CEN/TC 327/WG 3 - Feed additives and drugs

This document specifies the determination of phytase activity in feeding stuff samples, including feed raw materials from plant origin, compound feeds (complete, complementary, mineral feeds), premixtures and feed additives.
The method is applicable to, and is collaboratively validated for, the determination of phytase activity in complete feed, complementary feed including mineral feed, premixtures and feed additives.
The method does not distinguish between phytase added as a feed additive and endogenous phytase already present in the feed materials. Therefore, the method is also applicable for feed materials from plant origin.
The method does not apply to evaluating or comparing the in vivo efficacy of the phytase product. It is not a predictive method of the in vivo efficacy of phytases present on the market as they can develop different in vivo efficacy per unit of activity.

This analytical procedure specifies a reverse phase high performance liquid chromatographic with UV detection (RP-HPLC-UV) method for the simultaneous determination of four authorized carotenoids in fish compound feed and fish premix, namely astaxanthin (AXN), canthaxanthin (CXN), adonirubin (ADR) and astaxanthin dimethyldisuccinate (AXN DMDS), and of six authorized carotenoids in poultry feed and poultry premix, namely canthaxanthin (CXN); capsanthin (CSN), ethyl ester of beta-apo-8'-carotenoic acid (BACARE), citranaxanthin (CIXN), lutein (LUT) and zeaxanthin (ZEA) at levels ranging from approximately 2 mg/kg to approximately 4 500 mg/kg (depending on the carotenoid). Beta-carotene (BCAR), authorized in compound feed and premixes for all animal species, was also added to the scope. The analytical procedure is fit for the purpose of quantitation of declared carotenoids and labelling confirmation. This document is applicable to feed produced using natural and synthetic feed additives.
Xanthophyll esters like those of lutein, zeaxanthin and capsanthin that might be present in feed materials are not authorized feed additives and therefore not part of the scope of this document.

This document specifies general rules for the enumeration of lactobacilli in feeding stuffs (additives, premixtures and compound feeds excluding mineral feeds) that contain lactobacilli as a single microorganism component or in a mixture with other microorganisms. Applying the method to premixtures and compound feeds with critical amounts of copper demands a special procedure (see A.2). The document is not applicable to mineral feeds, which are defined as complementary feeding stuffs composed mainly of minerals and containing at least 40 % crude ash (Regulation (EC) No 767/2009) [3].
There are different categories of feed samples:
a)   Additives containing about 1010 colony forming units (CFU)/g;
b)   Premixtures containing about 1011 CFU/kg;
c)   Compound feeds, meal or pellets which contain about 109 CFU/kg.

This document specifies general rules for the enumeration of enterococci (E. faecium) in feeding stuffs (additives, premixtures and compound feeds excluding mineral feeds) that contain enterococci as a single microorganism component or in a mixture with other microorganisms. Applying the method to premixtures and compound feeds with critical amounts of copper demands a special procedure (see A.2). The document is not applicable to mineral feeds which are defined as complementary feeding stuffs composed mainly of minerals and containing at least 40 % crude ash (Regulation (EC) 767/2009) [4].
There are different categories of feed samples:
a)   Additives containing about 1010 colony forming units (CFU)/g;
b)   Premixtures containing 1011 CFU/kg;
c)   Compound feeds, meal or pellets which contain about 109 CFU/kg.

This document specifies general rules for the enumeration of pediococci in feeding stuffs (additives, premixtures and compound feeds excluding mineral feeds) that contain pediococci as a single microorganism component or in a mixture with other microorganisms. Applying the method to premixtures and compound feeds with critical amounts of copper demands a special procedure (see A.2). The document is not applicable to mineral feeds which are defined as complementary feeding stuffs composed mainly of minerals and containing at least 40 % crude ash (Regulation (EC) 767/2009) [3]. There are different categories of feed samples:
a)   Additives containing about 1010 colony forming units (CFU)/g;
b)   Premixtures containing about 1011 CFU/kg;
c)   Compound feeds, meal or pellets which contain about 109 CFU/kg.

