02009R0152 — EN — 16.11.2020 — 007.001

This text is meant purely as a documentation tool and has no legal effect. The Union's institutions do not assume any liability for its contents. The authentic versions of the relevant acts, including their preambles, are those published in the Official Journal of the European Union and available in EUR-Lex. Those official texts are directly accessible through the links embedded in this document

►B COMMISSION REGULATION (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis for the official control of feed (Text with EEA relevance) (OJ L 054 26.2.2009, p. 1)

Amended by:





Corrected by:









▼B

COMMISSION REGULATION (EC) No 152/2009

of 27 January 2009

laying down the methods of sampling and analysis for the official control of feed

(Text with EEA relevance)







▼M3

Article 1

Sampling for the official control of feed, in particular as regards the determination of constituents, including material which contains or consists of or is produced from genetically modified organisms (GMOs), feed additives as defined by Regulation (EC) No 1831/2003 of the European Parliament and of the Council ( 1 ), undesirable substances as defined by Directive 2002/32/EC of the European Parliament and of the Council ( 2 ) shall be carried out in accordance with the methods set out in Annex I.

The method of sampling set out in Annex I is applicable for the control of feed as regards the determination of pesticide residues as defined in Regulation (EC) No 396/2005 of the European Parliament and of the Council ( 3 ) and control of compliance with Regulation (EU) No 619/2011.

▼B

Article 2

Preparation of samples for analysis and expression of results shall be carried out in accordance with the methods set out in Annex II.

Article 3

Analysis for the official control of feed shall be carried out using the methods set out in Annex III (Methods of analysis to control the composition of feed materials and compound feed, Annex IV (Methods of analysis to control the level of authorised additives in feed), Annex V (Methods of analysis to control undesirable substances in feed) and Annex VI (Methods of analysis for the determination of constituents of animal origin for the official control of feed).

Article 4

The energy value of compound poultry feed shall be calculated in accordance with Annex VII.

Article 5

The methods of analysis to control illegal presence of no longer authorised additives in feed set out in Annex VIII shall be used for confirmatory purposes.

Article 6

Directives 71/250/EEC, 71/393/EEC, 72/199/EEC, 73/46/EEC, 76/371/EEC, 76/372/EEC, 78/633/EEC, 81/715/EEC, 84/425/EEC, 86/174/EEC, 93/70/EEC, 93/117/EC, 98/64/EC, 1999/27/EC, 1999/76/EC, 2000/45/EC, 2002/70/EC and 2003/126/EC are repealed.

References to the repealed Directives shall be construed as references to this Regulation and shall be read in accordance with the correlation tables in Annex IX.

Article 7

This Regulation shall enter into force on the twentieth day following that of 20th day following its publication in the Official Journal of the European Union.

It shall apply from 26 August 2009.

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

▼M3









ANNEX I

METHODS OF SAMPLING

1. PURPOSE AND SCOPE

Samples intended for the official control of feed shall be taken according to the methods described below. Samples thus obtained shall be considered as representative of the sampled portions.

The purpose of representative sampling is to obtain a small fraction from a lot in such a way that a determination of any particular characteristic of this fraction will represent the mean value of the characteristic of the lot. The lot shall be sampled by repeatedly taking incremental samples at various single positions in the lot. These incremental samples shall be combined by mixing to form an aggregate sample from which representative final samples shall be prepared by representative dividing.

If by a visual inspection, portions of the feed to be sampled show a difference in quality from the rest of the feed from the same lot, such portions shall be separated from the rest of the feed and treated as a separate sublot. If it is not possible to divide the feed into separate sublots, the feed shall be sampled as one lot. In such cases, mention shall be made of this fact in the sampling report.

Where a feed sampled in accordance with the provisions of this Regulation is identified as not satisfying EU requirements, is part of a lot of feed of the same class or description, it shall be presumed that all of the feed in that lot is so affected, unless following a detailed assessment there is no evidence that the rest of the lot fails to satisfy the EU requirements.

2. DEFINITIONS

— Lot (or batch) : an identified quantity of feed determined to have common characteristics, such as origin, variety, type of packaging, packer, consignor or labelling, and in case of a production process, a unit of production from a single plant using uniform production parameters or a number of such units, when produced in continuous order and stored together.

— Sampled portion : A lot or an identified part of the lot or sublot.

— Sealed sample : a sample sealed in such a manner as to prevent any access to the sample without breaking or removing the seal.

— Incremental sample : A quantity taken from one point in the sampled portion.

— Aggregate sample : An aggregate of incremental samples taken from the same sampled portion.

— Reduced sample : A part of the aggregate sample, obtained from the latter by a process of representative reduction.

— Final sample : A part of the reduced sample or of the homogenised aggregate sample.

— Laboratory sample : a sample intended for the laboratory (as received by the laboratory) and can be the final, reduced or aggregate sample.

3. GENERAL PROVISIONS

— Sampling personnel: the samples shall be taken by persons authorised for that purpose by the competent authority.

— The sample has to be sealed in such a manner as to prevent any access to the sample without breaking or removing the seal. The seal’s mark should be clearly identifiable and clearly visible. Alternatively, the sample can be put in a recipient which can be closed in such a manner that it cannot be opened without irreversibly damaging the receptacle or container, avoiding the re-use of the receptacle or container.

— Identification of the sample: the sample has to be indelibly marked and must be identified in such a way that there is an unambiguous link to the sampling report.

— From each aggregate sample at least two final samples are taken: at least one for control (enforcement) and one for the feed business operator (defence). Eventually, one final sample may be taken for reference. In case the complete aggregate sample is homogenized, the final samples are taken from the homogenized aggregate sample, unless such procedure conflicts with Member States’ rules as regards the right of the feed business operator.

4. APPARATUS

4.1. The sampling apparatus must be made of materials which cannot contaminate the products to be sampled. Apparatus which is intended to be used multiple times must be easy to clean to avoid any cross-contamination.

4.2. Apparatus recommended for the sampling of solid feed 4.2.1. Manual sampling 4.2.1.1. Flat-bottomed shovel with vertical sides 4.2.1.2. Sampling spear with a long split or compartments. The dimensions of the sampling spear must be appropriate to the characteristics of the sampled portion (depth of container, dimensions of sack, etc.) and to the particle size of the feed. In case the sampling spear has several apertures, in order to ensure that the sample is taken at the different locations alongside the spear, the apertures should be separated by compartments or sequentially staggered apertures. 4.2.2. Mechanical sampling Appropriate mechanical apparatus may be used for the sampling of moving feed. Appropriate means that at least the whole section of the flow is sampled. Sampling of feed in motion (at high flow rates) can be performed by automatic samplers. 4.2.3. Divider If possible and appropriate, apparatus designed to divide the sample into approximately equal parts should be used for the preparation of reduced samples in a representative way.

5. QUANTITATIVE REQUIREMENTS AS REGARDS NUMBER OF INCREMENTAL SAMPLES

— The quantitative requirements in points 5.1 and 5.2 as regards the number of incremental samples are applicable for sampled portion sizes up to a maximum of 500 tonnes and which can be sampled in a representative way. The sampling procedure described is equally valid for quantities larger than prescribed maximum sampled portion size provided that the maximum number of incremental samples given in the tables below is ignored, the number of incremental samples being determined by the square-root formula given in the appropriate part of the procedure (see point 5.3) and the minimum aggregate sample size increased proportionally. This does not prevent a large lot being divided into smaller sublots and each sublot sampled in accordance with the procedure described in points 5.1 and 5.2.

— The size of the sampled portion must be such that each of its constituent parts can be sampled.

— For very large lots or sublots (> 500 tonnes) and for lots which are transported or stored in such a way that sampling cannot be done in accordance with the sampling procedure provided for in points 5.1 and 5.2 of this chapter, the sampling procedure as provided for in point 5.3 is to be applied.

— In case the feed business operator is required by legislation to comply with this Regulation within the frame of a mandatory monitoring system, the feed business operator may deviate from the quantitative requirements as provided for in this chapter to take into account operational characteristics on the condition that the feed business operator has demonstrated to the satisfaction of the competent authority the equivalence of the sampling procedure as regards representativeness and after authorisation from the competent authority.

— In exceptional cases, if it is not possible to carry out the method of sampling set out as regards the quantitative requirements because of the unacceptable commercial damage to the lot (because of packaging forms, means of transport, way of storage etc.) an alternative method of sampling may be applied provided that it is as representative as possible and is fully described and documented.

5.1. Quantitative requirements as regards incremental samples in relation to the control of substances or products uniformly distributed throughout the feed

5.1.1. Loose solid feed







Size of sampled portion Minimum number of incremental samples ≤ 2,5 tonnes 7 > 2,5 tonnes √ 20 times the number of tonnes making up the sampled portion (*1), up to 40 incremental samples ( *1 ) Where the number obtained is a fraction, it shall be rounded up to the next whole number.

5.1.2. Loose liquid feed







Size of sampled portion Minimum number of incremental samples ≤ 2,5 tonnes or ≤ 2 500 litres 4 (*1) > 2,5 tonnes or > 2 500 litres 7 (*1) ( *1 ) In case it is not possible to make the liquid homogeneous, the number of incremental samples has to be increased.

5.1.3. Packaged feed

Feed (solid and liquid) can be packaged in bags, sacks, cans, barrels etc. which are referred to in the table as units. Large units (≥ 500 kg or litres) have to be sampled in accordance with the provisions foreseen for loose feed (see points 5.1.1 and 5.1.2).







Size of sampled portion Minimum number of units from which (at least) one incremental sample has to be taken (*1) 1 to 20 units 1 unit (*2) 21 to 150 units 3 units (*2) 151 to 400 units 5 units (*2) > 400 units ¼ of the √ number of units making up the sampled portion (*3), up to 40 units ( *1 ) In the case where opening of an unit might affect the analysis (e.g. perishable wet feeds) an incremental sample shall be the unopened unit. ( *2 ) For units whose contents do not exceed 1 kg or one litre, an incremental sample shall be the contents of one original unit. ( *3 ) Where the number obtained is a fraction, it shall be rounded up to the next whole number.

5.1.4. Feed blocks and mineral licks

Minimum one block or lick to be sampled per sampled portion of 25 units, up to a maximum of four blocks or licks.

For blocks or licks weighing not more than 1 kg each, an incremental sample shall be the contents of one block or one lick.

5.1.5. Roughages/forage







Size of sampled portion Minimum number of incremental samples (*1) ≤ 5 tonnes 5 > 5 tonnes √ 5 times the number of tonnes making up the sampled portion (*2), up to 40 incremental samples ( *1 ) It is acknowledged that in certain situations (e.g. silages) it is not possible to take the required incremental samples, without causing unacceptable damage to the lot. An alternative method of sampling may be applied in such situations and a guidance for sampling such lots will be elaborated before the entry into application of this Regulation. ( *2 ) Where the number obtained is a fraction, it shall be rounded up to the next whole number.

