‘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 or based on other relevant information, 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 the EU requirements and 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.

Sampling may also include feed offered for sale by feed business operators by means of distance communication in accordance with Article 11(3) of Regulation (EC) No 767/2009 of the European Parliament and of the Council (1). Sampling of feed offered for sale by means of distance communication shall in principle be subject to the points set out in this Annex. Specific aspects of the sampling of distance selling samples are described in point 11.

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 aggregate sample (mixed), of the reduced sample or of the homogenised aggregate sample, depending on the type of control (see point 9.4).

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

—	Distance selling sample: Sample of a lot or batch of feed offered for sale by means of distance communication.

3.   GENERAL PROVISIONS

—	The samples shall be taken by persons authorised for that purpose by the competent authority.

—	For a distance selling sample, a quantity of the feed shall be requested from the feed business operator by the competent authority by means of distance communication.

—	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.

—	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 or reduced sample, the following final samples are taken: one for control (enforcement) and one for the feed business operator (defence sample) must be taken. Eventually, one other final sample may be taken for reference. In case the complete aggregate sample is homogenised, the final samples are taken from the homogenised aggregate sample, unless such procedure conflicts with Member States’ rules as regards the right of the feed business operator.

—

In accordance with Article 15, paragraphs 1 and 2, of Regulation (EU) 2017/625, when it is necessary for the performance of official sampling, feed business operators shall, where required by the competent authorities: — give the staff of the competent authorities access to the equipment under their control, including, when necessary, making available the proper sampling equipment and personal protective equipment, — assist and cooperate with the staff of the competent authorities in order to enable the sampling, including making feed accessible to the staff of the competent authorities.

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 sampling 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. The mechanical apparatus shall be considered appropriate when 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 THE 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 following tables under points 5.1.1, 5.1.3 and 5.1.5 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 point, the sampling procedure as provided for in point 5.3 is to be applied.

—	For distance selling samples, the size of the lot from which the quantity is requested is usually not known by the competent authority. Therefore, the procedure referred to in points 5.1 and 5.2 cannot be used. In this case, the procedure described in point 11 shall 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 point 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

5.1.2.   Loose liquid feed

Size of sampled portion	Minimum number of incremental samples
≤ 2,5 tonnes or ≤ 2 500 litres	4  (*2)
> 2,5 tonnes or > 2 500 litres	7  (*2)

5.1.3.   Packaged feed

Feed (solid and liquid) can be packaged in bags, sacks, cans, barrels, etc. which are referred to in the following table as units. Large units (≥ 500 kg or litres) have to be sampled in accordance with the provisions foreseen for loose feed (see 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 (*3)
1 to 20 units	1 unit (*4)
21 to 150 units	3 units (*4)
151 to 400 units	5 units (*4)
> 400 units	¼ of the √ (number of units making up the sampled portion) (*5), up to 40 units

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 (*6)
≤ 5 tonnes	5
> 5 tonnes	√(5 times the number of tonnes making up the sampled portion) (*7), up to 40 incremental samples

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.

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 following table can be applied.

Size of sampled portion	Minimum number of incremental samples
< 80 tonnes	See quantitative requirements under 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.

6.   QUANTITATIVE REQUIREMENTS AS REGARDS AGGREGATE SAMPLE

A single aggregate sample per sampled portion is required.

	Nature of feed	Minimum size of aggregate sample (*8)  (*9)
6.1.	Loose feed	4 kg
6.2.	Packaged feed:	4 kg (*10)
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 (*11)

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 (*12)  (*13)  (*14)  (*15)
Liquid or semi-liquid feed	500 ml (*12)

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 point, 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 the operator’s 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 of part (sampled portion) of the whole lot is allowed to be undertaken. 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 provided 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, surfaces and containers intended to receive samples must be clean and dry.

9.2.   Incremental samples

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

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

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.

In case of distance selling samples, the size of the incremental sample depends on the size of the unit and may also contain less than 100 g or 100 ml in individual cases.

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 point 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 point 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 point 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 point 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 (2).

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.

9.4.1.   Uniformly distributed substances

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) (3) 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 aggregate sample or the reduced samples the final samples (for control, defence and possibly reference) shall then be taken of approximately the same amount and conforming to the quantitative requirements of point 7.

9.4.2.   Non-uniformly distributed substances

In case of the control of constituents, including genetically modified material, or substances likely to be distributed non-uniformly in feed, the aggregate sample shall be:

(i)	completely homogenised. Afterwards from the homogenised aggregate sample the final samples (for control, defence and possibly reference) shall then be taken of approximately the same amount and conforming to the quantitative requirements of point 7; or

(ii)

reduced to at least 2 kg or 2 litres (4) 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 possibly reference of at least 10 000 seeds grain (see footnote (**) in point 6 and footnote (*) in point 7).

From the reduced sample the final samples shall then be taken of approximately the same amount and conforming to the quantitative requirements of point 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. 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.

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.

11.   DISTANCE SELLING SAMPLE

—

For a distance selling sample, the feed shall be requested from the feed business operator by means of distance communication techniques. In this case, when requesting feed, the competent authority does not have to identify itself with an official identity to the feed business operator and may use a cover identity.

—

The aggregate sample and the final samples of the distance selling sample have to be taken immediately upon receipt of the consignment by persons authorised for this purpose. For generating the aggregate sample, an appropriate number of incremental samples have to be taken randomly and evenly distributed from the total quantity obtained and carefully mixed/homogenised, in accordance, as far as possible, with the principles laid down in point 5 and points 9.2 and 9.3. If the feed is packaged in individual units, at least 4 units should be obtained from which at least one incremental sample has to be taken. Should it be shown on a case-by-case basis that the units obtained come from different lots, the number of units to be sampled has to be reduced and limited to those units originating from the same lot. In case of analysing the distance selling sample for constituents or substances which are non-uniformly distributed in feed, the number of incremental samples has to be at least 2,5 times higher than that for samples analysed for substances uniformly distributed throughout the feed. From the aggregate sample, the corresponding final samples (for control, for defence and possibly reference) are then taken in accordance, as far as feasible, with the principles laid down in point 9.4 and the sampling record indicates that the sample is a distance selling sample. The competent authority then immediately informs the feed business operator of the sampling. The feed business operator is also notified that one sample (for defence) is kept, when possible, at their disposal by the competent authority, in a specified location, for defence purposes or sent to the feed business operator or sent to the laboratory designated by the feed business operator in accordance with the national rules in place. If the sample is sent directly to the official laboratory, the final sample must be prepared and sealed in the laboratory by persons authorised for this purpose or in the presence of persons authorised for this purpose. The sampling record of the distance selling sample has to be sent immediately after the final samples have been formed to the competent authority, which informs the feed business operator of the sampling. It is considered that the quantity supplied by the feed business operator to the competent authority represents a part of a lot of feed of the same class or description. In accordance with Article 15 of Regulation (EC) No 178/2002 of the European Parliament and of the Council (5), if that part of the lot has been identified as not satisfying EU requirements, it shall be presumed, also in the case of a distance selling sample, that all of the feed in that lot is so affected, unless following a detailed assessment (where appropriate in an on-the-spot inspection) there is no evidence that the rest of the lot fails to satisfy the EU requirements.

‘ANNEX II

GENERAL PROVISIONS ON METHODS OF ANALYSIS FOR FEED

A.   PREPARATION OF SAMPLES FOR ANALYSIS

1.   Purpose

The procedures described in this Annex 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.

In addition to the procedures described in this Annex, the guidelines for sample preparation as provided for by EN ISO 6498 shall be followed.

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 that the followed sample preparation procedure be 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.

Homogenisation of the sample by preparing a slurry by high shear mixing with water has proven to provide in certain cases more homogeneous sub-samples than dry homogenisation/grinding, in particular in case of heterogeneously distributed chemical substances. However also homogenisation by sufficient dry grinding might provide homogeneous subsamples.

In certain cases, such as for the determination of rye ergot, harmful botanical impurities, etc., the homogenisation of the sample cannot be done by grinding but by sufficiently mixing the sample.

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 homogenised 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 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 point 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 aggregate or final 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 possibly 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 homogenised, the final samples are taken from the homogenised aggregate sample.

—

For the determination of rye ergot and harmful botanical impurities, the final sample has to be divided into 2 subsamples of equal weight of approximately 500 grams. One subsample is examined. In case the result of the subsamples is equal or below 50 % (analytical threshold) of the maximum level, the sample is compliant with the maximum level. If the result is above 50 % of the maximum level, another subsample needs to be examined and the average of the result of the 2 subsamples is used for checking compliance with the maximum level.

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, is used for further assessment.

In case of the control of minimum or maximum levels of feed additives, if the results of the first determination is above the minimum level or below the maximum level 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 is used for further assessment.

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, is used for further assessment (the average analytical result falls or not within the acceptable range of variation of the declared content).

In some cases this acceptable range of variation is defined in legislation such as in Regulation (EC) No 767/2009 and Regulation (EU) 2019/4 of the European Parliament and of the Council (1).

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.