This document specifies general rules for the enumeration of Saccharomyces cerevisiae in feeding stuffs (additives, premixtures and compound feeds excluding mineral feeds) that contain Saccharomyces cerevisiae as a single microorganism component or in a mixture with other microorganisms. Applying the method to premixtures and compound feeds with critical amounts of copper demands a special procedure (see Annex A). The document is not applicable to mineral feeds, which are defined as complementary feeding stuffs composed mainly of minerals and containing at least 40 % crude ash (Regulation (EC) 767/2009) [3].
There are different categories of feed samples:
a)   Additives containing about 1010 colony forming units (CFU)/g;
b)   Premixtures containing about 1011 CFU/kg;
c)   Compound feeds, meal or pellets which contain about 109 CFU/kg.

This document specifies general rules for the enumeration of bacilli in feeding stuffs (additives, premixtures and compound feeds including mineral feeds) [4] that contain bacilli as a single microorganism component or in a mixture with other microorganisms. There are different categories of feed samples:
a)   Additives containing about 1010 colony forming units (CFU)/g;
b)   Premixtures containing about 1011 CFU/kg;
c)   Compound feeds, meal or pellets containing about 109 CFU/kg.

This document specifies a method for the determination of the content of the total vitamin A (retinol), vitamin E (α-tocopherol) and vitamin D3 (cholecalciferol) in animal feed using solid phase extraction (SPE) clean-up and high-performance liquid chromatography (HPLC).
NOTE   The procedure also enables determination of vitamin D2 but with the use of another internal standard. The method is fully validated only for vitamin D3.
The method has been successfully tested in collaborative trial for complete feed for broilers, pigs, and turkey, for premixture for broilers and piglets, for complementary feed for cows and mineral feed within the following ranges:
•   vitamin A: 4 365 IU/kg - 4 118 352 IU/kg;
•   vitamin E: 22 mg/kg - 13 800 mg/kg;
•   vitamin D3: 1 668 IU/kg - 1 638 150 IU/kg.
The limits of quantification were not determined within the validation study. Quantification limits of 1 100 IU for vitamin A/kg (using UV-detection), 4 mg for vitamin E/kg (using UV-detection), 2 mg for vitamin E/kg (using fluorescence detection) and 2 000 IU for vitamin D/kg (using UV-detection) should be normally achieved. Lower limits are possible provided they are validated by the user.

This document specifies a method for the determination of organic acids in animal feeding stuffs by Ion Chromatography with conductivity detection (IC-CD).
The method is intended to be used for the determination of formic acid, lactic acid, propionic acid, citric acid, fumaric acid and malic acid as active substances in feed additives, premixtures, feed materials, compound feed and water and for acetic acid in a limited manner in the same matrices. This method determines the total extractable concentration of the above mentioned organic acids and their salts.
It is advisable that the user of this standard determines the working range of the method for each organic acid. The lower limit of the working range depends on the matrix and the interferences encountered. It is advisable that a working range between 10 mg/l and 100 mg/l is achievable.
The method was successfully tested in an inter-laboratory study in concentrations between 0,02 % up to 27 % of the above mentioned organic acids.
NOTE   Limitation occurs during simultaneous determination of high concentration of lactic acid and low concentration of acetic acid. If the ratio of concentration of lactic acid to acetic acid exceeds factor 20, the determination of acetic acid is not guaranteed.
On the basis of the referred working range, sample weight and extraction volume, limits of quantification (LOQ), as calculated (Table 1) can be achievable.

This document specifies a method for the determination of benzoic acid and sorbic acid in animal feeding stuffs by high-performance liquid chromatography method with ultraviolet detection (HPLC-UV).
The method is intended to be used for the determination of benzoic acid and sorbic acid as active substances in feed additives, premixtures, feed materials and compound feed and for benzoic acid in water. This method determines the total extractable concentration of these organic acids and their salts.
It is advisable that the working range of the method is determined for each organic acid by the user of this standard. The lower limit of the working range depends on the matrix and the interferences encountered. It is advisable that a working range between 5 mg/l and 100 mg/l is accessible.
The method was successfully tested in an inter-laboratory study in concentrations between 0,02 % up to 9,0 %.
On the basis of the referred working range, sample weight and extraction volume, limits of quantification (LOQ), as calculated (Table 1) on the basis of a wavelength of 230 nm, can be achievable.