5.2. Quantitative requirements as regards incremental samples in relation to the control of constituents or substances likely to be distributed non-uniformly in feed

These quantitative requirements as regards incremental samples are to be used in the following situations:

— control of aflatoxins, rye ergot, other mycotoxins and harmful botanical impurities in feed materials;

— control of cross contamination by a constituent, including GM material, or substance for which non-uniform distribution is expected in feed materials.

In case the control authority has strong suspicion that such a non-uniform distribution occurs also in case of cross contamination by a constituent or substance in a compound feed, the quantitative requirements as provided for in the table below can be applied.







Size of sampled portion Minimum number of incremental samples < 80 tonnes See quantitative requirements under point 5.1. The number of incremental samples to be taken has to be multiplied by 2,5. ≥ 80 tonnes 100

5.3. Quantitative requirements as regards the incremental samples in the case of very large lots

In the case of large sampled portions (sampled portions > 500 tonnes), the number of incremental samples to be taken = 40 incremental samples + √ tonnes in relation to the control of substances or products uniformly distributed throughout the feed or 100 incremental samples + √ tonnes in relation to the control of constituents or substances likely to be distributed non-uniformly in feed materials.

6. QUANTITATIVE REQUIREMENTS AS REGARDS AGGREGATE SAMPLE







A single aggregate sample per sampled portion is required. Nature of feed Minimum size of aggregate sample (*1) (*2) 6.1. Loose feed 4 kg 6.2. Packaged feed: 4 kg (*3) 6.3. Liquid or semi-liquid feed: 4 litres 6.4. Feed blocks or mineral licks: 6.4.1. each weighing more than 1 kg 4 kg 6.4.2. each weighing not more than 1 kg weight of four original blocks or licks 6.5. Roughage/forage 4 kg (*4) ( *1 ) In case the sampled feed is of high value, a smaller quantity of aggregate sample can be taken on the condition this is described and documented in the sampling report. ( *2 ) In accordance with the provisions of Commission Regulation (EU) No 619/2011 of 24 June 2011 laying down the methods of sampling and analysis for the official control of feed as regards presence of genetically modified material for which an authorisation procedure is pending or the authorisation of which has expired (OJ L 166, 25.6.2011, p. 9), the aggregate sample for the control of the presence of genetically modified material must contain at least 35 000 seeds/grains. This means that for maize the size of the aggregate sample must be at least 10,5 kg and for soybean 7 kg. For other seeds and grains such as barley, millet, oat, rice, rye, wheat and rapeseed, the aggregate sample size of 4 kg corresponds to more than 35 000 seeds. ( *3 ) In case of packaged feed, it may also not be possible to achieve the size of 4 kg for the aggregate sample depending of the size of the individual units. ( *4 ) In case it concerns roughage or forage with a low specific gravity (e.g. hay, straw), the aggregate sample should have a minimum size of 1 kg.

7. QUANTITATIVE REQUIREMENTS AS REGARDS FINAL SAMPLES

Final samples

Analysis of at least one final sample is required. The amount in the final sample for analysis shall be not less than the following:







Solid feed 500 g (*1) (*2) (*3) Liquid or semi-liquid feed 500 ml (*1) ( *1 ) In accordance with the provisions of Regulation (EU) No 619/2011, the final sample for the control of the presence of genetically modified material must contain at least 10 000 seeds/grains. This means that for maize the size of the final sample must be at least 3 000 g and for soybean 2 000 g. For other seeds and grains such as barley, millet, oat, rice, rye, wheat and rapeseed, the final sample size of 500 g corresponds to more for 10 000 . ( *2 ) In case the size of the aggregate sample is significantly less than 4 kg or litre (see footnotes point (6), also a smaller quantity of final sample can be taken on the condition this is described and documented in the sampling report. ( *3 ) In case of sampling pulses, cereal grains and tree nuts for the determination of pesticide residues, the minimum size of the final sample shall be 1 kg in accordance with the provisions of Commission Directive 2002/63/EC (OJ L 187, 16.7.2002, p. 30).

8. METHOD OF SAMPLING FOR VERY LARGE LOTS OR LOTS STORED OR TRANSPORTED IN A WAY WHEREBY SAMPLING THROUGHOUT THE LOT IS NOT FEASIBLE

8.1. General principles

In case the way of transport or storage of a lot does not enable to take incremental samples throughout the whole lot, sampling of such lots should preferably be done when the lot is in flow.

In the case of large warehouses destined to store feed, operators should be encouraged to install equipment in the warehouse enabling (automatic) sampling across the whole stored lot.

In case of applying the sampling procedures as provided for in this chapter 8, the feed business operator or his representative is informed of the sampling procedure. In case this sampling procedure is questioned by the feed business operator or his representative, the feed business operator or his representative shall enable the competent authority to sample throughout the whole lot at his/her cost.

8.2. Large lots transported by ship

8.2.1. Dynamic sampling of large lots transported by ship

The sampling of large lots in ships is preferably carried out while the product is in flow (dynamic sampling).

The sampling is to be done per hold (entity that can physically be separated). Holds are however emptied partly one after the other so that the initial physical separation does no longer exist after transfer into storage facilities. Sampling can therefore be performed in function of the initial physical separation or in function of the separation after transfer into the storage facilities.

The unloading of a ship can last for several days. Normally, sampling has to be performed at regular intervals during the whole duration of unloading. It is however not always feasible or appropriate for an official inspector to be present for sampling during the whole operation of unloading. Therefore sampling is allowed to be undertaken of part (sampled portion) of the whole lot. The number of incremental samples is determined by taking into account the size of the sampled portion.

In the case of sampling a part of a lot of feed of the same class or description and that part of the lot has been identified as not satisfying EU requirements, it shall be presumed that all of the feed in that lot is so affected, unless following a detailed assessment there is no evidence that the rest of the lot fails to satisfy the EU requirements.

Even if the official sample is taken automatically, the presence of an inspector is necessary. However in case the automatic sampling is done with preset parameters which cannot be changed during the sampling and the incremental samples are collected in a sealed receptacle, preventing any possible fraud, then the presence of an inspector is only required at the beginning of the sampling, every time the receptacle of the samples needs to be changed and at the end of the sampling.

8.2.2. Sampling of lots transported by ship by static sampling

In case the sampling is done in a static way the same procedure as foreseen for storage facilities (silos) accessible from above has to be applied (see point 8.4.1).

The sampling has to be performed on the accessible part (from above) of the lot/hold. The number of incremental samples is determined by taking into account the size of the sampled portion. In the case of sampling a part of a lot of feed of the same class or description and that part of the lot has been identified as not satisfying EU requirements, it shall be presumed that all of the feed in that lot is so affected, unless following a detailed assessment there is no evidence that the rest of the lot fails to satisfy the EU requirements.

8.3. Sampling of large lots stored in warehouses

The sampling has to be performed on the accessible part of the lot. The number of incremental samples is determined by taking into account the size of the sampled portion. In the case of sampling a part of a lot of feed of the same class or description and that part of the lot has been identified as not satisfying EU requirements, it shall be presumed that all of the feed in that lot is so affected, unless following a detailed assessment there is no evidence that the rest of the lot fails to satisfy the EU requirements.

8.4. Sampling of storage facilities (silos)

8.4.1. Sampling of silos (easily) accessible from above

The sampling has to be performed on the accessible part of the lot. The number of incremental samples is determined by taking into account the size of the sampled portion. In the case of sampling a part of a lot of feed of the same class or description and that part of the lot has been identified as not satisfying EU requirements, it shall be presumed that all of the feed in that lot is so affected, unless following a detailed assessment there is no evidence that the rest of the lot fails to satisfy the EU requirements.

8.4.2. Sampling of silos not accessible from above (closed silos)

8.4.2.1. Silos not accessible from above (closed silos) with size > 100 tonnes

Feed stored in such silos cannot be sampled in a static way. Therefore in case the feed in the silo has to be sampled and there is no possibility to move the consignment, the agreement has to be made with the operator that he or she has to inform the inspector about when the silo will be unloaded in order to enable sampling when the feed is in flow.

8.4.2.2. Silos not accessible from above (closed silos) with size < 100 tonnes

Sampling procedure involves the release into a receptacle of a quantity of 50 to 100 kg and taking the sample from it. The size of the aggregate sample corresponds to the whole lot and the number of incremental samples relate to the quantity of the silo released in a receptacle for sampling. In the case of sampling a part of a lot of feed of the same class or description and that part of the lot has been identified as not satisfying EU requirements, it shall be presumed that all of the feed in that lot is so affected, unless following a detailed assessment there is no evidence that the rest of the lot fails to satisfy the EU requirements.

8.5. Sampling of loose feed in large closed containers

Such lots can often only be sampled when unloaded. It is in certain cases not possible to unload at the point of import or control and therefore the sampling should take place when such containers are unloaded.

9. INSTRUCTIONS FOR TAKING, PREPARING AND PACKAGING THE SAMPLES

9.1. General

The samples must be taken and prepared without unnecessary delay bearing in mind the precautions necessary to ensure that the product is neither changed nor contaminated. Instruments and also surfaces and containers intended to receive samples must be clean and dry.

9.2. Incremental samples

Incremental samples must be taken at random throughout the whole sampled portion and they must be of approximately equal sizes.

The incremental sample size is at least 100 grams or 25 grams in case of roughage or forage with low specific gravity.

In case that in accordance with the rules for the sampling procedure established in point 8 less than 40 incremental samples have to be taken, the size of the incremental samples shall be determined in function of the required size of the aggregate sample to be achieved (see point (6).

In case of sampling of small lots of packaged feed where according to the quantitative requirements a limited number of incremental samples have to be taken, an incremental sample shall be the contents of one original unit whose contents do not exceed 1 kg or one litre.

In case of sampling of packaged feed composed of small units (e.g. < 250 g), the size of the incremental sample depends on the size of the unit.

9.2.1. Loose feed

Where appropriate, sampling may be carried out when the sampled portion is being moved (loading or unloading).

9.2.2. Packaged feed

Having selected the required number of units for sampling as indicated in chapter 5, part of the contents of each unit shall be removed using a spear or shovel. Where necessary, the samples shall be taken after emptying the units separately.

9.2.3. Homogeneous or homogenisable liquid or semi-liquid feed

Having selected the required number of units for sampling as indicated in chapter 5, the contents shall be homogenised if necessary and an amount taken from each unit.

The incremental samples may be taken when the contents are being discharged.

9.2.4. Non-homogenisable, liquid or semi-liquid feed

Having selected the required number of units for sampling as indicated in chapter 5, samples shall be taken from different levels.

Samples may also be taken when the contents are being discharged but the first fractions shall be discarded.

In either case the total volume taken must not be less than 10 litres.

9.2.5. Feed blocks and mineral licks

Having selected the required number of blocks or licks for sampling as indicated in chapter 5, a part of each block or lick can be taken. In case of suspicion of a non-homogeneous block or lick, the whole block or lick can be taken as sample.

For blocks or licks weighing not more than 1 kg each, an incremental sample shall be the contents of one block or one lick.

9.3. Preparation of aggregate samples

The incremental samples shall be mixed to form a single aggregate sample.

