COMMISSION DIRECTIVE 98/64/EC of 3 September 1998 establishing Community methods of analysis for the determination of amino-acids, crude oils and fats, and olaquindox in feedingstuffs and amending Directive 71/393/EEC (Text with EEA relevance)

THE COMMISSION OF THE EUROPEAN COMMUNITIES,

Having regard to the Treaty establishing the European Community,

Having regard to Council Directive 70/373/EEC of 20 July 1970 on the introduction of Community methods of sampling and analysis for the official control of feedingstuffs (1), as last amended by the Act of Accession of Austria, Finland and Sweden, and in particular Article 2 thereof,

Whereas Directive 70/373/EEC stipulates that official controls of feedingstuffs for the purpose of checking compliance with the requirements arising under the laws, regulations and administrative provisions governing their quality and composition must be carried out using Community methods of sampling and analysis;

Whereas Council Directive 79/373/EEC of 2 April 1979 on the marketing of compound feedingstuffs (2), as last amended by Commission Directive 97/47/EC (3), and Council Directive 93/74/EEC of 13 September 1993 on feedingstuffs intended for particular nutritional purposes (4), as last amended by Directive 96/25/EC (5), stipulate that amino-acids and crude oils and fats are to be set out on the feedingstuffs label;

Whereas Council Directive 70/524/EEC of 23 November 1970 concerning additives in feedingstuffs (6), as last amended by Commission Directive 98/19/EC (7), stipulates that the olaquindox content must be indicated on the labelling where this substance is added to compound feedingstuffs;

Whereas the second Commission Directive 71/393/EEC of 18 November 1971 establishing Community methods of analysis for the official control of feedingstuffs (8), as last amended by Commission Directive 84/4/EEC (9), sets out methods for analysis for, inter alia, the determination of crude oils and fats; whereas it is appropriate to modify the method described;

Whereas Community methods of analysis must be established for checking these substances;

Whereas the measures provided for in this Directive are in accordance with the opinion of the Standing Committee for Feedingstuffs,

HAS ADOPTED THIS DIRECTIVE:

Article 1

The Member States shall provide that analyses conducted with a view to official controls of the amino-acid, crude oil and fat, and olaquindox content of feedingstuffs are carried out using the methods set out in the Annex hereto.

Article 2

In the Annex to Commission Directive 71/393/EEC, point '4. Determination of crude oils and fats` is replaced by part B of the annex to this Directive.

Article 3

1. Member States shall bring into force the laws, regulations or administrative provisions necessary to comply with this Directive by 31 December 1998. They shall immediately inform the Commission thereof.

They shall apply the measures from 1 January 1999.

When Member States adopt these measures, they shall contain a reference to this Directive or shall be accompanied by such reference at the time of their official publication. The procedure for such reference shall be adopted by Member States.

2. Member States shall communicate to the Commission the text of the main provisions of domestic law which they adopt in the field governed by this Directive.

Article 4

This Directive shall enter into force on the 20th day following its publication in the Official Journal of the European Communities.

Article 5

This Directive is addressed to the Member States.

Done at Brussels, 3 September 1998.

For the Commission

Franz FISCHLER

Member of the Commission

(1) OJ L 170, 3. 8. 1970, p. 2.

(2) OJ L 86, 6. 4. 1979, p. 30.

(3) OJ L 211, 5. 8. 1997, p. 45.

(4) OJ L 237, 22. 9. 1993, p. 23.

(5) OJ L 125, 23. 5. 1996, p. 35.

(6) OJ L 270, 14. 12. 1970, p. 1.

(7) OJ L 96, 28. 3. 1998, p. 39.

(8) OJ L 279, 20. 12. 1971, p. 7.

(9) OJ L 15, 18. 1. 1984, p. 28.

ANNEX

PART A

DETERMINATION OF AMINO ACIDS

1. Purpose and scope

This method is for the determination of free (synthetic and natural) and total (peptide bound and free) amino acids in feedingstuffs, using an amino acid analyzer. It is applicable to the following amino acids: cyst(e)ine, methionine, lysine, threonine, alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, phenylalanine, proline, serine, tyrosine and valine.

The method does not distinguish between the salts of amino acids and it cannot differentiate between D and L forms of amino acids. It is not valid for the determination of tryptophan or hydroxy analogues of amino acids.

