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
Date: 2001-01-09
Institutions: European Commission
Subjects: common organisation of markets, food inspection, milk, milk product, product quality

7.2.2001

EN

Official Journal of the European Communities

L 37/1

I

(Acts whose publication is obligatory)

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,

amended so that
the annexes to those Regulations
concerning analysis methods can be included in this
Regulation.

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 abovemen-
tioned Regulations
recast and brought
should be
together in a single text. For the same reason, Commis-
sion 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 inter-
vention 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
should be

such skimmed-milk powder (3)

(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.

(2)

(3)

The composition and quality requirements for milk and
milk products
the arrangements
provided for in Regulation (EC) No 1255/1999 must be
verified to ensure that they are strictly complied with.

laid down under

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
In order to ensure that reference
Community rules.
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)

(6)

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.

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.

L 37/2

EN

Official Journal of the European Communities

7.2.2001

(7)

(8)

(9)

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 concen-
trated butter for use in the manufacture of pastry prod-
ucts, ice-cream and other foodstuffs (1), as last amended
by Regulation (EC) No 635/2000 (2), 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.

Under Commission Regulation (EEC) No 3143/85 of 11
November 1985 on the sale at reduced prices of inter-
vention butter intended for direct consumption in the
form of concentrated butter (3), as last amended by
Regulation (EC) No 101/1999 (4), Commission Regula-
tion (EEC) No 429/90 of 20 February 1990 on the
granting by invitation to tender of an aid for concen-
trated butter intended for direct consumption in the
Community (5), as last amended by Regulation (EC) No
124/1999 (6), and Regulation (EC) No 2571/97, concen-
trated butter must have tracers added to it under super-
vision. 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.

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 prod-
ucts 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 casein-
ates (7), as last amended by Regulation (EC) No 2654/
1999 (8), the absence of coliforms must be detected.

(1) OJ L 350, 20.12.1997, p. 3.
(2) OJ L 76, 25.3.2000, p. 9.
(3) OJ L 298, 12.11.1985, p. 9.
(4) OJ L 11, 16.1.1999, p. 14.
(5) OJ L 45, 21.2.1990, p. 8.
(6) OJ L 16, 21.1.1999, p. 19.
(7) OJ L 279, 11.10.1990, p. 22.
(8) OJ L 325, 17.12.1999, p. 10.

internationally accepted reference method for
The
detecting coliforms in milk and milk products is Inter-
national 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 abovemen-
tioned standard.

(11)

Council Regulation (EEC) No 2658/87 of 23 July 1987
on the tariff and statistical nomenclature and on the
Common Customs Tariff (9), as last amended by Regula-
tion (EC) No 254/2000 (10), 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 there-
fore 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.

(9) OJ L 256, 7.9.1987, p. 1.
(10) OJ L 28, 3.2.2000, p. 16.

7.2.2001

EN

Official Journal of the European Communities

L 37/3

Article 2

List of methods

Annex I to this Regulation contains the list of reference

1.
methods applicable to analyses as referred to in Article 1.

A detailed description of the procedure applied must be avail-
able for consultation in the laboratory.

Laboratories

2.
establish their
shall
within a run for all methods, following:

in-house precision

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.

(b) a published, validated procedure with an established repeat-

ability.

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

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,
method shall be decisive.

the results obtained by the reference

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.

the

results of

3. Where
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 deter-
mine the critical difference from the limit concerned.

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 para-
graph 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 relia-
bility 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 refer-
ence 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 Commis-
sion.

The procedures described in Annex VI may be used to check
the performance of assessors and the reliability of results.

Article 7

Article 5

Admissibility of analysis results

Sampling and disputes over the results of analyses

Duplicate samples must be taken for analyses required

1.
under Community rules.

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.

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

L 37/4

EN

Official Journal of the European Communities

7.2.2001

CHAPTER II

that:

METHODS OF ANALYSIS

Article 8

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

(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.

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

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

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

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.

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

Article 11

Detection of coliforms

Article 9

Tracers

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

The analysis method described in Annex XIII shall be
2.
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.

Concentrated butter, butter and cream have been traced
4.
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
the reference sample
containing 1 % cows' milk as described in Annex XV.

than the content of

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

Routine methods for detecting coliforms may be used
2.
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.

The method for detecting rennet whey in skimmed-milk
feed is

2.
powder and mixtures intended for use as animal
described in Annex XIX.

Article 14

Detection of buttermilk

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

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

7.2.2001

EN

Official Journal of the European Communities

L 37/5

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
is
described in Annex XXIII.

and compound feedingstuffs

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)

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.’

is

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:

least

The skimmed-milk powder content of mixtures and
‘1.
compound feedingstuffs shall be determined by testing each
sample at
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 inspec-
tion shall be conclusive.

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

The starch content of compound feedingstuffs shall be
3.
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

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.

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

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’.

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.

L 37/6

EN

Official Journal of the European Communities

7.2.2001

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

ANNEX I

(Article 2)

LIST OF REFERENCE METHODS

Index

Min. = minimum, Max. = maximum, Annex = Annex to quoted Regulation, SNF = solids not fat, FFA = free fatty acids, PV = peroxide value, A = appear-
ance, F = flavour, C = consistency, TBC = total bacterial count, Therm = thermophilic bacterial count, MS = Member State, IDF = International Dairy
Federation, ISO = International Standards Organisation, IUPAC = International Union of Pure and Applied Chemistry, ADPI = American Dairy Products
Institute, SCM = sweetened condensed milk, EMC = evaporated milk or cream, MSNF = milk solids non fat.

PART A

Commission Regulation

Product

Parameter

Limit

Reference method

Remark

Regulation (EC) No 2771/1999
Public storage
(OJ L 333, 24.12.1999, p. 11)

Unsalted butter

Milk fats

Water

SNF

FFA (max.)

PV (max.)

Coliforms

Non-milk fat

Sterol tracers

Other tracers:

— vanillin

Min. 82 %

Up to 16 %

Up to 2 %

Annex XI

Annex IX

Annex X

1.2 mmole/100 g of fat

IDF Standard 6B:1989

0,3 meq. oxygen/1 000 g fat

IDF Standard 74A:1991
(English version)

Not detectable in 1 g

Annex XVII

Note 1

Note 3

detectable

Not
analysis

Not detectable

Not detectable

by

triglyceride

Annex XXVI

Annex XIV

Annex XII

Annex XIII

— ethyl ester of carotenic acid

Not detectable

— triglycerides of enanthic acid

Not detectable

IUPAC 2.301 sub 5

Sensory characteristics

At least 4 out of 5 points for A, G
and C

Annex VII

Water dispersion

At least 4 points

IDF Standard 112A:1989

Regulation (EC) No 2771/1999
Private storage

Unsalted butter

Regulation (EC) No 2771/1999
Private storage

Salted butter

Milk fats

Water

Milk fats

Water

Salt

Min. 82 %

Up to 16 %

Min. 80 %

Up to 16 %

Up to 2 %

Annex XI

Annex IX

Annex XI

Annex IX

IDF Standard 12B:1988

Note 6

Note 6

.

.

7
2
2
0
0
1

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n
C
o
m
m
u
n
i
t
i
e
s

L

3
7
/
7

Commission Regulation

Product

Parameter

Limit

Reference method

Remark

Regulation (EC) No 2571/97
(OJ L 350, 20.12.1997, p. 3)

Unsalted butter

Regulation (EC) No 2571/97

Salted butter

Min. 82 %

Up to 16 %

Min. 80 %

Up to 16 %

Up to 2 %

Milk fats

Water

Tracers:

— sterols

— vanillin

— ethyl ester of carotenic acid

— triglycerides of enanthic acid

Milk fats

Water

Salt

Tracers:

— sterols

— vanillin

— ethyl ester of carotenic acid

— triglycerides of enanthic acid

Annex XI

Annex IX

Annex XIV

Annex XII

Annex XIII

IUPAC 2.301 sub 5

Annex XI

Annex IX

IDF Standard 12B:1988

Annex XIV

Annex XII

Annex XIII

IUPAC 2.301 sub 5

Regulation (EC) No 2571/97

Concentrated butter

Milk fats

Moisture and MSNF

FFA

PV (max.)

Non-milk fat

Flavour

Smell

Other

Tracers:

— sterols

— vanillin

— ethyl ester of carotenic acid

— triglycerides of enanthic acid

Min. 99,8 %

Up to 0,2 %

IDF Standard 24:1964

IDF Standard 23A:1988 (moisture)
IDF Standard 24:1964 (MSNF)

Up to 0,35 % (oleic)

IDF Standard 6B:1989

IDF Standard 74A:1991
(English version)

Annex XXV

Note 1

0,5 meq. oxygen/1 000 g fat

Absence

Clean

Absence of foreign odours

Absence of neutralising agents, anti-
oxidants and preservatives

Annex XIV

Annex XII

Annex XIII

IUPAC 2.301 sub 5

L

3
7
/
8

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n
C
o
m
m
u
n
i
t
i
e
s

.

.

7
2
2
0
0
1

Commission Regulation

Product

Parameter

Limit

Reference method

Remark

Regulation (EC) No 2571/97

Cream

Fat

Tracers:

— sterols

— vanillin

— ethyl ester of carotenic acid

35 %

IDF Standard 16C:1987

Methods approved by the competent
authority

Note 2

Annex XII

Methods approved by the competent
authority

Note 2

— triglycerides of enanthic acid

IUPAC 2.301 sub 5

Regulation (EEC) No 429/90
(OJ L 45, 21.2.1990, p. 8)

Concentrated butter

Milk fats

SNF

Tracers:

Min. 96 %

Up to 2 %

Methods approved by the competent
authority

Note 2

Methods approved by the competent
authority

Note 2

— stigmasterol (95 %)

15 g/100 kg of concentrated butter Annex XIV

— stigmasterol (85 %)

17 g/100 kg of concentrated butter Annex XIV

— triglycerides of enanthic acid

1,1 kg/100 kg of concentrated butter

IUPAC 2.301 sub 5

— ethyl ester of butyric acid and

See Annex, point 1(c)

Annex XIV

Note 2

stigmasterol

— lecithin (E 322)

Up to 0,5 %

Methods approved by the competent
authority

Note 2

NaCl

FFA

PV (max.)

Flavour

Smell

Other

Milk fats

Water

Up to 0,75 %

IDF Standard 12B:1988

Up to 0,35 % (oleic)

IDF Standard 6B:1989

Up to 0,5 meq. oxygen/1 000 g fat

IDF Standard 74A:1991
(English version)

Note 1

Clean

Absence of foreign odours

Absence of neutralising agents, anti-
oxidants and preservatives

Min. 82 %

Up to 16 %

Annex XI

Annex IX

Regulation (EEC) No 2191/81
(OJ L 213, 1.8.1981. p. 20)

Unsalted butter

.

.

7
2
2
0
0
1

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n
C
o
m
m
u
n
i
t
i
e
s

L

3
7
/
9

Commission Regulation

Product

Parameter

Limit

Reference method

Remark

Regulation (EEC) No 2191/81

Salted butter

Milk fats

Water

Salt

Min. 80 %

Up to 16 %

Up to 2 %

Annex XI

Annex IX

IDF Standard 12B:1988

Article 9 and Title II of Regulation
(EC) No 1255/1999

Cheese made from ewes'
and/or goats' milk

Cows' milk

< 1 %

Annex XV

Regulation (EEC) No 2921/90

Annex I — Acid casein Water

Fat

Free acidity

Up to 12,00 %

Up to 1,75 %

IDF Standard 78C:1991

IDF Standard 127A:1988

Up to 0,30 % (lactic)

IDF Standard 91:1979

Regulation (EEC) No 2921/90

Annex I — Rennet-casein Water

Fat

Ash

Regulation (EEC) No 2921/90

Annex I — Caseinates

Water

Milk protein

Fat and ash

Up to 12,00 %

Up to 1,00 %

Min. 7,50 %

Up to 6,00 %

Min. 88,00 %

Up to 6,00 %

IDF Standard 78C:1991

IDF Standard 127A:1998

IDF Standard 90:1979

IDF Standard 78C:1991

IDF Standard 92:1979

IDF Standard 127A:1988
IDF Standard 89:1979 or
IDF Standard 90:1979

Regulation (EEC) No 2921/90

Annex II — Acid casein Water

Fat

Free acidity

TBC (max.)

Coliforms

Therm. (max.)

Regulation (EEC) No 2921/90

Annex II — Rennet-casein Water

Fat

Ash

TBC (max.)

Coliforms

Therm. (max.)

Up to 10,00 %

Up to 1,50 %

IDF Standard 78C:1991

IDF Standard 127A:1988

Up to 0,20 % (lactic)

IDF Standard 91:1979

30 000 l/g

Absence in 0,1 g

5 000 l/g

Up to 8,00 %

Up to 1,00 %

Min. 7,50 %

30 000 l/g

IDF Standard 100B:1991

Annex XVI

Note 3

Note 3

IDF Standard 100B:1991

Notes 3 and 4

IDF Standard 78C:1991

IDF Standard 127A:1988

IDF Standard 90:1979

IDF Standard 100B:1991

Absence in 0,1 g

Annex XVI

5 000 l/g

IDF Standard 100B:1991

Notes 3 and 4

Note 3

Note 3

L

3
7
/
1
0

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n
C
o
m
m
u
n
i
t
i
e
s

.

.

7
2
2
0
0
1

Commission Regulation

Product

Parameter

Limit

Reference method

Remark

Regulation (EEC) No 2921/90

Annex II — Caseinates

Water

Milk protein

Fat and ash

TBC (max.)

Coliforms

Therm. (max.)

Regulation (EEC) No 2921/90

Annex III — Caseinates

Water

Milk protein

Fat

Lactose

Ash

TBC (max.)

Coliforms

Therm. (max.)

Up to 6,00 %

Min. 88,00 %

Up to 6,00 %

IDF Standard 78C:1991

IDF Standard 92:1979

IDF Standard 127A:1988
IDF Standard 89:1979 or 90:1979

30 000 l/g

IDF Standard 100B:1991

Absence in 0,1 g

Annex XVI

Note 3

Note 3

5 000 l/g

IDF Standard 100B:1991

Notes 3 and 4

Up to 6,00 %

Min. 85,00 %

Up to 1,50 %

Up to 1,00 %

Up to 6,50 %

30 000 l/g

IDF Standard 78C:1991

IDF Standard 92:1979

IDF Standard 127A:1988

IDF Standard 106:1982

IDF Standard 89:1979 or 90:1979

IDF Standard 100B:1991

Absence in 0,1 g

Annex XVI

Note 3

Note 3

5 000 l/g

IDF Standard 100B:1991

Notes 3 and 4

Regulation (EC) No 2799/1999
(OJ L 340, 31.12.1999, p. 3)

Compound
and skimmed-milk powder
(SMP) (for use in feeding-
stuffs)

feedingstuffs Water (acid buttermilk powder)

Protein

Water (SMP)

Fats (SMP)

Rennet whey (SMP)

Starch (SMP)

Water (mixtures)

Fats (mixtures)

Up to 5 %

31,4 % (min.) of
matter)

Up to 5 %

Up to 11 %

Absence

Absence

the non-fat dry

IDF Standard 20B:1993

IDF Standard 26A:1993

IDF Standard 9C:1987

Annex XIX

Annex XXIII

Up to 5 % of the non-fat dry matter

IDF Standard 26A:1993

—

Commission Directive 84/4/EEC (OJ L
15, 18.1.1984, p. 28)

Rennet whey (mixtures)

Absence

SMP content (of end product)

Min. 50 %

Annex XIX

Annex XXII

Fats (in end product)

Min 2,5 % or 5 %

Commission Directive 84/4/EEC

Starch (in end product)

Copper (in end product)

Min 2 %

25 ppm

Annex XXIII

Commission Directive 78/633/EEC
(OJ L 206 26.7.1987, p. 43)

Note 7

Note 7

Note 8

Note 9

.

.

7
2
2
0
0
1

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n
C
o
m
m
u
n
i
t
i
e
s

L

3
7
/
1
1

Commission Regulation

Product

Parameter

Limit

Reference method

Remark

Regulation (EC) No 322/96 (OJ L
45, 23.2.96, p. 5)

SMP (spray)

Fat

Protein

Water

Acidity (N/10 NaOH)

Lactates

Phosphate

Solubility

Up to 1,0 %

31,4 % (min.) of
matter

Up to 3,5 %

Up to 19,5 ml

IDF Standard 9C:1987

the non-fat dry

IDF Standard 20B:1993

IDF Standard 26A:1993

IDF Standard 86:1981

Up to 150 mg/100 g

IDF Standard 69B:1987

Negative

ISO Standard 3356:1975

Up to 0,5 ml at 24 °C

IDF Standard 129A:1988

Scorched particles

Disk B min. (15,0 mg)

ADPI:1990

TBC

Coliforms

Buttermilk

Rennet whey

Acid whey

40 000/l g

Negative/0,1 g

Negative

Negative

Negative

IDF Standard 100B:1991

Annex XVI

Annex XX

Annex XVIII

Note 3

Note 3

Method approved by the competent
authority

Note 2

Anti-microbial agents

Annex XXI

The reference methods listed in Part B may be used for analysing products coverd by any of the Regulations listed in column 1.

PART B

Commission
Regulation

Regulation (EEC) No 2658/87
(OJ L 256, 7.9.1987, p. 1)
Regulation (EC) No 2414/98
(OJ L 299, 10.11.1998, p. 7)
Regulation (EC) No 1374/98
(OJ L 185, 30.6.1998, p. 21)
Regulation (EC) No 2508/97
(OJ L 345, 16.12.1997, p. 31)
Regulation (EC) No 174/1999
(OJ L 20, 27.1.1999, p. 8)

Product

CN code

Parameter

Limit

Reference method

Remark

Milk and cream, not con-
containing
centrated nor
added
other
or
sugar
sweetening matter

0401

Fat (≤ 6 %)

IDF Standard 1D:1996

The limits are those specified in the
description of the CN code for the
specific product, further specified,
where applicable,
in Commission
Regulation (EEC) No 3846/87 (OJ
L 366, 24.12.1987, p. 1) Part 9 of
the export nomenclature or Regu-
lation (EC) No 1374/98 (OJ L 185,
30.6.1998, p. 21)

Fat (> 6 %)

IDF Standard 16C:1987

L

3
7
/
1
2

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n
C
o
m
m
u
n
i
t
i
e
s

.

.

7
2
2
0
0
1

Commission
Regulation

Product

CN code

Parameter

Limit

Reference method

Remark

Milk and cream, concen-
trated or containing added
sugar or other sweetening
matter

0402

Fats (liquid form)

IDF Standard 13C:1987

.

.