9.4. Preparation of final samples

The material in the aggregate sample shall be carefully mixed ( 4 ).

— Each sample shall be put into an appropriate container/receptacle. All necessary precautions shall be taken to avoid any change of composition of the sample, contamination or adulteration which might arise during transportation or storage.

— In case of the control of constituents or substances uniformly distributed throughout the feed, the aggregate sample can be representatively reduced to at least 2,0 kg or 2,0 litres (reduced sample) ( 5 ) preferably either by using a mechanical or automatic divider. For the control of the presence of pesticide residues in pulses, cereal grains and tree nuts, the minimum size of the reduced sample shall be 3 kg. In case the nature of the feed does not allow using a divider or the divider is not available, then the sample can be reduced by the quartering method. From the reduced samples the final samples (for control, defence and reference) shall then be prepared of approximately the same amount and conforming to the quantitative requirements of chapter 7. In case of the control of constituents, including genetically modified material, or substances likely to be distributed non-uniformly in feed materials, the aggregate sample shall be: — completely homogenized and divided afterwards into final samples or — reduced to at least 2 kg or 2 litres ( 6 ) by using a mechanical or automatic divider. Only in the case that the nature of the feed does not allow for using a divider, the sample can, if necessary, be reduced by quartering method. For the control of the presence of genetically modified material in the frame of Regulation (EU) No 619/2011, the reduced sample must contain at least 35 000 seeds/grains to enable to obtain the final samples for enforcement, defence and reference of at least 10 000 seeds grain (see footnote (**) in chapter 6 and footnote (*) in chapter 7).

9.5. Packaging of samples

The containers or packages shall be sealed and labelled in such a manner that they cannot be opened without damaging the seal. The total label must be incorporated in the seal.

9.6. Sending of samples to the laboratory

The sample shall be sent without unnecessary delay to the designated analytical laboratory, together with the information necessary for the analyst.

10. SAMPLING RECORD

A record must be kept of each sample, permitting each sampled portion and its size to be identified unambiguously.

The record shall also mention any deviation of the sampling procedure as provided for in this Regulation.

Besides making the record available to the official control laboratory, the record shall be made available to the feed business operator and/or the laboratory designated by the feed business operator.









ANNEX II

GENERAL PROVISIONS ON METHODS OF ANALYSIS FOR FEED

A. PREPARATION OF SAMPLES FOR ANALYSIS

1. Purpose

The procedures described below concern the preparation for analysis of samples, sent to the control laboratories after sampling in accordance with the provisions laid down in Annex I.

The laboratory samples must be prepared in such a way that the amounts weighed out, as provided for in the methods of analysis, are homogeneous and representative of the final samples.

2. Precautions to be taken

The sample preparation procedure to be followed is dependent on the methods of analysis to be used and the constituents or substances to be controlled. It is therefore of major importance that it is ensured that the followed sample preparation procedure is appropriate for the used method of analysis and for constituents or substances to be controlled.

All the necessary operations must be performed in such a way as to avoid as far as possible contamination of the sample and changes of its composition.

Grinding, mixing and sieving shall be carried out without delay with minimal exposure of the sample to the air and light. Mills and grinders likely to appreciably heat the sample shall not be used.

Manual grinding is recommended for feed which are particularly sensitive to heat. Care shall also be taken to ensure that the apparatus itself is not a source of contamination.

If the preparation cannot be carried out without significant changes in the moisture content of the sample, determine the moisture content before and after preparation according to the method laid down in Part A of Annex III.

3. Procedure

3.1. General procedure

The test aliquot is taken from the final sample. Coning and quartering is not recommended because this might provide test aliquots with high splitting error.

3.1.1. Feed which can be ground as such

— Mix the sieved final sample and collect it in a suitable clean, dry container fitted with an air-tight stopper. Mix again in order to ensure full homogenisation, immediately before weighing out the amount for analysis (test aliquot).

3.1.2. Feed which can be ground after drying

— Unless otherwise specified in the methods of analysis, dry the final sample to bring its moisture content down to a level of 8 to 12 %, according to the preliminary drying procedure described under point 4.3 of the method of determination of moisture mentioned in Part A of Annex III). Then proceed as indicated in section 3.1.1.

3.1.3. Liquid or semi-liquid feed

— Collect the final sample in a suitable clean, dry container, fitted with an air-tight stopper. Mix thoroughly in order to ensure full homogenisation immediately before weighing out the amount for analysis (test aliquot).

3.1.4. Other feed

— Final samples which cannot be prepared according to one of the above procedures shall be treated by any other procedure which ensures that the amounts weighed out for the analysis (test aliquot) are homogeneous and representative of the final samples.

3.2. Specific procedure in case of examination by visual inspection or by microscopy or in cases where the whole aggregate sample is homogenised

— In case of an examination by visual inspection (without making use of microscope), the whole laboratory sample is used for examination.

— In case of a microscopic examination, the laboratory may reduce the aggregate sample, or further reduce the reduced sample. The final samples for defence and eventually reference purposes are taken following a procedure equivalent to the procedure followed for the final sample for enforcement.

— In case the whole aggregate sample is homogenized, the final samples are taken from the homogenized aggregate sample.

4. Storage of samples

Samples must be stored at a temperature that will not alter their composition. Samples intended for the analysis of vitamins or substances which are particularly sensitive to light shall be stored in such conditions that the sample is not adversely affected by light.

B. PROVISIONS RELATING TO REAGENTS AND APPARATUS USED IN METHODS OF ANALYSIS

1. Unless otherwise specified in the methods of analysis, all analytical reagents must be analytically pure (a.p.). When trace analysis is carried out, the purity of the reagents must be checked by a blank test. Depending upon the results obtained, further purification of the reagents may be required.

2. Any operation involving preparation of solutions, dilution, rinsing or washing, mentioned in the methods of analysis without indication as to the nature of the solvent or diluent employed, implies that water must be used. As a general rule, water shall be demineralised or distilled. In particular cases, which are indicated in the methods of analysis, it must be submitted to special procedures of purification.

3. In view of the equipment normally found in control laboratories, only those instruments and apparatus which are special or require specific usage are referred to in the methods of analysis. They must be clean, especially when very small amounts of substances have to be determined.

C. APPLICATION OF METHODS OF ANALYSIS AND EXPRESSION OF THE RESULTS

1. Extraction procedure

Several methods determine a specific extraction procedure. As a general rule, other extraction procedures than the procedure referred to in the method can be applied on the condition that the used extraction procedure has been proven to have the equivalent extraction efficiency for the matrix analysed as the procedure mentioned in the method.

2. Clean-up procedure

Several methods determine a specific clean-up procedure. As a general rule, other clean-up procedures than the procedure referred to in the method can be applied on the condition that the used clean-up procedure has been proven to result in equivalent analytical results for the matrix analysed as the procedure mentioned in the method.

3. Number of determinations

In case of the analysis of undesirable substances, if the result of the first determination is significantly (> 50 %) lower than the specification to be controlled, no additional determinations are necessary, on the condition that the appropriate quality procedures are applied. In other cases a duplicate analysis (second determination) is necessary to exclude the possibility of internal cross-contamination or an accidental mix-up of samples. The mean of the two determinations, taking into account the measurement uncertainty is used for verification of compliance.

In case of the control of the declared content of a substance or ingredient, if the result of the first determination confirms the declared content, i.e. the analytical result falls within the acceptable range of variation of the declared content, no additional determinations are necessary, on the condition that the appropriate quality procedures are applied. In other cases a duplicate analysis (second determination) is necessary to exclude the possibility of internal cross-contamination or an accidental mix-up of samples. The mean of the two determinations, taking into account the measurement uncertainty is used for verification of compliance.

In some cases this acceptable range of variation is defined in legislation such as in Regulation (EC) No 767/2009 of the European Parliament and of the Council of 13 July 2009 on the placing on the market and use of feed, amending European Parliament and Council Regulation (EC) No 1831/2003 and repealing Council Directive 79/373/EEC, Commission Directive 80/511/EEC, Council Directives 82/471/EEC, 83/228/EEC, 93/74/EEC, 93/113/EC and 96/25/EC and Commission Decision 2004/217/EC ( 7 ).

4. Reporting of the method of analysis used

The analysis report shall mention the method of analysis used.

5. Reporting of the analytical result

The analytical result shall be expressed in the manner laid down in the method of analysis to an appropriate number of significant figures and shall be corrected, if necessary, to the moisture content of the final sample prior to preparation.

6. Measurement uncertainty and recovery rate in case of analysis of undesirable substances

As regards undesirable substances within the meaning of Directive 2002/32/EC, a product intended for animal feed shall be considered as non-compliant with the established maximum content, if the analytical result, relative to a feed with a moisture content of 12 %, is deemed to exceed the maximum content taking into account expanded measurement uncertainty and correction for recovery. In order to assess compliance, the analysed concentration is used after being corrected for recovery and after deduction of the expanded measurement uncertainty. This procedure is only applicable in cases where the method of analysis enables the estimation of measurement uncertainty and correction for recovery (e.g. not possible in case of microscopic analysis).

The analytical result shall be reported as follows (in so far the used method of analysis enables to estimate the measurement uncertainty and recovery rate):

(a) corrected for recovery, the level of recovery being indicated. The correction for recovery is not necessary in case the recovery rate is between 90-110 %.

(b) as ‘x +/– U’, whereby x is the analytical result and U is the expanded measurement uncertainty, using a coverage factor of 2 which gives a level of confidence of approximately 95 %.

However, if the result of the analysis is significantly (> 50 %) lower than the specification to be controlled, and on the condition that the appropriate quality procedures are applied and the analysis serves only the purpose of checking compliance with legal provisions, the analytical result might be reported without correction for recovery and the reporting of the recovery rate and measurement uncertainty might be omitted in these cases.

▼B









ANNEX III

METHODS OF ANALYSIS TO CONTROL THE COMPOSITION OF FEED MATERIALS AND COMPOUND FEED

A. DETERMINATION OF MOISTURE

1. Purpose and Scope

This method makes it possible to determine the moisture content of feed. In case of feed containing volatile substances, such as organic acids, it is to be observed that also significant amount of volatile substances are determined together with the moisture content.

It does not cover the analysis of milk products as feed materials, the analysis of mineral substances and mixtures composed predominantly of mineral substances, the analysis of animal and vegetable fats and oils or the analysis of the oil seeds and oleaginous fruit.

2. Principle

The sample is desiccated under specified conditions which vary according to the nature of the feed. The loss in weight is determined by weighing. It is necessary to carry out preliminary drying when dealing with solid feed which has high moisture content.

3. Apparatus

3.1. Crusher of non-moisture-absorbing material which is easy to clean, allows rapid, even crushing without producing any appreciable heating, prevents contact with the outside air as far as possible and meets the requirements laid down in 4.1.1 and 4.1.2 (e.g. hammer or water cooled micro-crushers, collapsible cone mills, slow motion or cog wheeled crushers).

3.2. Analytical balance, accurate to 1 mg.

3.3. 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.

3.4. Electrically heated isothermal oven (± 2 oC) properly ventilated and ensuring rapid temperature regulation ( 8 ).

3.5. 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).