This document specifies requirements for light fire storage units providing protection against fire.
The method of test is specified to determine the ability of light fire storage units to protect paper media from the effects of fire. Two levels of fire exposure periods (LFS 30 and LFS 60) are specified using the maximum temperature increase permitted within the storage space of the light fire storage unit.
Protection after the fire exposure of 30 min (LFS 30) or 60 min (LFS 60) is not ensured by this document, but by European Standard EN 1047-1. Requirements are also specified for the test specimen, the technical documentation for the test specimen, correlation of the test specimen with the technical documentation, preparation for type testing and test procedures.
A scheme to classify the light fire storage units from the test results is also given (see Table 1).

This European Standard specifies a high performance liquid chromatography - mass spectrometry (LC-MS/MS) method for the identification of tylosin, spiramycin, virginiamycin, carbadox and olaquindox in animal feeds.
The method is suitable for the identification of low concentrations of tylosin, spiramycin, virginiamycin, carbadox and olaquindox in compound animal feeds. A limit of identification of 1 mg/kg for tylosin, spiramycin and virginiamycin, 4 mg/kg for carbadox and 3 mg/kg for olaquindox should be obtained by using the described method. The method was fully validated during a collaborative study (see Annex A).
Since tylosin, spiramycin and virginiamycin are fermentation products consisting of a mixture of several closely related compounds, the analysis is based on detection and identification of the most abundant constituents. For tylosin the marker is tylosin A, for spiramycin the marker is spiramycin I and II and for virginiamycin the marker is virginiamycin M1 and S1. The other isomers and forms can be readily detected with the same method but adjustment of the MS parameters according to the molecular mass of precursor and product ions need to be made. Carbadox and olaquindox are analysed as such.

The method makes it possible to detect and identify spiramycin, tylosin and virginiamycin in animal feeding stuffs (feed raw materials of mainly plant origin and compound feeds) excluding mineral feeds and premixtures. The limit of detection is about 2 mg/kg for spiramycin, 1 mg/kg for tylosin and 1 mg/kg for virginiamycin. In some milk replacers, it can be slightly higher than 1 mg/kg for virginiamycin.
Reported limits of detection are probably little overestimated but were fully validated during the collaborative study (see Annex B). In each laboratory, each day of analysis, spiked blank samples at 1 mg/kg for spiramycin and virginiamycin and at 0,5 mg/kg for tylosin are analysed for checking lower detection limits (see 9.2 and 9.3). These lower limits of detection are achievable, but should be established with an in-house validation first.
Some other antibiotics can interfere in the detection of these 3 specific macrolide antibiotics. The known interferences are specified in Annex A of the method.
That method should be used as a qualitative screening and/or a post-screening method (after microbiological plate test, for example). The follow-up of the antibiotics presence may be done by other analytical technics (LC and/or LC-MS technics) ([4], [10]). For confirmatory purposes, LCMS is required.

This European Standard specifies a high performance liquid chromatographic - UV detection (HPLC-UV) method for the simultaneous determination of two growth promoters Carbadox and Olaquindox contents in compound feeds and raw materials at levels ranging from the limit of quantification to 100 mg/kg.
The limit of quantification of the method has been demonstrated to be lower than 3 mg/kg for olaquindox and 4 mg/kg for carbadox.

This European Standard presents a method describing the screening on the antibiotics tylosin, virginiamycin, spiramycin, bacitracin-zinc and avoparcin at sub-additive levels in complete feeding stuffs and milk replacers by a microbiological 3-plate test.
The limit of detection of the method is 1 mg/kg for avoparcin, tylosin, spiramycin and virginiamycin, and 5 mg/kg for zinc bacitracin. The presence of other (veterinary) antibiotics may interfere with the method.
Furthermore, high concentrations of metals (Cu, Zn) may interfere. The method should be used as a qualitative screening method. Positive results can be analysed further by TLC; for confirmatory purposes LC-MS is required [1].
A lower limit of detection for zinc bacitracin (3 mg/kg) is achievable (see Table 2), but should be established with an in house validation first.