7
2
2
0
0
1

0403

Buttermilk,
fermented or
acidified milk and cream,
or
concentrated
not
containing
concentrated,
added
other
or
sugar
sweetening matter

Fats (solid form)

Protein

Sucrose (normal content)
Sucrose (low content)

Solids (SCM)
Solids (EMC)

(milk and cream

Water
powder)

Fat

Protein
Sucrose (normal content)
Sucrose (low content)

(acid

buttermilk

(sweet buttermilk

Water
powder)

Water
powder)

Solids (other products)

0404

Fat

Whey, whether or not
concentrated or containing
other
added
sweetening matter; prod-
ucts consisting of natural
milk constituents

sugar

or

0404 90

and

other

Butter
derived from milk; dairy
spreads

fats 0405

Butter

Butteroil

Protein
Sucrose (normal content)
Sucrose (low content)

Protein
Water
Solids
(Concentrated products)

Fat (if ≤ 85 %)
Water
SNF
NaCl
Fat (if > 99 %)
Water (if fat < 99 %)

IDF Standard 9C:1987

IDF Standard 20B:1993

IDF Standard 35A:1992
Methods
approved
competent authority
IDF Standard 15B:1991
IDF Standard 21B:1987

by

IDF standard 26A:1993

the

Note 2

IDF Standards 1D:1996,
9C:1987, 16C:1987, 22B:1987,
126A:1988
IDF Standard 20B:1993
IDF Standard 35A:1992
Methods
approved
competent authority

the

by

Note 2

Annex XXIV

IDF Standard 26A:1993

Methods
approved
competent authority

by

the

the

Note 2

IDF Standards 9C:1987,
16C:1987
22B:1987
IDF Standard 20B:1993
IDF Standard 35A:1992
Methods
approved
competent authority
IDF Standard 20B:1993
IDF Standard 26A:1993
IDF Standard 15B:1991
IDF Standard 21B:1987

by

Annex XI
Annex IX
Annex X
IDF Standard 12B:1998
IDF Standard 24:1964
IDF Standard 23A:1988

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n
C
o
m
m
u
n
i
t
i
e
s

L

3
7
/
1
3

Commission
Regulation

Product

CN code

Parameter

Limit

Reference method

Remark

Cheese and curd

0406

Fat
Solids
Solids (Ricotta)
NaCl
Lactose

Regulation (EEC) No 2658/87

Compound feedingstuffs

2309

Lactose

IDF Standard 5B:1986
IDF Standard 4A:1982
IDF Standard 58:1970
IDF Standard 88A:1988
IDF Standard 79B:1991

Annex XVII

Notes 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:
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.

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

L

3
7
/
1
4

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n
C
o
m
m
u
n
i
t
i
e
s

.

.

7
2
2
0
0
1

7.2.2001

EN

Official Journal of the European Communities

L 37/15

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

if R

/R

Rout

Ref

≤ 1

R

Rout:
R
:

Ref

If m
o

Reproducibility limit of the routine method

Reproducibility limit of the reference method

is an upper limit and R

/R

> 1, this decision limit is obtained using the formula

Ref

Rout

If, under the same conditions, m
o

is a lower limit, the decision limit is obtained using the formula

where CrD95 is the critical difference of the reference method (see Annex IV).
Where m
o 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 m
o
a routine method.

is a lower limit, the same procedure must be followed for a final result below the decision limit obtained using

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 (w
results obtained by the reference and the routine method is determined.

i) between the

The standard deviation calculation using the formula

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

A matrix effect cannot be excluded,

if

where

f =

α =

m (f: number of degrees of freedom)

error probability; α = 0,05.

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

L 37/16

EN

Official Journal of the European Communities

7.2.2001

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:

where:

:

1

CrD95
y–
:
y–
:
2
R:
r:

critical difference (P = 0.95)
arithmetic mean of two results obtained in laboratory 1
arithmetic mean of two results obtained in laboratory 2
reproducibility limit: to be determined by interpolation,
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:

where:
y–
:
y–
2:
r:

1

Remarks:

1. Rprov

2. R

prov

mean of two results obtained in laboratory 1
mean of two results obtained in laboratory 2 (see Annex IIIa)
repeatability limit or provisional repeatability limit.

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

is fixed at 2r if the calculated value for R

prov

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 (*), then

R

prov 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
the mean value of R

must be used for the calculation of critical differences. The rules given under 2 and 3 apply for
.
prov

(*) Horwitz equation:

where:

:
RSDR
c:

relative standard deviation of reproducibility
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).

(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.

7.2.2001

EN

Official Journal of the European Communities

L 37/17

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

x–:

arithmetic mean of the results obtained)

Some calculated RSD
R

values (examples)

Concentration

RSD

R

(%)

1 g/100 g

0,01 g/100 g

1 mg/1 000 g

4

8

16

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

R = 0.0283 * 1 * 4 = 0,11 g/100 g.

L 37/18

EN

Official Journal of the European Communities

7.2.2001

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:

where:
y–:

arithmetic mean of the results obtained

mo:
n:

limit

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 m0
— within the range m0 and m0 + CrD95 (y––m0|),
should therefore occur only exceptionnaly.

and m
0

+ CrD

95

0

(y––m

if the limit is a maximum;
if the limit is a minimum

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

(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 r

x and Rx of the final results x are calculated as:

± y

2

where r1 and r2 are the repeatability limits and R1 and R2 the reproducibility limits of y1 and y2,, respectively.
following the rules specified under 1 and 2. The critical difference is determined using
x is compared with the limit m0
the formula:

where x is the arithmethic mean of the results x
1

obtained.

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

(example: fat in dry matter content of cheese)

7.2.2001

EN

Official Journal of the European Communities

L 37/19

where y1 and y2 are the final results of a single type of analysis, then the overall repeatability and reproducibility limits
r
x

can be calculated as:

and R

x

µ

µ

:
1
:
2

limit or target value for y
1
limit or target value for y
2

(example: fat)

(example: dry matter)

where

r
:
1
r2:

repeatability limit, y
1
repeatability limit, y2

where
R1:
R

2

reproducibility limit, y1
reproducibility limit, y
2

2; R*1; R*2) are smaller than or equal to 0,15.

The procedures for calculating rx and Rx are applicable only if the relative repeatability and reproducibility limits (r*1,
r*
x is compared with the limit µx following the rules specified under 1 and 2. The critical difference is determined using
the formula:

Where x– is the arithmetic mean of the results x obtained in chronological order (*).

(*) Note: If, for example, the results y
must be calculated.

11,

y
12,

y
21

and y

22,

are obtained, the arithmetic mean of y

11

/y

21

and y

12

/y

22

L 37/20

EN

Official Journal of the European Communities

7.2.2001

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

laboratories’ (1).

in analytical

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 repeat-
ability 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

x ± 3s
t,

where s
t

is the total standard deviation,

warning limits at

x ± 2st

Total standard deviation:

in which:

s

:
s
b
w:
n:

between-run standard deviation

within-run standard deviation

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
, is the within-run standard deviation.
, where s
, the lower limit is 0, the upper limit (action limit) is 3.686 s
1.128 sw
w
w

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

(1) M. Thompson and R. Wood:

‘Pure and Applied Chemistry’ 67 (4), 649-666 (1995).

7.2.2001

EN

Official Journal of the European Communities

L 37/21

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,

if appropriate, different analysts.

instruments re-calibration and,

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.

L 37/22

EN

Official Journal of the European Communities

7.2.2001

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

, x
i2

of the p duplicate data, the sum of the duplicates,

and the difference of the duplicates,

are computed and summed up to

An estimate of the within-run standard deviation is

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
regarded as provisional and subject to review.

may be used to construct control charts (see Annex II). The limits set should be

7.2.2001

EN

Official Journal of the European Communities

L 37/23

2.2. Determining between-run precision

Compute the average value (s
1/2) 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 s
b

is calculated.

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

The total standard deviation s
limits set should be regarded as provisional and subject to review.

t is used to construct control charts for the average of n determinations (see Annex II). The

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
is being brought into question must be thoroughly investigated.
indication that the validity of the control material

4. Reporting of precision data

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

— the method employed,
— the within-run standard deviation s
— the between-run standard deviation s
b,
— the total standard deviation s
1,
— the number of analyses involved in obtaining precision data.

w, and in-house precision limit,

L 37/24

EN

Official Journal of the European Communities

7.2.2001

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:

where:

w

I:
x
i1:
:
x
i2

n:

repeatability index

score for the first evaluation of sample xi
score for the second evaluation of sample x
i

number of samples

The samples to be evaluated should reflect a broad quality range. w

I should not exceed 1.5 (5-point scales).

(b) Determining the ‘deviation index’

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

The following formula is used for the evaluation of results:

where:

x
i1; xi2:
x–
il; x–
i2:
n:

see section (a)

average score of the assessor group for the first and second evaluation respectively of sample xi
number of samples (at least ten per 12 months).

The samples to be evaluated should reflect a broad quality range. D

I 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.

7.2.2001

EN

Official Journal of the European Communities

L 37/25

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.

L 37/26

EN

Official Journal of the European Communities

7.2.2001

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:

Appearance

Consistency

Flavour

Maximum

Required

5

5

5

4

4

4

Where the required score is not obtained, a description of the defect has to be given. The score given by each
assessor for each attribute must be recorded in the control document. The product is accepted or rejected on the
basis of a majority decision. Cases where differences between the individual scoring for each attribute are wider
than adjacent points should not occur frequently (not more than once per 20 samples). Otherwise the compet-
ence 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.

7.2.2001

EN

Official Journal of the European Communities

L 37/27

Table 1: Butter scoring

Appearance

Consistency

Flavour + aroma

Points

No (1)

Remarks

Points
(quality
class)

No (1)

Remarks

Points
(quality
class)

No (1)

Remarks

5

4

3

2

Very good
ideal type
highest quality
(equal dry)

Good (2)
No evident defects

Fair (slight defects)
loose (free), moisture
not uniform, two coloured
streaky
mottled, marbled
speckled
oil separation
overcoloured
weak, open texture

Poor (evident defects)
loose (free) moisture
streaky
mottled, marbled
speckled
oil separation
foreign matter
mouldy
undissolved salt

1
2
3
4
5
6
7
8

1
3
4
5
6
10
11
12

5

4

3

2

Very good
ideal type
highest quality
(well spreadable)

Good (2)
hard
soft

Fair (slight defects)
short, brittle, crumbly
pasty, doughy, greasy
sticky
hard
soft

Poor (evident defects)
short, brittle, crumbly
pasty, doughy, greasy
sticky
hard
soft

17
18

14
15
16
17
18

14
15
16
17
18

5

4

3

2

1

Very poor (strong defects)

1

Very poor (strong defects)

1

1
3
4
5
6
7
9
10
11
12

loose (free) moisture
streaky
mottled, marbled,
speckled
oil separation
overcoloured
granular
foreign matter
mouldy
undissolved salt

14
15
16
17
18

short, brittle, crumbly
pasty, doughy, greasy
sticky
hard
soft

Very good
ideal type
highest quality
(absolutely pure finest
aroma)

Good (2)
No evident defects

Fair (slight defects)
unclear
foreign flavour
acid
cooked flavour, scorched
flavour

feed flavour
coarse, bitter
oversalted

Poor (evident defects)
unclean
foreign flavour
stale
acid
oxidized flavour, metallic
flavour

feed flavour
coarse, bitter
oversalted
musty-flat, putrid
chemical flavour

Very poor (strong defects)

foreign flavour
cheesy, lactic cheese flavour
acid
yeasty
mouldy flavour
rancid
oily, fishy
tallowy
oxidized flavour, metallic
flavour

coarse, bitter
musty-flat, putrid
malty
chemical flavour

21
22
25
27

33
34
35

21
22
23
25
32

33
34
35
36
38

22
24
25
26
28
29
30
31
32

34
36
37
38

(1) Table 2.
(2) The defects mentioned under ‘good’ are only very small deviations from the ideal type.

L 37/28

EN

Official Journal of the European Communities

7.2.2001

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.

7.2.2001

EN

Official Journal of the European Communities

L 37/29

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:

where
y–: arithmetic mean of all results obtained by both laboratories
mo
R: reproducibility

limit

:

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

(see Annex IV(3) and (4), respectively), R2

and r2

x

x

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.

L 37/30

EN

Official Journal of the European Communities

7.2.2001

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.

7.2.2001

EN

Official Journal of the European Communities

L 37/31

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:

where

m

m

m

0

1

2

is the mass in grams of the dish with the pumice (7.2.3)

is the mass in grams, of the test portion, dish and pumice before drying (7.2.4)

is the mass in grams, of the test portion, dish and pumice after drying (7.2.7)

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.

L 37/32

EN

Official Journal of the European Communities

7.2.2001

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 (m
).
0

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 (m
1

).

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) (m
).
2

7.2.2001

EN

Official Journal of the European Communities

L 37/33

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 (m
).
3

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:

where
m0
m

1

is the mass,

is the mass,

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

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

2

is the mass,

m
m3
Report the result to the first decimal place.

is the final mass,

in grams,

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

in the dish with the rod and the crucible containing sediment (8.2.11)

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:

If salted butter is analysed, added salt is determined as solids-non-fat. For the determination of the milk solids-non-fat
content, the content of added salt has to be subtracted from the solids-non-fat content. The calculated precision
figures for the determination of milk solids-non-fat are:

Repeatability:

Reproducibility:

r = 0,104 %

R = 0,206 %.

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.

L 37/34

EN

Official Journal of the European Communities

7.2.2001

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

where

W:

SNF:

is the percentage, by mass, of water

is the percentage, by mass, of solids-non-fat

The calculated precision figures for the determination of the fat content are:

Repeatability:

Reproducibility:

r = 0,22 %

R = 0,36 %.

7.2.2001

EN

Official Journal of the European Communities

L 37/35

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 × 4,6 mm ID) packed with LiChrospher RP 18 (Merck, 5 µm) or equivalent;

3.6.

guard column (ca. 20 mm × 3 mm ID) dry-packed with 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

L 37/36

EN

Official Journal of the European Communities

7.2.2001

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
1 000 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. B u t t e r

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. C o n c e n t r a t e d b u t t e r

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. C r e a m

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

5.2.

Preparation of the test solution

Add about 75 ml extraction solution (4.4) to the test portion (5.1.1, 5.1.2 or 5.1.3), stir, or shake vigorously,
for about 15 minutes and make up with extraction solution (4.4). Transfer about 10 ml of this extract to a test
tube fitted with stopper. Place the test tube in the freezer (3.1 ) and allow it to stand for about 30 minutes.
Centrifuge the cold extract for 5 minutes at approximately 2 000 rpm and decant immediately. Allow the
decanted solution to 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)
injections for each batch of test solutions (four areas or heights in total).

(AC), of the vanillin peaks associated with the bracketing duplicate

Calculate the response factor (R):

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

R = AC/CM

7.2.2001

EN

Official Journal of the European Communities

L 37/37

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

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
— 221,0 mg/kg (95 % of the minimum incorporation rate),
— 159,0 mg/kg (70 % of the minimum incorporation rate).

) taken into consideration):

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 (DCr

) taken into consideration):

95

— 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 %.

L 37/38

EN

Official Journal of the European Communities

7.2.2001

9.2.

9.3.

9.4.

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 %.

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 %.

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.

7.2.2001

EN

Official Journal of the European Communities

L 37/39

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).

A

max

=

A =

absorbance of the measuring solution at the maximum

weight of sample (g)

2 550 =

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.

Petroleum spirit (40 — 60 °C).

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

Chloroform.

Cyclohexane.

4.2.

4.3.

4.4.

4.5.

L 37/40

EN

Official Journal of the European Communities

7.2.2001

5. Procedure

5.1.

Preparation of the test sample

5.1.1. C o n c e n t r a t e d b u t t e r

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

5.1.2. B u t t e r

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)
volumetric flasks. Dilute to volume with petroleum spirit (4.2) and mix.

into five 100 ml

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=

V =

M=

apo-carotenic ester content, µg/ml, read from the calibration graph (5.3)

volume (ml) of the test solution (5.2)

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. B u t t e r a n a l y s i s

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. C o n c e n t r a t e d b u t t e r a n a l y s i s

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. B u t t e r a n a l y s i s

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.

7.2.2001

EN

Official Journal of the European Communities

L 37/41

7.2.2. C o n c e n t r a t e d b u t t e r a n a l y s i s

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 (DCr

95) 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 (DCr

95) 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.

L 37/42

EN

Official Journal of the European Communities

7.2.2001

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.

3.2.

3.3.

3.4.

3.5.

3.6.

3.7.

3.8.

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

500 ml separating funnels.

250 ml flasks.

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

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

Waterbath or isomantle.

Reaction vials, 2 ml.

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.

4.2.

Ethanol, at least 95 % pure.

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.

7.2.2001

EN

Official Journal of the European Communities

L 37/43

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.

5.1.2.

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.

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.

L 37/44

EN

Official Journal of the European Communities

7.2.2001

5.3.

Preparation of trimethyl silyl ethers

5.3.1.

5.3.2.

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.

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.

7.2.2001

EN

Official Journal of the European Communities

L 37/45

6. CALCULATION OF RESULTS

6.1.

Determine the area of the sterol peaks and betulin peaks in both standards bracketing a batch, and calculate
R
1:

Determine the area of the sterol peak (stigmasterol and sitosterol) and betulin peak in the sample and
calculate R
2:

W

= sterol content of the standard (mg) contained in 1 ml of standard solution (4.8.1 or 4.9.1)
1

W

2 = weight of sample (g) (5.2.1)

P = purity of standard sterol (4.8 or 4.9)

7. ACCURACY OF THE METHOD

7.1.

Butter

7.1.1.

R e p e a t a b i l i t y

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.

R e p r o d u c i b i l i t y

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.

S o u r c e o f p r e c i s i o n d a t a

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.

L 37/46

EN

Official Journal of the European Communities

7.2.2001

7.2.

Concentrated butter

7.2.1.

R e p e a t a b i l i t y

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.

R e p r o d u c i b i l i t y

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.

S o u r c e o f p r e c i s i o n d a t a

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.

S t i g m a s t e r o l

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 (DCr

) taken into consideration):

95

— 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.

S i t o s t e r o l

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.

7.2.2001

EN

Official Journal of the European Communities

L 37/47

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 (DCr

) taken into consideration):

95

— 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.

S t i g m a s t e r o l

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 (DCr

) taken into consideration):

95

— 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.

S i t o s t e r o l

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 (DCr

) taken into consideration):

95

— 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.

L 37/48

EN

Official Journal of the European Communities

7.2.2001

7.2.2001

EN

Official Journal of the European Communities

L 37/49

L 37/50

EN

Official Journal of the European Communities

7.2.2001

7.2.2001

EN

Official Journal of the European Communities

L 37/51

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.