3.6. Desiccator with a thick perforated metal or porcelain plate, containing an efficient drying agent.

4. Procedure

N.B. The operations described in this section must be carried out immediately after opening the packages of samples. Analysis must be carried out at least in duplicate.

4.1. Preparation

4.1.1. Feed other than those coming under 4.1.2 and 4.1.3

Take at least 50 g of the sample. If necessary, crush or divide in such a way as to avoid any variation in moisture content (see 6).

4.1.2. Cereals and groats

Take at least 50 g of the sample. Grind into particles of which at least 50 % will pass through a 0,5 mm mesh sieve and will leave no more than 10 % reject on a 1 mm round-meshed sieve.

4.1.3. Feed in liquid or paste form, feed predominantly composed of oils and fats

Take about 25 g of the sample, weigh to the nearest 10 mg, add an appropriate quantity of anhydrous sand weighed to the nearest 10 mg and mix until a homogeneous product is obtained.

4.2. Drying

4.2.1. Feed other than those coming under 4.2.2 and 4.2.3

Weigh a container (3.3) with its lid to the nearest 1 mg. Weigh into the weighed container, to the nearest 1 mg, about 5 g of the sample and spread evenly. Place the container, without its lid, in the oven preheated to 103 oC. To prevent the oven temperature from falling unduly, introduce the container as rapidly as possible. Leave to dry for four hours reckoned from the time when the oven temperature returns to 103 oC. Replace the lid on the container, remove the latter from the oven, leave to cool for 30 to 45 minutes in the desiccator (3.6) and weigh to the nearest 1 mg.

For feed composed predominantly of oils and fats, dry in the oven for an additional 30 minutes at 130 oC. The difference between the two weighings must not exceed 0,1 % of moisture.

4.2.2. Cereals, flour, groats and meal

Weigh a container (3.3) with its lid to the nearest 0,5 mg. Weigh into the weighed container, to the nearest 1 mg, about 5 g of the crushed sample and spread evenly. Place the container, without its lid, in the oven preheated to 130 oC. To prevent the oven temperature from falling unduly, introduce the container as rapidly as possible. Leave to dry for two hours reckoned from the time when the oven temperature returns to 130 oC. Replace the lid on the container, remove the latter from the oven, leave to cool for 30 to 45 minutes in the desiccator (3.6) and weigh to the nearest 1 mg.

4.2.3. Compound feed containing more than 4 % of sucrose or lactose: feed materials such as locust beans, hydrolysed cereal products, malt seeds, dried beet chips, fish and sugar solubles; compound feed containing more than 25 % of mineral salts including water of crystallisation.

Weigh a container (3.3) with its lid to the nearest 0,5 mg. Weigh into the weighed 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 (3.5) preheated to between 80 oC and 85 oC. To prevent the oven temperature from falling unduly, introduce the container as rapidly as possible.

Bring the pressure up to 100 Torr 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 80 oC to 85 oC. 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 (3.6) and weigh to the nearest 1 mg. Dry for an additional 30 minutes in the vacuum oven at 80 oC to 85 oC and reweigh. The difference between the two weighings must not exceed 0,1 % of moisture.

4.3. Preliminary drying

4.3.1. Feed other than those coming under 4.3.2

Solid feed with a high moisture content which makes crushing difficult must be subjected to preliminary drying as follows:

Weigh 50 g of uncrushed sample to the nearest 10 mg (compressed or agglomerated feed may be roughly divided if necessary) in a suitable container (e.g. a 20 × 12 cm aluminium plate with a 0,5 cm rim). Leave to dry in an oven from 60 oC to 70 oC until the moisture content has been reduced to between 8 % and 12 %. Remove from the oven, leave to cool uncovered in the laboratory for one hour and weigh to the nearest 10 mg. Crush immediately as indicated in 4.1.1 and dry as indicated in 4.2.1 or 4.2.3 according to the nature of the feed.

4.3.2. Cereals

Grain with a moisture content of over 17 % must be subjected to preliminary drying as follows:

Weigh 50 g of unground grain to the nearest 10 mg in a suitable container (e.g. a 20 × 12 cm aluminium plate with a 0,5 cm rim). Leave to dry for 5 to 7 minutes in an oven at 130 oC. Remove from the oven, leave to cool uncovered in the laboratory for two hours and weigh to the nearest 10 mg. Grind immediately as indicated in 4.1.2 and dry as indicated in 4.2.2.

5. Calculation of results

The moisture content (X), as a percentage of the sample, is calculated by using the following formulae:

5.1. Drying without preliminary drying

where:

m = initial weight, in grammes, of the test sample,

m0 = weight, in grammes, of the dry test sample.

5.2. Drying with preliminary drying

where:

m = initial weight, in grammes, of the test sample,

m1 = weight, in grammes, of the test sample after preliminary drying,

m2 = weight, in grammes, of the test sample after crushing or grinding,

m0 = weight, in grammes, of the dry test sample.

5.3. Repeatability

The difference between the results of two parallel determinations carried out on the same sample shall not exceed 0,2 % of the absolute value of moisture.

6. Observation

If crushing proves necessary and if this is seen to alter the moisture content of the product, the results of the analysis of the components of the feed must be corrected on the basis of the moisture content of the sample in its initial state.

B. DETERMINATION OF MOISTURE IN ANIMAL AND VEGETABLE FATS AND OILS

1. Purpose and scope

This method makes it possible to determine the water and volatile substances content of animal and vegetable fats and oils.

2. Principle

The sample is dried to constant weight (loss in weight between two successive weighings must be less than or equal to 1 mg) at 103 oC. The loss in weight is determined by weighing.

3. Apparatus

3.1. Flat-bottomed dish, of a corrosion-resistant material, 8 to 9 cm in diameter and approximately 3 cm high.

3.2. Thermometer with a strengthened bulb and expansion tube at the top end, graduated from approximately 80 oC to at least 110 oC, and approximately 10 cm in length.

3.3. Sand bath or electric hot-plate.

3.4. Desiccator, containing an efficient drying agent.

3.5. Analytical balance.

4. Procedure

Weigh out to the nearest mg approximately 20 g of the homogenised sample into the dry, weighed dish (3.1) containing the thermometer (3.2). Heat on the sand bath or hot-plate (3.3), stirring continuously with the thermometer, so that the temperature reaches 90 oC in about 7 minutes.

Reduce the heat, watching the frequency with which bubbles rise from the bottom of the dish. The temperature must not exceed 105 oC. Continue to stir, scraping the bottom of the dish, until bubbles stop forming.

In order to ensure complete elimination of moisture, reheat several times to 103 oC ± 2 oC, cooling to 93 oC between successive heatings. Then leave to cool to room temperature in the desiccator (3.4) and weigh. Repeat this operation until the loss in weight between two successive weighings no longer exceeds 2 mg.

N.B : An increase in the weight of the sample after repeated heating indicates an oxidation of the fat, in which case calculate the result from the weighing carried out immediately before the weight began to increase.

5. Calculation of results

The moisture content (X), as a percentage of the sample, is given by the following formula:

where:

m = weight, in grammes, of the test sample,

m1 = weight, in grammes, of the dish with its contents before heating,

m2 = weight, in grammes, of the dish with its contents after heating.

Results lower than 0,05 % must be recorded as ‘lower than 0,05 %’.

Repeatability

The difference in moisture between the results of two parallel determinations carried out on the same sample must not exceed 0,05 %, in absolute value.

C. DETERMINATION OF THE CONTENT OF CRUDE PROTEIN

1. Purpose and scope

This method makes it possible to determine the crude protein content of feed on the basis of the nitrogen content, determined according to the Kjeldahl method.

2. Principle

The sample is digested by sulphuric acid in the presence of a catalyst. The acid solution is made alkaline with sodium hydroxide solution. The ammonia is distilled and collected in a measured quantity of sulphuric acid, the excess of which is titrated with a standard solution of sodium hydroxide.

Alternatively, the liberated ammonia is distilled into an excess of boric acid solution, followed by titration with hydrochloric acid or sulphuric acid solution.

3. Reagents

3.1. Potassium sulphate.

3.2. Catalyst: copper (II) oxide CuO or copper (II) sulphate pentahydrate, CuSO4 5H2O.

3.3. Granulated zinc.

3.4. Sulphuric acid, ρ20 = 1,84 g/ml.

3.5. Sulphuric acid, standard volumetric solution, c(H2SO4) = 0,25 mol/l.

3.6. Sulphuric acid, standard volumetric solution, c(H2SO4) = 0,10 mol/l.

3.7. Sulphuric acid, standard volumetric solution, c(H2SO4) = 0,05 mol/l.

3.8. Methyl red indicator; dissolve 300 mg of methyl red in 100 ml of ethanol, σ = 95 %-96 % (v/v).

3.9. Sodium hydroxide solution (Technical grade may be used) β = 40 g/100 ml (m/v: 40 %).

3.10. Sodium hydroxide, standard volumetric solution c(NaOH) = 0,25 mol/l.

3.11. Sodium hydroxide, standard volumetric solution c(NaOH) = 0,10 mol/l.

3.12. Granulated pumice stone, washed in hydrochloric acid and ignited.

3.13. Acetanilide (m.p. = 114 oC, N-content = 10,36 %).

3.14. Sucrose (nitrogen free).

3.15. Boric acid (H3BO3).

3.16. Methyl red indicator solution: dissolve 100 mg methyl red in 100 ml ethanol or methanol.

3.17. Bromocresol green solution: dissolve 100 mg bromocresol green in 100 ml ethanol or methanol.

3.18. Boric acid solution (10 g/l to 40 g/l depending on the apparatus used).

When colorimetric end-point detection is applied, methyl red and bromocresol indicators must be added to the boric acid solutions. If 1 litre of the boric acid solution is prepared, before adjusting to volume, 7 ml methyl red indicator solution (3.16) and 10 ml bromocresol green solution (3.17) shall be added.

Dependent on the water used, the pH of the boric acid solution might differ from batch to batch. Often an adjustment with a small volume of alkali is necessary to obtain a positive blank.

Note : The addition of about 3 ml to 4 ml of NaOH (3.11) into 1 litre of 10 g/l boric acid usually gives good adjustments. Store the solution at room temperature and protect the solution from light and sources of ammonia fumes during storage.

3.19. Hydrochloric acid standard volumetric solution c(HCl) = 0,10 mol/l.

Note: Other concentrations of volumetric solutions (3.5, 3.6, 3.7, 3.10, 3.11, and 3.19) can be used, if this is corrected for in the calculations. The concentrations shall always be expressed to four decimal places.

4. Apparatus

Apparatus suitable for performing digestion, distillation and titration according to the Kjeldahl procedure.

5. Procedure

5.1. Digestion

Weigh 1 g of the sample to the nearest 0,001 g and transfer the sample to the flask of the digestion apparatus. Add 15 g of potassium sulphate (3.1), an appropriate quantity of catalyst (3.2) (0,3 to 0,4 g of copper (II) oxide or 0,9 to 1,2 g of copper (II) sulphate pentahydrate), 25 ml of sulphuric acid (3.4) and if required, a few granules of pumice stone (3.12) and mix.

