limit shall be checked at least once a year.

3. Where the results of applying validated reference methods or methods with provisional precision figures show that a limit has been exceeded, the analytical results may be evaluated using the method described in Annex IV to determine the critical difference from the limit concerned.

Article 5

Admissibility of analysis results

1. Analyses shall be performed in laboratories operating an internal quality control procedure in accordance with that described in Annex V(a) or a procedure of an equivalent standard.

A detailed description of the procedure applied must be available for consultation in the laboratory.

2. Laboratories shall establish their in-house precision within a run for all methods, following:

(a) the procedure described in Annex V(b), or

(b) a published, validated procedure with an established repeatability.

Compliance with the reproducibility limit must be checked at least once per year using the procedure described in Annex III(a).

The second subparagraph shall not apply to laboratories that have participated in a proficiency testing scheme during the year.

3. 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 IV and Annex VIII.

4. Analysis results shall be considered admissible if they have been obtained in accordance with the acceptability criteria described in the internal quality control procedure referred to in paragraph 1 and the in-house precision referred to in paragraph 2.

Article 6

Sensory evaluation

1. For butter, the procedures described in Annex VI shall be applied to check the performance of assessors and the reliability of results. The procedure described in Annex VII shall be applied as a reference method for sensory evaluation.

2. For milk and milk products other than butter, 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 VI may be used to check the performance of assessors and the reliability of results.

Article 7

Sampling and disputes over the results of analyses

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

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

CHAPTER II

METHODS OF ANALYSIS

Article 8

Water/solids-non-fat/fat content of butter

1. The analysis method described in Annex IX shall be used as the reference method for determining the water content of butter.

2. The analysis method described in Annex X shall be used as the reference method for determining the solids-non-fat content of butter.

3. The analysis method described in Annex XI shall be used as the reference method for determining the fat content of butter.

Article 9

Tracers

1. The analysis method described in Annex XII shall be used as the reference method for determining vanillin in concentrated butter, butter and cream.

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

3. The analysis method described in Annex XIV shall be used as the reference method for determining the β-sitosterol or stigmasterol content of butter and concentrated butter.

4. Concentrated butter, butter and cream have been traced in conformity with the relevant Community rules if the results obtained are in accordance with the specifications of paragraph 8 of the Annexes referred to in paragraphs 1, 2 and 3.

Article 10

Detection of cows' milk casein

1. The reference method of analysis described in Annex XV shall be used to ensure that cheese which must be made exclusively from ewes' milk, goats' milk or buffalos' milk or from a mixture of ewes', goats' and buffalos' milk does not in fact contain cows' milk casein.

Cows' milk casein shall be held to be present if the apparent cows' milk casein content of the sample to be analysed is equal to or higher than the content of the reference sample containing 1 % cows' milk as described in Annex XV.

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

(a) the detection limit is 0,5 % or lower,

(b) there are no false-positive results,

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

If the requirement provided for in (b) is not met, any sample giving a positive result must be analysed using the reference method.

If the requirement provided for in (c) is not met for one of the types of cheese referred to in paragraph 1, that cheese must be analysed using the reference method.

Article 11

Detection of coliforms

1. The reference method of analysis described in Annex XVI shall be used to detect the presence of coliforms in butter, skimmed-milk powder, casein and caseinates.

2. Routine methods for detecting coliforms may be used provided that the results obtained are comparable with the results obtained by the reference method described in the said Annex. Routine methods must in particular have an adequate detection limit. False-negative results must not occur. If the occurrence of false-positive results cannot be excluded, any positive result must be confirmed using the reference method.

Article 12

Lactose content

The method for determining the lactose content of products falling within CN code 2309 is described in Annex XVII.

Article 13

Detection of rennet whey

1. The method for detecting rennet whey in skimmed-milk powder intended for public storage is described in Annex XVIII.

2. The method for detecting rennet whey in skimmed-milk powder and mixtures intended for use as animal feed is described in Annex XIX.

Article 14

Detection of buttermilk

The method for detecting buttermilk in skimmed-milk powder is described in Annex XX.

Article 15

Antimicrobiotic residues

The method for detecting antibiotic and sulphonamide/dapson residues in skimmed-milk powder is described in Annex XXI.

Article 16

Skimmed-milk powder content

The method for determining the skimmed-milk powder content in compound feedingstuffs is described in Annex XXII.

Article 17

Detection of starch

The method for detecting starch in skimmed-milk powder, denatured milk powder and compound feedingstuffs is described in Annex XXIII.

Article 18

Moisture content of acid buttermilk powder

The method for determining the moisture content of acid buttermilk powder intended for use in feedingstuffs is described in Annex XXIV.

Article 19

Detection of foreign fats

The method for detecting foreign fats in milk fats is described in Annex XXV.

CHAPTER III

FINAL PROVISIONS

Article 20

Amendments to Regulation (EC) No 2771/1999

Regulation (EC) No 2771/1999 is amended as follows:

1. The first sentence of Article 4(1) is replaced by the following: "The competent authorities shall check the quality of butter using the methods described in Annex I and on the basis of samples taken in accordance with the rules set out in Annex IV."

2. In Annex I, footnote 2 is replaced by the following: "See Annex I to Regulation (EC) No 213/2001".

3. Annexes II and III are deleted.

4. In the penultimate sentence of Annex IV.2, the words "with Annex III" are replaced by "with Annex VII to Regulation (EC) No 213/2001".

Article 21

Amendments to Regulation (EC) No 2799/1999

Regulation (EC) No 2799/1999 is amended as follows:

1. Article 20(1), (2), (3) and (4) is replaced by the following: "1. The skimmed-milk powder content of mixtures and compound feedingstuffs shall be determined by testing each sample at least in duplicate using the analysis method described in Annex XXII to Regulation (EC) No 213/2001, supplemented by the checks provided for in Article 17(3) of this Regulation. Should there be a discrepancy between the results of these checks, the result of the on-the-spot inspection shall be conclusive.