3.2.

3.3.

3.4.

Isolation of caseins from cheese and the reference standards.

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

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

Evaluation of stained γ
-casein patterns (evidence of cows' milk) by comparison of the pattern obtained
2
from the sample with those obtained in the same gel from the reference standards containing 0 % and 1 %
cows' milk.

and γ

3

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 × 125 × 0,25 mm. Where other sizes and types of gel are used, the separation conditions may have to be
adjusted.

I s o e l e c t r i c f o c u s i n g

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.

L 37/52

EN

Official Journal of the European Communities

7.2.2001

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 (1)

250 µl ampholyte pH 6-8 (1).

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.

S a m p l e p r e p a r a t i o n

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 ammonium hydrogencarbonate solution (1,58 g/100 ml water) containing 0,05 mol/l
ethylenediaminetetraacetic acid (EDTA, 1,46 g/100 ml with a 0,2 mol/l ammonium carbonate solution
(1,92 g/100 ml water) containing 0,05 mol/l EDTA to ph 8.

4.7.2.

Bovine plasmin (EC. 3.4.21.7), activity at least 5 U/ml.

4.7.3.

ε-Aminocaproic acid solution for enzyme inhibition

Dissolve 2,624 g ε-aminocaproic acid (6 amino-n-hexanoic acid) in 100 ml of 40 % (v/v) ethanol.

(1) The produce Ampholine pH 3,5-9,5 (Pharmacia) and Resolyte pH 5-7 and pH 6-8 (BDH, Merck) have proved particularly

suitable for obtaining the required separation of γ-caseins.

7.2.2001

EN

Official Journal of the European Communities

L 37/53

4.8.

Standards

4.8.1.

Certified reference standards of a mixture of renneted ewes' and goats' skimmed milk containing 0 % and 1 %
of cows' milk are available from the Commission's Institute for Reference Materials and Measurements,
B-2440 Geel, Belgium.

4.8.2.

Preparation of laboratory interim-standards of buffalos' renneted milk containing 0 % and 1 % of cows' milk

Skimmed milk is prepared by centrifuging of either buffalo or bovine raw bulk milk at 37 °C at 2 500 g for
20 minutes. After cooling the tube and contents rapidly to 6 to 8 °C, the upper fat layer is removed
completely. For the preparation of the 1 % standard add 5,00 ml of bovine skimmed milk to a 495 ml of
buffalos' skimmed milk in a 1 l beaker, adjust the pH to 6,4 by the addition of dilute lactic acid (10 % w/v).
Adjust the temperature to 35 °C and add 100 µl of calf rennet (rennet activity 1: 10 000, c. 3 000 U/ml), stir
for 1 minute and then leave the beaker covered with an aluminium foil at 35 °C for one hour to allow
formation of the curd. After the curd has formed, the whole renneted milk is freeze-dried without prior
homogenization or draining of the whey. After freeze-drying it is finely ground to produce a homogeneous
powder. For the preparation of the 0 % standard, carry out the same procedure using genuine buffalo
skimmed milk. The standards must be stored at – 20 °C.

Note: It is advisable to check the purity of the buffalos' milk by isoelectric focusing of the plasmin-treated

caseins before preparation of the standards.

R e a g e n t s f o r p r o t e i n s t a i n i n g

4.9.

Fixative

Dissolve 150 g trichloroacetic acid in water and make up to 1 000 ml.

4.10.

Destaining solution

Dilute 500 ml methanol and 200 ml glacial acetic acid to 2 000 ml with distilled water.

Note: Prepare the destaining solution fresh every day; it can be prepared by mixing equal volumes of stock

solutions of 50 % (v/v) methanol and 20 % (v/v) glacial acetic acid.

4.11.

Staining solutions

4.11.1. Staining solution (stock solution 1)

Dissolve 3,0 g Coomassie Brilliant Blue G-250 (C.I. 42655) in 1 000 ml 90 % (v/v) methanol using a
magnetic stirrer (approximately 45 minutes), filter through two medium-speed folded filters.

4.11.2. Staining solution (stock solution 2)

Dissolve 5,0 g copper sulphate pentahydrate in 1 000 ml 20 % (v/v) acetic acid.

4.11.3. Staining solution (working solution)

Mix together 125 ml of each of the stock solutions (4.11.1, 4.11.2) immediately prior to staining.

Note: The staining solution should be prepared on the day that it is used.

5. Equipment

5.1.

5.2.

5.3.

5.4.

5.5.

5.6.

5.7.

Glass plates (265 × 125 × 4 mm); rubber roller (width 15 cm);

levelling table

Gel carrier sheet (265 × 125 mm).

Covering sheet (280 × 125 mm). Stick on strip of adhesive tape (280 × 6 × 0,25 mm) to each long edge (see
Figure 1).

Electrofocusing chamber with cooling plate (e.g. 265 × 125 mm) and suitable power supply (≥ 2,5 kV) or
automatic electrophoresis device.

Circulation cryostat, thermostatically controlled at 12 ± 0,5 °C.

Centrifuge, adjustable to 3 000 g.

Electrode strips (≥ 265 mm long).

L 37/54

EN

Official Journal of the European Communities

7.2.2001

5.8.

5.9.

Plastic dropping bottles fot the anode and cathode solutions.

Sample applicators (10 × 5 mm, viscose or low protein-adsorption filter paper).

5.10.

Stainless steel scissors, scalpels and tweezers.

5.11.

Stainless steel or glass staining and destaining dishes (e.g. 280 × 150 mm instrument trays).

5.12.

Adjustable rod homogenizer (10 mm shaft diameter), rpm range 8 000 to 20 000.

5.13. Magnetic stirrer.

5.14.

Ultrasonic bath.

5.15.

Film welder.

5.16.

25 µl micropipettes.

5.17.

Vacuum concentrator or freeze-dryer.

5.18.

Thermostatically controlled water bath adjustable to 35 and 40 ± 1 °C with shaker.

5.19.

Densitometer equipment reading at λ = 634 nm.

6. Procedure

6.1.

Sample preparation

6.1.1.

Isolation of caseins

Weigh the amount equivalent to 5 g dry mass of cheese or the reference standards into a 100 ml centrifuge
tube, add 60 distilled water and homogenize with a rod homogenizer (8 000 to 10 000 rpm). Adjust to pH
4,6 with dil. acetic acid (4.5.1) and centrifuge (5 minutes, 3 000 g). Decant the fat and whey, homogenize the
residue at 20 000 rpm in 40 ml distilled water adjusted to pH 4,5 with dil. acetic acid (4.5.1), add 20 ml
dichloromethane (4.5.2), homogenize again and centrifuge (5 minutes, 3 000 g). Remove the casein layer
that lies between the aqueous and organic phases (see Figure 2) with a spatula and decant off both phases.
Rehomogenize the casein in 40 ml distilled water (see above) and 20 ml dichloromethane (4.5.2) and
centrifuge. Repeat this procedure until both extraction phases are colourless (two to three times). Homogenize
the protein residue with 50 ml acetone (4.53) and filter through a medium-speed folded filter paper. Wash
the residue on the filter with two separate 25 ml portions of acetone each time and allow to dry in the air or
a stream of nitrogen, then pulverize finely in a mortar.

Note: Dry casein isolates should be kept at – 20 °C.

6.1.2.

Plasmin cleavage of β-caseins to intensify γ-caseins

Disperse 25 mg of isolated caseins (6.1.1) in 0,5 ml ammonium carbonate buffer (4.7.1) and homogenize
for 20 minutes by e.g. using ultrasonic treatment. Heat to 40 °C and add 10 µl plasmin (4.7.2), mix and
incubate for one hour at 40 °C with continuous shaking. To inhibit the enzyme add 20 µl ε-aminoproic acid
solution (4.7.3), then add 200 mg of solid urea and 2 mg of dithiothreitol.

Note: To obtain more symmetry in the focused casein bands it is advisable to freeze-dry the solution after
adding the ε-aminocaproic acid and then dissolving the residues in 0,5 ml protein dissolving buffer
(4.6).

6.2.

Preparation of the urea containing polyacrylamide gels

With the aid of a few drops of water roll the gel carrier sheet (5.2) onto a glass plate (5.1 ), removing any
extraneous water with paper towel or tissue. Roll the cover sheet (5.3) with spacers (0,25 mm) onto another
glass plate in the same way. Lay the plate horizontally on a levelling table.

Add 10 µl Temed (4.1.3.1 ) to the prepared and de-aerated gel solution (4.1.2), stir and add 10 µl
PER-solution (4.1.3.2), mix thoroughly and immediately pour out evenly onto the centre of the cover sheet.
Place one edge of the gel carrier plate (sheet side down) on the cover sheet plate and lower it slowly so that a
gel film forms between the sheets and spreads out regularly and free of bubbles (Figure 3). Carefully lower the
gel carrier plate completely using a thin spatula and place three more glass plates on top of it to act as
weights. After polymerization is complete (about 60 minutes) remove the gel polymerized onto the gel carrier
sheet along with the cover sheet by tipping the glass plates. Clean the reverse of the carrier sheet carefully to
remove gel residues and urea. Weld the gel sandwich into a film tube and store in a refrigerator (maximum
six weeks).

Note: The cover sheet with the spacers can be re-used. The polyacrylamide gel can be cut to smaller sizes,
recommended when there are few samples or if an automatic electrophoresis device is used (two gels,
size 4,5 × 5 cm).

7.2.2001

EN

Official Journal of the European Communities

L 37/55

6.3.

lsoelectric focusing

Set the cooling thermostat to 12 °C. Wipe off the reverse of the gel carrier sheet with kerosene, then drip a
few drops of kerosene (4.2) onto the centre of the cooling block. Then roll the gel sandwich, carrier side
down, onto it, taking care to avoid bubbles. Wipe off any excess kerosene and remove the cover sheet. Soak
length and place in the positions
the electrode strips with the electrode solutions (4.3, 4.4), cut to gel
provided (distance of electrodes 9,5 cm).

C o n d i t i o n s f o r

i s o e l e c t r i c f o c u s i n g :

6.3.1. Gel size 265 × 125 × 0,25 mm

Step

Time
(min.)

Voltage
(V)

Current
(mA)

Power
(W)

Volt-hours
(Vh)

1. Pre-focusing

2. Sample focusing (1)

3. Final focusing

30

60

60

40

30

maximum 2 500 maximum 15

constant 4

c. 300

maximum 2 500 maximum 15

constant 4

c. 1 000

maximum 2 500 maximum 5

maximum 20

maximum 2 500 maximum 6

maximum 20

maximum 2 500 maximum 7

maximum 25

c. 3 000

c. 3 000

c. 3 000

(1) Sample application: After pre-focusing (step 1), pipette 18 µl of the sample and standard solutions onto the sample
applicators (10 × 5 mm), place them on the gel at 1 mm intervals from each other and 5 mm longitudinally from the
anode and press lightly. Carry out focusing using the above conditions, carefully removing the sample applicators after the
60 minutes of sample focusing.

Note: If thickness or width of the gels are changed, the values for current and power have to be suitably
adjusted (e.g. double the values for electric current and power if a 265 × 125 × 0,5 mm gel is used).

6.3.2.

Example of a voltage programme for an automatic electrophoresis device (2 gels of 5,0 × 4,5 cm), electrodes
without strips applied directly to the gel.

Step

Voltage

Current

Power

Temp.

Volt-hours

1. Pre-focusing

1 000 V

10,0 mA

2. Sample focusing

250 V

5,0 mA

3. Focusing

4. Focusing

1 200 V

10,0 mA

1 500 V

5,0 mA

3,5 W

2,5 W

3,5 W

7,0 W

8 °C

8 °C

8 °C

8 °C

85 Vh

30 Vh

80 Vh

570 Vh

Place sample applicator in step 2 at 0 Vh.

Remove sample applicator in step 2 at 30 Vh.

6.4.

Protein staining

6.4.1.

Protein fixation

Remove the electrode strips immediately after turning off the power and put the gel immediately into a
staining/destaining dish filled with 200 ml fixative (4.9);

leave for 15 minutes, shaking continuously.

6.4.2. Washing and staining the gel plate

Thoroughly drain off the fixative and wash the gel plate twice for 30 seconds each time with 100 ml
destaining solution (4.10). Pour off the destaining solution and fill the dish with 250 ml staining solution
(4.11.3); allow to stain for 45 minutes with gentle shaking.

L 37/56

EN

Official Journal of the European Communities

7.2.2001

6.4.3. Destaining the gel plate

Pour off the staining solution, wash the gel plate twice using a 100 ml destaining solution (4.10) each time,
then shake with 200 ml destaining solution for 15 minutes and repeat the destaining step at least two or
three times until the background is clear and uncoloured. Then rinse the gel plate with distilled water (2 × 2
minutes) and dry in the air (2 to 3 hours) or with a hairdryer (10 to 15 minutes).

Note 1: Carry out fixing, washing, staining and destaining at 20 °C. Do not use elevated temperatures.

Note 2: If more sensitive silver staining (e.g. Silver Staining Kit, Protein, Pharmacia Biotech, Code No

17-1150-01) is preferred, plasmin-treated casein samples have to be diluted to 5 mg/ml.

7. Evaluation

Evaluation is performed by comparing the protein patterns of the unknown sample with reference standards on the
same gel. Detection of cows' milk in cheeses from ewes' milk, goats' milk and buffalos' milk and mixtures of ewes',
goats' and buffalos' milk is done via the γ
-caseins, whose isolelectric points range between pH 6,5 and pH 7,5
2
(Figures 4 a, b, Figure 5). The detection limit is less than 0,5 %.

- and γ
3

7.1.

Visual estimation

For visual evaluation of the amount of bovine milk it is advisable to adjust the concentrations of samples and
standards to obtain the same level of intensity of the ovine, caprine and/or buffalo γ
2- and γ3-caseins (see ‘γ2
E,G,B’ in Figures 4 a, b and Figure 5). After which the amount of bovine milk (less than, equal
E,G,B’ and ‘γ
to or greater than 1 %) in the unknown sample can be judged directly by comparing the intensity of the
bovine γ
3- and γ2-caseins (see ‘γ3 C’ and ‘γ2 C’ in Figures 4 a, b and Figure 5) to those of the 0 % and 1 %
reference standards (ewe, goat) or,

laboratory interim-standards (buffalo).

3

7.2.

Densitometric estimation

If available, apply densitometry (5.19) for the determination of the peak area ratio of bovine to ovine, caprine
and/or buffalo γ
-casein peak area ratio of the
- and γ
-caseins (see Figure 5). Compare this value to γ
- and γ
3
2
3
2
1 % reference standard (ewe, goat) or laboratory interim-standard (buffalo) analysed on the same gel.

Note: The method is operating satisfactorily,

- and
2
γ
3-caseins in the 1 % reference standard but not in the 0 % reference standard. If not, optimize the
procedure following the details of the method precisely.

if there is a clear positive signal for both bovine γ

A sample is judged as being positive, if both bovine γ
ratios are equal to or greater than the level of the 1 % reference standard.

-caseins or the corresponding peak area
- and γ
3

2

8. References

1.

2.

3.

4.

5.

-caseins. Milchwissenschaft 45, 708-711 (1990).

Addeo F., Moio L., Chianese L., Stingo C., Resmini P., Berner I, Krause I., Di Luccia A., Bocca A.: Use of plasmin
to increase the sensitivity of the detection of bovine milk in ovine and/or caprine cheese by gel isoelectric focusing of
γ2
Addeo F., Nicolai M.A., Chianese L., Moio L., Spagna Musso S., Bocca A., Del Giovine L.: A control method to
detect bovine milk in ewe and water buffalo cheese using immunoblotting. Milchwissenschaft 50, 83-85 (1995).

Krause I., Berner I, Klostermeyer H.: Sensitive detection of cow milk in ewe and goat milk and cheese by carrier
ampholyte — and carrier ampholyte/immobilized pH gradient — isoelectric focusing of γ-caseins using plasmin as
signal amplifier. in: Electrophoresis-Forum 89 (B. J. Radola, ed.) pp 389-393, Bode-Verlag, München (1989).

Krause Ι., Belitz H.-D., Kaiser K.-P.: Nachweis von Kuhmilch in Schaf and Ziegenmilch bzw. -käse durch isoelektrische
Fokussierung in harnstoffhaltigen Polyacrylamidgelen. Z. Lebensm. Unters. Forsch. 174, 195-199 (1982).

Radola B.J.: Ultrathin-layer isoelectric focusing in 50-100 µm polyacrylamide gels on silanised glass plates or polyester
films. Electrophoresis 1, 43-56 (1980).

7.2.2001

EN

Official Journal of the European Communities

L 37/57

Figure 1: Schematic drawing of the covering sheet

Figure 2: Casein layer floating between aqueous and organic phases after centrifugation

Figure 3: Flapping technique for casting of ultrathin polyacrylamide gels

a = spacer tape (0,25 mm); b = covering sheet
f = gel carrier sheet (5.2)

(5.3); c, e = glass plates (5.1); d = gel solution (4.1.2);

L 37/58

EN

Official Journal of the European Communities

7.2.2001

Figure 4a: Isoelectric focusing of plasmin-treated caseins from ewes' and goats' milk cheese containing different amounts

of cows' milk.
% CM = percentage of cows' milk, C = cow, E = ewe, G = goat
is shown.
Upper half of the IEF gel

Figure 4b: Isoelectric focusing of plasmin treated caseins from cheese made from mixtures of ewes', goats' and buffalos'

milk containing different amounts of cows' milk.
% CM = percentage of cows' milk; 1 + = sample containing 1 % of cows' milk and spiked with pure bovine
casein at the middle of the track. C = cow, E = ewe, G = goat, B = buffalo.
Total separation distance of the IEF gel

is shown.

7.2.2001

EN

Official Journal of the European Communities

L 37/59

Figure 5: Surperposition of densitograms of standards (STD) and cheese samples made from a mixture of ewes' and

goats' milk after isoelectric focusing.

a,b = standards containing 0 and 1 % of cows' milk; c-g = cheese samples containing 0, 1, 2, 3 and 7 % of
cows' milk. C = cow, E = ewe, G = goat.

Upper half of the IEF gel was scanned at λ = 634 nm.

L 37/60

EN

Official Journal of the European Communities

7.2.2001

ANNEX XVI

(Article 11)

REFERENCE METHOD FOR DETECTING COLIFORMS IN BUTTER, SKIMMED-MILK POWDER, CASEIN AND
CASEINATES

Samples corresponding to 1 g butter are inoculated into the culture medium, if butter is tested for the presence of
coliforms.

Where skimmed-milk powder or casein/caseinates is tested for the presence of coliforms, 0,1 g samples are inoculated
into the culture medium.