Most regulatory levels (e.g. maximum level, minimum level) in EU animal feed legislation are established relative to a feed with a moisture content of 12 %. Therefore, in these cases, in order to assess the analytical result measured on the sample against the regulatory level, the analytical result first needs to be divided by the dry matter content of the sample (in %) multiplied by 88, as indicated in the following formula:

where:

Mc	:	moisture content of the sample (in %). 100 – Mc therefore represents the dry matter content of the sample (in %).
Rana	:	analytical result as measured on the sample.
R12 %	:	result for a feed with a moisture content of 12 %; to be assessed against the regulatory level.

In addition, if the following conditions are met:

—	the result of the analysis is significantly (> 50 %) lower or higher than the labelling information/specification to be controlled (depending on whether the labelling information/specification is a maximum or a minimum level),

—

the moisture content of the sampled feed is known and it can be determined that correction to the moisture content will not change the assessment, then, on the condition that the appropriate quality procedures are applied and the analysis serves only the purpose of checking compliance with legal provisions, the correction to the moisture content might be omitted (e.g. in cases there is no specification or regulatory level), unless it is required for interpretation.

If the analytical result is corrected to the moisture content, the corresponding measurement uncertainty must also be corrected in the same procedure.

In case of the determination of rye ergot or harmful botanical impurities by visual/microscopic examination correction to the moisture content is not necessary.

6.   Analytical 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 as a mean of two independent determinations, relative to a feed with a moisture content of 12 %, is deemed to exceed the maximum content taking into account expanded analytical measurement uncertainty using a coverage factor of 2 which gives a level of confidence of approximately 95 % and correction for recovery. This means, in order to assess compliance, the analysed concentration is used after being corrected for recovery and after deduction of the expanded analytical measurement uncertainty. This procedure is only applicable in cases where the method of analysis enables the estimation of the expanded analytical measurement uncertainty and correction for recovery (e.g. not required in case of visual/microscopic examination).

If the analytical result of the sample taken for defence exceeds the maximum content (without taking into account the expanded analytical measurement uncertainty), this confirms the non-compliance established with the control sample, in the absence of specific national rules on this.

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

(a)

corrected for recovery, where appropriate and relevant, and when corrected it has to be so stated. The recovery rate is to be quoted unless intrinsic correction for bias is part of the procedure, whereby bias is the difference between the measured value and the reference concentration. 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 analytical measurement uncertainty, using a coverage factor of 2 (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 reporting of the recovery rate and expanded analytical measurement uncertainty might be omitted (e.g. in cases there is no specification or regulatory level), unless the measurement uncertainty is required for interpretation.

7.   Analytical measurement uncertainty and recovery rate in case of analysis of content of feed additives

In order to check compliance with authorised minimum and maximum content of feed additives, the presence of a feed additive shall be considered as non-compliant with the established minimum and maximum content, if the analytical result as mean of two independent determinations, relative to a feed with a moisture content of 12 %, is deemed to:

—

exceed the maximum content taking into account expanded analytical measurement uncertainty and correction for recovery. This means, in order to assess compliance, the analysed concentration (i.e. mean of two determinations) is used after being corrected for recovery and after deduction of the expanded analytical measurement uncertainty,

—

be lower than the minimum content taking into account the expanded analytical measurement uncertainty and correction for recovery. This means, in order to assess compliance, the analysed concentration (i.e. mean of two determinations) is used after being corrected for recovery and after the addition of the expanded analytical measurement uncertainty.

If the analytical result of the sample taken for defence exceeds the maximum content (without taking into account the expanded analytical measurement uncertainty), this confirms the non-compliance established with the control sample, in the absence of specific national rules on this.

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

(a)

corrected for recovery, where appropriate and relevant, and when corrected it has to be so stated. The recovery rate is to be quoted unless intrinsic correction for bias is part of the procedure, whereby bias is the difference between the measured value and the reference concentration. 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 (mean of two determinations) and U is the expanded analytical measurement uncertainty, using a coverage factor of 2 (3) which gives a level of confidence of approximately 95 %.

‘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 a significant number of volatile substances is determined together with the moisture content.

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

The determination of moisture content in oilseeds is to be determined by the method as provided for by EN ISO 665 Determination of moisture and volatile matter content, with the understanding that soybeans have to be ground before determination of moisture content.

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 °C) properly ventilated and ensuring rapid temperature regulation (1).

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

NB:	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 point 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

Dry a container (point 3.3) with its lid in the oven set at 103 °C for 30 min +/- 1 min. Remove from the oven and allow to cool to ambient temperature in the desiccator (point 3.6).

4.2.1.   Feed other than those coming under points 4.2.2 and 4.2.3

Weigh the container 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 °C. 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 °C. Open the oven, replace the lid on the container immediately, remove the latter from the oven, leave to cool for 30 to 45 minutes in the desiccator (point 3.6) and weigh to the nearest 1 mg.

For feed composed predominantly (> 50 %) of oils and fats of animal and plant origin, dry in the oven for an additional 30 minutes at 103 °C. The difference between the two weighings must not exceed 0,1 % of moisture.

4.2.2.   Cereals, flour, groats and meal

Weigh the container 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 °C. 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 °C. Open the oven, replace the lid on the container immediately, remove the latter from the oven, leave to cool for 30 to 45 minutes in the desiccator (point 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

Weigh the container 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 (point 3.5) preheated to between 80 °C and 85 °C. 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 °C to 85 °C. Carefully bring the oven back to atmospheric pressure. Open the oven, replace the lid on the container immediately, remove the container from the oven, leave to cool for 30 to 45 minutes in the desiccator (point 3.6) and weigh to the nearest 1 mg. Dry for an additional 30 minutes in the vacuum oven at 80 °C to 85 °C and reweigh. The difference between the two weighings must not exceed 0,1 % of moisture.

4.3.   Preliminary (partial) drying

It is necessary to partially dry “wet” feeds with a mass fraction of less than 85 % dry matter (e.g. forages, total mixed rations, (non)-liquid feed) prior to fine grinding in order to analyse their stable substances; for unstable substances, partial drying is not possible.

Partial drying can be performed using either a forced-air oven or a microwave oven or by freeze drying. With the exception of partial drying by freeze drying, the aim is to dry the feed while keeping sample temperature below 60 °C so that chemical composition is minimally affected. Drying at temperatures greater than 60 °C causes chemical changes in the feed (e.g. protein degradation). The dried feed shall be equilibrated at room temperature for about 15 minutes before measuring partial dry matter so as to minimise the potential change in moisture that can occur during grinding and storage. Drying at temperatures lower than 60 °C does not remove all of the water from the feed; therefore, (initial) partial drying does not represent the total dry matter of the feed. Following drying, the subsample is ground and analysed for (final) dry matter of the partially dry sample (the remaining 3 % to 15 % moisture) when other chemical constituents are determined.

Therefore, a two-step procedure for determining dry matter is recommended. First determine the partial dry matter content (if less than 85 % dry matter), then determine the remaining dry matter content on a ground test sample and multiply partial dry matter by the remaining dry matter to determine the total dry matter content.

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 (2)

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, except for wet pet food and dog chews, where the difference shall not exceed 0,5 % 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 °C. 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 °C to at least 110 °C, 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 (point 3.1) containing the thermometer (point 3.2). Heat on the sand bath or hot-plate (point 3.3), stirring continuously with the thermometer, so that the temperature reaches 90 °C 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 °C. 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 °C ± 2 °C, cooling to 93 °C between successive heatings. Then leave to cool to room temperature in the desiccator (point 3.4) and weigh. Repeat this operation until the loss in weight between two successive weighings no longer exceeds 2 mg.

NB:	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 0,1 %, in absolute value.

C.   DETERMINATION OF THE NITROGEN CONTENT AND CALCULATION OF CRUDE PROTEIN CONTENT

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 (3).

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 °C, 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 (point 3.16) and 10 ml bromocresol green solution (point 3.17) shall be added. Dependent on the water used, the pH of the boric acid solution might differ from batch to batch. The pH of the boric acid solution has to be between 4,3 and 4,7. 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 (point 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 (points 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 (point 3.1), an appropriate quantity of catalyst (point 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 (point 3.4) and if required , a few granules of pumice stone (point 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 (point 3.3), if required. Proceed according to point 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 (point 3.5) or (point 3.7) depending on the presumed nitrogen content. Add a few drops of methyl red indicator (point 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 point 8.3). Slowly pour 100 ml of sodium hydroxide solution (point 3.9) into the digestion flask without loss of ammonia (see observation point 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 (point 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 (point 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 point 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 (point 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 (point 3.19) or with the sulphuric acid standard volumetric solution (point 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 (point 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 (point 3.14) in place of the sample.

6.   Calculation of results

Calculations are performed according to point 6.1 or 6.2.

6.1.   Calculation for titration according to point 5.3.1

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

where:

V0	=	is the volume (ml) of NaOH (point 3.10 or 3.11) used in the blank test
V1	=	is the volume (ml) of NaOH (point 3.10 or 3.11) used in the sample titration
c	=	is the concentration (mol/l) of sodium hydroxide (point 3.10 or 3.11)
m	=	is the weight (g) of sample.

6.2.   Calculation for titration according to point 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 (point 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 (point 3.6)
V0	=	is the volume (in ml) of sulphuric acid (point 3.6) used for the blank test
V1	=	is the volume (in ml) of sulphuric acid (point 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:

—	0,4 % in absolute value, for crude protein contents of less than 20 %,

—	2,0 % relative to the higher value, for crude protein contents from 20 % to 40 %,

—	0,8 % in absolute value, for crude protein contents of more than 40 %.