2. The absence of rennet whey shall be proven using the method described in Annex XIX to Regulation (EC) No 213/2001.

3. The starch content of compound feedingstuffs shall be determined by the checks provided for in Article 17(3) of this Regulation, which must be supplemented with the analysis method described in Annex XXIII to Regulation (EC) No 213/2001.

4. The moisture content of acid buttermilk powder shall be determined using the analysis method described in Annex XXIV to Regulation (EC) No 213/2001."

2. Annexes III, IV, V and VI are deleted.

Article 22

Repeals

Regulations (EEC) No 1216/68, (EEC) No 3942/92, (EC) No 86/94, (EC) No 2721/95, (EC) No 1854/96, (EC) No 1080/96, (EC) No 1081/96, (EC) No 1082/96, (EC) No 880/98 and (EC) No 1459/98 are repealed.

References to the repealed Regulations shall be construed as references to this Regulation.

Article 23

Entry into force

This Regulation shall enter into force on the seventh day following its publication in the Official Journal of the European Communities.

However, the methods described in Annexes III, IV.4, V, VI and VIII shall apply 18 months after the entry into force of this Regulation.

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

Done at Brussels, 9 January 2001.

For the Commission

Franz Fischler

Member of the Commission

(1) OJ L 160, 26.6.1999, p. 48.

(2) OJ L 333, 24.12.1999, p. 11.

(3) OJ L 340, 31.12.1999, p. 3.

(4) OJ L 350, 20.12.1997, p. 3.

(5) OJ L 76, 25.3.2000, p. 9.

(6) OJ L 298, 12.11.1985, p. 9.

(7) OJ L 11, 16.1.1999, p. 14.

(8) OJ L 45, 21.2.1990, p. 8.

(9) OJ L 16, 21.1.1999, p. 19.

(10) OJ L 279, 11.10.1990, p. 22.

(11) OJ L 325, 17.12.1999, p. 10.

(12) OJ L 256, 7.9.1987, p. 1.

(13) OJ L 28, 3.2.2000, p. 16.

ANNEX I

(Article 2)

LIST OF REFERENCE METHODSNotes to list of European Union reference methods

Note 1: Milk fat isolation as described in IDF Standard 6B:1989 (protection from light).

Note 2: No reference method has been established.

Note 3: Sample to be prepared according to IDF Standard 122C:1996 or IDF Standard 73A:1985.

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: % SNF = % solids - % fat.

Note 6: Butter must correspond to the national quality class of the Member State of production referred to in Annex V to Commission Regulation (EC) No 2771/1999.

Note 7: Commission Directive 84/8/EEC.

Note 8: Commission Regulation (EC) No 1758/94 (OJ L 183, 19.7.1994, p. 14).

Note 9: Commission Directive 78/633/EEC.

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ANNEX II

(Article 3)

CHECKING THE RESULTS OBTAINED BY ROUTINE METHODS WHICH ARE CLOSE TO THE LIMITS SPECIFIED IN THE REGULATIONS FOR COMPOSITION AND QUALITY REQUIREMENTS

If mo is a limit for composition and quality requirements specified in a Regulation, the decision limit (L) is

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if RRout/RRef <= 1RRout: Reproducibility limit of the routine method

RRef: Reproducibility limit of the reference method

If mo is an upper limit and RRout/RRef > 1, this decision limit is obtained using the formula

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If, under the same conditions, mo is a lower limit, the decision limit is obtained using the formula

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where CrD95 is the critical difference of the reference method (see Annex IV).

Where mo is an upper limit, a final result above the decision limit obtained using a routine method must be substituted with a final result obtained using the reference method. This final result must be based on at least the same number of analyses/samples as the final result obtained using the routine method.

Where mo is a lower limit, the same procedure must be followed for a final result below the decision limit obtained using a routine method.

Remark

The procedure described above can be followed if there are no detectable matrix effects.

Matrix effects can be detected in the following way: for each sample used for calibration, the difference (wi) between the results obtained by the reference and the routine method is determined.

The standard deviation calculation using the formula

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m: Number of samples used for calibration

is compared with the arithmetic mean of the repeatability standard deviation of the reference and the routine method

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A matrix effect cannot be excluded, if

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where

f= m (f: number of degrees of freedom)

α= error probability; α = 0,05.

In this case, further investigations are necessary before a decision limit can be fixed.

ANNEX III

(Articles 4 and 5)

(a) Procedure for determining compliance with an established reproducibility limit (Chemical analysis)

Compliance with the reproducibility limit is checked by comparing the laboratory results with the results of an experienced laboratory(1), obtained using an identical sample. A double determination is carried out in both laboratories and the results are evaluated using the formula:

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where:

CrD95: critical difference (P = 0.95)

>REFERENCE TO A GRAPHIC>: arithmetic mean of two results obtained in laboratory 1

>REFERENCE TO A GRAPHIC>: arithmetic mean of two results obtained in laboratory 2

R: reproducibility limit: to be determined by interpolation,

r: repeatability limit: if precision varies with level.

If the critical difference is exceeded, another experiment must be performed within the next two months. If the results of the second experiment do not comply with the reproducibility limit, the competent authorities must take the necessary steps.

(b) Procedure for obtaining a provisional reproducibility limit (Chemical analysis)

A provisional reproducibility limit (Rprov) is obtained using the following equation:

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where:

>REFERENCE TO A GRAPHIC>: mean of two results obtained in laboratory 1

>REFERENCE TO A GRAPHIC>: mean of two results obtained in laboratory 2 (see Annex IIIa)

r: repeatability limit or provisional repeatability limit.