Heat the flask moderately at first, swirling from time to time if necessary until the mass has carbonised and the foam has disappeared; then heat more intensively until the liquid is boiling steadily. Heating is adequate if the boiling acid condenses on the wall of the flask. Prevent the sides from becoming overheated and organic particles from sticking to them.

When the solution becomes clear and light green continue to boil for another two hours, then leave to cool.

5.2. Distillation

Add carefully enough water to ensure complete dissolution of the sulphates. Allow to cool and then add a few granules of zinc (3.3), if required. Proceed according to 5.2.1 or 5.2.2.

5.2.1. Distillation into sulphuric acid

Place in the collecting flask of the distillation apparatus an exactly measured quantity of 25 ml of sulphuric acid (3.5) or (3.7) depending on the presumed nitrogen content. Add a few drops of methyl red indicator (3.8).

Connect the digestion flask to the condenser of the distillation apparatus and immerse the end of the condenser in the liquid contained in the collecting flask to a depth of at least 1 cm (see observation 8.3). Slowly pour 100 ml of sodium hydroxide solution (3.9) into the digestion flask without loss of ammonia (see observation 8.1). Heat the flask until the ammonia has distilled over.

5.2.2. Distillation into boric acid

Where titration of the ammonia content of the distillate is performed manually, the procedure mentioned below applies. Where the distillation unit is fully automated to include titration of the ammonia content of the distillate, follow the manufacturer's instructions for operation of the distillation unit.

Place a collecting flask containing 25 ml to 30 ml of the boric acid solution (3.18) under the outlet of the condenser in such a way that the delivery tube is below the surface of the excess boric acid solution. Adjust the distillation unit to dispense 50 ml of sodium hydroxide solution (3.9). Operate the distillation unit in accordance with the manufacturer's instructions and distil off the ammonia liberated by the addition of the sodium hydroxide solution. Collect distillate in the boric acid receiving solution. The amount of distillate (time of steam distillation) depends on the amount of nitrogen in the sample. Follow the instructions of the manufacturer.

Note: In a semi-automatic distillation unit, the addition of excess sodium hydroxide and the steam distillation are performed automatically.

5.3. Titration

Proceed according to 5.3.1 or 5.3.2.

5.3.1. Sulphuric acid

Titrate the excess sulphuric acid in the collecting flask with sodium hydroxide solution (3.10 or 3.11) depending on the concentration of the sulphuric acid used, until the end-point is reached.

5.3.2. Boric acid

Titrate the contents of the collecting flask with the hydrochloric acid standard volumetric solution (3.19) or with the sulphuric acid standard volumetric solution (3.6) using a burette and read the amount of titrant used.

When colorimetric end-point detection is applied, the end-point is reached at the first trace of pink colour in the contents. Estimate the burette reading to the nearest 0,05 ml. An illuminated magnetic stirrer plate or a photometric detector may aid visualisation of the end-point.

This can be done automatically using a steam distiller with automatic titration.

Follow the manufacturers' instructions for operation of the specific distiller or distiller/titrator.

Note : When an automatic titration system is used, titration begins immediately after distillation starts and the 1 % boric acid solution (3.18) is used. Where a fully automatic distillation unit is employed, the automatic titration of the ammonia can also be carried out with end-point detection using a potentiometric pH system. In this case an automatic titrator, with a pH-meter is used. The pH-meter shall be calibrated properly in the range of pH 4 to pH 7 following normal laboratory pH-calibration procedures. The pH end-point of the titration is reached at pH 4,6 , being the steepest point in the titration curve (inflection point).

5.4. Blank test

To confirm that the reagents are free from nitrogen, carry out a blank test (digestion, distillation and titration) using 1 g of sucrose (3.14) in place of the sample.

6. Calculation of results

Calculations are performed according to 6.1 or 6.2.

6.1. Calculation for titration according to 5.3.1

The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:

where:

Vo = is the volume (ml) of NaOH (3.10 or 3.11) used in the blank test,

V1 = is the volume (ml) of NaOH (3.10 or 3.11) used in the sample titration,

c = is the concentration (mol/l) of sodium hydroxide (3.10 or 3.11),

m = is the weight (g) of sample.

6.2. Calculation for titration according to 5.3.2

6.2.1. Titration with hydrochloric acid

The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:

where:

m = is the weight (g) of the test portion,

c = is the concentration (mol/l) of the standard volumetric solution of the hydrochloric acid (3.19),

V0 = is the volume (in ml) of hydrochloric acid used for the blank test,

V1 = is the volume (in ml) of hydrochloric acid used for the test portion.

6.2.2. Titration with sulphuric acid

The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:

where:

m = is the weight (g) of the test portion,

c = is the concentration (mol/l) of the standard volumetric solution of sulphuric acid (3.6),

V0 = is the volume (in ml) of sulphuric acid (3.6) used for the blank test,

V1 = is the volume (in ml) of sulphuric acid (3.6) used for test portion.

7. Verification of the method

7.1. Repeatability

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

— 0,2 % in absolute value, for crude protein contents of less than 20 %,

— 1,0 % relative to the higher value, for crude protein contents from 20 % to 40 %,

— 0,4 % in absolute value, for crude protein contents of more than 40 %.

7.2. Accuracy

Carry out the analysis (digestion, distillation and titration) on 1,5 to 2,0 g of acetanilide (3.13) in the presence of 1 g of sucrose (3.14); 1 g acetanilide consumes 14,80 ml of sulphuric acid (3.5). Recovery must be at least 99 %.

8. Observations

8.1. Apparatus may be of the manual, semi-automatic or automatic type. If the apparatus requires transference between the digestion and distillation steps, this transfer must be carried out without loss. If the flask of the distillation apparatus is not fitted with a dropping funnel, add the sodium hydroxide immediately before connecting the flask to the condenser, pouring the liquid slowly down the side.

8.2. If the digest solidifies, recommence the determination using a larger amount of sulphuric acid (3.4) than that specified above.

8.3. For products with a low nitrogen content, the volume of sulphuric acid (3.7) to be placed in the collecting flask may be reduced, if necessary, to 10 or 15 ml and made up to 25 ml with water.

8.4. For routine analysis, alternative methods of analysis can be applied for the determination of crude protein but the Kjeldahl method described in this Part C is the reference method. The equivalence of the results obtained with the alternative method (e.g. DUMAS) compared to the reference method must be demonstrated for each matrix individually. As the results obtained with an alternative method, even after having verified the equivalency, might deviate slightly from the results obtained with the reference method, it is necessary to mention in the analytical report the method of analysis used for the determination of crude protein.

D. DETERMINATION OF UREA

1. Purpose and scope

This method makes it possible to determine the level of urea in feed.

2. Principle

The sample is suspended in water with a clarifying agent. The suspension is filtered. The urea content of the filtrate is determined after the addition of 4-dimethylaminobenzaldehyde (4-DMAB) by measuring the optical density at a wavelength of 420 nm.

3. Reagents

3.1. Solution of 4-dimethylaminobenzaldehyde: dissolve 1,6 g of 4-DMAB in 100 ml of 96 % ethanol and add 10 ml of hydrochloric acid (ρ201,19 g/ml). This reagent keeps for a maximum period of two weeks.

3.2. Carrez solution I: dissolve in water 21,9 g of zinc acetate, Zn(CH3COO)2 2H2O and 3 g of glacial acetic acid. Make up to 100 ml with water.

3.3. Carrez solution II: dissolve in water 10,6 g of potassium ferrocyanide, K4 Fe (CN)6 3H2O. Make up to 100 ml with water.

3.4. Active carbon which does not absorb urea (to be checked).

3.5. Urea, 0,1 % solution (w/v).

4. Apparatus

4.1. Mixer (tumbler): approximately 35 to 40 r.p.m.

4.2. Test tubes: 160 × 16 mm with ground-glass stoppers.

4.3. Spectrophotometer.

5. Procedure

5.1. Analysis of sample

Weigh out 2 g of the sample to the nearest mg and place with 1 g of active carbon (3.4) in a 500 ml volumetric flask. Add 400 ml of water and 5 ml of Carrez solution I (3.2), mix for approximately 30 seconds and add 5 ml of Carrez solution II (3.3). Mix for 30 minutes in the tumbler. Make up to volume with water, shake and filter.

Remove 5 ml of the transparent colourless filtrates, place in test tubes with ground-glass stoppers, add 5 ml of 4-DMAB solution (3.1) and mix. Place the tubes in a water bath at 20 oC (+/- 4 oC). After 15 minutes measure the optical density of the sample solution with the spectrophotometer at 420 nm. Compare with the blank test solution of the reagents.

5.2. Calibration curve

Remove volumes of 1, 2, 4, 5 and 10 ml of the urea solution (3.5), place in 100 ml volumetric flasks and make up the volume with water. Remove 5 ml from each solution, add 5 ml of 4-DMAB solution (3.1) to each of them, homogenise and measure the optical density as shown above in comparison with a control solution containing 5 ml of 4-DMAB and 5 ml of water free from urea. Plot the calibration curve.

6. Calculation of results

Determine the amount of urea in the sample using the calibration curve.

Express the result as a percentage of the sample.

7. Observations

7.1. In the case of contents of urea exceeding 3 %, reduce the sample to 1 g or dilute the original solution so that there are not more than 50 mg of urea in 500 ml.

7.2. In the case of low contents of urea, increase the sample as long as the filtrate remains transparent and colourless.

7.3. If the sample contains simple nitrogenous compounds such as amino acids, the optical density shall be measured at 435 nm.

E. DETERMINATION OF VOLATILE NITROGENOUS BASES

I. BY MICRODIFFUSION

1. Purpose and scope

This method makes it possible to determine the content of volatile nitrogenous bases, expressed as ammonia, in feed.

2. Principle

The sample is extracted with water and the solution clarified and filtered. The volatile nitrogenous bases are displaced by microdiffusion using a solution of potassium carbonate, collected in a solution of boric acid and titrated with sulphuric acid.

3. Reagents

3.1. Trichloroacetic acid, solution 20 % (w/v).

3.2. Indicator: dissolve 33 mg of bromocresol green and 65 mg of methyl red in 100 ml of 95 %-96 % (v/v) of ethanol.

3.3. Boric acid solution: in a 1 litre graduated flask dissolve 10 g of boric acid in 200 ml of 95 %-96 % (v/v) ethanol and 700 ml of water. Add 10 ml of indicator (3.2). Mix and, if necessary, adjust the colour of the solution to light red by adding a solution of sodium hydroxide. 1 ml of this solution will fix a maximum of 300 μg of NH3.

3.4. Saturated potassium carbonate solution: dissolve 100 g of potassium carbonate in 100 ml of boiling water. Leave to cool, filter.