7.2.   Reproducibility

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

—	1,8 % in absolute value, for crude protein contents of less than 20 %,

—	9,0 % relative to the higher value, for crude protein contents from 20 % to 40 %,

—	3,6 % in absolute value, for crude protein contents of more than 40 %.

7.3.   Accuracy

Carry out the analysis (digestion, distillation and titration) on an appropriate quantity of acetanilide (point 3.13) (e.g. 0,2 to 0,3 g) in the presence of 1 g of sucrose (point 3.14); 1 g acetanilide consumes 14,80 ml of sulphuric acid (point 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 (point 3.4) than that specified in point 5.1.

8.3.

For products with a low nitrogen content, the volume of sulphuric acid (point 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 used as feed additive in ruminant 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 (ρ20 1,19 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, K4Fe(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 rpm.

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 (point 3.4) in a 500 ml volumetric flask. Add 400 ml of water and 5 ml of Carrez solution I (point 3.2), mix for approximately 30 seconds and add 5 ml of Carrez solution II (point 3.3). Mix for thirty 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 (point 3.1) and mix. Place the tubes in a water bath at 20 °C (+/– 4 °C). After fifteen 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 (point 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 (point 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 in mg urea per kg sample.

7.   Evaluation of the method

7.1.   Repeatability

The difference between the results of two determinations carried out on the same sample in the same laboratory and by the same operator must not exceed:

—	At 420 nm: — 50 % relative to the higher value, for urea contents from 3 000 mg/kg to lower than 5 000 mg/kg, — 25 % relative to the higher value, for urea contents from 5 000 mg/kg to lower than 7 000 mg/kg, — 20 % relative to the higher value, for urea contents of 7 000 mg/kg or more.

—	At 435 nm: — 40 % relative to the higher value, for urea contents from 3 000 mg/kg to lower than 5 000 mg/kg, — 25 % relative to the higher value, for urea contents from 5 000 mg/kg to lower than 9 000 mg/kg, — 5 % relative to the higher value, for urea contents of 9 000 mg/kg or more.

7.2.   Reproducibility

The difference between the results of two determinations carried out on the same sample in different laboratories and/or by different operators must not exceed:

—	At 420 nm: — 3 000 mg/kg, in absolute value, for urea contents from 3 000 mg/kg to lower than 12 000 mg/kg, — 4 500 mg/kg, in absolute value, for urea contents of 12 000 mg/kg or more.

—	At 435 nm — 50 % relative to the higher value, for urea contents from 3 000 mg/kg to lower than 8 000 mg/kg, — 25 % relative to the higher value, for urea contents of 8 000 mg/kg or more.

8.   Results of a collaborative study

An EU interlaboratory comparison exercise was organised in which 18 laboratories took part. Five positive ruminant compound feed samples (in Tables 1 and 2 referred to as MAT) were analysed (1 analysis) in blind duplicates while one blank compound ruminant feed was analysed once.

Calculations for repeatability (r) and reproducibility (R) limits as defined by international guidelines were carried out after the removal of outliers using Analysis of Variance of the valid values.

The calculated method performance figures (repeatability, reproducibility) are presented in the following tables. Over all tested samples including the blank material, no false positives or false negatives were found.

Table 1

Method performance characteristics for urea measured at λ = 420 nm in all materials

	MAT 2	MAT 5	MAT 3	MAT 4	MAT 6
	Sheep	Cattle	Sheep	Sheep	Cattle
Target mass fraction (mg kg-1)	3 000	5 000	7 001	9 036	11 000
Average mass fraction. (mg kg-1)	4 241	6 993	7 830	9 962	12 071
Reproducibility standard deviation sR (mg kg-1)	1 141	1 303	985	994	1 711
Repeatability standard deviation sr (mg kg-1)	723	601	549	712	737
Reproducibility relative standard deviation RSDR (%)	27	19	13	10	14
Repeatability relative standard deviation RSDr (%)	17	9	7	7	6
Limit of reproducibility, R [R = 2,8 × sR ]	3 195	3 649	2 759	2 784	4 790
Limit of repeatability, r [r = 2,8 × sr ]	2 024	1 684	1 536	1 994	2 064

Table 2

Method performance characteristics for urea measured at λ = 435 nm in all materials

	MAT 2	MAT 5	MAT 3	MAT 4	MAT 6
	Sheep	Cattle	Sheep	Sheep	Cattle
Target mass fraction (mg kg-1)	3 000	5 000	7 001	9 036	11 000
Average mass fraction. (mg kg-1)	4 101	6 467	7 890	10 062	11 642
Reproducibility standard deviation sR (mg kg-1)	706	1 194	675	745	1 378
Repeatability standard deviation sr (mg kg-1)	570	628	613	196	167
Reproducibility relative standard deviation RSDR (%)	17	18	9	7	12
Repeatability relative standard deviation RSDr (%)	14	10	8	2	1
Limit of reproducibility, R [R = 2,8 × sR ]	1 977	3 344	1 889	2 087	3 859
Limit of repeatability, r [r = 2,8 × sr ]	1 596	1 759	1 715	549	467

9.   Observations

9.1.

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.

9.2.

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

9.3.

The above results from collaborative trials do not indicate a significant difference in precision between urea measured at 420 nm or at 435 nm.

E.   DETERMINATION OF AMINO ACIDS (EXCEPT TRYPTOPHAN)

The methods of analysis to be used for the determination of amino acids (except tryptophan) are:

—	EN ISO 13903 Animal feeding stuffs – Determination of amino acids content,

—	EN ISO 17180 Animal feeding stuffs – Determination of lysine, methionine, and threonine in commercial amino acid products and premixtures (4),

—	the method of analysis as described in points 1 to 10 hereafter.

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 materials, compound feeds and premixtures containing less than 10 % (5) of each amino acid, 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 various 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 point 1 (Purpose and scope) can be determined in either the oxidised or unoxidised sample.

Oxidation is performed at 0 °C with a performic acid/phenol mixture. Excess oxidation reagent is decomposed with sodium disulphite. The oxidised or unoxidised sample is hydrolysed with hydrochloric acid (point 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 °C.

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 of the amino acids listed under point 1 (Purpose and scope). 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 (point 3.5) in water and make up to 1 litre.

3.18.

Sodium hydroxide solution, c = 1 mol/l: Dissolve 40 g NaOH (point 3.5) in water and make up to 1 litre.

3.19.

Formic acid – phenol solution: Mix 889 g formic acid (point 3.2) with 111 g water and add 4,73 g phenol (point 3.3).

3.20.

Hydrolysis mixture, c = 6 mol HCl/l containing 1 g phenol/l: Add 1 g phenol (point 3.3) to 492 ml HCl (point 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 (point 3.7), dilute with approximately 900 ml water, add 20 ml thiodiglycol (point 3.9) and make up to 1 litre with water (do not mix points 3.7 and 3.9 directly).

3.22.

5-Sulfosalicylic acid, ß = 6 %: Dissolve 60 g 5-sulfosalicylic acid (point 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 (point 3.1) with 4,5 ml formic acid-phenol solution (point 3.19) in a small beaker. Incubate at 20–30 °C 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 (point 3.8), 5 ml thiodiglycol (point 3.9), 1 g phenol (point 3.3) and 16,50 ml HCl (point 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 (point 4.9).

3.26.

Ninhydrin reagent, prepared according to conditions for the analyser used (point 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 (point 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 (point 3.16.2) and 0,2265 g methionine sulphone (point 3.16.3) in citrate buffer (point 3.24) in a 1 litre graduated flask and make up to mark with citrate buffer. Store below 5 °C for not more than 12 months. This solution is not used if the stock standard solution (point 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 (point 3.13) in citrate buffer (point 3.24) in a graduated flask and make up to 250 ml with citrate buffer. Store below 5 °C 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 (point 3.10) in 100 ml beaker with 30 ml citrate buffer (point 3.24). Add 4,00 ml stock standard solution of amino acids (point 3.27.1), 4,00 ml stock standard solution of cysteic acid and methionine sulphone (point 3.27.2) and 0,50 ml stock standard solution of internal standard (point 3.27.3) if used. Adjust pH to 2,20 with sodium hydroxide (point 3.18). Transfer quantitatively to a 50 ml graduated flask and make up to the mark with citrate buffer (point 3.24) and mix. Store below 5 °C for not more than 3 months. See also observations point 9.1.

3.27.5.

Calibration solution of standard amino acids for use with hydrolysates prepared according to point 5.3.3.1 and for use with extracts (point 5.2). The calibration solution is prepared according to point 3.27.4 but omitting sodium chloride. Store below 5 °C 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 °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 °C or freeze dried prior to grinding. Samples with a high fat content shall be extracted with light petroleum (point 3.12) prior to grinding.

5.2.   Determination of free amino acids

Weigh to the nearest 0,2 mg an appropriate amount (1-5 g) of the prepared sample (point 5.1), into a conical flask and add 100,0 ml of extraction mixture (point 3.21). Shake the mixture for 60 min using a mechanical shaker or a magnetic stirrer (point 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 (point 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 (point 3.18), transfer to a volumetric flask of appropriate volume using citrate buffer (point 3.24), and make up to the mark with the buffer solution (point 3.24).

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

Proceed to the chromatography step according to point 5.4.