Remarks:

1. Rprov can be used to calculate critical differences (see Annex VI).

2. Rprov is fixed at 2r if the calculated value for Rprov is smaller than 2r.

3. If the calculated value is larger than 3r or greater than twice the R-value predicted from the Horwitz equation(2), then Rprov is unacceptably high and cannot be used to calculate the critical difference.

4. Rprov should be determined at least once per year on the basis of results obtained in two laboratories (see Annex IV).

5. The mean value of Rprov must be used for the calculation of critical differences. The rules given under 2 and 3 apply for the mean value of Rprov.

The reproducibility limit (R-value) is obtained from the calculated RSDR-value as follows

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>REFERENCE TO A GRAPHIC>: arithmetic mean of the results obtained)

Some calculated RSDR values (examples)

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A concentration of the analyte of 1 g/100 g will obtain:

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(1) The experienced laboratory should generally be one which has participated successfully either in the validation of the test method or in a proficiency test.

(2) Horwitz equation:

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where:

RSDR: relative standard deviation of reproducibility

c: concentration expressed as a decimal fraction (example: 10 g/100 g = 0,1).

Reference:

Peeler, J.T., Horwitz, W. and Albert, R.

J.Ass. Off. Anal. Chem.

72(5), 784-806 (1989).

ANNEX IV

(Article 4)

EVALUATION OF ANALYTICAL RESULTS OBTAINED USING VALIDATED METHODS

If the analytical result shows that a limit has been exceeded, the arithmetic mean of two or more results is calculated. The following procedure is to be followed:

1. In cases where the analytical result represents a single result, a second analysis must be carried out under repeatability conditions. If the two analyses cannot be carried out under repeatability conditions, carry out a further duplicate analysis under repeatability conditions and use those results for the assessment of critical difference compliance.

2. The absolute value of the difference between the arithmetic mean of the results obtained under repeatability conditions and the limit is determined. An absolute value of the difference greater than the critical difference means that the sample which has been analysed does not fulfil the requirements.

The critical difference is determined using the following formula:

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where:

>REFERENCE TO A GRAPHIC>: arithmetic mean of the results obtained

mo: limit

n: number of analyses/sample

If precision varies with level, it may be necessary to determine r and R by interpolation.

Normally, a final result reported for a sample must show that a limit has been comlied with.

Final results

- within the range

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and

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- within the range

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and

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should therefore occur only exceptionnaly.

Final results within the ranges mentioned are acceptable only if they occur no more than once for every five samples analysed per consignment. If less than five samples are analysed per consignment, one result within the range mentioned is acceptable. However, the rule that only one result within the ranges mentioned is obtained per five samples analysed must be adhered to if consignments are repeatedly offered by a producer.

3. If the final result x is calculated using a formula of the form x = y1 +- y2 (example: water + solids-non-food-fat content of butter to calculate the fat content) where y1 and y2 are the final results of a single type of analysis, then the overall repeatability and reproducibility limits rx and Rx of the final results x are calculated as:

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where r1 and r2 are the repeatability limits and R1 and R2 the reproducibility limits of y1 and y2,, respectively.

x is compared with the limit m0 following the rules specified under 1 and 2. The critical difference is determined using the formula:

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where x is the arithmethic mean of the results x1 obtained.

4. If the final result is calculated using as formula of the form

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(example: fat in dry matter content of cheese)

where y1 and y2 are the final results of a single type of analysis, then the overall repeatability and reproducibility limits rx and Rx can be calculated as:

>REFERENCE TO A GRAPHIC>

>REFERENCE TO A GRAPHIC>

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μ1: limit or target value for y1 (example: fat)

μ2: limit or target value for y2 (example: dry matter)

>REFERENCE TO A GRAPHIC>

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where

r1: repeatability limit, y1

r2: repeatability limit, y2

>REFERENCE TO A GRAPHIC>

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where

R1: reproducibility limit, y1

R2: reproducibility limit, y2

The procedures for calculating rx and Rx are applicable only if the relative repeatability and reproducibility limits (r*1, r*2; R*1; R*2) are smaller than or equal to 0,15.

x is compared with the limit μx following the rules specified under 1 and 2. The critical difference is determined using the formula:

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Where

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is the arithmetic mean of the results x obtained in chronological order(1).

(1) Note:

If, for example, the results y11, y12, y21 and y22, are obtained, the arithmetic mean of y11/y21 and y12/y22 must be calculated.

ANNEX V

INTERNAL CONTROL

(Article 5)

(a) Internal quality control (IQC) procedure (chemical analysis)

Definition of control material

A material used for the purpose of IQC is subjected to the same, or part of the same, procedure as tested materials.

A control material may be:

- certified reference material,

- in-house reference material,

- material validated by an interlaboratory test,

- fortified material.

Procedure for establishing IQC

The laboratory should introduce IQC following the procedure described in the IUPAC document "Harmonized Guidelines for Internal Quality Control in analytical laboratories"(1).

IQC involves including control materials in the analytical sequence or replicating analysis of the test sample. Control materials must be similar in chemical composition to test samples and be adequately stable over the period of interest. It must be demonstrated that they can be suitably divided into identical portions for analysis, and are of concentration of the analyte appropriate to the range of interest.

Control material must be inserted at least once in each analytical run and the value obtained plotted on a control chart in order to measure long-term errors. In addition, the laboratory should periodically demonstrate compliance with repeatability conditions within the run. This may be achieved by duplicate analysis of control and/or test materials. Results of these analyses should be compared with any published repeatability limits and existing data on in-house precision.

Where control materials are used, the values obtained for the between-run analysis of the control material must be plotted on a Shewhart chart (ISO 8258 (1991)) with appropriate control limits. Action limits must be set at

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where st is the total standard deviation,

warning limits at

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Total standard deviation:

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in which:

sb: between-run standard deviation

sw: within-run standard deviation

n: number of determinations

In cases where control materials are not used (e.g. because of lack of stability), at least one of the test materials must be analysed in duplicate in each run.