2. Principle

2.1. Free amino acids

The free amino acids are extracted with diluted hydrochloric acid. Coextracted nitrogenous macromolecules are precipitated with sulfosalicylic acid and removed by filtration. The filtered solution is adjusted to pH 2,20. The amino acids are separated by ion exchange chromatography and determined by reaction with ninhydrin with photometric detection at 570 nm.

2.2. Total amino acids

The procedure chosen depends on the amino acids under investigation. Cyst(e)ine and methionine must be oxidised to cysteic acid and methionine sulphone respectively prior to hydrolysis. Tyrosine must be determined in hydrolysates of unoxidized samples. All the other amino acids listed in paragraph 1 can be determined in either the oxidised or unoxidised sample.

Oxidation is performed at 0 °C with a performic acid/phenol mixture. Excess oxidation reagent is decomposed with sodium disulphite. The oxidised or unoxidised sample is hydrolysed with hydrochloric acid (c = 6 mol/l) for 23 hours. The hydrolysate is adjusted to pH 2,20. The amino acids are separated by ion exchange chromatography and determined by reaction with ninhydrin using photometric detection at 570 nm (440 nm for proline).

3. Reagents

Double distilled water or water of equivalent quality must be used (conductivity NUM>A × E × MW × F

>DEN>B × W × 1 000

= g amino acid per kg sample

If an internal standard is used multiply by:

>NUM>D

>DEN>C

>TABLE>

Cystine and cysteine are both determined as cysteic acid in hydrolysates of oxidized sample, but calculated as cystine (C6H12N2O4S2, MW 240,30 by using MW 120,15 (= 0,5 × 240,30).

Methionine is determined as methionine sulphone in hydrolysates of oxidized sample, but calculated as methionine by using MW of methionine: 149,21.

Added free methionine is determined after extraction as methionine, for the calculation the same MW is used.

6.1. The total dilution volume of extracts (F) for determination of free amino acids (5.2) is calculated as following:

F = 100 ml × >NUM>(10 ml + 5ml)

>DEN>10 ml

× >NUM>Vml

>DEN>10 ml

V = volume of final extract

7. Evaluation of the method

The method has been tested in an intercomparison made at international level in 1990 using four different feedingstuffs (mixed pig feed, broiler compound, protein concentrate, premixture). The results, after elimination of outliers, of mean and standard deviation are given in the table below:

>TABLE>

7.1. Repeatability

The repeatability expressed as 'within laboratory standard deviation` of the above mentioned intercomparison is given in the tables below:

>TABLE>

>TABLE>

7.2. Reproducibility

The results for between laboratory standard deviation by the above mentioned intercomparison are given in the table below:

>TABLE>

>TABLE>

8. Use of reference materials

The correct application of the method shall be verified by making replicate measurements of certified reference materials when available. Calibration with certified amino acid calibration solution is recommended.

9. Observations

9.1. Because of differences between amino acid analysers the final concentrations of the calibration solutions of standard amino acids (see 3.27.4 and 3.27.5) and of the hydrolysate (see 5.3.4) should be taken as a guideline.

The range of linear response of the apparatus has to be checked for all amino acids.

The standard solution is diluted with citrate buffer to give peak areas in the middle of the range.

9.2. Where high performance liquid chromatographic equipment is used to analyse the hydrolysates, the experimental conditions must be optimised in accordance with the manufacturer's recommendations.

9.3. By applying the method to feedingstuffs containing more than 1 % chloride (concentrate, mineral feeds, supplementary feeds) underestimation of methionine could occur and special treatment has to be done.

PART B

DETERMINATION OF CRUDE OILS AND FATS

1. Purpose and scope

This method is for the determination of crude oils and fats in feedingstuffs. It does not cover the analysis of oil seeds and oleaginous fruit defined in Council Regulation 136/66/EEC of 22 September 1966.

The use of the two procedures described below depends on the nature and composition of the feedingstuff and the reason for carrying out the analysis.

1.1. Procedure A - Directly extractable crude oils and fats

This method is applicable to feed materials of plant origin, except those included within the scope of Procedure B.

1.2. Procedure B - Total crude oils and fats

This method is applicable to feed materials of animal origin and to all compound feeds. It is to be used for all materials from which the oils and fats cannot be completely extracted without prior hydrolysis (eg glutens, yeast, potato proteins and products subject to processes such as extrusion, flaking and heating).

