1987R4154 — EN — 18.02.1998 — 001.001 — 1
This document is meant purely as a documentation tool and the institutions do not assume any liability for its contents
►B COMMISSIONREGULATION(EEC)No4154/87
of22December 1987
layingdownthemethodsofanalysisandothertechnicalprovisionsnecessaryfortheimplementa-
tion of Regulation (EEC) No 3033/80 laying down the trade arrangements applicable to certain
goodsresultingfromtheprocessingofagricultural products
(OJ L 392, 31.12.1987, p. 19)
Amended by:
Official Journal
No page date
►M1CommissionRegulation(EC) No 203/98 of 26 Jan uary 1998 L 21 6 28.1.1998
▼B
COMMISSIONREGULATION(EEC)No4154/87
of22December 1987
layingdownthemethodsofanalysisandothertechnical provisions
necessary for the implementation of Regulation (EEC) No 3033/80laying down the trade arrangements applicable to certain goods
resultingfromtheprocessingofagricultural products
THE COMMISSION OF THE EUROPEANCOMMUNITIES,
Having regard to the Treaty establishing the European EconomicCommunity,
Having regard to Council Regulation (EEC) No 2658/87 of 23 July
1987 on the tariff and statistical nomenclature and on the Common
Customs Tariff(
1) as amended by Regulation (EEC) No 3985/87(2)
and in particular Article 9 thereof,
Whereas CommissionRegulation(EEC) No 1061/69(3), as last
amended by Regulation (EEC) No 1822/86(4), defined the analytical
methods to be used for the applicationof Coun cil Regulation(EEC)No 1059/69(
5); whereas Regulation(EEC) No 1059/69 was repealed
and replaced by Council Regulation (EEC) No 3033/80(6), as last
amended by Regulation (EEC) No 3743/87(7);
Whereas the procedures for implementing Regulation (EEC) No 3033/
80 for imports, were prescribed by Council Regulation (EEC) No 3034/80, of 11 November 1980, fixing the quantities of basic products
considered to have been used in the manufacture of goods covered by
Regulation(EEC) No 3033/80(
8) amending Regulation (EEC) No 950/
68 onthe CommonCustoms Tariff, as last amen ded by Regulation(EEC) No 4091/87(
9);
Whereas in order to take account of the scientific and technological
evolution of analytical methods and to continue to ensure the uniform
treatment on import into the Community of goods covered by Regula-tion(EEC) No 3033/80, it is appropriate to repeal an d replaceRegulation(EEC) No 1061/69;
Whereas the measures provided for inthis Regulationare inaccor-
dance with the opinion of the Nomenclature Committee,
HAS ADOPTED THIS REGULATION:
Article 1
This Regulation lays down the methods of analysis necessary for the
implementation of Regulations (EEC) No 3033/80 (as far as importsare concerned) and (EEC) No 3034/80, or, in the absence of a method
of analysis, the nature of the analytical operations to be carried out or
the principle of a method to be applied.
Article 2
In accordance with the definitions set out in Annex III to Regulation
(EEC) No 3034/80 concerning:
— starch/glucose, content, and
— sucrose/invert sugar/isoglucose content1987R4154 — EN — 18.02.1998 — 001.001 — 2
(1) OJ No L 256, 7. 9. 1987, p. 1.
(2) OJ No L 376, 31. 12. 1987, p. 1.
(3) OJ No L 141, 12. 6. 1969, p. 24.
(4) OJ No L 158, 13. 6. 1986, p. 1.
(5) OJ No L 141, 12. 6. 1969, p. 1.
(6) OJ No L 323, 29. 11. 1980, p. 1.
(7) OJ No L 352, 15. 12. 1987, p. 29.
(8) OJ No L 323, 29. 11. 1980, p. 7.
(9) OJ No L 382, 31. 12. 1987, p. 27.
▼B
and for the purpose of implementing Annexes II and III to that Regula-
tion, the following formulae, procedures and methods shall be used:
1.Starch/glucose content:
expressed as 100 % anhydrous starch content of the goods as
presented)
(a) (Z − F) × 0,9,
if the glucose content is not less than the fructose content; or
(b) (Z − G) × 0,9,
if the glucose content is less than the fructose content
where:
Z is the glucose content determined by the method set out in
Annex I of this Regulation;
F is the fructose content determined by HPLC (high performance
liquid chromatography);
G is the glucose content determined by HPLC.