The absolute difference obtained from duplicate analyses within a run (see Annex III) must be plotted. The centre line is 1.128 sw, the lower limit is 0, the upper limit (action limit) is 3.686 sw, where sw, is the within-run standard deviation.

The control procedure should include materials of low and high levels when the concentration range is large.

If the test materials cover a wide range of analyte concentrations, the laboratory should establish the relationship between precision and level. If precision is proportional to level, subsequent control should be based on relative precision (i.e. absolute difference as a percentage of mean value).

An out-of-control condition in the analytical system is signalled if any of the following occur:

A. the current plot value falls outside the action limits,

B. the current value and the previous value fall outside the warning limits but within the action limits,

C. where control materials are used, nine successive values fall on the same side of the mean line.

The laboratory should respond to an out-of-control condition by:

A. stopping analysis pending diagnostic tests and remedial action, and

B. rejecting the run of results and re-analysing the test materials.

(b) Procedure for selecting in-house control material and determining "in-house" precision limits (chemical analysis)

Data on within-laboratory precision may be obtained by replicate analysis of control materials and/or by replicate analysis of test samples.

Laboratories should use the following procedure to establish precision parameters for within-run and between-run variation for subsequent use in constructing control charts. Laboratories may adopt alternative procedures provided that they can adequately demonstrate that reliable precision data have been derived.

1. Selection of control materials

Where it is appropriate for the laboratory to use a control material, data must first be gathered in order to set limits. Where possible, certified reference materials (CRMs) should be used. Candidate control materials should be analysed under repeatability conditions within a run including suitable CRMs with replication and randomisation. Where this approach is not possible, laboratories should seek to participate in proficiency testing and to establish consensus means (assigned values) which may be regarded as a conventional true mean to which a meaningful uncertainty could be attached. Other procedures include assigning true value by formulation or the use of spiked control materials.

In addition, where the laboratory regularly carries out this type of analysis and has already established statistical control, any new control materials (e.g. required because stocks have run out) must be obtained with reference to analyses which are under control using the existing materials.

2. Assignment of limits

Having selected a control material, the laboratory should use it to establish within-run and between-run precision figures.

As a minimum requirement for establishing within-run precision, the control material should be analysed in duplicate on 12 occasions. Duplicate analysis should be performed under repeatability conditions, i.e. same operator, same reagents etc. Duplicate analysis of the control material should be randomised within an analytical run. Each duplicate analysis should be undertaken on a separate day over a period of time so as to reflect reasonable run-to-run variation, taking into account normal variations e.g. reagents, instruments re-calibration and, if appropriate, different analysts.

Note:

The use of data which are not fully representative of between-run variation may result in unnecessary replication of analysis because excessively tight limits have been set. Conversely, a laboratory presenting precision data which are too imprecise may be unable to meet prescribed limits in reference methods, could expect to show a poor performance when compared with peer laboratories and may not be producing data which is fit for the intended purpose.

2.1. Determining within-run precision

2.1.1. Within-run precision where a control material is available

Duplicate data (minimum of 12 duplicates) should first be subjected to Cochran's maximum variance test. This involves comparing the squared maximum duplicate range with the sum of square ranges.

>REFERENCE TO A GRAPHIC>

where

di= the difference between duplicates.

The value of Cochran's criterion, C, is compared with tabulated values (ISO 5725 (1994)). Where a value can be classified as a straggler or outlier, the result should be investigated for an explanation, e.g. technical error, computational error, slip in performing the test, analysis of wrong sample. If the explanation of the technical error is such that it proves impossible to replace the suspect result, it should be discarded as a real outlier. When any stragglers or outliers remain that cannot be explained, the stragglers are retained as correct and the statistical outliers are discarded. The laboratory should seek to obtain replacement values.

Once the laboratory is satisfied that data are free from outliers, the within-run standard deviation sw is obtained as follows: for each pair xi1, xi2 of the p duplicate data, the sum of the duplicates,

>REFERENCE TO A GRAPHIC>

and the difference of the duplicates,

>REFERENCE TO A GRAPHIC>

are computed and summed up to

>REFERENCE TO A GRAPHIC>

>REFERENCE TO A GRAPHIC>

>REFERENCE TO A GRAPHIC>

An estimate of the within-run standard deviation is

>REFERENCE TO A GRAPHIC>

The in-house precision limit is 2.8. sw.

If a reference method is used, the in-house precision limit should be compared with the published repeatability limit. The laboratory should comply with the requirement of the reference method. Failure to meet this requirement must be investigated.

The limits set should be regarded as provisional and subject to review.

2.1.2. Within-run precision where a control material is not available

The laboratory may choose to establish within-run precision by duplicate analysis of representative test samples (minimum of 12 duplicate analyses). In cases where it is not possible to use control materials, e.g. because of instability, duplicate data must be accumulated by this method.

Note:

It is assumed that the analyses cover a relatively narrow range of values and therefore a single value can be applied to all samples. In cases where the range of results is wider, e.g. over an order of magnitude, and precision is dependent on level, laboratories should investigate use of relative standard deviations.

Data should be subjected to Cochran's test, as in 2.1.1. Once the laboratory is satisfied that data are free from outliers, the within-run standard deviation and in-house precision limit may be obtained as in section 2.1.1.

The within-run standard deviation sw may be used to construct control charts (see Annex II). The limits set should be regarded as provisional and subject to review.

2.2. Determining between-run precision

Compute the average value (s1/2) for each pair and subject these to Grubbs test (ISO 5725 (1994)). Rejection/acceptance criteria for outliers or stragglers are as described in 2.1.1. The laboratory should seek to obtain a replacement value for any discarded result. Once the laboratory is satisfied that the data are free from outliers, the between-run standard deviation sb is calculated.

>REFERENCE TO A GRAPHIC>

or 0 if the expression under the square root sign is negative.