1.3. Interpretation of results

In all cases where a higher result is obtained by using Procedure B than by Procedure A, the result obtained by Procedure B shall be accepted as the true value.

2. Principle

2.1. Procedure A

The sample is extracted with light petroleum. The solvent is distilled off and the residue dried and weighed.

2.2. Procedure B

The sample is treated under heating with hydrochloric acid. The mixture is cooled and filtered. The residue is washed and dried and submitted to the determination according to Procedure A.

3. Reagents

3.1. Light petroleum, boiling range: 40 to 60 °C. The bromine value must be less than 1 and the residue on evaporation less than 2 mg/100 ml.

3.2. Sodium sulfate, anhydrous.

3.3. Hydrochloric acid, c = 3 mol HCl/l

3.4. Filtration aid, e.g. Kieselguhr, Hyflo-supercel.

4. Apparatus

4.1. Extraction apparatus. If fitted with a siphon (Soxhlet apparatus), the reflux rate should be such as to produce about 10 cycles per hour; if of the non-siphoning type, the reflux rate should be about 10 ml per minute.

4.2. Extraction thimbles, free of matter soluble in light petroleum and having a porosity consistent with the requirements of point 4.1.

4.3. Drying oven, either a vacuum oven set at 75 °C ± 3 °C or an air-oven set at 100 °C ± 3 °C.

5. Procedure

5.1. Procedure A (see point 8.1)

Weigh 5 g of the sample to the nearest 1 mg, transfer it to an extraction thimble (4.2) and cover with a fat-free wad of cotton wool.

Place the thimble in an extractor (4.1) and extract for six hours with light petroleum (3.1). Collect the light petroleum extract in a dry, weighed flask containing fragments of pumice stone (1).

Distil off the solvent. Dry the residue maintaining the flask for one and a half hours in the drying oven (4.3). Leave to cool in a desiccator and weigh. Dry again for 30 minutes to ensure that the weight of the oils and fats remains constant (loss in weight between two successive weighings must be less than 1 mg).

5.2. Procedure B

Weigh 2,5 g of the sample to the nearest 1 mg (see point 8.2), place in a 400 ml beaker or a 300 ml conical flask and add 100 ml of hydrochloric acid (3.3) and fragments of pumice stone. Cover the beaker with a watch glass or fit the conical flask with a reflux condenser. Bring the mixture to a gentle boil over a low flame or a hot-plate and keep it there for one hour. Do not allow the product to stick to the sides of the container.

Cool and add a quantity of filtration aid (3.4) sufficient to prevent any loss of oil and fat during filtration. Filter through a moistened, fat-free, double filter paper. Wash the residue in cold water until a neutral filtrate is obtained. Check that the filtrate does not contain any oil or fats. Their presence indicates that the sample must be extracted with light petroleum, using Procedure A, before hydrolysis.

Place the double filter paper containing the residue on a watch glass and dry for one and a half hours in the air oven (4.3) 100 °C ± 3 °C.

Place the double filter paper containing the dry residue in an extraction thimble (4.2) and cover with a fat-free wad of cotton wool. Place the thimble in an extractor (4.1) and proceed as indicated in the second and third paragraphs of point 5.1.

6. Expression of result

Express the weight of the residue as a percentage of the sample.

7. Repeatability

The difference between the results of two parallel determinations carried out on the same sample by the same analyst should not exceed:

- 0,2 % in absolute value, for contents of crude oils and fats lower than 5 %,

- 4,0 % relative to the highest result for contents of 5 to 10 %,

- 0,4 % in absolute value, for contents above 10 %.

8. Observations

8.1. For products with a high content of oils and fats, which are difficult to crush or unsuitable for drawing a homogeneous reduced test sample, proceed as follows.

Weigh 20 g of the sample to the nearest 1 mg and mix with 10 g or more of anhydrous sodium sulfate (3.2). Extract with light petroleum (3.1) as indicated in point 5.1. Make up the extract obtained to 500 ml with light petroleum (3.1) and mix. Take 50 ml of the solution and place in a small, dry, weighed flask containing fragments of pumice stone (2). Distil off the solvent, dry and proceed as indicated in the last paragraph of point 5.1.

Eliminate the solvent from the extraction residue left in the thimble, crush the residue to a fineness of 1 mm, return it to the extraction thimble (do not add sodium sulfate) and proceed as indicated in the second and third paragraphs of point 5.1.