Incase 1 (a), where the presen ce of a lactose hydrolysate is declared
and/or quantities of lactose and galactose are detected, a glucosecontent equivalent to the galactose content (determined by HPLC),shall be deducted from the glucose content (Z) before any othercalculationis made.
2.Sucrose/invert sugar/isoglucose content:
(expressed as sucrose content of the goods as presented)
(a) S + (2F) × 0,95,
if the glucose content is not less than the fructose content
(b) S + (G + F) × 0,95,
if the glucose content is less than the fructose content
where:
S is the sucrose content determined by HPLC;
F is the fructose content determined by HPLC;
G is the glucose content determined by HPLC.
Where the presence of a lactose hydrolysate is declared and/or quan-
tities of lactose and galactose are detected, a glucose content,equivalent to the galactose content (determined by HPLC), shall bededucted from the glucose (G) content before any other calculationis made.
3.Milk fat content:
(a) Save as otherwise provided inparagraph (b), the milk fat
content by weight shall be determined by extraction with lightpetroleum after hydrolysis with hydrochloric acid.
(b) Where fats other thanmilk fats are also declared inthe compo-
sitionof the goods, the followin g procedure shall be applied:
— the percentage of weight of the total fats in the goods shall
be determined as mentioned in paragraph (a),
— for the purposes of determining the milk fat content, a
method based onextractionwith light petroleum, preceded
by hydrolysis with hydrochloric acid and followed by gaschromatography of the methyl esters of the fatty acids, shallbe used. If the presence of milk fats is detected, the percen-tage proportionthereof shall be calculated by multiplyin g the
percentage concentration of methyl butyrate by 25, multi-
plying the product by the total percentage fat content byweight of the goods and dividing by 100.
4.Milk protein content:
(a) Save as otherwise provided in(b) below, the milk protein
content of the goods shall be calculated by multiplying the1987R4154 — EN — 18.02.1998 — 001.001 — 3
▼M1
▼B
▼B
nitrogen content (determined by the Kjeldahl method) by the
factor 6,38.
(b) Where components containing proteins other than milk proteins
are also declared inthe compositionof the goods:
(i) the total nitrogen content shall be determined by the Kjel-
dahl method;
(ii) the milk protein content shall be calculated as mentioned in
(a) above by subtracting from the total nitrogen content thenitrogen content corresponding to the non-milk proteins.
Article 3
For the purposes of implementing Annex I to Regulation (EEC) No
3034/80, the following methods and/or procedures shall be used:
1. For the purposes of classifying goods falling within subheadings
0403 10 51 to 0403 10 59, 0403 1091 to 0403 1099, 040390 71 to
0403 90 79 and 0403 9091 to 0403 90 99 of the combined nomen-clature, the milk fat content shall be determined by the methodreferred to inArticle 2 (3);
2. For the purposes of classifying goods falling within subheadings
1704 10 11 to 170410 99 and 190520 10 to 1905 2090 of thecombined nomenclature, the sucrose content, including invert sugar
expressed as sucrose, shall be determined by an HPLC method;
(invert sugar expressed as sucrose means the sum of equal amountsof glucose and fructose multiplied by 0,95);
3. For the purposes of classifying goods falling within subheadings
1806 10 10 to 1806 1090 of the combined nomenclature, thesucrose/invert sugar/isoglucose content shall be determined in accor-
dance with the formulae, method and procedures set out in Article 2
(2);
4. For the purposes of classifying goods falling within subheadings
3505 20 10 to 3505 20 90 of the combined nomenclature, the starch,dextrin or other modified starch content shall be determined by themethod set out in Annex II of this Regulation;
5. For the purposes of classifying goods falling within subheadings
3809 10 10 to 3809 1090 of the combined nomenclature, the
amylaceous substances shall be determined by the method set out
in Annex II to this Regulation;
6. For the purposes of classifying goods falling within either
subheading 1901 9011 or subheading 1901 9019 of the combinednomenclature, the distinction shall be drawn on the basis of the dryextract determined by drying at a temperature of 103 ± 2 ºC to
constant weight;
7. For the purposes of classifying goods falling within subheadings
1902 19 10 and 1902 19 90 of the combined nomenclature, themethod set out in Annex III to this Regulation shall be used to testfor the presence of common wheat flours and semolinas in pasta;
8. The content of mannitol and D-glucitol (sorbitol) of the goods
falling within subheadings 2905 44 11 to 2905 44 99 and3823 60 11 to 3823 6099 of the combined nomenclature, is deter-
mined using a method based on HPLC.