The total standard deviation st is used to construct control charts for the average of n determinations (see Annex II). The limits set should be regarded as provisional and subject to review.

3. Review of initial limits

Control limits set as described above must be regarded as initial estimates.

In order to update limits set on the basis of acceptable within-run precision (section 2.1.2), further duplicate data on test samples should be collected. The interval before review will depend on the frequency of analysis. As a guideline, data should be reviewed after a further 10 duplicates have been obtained. All the data should then be subjected to Cochran's test and limits reset on the basis of the new standard deviation figure. Subsequent decisions on the validity of controls limits must be made in the light of further data.

Review of initial data obtained for between-run precision also depends on the frequency of analysis. As a guideline, after a further ten data points have been obtained from analysis of the control material, at a frequency of one analysis per batch, the initial assumptions made on standard deviation and mean should be reviewed.

All the data should be subjected to Grubbs test for outliers. The mean and standard deviation should be recalculated on the basis of the new data.

In addition, at this stage, the laboratory should apply a Cusum chart (BS S700: (1984) and amendment 5480 (1987)) to investigate any problems which may be associated with e.g. ageing of reagents. Any single result falling outside the Cusum "V-mask" limits must be investigated.

The new limits (mean and standard deviation) must be subject to regular inspection using the Cusum technique. Any indication that the validity of the control material is being brought into question must be thoroughly investigated.

4. Reporting of precision data

Laboratories must send the following information to the competent national authority:

- the method employed,

- the within-run standard deviation sw, and in-house precision limit,

- the between-run standard deviation sb,

- the total standard deviation s1,

- the number of analyses involved in obtaining precision data.

(1) M. Thompson and R. Wood: "Pure and Applied Chemistry" 67 (4), 649-666 (1995).

ANNEX VI

(Article 6)

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:

>REFERENCE TO A GRAPHIC>

where:

wI: 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:

>REFERENCE TO A GRAPHIC>

where:

xi1; xi2: see section (a)

>REFERENCE TO A GRAPHIC>;

>REFERENCE TO A GRAPHIC>: average score of the assessor group for the first and second evaluation respectively of sample xi

n: number of samples (at least ten 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.

(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 VII

(Article 6)

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.

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.

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

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

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

3.3. 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 temperature control.

4. Selection of assessors

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

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 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-favoured (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. Assessment of the value of each attribute

6.1. 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, mould growth and water dispersion. Water dispersion is tested according to IDF-Standard 112A/1989.

Consistency involves the following features: firmness and spreadability.

Physcial methods may be applied for the evaluation of butter consistency. The Commission envisages the future harmonisation of these methods.

Flavour involves the following features: taste and odour.

A significant deviation from the recommended temperature prevents a reliable evaluation of consistency and flavour. The temperature is of paramount importance.

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

6.3. 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.

6.4. Scoring for acceptance is as follows:

>TABLE>

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.

7. 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 must record the individual scores for each attribute in the control document and certify whether the product is accepted or rejected.

8. Sampling and preparation of the sample

8.1. - It is desirable that the identity of the samples are concealed during the assessment so that any possible bias is avoided.

- This should be organized by the panel leader prior to the evaluation without the presence of the other panel members.

8.2. When the sensory evaluation is carried out at the 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.

8.3. During the evaluation, the butter should have the temperature 10 to 12 °C. Large deviations should be avoided at all cost.

9. Nomenclature

Refer to the appended table 2.

Table 1: Butter scoring

>TABLE>

>TABLE>

>TABLE>

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 & 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 VIII

(Article 7)

PROCEDURE APPLICABLE WHEN THE RESULTS OF AN ANALYSIS ARE DISPUTED (chemical analysis)

1. A further analysis may be carried out at the request of the operator, within seven working days of notification of the results of the first analysis, provided that sealed duplicate samples of the product are available and have been stored appropriately with the competent authorities.

2. The competent authority will send these samples to a second laboratory, at the request and expense of the operator. This laboratory must be authorised to carry out official analyses and must have a proven competence for the analyses under question. This competence should be documented by a successful participation in collaborative studies, proficiency tests or interlaboratory comparisons. The second laboratory must use the reference method. The results obtained by the two laboratories must be evaluated as follows:

(a) Where both laboratories meet the repeatability requirement and the reproducibility requirement

The arithmetic mean of the test results obtained by both laboratories is reported as the final result. This final result is evaluated taking the critical difference into consideration, using the following formula:

>REFERENCE TO A GRAPHIC>

where

>REFERENCE TO A GRAPHIC>: arithmetic mean of all results obtained by both laboratories

mo: limit

R: reproducibility

r: repeatability

n1: number of results obtained by laboratory 1

n2: number of results obtained by laboratory 2.

Note:

If the final result is calculated using the formulae

>REFERENCE TO A GRAPHIC>

or

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(see Annex IV(3) and (4), respectively), R2x and r2x should be inserted into the formulae instead of R2 and r2.

(b) Where both laboratories meet the repeatability requirement but not the reproducibility requirement

If the second analysis confirms the first, the quantity under analysis should be rejected as non-compliant. Otherwise, the quantity should be accepted.

(c) Where only one laboratory meets the repeatability requirement

The final result of the laboratory meeting the repeatability requirement is to be used to decide whether to accept the quantity under analysis.

(d) Where neither laboratory meets the repeatability requirement but the reproducibility requirement is met

(a) applies.

(e) Where neither laboratory meets either the repeatability requirement or the reproducibility requirement

The quantity under analysis should be accepted if the results obtained by one laboratory lead to this conclusion.

(f) Where the results have been obtained using non-validated methods

The quantity under analysis should be accepted if the results obtained by one laboratory lead to this conclusion.

3. The results of the second analysis must be notified by the competent authority to the operator as soon as possible. The costs of the second analysis are to be borne by the operator if the quantity under analysis is rejected.