Calculate the content of oils and fats as a percentage of the sample by using the following formula:

(10 a + b) × 5

where:

a = mass in grams of the residue after the first extraction (aliquot part of the extract),

b = mass in grams of the residue after the second extraction.

8.2. For products low in oils and fats the test sample may be increased to 5 g.

8.3. Pet foods containing a high content of water may need to be mixed with anhydrous sodium sulfate prior to hydrolysis and extraction as per Procedure B.

8.4. In paragraph 5.2 it may be more effective to use hot water in place of cold water to wash the residue after filtration.

8.5. The drying time of 1,5 h may need to be extended for some feedingstuffs. Excessive drying should be avoided as this can lead to low results. A microwave oven can also be used.

8.6. Pre-extraction by Procedure A prior to hydrolysis and re-extraction by Procedure B is recommended if the crude oil/fat content is greater than 15 %. To some extent this depends on the nature of the feedingstuff and the nature of the oil-fat in the feedingstuff.

PART C

DETERMINATION OF OLAQUINDOX (2-[N-2'-(hydroxyethyl)carbamoyl]-3-methylquinoxaline-N1,N4-dioxyde)

1. Purpose and scope

This method is for the determination of olaquindox in feedingstuffs. The lower limit of determination is 5 mg/kg.

2. Principle

The sample is extracted by a water-methanol mixture. The content of olaquindox is determined by reversed-phase high-performance liquid chromatography (HPLC) using an UV detector.

3. Reagents

3.1. Methanol.

3.2. Methanol, HPLC grade

3.3. Water, HPLC grade

3.4. Mobile phase for HPLC:

Water (3.3) -methanol (3.2) mixture, 900 + 100 (V + V).

3.5. Standard substance: pure olaquindox 2-[N-2'-(hydroxyethyl)carbamoyl]-3-methylquinoxaline-N1,N4-dioxyde, E 851.

3.5.1. Olaquindox stock standard solution, 250 ìg/ml

Weigh to the nearest 0,1 mg 50 mg of olaquindox (3.5) in a 200 ml graduated flask and add ca. 190 ml water. Then place the flask for 20 min into an ultrasonic bath (4.1). After ultrasonic treatment bring the solution to room temperature, make up to the mark with water and mix. Wrap the flask with aluminum foil and store in a refrigerator. This solution must be prepared fresh each month.

3.5.2. Olaquindox intermediate standard solution, 25 ìg/ml

Transfer 10,0 ml of the stock standard solution (3.5.1) into a 100 ml graduated flask, make up to the mark with the mobile phase (3.4) and mix. Wrap the flask with aluminium foil and store in a refrigerator. This solution must be prepared fresh each day.

3.5.3. Calibration solutions:

Into a series of 50 ml graduated flask transfer 1,0, 2,0, 5,0, 10,0, 15,0 and 20,0 ml of the intermediate standard solution (3.5.2). Make up to the mark with the mobile phase (3.4) and mix. Wrap the flask with aluminium foil. These solutions correspond to 0,5, 1,0, 2,5, 5,0, 7,5 and 10,0 ìg of olaquindox per ml respectively.

These solutions must be prepared fresh each day.

4. Apparatus

4.1. Ultrasonic bath

4.2. Mechanical shaker

4.3. HPLC equipment with variable wavelength ultraviolet detector or diode array detector

4.3.1. Liquid chromatographic column, 250 mm × 4 mm, C18, 10 ìm packing, or equivalent

4.4. Membrane filters, 0,45 ìm

5. Procedure

Note: Olaquindox is light sensitive. Carry out all procedures under subdued light or use amber glassware.

5.1. General

5.1.1. A blank feed should be analysed to check that neither olquindox nor interfering substances are present.

5.1.2. A recovery test should be carried out by analysing the blank feed which has been fortified by addition of a quantity of olaquindox, similar to that present in the sample. To fortify at a level of 50 mg/kg, transfer 10,0 ml of the stock standard solution (3.5.1) to a 250 ml conical flask and evaporate the solution to ca. 0,5 ml. Add 50 g of the blank feed, mix thoroughly and leave for 10 min mixing again several times before proceeding with the extraction step (5.2).

Note: For the purpose of this method the blank feed should be similar in type to that of the sample and olaquindox should not be detected.