Article 4
1. A test report shall be drawnup.
2. The test report shall include the following particulars:
— all the information necessary for identifying the sample,
— the method used and precise reference to the legal instrument in
which it is laid down, or, where appropriate, detailed reference to
a method, specifying the nature of the analytical operations to becarried out, or the principle of the method to be applied, as indi-cated inthis Regulation ,
— any factors liable to have influenced the results,1987R4154 — EN — 18.02.1998 — 001.001 — 4
▼B
— the results of the analysis, with due regard to the way in which they
are expressed in the method used and the means of expressiondictated by the needs of the customs or administrative departments
that requested the analysis.
Article 5
Regulation(EEC) No 1061/69 is hereby repealed.
Article 6
This Regulation shall enter into force on 1 January 1988.
This Regulation shall be binding in its entirety and directly applicable
inall Member States.1987R4154 — EN — 18.02.1998 — 001.001 — 5
▼B
ANNEX I
DETERMINATION OF STARCH CONTENT AND ITS DEGRADATION
PRODUCTSINCLUDINGGLUCOSE
1.Purposeandfieldofapplication
(a) The method permits the determination of the starch content, its degra-
dation products including glucose, hereafter referred to as ‘starch’.
(b) ‘Starch’ content referred to in 1 (a) is equal to value E, as calculated
inparagraph 6 of the presen t method.
2.Principle
The sample is broken down by means of sodium hydroxide and the starch
divided into glucose units with amyloglucosidase. The glucose determina-
tionis performed by the en zymatic route.
3.Reagents
(Use double-distilled water.)
3.1. 0,5 N Sodium hydroxide solution(0,5 mol/l).
3.2. 96 % (minimum) glacial acetic acid.
3.3. Solutionof amyloglucosidase:
Immediately before use, dissolve approximately 10 mg of amyloglucosi-
dase (EC 3.2.1.3) (6 U per mg) inon e ml of water(1).
3.4. Triethanolamine buffer solution:
Dissolve 14,0 g of triethanolamine hydrochloride [tris(2-hydroxyethy-
l)ammonium chloride] and 0,25 g of magnesium sulphate MgSO47H2O)
in80 ml of water, add approximately 5 ml of 5 N sodium hydroxyde
solution (5 mol/l) and adjust the pH to 7,6, using a 1 N sodium hydroxide
solution(1 mol/l). Make up to 100 ml with water. This buffer solution
canbe kept for at least four weeks at 4 ºC.
3.5. NADP (nicotinamide adenine dinucleotide phosphate, disodium salt) solu-
tion:
Dissolve 60 mg of NADP in6 ml of water. This solutioncanbe kept for
at least four weeks at 4 ºC.
3.6. ATP (adenosine-5'-triphosphate, disodium salt) solution:
Dissolve 300 mg of ATP.3H2O and 300 mg of sodium hydrogen carbo-
nate (NaHCO3) in6 ml of water.
This solutioncanbe kept for at least four weeks at 4 ºC.
3.7. Suspension HK/G6P-DH [Hexokinase (EC 2.7.1.1) and glucose-6-phos-
phate-dehydrogenase (EC 1.1.1.49)]:
suspend 280 U HK and 140 U of G6P-DH in 1 ml of 3,2 M ammonium
sulphate solution. This suspension can be kept for at least one year at 4
ºC.
4.Apparatus
4.1. Magnetic stirrer water bath at 60 ºC.4.2. Magnetic rods.
4.3. UV spectrophotometer with 1 cm optical cells.
4.4. Pipettes for enzymatic analysis.1987R4154 — EN — 18.02.1998 — 001.001 — 6
(1) U is the international unit of enzyme activity.