4. If the operator 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 quantity under analysis must be accepted.

ANNEX IX

(Article 8)

DETERMINATION OF THE WATER CONTENT OF BUTTER

1. Scope and field of application

This reference method specifies a method for the determination of the water content of butter.

2. Reference

IDF standard 50 C: 1995 - Milk and milk products - Methods of sampling

3. Definition

Water content of butter: the loss of mass after completion of the heating process specified in this standard. It is expressed in grams per 100 grams.

4. Principle

Evaporation of the water from a test portion in the presence of pumice at a temperature of 102 °C in a drying oven.

5. Apparatus and materials

Usual laboratory apparatus, and in particular:

5.1. Analytical balance, sensitivity 1 mg.

5.2. Desiccator provided with efficient drying agent (for example, freshly dried silica gel with hygroscopic indicator).

5.3. Drying oven, ventilated, thermostatically controlled, operating at 102 ± 2 °C throughout the total working space.

5.4. Glass, porcelain or non-corrosive metal dishes, height of about 20 mm, diameter 60 to 80 mm.

5.5. Pumice stone, granulated, washed with a diameter of 0,8-10 mm.

6. Sampling

See IDF 50 C: 1995

7. Procedure

7.1. Preparation of the test sample

Warm the laboratory sample in the closed glass or suitable plastic container, which should be from one-half to two-thirds full, to a temperature at which the sample will be soft enough to facilitate thorough mixing to a homogeneous state (either by a mechanical shaker or by hand). The temperature of mixing should normally not exceed 35 °C. Cool the sample to ambient temperature. As soon as possible after cooling open the sample container and stir briefly (not longer than 10 seconds) with a suitable device, for example a spoon or spatula, before weighing.

7.2. Determination of the water content

7.2.1. Place approximately 10 g of pumice in the dish (5.4).

7.2.2. Dry the dish with the pumice in the oven (5.3) at 102 ± 2 °C for at least one hour.

Note:

the drying periods mentioned in 7.2.2, 7.2.5 and 7.2.7 start when the temperature of the oven reaches 102 ± 2 °C.

7.2.3. Allow the dish to cool in the desiccator (5.2) to the temperature of the balance room and weigh to the nearest 1 mg.

7.2.4. Weigh into the dish, to the nearest 1 mg, a test portion of approximately 5 g of the test sample.

7.2.5. Place the dish in the oven at 102 ± 2 °C and leave it for three hours.

7.2.6. Allow the dish to cool in the desiccator to the temperature of the balance room and weigh to the nearest 1 mg.

7.2.7. Repeat the drying process for additional periods of one hour, cooling and weighing each time as specified in 7.2.6 until constant mass (mass change not exceeding 1 mg) is reached.

In the event of an increase in mass, take for calculation the lowest mass recorded.

8. Expression of results

8.1. Method of calculation and formula

Calculate the water content, W, as a percentage by mass using the following formula:

>REFERENCE TO A GRAPHIC>

where

>TABLE>

Report the result to the first decimal place.

8.2. Repeatability

The absolute difference between the results of two single determinations, carried out simultaneously or in rapid succession by the same operator under the same conditions on identical test material, shall not exceed 0,2 %.

8.3. Reproducibility

The absolute difference between two single and independent results, obtained by two operators working in different laboratories on identical test material, shall not exceed 0,3 %.

9. Test report

The test report shall specify the method used and the results obtained. It shall also mention all operating details not specified in this international standard or regarded as optional, together with details of any incidents which may have influenced the results. The test report shall include all information necessary for the complete identification of the sample.

ANNEX X

(Article 8)

BUTTER: DETERMINATION OF SOLIDS-NON-FAT CONTENT

1. Scope and field of application

This standard species a method for the determination of the solids-non-fat content of butter.

2. References

IDF Standard 50 C: 1995 - Milk and Milk products - Methods of sampling

3. Definitions

Solids-non-fat content of butter: The percentage by mass of substances as determined by the procedure specified. It is expressed in grams per 100 grams.

4. Principle

Evaporation of water from a known mass of butter, extraction of the fat with light petroleum and weighing of the residue.

5. Reagent

Light petroleum with a boiling range between 30 and 60 °C. The reagent shall not leave more than 1 mg of residue after evaporation of 100 ml.

6. Apparatus and materials

6.1. Analytical balance sensitivity 1 mg.

6.2. Desiccator provided with efficient drying agent (for example, freshly dried silica gel with hygroscopic indicator).

6.3. Drying oven, ventilated, thermostatically controlled, operating at 102 ± 2 °C throughout the total working space.

6.4. Glass, porcelain or non-corrosive metal dishes, with a spout height of about 20 mm, diameter 60 to 80 mm, provided with a glass stirring rod.

6.5. Filter crucible, sintered glass, pore diameter 16 to 40 μm, with suction flask.

7. Sampling

See IDF standard 50 C: 1995

8. Procedure

8.1. Preparation of the test sample:

Warm the laboratory sample in the closed glass or suitable plastic container, which should be from one-half to two-thirds full, to a temperature at which the sample will be soft enough to facilitate thorough mixing to a homogeneous state (either by a mechanical shaker or by hand). The temperature of mixing should normally not exceed 35 °C. Cool the sample to ambient temperature. As soon as possible after cooling open the sample container and stir briefly (not longer than 10 seconds) with a suitable device, for example a spoon or spatula, before weighing.

8.2. Determination

8.2.1. Dry the dish with the rod (6.4) and the crucible (6.5) in the oven (6.3) for 1 hour. Allow these objects to cool in the desiccator and weigh them together (i.e. dish, rod and crucible) to the nearest 1 mg (m0).

Notes:

- As a rule, a cooling time of 45 minutes is sufficient,

- It is important that the same combination of dish, rod and crucible is used for each test portion if more than one test portion is being analysed in the batch.