ISO 17180:2013 specifies a method for the quantitative determination of free (non-protein-bound) lysine, methionine, and threonine in commercial products and premixtures containing more than about 10 % mass fraction of the respective amino acid. It does not distinguish between d- and l-forms.

ISO 6498:2012 specifies guidelines for the preparation of test samples from laboratory samples of animal feeding stuffs, including pet foods.
The guidelines are overruled by special instructions and regulations for sample preparation demanded by specific analysis methods.

This European Standard specifies a method for the determination of decoquinate. This high-performance liquid chromatographic (HPLC) method with a fluorescence detection is applicable to the quantification of decoquinate content in complete and complementary compound feeds, medicated feeds, semi-liquid feeds, premixtures and feed additives.
The method was fully validated from LOQ to 60 000 mg/kg on different matrices during an international collaborative study [11], especially on complete compound feeds for poultry, at trace contamination level of 3 mg/kg and at European authorized level of 20 mg/kg to 40 mg/kg [12].
The limit of detection is between 0,1 mg/kg and 0,3 mg/kg and the limit of quantification is around 0,5 mg/kg. These limits were validated during the collaborative study [11], from results on the blank feed. Lower limits of detection or quantification could be reached but a single laboratory validation is then requested.

This European standard specifies a high-performance liquid chromatographic (HPLC) method for the determination of the semduramicin content at authorized level in animal feeding stuffs [2], using mass spectrometry detection or post-column derivatization and (UV)-VIS detection (hereinafter UV detection). This method is applicable to poultry feed. The limit of quantitation is 1,0 mg/kg when mass spectrometry is used for detection and 3,0 mg/kg when the detection is performed by UV with post-column derivatization. Lower limits of quantitation are achievable but this is to be validated by the user.
The method allows the discrimination of semduramicin from monensin, salinomycin, narasin, maduramicin and lasalocid.

This European Standard defines general rules for the enumeration of probiotic bifidobacteria in feed samples (additives, premixtures and feeding stuffs) that contain bifidobacteria as a single bacterial component or in a mixture with other microorganisms. This standard is not applicable for mineral feeds which are defined as complementary feeding stuffs composed mainly of minerals and containing at least 40% crude ash (Council Directive 79/373/EEC) [3].
There are different categories of feed samples:
a)   Additives containing about 1010 colony forming units (CFU)/g
b)   Premixtures containing  about 108 CFU/g
c)   Feeds, meal or pellets, which contain about 106 CFU/g and include complete feeding stuffs, and milk replacers.
The detection limit is as defined in ISO 7218.

This European Standard specifies a method for the determination of additive use of nicarbazin in animal feeding stuffs and premixtures (maximum concentration 2,5% nicarbazin) using high performance liquid chromatography. Nicarbazin is a 1:1 equimolar mixture of 4,4’-dinitrocarbanilide (DNC) and 4,6-dimethyl-2-pyriminol (HDP). Nicarbazin is generally determined by using DNC as the target compound. In this method the DNC moiety of nicarbazin is detected.
The limit of quantitation is 20 mg/kg. The limit of detection is 0,5 mg/kg
NOTE   A lower limit of quantitation may be achievable but should be validated by the user.

This European Standard specifies a high performance liquid chromatography (HPLC) method for the
determination of the content of maduramicin in feeding stuffs and premixtures.
The usual concentration of maduramicin in feedstuffs is 5 mg/kg, in premixtures 500 mg/kg. The limit of
quantification is 2 mg/kg. The limit of detection is 0,5 mg/kg.
NOTE A lower limit of quantification may be achievable but shall be validated by the user.

ISO 14183:2005 specifies a high-performance liquid chromatographic (HPLC) method for the determination of the monensin, narasin and salinomycin contents of animal feeding stuffs, supplements (dry and liquid) and mineral premixtures. The method is not applicable to drug premixes (pharmaceutical products). Lasalocid and semduramicin cannot be determined by this method.
The limit of quantitation is approximately 1 mg/kg, 2 mg/kg and 2 mg/kg for monensin, salinomycin and narasin, respectively. A lower limit of quantitation can be achievable but this is to be validated by the user.