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

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 (point 5.1) into:

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

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

—	a 100 ml bottle fitted with a screw cap (point 4.2) (for closed hydrolysis point 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 °C, add 5 ml of oxidation mixture (point 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 °C 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 (point 3.4).

Proceed to point 5.3.2.1.

5.3.2.   Hydrolysis

5.3.2.1.

Hydrolysis of oxidised samples To the oxidised sample prepared according to point 5.3.1 add 25 ml of hydrolysis mixture (point 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 point 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-bottomed flask (point 4.1) or a 100 ml bottle fitted with a screw cap (point 4.2), to the nearest 0,2 mg, from 0,1 to 1 g of the prepared sample (point 5.1). The weighed sample portion must have a nitrogen content of about 10 mg. Add carefully 25 ml of hydrolysis mixture (point 3.20) and mix with the sample. Proceed according to either point 5.3.2.3 or point 5.3.2.4.

5.3.2.3.

Open hydrolysis Add 3 glass beads to the mixture in the flask (prepared in accordance with point 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 (point 3.24). Disconnect the flask and cool it in an ice bath. Proceed according to point 5.3.3.

5.3.2.4.

Closed hydrolysis Place the bottle containing the mixture prepared in accordance with point 5.3.2.1 or 5.3.2.2 in an oven (point 4.3) at 110 °C. 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 (point 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 (point 5.3.3), quantitatively transfer the contents of the bottle to a 250 ml beaker or a 250 ml round-bottomed flask, using citrate buffer (point 3.24). Proceed according to point 5.3.3.

5.3.3.   Adjustment of pH

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

5.3.3.1.

For chromatographic systems (point 4.9) requiring a low sodium concentration. It is advisable to use an internal stock standard solution (point 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 (point 3.27.3) to the hydrolysate before the evaporation. Add 2 drops of 1-octanol (point 3.15) to the hydrolysate obtained in accordance with point 5.3.2.3 or 5.3.2.4. Using a rotary evaporator (point 4.7) reduce the volume to 5-10 ml under vacuum at 40 °C. 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 (point 3.18) and proceed to point 5.3.4.

5.3.3.2.

For all other amino acid analysers (point 4.9) Take the hydrolysates obtained in accordance with point 5.3.2.3 or 5.3.2.4 and partly neutralise them by carefully adding with stirring, 17 ml of sodium hydroxide solution (point 3.17), ensuring that the temperature is kept below 40 °C. Adjust the pH to 2,20 at room temperature using sodium hydroxide solution (point 3.17) and finally sodium hydroxide solution (point 3.18). Proceed to point 5.3.4.

5.3.4.   Sample solution for chromatography

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

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

Proceed to the chromatography step (point 5.4).

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

5.4.   Chromatography

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

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 (point 5.3.4) or ml calculated total dilution volume of extract (point 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 × 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 (point 5.2) is calculated as following: 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 feeds (mixed pig feed, broiler compound, protein concentrate, premixture).

Note:

The method has been tested during a second international intercomparison study in 2003 by using blind duplicate pairs of broiler finisher feed, broiler starter feed, corn, fishmeal and poultry meal samples. For details see EN ISO 13903.

The results of 1990 intercomparison, 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 Feed	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 intercomparison of the previous table is given in the following table:

Coefficient of variation (%) for repeatability (CVr)

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 Feed	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 above mentioned intercomparison are given in the table below:

Coefficient of variation (%) for reproducibility (CVR)

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 feed	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 points 3.27.4 and 3.27.5) and of the hydrolysate (see point 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 compound feed or premixtures containing more than 1 % chloride (concentrate, mineral feeds, complementary feeds) underestimation of methionine could occur and special treatment shall be done.

10.   Performance criteria

Compilation of the results (except for tyrosine) coming from the 2 collaborative studies (from 1990 reported in point 7 above and from 2005 reported in EN/ISO 13903) gives the following criteria for repetability and reproducibility. The values derived from these 2 interlaboratory tests may not be applicable to concentration ranges and matrices other than those given.

10.1.   Repeatability

The difference between the results of two determinations carried out on the same sample in the same laboratory and by the same operator must not exceed:

—	6 % relative to the higher value, for total amino acids in case of glycine, alanine, lysine, proline, glutamic acid, isoleucine and histidine,

—	8 % relative to the higher value, for total amino acids in case of threonine, phenylalanine, methionine, aspartic acid and leucine,

—	10 % relative to the higher value, for total amino acids in case of arginine and valine,

—	12 % relative to the higher value, for total serine amino acid,

—	15 % relative to the higher value, for total cyst(e)ine amino acid.

10.2.   Reproducibility

The difference between the results of two determinations carried out on the same sample in different laboratories and/or by different operators must not exceed:

—	15 % relative to the higher value, for total amino acids in case of glycine, alanine and threonine,

—	20 % relative to the higher value, for total amino acids in case of lysine, proline, phenylalanine, methionine and aspartic acid,

—	22 % relative to the higher value, for total amino acids in case of glutamic acid and leucine,

—	27 % relative to the higher value for total arginine amino acid,

—	32 % relative to the higher value, for total isoleucine amino acid,

—	35 % relative to the higher value, for total amino acids in case of valine and serine,

—	40 % relative to the higher value, for total histidine amino acid,

—	50 % relative to the higher value, for total cyst(e)ine amino acid.

F.   DETERMINATION OF TRYPTOPHAN

The methods of analysis to be used for the determination of tryptophan are:

—	EN ISO 13904 Animal feeding stuffs – Determination of tryptophan content,

—	the method of analysis as described in points 1 to 9 hereafter.

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 °C 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 point 9.3).

3.5.

Sodium hydroxide.

3.6.

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

3.7.

Hydrochloric acid, ρ20 1,19 g/ml.

3.8.

Methanol, equivalent to HPLC grade.

3.9.

Light petroleum, boiling range 40–60 °C.

3.10.

Sodium hydroxide solution, c = 1 mol/l: Dissolve 40,0 g NaOH (point 3.5) in water and make up to 1 litre with water (point 3.1).

3.11.

Hydrochloric acid, c = 6 mol/l: Take 492 ml HCl (point 3.7) and make up to 1 litre with water.

3.12.

Hydrochloric acid, c = 1 mol/l: Take 82 ml HCl (point 3.7) and make up to 1 litre with water.

3.13.

Hydrochloric acid, c = 0,1 mol/l: Take 8,2 ml HCl (point 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 (point 3.6) and make up to 1 litre with water (point 3.1).

3.15.

Concentrated solution of tryptophan (point 3.2), c = 2,50 μmol/ml: In a 500 ml volumetric flask dissolve 0,2553 g tryptophan (point 3.2) in hydrochloric acid (point 3.13) and make up to the mark with hydrochloric acid (point 3.13). Store at – 18 °C 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 (point 3.3) in hydrochloric acid (point 3.13) and make up to the mark with hydrochloric acid (point 3.13). Store at – 18 °C for a maximum of 4 weeks.

3.17.

Calibration standard solution of tryptophan and internal standard: Take 2,00 ml concentrated solution of tryptophan (point 3.15), and 2,00 ml of concentrated internal standard (α-methyl-tryptophan) solution (point 3.16). Dilute with water (point 3.1) and methanol (point 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 (point 3.19) in 100 ml methanol (point 3.8).

3.22.

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

4.   Apparatus

4.1.

HPLC equipment with a spectrofluorometric detector.

4.2.

Liquid chromatographic column, 125 mm × 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) °C, 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 °C or freeze dried prior to grinding. Samples with high fat content shall be extracted with light petroleum (point 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 (point 5.1), into a conical flask. Add 100,0 ml hydrochloric acid (point 3.13) and 5,00 ml concentrated internal standard solution (point 3.16). Shake or mix for 60 min. using a mechanical shaker or a magnetic stirrer (point 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 (point 3.14). Adjust the pH to 3 using sodium hydroxide (point 3.10). Add sufficient methanol (point 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 (point 3.17)).

Filter a few ml of the solution through a 0,45 μm membrane filter (point 4.5) before injection on the HPLC column. Proceed to the chromatography step according to point 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 °C 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 (point 5.1) into the polypropylene flask (point 4.4). The weighed sample portion shall have a nitrogen content of about 10 mg. Add 8,4 g barium hydroxide octa-hydrate (point 3.4) and 10 ml water. Mix on a vortex mixer (point 4.8) or magnetic stirrer (point 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 (point 4.6) with boiling water and let it steam for 30-60 minutes. Close the autoclave and autoclave at 110 (± 2) °C for 20 hours.

Before opening the autoclave reduce the temperature to just under 100 °C. 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 (point 3.16). Cool the vessels on water/ice bath for 15 minutes.

Then, add 5 ml ortho-phosphoric acid (point 3.14). Keep the vessel in the cooling bath and neutralise with HCl (point 3.11) whilst stirring and adjust the pH to 3,0 using HCl (point 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 (point 4.5) before injection on the HPLC column. Proceed to the chromatography step according to point 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 °C 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 point 9.1 and 9.2):

Liquid chromatographic column (point 4.2):	125 mm × 4 mm, C18, 3 μm packing or equivalent
Column temperature:	Room temperature
Mobile phase (point 3.22):	3,00 g acetic acid (point 3.18) + 900 ml water (point 3.1) + 50,0 ml solution (point 3.21) of 1,1,1-trichloro-2-methyl-2-propanol (point 3.19) in methanol (point 3.8) (1 g/100 ml). Adjust pH to 5,00 using ethanolamine (point 3.20). Make up to 1 000 ml with water (point 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.