3.5. Sulphuric acid 0,01 mol/litre.

4. Apparatus

4.1. Mixer (tumbler): approximately 35 to 40 r.p.m.

4.2. Glass or plastic Conway cells (see diagram).

4.3. Microburettes graduated in 1/100 ml.

5. Procedure

Weigh 10 g of sample to the nearest 1 mg and place with 100 ml of water in a 200 ml graduated flask. Mix or stir in the tumbler for 30 minutes. Add 50 ml of trichloroacetic acid solution (3.1), make up to volume with water, shake vigorously and filter through a pleated filter.

Using a pipette, introduce 1 ml of boric acid solution (3.3) into the central part of the Conway cell and 1 ml of the sample filtrate into the crown of the cell. Cover partially with the greased lid. Drop 1 ml of saturated potassium carbonate solution (3.4) quickly into the crown and close the lid so that the cell is airtight. Turn the cell carefully rotating it in a horizontal plane so that the two reagents are mixed. Leave to incubate either for at least four hours at room temperature or for one hour at 40 oC.

Using a microburette (4.3), titrate the volatile bases in the boric acid solution with sulphuric acid (3.5).

Carry out a blank test using the same procedure but without a sample to be analysed.

6. Calculation of results

1 ml of H2SO40,01 mol/litre corresponds to 0,34 mg of ammonia.

Express the result as a percentage of the sample.

Repeatability

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

— 10 %, in relative value, for ammonia contents of less than 1,0 %,

— 0,1 %, in absolute value, for ammonia contents of 1,0 % or more.

7. Observation

If the ammonia content of the sample exceeds 0,6 %, dilute the initial filtrate.

CONWAY CELL

Scale 1/1

II. BY DISTILLATION

1. Purpose and Scope

This method makes it possible to determine the content of volatile nitrogenous bases, expressed as ammonia, in fish-meal containing practically no urea. It is applicable only to ammonia contents of less than 0,25 %.

2. Principle

The sample is extracted with water and the solution clarified and filtered. The volatile nitrogenous bases are displaced at boiling point by adding magnesium oxide and collected in a specific quantity of sulphuric acid, the excess of which is back-titrated with a solution of sodium hydroxide.

3. Reagents

3.1. Trichloroacetic acid, solution 20 % (w/v).

3.2. Magnesium oxide.

3.3. Anti-foaming emulsion (e.g. silicone).

3.4. Sulphuric acid 0,05 mol/litre.

3.5. Sodium hydroxide solution 0,1 mol/litre.

3.6. Methyl red solution 0,3 % in 95 %-96 % (v/v) ethanol.

4. Apparatus

4.1. Mixer (tumbler): approximately 35 to 40 r.p.m.

4.2. Distilling apparatus of the Kjeldahl type.

5. Procedure

Weigh 10 g of the sample to the nearest 1 mg and place with 100 ml of water in a 200 ml graduated flask. Mix or stir in the tumbler for 30 minutes. Add 50 ml of trichloroacetic acid solution (3.1), make up to volume with water, shake vigorously and filter through a pleated filter.

Take a quantity of clear filtrate appropriate for the presumed content of volatile nitrogenous bases (100 ml is usually suitable). Dilute to 200 ml and add 2 g of magnesium oxide (3.2) and a few drops of anti-foaming emulsion (3.3). The solution must be alkaline to litmus paper; otherwise add some magnesium oxide (3.2). Proceed according to 5.2 and 5.3 of the method of analysis for the determination of the crude protein content (Part C of this Annex).

Carry out a blank test using the same procedure but without a sample to be analysed.

6. Calculation of results

1 ml of H2SO40,05 mol/litre corresponds to 1,7 mg of ammonia.

Express the result as a percentage of the sample.

Repeatability

The difference between the results of two parallel determinations carried out on the same sample shall not exceed, in relative value, 10 % of ammonia.

F. DETERMINATION OF AMINO ACIDS (EXCEPT TRYPTOPHANE)

1. Purpose and scope

This method makes the determination possible of free (synthetic and natural) and total (peptide bound and free) amino acids in feed, using an amino acid analyser. 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. Co-extracted 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 unoxidised 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 oC with a performic acid/phenol mixture. Excess oxidation reagent is decomposed with sodium disulphite. The oxidised or unoxidised sample is hydrolysed with hydrochloric acid (3.20) 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 < 10 μS).

3.1. Hydrogen peroxide, w (w/w) = 30 %.

3.2. Formic acid, w (w/w) = 98 %-100 %.

3.3. Phenol.

3.4. Sodium disulphite.

3.5. Sodium hydroxide.

3.6. 5-Sulfosalicylic acid dihydrate.

3.7. Hydrochloric acid, density approximately 1,18 g/ml.

3.8. tri-Sodium citrate dihydrate.

3.9. 2,2 '-Thiodiethanol (thiodiglycol).

3.10. Sodium chloride.

3.11. Ninhydrin.

3.12. Light petroleum, boiling range 40-60 oC.

3.13. Norleucine, or other compound suitable for use as internal standard.

3.14. Nitrogen gas (< 10 ppm oxygen).

3.15. 1-Octanol.

3.16. Amino acids.

3.16.1. Standard substances listed under paragraph 1. Pure compounds containing no water of crystallisation. Dry under vacuum over P2O5 or H2SO4 for 1 week prior to use.

3.16.2. Cysteic acid.

3.16.3. Methionine sulphone.

3.17. Sodium hydroxide solution, c = 7,5 mol/l:

Dissolve 300 g NaOH (3.5) in water and make up to 1 litre.

3.18. Sodium hydroxide solution, c = 1 mol/l:

Dissolve 40 g NaOH (3.5) in water and make up to 1 litre.

3.19. Formic acid — phenol solution:

Mix 889 g formic acid (3.2) with 111 g water and add 4,73 g phenol (3.3).

3.20. Hydrolysis mixture, c = 6 mol HCl/l containing 1 g phenol/l:

Add 1 g phenol (3.3) to 492 ml HCl (3.7) and make up to 1 litre with water.

3.21. Extraction mixture, c = 0,1 mol HCl/l containing 2 % thiodiglycol: Take 8,2 ml HCl (3.7), dilute with approximately 900 ml water, add 20 ml thiodiglycol (3.9) and make up to 1 litre with water, (do not mix 3.7 and 3.9 directly).

3.22. 5-Sulfosalicylic acid, ß = 6 %:

Dissolve 60 g 5-sulfosalicylic acid (3.6) in water and make up to 1 l with water.

3.23. Oxidation mixture (Performic acid — phenol):

Mix 0,5 ml hydrogen peroxide (3.1) with 4,5 ml formic acid-phenol solution (3.19) in a small beaker. Incubate at 20-30 oC for 1 hour in order to form performic acid, then cool on an ice-water bath (15 min.) before adding to the sample.

Caution: Avoid contact with skin and wear protective clothing.

3.24. Citrate buffer, c = 0,2 mol Na+/l, pH 2,20 :

Dissolve 19,61 g sodium citrate (3.8), 5 ml thiodiglycol (3.9), 1 g phenol (3.3) and 16,50 ml HCl (3.7) in approximately 800 ml water. Adjust pH to 2,20 . Make up to 1 litre with water.

3.25. Elution buffers, prepared according to conditions for the analyser used (4.9).

3.26. Ninhydrin reagent, prepared according to conditions for the analyser used (4.9).

3.27. Standard solutions of amino acids. These solutions shall be stored below 5 oC.

3.27.1. Stock standard solution of amino acids (3.16.1).

c = 2,5 μmol/ml of each in hydrochloric acid.

May be obtained commercially.

3.27.2. Stock standard solution of cysteic acid and methionine sulphone, c = 1,25 μmol/ml.

Dissolve 0,2115 g cysteic acid (3.16.2) and 0,2265 g methionine sulphone (3.16.3) in citrate buffer (3.24) in a 1 litre graduated flask and make up to mark with citrate buffer. Store below 5 oC for not more than 12 months. This solution is not used if the stock standard solution (3.27.1) contains cysteic acid and methionine sulphone.

3.27.3. Stock standard solution of the internal standard e.g. norleucine, c = 20 μmol/ml.

Dissolve 0,6560 g norleucine (3.13) in citrate buffer (3.24) in a graduated flask and make up to 250 ml with citrate buffer. Store below 5 oC for no more than 6 months.

3.27.4. Calibration solution of standard amino acids for use with hydrolysates, c = 5 nmol/50 μl of cysteic acid and methionine sulphone and c = 10 nmol/50 μl of the other amino acids. Dissolve 2,2 g sodium chloride (3.10) in 100 ml beaker with 30 ml citrate buffer (3.24). Add 4,00 ml stock standard solution of amino acids (3.27.1), 4,00 ml stock standard solution of cysteic acid and methionine sulphone (3.27.2) and 0,50 ml stock standard solution of internal standard (3.27.3) if used. Adjust pH to 2,20 with sodium hydroxide (3.18).

Transfer quantitatively to a 50 ml graduated flask and make up to the mark with citrate buffer (3.24) and mix.

Store below 5 oC for not more than 3 months.

See also observation 9.1.

3.27.5. Calibration solution of standard amino acids for use with hydrolysates prepared according to paragraph 5.3.3.1 and for use with extracts (5.2). The calibration solution is prepared according to 3.27.4 but omitting sodium chloride.

Store below 5 oC for not more than 3 months.

4. Apparatus

4.1. 100 or 250 ml round bottomed flask fitted with a reflux condenser.

4.2. 100 ml borosilicate glass bottle with screw cap with rubber/teflon liner (e.g. Duran, Schott) for use in the oven.

4.3. Oven with forced ventilation and a temperature regulator with an accuracy better than ± 2 oC.

4.4. pH-meter (three decimal places).

4.5. Membrane filter (0,22 μm).

4.6. Centrifuge.

4.7. Rotary vacuum evaporator.

4.8. Mechanical shaker or magnetic stirrer.

4.9. Amino acid analyser or HPLC equipment with ion exchange column, device for ninhydrin, post column derivatisation and photometric detector.

The column is filled with sulfonated polystyrene resins capable of separating the amino acids from each other and from other ninhydrin-positive materials. The flow in the buffer and ninhydrin lines is provided by pumps having a flow stability of ±0,5 % in the period covering both the standard calibration run and the analysis of the sample.

With some amino acid analysers hydrolysis procedures can be used in which the hydrolysate has a sodium concentration of c = 0,8 mol/l and contains all the residual formic acid from the oxidation step. Others do not give a satisfactory separation of certain amino acids if the hydrolysate contains excess formic acid and/or high sodium ion concentrations. In this case the volume of acid is reduced by evaporation to approx. 5 ml after the hydrolysis and prior to pH adjustment. The evaporation shall be performed under vacuum at 40 o C maximum.

5. Procedure

5.1. Preparation of the sample

The sample is ground to pass through a 0,5 mm sieve. Samples high in moisture must be either air-dried at a temperature not exceeding 50 oC or freeze dried prior to grinding. Samples with a high fat content shall be extracted with light petroleum (3.12) prior to grinding.

5.2. Determination of free amino acids in feed and premixtures

Weigh to the nearest 0,2 mg an appropriate amount (1-5 g) of the prepared sample (5.1), into a conical flask and add 100,0 ml of extraction mixture (3.21). Shake the mixture for 60 min. using a mechanical shaker or a magnetic stirrer (4.8). Allow the sediment to settle and pipette 10,0 ml of the supernatant solution into a 100 ml beaker.