IDF Standard 73A: 1985, method B,

is applied with the following modifications:

1) Sample preparation is according to IDF Standard 122B:1992. For acid casein, the sample preparation procedure

described in IDF Standard 73A:1985 may be used alternatively.

2) Only tubes inoculated with 1 g samples (butter) or 0,1 g samples (skimmed-milk powder, casein/caseinates) respec-

tively, are incubated and evaluated. No decimal dilutions are made.

Evaluation of the results

Three negative results:

Requirement fulfilled

Two or three positive results:

Requirement not fulfilled

Two negative results:

Remark

Two further samples should be analysed, weighing 1 g (butter) and 0,1 g
(skimmed-milk powder, casein/caseinates).
If the last two results are negative, the requirement is fulfilled; otherwise the
requirement is not fulfilled.

Coliform content: 1/10 g for butter; 1/g for skimmed-milk powder, casein/caseinates, on average.

Results indicating that the requirement is fulfilled are obtained with a probability of 93 %.

Coliform content: 1/g for butter; 1/0,1 g for skimmed milk powder or casein/caseinates on average.

Results indicating that the requirement is not fulfilled are obtained with a probability of 91 %.

(Assumption: Poisson distribution)

7.2.2001

EN

Official Journal of the European Communities

L 37/61

ANNEX XVII

(Article 12)

METHOD FOR DETERMINING THE LACTOSE CONTENT OF PRODUCTS FALLING WITHIN CN CODE
2309 (1)

PART I

1. Field of application

The method should be applied in cases where the lactose content exceeds 0,5 %.

2. Principle

Dissolve sugars in water. Allow the yeast (Saccharomyces cerevisiae) to act; this will leave the lactose intact. Determine
the lactose content of that solution, by the Luff-Schoorl method, after clarification and filtering.

3. Reagents

Sodium thiosulphate 0,1 N

Indicator: starch solution. Add a mixture of 5 g of soluble starch (10 mg of mercuric iodide may be added as a
preservative agent) and 30 ml of water to 1 litre of boiling water; keep the mixture boiling for three minutes; leave to
cool.

Potassium iodide solution AR at 30 % (w/v).

Sulphuric acid solution 6 N

Luff-Schoorl reagent:

(a) Dissolve 25 g of iron-free copper II sulphate pentahydrate (CuSO4
(b) Dissolve 50 g of citric acid monohydrate (C6
(c) Dissolve 143,8 g of anhydrous sodium carbonate (Na2

CO
2

O
8

H
7

H

O) in 50 ml of water
2

5H
2

O) in 100 ml water

) in about 300 ml of hot water and allow to cool.

Add (b) to (c), shaking gently, then add (a). Make up to 1 litre, allow to stand overnight and then filter. Check the
normality of the reagent thus obtained (Cu 0.1 N, Na2CO3 2 N). The pH must be approximately 9.4.

Carrez I solution: dissolve 23,8 g Zn (C2H3O2)2.2H2O and 3 g glacial acetic acid in water and make up to 100 ml.

F
Carrez II: dissolve 10,6 g de K4

(CN)
2

.3H
6

O in water and make up to 100 ml.
2

Grains of pumice stone, treated when boiling with hydrochloric acid, washed with water and dried. Suspension of
Saccharomyces cerevisae: 25 g of fresh yeast in 100 ml of water (do not keep for more than one week in a
refrigerator).

4. Procedure

Weigh, to the nearest 1 mg, 1 g of the sample for analysis; place this in a calibrated 100 ml flask. Add 25 to 30 ml
of water. Place the flask in a boiling waterbath for thirty minutes, then cool to approximately 35 °C.

Add 5 ml of the yeast suspension (2) and shake. Leave the calibrated flask and its contents in a waterbath at a
temperature of 38 to 40 °C for two hours.

After fermentation, cool to a temperature of approximately 20 °C. Add 2,5 ml of the Carrez I solution and shake for
thirty seconds; then add 2,5 ml of the Carrez II solution and shake again for thirty seconds. Make up to 100 ml with
water, mix and filter. Pipette a quantity of the filtrate of not more than 25 ml, preferably containing 40 to 80 mg
lactose; if necessary, make up to 25 ml with water and determine the anhydrous lactose content by the Luff-Schoorl
method.

Carry out complete blank test with yeast only.

(1) Regulation (EEC) No 222/88.
(2) For products containing more than 40 % fermentable sugar,

increase the quantity of suspension.

L 37/62

EN

Official Journal of the European Communities

7.2.2001

1. Determining the lactose content by the Luff-Schoorl method

PART II

Pipette 25 ml of Luff-Schoorl reagent and place this in an Erlenmeyer flask of 300 ml; add 25 ml of the clarified
solution measured exactly.

After adding two grains of pumice stone, heat, shaking by hand over a naked flame of average height and bring the
liquid to the boil for approximately two minutes. Immediately place the Erlenmeyer flask on a wire gauze with an
asbestos screen, under which a flame has previously been lit. This is so regulated that the Erlenmeyer flask is heated
solely at the bottom; then fit a reflux condenser. From that moment, boil for ten minutes exactly. Cool immediately in
cold water and after approximately five minutes test as follows:

Add to the liquid 10 ml of potassium iodide and immediately afterwards, but with caution (since considerable
foaming may occur), 25 ml of sulphuric acid 6 N.

The test with sodium thiosulphate until a dull yellow colour appears and towards the end of the test add the starch
indicator.

Carry out the same test with a mixture of precisely 25 ml of Luff-Schoorl reagent and 25 ml of water, after adding
10 ml of potassium iodide and 25 ml of sulphuric acid 6 N, this time without bringing to the boil.

Using the following table, establish the amount in mg of lactose corresponding to the difference between the results of
the two tests (expressed in ml of sodium thiosulphate 0,1 N).

TABLE

Table for 25 ml de Luff-Schoorl reagent

(see conditions in the text)

1. Sodium thiosulphate 0,1 N

2. Lactose C

12H22O11

1

ml

2

mg

Difference

1

ml

2

mg

Difference

1

2

3

4

5

6

7

8

9

10

11

3,6

7,3

11,0

14,7

18,4

22,1

25,8

29,5

33,2

37,0

40,8

3,7

3,7

3,7

3,7

3,7

3,7

3,7

3,7

3,8

3,8

3,8

12

13

14

15

16

17

18

19

20

21

22

23

44,6

48,4

52,2

56,0

59,9

63,8

67,7

71,7

75,7

79,8

83,9

88,0

3,8

3,8

3,8

3,9

3,9

3,9

4,0

4,0

4,1

4,1

4,1

7.2.2001

EN

Official Journal of the European Communities

L 37/63

ANNEX XVIII

(Article 13)

DETECTION OF RENNET WHEY IN SKIMMED-MILK POWDER FOR PUBLIC STORAGE BY DETERMINATION
OF GLYCOMACROPEPTIDES HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

1. Scope and field of application

This method allows detection of rennet whey in skimmed-milk powder intended for public storage by determination
of the glycomacropeptides.

2. Reference

International Standard ISO 707 — Milk and Milk Products — Methods of sampling, conforming to the guidelines
contained in Annex I(2)(c) last paragraph.

3. Definition

Glycomacropeptide content of skimmed-milk powder: the content of substances determined by the method set out
below, expressed as a percentage by mass.

4. Principle

— Reconstitution of the skimmed-milk powder, removal of fat and proteins with trichloroacetic acid, followed by

centrifugation;

— Determination of the quantity of glycomacropeptides (GMP)

in the supernatant by high-performance liquid

chromatography (HPLC);

— Evaluation of the result obtained for the samples by reference to standard samples consisting of skimmed-milk

powder with or without the addition of a known percentage of whey powder.

5. Reagents

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

5.1.

Tricloroacetic acid solution

Dissolve 240 g of trichloroacetic acid (Cl3

CCOOH) in water and make up to 1 000 ml.

5.2.

Eluent solution, pH 6,0

), 12,37 g of potassium dihydrogen phosphate
2PO4) and 21,41 g of sodium sulphate (Na2SO4) in about 700 ml of water. Adjust, if necessary, to pH

Dissolve 1,74 g of dipotassium hydrogen phosphate (K
2
(KH
6,0, using a solution of phosphoric acid or potassium hydroxide.

HPO
4

Make up to 1 000 ml with water and homogenise.

Filter the eluent solution, prior to use, through a membrane filter with a 0,45 µm pore diameter.

5.3.

Flushing solvent

Mix one volume acetonitrile (CH3
membrane filter with a 0,45 µm pore diameter.

CN) with nine volumes water. Filter the mixture prior to use through a

Note: Any other flushing solvent with a bactericidal effect which does not impair the columns' resolution

efficiency may be used.

5.4.

Standard samples

5.4.1.

Skimmed-milk powder meeting the requirements of this Resolution (i.e. [0]).

5.4.2.

The same skimmed-milk powder adulterated with 5 % (m/m) rennet-type whey powder of standard com-
position (i.e. [5]).

L 37/64

EN

Official Journal of the European Communities

7.2.2001

6. Apparatus

6.1.

6.2.

6.3.

6.4.

6.5.

6.6.

6.7.

6.8.

6.9.

Analytical balance.

Centrifuge capable of attaining a centrifugal force of 2 200 g, fitted with stoppered centrifuge tubes of about
25 ml capacity.

Mechanical shaker.

Magnetic stirrer.

Glass funnels, diameter about 7 cm.

Filter papers, medium filtration, diameter about 12,5 cm.

Glass filtration equipment with 0,45 µm pore diameter membrane filter.

Graduated pipettes allowing delivery of 10 ml (ISO 648, Class A, or ISO/R 835).

Thermostatic waterbath, set at 25 ± 0,5 °C.

6.10.

HPLC equipment, consisting of:

6.10.1.

Pump.

6.10.2.

Injector, hand or automatic, with a 15 to 30 µl capacity.

6.10.3.

Two TSK 2 000-SW columns in series (length 30 cm, internal diameter 0,75 cm) or equivalent columns
and a precolumn (3 cm × 0,3 cm) packed with I 125 or material of equivalent effectiveness.

6.10.4.

Thermostatic column oven, set at 35 ± 1 °C.

6.10.5.

Variable wavelength UV detector, permitting measurements at 205 nm with a sensitivity of 0,008 A.

6.10.6.

Integrator capable of valley-to-valley integration.

Note: Working with columns kept at room temperature is possible, but their power of resolution in slightly
lower. In that case, the temperature should vary by less than ± 5 °C in any one range of analyses.

7. Sampling

7.1.

International Standard ISO 707 — ‘Milk and Milk Products — Methods of sampling’, conforming to the
guidelines contained in Annex I(2)(c) last paragraph.

7.2.

Store the sample in conditions which preclude any deterioration or change in composition.

8. Procedure

8.1.

Preparation of the test sample

Transfer the milk powder into a container with a capacity of about twice the volume of the powder, fitted
with an airtight lid. Close the container immediately. Mix the milk powder well by means of repeated
inversion of the container.

8.2.

Test portion

Weight 2,000 + 0,001 g of test sample into a centrifuge tube (6.2).

8.3.

Removal of fat and proteins

8.3.1.

8.3.2.

Add 20 g of warm water (50 °C) to the test portion. Dissolve the powder by shaking for five minutes using
a mechanical shaker (6.3). Cool the tube to 25 °C.

Add 10,0 ml of the trichloroacetic acid solution (5.1) in two minutes, while stirring with the aid of the
magnetic stirrer (6.4). Place the tube in a waterbath (6.9) and leave for 60 minutes.

8.3.3.

Centrifuge (6.2) for 10 minutes at 2 200 g, or filter through paper (6.6), discarding the first 5 ml of filtrate.

7.2.2001

EN

Official Journal of the European Communities

L 37/65

8.4.

8.4.1.

Chromatographic determination

Inject 15 to 30 µl of accurately measured supernatant or filtrate (8.3.3) into the HPLC apparatus (6.10)
operating at a flow rate of 1,0 ml of eluent solution (5.2) per minute.

Notes:

1. Keep the eluent solution (5.2) at 85 °C throughout the chromatographic analysis in order to keep the
eluent degassed and to prevent bacterial growth. Any precaution with a similar effect may be used.

2. Rinse the columns with water during each interruption. Never leave the eluent solution in them (5.2).

Prior to any interruption of more than 24 hours, rinse the columns with water then wash them with
solution (5.3) for at least three hours at a flow rate of 0,2 ml per minute.

8.4.2.

The results of chromatographic analysis of the test sample [E] are obtained in the form of chromatogram in
which each peak is identified by its retention time RT as follows:

Peak II:

The second peak of the chromatogram which an RT of about 12,5 minutes.

Peak III:

The third peak of the chromatogram, corresponding to the GMP, with an RT of 15,5 ± 1,0
minutes.

Peak IV:

The fourth peak of the chromatogram with an RT of about 17,5 minutes.

The quality of the columns can effect the retention times of the individual peaks.

The integrator (6.10.6) automatically calculates the area A of each peak:

:

AII
AIII
AIV

:

area of peak II,

area of peak III,

:

area of peak IV.

It is essential to examine the appearance of each chromatogram prior to quantitative interpretation, in order
to detect any abnormalities due either to malfunctioning of the apparatus or the columns, or to the origin
and nature of the sample analysed.

If in doubt, repeat the analysis.

8.5.

Calibration

8.5.1.

Apply exactly the procedure described from point 8.2 to point 8.4.2 to the standard samples (5.4).

Use freshly prepared solutions, because GMP degrade in an 8 % trichloroacetic environment. The loss is
estimated at 0,2 % per hour at 30 °C.

8.5.2.

Prior to chromatographic determination of the samples, condition the columns by repeatedly injecting the
standard sample (5.4.2) in solution (8.5.1) until the area and retention time of the peak corresponding to the
GMP are constant.

8.5.3.

Determine the response factors R by injecting the same volume of filtrates (8.5.1) as used for the samples.

9. Expression of results

9.1.

Method of calculation and formulae

9.1.1.

Calculation of the response factors R:

Peak II:

Peak IV:

where:

R

II
A
II

and R

=

IV

[0] and A

IV

Peak III:

where:

=

R

III
A
III
W =

[0] and A
III

the response factors of peaks II and IV respectively,

[0] = the areas of peaks II and IV respectively of the standard sample [0] obtained in 8.5.3.

the response factor of peak III,

[5] = the areas of peak III in standard samples [0] and [5] respectively obtained in 8.5.3,

the quantity of whey in standard sample [5],

i.e. 5.

L 37/66

EN

Official Journal of the European Communities

7.2.2001

9.1.2.

Calculation of the relative area of the peaks in the sample [E]

SII

[E] = R

× A

II

II

[E]

S

III

[E] = R

× A
III

III

[E]

S
IV [E] = RIV × AIV [E]

where:

S

II

[E], S
III

[E], S

IV

[E] =

the relative areas of peaks II, III and IV respectively in the sample [E],

A
II

[E], A
III

[E], A

IV

R
II

, R
III

, R

IV

=

[E] = the areas of peaks II, III and IV respectively in the sample [E] obtained in 8.4.2,

the response factors calculated in 9.1.1.

9.1.3.

Calculation of the relative retention time of peak III in sample [E]:

where:

RRT

III

[E] = the relative retention time of peak III in sample [E],

RT

RT
III

III [E] =
[5] =

the retention time of peak III in sample [E] obtained in 8.4.2,

the retention time of peak III in control sample [5] obtained in 8.5.3.

9.1.4.

Experiments have shown that there is a linear relation between the relative retention time of peak III, i.e.
RRT

[E] and the percentage of whey powder added up to 10 %.

III

— The RRTIII
— The RRT

[E] is < 1,000 when the whey content is > 5 %;
III [E] is ≥ 1,000 when the whey content is ≤ 5 %.

The uncertainty allowed for the values of RRT
III

is ± 0,002.

Normally the value of RRTIII
value may approach 1,000, but it must always be greater.

[0] deviates little from 1,034. Depending on the condition of the columns, the

9.2.

Calculation of the percentage of rennet whey powder in the sample:

where:

W =

S

III [E] =
1,3

S
III

[0]

the percentage m/m of rennet whey in the sample [E];

the relative area of peak III of test sample [E] obtained as in 9.1.2;

represents the relative average area of peak III expressed in grams of rennet whey per
100 g determined in non-adulterated skimmed-milk powder of various origins. This
figure was obtained experimentally;

represents the relative area of peak III which is equal to R
III
obtained in 9.1.1 and 8.5.3 respectively;

× A

III

[0]. These values are

(S
III

[0] – 0,9)

represents the correction to be made to the relative average area 1,3 when S
[0] is not
III
equal to 0,9. Experimentally the relative average area of peak III of the control sample
[0] is 0,9.

9.3.

Accuracy of the procedure

9.3.1.

R e p e a t a b i l i t y

The difference between the results of two determinations carried out simultaneously or in rapid succession
by the same analyst using the same apparatus on identical test material shall not exceed 0,2 % m/m.

9.3.2.

R e p r o d u c i b i l i t y

The difference between two single and independent results, obtained in two different laboratories on
identical test material shall not exceed 0,4 % m/m.

7.2.2001

EN

Official Journal of the European Communities

L 37/67

9.4.

9.4.1.

9.4.2.

9.4.3.

Interpretation
Assume the absence of whey if the relative area of peak III, SIII
100 g of the product is ≤ 2,0 + (S

[0] – 0,9)

III

[E] expressed in grams of rennet whey per

where

2,0

is the maximum value allowed for the relative area of peak III taking into account the
relative area of peak III, i.e. 1,3, the uncertainty due to variations in the composition of
skimmed-milk powder and the reproducibility of the method (9.3.2),

(S

III

[0] — 0,9)

is the correction to be made when the area S
III

[0] is different from 0,9 (see point 9.2)

If the relative area of peak III, S
III
determine the rennet whey content as indicated in point 9.2.

[E] is > 2,0 + (S
III

[0] – 0,9) and the relative area of peak II, S
II

If the relative area of peak III, S
III
determine the total protein content (P %); then examine graphs 1 and 2.

[E] is > 2,0 + (S
III

[0] – 0,9) and the relative area of peak II, S
II

[E] ≤ 160,

[E] ≤ 160,

9.4.3.1. The data obtained after analysis of samples of unadulterated skimmed-milk powders with a high total

protein content have been assembled in graphs 1 and 2.

The continuous line represents the linear regression, the coefficients of which are calculated by the least
squares method.

The dashed straight line fixes the upper limit of the relative area of peak III with a probability of not being
exceeded in 90 % of cases.

The equations for the dashed straight lines of graphs 1 and 2 are:

S
III = 0,376 P % – 10,7
SIII

= 0,0123 S
II

[E] + 0,93

(graph 1),

(graph 2),

respectively where:

SIII

P %
(SII

[E]

is the relative area of peak III calculated either according to total protein content or
according to the relative area of peak S
II

[E],

is the total protein content expressed as a percentage, by weight,

is the relative area of sample calculated in point 9.1.2.