This Technical Specification defines a polymerase chain reaction (PCR) methodology for the identification of S. cerevisiae probiotic yeast strains. Additionally, a method for the extraction of high quality DNA from yeast is suggested.

ISO 30024:2009 specifies the determination of phytase activity in feed samples.
The method does not distinguish between phytase added as a feed additive and endogenous phytase already present in the feed materials.
The method cannot be used to evaluate or compare the in vivo efficacy of the phytase product. It is not a predictive method of the in vivo efficacy of phytases present on the market as they can develop different in vivo efficacy per unit of activity.
The method is suitable and validated exclusively for the determination of phytase activity and exclusively in complete feeds.

This European Standard defines general rules for the enumeration of probiotic lactobacilli in feed samples (additives, premixtures and feeding stuffs) that contain lactobacilli as a single bacterial component or in a mixture with other microorganisms. This standard is not applicable to mineral feeds, which are defined as complementary feeding stuffs composed mainly of minerals and containing at least 40% crude ash (Council Directive 79/373/EEC [3]).
There are different categories of feed samples:
a)   Additives containing about 1010 colony forming units (CFU)/g
b)   Premixtures containing about 108 CFU/g
c)   Feeds, meal or pellets, which contain about 106 CFU/g and include complete feeding stuffs and milk replacers.
The detection limit is as defined in ISO 7218.

This European Standard defines general rules for the enumeration of probiotic pediococci in feed samples (additives, premixtures and feeding stuffs) that contain pediococci as a single bacterial component or in a mixture with other microorganisms. This standard is not applicable for mineral feeds which are defined as complementary feeding stuffs composed mainly of minerals and containing at least 40% crude ash (Council Directive 79/373/EEC) [3].
There are different categories of feed samples:
a)   Additives containing about 1010 (colony forming units) CFU/g
b)   Premixtures containing  about 108 CFU/g
c)   Feeds, meal or pellets, which contain about 106 CFU/g and include complete feeding stuffs, and milk replacers.
The detection limit is as defined in ISO 7218.

This European Standard defines general rules for the enumeration of enterococci in feed samples (additives, premixtures and feeding stuffs) that contain enterococci (E. faecium) as a single microorganism component or in a mixture with other microorganisms. This standard is not applicable to mineral feeds which are defined as complementary feedingstuffs composed mainly of minerals and containing at least 40% crude ash (Council Directive 79/373/EEC) [3].
There are different categories of feed samples:
a)   Additives containing about 1010 colony forming units (CFU)/g;
b)   Premixtures containing 108 CFU/g;
c)   Feeds, meal or pellets which contain about 106 CFU/g and include complete feeding stuffs,and milk replacers.
The detection limit is as defined in ISO 7218.

This European Standard defines general rules for the enumeration of probiotic yeasts in feed samples (additives, premixtures and feeding stuffs) that contain yeast as a single microorganism component or in a mixture with other microorganisms. The standard is not applicable to mineral feeds which are defined as complementary feedingstuffs composed mainly of minerals and containing at least 40% crude ash (Council Directive 79/373/EEC) [4].
There are different categories of feed samples:
a)   Additives which contain about 109 CFU/g to 1010 CFU/g (CFU = colony forming units).
b)   Premixtures which contain about 108 CFU/g
c)   Feeds, meal or pellets, which contain about 106 CFU/g and include complete feedingstuffs, and milk replacers.
The detection limit is as defined in ISO 7218.

This European Standard defines general rules for the enumeration of probiotic bacilli in feeds containing bacilli (Bacillus species) as a single microorganism, component or mixed with other microorganisms. This method is not applicable to mineral feeds which are defined as complementary feeding stuffs composed mainly of minerals and containing at least 40% crude ash (Council Directive 79/373/EEC) [3].
There are different categories of feed samples:
a)   Additives containing about 1010 colony forming units (CFU)/g;
b)   Premixtures containing about 108 CFU/g;
c)   Feeds, meal or pellets, which contain about 106 CFU/g and include complete feeding stuffs, and milk replacers.
The detection limits are 500 (5 x 102) colony forming units per gram (CFU/g). The limits of determination are 2 x 104 CFU/g.