Add 5,0 ml of sulfosalicylic acid solution (3.22), with stirring and continue to stir with the aid of magnetic stirrer for 5 min. Filter or centrifuge the supernatant in order to remove any precipitate. Place 10,0 ml of the resulting solution into a 100 ml beaker and adjust the pH to 2,20 using sodium hydroxide solution (3.18), transfer to a volumetric flask of appropriate volume using citrate buffer (3.24), and make up to the mark with the buffer solution (3.24).

If an internal standard is being used add 1,00 ml of internal standard (3.27.3) for each 100 ml final solution and make up to the mark with the buffer solution (3.24).

Proceed to the chromatography step according to paragraph 5.4.

If the extracts are not being examined the same day, they must be stored below 5 oC.

5.3. Determination of total amino acids

5.3.1. Oxidation

Weigh to the nearest 0,2 mg from 0,1 to 1 g of the prepared sample (5.1) into:

— a 100 ml round-bottomed flask (4.1) for open hydrolysis (5.3.2.3) or,

— a 250 ml round-bottomed flask (4.1) if a low sodium concentration is required (5.3.3.1) or,

— a 100 ml bottle fitted with a screw cap (4.2), for closed hydrolysis (5.3.2.4).

The weighed sample portion must have a nitrogen content of about 10 mg and a moisture content not exceeding 100 mg.

Place the flask/bottle in an ice-water bath and cool to 0 oC, add 5 ml of oxidation mixture (3.23) and mix using a glass spatula with a bent tip. Seal the flask/bottle containing the spatula with an air-tight film, place the ice-water bath containing the sealed container in a refrigerator at 0 oC and leave for 16 hours. After 16 hours remove from the refrigerator and decompose the excess oxidation reagent by the addition of 0,84 g of sodium disulphite (3.4).

Proceed to 5.3.2.1.

5.3.2. Hydrolysis

5.3.2.1. Hydrolysis of oxidised samples

To the oxidised sample prepared according to 5.3.1 add 25 ml of hydrolysis mixture (3.20) taking care to wash down any sample residue adhering to the sides of the vessel and the spatula.

Depending on the hydrolysis procedure being used, proceed according to 5.3.2.3 or 5.3.2.4.

5.3.2.2. Hydrolysis of unoxidised samples

Weigh into either a 100 ml or a 250 ml round-bottom flask (4.1) or a 100 ml bottle fitted with a screw cap (4.2), to the nearest 0,2 mg, from 0,1 to 1 g of the prepared sample (5.1). The weighed sample portion must have a nitrogen content of about 10 mg. Add carefully 25 ml of hydrolysis mixture (3.20) and mix with the sample. Proceed according to either 5.3.2.3 or 5.3.2.4.

5.3.2.3. Open hydrolysis

Add 3 glass beads to the mixture in the flask (prepared in accordance with 5.3.2.1 or 5.3.2.2) and boil with continuous bubbling under reflux for 23 hours. On completion of hydrolysis, wash the condenser down with 5 ml of citrate buffer (3.24). Disconnect the flask and cool it in an ice bath.

Proceed according to 5.3.3.

5.3.2.4. Closed Hydrolysis

Place the bottle containing the mixture prepared in accordance with 5.3.2.1 or 5.3.2.2 in an oven (4.3) at 110 oC. During the first hour in order to prevent a build up of pressure (due to the evolution of gaseous substances) and to avoid explosion, place the screw cap over the top of the vessel. Do not close the vessel with the cap. After one hour close the vessel with the cap and leave in the oven (4.3) for 23 hours. On completion of hydrolysis, remove the bottle from the oven, carefully open the cap of the bottle and place the bottle in an ice-water bath. Leave to cool.

Depending on the procedure for pH adjustment (5.3.3), quantitatively transfer the contents of the bottle to a 250 ml beaker or a 250 ml round-bottom flask, using citrate buffer (3.24).

Proceed according to 5.3.3.

5.3.3. Adjustment of pH

Depending on the sodium tolerance of the amino acid analyser (4.9) proceed according to 5.3.3.1 or 5.3.3.2 for the pH adjustment.

5.3.3.1. For Chromatographic Systems (4.9) requiring a low sodium concentration

It is advisable to use an internal stock standard solution (3.27.3) when amino acid analysers requiring a low sodium concentration are employed (when the acid volume has to be reduced).

In this case add 2,00 ml of the internal stock standard solution (3.27.3) to the hydrolysate before the evaporation.

Add 2 drops of 1-octanol (3.15) to the hydrolysate obtained in accordance with paragraph 5.3.2.3 or 5.3.2.4.

Using a rotary evaporator (4.7) reduce the volume to 5-10 ml under vacuum at 40 oC. If the volume is accidentally reduced to less than 5 ml the hydrolysate must be discarded and the analysis recommenced.

Adjust the pH to 2,20 with sodium hydroxide solution (3.18) and proceed to paragraph 5.3.4.

5.3.3.2. For all other Amino Acid Analysers (4.9)

Take the hydrolysates obtained in accordance with 5.3.2.3 or 5.3.2.4 and partly neutralise them by carefully adding with stirring, 17 ml of sodium hydroxide solution (3.17), ensuring that the temperature is kept below 40 oC.

Adjust the pH to 2,20 at room temperature using sodium hydroxide solution (3.17) and finally sodium hydroxide solution (3.18). Proceed to 5.3.4.

5.3.4. Sample solution for chromatography

Quantitatively transfer the pH adjusted hydrolysate (5.3.3.1 or 5.3.3.2) with citrate buffer (3.24) to a 200 ml graduated flask, and make up to the mark with buffer (3.24).

If an internal standard has not already been used, add 2,00 ml of internal standard (3.27.3) and make up to the mark with citrate buffer (3.24). Mix thoroughly.

Proceed to the chromatography step (5.4).

If the sample solutions are not being examined the same day they must be stored below 5 oC.

5.4. Chromatography

Before chromatography bring the extract (5.2) or hydrolysate (5.3.4) to room temperature. Shake the mixture and filter a suitable amount through a 0,22 μm membrane filter (4.5). The resulting clear solution is subjected to ion exchange chromatography, using an amino acid analyser (4.9).

The injection may be performed manually or automatically. It is important that the same quantity of solution ± 0,5 % is added to the column for the analysis of standards and samples except when an internal standard is used, and that the sodium:amino acid ratios in the standard and sample solutions are as similar as is practicable.

In general the frequency of calibration runs depends on the stability of the ninhydrin reagent and the analytical system. The standard or sample is diluted with citrate buffer (3.24) to give a peak area of the standard of 30 %-200 % of the sample amino acid peak area.

The chromatography of amino acids will vary slightly according to the type of analyser employed and resin used. The chosen system must be capable of separating the amino acids from each other and from the ninhydrin-positive materials. In the range of operation the chromatographic system must give a linear response to changes in the amounts of amino acids added to the column.

During the chromatography step the valley:peak height ratios mentioned below apply, when an equimolar solution (of the amino acids being determined) is analysed. This equimolar solution must contain at least 30 % of the maximum load of each amino acid which can be accurately measured with the amino acid analyser system (4.9).

For separation of threonine-serine the valley:peak height ratio of the lower of the two overlapping amino acids on the chromatogram must not exceed 2:10. (if only cyst(e)ine, methionine, threonine and lysine are determined, insufficient separation from adjoining peaks will adversely influence the determination). For all other amino acids the separation must be better than 1:10.

The system must ensure that lysine is separated from ‘lysine artifacts’ and ornithine.

6. Calculation of results

The area of the sample and standard peaks is measured for each individual amino acid and the amount (X), in g amino acid per kg sample, is calculated as follows:

If an internal standard is used multiply by:

A = peak area, hydrolysate or extract

B = peak area, calibration standard solution

C = peak area, internal standard in hydrolysate or extract

D = peak area, internal standard, calibration standard solution

M = molar weight of the amino acid being determined

c = concentration of standard in μmol/ml

m = sample weight (g) (corrected to original weight if dried or defatted)

V = ml total hydrolysate (5.3.4) or ml calculated total dilution volume of extract (6.1)

Cystine and cysteine are both determined as cysteic acid in hydrolysates of oxidised sample, but calculated as cystine (C6H12N2O4S2, M 240,30 g/mol) by using M 120,15 g/mol (= 0,5 x 240,30 g/mol).

Methionine is determined as methionine sulphone in hydrolysates of oxidised sample, but calculated as methionine by using M of methionine: 149,21 g/mol.

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

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

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 feed (mixed pig feed, broiler compound, protein concentrate, premixture). The results, after elimination of outliers, of mean and standard deviation are given in the tables in this point:







Means in g/kg Reference material Amino Acid Threonine Cyst(e)ine Methionine Lysine Mixed Pig Feed 6,94 n = 15 3,01 n = 17 3,27 n = 17 9,55 n = 13 Broiler Compound 9,31 n = 16 3,92 n = 18 5,08 n = 18 13,93 n = 16 Protein Concentrate 22,32 n = 16 5,06 n = 17 12,01 n = 17 47,74 n = 15 Premixture 58,42 N = 16 — 90,21 n = 16 98,03 n = 16 n = Number of participating laboratories.

7.1. Repeatability

The repeatability expressed as ‘within laboratory standard deviation’ of the abovementioned intercomparison is given in the tables below:







Within Laboratory Standard Deviation (Sr) in g/kg Reference material Amino Acid Threonine Cyst(e)ine Methionine Lysine Mixed Pig Feed 0,13 n = 15 0,10 n = 17 0,11 n = 17 0,26 n = 13 Broiler Compound 0,20 n = 16 0,11 n = 18 0,16 n = 18 0,28 n = 16 Protein Concentrate 0,48 n = 16 0,13 n = 17 0,27 n = 17 0,99 n = 15 Premixture 1,30 N = 16 — 2,19 n = 16 2,06 n = 16 n = Number of participating laboratories.







Coefficient of Variation (%) for Within Laboratory Standard Deviation (Sr) Reference material Amino Acid Threonine Cyst(e)ine Methionine Lysine Mixed Pig Feed 1,9 n = 15 3,3 n = 17 3,4 n = 17 2,8 n = 13 Broiler Compound 2,1 n = 16 2,8 n = 18 3,1 n = 18 2,1 n = 16 Protein Concentrate 2,7 n = 16 2,6 n = 17 2,2 n = 17 2,4 n = 15 Premixture 2,2 n = 16 — 2,4 n = 16 2,1 n = 16 n = Number of participating laboratories.

7.2 Reproducibility

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







Between Laboratory Standard Deviation (SR) in g/kg Reference material Amino Acid Threonine Cyst(e)ine Methionine Lysine Mixed Pig Feed 0,28 n = 15 0,30 n = 17 0,23 n = 17 0,30 n = 13 Broiler Compound 0,48 n = 16 0,34 n = 18 0,55 n = 18 0,75 n = 16 Protein Concentrate 0,85 n = 16 0,62 n = 17 1,57 n = 17 1,24 n = 15 Premixture 2,49 n = 16 — 6,20 n = 16 6,62 n = 16 n = Number of participating laboratories.