These equations are equivalent to the figure of 1,3 mentioned in point 9.2.

The discrepancy (T
1
of the following:

) between the relative area S
and T
III
2

[E] found and the relative area S
III

is given by means

9.4.3.2.

If T

1 and/or T2

are zero or less, the presence of rennet whey cannot be determined.

If T

1 and T2

exceed zero, rennet whey is present.

The rennet whey content is calculated according to the following formula:

where:

0,91 is the distance on the vertical axis between the continuous and dotted straight lines.

L 37/68

EN

Official Journal of the European Communities

7.2.2001

7.2.2001

EN

Official Journal of the European Communities

L 37/69

ANNEX XIX

(Article 13)

DETERMINING RENNET WHEY SOLIDS IN SKIMMED-MILK POWDER AND THE MIXTURES REFERRED TO
IN REGULATION (EC) No 2799/1999

1. Purpose: Detecting the addition of rennet whey solids to:

(a) skimmed-milk powder as defined in Article 2 of Regulation (EC) No 2799/1999, and

(b) mixtures as defined in Article 4 of Regulation (EC) No 2799/1999

2. References: International standard ISO 707

3. Definition

The content of rennet whey solids is defined as the percentage by mass as determined by the procedure described.

4. Principle

Glycomacropeptide A content is determined in accordance with Annex XVIII. Samples giving positive results are
analysed for glycomacropeptide A by a reversed-phase high-performance liquid chromatography procedure (HPLC
procedure). Evaluation of the result is obtained by reference to standard samples consisting of skimmed-milk powder
with and without a known percentage of whey powder. Results higher than 1 % (m/m) show that rennet whey solids
are present.

5. Reagents

All reagents must be of recognised analytical grade. The water used must be distilled water or water of at least
equivalent purity. Acetonitrile should be of spectroscopic or HPLC quality.

Reagents needed for the procedure are described in Annex XVIII to this Regulation.

Reagents for reversed phase HPLC.

5.1.

Trichloroacetic acid solution

Dissolve 240 g of trichloroacetic acid (CCI3CCOOH) in water and make up to 1 000 ml.

5.2.

Eluents A and B

Eluent A: 150 ml of acetonitrile (CH3CN), 20 ml of isopropanol (CH3CHOHCH3), and 1,00 ml of trifluoro-
acetic acid (TFA, CF3COOH) are placed in a 1 000 ml volumetric flask. Make up to 1 000 ml with water.
Eluent B: 550 ml of acetonitrile, 20 ml of isopropanol and 1,00 ml of TFA are placed in a 1 000 ml
volumetric flask. Make up to 1 000 ml with water. Filter the eluent solution, prior to use, through a
membrane filter with a 0,45 µm pore diameter.

5.3.

Conservation of the column

After the analyses the column is flushed with eluent B (via a gradient) and subsequently flushed with
acetonitrile (via a gradient for 30 minutes). The column is stored in acetonitrile.

5.4.

Standard samples

5.4.1.

Skimmed-milk powder meeting the requirements for public storage (i. e. (0)).

5.4.2. The same skimmed-milk powder adulterated with 5 % (m/m) rennet-type whey powder of standard composi-

tion (i.e. (5)).

5.4.3. The same skimmed-milk powder adulterated with 50 % (m/m) rennet-type whey powder of standard composi-

tion (i.e. (50)) (1).

6. Apparatus

Apparatus needed for the procedure described is described in Annex XVIII to this Regulation.

6.1.1. Analytical balance.

6.2.

Centrifuge capable of attaining a centrifugal force of 2 200 g, fitted with stoppered centrifuge tubes of about
50 ml capacity.

(1) Rennet-type whey powder of standard composition and also the adulterated skimmed-milk powder are available from NIZO, Kern-
hemseweg 2, PO Box 20 — NL-6710 BA Ede. However, powders giving equivalent results to the NIZO powders may also be used.

L 37/70

EN

Official Journal of the European Communities

7.2.2001

6.3.

Mechanical shaker with a provision to shake at 50 °C.

6.4.

Magnetic stirrer.

6.5.

Glass funnels, diameter about 7 cm.

6.6.

Filter papers, medium filtration, diameter about 12,5 cm.

6.7.

Glass filtration equipment with 0,45 µm pore diameter membrane filter.

6.8.

Graduated pipettes, allowing delivery of 10 ml (ISO 648, Class A, or ISO/R 835), or a system capable of
delivering 10,0 ml

in two minutes.

6.9.

Thermostatic waterbath, set at 25 ± 0,5 °C.

6.10. HPLC equipment, consisting of:

6.10.1. Binary gradient pumping system.

6.10.2. Injector, hand or automatic, with a 100 µl capacity.

6.10.3. Dupont Protein Plus column (length 25 cm, 0,46 cm internal diameter) or an equivalent wide-pore silica

based reversed-phase column.

6.10.4. Thermostatic column oven, set at 35 ± 1 °C.

6.10.5. Variable wavelength UV detector, permitting measurements at 210 nm (if necessary, a higher wavelength up

to 220 nm may be used) with a sensitivity of 0,02 Å.

6.10.6. Integrator capable of valley-to-valley integration.

Note

Operation of the column at room temperature is possible, provided that the room temperature does not
fluctuate more than 1 °C, otherwise too much variation in the retention time of GMPÅ

takes place.

7. Sampling

7.1.

Samples must be taken in accordance with the procedure laid down in International Standard ISO 707.
However, Member States may use another method of sampling provided that it complies with the principles of
the abovementioned standard.

7.2.

Store the sample in conditions which preclude any deterioration or change in composition.

8. Procedure

8.1.

Preparation of the test sample

Transfer the milk powder into a container with a capacity of about twice the volume of the powder, fitted
with an airtight lid. Close the container immediately. Mix the milk powder well by means of repeated
inversion of the container.

8.2.

Test portion

Weigh 2,00 ± 0,001 g of test sample into a centrifuge tube (6.2) or suitable stoppered flask (50 ml).

8.3.

Removal of fat and proteins

8.3.1. Add 20,0 g of warm water (50 °C) to the test portion. Dissolve the powder by shaking for five minutes or 30
minutes in the case of acid buttermilk using a mechanical shaker (6.3). Place the tube into the waterbath (6.9)
and allow to equilibrate to 25 °C.

8.3.2. Add 10,0 ml of the trichloroacetic acid solution at 25 °C (5.1) constantly over two minutes, while stirring
vigorously with the aid of the magnetic stirrer (6.4). Place the tube in a waterbath (6.9) and leave for 60
minutes.

8.3.3. Centrifuge (6.2) 2,200 g for 10 minutes, or filter through paper (6.6), discarding the first 5 ml of filtrate.

8.4.

Chromatographic determination

8.4.1.

Perform HPLC-analysis as described in Annex XVIII. If a negative result is obtained, the sample analysed does
not contain rennet-whey solids in detectable amounts. If the result is positive, the reversed-phase HPLC
procedure described below must be applied. The presence of acid buttermilk powder may give rise to
false-positive results. The reversed-phase HPLC method excludes this possibility.

7.2.2001

EN

Official Journal of the European Communities

L 37/71

8.4.2. Before the reversed phase HPLC-analysis is carried out, the gradient conditions should be optimised. A
retention time of 26 ± 2 minutes for GMPA is optimal for gradient systems with a dead volume of about 6 ml
(volume from the point where the solvents come together to the volume of the injector loop, inclusive).
Gradient systems with a lower dead volume (e.g. 2 ml) should use 22 minutes as an optimal retention time.

Take solutions of the standard samples (5.4) without and with 50 °6 rennet whey.

Inject 100 µl of supernatant or filtrate (8.3.3) into the HPLC apparatus operating at the scouting gradient
conditions given in Table 1.

Scouting gradient conditions for optimisation of the chromatography

Table 1

Time
(minutes)

Flow
(ml/minutes)

Init

27

32

37

42

1,0

1,0

1,0

1,0

1,0

% A

90

60

10

10

90

% B

10

40

90

90

10

Curve

*

lin

lin

lin

lin

Comparison of the two chromatograms should reveal the location of the (peak of GMP

Α).

Using the formula given below, the initial solvent composition to be used for the normal gradient (see 8.4.3)
can be calculated

Where:

RTgmpA:

10:

2,5:

13,5:

26:

6:

30:

27:

retention time of GMPΑ in the scouting gradient
the initial % B of the scouting gradient

% B at midpoint minus % B at initial

in the normal gradient

midpoint time of the scouting gradient

required retention time of GMP
Α

ratio of slopes of the scouting and normal gradient

% B at initial minus % B at 27 minutes in the scouting gradient

run-time of the scouting gradient.

8.4.3. Take solutions of the test samples

Inject 100 µl of accurately measured supernatant or filtrate (8.3.3) into the HPLC apparatus operating at a flow
rate of 1,0 ml of eluent solution (5.2) per minute.

The composition of the eluent of the start of the analysis is obtained from 8.4.2. It is normally close to
A:B = 76:24 (5.2). Immediately after the injection a linear gradient is started, which results in a 5 % higher
percentage of B after 27 minutes. Subsequently a linear gradient is started, which brings the eluent composi-
tion to 90 % B in five minutes. This composition is maintained for five minutes, after which the composition
is changed, via a linear gradient in five minutes to the initial composition. Depending on the internal volume
of the pumping system, the next injection can be made 15 minutes after reaching the initial conditions.

Remarks

1. The retention time of the glycomacropeptide should be 26 ± two minutes. This can be achieved by varying
the initial and end conditions of the first gradient. However, the difference in the % B for the initial and end
conditions of the first gradient must remain 5 % B.

2. The eluents should be degassed sufficiently and should also remain degassed. This is essential for proper
functioning of the gradient pumping system. The standard deviation for the retention time of the GMP
peak should be smaller than 0,1 minutes (n = 10).

3. Every five samples the reference sample (5) should be injected and used to calculate a new response factor

R. (9.1.1).

L 37/72

EN

Official Journal of the European Communities

7.2.2001

8.4.4. The results of the chromatographic analysis of the test sample (E) are obtained in the form of a chromatogram

in which the GMP peak is identified by its retention time of about 26 minutes.

The integrator (6.40.6) automatically calculates the peak height H of the GMP peak. The baseline location
should be checked in every chromatogram. The analysis or the integration should be repeated if the baseline
was incorrectly located.

It is essential to examine the appearance of each chromatogram prior to quantitative interpretation, in order to
detect any abnormalities due either to malfunctioning of the apparatus or the column, or to the origin and
nature of the sample analysed. If in doubt, repeat the analysis.

8.5.

Calibration

8.5.1. Apply exactly the procedure described from point 8.2 to point 8.4.4 to the standard samples (5.4.1 to 5.4.2).
Use freshly prepared solutions, because GMP degrades in an 8 % trichloroacetic acid environment at room
temperature. At 4 °C the solution remains stable for 24 hours. In the case of long series of analyses the use of
a cooled sample tray in the automatic injector is desirable.

Note

8.4.2. may be omitted if the % B at initial conditions is known from previous analyses.

The chromatogram of the reference sample (5) should be analogous to Figure. 1. In this figure the GMPA peak
is preceded by two small peaks. It is essential to obtain a similar separation.

8.5.2.

Prior to chromatographic determination of the samples inject 100 µl of the standard sample without rennet
whey (0) (5.4.1).

The chromatogram should not show a peak at the retention time of the GMPA peak.

8.5.3. Determine the response factors R by injecting the same volume of filtrate (8.5.1) as used for the samples.

9. Expression of results

9.1.

Method of calculation and formulae

9.1.1. Calculation of the response factor R:

GMP peak: R = W/H

Where:

R =

H =

W =

the response factor of the GMP peak

the height of the GMP peak

the quantity of whey in the standard sample (5).

9.2.

Calculation of the percentage of rennet whey powder in the sample

W(E) = R × H(E)

Where:

W(E) =

the percentage (m/m) of rennet whey in the sample (E).

R =

the response factor of the GMP peak (9.1.1)

H(E) =

the height of the GMP peak of the sample (E)

If W(E) is greater than 1 % and the difference between the retention time and that of the standard sample (5) is
smaller than 0,2 minutes then rennet whey solids are present.

9.3.

Accuracy of the procedure

9.3.1. Repeatability

The difference between the results of two determinations carried out simultaneously or in rapid succession by
the same analyst using the same apparatus on identical test material shall not exceed 0,2 % m/m.

7.2.2001

EN

Official Journal of the European Communities

L 37/73

9.3.2. Reproducibility

Not yet determined.

9.3.3.

Linearity

From 0 to 16 % of rennet whey a linear relationship should be obtained with a coefficient of correlation
> 0,99.

9.4.

Interpretation

9.4.1. Whey is considered to be present if the result obtained in 9.2 is higher than 1 % m/m and the retention time
of the GMP peak differs less than 0,2 minutes from that of the standard sample (5). The 1 % limit is fixed in
agreement with the provisions of points 9.2 and 9.4.1 of Annex V to Regulation (EEC) No 625/78.

L 37/74

EN

Official Journal of the European Communities

7.2.2001

ANNEX XX

(Article 14)

SKIMMED-MILK POWDER: QUANTITATIVE DETERMINATION OF PHOSPHATIDYLSERINE AND PHOSPHA-
TIDYLETHANOLAMINE

Method: reversed-phase HPLC

1. Purpose and field of application

The method describes a procedure for the quantitative determination of phosphatidylserine (PS) and phosphatidyle-
thanolamine (PE) in skimmed-milk powder (SMP) and is suitable for detecting buttermilk solids in SMP.

2. Definition

PS +PE content: the mass fraction of substance determined using the procedure here specified. The result is expressed
as milligrams of phosphatidylethanolamine dipalmitoyl (PEDP) perr 100 g powder.

3. Principle

Extraction of aminophospholipids by methanol from reconstituted milk powder. Determination of PS and PE as
o-phthaldialdehyde (OPA) derivatives by reversed-phase (RP) HPLC and fluorescence detection. Quantitification of PS
and PE content in the test sample by reference to a standard sample containing a known amount of PEDP.

4. Reagents

All reagents must be of recognised analytical grade. Water must be distilled or of at least equivalent purity unless
otherwise specified.

4.1.

Standard material: PEDP, at least 99 % pure

Note: Standard material must be stored at – 18 °C.

4.2.

Reagents for standard sample and test sample preparation

4.2.1. HPLC-grade methanol

4.2.2. HPLC-grade chloroform

4.2.3. Tryptamine-monohydrochloride

4.3.

Reagents for o-phthaldialdehyde derivatisation

4.3.1.

Sodium hydroxide, 12 M water solution

4.3.2. Boric acid, 0,4 M water solution adjusted to pH 10,0 with sodium hydroxide (4.3.1)

4.3.3. 2-mercaptoethanol

4.3.4. o-phthaldialdehyde (OPA)

4.4.

HPLC elution solvents

Elution solvents must be prepared using HPLC-grade reagents.

4.4.1. HPLC-grade water

4.4.2. Methanol of fluorimetric tested purity

4.4.3. Tetrahydrofuran

4.4.4.

Sodium dihydrogen phosphate

4.4.5.

Sodium acetate

4.4.6. Acetic acid

5. Apparatus

5.1.

5.2.

5.3.

5.4.

Analytical balance

Beakers, 25 and 100 ml capacity

Pipettes, delivering 1 and 10 ml

Magnetic stirrer

7.2.2001

EN

Official Journal of the European Communities

L 37/75

5.5.

Graduated pipettes, delivering 0,2, 0,5 and 5 ml

5.6.

Volumetric flasks, 10, 50 and 100 ml capacity

5.7.

Syringes, 20 and 100 µl capacity

5.8.

Ultrasonic bath

5.9.

Centrifuge operating at 27 000 × g

5.10. Glass vials, about 5 ml capacity

5.11. Graduated cylinder, 25 ml capacity

5.12.

pH-meter

5.13. HPLC equipment

5.13.1. Gradient pumping system, capable of operating at 1,0 ml/min at 200 bar

5.13.2. Autosampler with derivatisation capability

5.13.3. Column heater set at 30 °C

5.13.4. Fluorescence detector set at 330 nm excitation wavelength and 440 nm emission wavelength

5.13.5. Integrator or data processing software capable of peak area measurement

5.13.6. A Lichrosphere — 100 column (250 × 4,6 mm) or an equivalent column packed with octadecylsilane (C

18), 5 µm particle size

6. Sampling

Sampling must be carried out in accordance with IDF Standard 50B:1985.

7. Procedure

7.1.

Preparation of the internal standard solution

Weigh 30,0 ± 0,1 mg of tryptamine-monohydrochloride (4.2.3) into a 100 ml volumetric flask (5.6) and
make up to the mark with methanol (4.2.1). Pipette 1 ml (5.3) of this solution into a 10 ml volumetric
flask (5.6) and make up to the mark with methanol (4.2.1) in order to obtain a 0,15 mM tryptamine
concentration.

7.2.

Preparation of the test sample solution

Weigh 1,000 ± 0,001 g of the SMP sample into a 25 ml beaker (5.2). Add 10 ml of distilled water at 40 °C
by a pipette (5.3) and stir with a magnetic stirrer (5.4) for 30 minutes in order to dissolve any lumps. Pipette
0,2 ml (5.5) of the reconstituted milk into a 10 ml volumetric flask (5.6), add 100 µl of the 0,15 mM
tryptamine solution (7.1) using a syringe (5.7) and make up to the volume with methanol (4.2.1). Mix
carefully by inversion and sonicate (5.8) for 15 min. Centrifuge (5.9) at 27 000 × g for 10 minutes and
collect the supernatant in a glass vial (5.10).

Note: Test sample solution should be stored at 4 °C until the HPLC analysis is performed.

7.3.

Preparation of the external standard solution

Weigh 55,4 mg PEDP (4.1 ) into a 50 ml volumetric flask (5.6) and add about 25 ml of chloroform (4.2.2)
using a graduated cylinder (5.11). Heat the stoppered flask to 50 °C and mix carefully till the PEDP dissolves.
Cool the flask to 20 °C, make up to the volume with methanol (4.2.1) and mix by inversion. Pipette 1 ml
(5.3) of this solution into a 100 ml volumetric flask (5.6) and make up to the volume with methanol (4.2.1).
Pipette 1 ml (5.3) of this solution into a 10 ml volumetric flask (5.6), add 100 µl (5.7) of 0,15 mM
tryptamine solution (7.1) and make up to the volume with methanol (4.2.1). Mix by inversion.

Note: Reference sample solution should be stored at 4 °C until the HPLC analysis is performed.

7.4.