5.2. Extraction

Weigh to the nearest 0,01 g, approximately 50 g of the sample. Transfer to a 1 000 ml conical flask, add 100 ml of methanol (3.1) and place the flask for 5 minutes in the ultrasonic bath (4.1). Add 410 ml water and leave in the ultrasonic bath for further 15 minutes. Remove the flask from the ultrasonic bath, shake it for 30 minutes on the shaker (4.2) and filter through a folded filter. Transfer 10,0 ml of the filtrate into a 20 ml graduated flask, make up to the mark with water and mix. An aliquot is filtered through a membrane filter (4.4). (see 9. Observation) Proceed to the HPLC determination (5.3).

5.3. HPLC determination

5.3.1. Parameters:

The following conditions are offered for guidance, other conditions may be used provided that they give equivalent results.

>TABLE>

Check the stability of the chromatographic system, injecting several times the calibration solution (3.5.3) containing 2,5 ìg/ml, until constant peak heights and retention times are achieved.

5.3.2. Calibration graph

Inject each calibration solution (3.5.3) several times and determine the mean peak height (areas) for each concentration. Plot a calibration graph using the mean peak heights (areas) of the calibration solutions as the ordinates and the corresponding concentrations in ìg/ml as the abscissae.

5.3.3. Sample solution

Inject the sample extract (5.2) several times using the same volume as taken for the calibration solutions and determine the mean peak height (area) of the olaquindox peaks.

6. Calculation of the results

From the mean height (area) of the olaquindox peaks of the sample solution determine the concentration of the sample solution in ìg/ml by reference to the calibration graph (5.3.2).

The olaquindox content w in mg/kg of the sample is given by the following formula:

w = >NUM>c × 1 000

>DEN>m

in which:

c = olaquindox concentration of the sample extract (5.2) in ìg/ml

m = mass of the test portion in g (5.2).

7. Validation of the results

7.1. Identity

The identity of the analyte can be confirmed by co-chromatography, or by using a diode-array detector by which the spectra of the sample extract (5.2) and the calibration solution (3.5.3) containing 5,0 ìg/ml are compared.

7.1.1. Co-chromatography

A sample extract (5.2) is fortified by addition of an appropriate amount of calibration solution (3.5.3). The amount of added olaquindox should be similar to the amount of olaquindox found in the sample extract.

Only the height of the olaquindox peak should be enhanced after taking into account both the amount added and the dilution of the extract. The peak width, at half of its height, must be within ± 10 % of the original width of the olaquindox peak of the unfortified sample extract.

7.1.2. Diode array detection

The results are evaluated according to the following criteria:

(a) The wavelength of maximum absorption of the sample and of the standard spectra, recorded at the peak apex on the chromatogram, must be within a margin determined by the resolving power of the detection system. For diode-array detection this is typically within ± 2 nm.

(b) Between 220 and 400 nm, the sample and standard spectra recorded at the peak apex of the chromatogram, must not be different for those parts of the spectrum within the range 10-100 % of relative absorbance. This criterion is met when the same maxima are present and at no observed point the deviation between the two spectra exceeds 15 % of the absorbance of the standard analyte.

(c) Between 220 and 400 nm, the spectra of the upslope, apex and downslope of the peak produced by the sample extract must not be different from each other for those parts of the spectrum within the range 10-100 % of relative absorbance. This criterion is met when the same maxima are present and when at all observed points the deviation between the spectra does not exceed 15 % of the absorbance of the spectrum of the peak apex.

If one of these criteria is not met the presence of the analyte has not been confirmed.

7.2. Repeatability

The difference between the results of two parallel determinations carried out on the same sample must not exceed 15 % relative to the higher result for olaquindox contents between 10 and 20 mg/kg.

7.3. Recovery

For a fortified blank sample the recovery should be at least 90 %.

8. Results of a collaborative study

An EC collaborative study was arranged in which four piglet feed samples including one blank feed were analysed by up to 13 laboratories. The results are given below:

>TABLE>

9. Observation

Although the method has not been validated for feeds containing more than 100 mg/kg of olaquindox, it may be possible to obtain satisfactory results by taking a smaller sample weight and/or diluting the extract (5.2) to reach a concentration within the range of the calibration graph (5.3.2).

(1) Where the oil or fat has to undergo subsequent quality tests, replace the fragments of pumice stone by glass beads.