Coefficient of Variation (%) for Between Laboratory Standard Deviation (SR) Reference material Amino Acid Threonine Cyst(e)ine Methionine Lysine Mixed Pig Feed 4,1 n = 15 9,9 n = 17 7,0 n = 17 3,2 n = 13 Broiler Compound 5,2 n = 16 8,8 n = 18 10,9 n = 18 5,4 n = 16 Protein Concentrate 3,8 n = 16 12,3 n = 17 13,0 n = 17 3,0 n = 15 Premixture 4,3 n = 16 — 6,9 n = 16 6,7 n = 16 n = Number of participating laboratories.

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) shall 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 feed containing more than 1 % chloride (concentrate, mineral feeds, supplementary feeds) underestimation of methionine could occur and special treatment has to be done.

G. DETERMINATION OF TRYPTOPHAN

1. Purpose and scope

The method makes the determination possible of the total and free tryptophan in feed. It does not distinguish between D- and L- forms.

2. Principle

For the determination of the total tryptophan, the sample is hydrolysed under alkaline conditions with saturated barium hydroxide solution and heated to 110 oC for 20 hours. After hydrolysis internal standard is added.

For the determination of free tryptophan, the sample is extracted under mild acidic conditions in the presence of internal standard.

The tryptophan and the internal standard in the hydrolysate or in the extract are determined by HPLC with fluorescence detection.

3. Reagents

3.1. Double distilled water or water of equivalent quality must be used (conductivity < 10 μS/cm).

3.2. Standard substance: tryptophan (purity/content ≥ 99 %) dried under vacuum over phosphorous pentoxide.

3.3. Internal standard substance: α-methyl-tryptophan (purity/content ≥ 99 %), dried under vacuum over phosphorous pentoxide.

3.4. Barium hydroxide octa-hydrate (care shall be taken not to expose the Ba(OH)2 .8 H2O excessively to air in order to avoid formation of BaCO3, which could disturb the determination) (see observation 9.3).

3.5. Sodium hydroxide.

3.6. Ortho-phosphoric acid, w (w/w) = 85 %.

3.7. Hydrochloric acid, ρ201,19 g/ml.

3.8. Methanol, equivalent to HPLC grade.

3.9. Light petroleum, boiling range 40-60 oC.

3.10. Sodium hydroxide solution, c = 1 mol/l:

Dissolve 40,0 g NaOH (3.5) in water and make up to 1 litre with water (3.1).

3.11. Hydrochloric acid, c = 6 mol/l:

Take 492 ml HCl (3.7) and make up to 1 litre with water.

3.12. Hydrochloric acid, c = 1 mol/l:

Take 82 ml HCl (3.7) and make up to 1 litre with water.

3.13. Hydrochloric acid, c = 0,1 mol/l:

Take 8,2 ml HCl (3.7) and make up to 1 litre with water.

3.14. Ortho-phosphoric acid, c = 0,5 mol/l:

Take 34 ml ortho-phosphoric acid (3.6) and make up to 1 litre with water (3.1).

3.15. Concentrated solution of tryptophan (3.2), c = 2,50 μmol/ml:

In a 500 ml volumetric flask dissolve 0,2553 g tryptophan (3.2) in hydrochloric acid (3.13) and make up to the mark with hydrochloric acid (3.13). Store at - 18 oC for a maximum of 4 weeks.

3.16. Concentrated internal standard solution, c = 2,50 μmol/ml:

In a 500 ml volumetric flask dissolve 0,2728 g α-methyl-tryptophan (3.3) in hydrochloric acid (3.13) and make up to the mark with hydrochloric acid (3.13). Store at - 18 oC for a maximum of 4 weeks.

3.17. Calibration standard solution of tryptophan and internal standard:

Take 2,00 ml concentrated solution of tryptophan (3.15), and 2,00 ml of concentrated internal standard (α-methyl-tryptophan) solution (3.16). Dilute with water (3.1) and methanol (3.8) to approximately the same volume and to approximately the same concentration of methanol (10 %-30 %) as the finished hydrolysate.

This solution must be prepared freshly before use.

Protect from direct sunlight during preparation.

3.18. Acetic acid

3.19. 1,1,1-trichloro-2-methyl-2-propanol.

3.20. Ethanolamine w (w/w) > 98 %.

3.21. Solution of 1 g 1,1,1-trichloro-2-methyl-2-propanol (3.19) in 100 ml methanol (3.8).

3.22. Mobile phase for HPLC: 3,00 g acetic acid (3.18) + 900 ml water (3.1) +50,0 ml solution (3.21) of 1,1,1-trichloro-2-methyl-2-propanol (3.19) in methanol (3.8) (1g/100ml). Adjust pH to 5,00 using ethanolamine (3.20). Make up to 1 000 ml with water (3.1).

4. Apparatus

4.1. HPLC equipment with a spectrofluorometric detector.

4.2. Liquid chromatographic column, 125 mm x 4 mm, C18, 3 μm packing, or equivalent.

4.3. pH-meter.

4.4. Polypropylene flask, capacity 125 ml, with wide neck and screw cap.

4.5. Membrane filter, 0,45 μm.

4.6. Autoclave, 110 (± 2) oC, 1,4 (±0,1 ) bar.

4.7. Mechanical shaker or magnetic stirrer.

4.8. Vortex mixer.

5. Procedure

5.1. Preparation of samples

The sample is ground to pass through a 0,5 mm sieve. Samples high in moisture must be either air-dried at a temperature not exceeding 50 oC or freeze dried prior to grinding. Samples with high fat content shall be extracted with light petroleum (3.9) prior to grinding.

5.2. Determination of free tryptophan (extract)

Weigh to the nearest 1 mg an appropriate amount (1-5 g) of the prepared sample (5.1), into a conical flask. Add 100,0 ml hydrochloric acid, (3.13) and 5,00 ml concentrated internal standard solution (3.16). Shake or mix for 60 min. using a mechanical shaker or a magnetic stirrer (4.7). Allow the sediment to settle and pipette 10,0 ml of the supernatant solution into a beaker. Add 5 ml ortho-phosphoric acid (3.14). Adjust the pH to 3 using sodium hydroxide (3.10). Add sufficient methanol (3.8) to give a concentration of between 10 % and 30 % of methanol in the final volume. Transfer to a volumetric flask of appropriate volume and dilute with water to a volume necessary for the chromatography (approx. the same volume as the calibration standard solution (3.17)).

Filter a few ml of the solution through a 0,45 μm membrane filter (4.5) before injection on the HPLC column. Proceed to the chromatography step according to paragraph 5.4.

Protect standard solution and extracts against direct sunlight. If it is not possible to analyse the extracts the same day, the extracts may be stored at 5 oC for a maximum of 3 days.

5.3. Determination of total tryptophan (hydrolysate)

Weigh to the nearest 0,2 mg from 0,1 to 1 g of the prepared sample (5.1) into the polypropylene flask (4.4). The weighed sample portion shall have a nitrogen content of about 10 mg. Add 8,4 g barium hydroxide octa-hydrate (3.4) and 10 ml water. Mix on a vortex mixer (4.8) or magnetic stirrer (4.7). Leave the teflon coated magnet in the mixture. Wash down the walls of the vessel with 4 ml water. Put on the screw cap and close the flask loosely. Transfer to an autoclave (4.6) with boiling water and steam for 30-60 minutes. Close the autoclave and autoclave at 110 (± 2) oC for 20 hours.

Before opening the autoclave reduce the temperature to just under 100 oC. In order to avoid crystallisation of Ba(OH)2 · 8 H2O, add to the warm mixture 30 ml water which is at room temperature. Shake or stir gently. Add 2,00 ml concentrated internal standard (α-methyl-tryptophan) solution (3.16). Cool the vessels on water/ice bath for 15 minutes.

Then, add 5 ml ortho-phosphoric acid (3.14). Keep the vessel in the cooling bath and neutralise with HCl (3.11) whilst stirring and adjust the pH to 3,0 using HCl (3.12). Add sufficient methanol to give a concentration of between 10 % and 30 % of methanol in the final volume. Transfer to a volumetric flask of appropriate volume and dilute with water to the defined volume necessary for the chromatography (for example 100 ml). The addition of methanol shall not cause precipitation.

Filter a few ml of the solution through a 0,45 μm membrane filter (4.5) before injection on the HPLC column. Proceed to the chromatography step according to paragraph 5.4.

Protect standard solution and hydrolysates against direct sunlight. If it is not possible to analyse the hydrolysates the same day, they may be stored at 5 oC for a maximum of 3 days.

5.4. HPLC determination

The following conditions for isocratic elution are offered for guidance; other conditions may be used, provided they yield equivalent results (see also observations 9.1 and 9.2):







Liquid chromatographic column (4.2): 125 mm x 4 mm, C18, 3 μm packing or equivalent Column temperature: Room temperature Mobile phase (3.22): 3,00 g acetic acid (3.18) + 900 ml water (3.1) +50,0 ml solution (3.21) of 1,1,1-trichloro-2- methyl-2-propanol (3.19) in methanol (3.8) (1 g/100 ml). Adjust pH to 5,00 using ethanolamine (3.20). Make up to 1 000 ml with water (3.1) Flow rate: 1 ml/min. Total run time: approx. 34 min. Detection wavelength: excitation: 280 nm, emission: 356 nm. Injection volume 20 μl

6. Calculation of results

The amount of tryptophane (X), in g per 100g sample, is calculated as follows:

A = peak area of internal standard, calibration standard solution (3.17)

B = peak area of tryptophan, extract (5.2) or hydrolysate (5.3)

V1 = volume in ml (2 ml) of concentrated tryptophan solution (3.15) added to the calibration solution (3.17)

c = concentration in μmol/ml (= 2,50 ) of concentrated tryptophan solution (3.15) added to calibration solution (3.17)

V2 = volume in ml of concentrated internal standard solution (3.16) added at the extraction (5.2) (= 5,00 ml) or to the hydrolysate (5.3) (= 2,00 ml)

C = peak area of internal standard, extract (5.2) or hydrolysate (5.3)

D = peak area of tryptophan, calibration standard solution (3.17)

V3 = volume in ml (= 2,00 ml) of concentrated internal standard solution (3.16) added to calibration standard solution (3.17)

m = sample weight in g (corrected to original weight if dried and/or defatted)

M = molar weight of tryptophan (= 204,23 g/mol)

7. Repeatability

The difference between the results of two parallel determinations carried out on the same sample must not exceed 10 % relative to the highest result.

8. Results of a collaborative study

An EC collaborative study (4th intercomparison) was arranged in which three samples were analysed by up to 12 laboratories to certify the method for hydrolysis. Replicate (5) analyses were performed on each sample. The results are given in the following table:







Sample 1 Pig feed Sample 2 Pig feed supplemented with L-tryptophan Sample 3 Feed concentrate f