A	=	peak area of internal standard, calibration standard solution (point 3.17)
B	=	peak area of tryptophan, extract (point 5.2) or hydrolysate (point 5.3)
V1	=	volume in ml (2 ml) of concentrated tryptophan solution (point 3.15) added to the calibration solution (point 3.17)
c	=	concentration in μmol/ml (= 2,50) of concentrated tryptophan solution (point 3.15) added to calibration solution (point 3.17)
V2	=	volume in ml of concentrated internal standard solution (point 3.16) added at the extraction (point 5.2) (= 5,00 ml) or to the hydrolysate (point 5.3) (= 2,00 ml)
C	=	peak area of internal standard, extract (point 5.2) or hydrolysate (point 5.3)
D	=	peak area of tryptophan, calibration standard solution (point 3.17)
V3	=	volume in ml (= 2,00 ml) of concentrated internal standard solution (point 3.16) added to calibration standard solution (point 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 EU 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 for pigs
L	12	12	12
n Mean [g/kg]	50 2,42	55 3,40	50 4,22
sr [g/kg]	0,05	0,05	0,08
r [g/kg]	0,14	0,14	0,22
CVr [%]	1,9	1,6	1,9
SR [g/kg]	0,15	0,20	0,09
R [g/kg]	0,42	0,56	0,25
CVR [%]	6,3	6,0	2,2
L = number of laboratories submitting results n = number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr = standard deviation of repeatability SR = standard deviation of reproducibility r = repeatability R = reproducibility CVr = coefficient of variation of repeatability, % CVR = coefficient of variation of reproducibility, %.

Another EU collaborative study (3rd intercomparison) was arranged in which two samples were analysed by up to 13 laboratories to certify the method for extraction of free tryptophan. Replicate (5) analyses were performed on each sample. The results are given in the following table:

	Sample 4 Wheat and soya mixture	Sample 5 Wheat and soya mixture (= sample 4) with added tryptophan (0,457 g/kg)
L n	12 55	12 60
Mean [g/kg]	0,391	0,931
sr [g/kg]	0,005	0,012
r [g/kg]	0,014	0,034
CVr [%]	1,34	1,34
SR [g/kg]	0,018	0,048
R [g/kg]	0,050	0,134
CVR [%]	4,71	5,11
L = number of laboratories submitting results n = number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr = standard deviation of repeatability SR = standard deviation of reproducibility r = repeatability R = reproducibility CVr = coefficient of variation of repeatability, % CVR = coefficient of variation of reproducibility, %.

Another EU intercomparison study was arranged in which four samples were analysed by up to 7 laboratories with the aim of a tryptophan certification for hydrolysis. The results are given below Replicate (5) analyses were performed on each sample.

	Sample 1 Mixed pig feed(CRM 117)	Sample 2 Low fat fish meal (CRM 118)	Sample 3 Soybean meal (CRM 119)	Sample 4 Skimmed milk powder (CRM 120)
L	7	7	7	7
n	25	30	30	30
Mean [g/kg]	2,064	8,801	6,882	5,236
sr [g/kg]	0,021	0,101	0,089	0,040
r [g/kg]	0,059	0,283	0,249	0,112
CVr [%]	1,04	1,15	1,30	0,76
SR [g/kg]	0,031	0,413	0,283	0,221
R [g/kg]	0,087	1,156	0,792	0,619
CVR [%]	1,48	4,69	4,11	4,22
L = number of laboratories submitting results n = number of single results retained after eliminating outliers (identified by Cochran, Dixon outlier test) sr = standard deviation of repeatability SR = standard deviation of reproducibility r = repeatability R = reproducibility CVr = coefficient of variation of repeatability, % CVR = coefficient of variation of reproducibility, %.

9.   Observations

9.1.

Following special chromatographic conditions may give better separation between tryptophan and α-methyl-tryptophan. Isocratic elution followed by gradient column cleaning: Liquid chromatographic column: 125 mm × 4 mm, C18, 5 μm packing or equivalent Column temperature: 32 °C Mobile phase: A: 0,01 mol/l KH2PO4/Methanol, 95 + 5 (V + V) B: Methanol Gradient programme: 0 min 100 % A 0 % B 15 min 100 % A 0 % B 17 min 60 % A 40 % B 19 min 60 % A 40 % B 21 min 100 % A 0 % B 33 min 100 % A 0 % B Flow rate: 1,2 ml/min Total run time: approx. 33 min.

9.2.

The chromatography will vary according to the type of HPLC and column packing material used. The chosen system must be capable of giving baseline separation between the tryptophan and the internal standard. Moreover, it is important that degradation products are well separated from the tryptophan and the internal standard. Hydrolysates without internal standard shall be run in order to check the base line under the internal standard for impurities. It is important that the run time is sufficiently long for the elution of all the degradation products, otherwise late eluting peaks may interfere with subsequent chromatographic runs. In the range of operation, the chromatographic system shall give linear response. The linear response shall be measured with a constant (the normal) concentration of the internal standard and varying concentrations of tryptophan. It is of importance that the size of both the tryptophan and internal standard peaks are within the linear range of the HPLC/fluorescence system. If either the tryptophan and/or the internal standard peak(s) is (are) too small or too high the analysis shall be repeated with another sample size and/or a changed final volume.

9.3.   Barium hydroxide

With age barium hydroxide becomes more difficult to dissolve. This results in an unclear solution for the HPLC determination, which may produce low results for tryptophan.

G.   DETERMINATION OF CRUDE OILS AND FATS

1.   Purpose and scope

This method is for the determination of crude oils and fats in feed.

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

For the determination of crude oils and fats in oil seeds and oleaginous fruit as well in feed in which the crude oil/fat content is higher than 15 %, the extraction should be performed by Procedure A and re-extraction by Procedure B (point 5.3).

1.1.   Procedure A – Directly extractable crude oils and fats

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

1.2.   Procedure B – Total crude oils and fats

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

1.3.   Interpretation of results

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

2.   Principle

2.1.   Procedure A

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

2.2.   Procedure B

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

3.   Reagents

3.1.

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

3.2.

Sodium sulfate, anhydrous.

3.3.

Hydrochloric acid, c = 3 mol/l.

3.4.

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

4.   Apparatus

4.1.

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

4.2.

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

4.3.

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

5.   Procedure

5.1.   Procedure A (see point 8.1)

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

Place the thimble in an extractor (point 4.1) and extract for six hours with light petroleum (point 3.1). Collect the light petroleum extract in a dry, weighed flask containing fragments of pumice stone (6).

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

5.2.   Procedure B

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

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

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

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

5.3.   Procedure A and re-extraction by Procedure B

For the determination of crude oils and fats in oil seeds and oleaginous fruit as well in feed in which the crude oil/fat content is higher than 15 % the extraction should be performed by Procedure A and re-extraction by Procedure B.

This means after the extraction with light petroleum (procedure A), the residue or a portion of the residue is re-extracted with hydrochloric acid (procedure B). The crude oil and fat content is the sum of the result of procedure A and B.

6.   Expression of result

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

7.   Repeatability

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

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

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

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

8.   Observations

8.1.

For products with a high content of oils and fats, which are difficult to crush or unsuitable for drawing a homogeneous reduced test sample, proceed as follows. Weigh 20 g of the sample to the nearest 1 mg and mix with 10 g or more of anhydrous sodium sulfate (point 3.2). Extract with light petroleum (point 3.1) as indicated in point 5.1. Make up the extract obtained to 500 ml with light petroleum (point 3.1) and mix. Take 50 ml of the solution and place in a small, dry, weighed flask containing fragments of pumice stone. Distil off the solvent, dry and proceed as indicated in the last paragraph of point 5.1. Eliminate the solvent from the extraction residue left in the thimble, crush the residue to a fineness of 1 mm, return it to the extraction thimble (do not add sodium sulfate) and proceed as indicated in the second and third paragraphs of point 5.1. Calculate the content of oils and fats as a percentage of the sample by using the following formula: where: m1 = weight in grams of the residue after the first extraction (aliquot part of the extract), m2 = weight in grams of the residue after the second extraction.

8.2.

For some products (e.g. low in oils and fats) the test sample may be increased.

8.3.

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

8.4.

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

8.5.

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

H.   DETERMINATION OF CRUDE FIBRE

1.   Purpose and scope

This method makes it possible to determine fat-free organic substances in feed which are insoluble in acid and alkaline media and are conventionally described as crude fibre.

The method is not applicable in the case of lignocellulose and vegetable carbon (particles too fine).

2.   Principle

The sample, defatted where necessary, is treated successively with boiling solutions of sulphuric acid and potassium hydroxide of specified concentrations. The residue is separated by filtration on a sintered-glass filter, washed, dried, weighed and ashed within a range of 475 to 500 °C. The loss of weight resulting from ashing corresponds to the crude fibre present in the test sample.

3.   Reagents

3.1.

Sulphuric acid, c = 0,13 mol/l.

3.2.

Anti-foaming agent (e.g. n-octanol).

3.3.