▼B
5.Method
5.1.The sample is washed with ethanol, digested in sodium hydroxide and the
‘starch’ subject to enzymatic hydrolysis:
5.1.1. Select the weight of sample as follows, according to the presumed
‘starch’ content (the ‘starch’ content must not exceed 0,4 g per sample)
as follows:
Presumed ‘starch’
contentof product
ing/100 gApproximate weight of
sample ing
(p)Volume of graduated
flask
inmlDilutionfactorup to 1
litre
(f)
> 70 0,35-0,4 500 2
20-70 max 0,5 500 2
5-20 max 1 250 4
< 5 max 2 200 5
5.1.2. Weigh the sample exactly to 0,1 mg.
5.1.3. Add 50 ml of 0,5 N sodium hydroxide solution (3.1) and stir continuously
for 30 minutes in the water bath (4.1) with a magnetic stirrer at 60 ºC.
5.1.4. Add a few ml of concentrated acetic acid (3.2) and bring the pH to 4,6 to
4,8.
5.1.5. Place inthe water bath with the magn etic stirrer (4.1) at 60 ºC, add 1,0
ml of enzyme solution (3.3) and allow to react whilst stirring continu-
ously for 30 minutes.
5.1.6. After cooling, transfer quantitatively to a graduated flask (5.1.1) and
make up to the mark with water.
5.1.7. If necessary, filter through a fluted filter (see Note 1).
5.2.Quantitative determination of glucose:
5.2.1. The test solutionmust con tain100 to 1 000 mg of glucose per litre,
which corresponds to a ∆Ε340of between0,1 an d 1,0.
The absorbence of the test solution in a 1 + 30 dilution with water must
not exceed 0,4 (measured against air), at 340 nm.
5.2.2. Bring the buffer solution (3.4) to ambient temperature (20 ºC).5.2.3. The temperature of the reagents and of the sample must be between 20
and 25 ºC.
5.2.4. Measure the absorbence at 340 nm against air (i. e. with no optical cell in
the reference path).
5.2.5. Proceed in accordance with the pipetting chart below:
Pour into the optical cellsControl
(ml)Test
(ml)
Buffer (reagent 3.4) 1,00 1,00
NADP (reagent 3.5) 0,10 0,10ATP (reagent 3.6) 0,10 0,10Test solution(5.1.6 or 5.1.7) — 0,10Double-destilled water 2,00 1,90
Mix and, after about three minutes, measure the absorbence of the solutions (E
1). Initiate the
reactionby addin g:
HK/G6P.DH (reagent 3.7) 0,02 0,02Mix, allow the reaction to proceed to completion (approximately 15 minutes) and measure the
absorbence of the solutions (E
2). If the reaction has not stopped after 15 minutes read absor-
bences at five-minute invervals until the rate of increase is constant. Then extrapolate
backwards to the time of additionof suspen sion(3.7) (see Note 2).
5.2.6. Calculate the absorbence differences for reagent blank and sample (E2—
E1).1987R4154 — EN — 18.02.1998 — 001.001 — 7
▼B
Subtract absorbence difference of the reagent blank ( ∆Εreagent blank)
from that of the sample ( ∆Ε)sample):
∆Ε=∆Εsample−∆Εreagent blank
This difference gives the glucose content of the test solution:
Glucose content contained in test solution, g/l
Gl¼3;22/C2180 ;16
6;3/C21/C20;1/C21000/C2∆Ε340¼0;921/C2∆Ε340
(3,22: volume of the solutionto be measured; 1: cell thickn ess; 0,1:
volume of the sample solution; The molecular weight of glucose is
180,16 (g/mol).
5.2.7. If, for any reason, a measurement cannot be made of 340 nm, the
measurement may be made at a wavelength of 365 nm or 334 nm, the
figure 6,3 inthe formula for Gl above should be replaced by the figure
3,5 or 6,18 respectively.
6.Calculationandexpressionofresults
(a) E = ‘Starch’ content in g/100 g:
E¼100/C20;9/C2Gl
p/C2f
(b) Z = ‘Glucose’ content in g/100 g:
Z¼100/C2Gl
p/C2f
where:
Gl = glucose inG/l (5.2.6);
f = dilutionfactor (5.1.1);p = weight of sample ing;0,9 = glucose conversion factor for starch.
Notes:
(1) Where a test solution cannot be filtered according to 5.1.7 appropriate
methods should be takeninorder to obtaina clear solution .