Preparation of the derivatising reagent

Weigh 25,0 ± 0,1 mg of OPA (4.3.4) into a 10 ml volumetric flask (5.6), add 0,5 ml (5.5) of methanol
(4.2.1) and mix carefully to dissolve the OPA. Make up to the mark with boric acid solution (4.3.2) and add
20 µl of 2-mercaptoethanol (4.3.3) by syringe (5.7).

Note: The derivatising reagent should be stored at 4 °C in a dark vial and is stable for one week.

L 37/76

EN

Official Journal of the European Communities

7.2.2001

7.5.

Determination by HPLC

7.5.1.

Elution solvents (4.4)

Solvent A:

Solution of 0,3 mM sodium dihydrogen phosphate and 3 mM sodium acetate solution (adjusted to pH 6,5
with acetic acid): methanol: tetrahydrofuran = 558:440:2 (v/v/v)

Solvent B:

methanol

7.5.2.

Suggested eluting gradient:

Time (min)

Solvent A (%)

Solvent B (%)

Flow rate (ml/min)

Initial

0,1

5,0

6,0

6,5

9,0

10,0

11,5

12,0

16,0

19,0

20,0

21,0

29,0

30,0

40

40

40

40

40

36

20

16

16

10

0

0

40

40

40

60

60

60

60

60

64

80

84

84

90

100

100

60

60

60

0

0,1

0,1

1,0

1,0

1,0

1,0

1,0

1,0

1,0

1,0

1,0

1,0

1,0

0

Note: The eluting gradient may require slight modification in order to achieve the resolution shown in

figure 1.

Column temperature: 30 °C.

7.5.3.

Injection volume: 50 µl derivatising reagent and 50 µl sample solution.

7.5.4. Column equilibration

Starting up the system on a daily basis, flush the column with 100 % solvent B for 15 minutes, then set at
A : B = 40 : 60 and equilibrate at 1 ml/min for 15 minutes. Perform a blank run by injecting methanol
(4.2.1).

Note: Before long-term storage flush the column with methanol : chloroform = 80 : 20 (v/v) for 30 minutes.

7.5.5. Determination of the PS + PE content in the test sample

7.5.6.

Perform the sequence of the chromatographic analyses keeping constant the run-to-run time in order to
obtain constant retention times. Inject the external standard solution (7.3) every 5-10 test sample solutions
in order to evaluate the response factor.

Note: The column must be cleaned by flushing with 100 % solvent B (7.5.1) for at least 30 minutes every

20-25 runs.

7.6.

Integration mode

7.6.1.

PEDP peak

PEDP is eluted as a single peak. Determine the peak area by valley-to-valley integration.

7.6.2. Tryptamine peak

Tryptamine is eluted as a single peak (Figure 1). Determine the peak area by valley-to-valley integration.

7.2.2001

EN

Official Journal of the European Communities

L 37/77

7.6.3.

PS and PE peaks groups
Under the described conditions (Figure 1), PS elutes as two main partially unresolved peaks preceded by a
minor peak. PE elutes as three main partially unresolved peaks. Determine the whole area of each peak
cluster setting the baseline as reported in Figure 1.

8. Calculation and expression of results

PS and PE content in the test sample shall be calculated as follows:

where:
C =
A1 =
A2 =
=
T1
=
T2

9. Precision

PS or PE content (mg/100 g powder) in the test sample
PEDP peak area of the standard sample solution (7.3)
PS or PE peak area of the test sample solution (7.2)
Tryptamine peak area of the standard sample solution (7.3)
Tryptamine peak area of the test sample solution (7.2).

Note: The values for repeatability were calculated according to the IDF International Standard (1). The provisional

reproducibility limit was calculated according to the procedure defined in Annex III(b) hereto.

9.1.

Repeatability

The relative standard derivation of the repeatability, which expresses the variability of independent analytical
results obtained by the same operator using the same apparatus under the same conditions on the same test
sample and in a short interval of time, should not exceed 2 % relative. If two determinations are obtained
under these conditions, the relative difference between the two results should not be greater than 6 % of the
arithmetic mean of the results.

9.2.

Reproducibility

If two determinations are obtained by operators in different laboratories using different apparatus under
different conditions for the analysis on the same test sample, the relative difference between the two results
should not be greater than 11 % of the arithmetic mean of the results.

10. References

10.1.

Resmini P., Pellegrino L., Hogenboom J.A., Sadini V., Rampilli M., ‘Detection of buttermilk solids in skimmilk
aminophospholipids.’ Sci. Tecn. Latt.-Cas., 39,395 (1988).
powder by HPLC quantification of

(1) International IDF Standard 135B/1991. Milk and milk products. Precision characteristics of analytical methods. Outline of collabora-

tive study procedure.

L

3
7
/
7
8

E
N

O

f
f
i
c
i
a
l

J
o
u
r
n
a
l

o
f

t
h
e

E
u
r
o
p
e
a
n
C
o
m
m
u
n
i
t
i
e
s

Figure 1: HPLC pattern of OPA-derivatives of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in methanol extract of reconstituted skim-milk powder. Integration mode for the peaks of PS, PE and

tryptamine (internal standard) is reported.

.

.

7
2
2
0
0
1

7.2.2001

EN

Official Journal of the European Communities

L 37/79

ANNEX XXI

(Article 15)

DETECTION OF ANTIBIOTIC AND SULFONAMIDE/DAPSON RESIDUES IN SKIMMED MILK POWDER

A microbial inhibitor screening test using Bacillus stearothermophilus var. calidolactis C953 as test micro-organism and being
sufficiently sensitive to detect 4 µg Benzylpenicillin per milk and 100 µg sulfadimidine per milk shall be used.
Commercial tests kits are available and can be used if they have the required sensitivity for Benzylpencillin and
sulfadimidine (1).

For the test, reconstituted skimmed milk powder (1 g powder + 9 ml aqua dest) is used. The test is carried out as
described in IDF — Bulletin No 258/1991, section 1, Chapter 2, or according to the instructions of the test kit
manufacturer.

Positive results are to be interpreted as follows:

1. Repeat the test adding penicillinase to the test system:

Positive result: Inhibiting substance cannot be identified by this procedure.

Negative result: Inhibiting substance is a β-lactam antibiotic.

2. Repeat the test adding p-amino benzoic acid to the test system:

Positive result: Inhibiting substance cannot be identified by this procedure.

Negative result: Inhibiting substance is a sulfonamide/dapson.

3. Repeat the test adding pencillinase + p-amino benzoic acid to the test system:

Positive result: Inhibiting substance cannot be identified by this procedure.

Negative result: Inhibiting substances are a β-lactam antibiotic and a sulfonamide/dapson.

(1) Important notice: False-positive results may be obtained, when skimmed milk powder is analysed. It is important, therefore, to verify

that the test system used does not yield false-positive results.

L 37/80

EN

Official Journal of the European Communities

7.2.2001

ANNEX XXII

(Article 16)

QUANTITATIVE DETERMINATION OF SKIMMED-MILK POWDER IN COMPOUND FEEDINGSTUFFS BY
ENZYMATIC COAGULATION OF PARA-CASEIN

1. Purpose

Quantitative determination of skimmed-milk powder in compound feedingstuffs by enzymatic coagulation of
para-casein.

2. Scope

This method applies to compound feedingstuffs containing at least 10 % skimmed-milk powder; large quantities of
buttermilk and/or of certain non-milk proteins may lead to interferences.

3. Principle of the method

3.1. Dissolving of casein contained in the compound feedingstuff by extraction with sodium citrate solution.

3.2. Adjustment of the calcium ion concentration to the required level to precipitate para-casein; by addition of

rennet para-casein is obtained from casein.

3.3.

The nitrogen content of the para-casein precipitate is determined by the Kjeldahl method as described by IDF
standard 20A 1986; the quantity of skimmed-milk powder is calculated on the basis of a minimum casein
content of 27,5 % (see 9.1).

4. Reagents

The reagents used must be of analytical grade. The water used must be distilled water or water of equivalent purity.
With the exception of the rennet (4.5), all the reagents and solutions must be free of nitrogenous substances.

4.1.

Trisodium citrate, dihydrate (1 % w/v solution).

4.2.

Calcium chloride (2M solution). Weigh 20,018 g of CaCO
(analytical grade) in a porcelain vessel of suitable
3
size (150 to 200 ml) or in a beaker. Cover with distilled water and transfer onto a boiling water bath. Add
slowly 50 to 60 ml of HCl solution (conc. HCl:water = 1:1) to solubilise the carbonate completely. Keep on
the boiling water bath until the CaCl
2 is dried, to eliminate the HCl which has not reacted. Transfer with
distilled water to a 100 ml measuring flask and dilute to the mark. Measure the pH value, which must be not
lower than 4,0. Store the solution in a refrigerator.

4.3.

0,1 N sodium hydroxide.

4.4.

0,1 N hydrochloric acid.

4.5.

Liquid calf rennet (standard strength of 1:10 000). Store in a refrigerator at 4 to 6 °C.

4.6.

Reagents for the quantitative determination of nitrogen according to the Kjeldahl method as described by IDF
standard 20A 1986.

5. Apparatus

Common laboratory apparatus,

including:

5.1. Mortar or homogeniser

5.2. Analytical balance

5.3.

Bench-top centrifuge (2 000 to 3 000 rpm) with 50 ml tubes

5.4. Magnetic stirrer with (10 to 15 mm) followers

5.5.

150 to 200 ml beakers

5.6.

250 and 500 ml flasks

5.7. Glass funnels of 60 to 80 mm diameter

5.8.

Fast-filtering ashless filters of diameter 150 mm (S.S. 5892, S.S. 595 1/2)

5.9.

Pipettes of various nominal volume

7.2.2001

EN

Official Journal of the European Communities

L 37/81

5.10. Thermostatically controlled water bath at 37 °C

5.11. pH meter

5.12. Kjeldahl digestion and distillation assembly with fittings

5.13. 25 ml graduated burette

5.14. Plastic wash bottle for distilled water

5.15. Stainless steel spatulas

5.16. Thermometers

5.17. Temperature-controlled drying oven.

6. Procedure

6.1.

Preparation of the sample.

Grind in the mortar or homogenise in the mill 10 to 20 g of the sample to obtain a homogeneous mixture.

6.2. Dissolving of milk powder and separation of the insoluble residue.

6.2.1. Weigh 1,000 ± 0,002 g of well-homogenised compound feedingstuff (6.1) directly into a 50 ml centrifuge
tube. Add 30 ml of trisodium citrate solution (4.1) previously heated to 45 °C. Mix with the aid of the
magnetic stirrer for at least five minutes.

6.2.2. Centrifuge at 500 g (2 000 to 3 000 rpm) for 10 minutes and decant the clear aqueous supernatant into a

150 to 200 ml beaker, taking care that no loose material on the bottom goes over.

6.2.3. Carry out two further extractions on the residue, according to the same procedure, adding the extracts to the

first one.

6.2.4. If a layer of oil forms at the surface, cool in the refrigerator until the fat solidifies and remove the solid layer

with a spatula.

6.3.

Coagulation of casein with the enzymes of rennet.

6.3.1. While stirring continuously, add dropwise 3,4 ml of a saturated solution of calcium chloride (4.2) to the total
aqueous extract (about 100 ml). Adjust the pH to 6,4-6,5 with solutions of NaOH (4.3) or HCl (4.4). Place in
the thermostatically-controlled water bath at 37 °C for 15 to 20 minutes to obtain saline balance. It becomes
more evident by the formation of a light turbidity.

6.3.2. Transfer the liquid into one (or two) centrifuge tubes and centrifuge at 2 000 g for 10 minutes in order to
remove the precipitated material. Transfer the supernatant, without washing the sediment, into one (or two)
centrifuge tubes.

6.3.3. Bring the temperature of the supernatant back to 37 °C. While stirring the extract, add, dropwise, 0,5 ml of

the liquid rennet (4.5). Coagulation occurs in one or two minutes.

6.3.4. Return the sample to the water bath and leave at a temperature of 37 °C for 15 minutes. Remove the sample
from the bath and break the coagulum by stirring. Centrifuge at 2 000 g for 10 minutes. Filter the
supernatant through a suitable filter paper (1) (Whatman No 541 or equivalent) and retain the filter paper.
Wash the precipitate in the centrifuge tube with 50 ml of water at approximately 35 °C by stirring the
precipitate.

Centrifuge again at 2 000 g for 10 minutes. Filter the supernatant through the filter paper retained previously.

6.4. Determination of casein nitrogen.

6.4.1. After washing, transfer quantitatively the precipitate to the filter paper retained from 6.3.4 using distilled
water. Transfer the filter paper to the Kjeldahl flask. Determine the nitrogen by the Kjeldahl method as
described by IDF standard 20A 1986.

7. Blank test

7.1. A blank test shall be made regularly using an ashless filter paper (5.8) moistened with a mixture of 90 ml
(4.1) sodium citrate solution, 1 ml saturated solution of calcium chloride (4.2), 0,5 ml of liquid rennet (4.5),
and washed with 3 × 15 ml of distilled water before mineralisation by the Kjeldahl method as described at IDF
standard 20A 1986.

7.2.

The volume of acid used for the blank test must be subtracted from the volume of acid (4.4) used for titration
of the sample.

(1) As fast filtering ashless paper should be used.

L 37/82

EN

Official Journal of the European Communities

7.2.2001

8. Control test

8.1.

To test
the abovementioned procedure and reagents, make a determination on a standard compound
feedingstuff with a known skimmed-milk powder content as established by collaborative study. The average
result of a duplicate determination should not differ by more than 1 % from that of the collaborative study.

9. Expression of results

9.1.

The percentage of skimmed-milk powder in the compound feedingstuff is calculated by the following formula:

where N is the percentage of para-casein nitrogen; 27,5 is the factor for converting determined casein into the
percentage of skimmed-milk powder; 2,81 and 0,908 are correction factors obtained from regression analysis.

10. Accuracy of the method

10.1. Repeatability

In at least 95 % of the cases studied, duplicate analysis of the same sample by the same operator in the same
laboratory must give differences in the results equivalent to no greater than 2,3 g of skimmed-milk powder in
100 g of compound feedingstuff.

10.2. Reproducibility

In at least 95 % of the cases studied, the same sample analysed by two laboratories, must give differences in
the results no greater than 6,5 g of skimmed-milk powder in 100 g of compound feedingstuff.

11. Tolerance limit

The CrD

95

value (critical difference; 95 % confidence limit) is calculated using the formula (ISO 5725):

(R: reproducibility; r: repeatability)

Double determination: CrD

95

= 4,5 g

Where the result of the chemical analysis differs from the declared content of skimmed-milk powder by not more
than 4,5 g (double determination) the consignment of compound feedingstuff is deemed to comply with this
provision of the Regulation.

12. Observations

12.1. The addition of large percentages of certain non-milk proteins and especially of soya proteins, when heated
together with skimmed-milk powder, may lead to too high results due to co-precipitation with the para-casein
of milk.

12.2. Addition of buttermilk may lead to somewhat low figures, due to the fact that only the non-fat portion is
determined. Addition of certain acid buttermilk may give considerably low figures, due to incomplete
dissolving in the citrate solution.

12.3. Lecithin additions of 0,5 % or more may also lead to low results.

12.4.

Incorporation of high-heat skimmed-milk powder may lead to too high figures due to the co-precipitation of
certain whey proteins with the para-casein of milk.

7.2.2001

EN

Official Journal of the European Communities

L 37/83

ANNEX XXIII

(Article 17)

QUALITATIVE DETERMINATION OF STARCH IN SKIMMED-MILK POWDER, DENATURED MILK POWDER
AND COMPOUND FEEDINGSTUFFS

1. Scope

This method is for the detection of starch which is issued as a tracer in denatured milk powders.

Limit of detection of the method is approximately 0,05 g of starch per 100 g of sample.

2. Principle

The reaction is based on the one used in iodometry:

— fixation by the colloids of the free iodine in aqueous solution,

— absorption by the starch micelles and by colour formation.

3. Reagents

3.1. Iodine solution

— iodine: 1 g,

— potassium iodine: 2 g,

— distilled water: 100 ml.

4. Apparatus

4.1. Analytical balance

4.2. Water bath

4.3. Test tubes, 25 mm × 200 mm

5. Procedure

Weight 1 g of the sample and transfer it into the test tube (4.3).

Add 20 ml of distilled water and shake in order to disperse the sample.

Place in the boiling water bath (4.2) and leave for 5 minutes.

Remove from the bath and cool to room temperature.

Add 0,5 ml of the iodine solution (3.1), shake and observe the resulting colour.

6. Expression of results

A blue colouration indicates the presence of native starch in the sample.

When the sample contains modified starch the colour may not be blue.

7. Remarks

The colour, the intensity of the colour and the microscopic appearance of the starch, will vary depending on the origin
of native starch (e.g. maize or potato) and the type of modified starch present in the sample.

In the presence of modified starches the colour produced turns violet, red or brown, according to the degree of
modification of the crystalline structure of native starch.

L 37/84

EN

Official Journal of the European Communities

7.2.2001

ANNEX XXIV

(Article 18)

DETERMINATION OF MOISTURE IN ACID BUTTERMILK POWDER

1. Scope

To determine the moisture content of acid buttermilk powder intended for animal feedingstuffs.

2. Principle

The sample is dried under vacuum. The loss of mass is determined by weighing.

3. Apparatus

3.1. Analytical balance

3.2. Dry containers of non-corrodible metal or of glass with lids ensuring airtight closure; working surface allowing

the test sample to be spread at about 0,3 g/cm2.

3.3. Adjustable electrically heated vacuum oven fitted with an oil pump and either a mechanism for introducing hot

dried air or a drying agent (e.g. calcium oxide).

3.4. Desiccator with an efficient drying agent.

3.5. Drying oven ventilated, thermostatically controlled, at 102 ± 2 °C.

4. Procedure

Heat a container (3.2) with its lid in the oven (3.5) for at least one hour. Place the lid on the container, immediately
transfer to a desiccator (3.4) allow to cool to room temperature and weigh to the nearest 0,5 mg.

Weigh a container (3.2) with its lid to the nearest 0,5 mg. Weight in the weighted container, to the nearest 1 mg,
about 5 g of the sample and spread evenly. Place the container without its lid, in the vacuum oven (3.3) preheated to
83 °C. To prevent the oven temperature from falling unduly,

introduce the container as rapidly as possible.