Filter aid (Celite 545 or equivalent), heated at 500 °C for four hours (point 8.6).

3.4.

Acetone.

3.5.

Light petroleum boiling-range 40 to 60 °C.

3.6.

Hydrochloric acid, c = 0,5 mol/l.

3.7.

Potassium hydroxide solution, c = 0,23 mol/l.

4.   Apparatus

4.1.

Heating unit for digestion with sulphuric acid and potassium hydroxide solution, equipped with a support for the filter crucible (point 4.2) and provided with an outlet tube with a tap to the liquid outlet and vacuum, possibly with compressed air. Before use each day preheat the unit with boiling water for five minutes.

4.2.

Glass filter crucible with fused sintered glass filter plate pore size 40-90 μm. Before first use, heat to 500 °C for a few minutes and cool (point 8.6).

4.3.

Cylinder of at least 270 ml with a reflux condenser, suitable for boiling.

4.4.

Drying oven with thermostat.

4.5.

Muffle furnace with thermostat.

4.6.

Extraction unit consisting of a support plate for the filter crucible (point 4.2) and with a discharge pipe with a tap to the vacuum and liquid outlet.

4.7.

Connecting rings to assemble the heating unit (point 4.1), crucible (point 4.2) and cylinder (point 4.3) and to connect the cold extraction unit (point 4.6) and crucible.

5.   Procedure

Weigh out 1 g of the prepared sample to the nearest 1 mg and place it in the crucible (point 4.2), (see observations points 9.1, 9.2 and 9.3) and add 1 g of filter aid (point 3.3).

Assemble the heating unit (point 4.1) and the filter crucible (point 4.2), then attach the cylinder (point 4.3) to the crucible. Pour 150 ml of boiling sulphuric acid (point 3.1) into the assembled cylinder and crucible and if necessary add a few drops of antifoaming agent (point 3.2).

Bring the liquid to the boil within 5 ± 2 minutes and boil vigorously for exactly 30 minutes.

Open the tap to the discharge pipe (point 4.1) and, under vacuum, filter the sulphuric acid through the filter crucible and wash the residue with three consecutive 30 ml portions of boiling water, ensuring that the residue is filtered dry after each washing.

Close the outlet tap and pour 150 ml boiling potassium hydroxide solution (point 3.7) to the assembled cylinder and crucible and add a few drops of antifoaming agent (point 3.2). Bring the liquid to boiling point within 5 ± 2 minutes and boil vigorously for exactly 30 minutes. Filter and repeat the washing procedure used for the sulphuric acid step.

After the final washing and drying, disconnect the crucible and its contents and reconnect it to the cold extraction unit (point 4.6). Apply the vacuum and wash the residue in the crucible with three consecutive 25 ml portions of acetone (point 3.4) ensuring that the residue is filtered dry after each washing.

Dry the crucible to constant weight in the oven at 130 °C. After each drying cool in the desiccator and weigh rapidly. Place the crucible in a muffle furnace and ash to constant weight (loss in weight between two successive weighings must be less than or equal to 2 mg) at 475 °C to 500 °C for at least 30 minutes.

After each heating cool first in the furnace and then in the desiccator before weighing.

Carry out a blank test without the sample. Loss of weight resulting from ashing must not exceed 4 mg.

6.   Calculation of results

The crude fibre content as a percentage of the sample is given by the expression:

where:

m	=	weight of sample in g;
m0	=	loss of weight after ashing during the determination, in g;
m1	=	loss of weight after ashing during the blank test, in g.

7.   Repeatability

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

—	0,6 % in absolute value for crude fibre contents lower than 10 %,

—	6 % relative to the higher result, for crude fibre contents equal to or greater than 10 %.

8.   Reproducibility

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

—	1,0 % in absolute value for crude fibre contents lower than 10 %,

—	10 % relative to the higher result, for crude fibre contents equal to or greater than 10 %.

9.   Observations

9.1.

Feed containing more than 10 % crude fat must be defatted prior to analysis with light petroleum (point 3.5). Connect the filter crucible (point 4.2) and its contents to the cold extraction unit (point 4.6) and apply vacuum and wash the residue with three consecutive 30 ml portions of light petroleum, ensuring that the residue is dry. Connect the crucible and its contents to the heating unit (point 4.1) and continue as described under point 5.

9.2.

Feed containing fats which cannot be extracted directly with light petroleum (point 3.5) must be defatted as shown in point 8.1 and defatted once more after boiling with acid. After boiling with acid and the subsequent washing connect the crucible and its contents to the cold extraction unit (point 4.6) and wash three times with 30 ml acetone followed by three further washings with 30 ml portions of light petroleum. Filter under vacuum until dry and continue the analysis as described under point 5, beginning with potassium hydroxide treatment.

9.3.

If the feed contain over 5 % of carbonates, expressed as calcium carbonate, connect the crucible (point 4.2) with the weighed sample to the heating unit (point 4.1). Wash the sample three times with 30 ml hydrochloric acid (point 3.6). After each addition let the sample stand for about one minute before filtering. Wash once with 30 ml water and then continue as described under point 5.

9.4.

If an apparatus in the form of a stand is used (several crucibles attached to the same heating unit) no two individual determinations on the same sample for analysis may be carried out in the same series.

9.5.

If after boiling it is difficult to filter the acidic and basic solutions, use compressed air through the discharge pipe of the heating unit and then continue filtering.

9.6.

The temperature for ashing shall not be higher than 500 °C in order to extend the lifetime of the glass filter crucibles. Care must be taken to avoid excessive thermal shock during heating and cooling cycles.

I.   DETERMINATION OF SUGAR

1.   Purpose and scope

This method makes it possible to determine the amount of reducing sugars and total sugars after inversion, expressed as glucose or where appropriate as sucrose, converting by the factor 0,95. It is applicable to compound feed. Special methods are provided for other feed. Where necessary, lactose shall be measured separately and taken into account when calculating the results.

This method is to be used for the determination of the sugar content for use in energy value calculation of the feed.

In case the sugar content is to be determined for other purposes, other methods of analysis can be used.

2.   Principle

The sugars are extracted in dilute ethanol; the solution is clarified with Carrez solutions I and II. After eliminating the ethanol, the quantities before and after inversion are determined by the Luff-Schoorl method.

3.   Reagents

3.1.

Ethanol solution 40 % (v/v) density: 0,948 g/ml at 20 °C, neutralised to phenolphthalein.

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 K4Fe (CN)6 3H2O. Make up to 100 ml with water.

3.4.

Methyl orange, solution 0,1 % (w/v).

3.5.

Hydrochloric acid 4 mol/litre.

3.6.

Hydrochloric acid 0,1 mol/litre.

3.7.

Sodium hydroxide solution 0,1 mol/litre.

3.8.

Luff-Schoorl reagent: Stirring carefully, pour the citric acid solution (point 3.8.2) into the sodium carbonate solution (point 3.8.3). Add the copper sulphate solution (point 3.8.1) and make up to 1 litre with water. Leave to settle overnight and filter. Check the concentration of the reagent thus obtained (Cu 0,05 mol/litre; Na2 CO3 1 mol/litre), see point 5.4, last paragraph. The solution’s pH shall be approximately 9,4.

3.8.1.

Copper sulphate solution: dissolve 25 g of copper sulphate, Cu SO4 5H2O, free from iron, in 100 ml of water.

3.8.2.

Citric acid solution: dissolve 50 g of citric acid, C6H8O7•H2O in 50 ml of water.

3.8.3.

Sodium carbonate solution: dissolve 143,8 g of anhydrous sodium carbonate in approximately 300 ml of warm water. Leave to cool.

3.9.

Sodium thiosulphate solution 0,1 mol/litre.

3.10.

Starch solution: add a mixture of 5 g of soluble starch in 30 ml of water to 1 litre of boiling water. Boil for three minutes, leave to cool and if necessary add 10 mg of mercuric iodide as a preservative.

3.11.

Sulphuric acid 3 mol/litre.

3.12.

Potassium iodide, solution 30 % (w/v).

3.13.

Granulated pumice stone boiled in hydrochloric acid, washed in water and dried.

3.14.

3-methylbutan-1-ol.

4.   Apparatus

Mixer (tumbler): approximately 35 to 40 rpm.

5.   Procedure

5.1.   Extraction of sample

Weigh 2,5 g of the sample to the nearest mg and place in a 250 ml volumetric flask. Add 200 ml of ethanol (point 3.1) and mix in the tumbler for one hour. Add 5 ml of Carrez solution I (point 3.2) and stir for approximately 30 seconds. Add 5 ml of Carrez solution II (point 3.3) and again stir for one minute. Make up to volume with ethanol (point 3.1), homogenise and filter. Remove 200 ml of the filtrate and evaporate to approximately half volume in order to eliminate most of the ethanol. Transfer the evaporation residue quantitatively to a 200 ml volumetric flask using warm water, cool, bring up to volume with water, homogenise and filter if necessary. This solution will be used to determine the amount of reducing sugars and, after inversion, of total sugars.

5.2.   Determination of reducing sugars

Using a pipette, remove not more than 25 ml of the solution containing less than 60 mg of reducing sugars expressed as glucose. If necessary, make up to 25 ml with distilled water and determine the content of reducing sugars by the Luff-Schoorl method. The result is expressed as the percentage content of glucose in the sample.