8.2.2. Remove the crucible, record the weight of the dish and rod together, to the nearest 1 mg (m1).

8.2.3. Weigh into the dish, to the nearest 1 mg, a test portion of about 5 g of the test sample (8.1) (m2).

8.2.4. Place the dish (containing the rod and the butter) in the oven at 102 ± 2 °C and leave it over night.

8.2.5. Allow the dish (8.2.3) to cool to room temperature.

8.2.6. Add 15 ml of warm (approximately 25 °C) light petroleum to the dish and detach as much as possible of the sediment adhering to the dish using the glass rod. Transfer the solvent into the crucible and allow it to filter in the suction flask.

8.2.7. Carry out operation 8.2.6 four additional times. If there are no traces of fat on the surface of the dish, quantitatively transfer, during the fourth washing, as much as possible of the sediment into the crucible. Otherwise, repeat operation 8.2.6 till complete elimination of all traces of fat.

8.2.8. Wash the sediment in the crucible with 25 ml of warm light petroleum.

8.2.9. Dry the dish and the glass rod, and the crucible together in the oven at 102 +- 2 °C for 30 minutes.

8.2.10. Allow to cool in the desiccator to room temperature and weigh to the nearest 1 mg.

8.2.11. Repeat operations 8.2.9 and 8.2.10 until constant mass (mass change not exceeding 1 mg) is reached for the dish, rod and crucible together (m3).

9. Expression of results

9.1. Calculation of solids-non-fat content

Calculate the solids-non-fat content SNF, as a percentage by mass, using the following formula:

>REFERENCE TO A GRAPHIC>

where

m0 is the mass, in grams, of the empty dish with the glass rod and the crucible (8.2.1)

m1 is the mass, in grams, of the empty dish with the glass rod (8.2.2)

m2 is the mass, in grams, of the test portion and dish with the glass rod (8.2.3)

m3 is the final mass, in grams, in the dish with the rod and the crucible containing sediment (8.2.11)

Report the result to the first decimal place.

9.2. Repeatability

The absolute difference between the results of two single determinations, carried out simultaneously or in rapid succession by the same operator under the same conditions on identical test material, shall not exceed 0,1 %.

9.3. Reproducibility

The absolute difference between two single and independent results, obtained by two operators working in different laboratories on identical test material, shall not exceed 0,2 %.

10. Test report

The test report shall specify the method used and the results obtained. It shall also mention all operating details not specified in this international standard or regarded as optional, together with details of any incidents which may have influenced the results. The test report shall include all information necessary for the complete identification of the sample.

Note:

>TABLE>

It can be concluded that the precision figures obtained for the solids-non-fat determination are valid for the determination of the milk solids-non-fat content.

ANNEX XI

(Article 8)

DETERMINATION OF THE FAT CONTENT OF BUTTER

The fat content is obtained indirecly by determination of the water content and the solids-non-fat content according to Annex IX and Annex X, respectively. The percentage, by mass, of fat is equal to

>REFERENCE TO A GRAPHIC>

>TABLE>

>TABLE>

ANNEX XII

(Article 9)

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 AU full scale), a recorder or integrator and a column thermostat operating at 25 °C;

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

3.6. guard column (ca. 20 mm x 3 mm ID) dry-packed with Perisorb RP 18 (30 to 40 μm) or equivalent.

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)

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.

4.6. Methanol, HPLC quality

4.7. Acetic acid, glacial

4.8. Water, HPLC quality

4.9. HPLC mobile phase

Mix 300 ml methanol (4.6) with about 500 ml water (4.8) and 20 ml acetic acid (4.7) in a volumetric flask of 1000 ml and make up with water (4.8). 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 above 40 °C. 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 2000 rpm and decant immediately. Allow the decanted solution to cool 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). 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) alter 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):

>REFERENCE TO A GRAPHIC>

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

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

>REFERENCE TO A GRAPHIC>

where:

AS= peak area 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).

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 carned 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, tbe 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 (critical difference for a 95 % probability level (DCr95) taken into consideration):

- 221,0 mg/kg (95 % of the minimum incorporation rate),

- 159,0 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 221,0 mg/kg and 159,0 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 (critical difference for a 95 % probability level (DCr95) taken into consideration):

- 79,0 mg/kg (95 % of the minimum incorporation rate),

- 54,0 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 79,0 mg/kg and 54,0 mg/kg.

9. Notes

9.1. Repeatability r is the value below which the absolute difference between two single test results obtained with the same method on identical test material, under the same conditions (same apparatus, same laboratory, and in a short interval of time), may be expected to lie with a specified probability; in the absence of other indications, the probability is 95 %.

9.2. Reproducibility R is the value below which the absolute difference between two single test results obtained with the same method on identical test material, under different conditions (different operators, different apparatus, different laboratories and/or different time), may be expected to lie with a specified probability; in the absence of other indications, the probability is 95 %.

9.3. 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 %.

9.4. 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.

9.5. The analysis is based an a linear calibration line with a zero intercept.

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.

ANNEX XIII

(Article 9)

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.

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:

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).

>REFERENCE TO A GRAPHIC>

Amax= absorbance of the measuring solution at the maximum

A= weight of sample (g)

2550= reference A (1 %, 1 cm) value

The purity of the suspension is P (%).

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 0.1 mg about 0.100 g of apo-carotenic ester suspension (4.1.1) (Wg), 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)), (Mg). Transfer quantitatively to a 20 ml (Vml) 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 reference to the calibration graph (C μ/ml).

5.3. Calibration graph

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.

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.

7.2.2. 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.

7.3. 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 [critical difference for a 95 % probability level (DCr95) taken into consideration]:

- 18.0 mg/kg (95 % of the minimum incorporation rate),

- 13.0 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 18.0 mg/kg and 13.0 mg/kg.