(2) Where an inhibition of enzymes occurs, it is advisable to apply a method
which involves the addition of known amounts of pure starch.1987R4154 — EN — 18.02.1998 — 001.001 — 8
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ANNEX II
DETERMINATIONOF STARCHESOR DEXTRINSOR OTHER MODI-
FIED STARCHES CONTENT INTO GOODS OF SUBHEADINGS
35052010 TO 35052090 OF THE COMBINED NOMENCLATUREAND
OF AMYLACEOUS SUBSTANCES CONTENT INTO GOODS OF
SUBHEADINGS 38091010 TO 38091090 OF THE COMBINED
NOMENCLATURE
I.Principle
Starch is converted by acid hydrolysis into reducing sugars which are deter-
mined by volume using Fehling's solution.
II.Apparatusandreagents
1. 250 ml flask;
2. 200 ml graduated flask;3. 25 ml graduated burette;
4. Hydrochloric acid at 1,19 density;
5. Potassium hydroxide solution;6. Decolourizing charcoal;
7. Fehling's solution;
8. Methylene blue solution (1 %).
III.Method
Into a 250 ml flask place a sample containing about 1 g of starch. Add 100
ml of distilled water and 2 ml of hydrochloric acid. Bring to the boil and
reflux for three hours.
Transfer the contents of the flask and rinsings into a 200 ml graduated
flask. Cool and nearly neutralize with potassium hydroxide solution. Add
distilled water to 200 ml and filter through a little decolourizing charcoal.
Then pour the solution into a graduated burette and reduce 10 ml of Fehl-
ing's solution by the following method:
Into a flat-bottomed flask of about 250 ml pour 10 ml of Fehling's solution
(5 ml of solution A and 5 ml of solution B). Shake until clear and add 40
ml of distilled water and a small quantity of quartz or pumice.
Place the flask ona square asbestos plate with a roun d hole of about 6 cm
diameter in the centre, the asbestos in turn resting on a piece of wire gauze.
Heat the flask at such a rate that the liquid begins boiling after about two
minutes.
From the burette, add to the boiling liquid successive quantities of the sugar
solution until the blue colour of the Fehling's solution becomes hardly
discernible; then add 2 or 3 drops of methylene blue solution as indicator,
and complete the titration by adding further quantities of the sugar solution,
drop by drop, until the blue colour of the indicator disappears.
For greater accuracy repeat the titration under the same conditions, but
adding without a break almost all the sugar solution required to reduce the
Fehling's solution. In this second titration, the reduction of the Fehling's
solutionshould occur withinthree min utes.
Keep boiling for exactly two further minutes, adding the reagent within one
minute drop by drop to the boiling solution until the blue colour disappears.
The percentage by weight of starch in the sample is determined by means
of the following formula:
starch% ¼
T/C2200/C2100
n/C2p/C20;95
where:
T is the quantity in grammes of anhydrous dextrose corresponding to
10 ml of Fehlin g's solution(5 ml of solutionA an d 5 ml of solution
B). This titer corresponds to 0,04945 g of anhydrous dextrose when
solution A contains 17,636 g of copper per litre;
n is the number of ml of the sugar solution used for titration;
p is the weight of the sample amount;1987R4154 — EN — 18.02.1998 — 001.001 — 9
▼B
0,95 is the rate of conversion of anhydrous dextrose into starch
IV.PreparationofFehling'ssolutions
SolutionA: Ina graduated flask dissolve 69,278 g of pure crystallized
copper sulphate — Analytical Reagent (CuSO4·5H2O) —
free from iron in distilled water and bring the volume to 1
litre with distilled water. The correct strength of this solution
must be verified by a quantitative determination of the
copper.
SolutionB: Ina graduated flask dissolve 100 g of sodium hydroxide an d
346 g of double sodium potassium tartrate (Rochelle salt) in
distilled water and bring the volume to 1 litre with distilled
water.