Bring the pressure up to 100 Torr (13,3 kPa) and leave to dry for four hours at this pressure, either in a current of
hot, dry air or using a drying agent (about 300 g for 20 samples). In the latter instance, disconnect the vacuum pump
when the prescribed pressure has been reached. Reckon drying time from the moment when the oven temperature
returns to 83 °C. Carefully bring the oven back to atmospheric pressure. Open the oven, place the lid on the container
immediately, remove the container from the oven, leave to cool for 30 to 45 minutes in the desiccator (3.4) and weigh
to the nearest 1 mg. Dry for an additional 30 minutes in the vacuum oven (3.3) at 83 °C and reweigh. The difference
between the two weighings must not exceed 0,1 % of moisture.

5. Calculation

where

E = initial mass,

in grammes of the test sample,

m = mass,

in grammes, of the dry test sample.

6. Precision

6.1. Repeatability limit

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 shall not exceed 0,4 g water/100 g acid
buttermilk powder.

7.2.2001

EN

Official Journal of the European Communities

L 37/85

6.2. Reproducibility limit

The difference between the results of two determinations carried out by operators in different laboratories, using
different apparatus on identical test material shall not exceed 0,6 g water/100 g acid buttermilk powder.

6.3. Source of precision data

The precision data were determined from an experiment conducted in 1995 involving eight laboratories and 12
samples (6 blind duplicates).

L 37/86

EN

Official Journal of the European Communities

7.2.2001

ANNEX XXV

(Article 19)

REFERENCE METHOD FOR THE DETECTION OF FOREIGN FATS IN MILK FAT BY GAS CHROMATO-
GRAPHIC ANALYSIS OF TRIGLYCERIDES — REVISION 1

1. Scope and field of application

This standard lays down a method for the detection of foreign fats, of both vegetable fats and animal fats such as beef
tallow and lard in milk fat of milk products using gas chromatographic analysis of triglycerides.

Using defined triglyceride formulae vegetable and animal fats are qualitatively and quantitatively detected in pure milk
fat irrespective of feeding or lactation conditions.

Note 1: Although butyric acid (C4) occurring exclusively in milk fats enables quantitative estimations of low to mean
amounts of milk fat in vegetable fats to be made, qualitative and quantitative information can hardly be
provided in the range of an addition of up to at least 20 % (weight %) foreign fat to pure milk fat because of
the large variation of C4 ranging approximately between 3,5 to 4,5 % (weight %).

Note 2: Quantitative results can practically only be obtained by triglyceride analyses, because the sterol content of the

vegetable fats is different as a function of production and treatment conditions.

2. Definition

Foreign fats in milk fat: foreign fats as defined in this standard are all vegetable and animal fats except milk fat.

3. Principle of the method

After extraction of the milk fat a stock solution is prepared. From this solution the triglycerides (total carbon
numbers) are determined gas chromatographically on packed columns. By inserting the weight % of the fat molecules
of different size (C24 — C54 — only even numbers)
in the triglyceride formula the foreign fats are either
qualitatively detected or quantitatively determined.

Note: Observing the evaluation described here capillary gas chromatography can be used, if it is guaranteed that

comparable results are achieved (1).

4. Reagents

Analysis-grade chemicals must be used.

4.1. Carrier gas: nitrogen, purity degree (cid:6) 99,996 %.

4.2.

Standard triglycerides (2), saturated as well as cholesterol for standardizing a standard milk fat according to
section 6.5.4.

4.3. Methanol, water-free.

4.4. n-Hexane

4.5. n-Heptane

4.6. Toluene

4.7. Dimethylchlorosilane solution: 50 ml dimethylchlorosilane are dissolved in 283 ml toluene.

4.8. Combustible gas: hydrogen and synthetic air

4.9.

Stationary phase, 3–% OV-1 on 125/150 µm (100/120 mesh) Gas ChromQ. (3)

4.10. 10 % cocoa butter solution

5.

Instruments

Normal

laboratory apparatus and particularly the following:

5.1.

High temperature gas chromatograph suited for temperatures of at least 400 — 450 °C, equipped with a
flame ionization detector (FID) and constant mass flow controller for the carrier gas. Combustion gas:
30 ml/min H2

, 270 ml/min synthetic air.

(1) Suited methods have already been described, see D. Precht and J. Molkentin: Quantitative triglyceride analysis using short capillary

columns, Chrompack News 4, 16-17 (1993).
(2) Suitable products are commercially available.
(3) Trade names such as, e.g. Extrelut, Gas ChromQ, Chrompack are examples for suited products available in the specialised trade.
This information shall serve the purpose of easy handling of the standard by the user and does not represent a request of the
product. The indication of grain was transferred to the SI-unit µm according to BS 410:1988 ‘British Standard Specification for test
sieves’.

7.2.2001

EN

Official Journal of the European Communities

L 37/87

Given the high carrier gas flow, the flame jet should be particularly large.

Note: Because of the high temperatures occuring during triglyceride analyses the glass inserts in the FID or

in the injector system must be frequently cleaned.

The gas chromatograph must be equipped with septa, withstanding high temperatures, which can be
frequently used and exhibit generally a very low degree of ‘bleeding’

Note: Suited are Chromblue (tm) septa (Chrompack).

The septa must be exchanged at regular intervals, e.g. after roughly 100 injections or as soon as the
resolution deteriorates (see figure 4).

5.2.

Chromatography column

U-shaped glass column (inside diameter 2 mm, 500 mm in length), which is first silanized according to
section 6.1 with dimethylchlorosilane in order to deactivate the glass surface.

Note: Suited are also somewhat longer (80 — 200 mm in length) packed columns. With them a slightly
better reproducibility of the results can be achieved. On the other hand, the stationary phase exhibits
occasionally fractures after operation, which may lead, in turn, to worse quantitative results. Further,
the FID flame is easily extinguised as a result of the required extremely high carrier gas flow of 75 to
85 ml/min.

5.3.

Arrangement for filling the column (see figure 1)

Figure 1

Filling of the column

5.3.1.

5.3.2.

Plastic column with screwed-on end caps, provided with a mark up to which the required quantity of
stationary phase can be filled

Fine sieve (mesh size approximately 100 µm) with screw cap, suited for sealing the glass column according
to figure 1.

5.3.3. Deactivated, silanized glass wool

5.3.4.

Vibrator for uniform distribution of the stationary phase during filling

5.4.

1 to 3 ml Extrelut column (1) with silica gel. This column can alternatively be used for the extraction for
obtaining milk fat.

(1) See footnote 3 on page 86.

L 37/88

EN

Official Journal of the European Communities

7.2.2001

5.5.

5.6.

Graphite seal 6,4 mm (1/4(cid:7)) with 6 mm bore

Devices for silanizing the glass surface of the column according to section 6.1.

5.6.1. Woulff bottle

5.6.2. Water suction pump

5.7.

5.8.

5.9.

Water bath, adjustable to (50 ± 2) °C

Drying cabinet, adjustable to (50 ± 2) °C and (100 ± 2) °C

Microlitre pipette

5.10.

5 ml graduated pipette for dosing 1,5 ml methanol

5.11.

50 ml round-bottomed flask

5.12.

Erlenmeyer flask, nominal volume 50 ml

5.13.

Funnel

5.14.

Fine-pored filter

5.15.

Rotary evaporator

5.16.

Ampoules, nominal volume 1 ml, sealable with an aluminium cap, with a septum in the interior

5.17.

Injection syringe, the plunger of the syringe used must not reach into the tip of the needle.

Note: Such syringes allow a better reproducibility of the results to be obtained.

In oder to avoid deterioration of the septum, the tip of the needle should be checked at regular intervals (e.g.
with a stereomicroscope).

6. Procedure

6.1.

Preparation of the column (silanization)

After connecting the Woulff bottle, as shown in figure 2, with the water suction pump tube 2 is dipped into
the solution according to section 4.7. By closing the stopcock the column is filled; subsequently the two
tubes are removed.

Figure 2

Arrangement for silanisation

The column is fixed on a stand and completely filled with the dimethyldichlorosilane solution by means of a
pipette.

After 20-30 min the Woulff bottle is replaced by a filter flask and the column emptied by connecting it
with the water suction pump (see figure 3).

7.2.2001

EN

Official Journal of the European Communities

L 37/89

6.2.

Filling of the column

This is followed by successive rinsing using 75 ml toluene and 50 ml methanol; then the emptied column
is dried in the drying cabinet at 100 °C for approximately 30 minutes.

Figure 3

Arrangement for rinsing

For filling of the column the arrangement as represented in figure 1 is used. The stationary phase according
to 4.9 is filled into the plastic column up to the mark. The lower end of the glass column to be filled is
sealed with an approximately 1 cm long plug of glass wool, which had been silanized before, and which is
pressed in using a steel rod. Then the end of the column is closed with the sieve according to section 5.3.2.

The column is filled under pressure (3 bar, with N
2
continuous and firm packing, a vibrator is moved up and down the glass column during filling.

) with the stationary phase. To obtain a uniform,

After filling a solid plug of silanized glass wool is pressed into the other end of the packed column, the
protruding ends are cut off and the plug pressed into the column a few millimetres with a spatula.

6.3.

Preparation of the samples

For sample preparation one of the three following methods is used:

6.3.1.

Isolation of the milk fat from butter

5 to 10 g of butter is melted in a suitable vessel

in a water bath according to section 5.7 at 50 °C.

A 50 ml Erlenmeyer flask and a funnel with inserted filter according to section 5.14 are heated in the drying
cabinet to 50 °C. The fat layer of the molten butter sample is filtered using the preheated device.

Such a milk fat is almost phospholipid-free.

6.3.2.

Extraction of the fat fraction according to the Röse-Gottlieb method

Extraction is done either according to IDF Standard 1 C: 1987, 16C: 1987, 116A: 1987 or 22B: 1987.

With such a milk fat phospholipids allow a cholesterol peak to be obtained which is increased by
approximately 0,1 %.

The triglyceride spectrum standardized to 100 with the cholesterol is thereby influenced only to a negligible
extent.

6.3.3.

Extraction from milk using silica gel columns

0,7 ml of a milk sample tempered to 20 °C are applied to a 1 to 3 ml Extrelut column with a microlitre
pipette according to section 5.4 and allowed to distribute uniformly on the silica gel for approximately five
minutes.

For denaturing the protein-lipid complexes 1,5 ml of methanol is added by pipette. Subsequently the sample
is extracted with 20 ml n-hexane. The n-hexane is slowly added in small amounts and the solvent draining
off collected in a 50 ml round-bottomed flask that had been dried to a constant, known weight.

After extraction led the column drain until empty.

L 37/90

EN

Official Journal of the European Communities

7.2.2001

From the eluate the solvents are distilled off on a rotatory evaporator at a water bath temperature of 40 to
50 °C.

The flask is dried and the fat yield determined by weighing.

Note: Fat extractions according to Gerber, Weibull-Berntrop, Schmid-Bondzynski-Ratzlaff or isolation of
milk fat using detergents (BDI method) are not suited for triglyceride analysis, because with these
methods more or less large quantities of partial glycerides or phospholipids can pass into the fat
phase.

6.4.

Preparation of the sample solution

For gas chromatography a 5 % solution of the fat in n-heptane obtained according to section 6.3 is used. For
preparing this sample solution corresponding amounts of the sample material obtained according to the
sections 6.3.1 and 6.3.2 are weighed and dissolved in corresponding amounts of n-heptane.

With sample preparation according to section 6.3.3 the amount of n-heptane to be added to the sample
in the flask is calculated on the basis of weighing and the remainer dissolved in it.
material

Approximately 1 ml of the sample solution is filled into an ampule according to section 5.16.

6.5.

Chromatographic triglyceride determination

With the high temperatures of up to 350 °C for eluting the long-chain triglycerides C52-C56 an increase in
baseline occurs easily, particularly if the columns have not been adequately conditioned in the beginning.
This rise in baseline at high temperatures can be avoided completely by either combining two columns or
baseline subtraction.

With the compensating mode or operation with single columns, as well as for the glass inserts in the
injector and in the detector the graphite seals according to section 5.5 have to be used.

6.5.1.

Baseline correction

To avoid baseline rising one of the four methods is used:

6.5.1.1. Combination of columns

Two packed columns are used in compensating mode.

6.5.1.2. Baseline correction by the gas chromatograph

By application of a run by the gas chromatograph without injection of a fat solution and subsequent
subtraction of the stored baseline rising of the baseline can be avoided.

6.5.1.3. Baseline correction by integration software

By application of a run by the integration system without injection of a fat solution and subsequent
subtraction of the stored baseline rising of the baseline can be avoided.

6.5.1.4. Baseline correction by adequate conditioning

With adequate initial conditioning of the column and approximately 20 injections with milk fat solutions
baseline rising at high temperatures is frequently so low that baseline corrections are not necessary.

6.5.2.

Injection technique

To avoid discrimination effects the ‘hot injection’ technique is applied to achieve better quantitative results
with the high-boiling triglyceride components. Here, the fat solution is drawn up in the syringe and the cold
needle of the syringe warmed up prior to injection for approximately three seconds in the injector head.
Τhen, the syringe content is rapidly injected.

Note: With this injection technique the risk of fractionation phenomena inside the syringe or the injection
block is reduced. ‘On-column’ direct injection in the upper, extended heated part of the column is not
applied, because the fragments of the septum, which accumulate here, as well as contaminations can
be easily eliminated with the used technique by regularly changing an injector insert without
dismounting the column.

Bending of the tip of the needle caused by touching the bottom of the sample beaker (even if it is hardly
visible to the eye) must be alsolutely avoided in order not to damage the septum.

7.2.2001

EN

Official Journal of the European Communities

L 37/91

Figure 4

Triglyceride chromatogram of a milk fat sample

6.5.3.

Conditioning of a packed column

During steps (a) to (c) the top of the column is not connected to the detector to avoid contamination.

The columns filled according to section 6.2 are conditioned as follows:

(a) 15 min 40 ml/min N
2

-flow at 50 °C;

(b) Heating with 1 K/min up to 355 °C at 10 ml N

/min;
2

(c) Holding for 12 to 15 h at 355 °C;

(d) two injections of 1 µl of the cocoa butter solution according to section 4.10 and respective temperature

program;

(e) 20 injections of 0,5 µm of a milk fat solution for two to three days according to section 6.4.

Note: Cocoa butter consists almost exclusively of high-boiling C5O to C56 triglycerides. Injection with
cocoa butter serves the purpose of special conditioning in this long-chain range. With the high-boiling
triglycerides C50 to C54 partly response factors of up to approximately 1,20 may occur. Normally,
with repeated injection of a milk fat solution a reduction of the initially high response factors for C50
to C54 has to be expected. With triglycerides with low acyl-c number the factors approximate 1.
Three pairs, respectively, of the columns filled according to section 6.2 are prepared. The conditioned
pairs are checked, respectively, with a milk fat analysis for routine testing.

The pair with the best quantitative results (response factors almost 1) is used in the following. With response
factors > 1,20 the column is not used.

6.5.4.

Calibration

For calibration the response factors of the corresponding triglycerides, as well as of cholesterol of a milk fat
(standardized fat) should be determined using the standardized triglycerides (at least the saturated triglycer-
ides C24, C30, C36, C42, C48 and C54, as well as cholesterol; better still additionally C50 and C52).
Intermediate response factors can be found by mathematical

interpolation.

Using the standardized fat two to three calibrations have to be performed every day. If almost identical
results are obtained, well reproducible quantitative results are achieved with triglyceride analysis of the
samples.

The standardized milk fat has a stock life of several months at a storage temperature of maximally –18 °C
and can, thus, be used as a standard.

Note: The response factor of each constituent may also be determined using a standardised fat with a
certified triglyceride composition, such as CRM 519 (anhydrous milk fat) obtainable from the Institute
for Reference Materials and Measurements, Geel, Belgium.

L 37/92

EN

Official Journal of the European Communities

7.2.2001

6.5.5.

Temperature programme, carrier gas and other conditions for triglyceride analysis

Temperature programme:
6 °C/min to 350 °C and hold at final temperature for five minutes.

initial column temperature 210 °C, hold for one minute, then program at

Detector- and injector temperature: 370o C, respectively.

Note: Detector, injector, and oven temperatures (initial temperature) should be maintained at a constant level

(also overnight, during weekends and holidays).

Carrier gas: nitrogen, flow rate 40 ml/min.

Note: If 80 cm columns are used, the flow must be at least 75 ml/min N2

. The carrier gas flow must be
constantly maintained (also overnight, as well as during weekends and holidays). The exact carrier gas
flow should be adjusted in a manner that independent of column length C54 is eluted at 341 °C.

Duration of analysis : 29,3 minutes.

Injection volume : 0,5 µl.

Note: The syringe has to be rinsed several times with pure heptane after each injection.

FID conditions: according to section 5.1

Note: The flame ionization detector is ignited, respectively, at the beginning of each working day.

7.

Integration, evaluation and control of the measuring conditions

Triglycerides with odd acyl-c number (2n + 1) are combined with the preceding even-numbered triglyceride (2n).
The less reproducible low C56 contents are not taken into account. The remaining triglycerides (peak area) in the
chromatogram, including cholesterol (peak near to C24) are multiplied by the respective response factors of the
standard fat (last calibration) and altogether normalized to 100. Besides free cholesterol the triglycerides C24, C26,
C28, C30, C32, C34, C36, C38, C40, C42, C44, C46, C48, C50, C52 and C54 are, thus, evaluated. Results are given
in weight % (g/100 g).

Evaluation of the chromatogram peaks should be done with an integrator, with which the baseline can be plotted.
Reintegration with optimized integration parameters should be possible.

Figures 5 and 6 demonstrate two examples of triglyceride chromatograms. Figure 5 shows a chromatogram which
can be well evaluated, whilst figure 6 represents a sporadic error in the C50 to C54 range, the baseline running
incorrectly compared with figure 5. Such typical errors can be detected with a high degree of certainty and avoided
only by use of an integrator with which the baseline is plotted.

Easy-to-evaluate triglyceride chromatogram of a milk fat with baseline drawn in

Figure 5

7.2.2001

EN

Official Journal of the European Communities

L 37/93

Figure 6

Wrongly integrated chromatogram of milk fat

To control measuring conditions, the relative standard deviations (RSD: coefficient of variation × 100) given in Table
1 for the different triglycerides may be used. They were calculated from 19 consecutive analyses of the same milk fat.

Table 1

Relative standard deviations (RSD) of triglyceride contents (n=19)

Tryglyceride

C24

C26

C28

C30

C32

C34

C36

C38

C40

C42

C44

C46

C48

C50

C52

C54

RSD (%)

10,00

2,69

3,03

1,76

1,03

0,79

0,25

0,42

0,20

0,26

0,34

0,37

0,53

0,38

0,54

0,60

If the RSDs are considerably higher than the values in Table 1, the chromatographic conditions are not appropriate
and the septa or the carrier gas flow rate must be verified. Further, small particles of the septum may have formed
deposits on the glass wool at the entrance of the column or the column might have become unsuited for use as a
result of ageing, temperature influences, etc. (see Figure 3).