8.3. Concentrated butter

8.3.1. The incorporation rate for concentrated butter, taking into account background absorbance, is 24 mg/kg.

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 [critical difference for a 95 % probability level (DCr95) taken into consideration]:

- 20.0 mg/kg (95 % of the minimum incorporation rate),

- 14.0 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 20.0 mg/kg and 14.0 mg/kg.

ANNEX XIV

(Article 9)

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 x 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 in a uniform thickness 0,25 μm; the column must be capable of resolving trimethyl-silyl derivatives of lanosterol and sitosterol. A BP1, 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 a 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:

>REFERENCE TO A GRAPHIC>

Determine the area of the sterol peak (stigmasterol and sitosterol) and betulin peak in the sample and calculate R2:

>REFERENCE TO A GRAPHIC>

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)

>REFERENCE TO A GRAPHIC>

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 (critical difference for a 95 % probability level (DCr95) taken into consideration):

- 116,0 mg/kg (95 % of the minimum incorporation rate for 95 % pure stigmasterol),

- 118,0 mg/kg (95 % of the minimum incorporation rate for 85 % pure stigmasterol),

- 81,0 mg/kg (70 % of the minimum incorporation rate for 95 % pure stigmasterol),

- 82,0 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 116,0 mg/kg and 81,0 mg/kg or 118,0 mg/kg and 82,0 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 (critical difference for a 95 % probability level (DCr95) taken into consideration):

- 486,0 mg/kg (95 % of the minimum incorporation rate for 90 % pure sitosterol),

- 358,0 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 486,0 mg/kg and 358,0 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 (critical difference for a 95 % probability level (DCr95) taken into consideration):

- 120,0 mg/kg (95 % of the minimum incorporation rate for 95 % pure stigmasterol),

- 122,0 mg/kg (95 % of the minimum incorporation rate for 85 % pure stigmasterol),

- 84,0 mg/kg (70 % of the minimum incorporation rate for 95 % pure stigmasterol),

- 86,0 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 120,0 mg/kg and 84,0 mg/kg or 122,0 mg/kg and 86,0 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 (critical difference for a 95 % probability level (DCr95) taken into consideration):

- 486,0 mg/kg (95 % of the minimum incorporation rate for 90 % pure sitosterol),

- 358,0 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 486,0 mg/kg and 358,0 mg/kg.

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ANNEX XV

(Article 10)

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' and buffalos' milk. It is not suitable for the detection of milk and cheese adulteration by heat-treated bovine whey protein concentrates.

3. Principle of the method

3.1. Isolation of caseins from cheese and the reference standards.

3.2. Dissolving of the isolated caseins and submitting to plasmin (EC.3.4.21.7) cleavage.

3.3. Isoelectric focusing of plasmin-treated caseins in the presence of urea and staining of proteins.

3.4. Evaluation of stained γ3 and γ2-casein patterns (evidence of cows' milk) by comparison of the pattern obtained from the sample with those obtained in the same gel from the reference standards containing 0 % and 1 % cows' milk.

4. Reagents

Unless otherwise indicated, analytical grade chemicals must be used. Water must be double-distilled or of equivalent purity.

Note:

The following details apply to laboratory prepared polyacrylamide gels containing urea, of dimensions 265 x 125 x 0,25 mm. Where other sizes and types of gel are used, the separation conditions may have to be adjusted.

Isoelectric focusing

4.1. Reagents for production of the urea containing polyacrylamide gels

4.1.1. Stock gel solution

Dissolve:

4,85 g acrylamide

0,15 g N, N'-methylene-bis-acrylamide (BIS)

48,05 g urea

15,00 g glycerol (87 % w/w),

in water and make up to 100 ml and store in a brown glass bottle in the refrigerator.

Note:

A commercially available preblended acrylamide/BIS solution can be used in preference to the quoted fixed weights of the neurotoxic acrylamides. Where such a solution contains 30 % w/v acrylamide and 0,8 % w/v BIS, a volume of 16,2 ml must be used for the formulation instead of the fixed weights. The shelf life of the stock solution is a maximum of 10 days; if its conductivity is more than 5 μS, de-ionize by stirring with 2 g Amberlite MB-3 for 30 minutes, then filter through a 0,45 μm membrane.

4.1.2. Gel solution

Prepare a gel solution by mixing additives and ampholytes with the stock gel solution (see 4.1.l).

9,0 ml stock solution

24 mg β-alanine

500 μl ampholyte pH 3,5 - 9,5(1)

250 μl ampholyte pH 5-7(2)

250 μl ampholyte pH 6-8(3).

Mix the gel solution and de-gas for two to three minutes in an ultrasonic bath or in vacuo.

Note:

Prepare the gel solution immediately prior to pouring it (see 6.2).

4.1.3. Catalyst solutions

4.1.3.1. N, N, N' N' - tetramethylethylenediamine (Temed).

4.1.3.2. 40 % w/v ammonium persulphate (PER):

Dissolve 800 mg PER in water and make up to 2 ml.

Note:

Always use freshly prepared PER solution.

4.2. Contact fluid

Kerosene or liquid paraffin

4.3. Anode solution

Dissolve 5,77 g phosphoric acid (85 % w/w) in water and dilute to 100 ml.

4.4. Cathode solution

Dissolve 2,00 g sodium hydroxide in water and dilute to 100 ml with water.

Sample preparation

4.5. Reagents for protein isolation

4.5.1. Dilute acetic acid (25,0 ml of glacial acetic acid made up to 100 ml with water)

4.5.2. Dichloromethane

4.5.3. Acetone

4.6. Protein dissolving buffer

Dissolve

5,75 g glycerol (87 % w/w)

24,03 g urea

250 mg dithiothreitol,

in water and make up to 50 ml

Note:

Store in a refrigerator, maximum shelf-life one week.

4.7. Reagents for plasmin cleavage of caseins

4.7.1. Ammonium carbonate buffer

Titrate a 0,2 mol/l ammoni