The two solutions A and B must be mixed in equal quantities immediately
before use. 10 ml of Fehling's solution (5 ml of solution A and 5 ml of
solution B) is completely reduced, under the conditions described at III, by
0,04945 g of anhydrous dextrose.1987R4154 — EN — 18.02.1998 — 001.001 — 10
▼B
ANNEX III
DETECTION OF COMMON WHEAT FLOURORMEAL IN
MACARONI,SPAGHETTIANDSIMILARPRODUCTS(PASTA)
(by the Young and Gilles method, modified by Bernaerts and Gruner)
I.Principle
An extract of the sample of the pasta for analysis is prepared by using a
non-polar solvent.
This extract is chromatographed ona thinlayer of silica gel so as to sepa-
rate the sterols present in various band form fractions.
According to the number of brightly coloured bands it is possible to deter-
mine whether the product under examination has been manufactured
exclusively from durum wheat or commonwheat, or from a mixture of the
two. It is also possible to determine whether eggs have been added.
II.Apparatusandreagents
1. Homogenizer or grinder to obtain a grist that will pass through a stan-
dard sieve with a 0,200 mm mesh;
2. Standard sieve with a 0,200 mm mesh;3. Evaporator with a water bath for evaporationun der reduced pressure;4. Glass plate, aluminium sheet or other appropriate backing measuring 20
cm x 20 cm covered with a thinlayer of silica gel. If the thinlayer has
to be prepared, silica gel mixed with about 13 plaster should be used,
and it should be applied in a 0,25 mm layer with suitable apparatus in
accordance with the manufacturer's instructions;
5. Micropipette for measuring 20 microlitres;6. Container with lid suitable for the development of chromatograms;7. Atomizer;8. Petroleum ether with a boiling point between 40 and 60 ºC, redistilled
before use;
9. Anhydrous ethyl ether for analysis;
10. Carbontetrachloride for chromatography, redistilled before use;11. Phosphomolybdic acid for analysis;12. 94º ethyl alcohol.
III.Method
Grind about 20 g of the sample for analysis so that all of it passes through
the sieve. Put the sample in an Erlenmeyer flask and cover with 150 ml
petroleum ether. Leave at normal temperature until the following day.
Shake from time to time.
Then filter on a Buchner funnel fitted with a filtering aid or on a sintered
filter. Gradually transfer the clear solution thus obtained into a 100 ml cali-
brated flask. Evaporate the solvent under reduced pressure by heating the
flask ina water bath at 40 ºC to 50 ºC. Whenthe solven t has evaporated,heat under reduced pressure for a further ten minutes.
Whenthe flask has cooled, determin e the weight of the extract. Dilute the
extract inethyl ether onthe basis of 1 ml ethyl ether per 60 mg of extract.
Activate the thin layers by bringing them to 130 º C for three hours. Leave
to cool in a desiccator containing silica gel. Plates which are not used
immediately canbe preserved inthe same desiccator.
Apply, drop by drop, 20 microlitres of the clear solutionto form a ban d of
constant width and 3 cm in length on a layer preferably newly activated.
Let the solvent evaporate.
Develop the chromatogram under normal temperature with carbon tetra-
chloride using a chromatographic container the walls of which are covered
with filter paper soaked in solvent. After about an hour the solvent will
reach a height of 18 cm. Remove the plate and leave the solvent to evapo-
rate in the open. For better separation of the bands, develop the
chromatogram a second time. Again let the solvent evaporate in the open.1987R4154 — EN — 18.02.1998 — 001.001 — 11
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Spray the thinlayer of silica gel with a solutionof 20 % phosphomolybdic
acid inethyl alcohol. The colour of the layer must be a un iform yellow.
Develop the bands by the heating the sprayed plate at 110 ºC for five
minutes.
IV.Interpretationofthechromatogram
If the chromatogram shows a single main brightly coloured band with an Rf
of about 0,4 to 0,5, the wheat used for the manufacture of the pasta in ques-
tion is durum wheat. If, on the other hand, two main bands of equal
brightness appear, the raw material used is common wheat. Mixtures of
durum wheat and common wheat can be assessed by an evaluation of the
relative brightness of the two bands.
If there are three bands (two bands at the height where the main bands for
common wheat are to be found, with a further band between them) eggs
have beenadded to pasta. Inthis case, the raw material used is durum
wheat if the middle band is brighter than the upper band. On the other
hand, if the upper band is brighter than the middle band, the raw material
used is commonwheat.1987R4154 — EN — 18.02.1998 — 001.001 — 12