5.3.   Determination of total sugars after inversion

Using a pipette take 50 ml of the solution and transfer to a 100 ml volumetric flask. Add a few drops of methyl orange solution (point 3.4) then, carefully and stirring continuously, add hydrochloric acid (point 3.5) until the liquid turns a definite red. Add 15 ml of hydrochloric acid (point 3.6), immerse the flask in a fast boiling water bath and keep there for thirty minutes. Cool rapidly to approximately 20 °C and add 15 ml of sodium hydroxide solution (point 3.7). Make up to 100 ml with water and homogenise. Remove not more than 25 ml containing less than 60 mg of reducing sugars expressed as glucose. If necessary, make up to 25 ml with distilled water and determine the content of reducing sugars by the Luff-Schoorl method. The result is expressed as the percentage of glucose or, where appropriate, sucrose, by multiplying by the factor 0,95.

5.4.   Titration by the Luff-Schoorl method

Using a pipette, take 25 ml of Luff-Schoorl reagent (point 3.8) and transfer to a 300 ml Erlenmeyer flask; add exactly 25 ml of the clarified sugar solution. Add 2 granules of pumice stone (point 3.13), heat, stirring by hand, over a free flame of medium height and bring the liquid to the boil in approximately two minutes. Place the Erlenmeyer immediately on an asbestos-coated wire gauze with a hole approximately 6 cm in diameter under which a flame has been lit. The flame shall be regulated in such a way that only the base of the Erlenmeyer is heated. Fit a reflux condenser to the Erlenmeyer flask. Boil for exactly ten minutes. Cool immediately in cold water and after approximately five minutes titrate as follows:

Add 10 ml of potassium iodide solution (point 3.12) and immediately afterwards (carefully, because of the risk of abundant foaming), add 25 ml of sulphuric acid (point 3.11). Titrate with sodium thiosulphate solution (point 3.9) until a dull yellow colour appears, add the starch indicator (point 3.10) and complete titration.

Carry out the same titration on an accurately measured mixture of 25 ml of Luff-Schoorl reagent (point 3.8) and 25 ml of water, after adding 10 ml of potassium iodide solution (point 3.12) and 25 ml of sulphuric acid (point 3.11) without boiling.

6.   Calculation of results

Using the table establish the amount of glucose in mg which corresponds to the difference between the values of the two titrations, expressed in ml of sodium thiosulphate 0,1 mol/litre. Express the result as a percentage of the sample.

7.   Special procedures

7.1.

In the case of feed which are rich in molasses and other feed which are not particularly homogeneous, weigh out 20 g and place with 500 ml of water in a 1 litre volumetric flask. Mix for one hour in the tumbler. Clarify using Carrez solutions I (point 3.2) and II (point 3.3) reagents as described under point 5.1, this time however using four times the quantities of each reagent. Bring up to volume with 80 % ethanol (v/v). Homogenise and filter. Eliminate the ethanol as described under point 5.1. If there is no dextrinised starch, bring up to volume with distilled water.

7.2.

In the case of molasses and feed materials which are rich in sugar and almost starch-free (carobs, dried beetroot cossettes, etc.), weigh out 5 g, place in a 250 ml volumetric flask, add 200 ml of distilled water and mix in the tumbler for one hour, or more if necessary. Clarify using Carrez solutions I (point 3.2) and II (point 3.3) reagents as described under point 5.1. Bring up to volume with cold water, homogenise and filter. In order to determine the amount of total sugars, continue as described under point 5.3.

8.   Observations

8.1.

In order to prevent foaming it is advisable to add (irrespective of the volume) approximately 1 ml of 3-methylbutan-1-ol (point 3.14) before boiling with Luff-Schoorl reagent.

8.2.

The difference between the content of total sugars after inversion, expressed as glucose, and the content of reducing sugars, expressed as glucose, multiplied by 0,95, gives the percentage content of sucrose.

8.3.

In order to determine the content of reducing sugars, excluding lactose, two methods may be adopted:

8.3.1.

For an approximate calculation, multiply by 0,675 the lactose content established by a different method of analysis and subtract the result obtained from the content of reducing sugars.

8.3.2.

For an accurate calculation of reducing sugars, excluding lactose, the same sample must be used for the two final determinations. One of the analyses is carried out on part of the solution obtained under point 5.1, the other on part of the solution obtained during the determination of lactose by the method laid down for that purpose (after fermenting the other types of sugar and clarifying). In both cases the amount of sugar present is determined by the Luff-Schoorl method and calculated in mg of glucose. One of the values is subtracted from the other and the difference is expressed as a percentage of the sample. Example The two volumes taken correspond, for each determination, to a sample of 250 mg. In the first case 17 ml of sodium thiosulphate solution 0,1 mol/litre corresponding to 44,2 mg of glucose is consumed; in the second, 11 ml, corresponding to 27,6 mg of glucose. The difference is 16,6 mg of glucose. The content of reducing sugars (excluding lactose), calculated as glucose, is therefore: Table of values for 25 ml of Luff Schoorl reagent ml of Na2 S2 O3 0,1 mol/litre, two minutes’ heating, ten minutes’ boiling Na2 S2 O3 0,1 mol/litre Glucose, fructose invert sugars C6 H12 O6 Lactose C12 H22 O11 Na2 S2 O3 0,1 mol/litre ml mg difference mg difference ml 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2,4 4,8 7,2 9,7 12,2 14,7 17,2 19,8 22,4 25,0 27,6 30,3 33,0 35,7 38,5 41,3 44,2 47,1 50,0 53,0 56,0 59,1 62,2 2,4 2,4 2,5 2,5 2,5 2,5 2,6 2,6 2,6 2,6 2,7 2,7 2,7 2,8 2,8 2,9 2,9 2,9 3,0 3,0 3,1 3,1 3,6 7,3 11,0 14,7 18,4 22,1 25,8 29,5 33,2 37,0 40,8 44,6 48,4 52,2 56,0 59,9 63,8 67,7 71,7 75,7 79,8 83,9 88,0 3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,8 3,8 3,8 3,8 3,8 3,8 3,9 3,9 3,9 4,0 4,0 4,1 4,1 4,1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

J.   DETERMINATION OF LACTOSE

1.   Purpose and scope

This method makes it possible to determine the level of lactose in feed containing more than 0,5 % of lactose.

2.   Principle

The sugars are dissolved in water. The solution is subjected to fermentation by the yeast Saccharomyces cerevisiae which leaves the lactose intact. After clarification and filtration the lactose content of the filtrate is determined by the Luff-Schoorl method.

3.   Reagents

3.1.

Suspension of Saccharomyces cerevisiae: suspend 25 g of fresh yeast in 100 ml of water. The suspension will keep for a maximum period of one week in a refrigerator.

3.2.

Carrez solution I: dissolve in water 21,9 g of zinc acetate, Zn (CH3 COO)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 K4Fe (CN)6 3H2O. Make up to 100 ml with water.

3.4.

Luff-Schoorl reagent: Stirring carefully, pour the citric acid solution (point 3.4.2) into the sodium carbonate solution (point 3.4.3). Add the copper sulphate solution (point 3.4.1) and make up to 1 litre with water. Leave to settle overnight and filter. Check the concentration of the reagent thus obtained (Cu 0,05 mol/litre; Na2 CO3 1 mol/litre). The solution’s pH shall be approximately 9,4.

3.4.1.

Copper sulphate solution: dissolve 25 g of copper sulphate Cu SO4 5H2O, free from iron, in 100 ml of water.

3.4.2.

Citric acid solution: dissolve 50 g of citric acid C6H8O7 • H2O in 50 ml of water.

3.4.3.

Sodium carbonate solution: dissolve 143,8 g of anhydrous sodium carbonate in approximately 300 ml of warm water. Leave to cool.

3.5.

Granulated pumice stone boiled in hydrochloric acid, washed in water and dried.

3.6.

Potassium iodide, solution 30 % (w/v).

3.7.

Sulphuric acid 3 mol/litre.

3.8.

Solution of sodium thiosulphate 0,1 mol/litre.

3.9.

Starch solution: add a mixture of 5 g of soluble starch in 30 ml of water to 1 litre of boiling water. Boil for three minutes, leave to cool, and if necessary add 10 mg of mercuric iodide as a preservative.

4.   Apparatus

Water bath with thermostat set at 38–40 °C.

5.   Procedure

Weigh 1 g of the sample to the nearest mg and place this portion of the sample in a 100 ml volumetric flask. Add 25 to 30 ml of water. Place the flask in a boiling water bath for thirty minutes and then cool to approximately 35 °C. Add 5 ml of yeast suspension (point 3.1) and homogenise. Leave the flask to stand for two hours in a water bath, at a temperature of 38–40 °C. Cool to approximately 20 °C.

Add 2,5 ml of Carrez solution I (point 3.2) and stir for thirty seconds, then add 2,5 ml of Carrez solution II (point 3.3) and again stir for thirty seconds. Make up to 100 ml with water, mix and filter. Using a pipette, remove an amount of filtrate which does not exceed 25 ml and which preferably contains from 40 to 80 mg of lactose and transfer it to a 300 ml Erlenmeyer flask. If necessary, make up to 25 ml with water.