Note: The values given in Table 1 are not mandatory, but are indicative for quality control purposes. However, if
higher RSD values are accepted, the repeatability and reproducibility limits given in point 11 must nevertheless
be complied with.

L 37/94

EN

Official Journal of the European Communities

7.2.2001

8. Qualitative foreign fat detection

For the detection of foreign fats triglyceride formulae (Table 2) with limits S (Table 3) have been developed, in which
the S-values of pure milk fats can fluctuate. If these limits are transgressed, the presence of a foreign fat can be
assumed.

The most sensitive formula for the detection of lard addition is, e.g.

6,5125 · C26 + 1,2052 · C32 + 1,7336 · C34 + 1,7557 · C36 + 2,2325 · C42 + 2,8006 · C46 + 2,5432 · C52 +
0,9892 · C54 = S

Note: Using 755 different milk fat samples a 99 % confidence range of S = 97,96 – 102,04 was established for pure

milk fat samples with a standard deviation for all S-values SD = 0,39897.

Starting from the triglyceride composition of an unknown fat sample such a formula allows, without using a
the triglyceride contents stated here with the
computer,
corresponding factors falls outside the range of 97,96 – 102,04 and one has most probably to do with foreign fat
addition.

to verify in a simple manner whether the sum of

For detecting different foreign fats Table 2 shows different triglyceride formulae. For the detection of the foreign fats
soybean oil, sunflower oil, olive oil, rape-seed oil, linseed oil, wheat germ oil, maize germ oil, cotton seed oil, and
hydrogenized fish oil, for the vegetable fats coconut- and palm kernel fat, as well as for palm oil and beef tallow a
common formula can be used, respectively.

Since the triglyceride composition of the foreign fats is also subjected to fluctuations, up to four different,
experimentally measured foreign fat triglyceride data of the same type were used. (With the same foreign fat types the
least favourable limit has been considered, respectively (see Table 4)).

With the following ‘Total formula’ similarly good results can be obtained for all foreign fats:

– 2,7575 · C26 + 6,4077 · C28 + 5,5437 · C30 – 15,3247 · C32 + 6,2600 · C34 + 8,0108 · C40 – 5,0336 · C42 +
0,6356 · C44 + 6,0171 · C46 = S

Calculations for the detection of any foreign fat combination in milk fat have shown that, e.g., although with the
formula for lard given in Table 2 the limit for this foreign fat in low, namely 2,7 %, other fats such as coconut fat,
palm oil or palm kernel fat with detection limits of 26,8, 12,5 and 19,3 %, respectively, can, with this formula, only
be detected if extremely high amounts have been added to milk fat. This applies also to other formulae in Table 2.

Triglyceride formulae for detecting various foreign fats in milk fat, indicating the standard deviations
SD for S

Table 2

Formula for soybean, sunflower, olive, rape-seed,

linseed, wheat germ, maize germ, cotton seed and fish oil

2,0983 · C30 + 0,7288 · C34 + 0,6927 · C36 + 0,6353 · C38 + 3,7452 · C40 – 1,2929 C42 + 1,3544 · C44 +
1,7013 · C46 + 2,5283 · C50 = S; SD = 0,38157

Formula for coconut and palm kernel fat

3,7453 · C32 + 1,1134 · C36 + 1,3648 · C38 + 2,1544 · C42 + 0,4273 · C44 + 0,5809 · C46 + 1,1226 · C48 +
1,0306 · C50 + 0,9953 · C52 + 1,2396 · C54 = S; SD = 0,11323

3,6644 · C28 + 5,2297 · C30 – 12,5073 · C32 + 4,4285 · C34 – 0,2010 · C36 + 1,2791 · C38 + 6,7433 · C40 –
4,2714 · C42 + 6,3739 · C46 = S; SD = 0,81094

Formula for palm oil and beef tallow

6,5125 · C26 + 1,2052 · C32 + 1,7336 · C34 + 1,7557 · C36 + 2,2325 · C42 + 2,8006 · C46 + 2,5432 · C52 +
0,9892 · C54 = S; SD = 0,39897

Formula for lard

Therefore for checking an unknown fat sample all formulae given in Table 2 and the Total formula (2) must be used,
if the sample is likely to be a mixture of milk fat and one of the 14 different foreign fats or a combination of these
foreign fats. If, by inserting the triglyceride of a fat sample to be analysed an S-value is obtained, which falls outside
the ranges of Table 3 of only one of the five formulae, then the sample is most likely a modified milk fat. Detection
of a foreign fat in milk fat by means of one of the four formulae in Table 2 does not allow conclusions to be drawn
on the type of the foreign fat admixture.

7.2.2001

EN

Official Journal of the European Communities

L 37/95

Table 3

S-Limits for milk fats

Formula for detection of

S-range

Soybean, sunflower, olive, rape-seed,
germ, cotton, fish oil

linseed, wheat germ, maize

Coconut and palm kernel fat

Palm oil and beef tallow

Lard

Total formula

98,05 — 101,95

99,42 — 100,58

95,90 — 104,10

97,96 — 102,04

95,68 — 104,32

In Table 4 the detection limits for the different foreign fats with a 99 % confidence are given. The first column shows
the minimal detection limits for the best milk fat formulae in Table 2. In the second column the detection limits for
the total formula are given. Although the limits are somewhat higher, only this formula is necessary to detect a little
bit higher amounts of foreign fats. With all formulae also combinations of the different foreign fats can be detected.
The ranges of variation of the triglycerides of different foreign fats of one type have no considerable influence on the
detection limits.

99 % limits of detection by addition of foreign fat to milk fat in %

Table 4

Individual formula

Total formula

Soybean oil

Sunflower oil

Olive oil

Coconut fat

Palm oil

Palm kernel fat

Rape-seed oil

Linseed oil

What germ oil

Maize germ oil

Cotton seed oil

Lard

Beef tallow

Hydrogenised fish oil

2,1

2,3

2,4

3,5

4,4

4,6

2,0

2,0

2,7

2,2

3,3

2,7

5,2

5,4

4,4

4,8

4,7

4,3

4,7

5,9

4,4

4,0

6,4

4,5

4,4

4,7

5,4

6,1

Note: The S-ranges are calculated in that way, that a foreign fat is only assumed, if the limits of the individual

formula are exceeded (see Table 4).

9. Quantitative foreign fat determination

In order to obtain quantitative information on the foreign fat concentration in a milk fat sample, the following
formula is used

) |,
X (%) = 100 · | (100 – S)/(100 – S
F

X being the quantity of an unknown foreign fat or foreign fat mixture in an unknown milk fat. S results from
addition of an unknown foreign fat by inserting the triglycerides of the foreign fat/milk fat mixture in the above total
triglyceride formula. If an unknown foreign fat is added to milk fat, the mean S-value of the different foreign fats for
; this mean S-value is obtained by inserting the triglyceride data of the pure foreign
the Total formula is chosen for SF
= 7,46). Good quantitative results concerning any foreign fat
fats in this formula and by calculating a mean value (SF
-value of 10,57.
additions are also obtained using the palm oil/beef tallow formula (Table 2) and a mean S
F

With known foreign fat types the following SF
foreign fat formula from Table 2 has to be chosen:

-values must be inserted in the above formula and the respective

L 37/96

EN

Official Journal of the European Communities

7.2.2001

Table 5

-values of various foreign fats

SF

Foreign fat

S
F

8,18

9,43

12,75

118,13

7,55

112,32

3,30

4,44

27,45

9,29

41,18

177,55

17,56

64,12

Soybean oil

Sunflower oil

Olive oil

Coconut fat

Palm oil

Palm kernel oil

Rape-seed oil

Linseed oil

Wheat germ oil

Maize germ oil

Cotton seed oil

Lard

Beef tallow

Fish oil

10. Range of application of the detection method

The described method applies to bulk milks and is based on the representativeness of the milk fat samples.

Highly specific detection would be possible, if, for a representative number of milk fats, formulae as described above
were derived for different countries.

There could be particularly suited possibilities of detection obtained, if in the different countries formulae, as have
been described here, were set up of a representative number of milk fats. In this case, the use of complex computer
programmes is not required, if the triglyceride combinations used in Table 2 are applied and the factors redetermined
by using the method of least squares.

By applying the S-ranges as shown in Table 3 the formulae are, under particular feeding conditions as, for instance,
underfeeding or feeding of cows with feed yeast or Ca-soaps, generally applicable. Only in the case of extreme feeding
conditions (e.g. high uptake of pure feed oils, high administration of Ca-soaps combined with feed fat etc.) the
formulae partly indicate a modified milk fat.

Note: Fractionated milk fats are generally recognized as unmodified milk fat, if a modification is assumed only, when
the limits are exceeded. Only with fractionated milk fats with unusual milk fat composition, as it is, e.g., the
case with a hard fraction, obtained with fractionation by physical methods at high temperatures of approxi-
, the formulae indicate
mately 30 °C with low yields of a few percent or with fractionation with overcritical CO2
a modification.

Milk fat fractionation may, however, be detected using other procedures e.g. Differential-Scanning-Calorimetry.

11. Accuracy of the method

Determined using milk fat on the basis of the formulae from Table 2 and the S-ranges in Table 3.

11.1.

Repeatability

As difference of the S-values of two determinations carried out within the shortest feasible time interval by
one operator using the same procedure and identical sample material under the same conditions (same
person, same instruments/same device, same laboratory):

7.2.2001

EN

Official Journal of the European Communities

L 37/97

Table 6

Repeatability limits (r) for the different formulae

Formula for detection of

Soyabean, sunflower, olive, rape-seed,
cotton, fish oil

linseed, wheat germ, maize germ,

Coconut and palm kernel fat

Palm oil and beef tallow

Lard

Total formula

11.2.

Reproducibility

r

0,67

0,12

1,20

0,58

1,49

As difference of the S-values of two determinations carried out by operators in different laboratories,
according to the same procedure using identical sample material under different conditions (different person,
different instruments) at different times.

Table 7

Reproducibility limits (R) for the different formulae

Formula for detection of

Soybean, sunflower, olive, rape-seed,
cotton, fish oil

linseed, wheat germ, maize germ,

Coconut and palm kernel fat

Palm oil and beef tallow

Lard

Total formula

11.3.

Critical difference

R

1,08

0,40

1,81

0,60

2,07

With the repeatability (r) and the reproducibility limits (R) the critical differences for all S-ranges of Table 3
can be calculated (duplicate analyses). The respective values are given in Table 8.

Table 8

Critical differences for all triglyceride formulae

Formula for detection of

range

Soybean, sunflower, olive, rape-seed,
cotton, fish oil

linseed, wheat germ, maize germ,

Coconut and palm kernel fat

Palm oil and beef tallow

Lard

Total formula

97,43 — 102,57

99,14 — 100,86

94,91 — 105,09

97,65 — 102,35

94,58 — 105,42

L 37/98

EN

Official Journal of the European Communities

7.2.2001

11.4.

Acceptability of results

All calibrated with two rounded decimals calculated triglyceride contents of C24, C26, C28 to C54 as well as
cholesterol must be exactly normalized to 100.

The results of the duplicate analysis is used as a check on the repeatability. If the absolute difference between
the two S-results for all five triglyceride formulae do not transgress the repeatability limits r in Table 6, then
the repeatability requirement is met.

For control of optimal gas chromatographic conditions and especially of the quality of the column it should
be guaranteed that with 10 repetition runs the difference of the maximum and minimum S-values of all five
triglyceride formulae do not transgress the range x · r, with x = 1,58 (for 10 runs, see literature (16)), and
the repeatability limits r for the different formulae in Table 6.

12. Quoted standards

DIN 10 336: 1994

Nachweis and Bestimmung von Fremdfetten in Milchfett anhand einer gaschromato-
graphischen Triglyceridanalyse

IDF Standard 1 C: 1987

Milk. Determination of Fat Content — Röse Gottlieb Gravimetric Method

IDF Standard 16C: 1987

Cream. Determination of Fat Content — Röse Gottlieb Gravimetric Method

IDF Standard 116A: 1987 Milk-Based Edible Ices and Ice Mixes. Determination of Fat Content — Röse Gottlieb

Gravimetric Method

IDF Standard 22B: 1987

Skimmed Milk, Whey & Buttermilk. Determination of Fat Content — Röse Gottlieb
Gravimetric Method.

13. References

1. Commission of the European Communities: Detection of foreign fats in milk fat by means of gas chromatographic

triglyceride analysis, Doc. No VI/5202/90-EN, VI/2645/91.

2. Commission of the European Communities: Control of butter fat purity of 100 different samples of different feeding

periods from 11 EEC countries; Doc. No VI/4577/93.

3. Commission of the European Communities: Consideration of results from the first, second, third, fourth, fifth and sixth
EEC collaborative trial: Determination of triglycerides in milk fat; Doc. No VI/2644/91, VI/8.11.91, VI/1919/92,
VI/3842/92, VI/5317/92, VI/4604/93.

4. Timms, R. E.: Detection and quantification of non-milk fat in mixtures of milk and non-milk fats. Dairy Research

47 295-303 (1980).

5. Precht, D., Heine, K.: Nachweis von modifiziertem Milchfett mit der Triglyceridanalyse. 2. Fremdfettnachweis im Milchfett

mit Hilfe von Triglyceridkombinationen, 41 406-410 (1986).

6. Luf, W., Stock, A., Brandl, E.: Zum Nachweis von Fremdfett in Milchfett über die Trigylceridanalyse. Österr. Milch-

wirtsch. Wissensch. Beilage 5, 42 29-35 (1987).

7. Precht, D.: Bestimmung von pflanzlichen Fetten oder

tierischen Depotfetten in Milchfett. Kieler Milchwirtsch.

Forschungsber. 42 143-157 (1989).

8. Precht, D.: Schnelle Extraktion von Milchfett, Kieler Milchwirtsch. Forschungsber. 42 119-128 (1990).

9. Precht, D.: Schnelle gaschromatographische Triglyceridanalyse von Milchfett. Kieler Milchwirtsch. Forschungsber.

42 139-154 (1900).

10. Precht, D.: Control of milk fat purity by gas chromatographic triglyceride analysis. Kieler Milchwirtsch. Forschungsber.

43 (3) 219-242 (1991).

11. Precht, D.: Detection of adulterated milk fat by fatty acid and triglyceride analysis. Fat Sci. Technol. 93 538-544 (1991).

12. Precht, D.: Detection for foreign fat in milk fat. I. Qualitative detection by triacylglycerol formulae. II. Quantitative

evaluation of foreign fat mixtures. Z. Lebensm. Unters. Forsch. 194 1-8, 107-114 (1992).

13. Precht, D.: Gas chromatography of triacylglycerols and other lipids on packed columns in CRC Handbook of Chromato-
graphy: Analysis of lipids, p. 123-138, Ed. K.D. Mukherjee, N. Weber, J. Sherma, CRC Press, Boca Raton (1993).

14. Precht, D., Molkentin, J.: Quantitative triglyceride analysis using short capillary columns, Chrompack News 4 16-17

(1993).

15. Molkentin, J., Precht, D.: Comparison of packed and capillary columns for quantitative gas chromatography of triglycerides

in milk fat. Chromatographia 39 (5/6) 265-270 (1994).

16. Stange, K.: Angewandte Statistik, Erster Teil, Eindimensionale Probleme, Springer-Verlag, Berlin, P. 378 (1970).

7.2.2001

EN

Official Journal of the European Communities

L 37/99

ANNEX XXVI

LIST OF THE REGULATIONS REFERRED TO IN THE FIRST RECITAL

— Commission Regulation (EEC) No 1216/68 of 9 August 1968 laying down the method for determining the lactose
content of compound feeding-stuffs imported from third countries (1), as amended by Commission Regulation (EEC)
No 222/88 of 22 December 1987 amending certain measures on the application of the common market organisation
in the milk and milk products sector following the introduction of the combined nomenclature (2);

— Commission Regulation (EEC) No 3942/92 of 22 December 1992 establishing a reference method for the deter-
mination of sitosterol and stigmasterol in butteroil (3), as last amended by Commission Regulation (EC) No 175/1999
amending Regulations (EEC) No 3942/92, (EC) No 86/94, (EC) No 1082/96 and (EC) No 1459/98 establishing
reference methods for the determination of certain tracers in butter, butteroil and cream (4);

— Commission Regulation (EC) No 86/94 of 19 January 1994 establishing a reference method for the determination of

sitosterol and stigmasterol

in butter (5), as amended by Regulation (EC) No 175/1999;

— Commission Regulation (EC) No 2721/95 of 24 November 1995 establishing rules for the application of reference
and routine methods for the analysis and quality evaluation of milk and milk products under the common market
organisation (6);

— Commission Regulation (EC) No 1080/96 of 14 June 1996 establishing a reference method for the detection of

coliforms in butter, skimmed milk powder and casein/caseinates (7);

— Commission Regulation (EC) No 1081/96 of 14 June 1996 establishing a reference method for the detection of cows'
milk and caseinate in cheeses made from ewes' milk, goats' milk or buffalos' milk or mixtures of ewes', goats' and
buffalos' milk and repealing Regulation (EEC) No 690/92 (8);

— Commission Regulation (EC) No 1082/96 of 14 June 1996 establishing a reference method for the determination of
the ethyl ester of beta-apo-8' carotenic acid in concentrated butter and butter (9), as amended by Regulation (EC) No
175/1999;

— Commission Regulation (EC) No 1854/96 of 26 September 1996 establishing a list of reference methods to be applied
for the analysis and quality evaluation of milk and milk products under the common market organisation (10), as
amended by Regulation (EC) No 881/1999 (11);

— Commission Regulation (EC) No 880/98 of 24 April 1998 establishing reference methods for the determination of the

water, solids-non-fat and fat content of butter (12);

— Commission Regulation (EC) No 1459/98 of 8 July 1998 establishing a reference method for the determination of

vanillin in concentrated butter, butter or cream (13), as amended by Regulation (EC) No 175/1999.

(1) OJ L 198, 10.8.1968, p. 13.
(2) OJ L 28, 1.2.1988, p. 1.
(3) OJ L 399, 31.12.1992, p. 29.
(4) OJ L 20, 27.1.1999, p. 22.
(5) OJ L 17, 20.1.1994, p. 7.
(6) OJ L 283, 25.11.1995, p. 7.
(7) OJ L 142, 15.6.1996, p. 13.
(8) OJ L 142, 15.6.1996, p. 15.
(9) OJ L 142, 15.6.1996, p. 26.
(10) OJ L 246, 27.9.1996, p. 5.
(11) OJ L 111, 29.4.1999, p. 24.
(12) OJ L 124, 25.4.1998, p. 16.
(13) OJ L 193, 9.7.1998, p. 16.