Carry out a blank test in the same way with 5 ml of yeast suspension (point 3.1). Determine the lactose content according to Luff-Schoorl, as follows: add exactly 25 ml of Luff-Schoorl reagent (point 3.4) and two granules of pumice stone (point 3.5). Stir by hand while heating over a free flame of medium height and bring the liquid to the boil in approximately two minutes. Place the Erlenmeyer immediately on an asbestos-coated wire gauze with a hole approximately 6 cm in diameter under which a flame has been lit. The flame shall be regulated in such a way that only the base of the Erlenmeyer is heated. Fit a reflux condenser to the Erlenmeyer flask. Boil for exactly ten minutes. Cool immediately in cold water and after approximately five minutes titrate as follows:

Add 10 ml of potassium iodide solution (point 3.6) and immediately afterwards (carefully, because of the risk of abundant foaming) add 25 ml of sulphuric acid (point 3.7). Titrate with sodium thiosulphate solution (point 3.8) until a dull yellow colour appears, add the starch indicator (point 3.9) and complete titration.

Carry out the same titration on an accurately measured mixture of 25 ml of Luff-Schoorl reagent (point 3.4) and 25 ml of water, after adding 10 ml of potassium iodide solution (point 3.6) and 25 ml of sulphuric acid (point 3.7) without boiling.

6.   Calculation of results

Using the attached table, establish the amount of lactose in mg which corresponds to the difference between the results of the two titrations, expressed in ml of sodium thiosulphate 0,1 mol/litre.

Express the result of anhydrous lactose as a percentage of the sample.

7.   Observation

1.

For products containing more than 40 % of fermentable sugar, use more than 5 ml of yeast suspension (point 3.1).

2.

In “lactose reduced” feed (e.g. cat milk), lactose is converted into fructose, which is not completely fermented within 2 hours resulting in higher or false positive results (because residues of fructose remain in the extract). Table of values for 25 ml of Luff-Schoorl reagent ml of Na2 S2 O3 0,1 mol/litre, two minutes’ heating, ten minutes’ boiling Na2 S2 O3 0,1 mol/litre Glucose, fructose invert sugars C6 H12 O6 Lactose C12 H22 O11 Na2 S2 O3 0,1 mol/litre ml mg difference mg difference ml 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2,4 4,8 7,2 9,7 12,2 14,7 17,2 19,8 22,4 25,0 27,6 30,3 33,0 35,7 38,5 41,3 44,2 47,1 50,0 53,0 56,0 59,1 62,2 2,4 2,4 2,5 2,5 2,5 2,5 2,6 2,6 2,6 2,6 2,7 2,7 2,7 2,8 2,8 2,9 2,9 2,9 3,0 3,0 3,1 3,1 3,6 7,3 11,0 14,7 18,4 22,1 25,8 29,5 33,2 37,0 40,8 44,6 48,4 52,2 56,0 59,9 63,8 67,7 71,7 75,7 79,8 83,9 88,0 3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,7 3,8 3,8 3,8 3,8 3,8 3,8 3,9 3,9 3,9 4,0 4,0 4,1 4,1 4,1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

K.   DETERMINATION OF STARCH

POLARIMETRIC METHOD

1.   Purpose and scope

This method makes it possible to determine the levels of starch and of high molecular weight starch degradation products in feed for the purpose of checking compliance with the declared energy value (provisions in Annex VII) and Regulation (EC) No 767/2009.

This method is to be used for the determination of the starch content for use in energy value calculation of the feed.

In case the starch content is to be determined for other purposes, other methods of analysis can be used.

2.   Principle

The method comprises of two determinations. In the first one, the sample is treated with dilute hydrochloric acid. After clarification and filtration, the optical rotation of the solution is measured by polarimetry.

In the second one, the sample is extracted with 40 % ethanol. After acidifying the filtrate with hydrochloric acid, clarifying and filtering, the optical rotation is measured as in the first determination.

The difference between the two measurements, multiplied by a known factor, gives the starch content of the sample.

3.   Reagents

3.1.

Hydrochloric acid, solution 25 % (w/w) density: 1,126 g/ml.

3.2.

Hydrochloric acid. solution 1,13 % (w/v) The concentration must be checked by titration using a sodium hydroxide solution 0,1 mol/litre in the presence of 0,1 % (w/v) methyl red in 94 % (v/v) ethanol. For the neutralisation of 10 ml, 30,94 ml of NaOH 0,1 mol/litre is needed.

3.3.

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

3.4.

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

3.5.

Ethanol, solution 40 % (v/v), density: 0,948 g/ml at 20 °C.

4.   Apparatus

4.1.

250 ml Erlenmeyer flask with standard ground-glass joint and with reflux condenser.

4.2.

Polarimeter or saccharimeter.

5.   Procedure

5.1.   Preparation of the sample

Crush the sample until it is fine enough for all of it to pass through a 0,5 mm round-meshed sieve.

5.2.   Determination of the total optical rotation (P or S) (see observation point 7.1)

Weigh 2,5 g of the crushed sample to the nearest mg and place in a 100 ml graduated flask. Add 25 ml of hydrochloric acid (point 3.2), shake to obtain even distribution of the test sample and add a further 25 ml of hydrochloric acid (point 3.2). Immerse the flask in a boiling water bath shaking vigorously and steadily for the first three minutes to prevent the formation of agglomerates. The quantity of water in the water bath must be sufficient for the bath to remain at boiling point when the flask is introduced into it. The flask must not be taken out of the bath whilst being shaken. After exactly 15 minutes, remove from the bath, add 30 ml of cold water and cool immediately to 20 °C.

Add 5 ml of Carrez solution I (point 3.3) and shake for approximately 30 seconds. Then add 5 ml of Carrez solution II (point 3.4) and shake again for approximately 30 seconds. Make up to volume with water, mix and filter. If the filtrate is not perfectly clear (which is rare), repeat the determination using a larger quantity of Carrez solutions I and II, for example 10 ml.

Measure the optical rotation of the solution in a 200 mm tube with the polarimeter or saccharimeter.

5.3.   Determination of the optical rotation (P' or S') of substances soluble in 40 % ethanol

Weigh 5 g of the sample to the nearest mg, place in a 100 ml graduated flask and add about 80 ml of ethanol (point 3.5) (see observation point 7.2). Leave the flask to stand for 1 hour at room temperature; during this time, shake vigorously on six occasions so that the test sample is thoroughly mixed with the ethanol. Make up to volume with ethanol (point 3.5), mix and filter.

Pipette 50 ml of the filtrate (corresponds to 2,5 g of the sample) into a 250 ml erlenmeyer flask, add 2,1 ml of hydrochloric acid (point 3.1) and shake vigorously. Fit a reflux condenser to the erlenmeyer flask and immerse the latter in a boiling water bath. After exactly 15 minutes, remove the erlenmeyer flask from the bath, transfer the contents to a 100 ml graduated flask, rinsing with a little cold water, and cool to 20 °C.

Clarify using Carrez solutions I (point 3.3) and II (point 3.4), make up to volume with water, mix, filter and measure the optical rotation as indicated in point 5.2, second and third paragraphs.

6.   Calculation of results

The starch content (%) is calculated as follows:

6.1.   Measurement by polarimeter

P	=	Total optical rotation in angle degrees
P'	=	Optical rotation in angle degrees of the substances soluble in 40 % (V/V) ethanol
	=	Specific optical rotation of pure starch. The numerical values conventionally accepted for this factor are the following:

+ 185,9°	:	rice starch
+ 185,7°	:	potato starch
+ 184,6°	:	maize starch
+ 182,7°	:	wheat starch
+ 181,5°	:	barley starch
+ 181,3°	:	oat starch
+ 184,0°	:	other types of starch and starch mixtures in compound feed.

6.2.   Measurement by saccharimeter

S	=	Total optical rotation in saccharimeter degrees
S'	=	Optical rotation in saccharimeter degrees of the substances soluble in 40 % (v/v) ethanol
N	=	weight (g) of saccharose in 100 ml of water yielding an optical rotation of 100 saccharimeter degrees when measured using a 200 mm tube 16,29 g for the French saccharimeters 26,00 g for the German saccharimeters 20,00 g for mixed saccharimeters.
	=	Specific optical rotation of pure starch (see point 6.1).

6.3.   Repeatability

The difference between the results of two parallel determinations carried out on the same sample must not exceed 0,4 in absolute value for a starch content lower than 40 % and 1 % relative for starch contents equal to or greater than 40 %.

7.   Observations

7.1.

If the sample contains more than 6 % of carbonates, calculated in terms of calcium carbonate, they must be destroyed by treatment with an exactly appropriate quantity of dilute sulphuric acid before determination of the total optical rotation.

7.2.

In the case of products with a high lactose content, such as powdered milk serum or skimmed milk powder, proceed as follows after adding 80 ml of ethanol (point 3.5). Fit a reflux condenser to the flask and immerse the latter in a water bath at 50 °C for 30 minutes. Leave to cool and continue the analysis as indicated in point 5.3.

7.3.

The following feed materials, where they are present in significant amounts in feed, are known to give rise to interferences when determining the starch content by the polarimetric method and thereby incorrect results could be yielded: — (sugar) beet products such as (sugar)beet pulp, (sugar) beet molasses, (sugar) beet pulp – molassed, (sugar) beet vinasse, (beet) sugar, — citrus pulp, — linseed; linseed expeller; linseed extracted,