This document specifies a gas chromatographic method with electron capture detection (ECD) for the determination of organochlorine pesticides (OCPs) in compound feeds and oil and fats.
The method is applicable to animal compound feed, oils and fats and fish meals with a water content up to about 20 % by weight and oil/fatty samples containing residues of one or more of the following OCPs, toxaphene and some of their isomers and degradation products:
—   aldrin;
—   dieldrin;
—   dichlorodiphenyltrichloroethane (DDT) (the isomers op'-DDT, pp'-DDT, pp'-TDE (pp'-DDD), and pp'-DDE);
—   endosulfan (as the sum of α-/β-isomers);
—   endrin;
—   hexachlorobenzene (HCB);
—   hexachlorocyclohexane isomers α-HCH (α-BHC), β-HCH (β-BHC), γ-HCH (γ-BHC or lindane);
For the following OCPs, the method is considered a screening method. Additional in-house validation is required for reporting validated data.
—   chlordane (as the sum of chlordane isomers and oxychlordane);
—   endosulfan-sulphate;
—   delta-keto-endrin;
—   heptachlor (as the sum of heptachlor and heptachlor epoxide);
—   photo-heptachlor;
—   cis
—   and trans-nonachlor.
A limit of quantification (LOQ) for the mentioned OCPs of 5 µg/kg is intended to be obtained. However, 10 µg/kg applies for heptachlor, aldrin, endrin, dieldrin, and endosulfan (α-/β– and sulphate). Individual laboratories are responsible for ensuring that the equipment that they use, achieves these limits of quantifications. The LOQs apply to the individual OCPs.

  • Standard
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This document specifies a gas chromatographic mass spectrometric (GC/MS) method for the determination of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in animal feeding stuffs and oil.
The method is applicable to animal feeding stuffs consisting of less than 20 % by mass and oil/fatty samples containing residues of one or more of the following OCPs and PCBs and some of their isomers and degradation products:
   aldrin;
   dieldrin;
   chlordane, as the sum of chlordane isomers and oxychlordane;
   dichlorodiphenyltrichloroethane (DDT), as the sum of isomers op'-DDT, pp'-DDT, pp'-TDE (pp'-DDD), and pp'-DDE;
   endosulfan, as the sum of α-/β-isomers and endosulfan-sulphate;
   endrin, as the sum of endrin and delta-keto-endrin;
   heptachlor, as the sum of heptachlor and heptachlor epoxide;
   hexachlorobenzene (HCB);
   hexachlorocyclohexane isomers α-HCH (α-BHC), β-HCH (β-BHC), γ-HCH (γ-BHC or lindane);
   photo heptachlor;
   cis- and trans-nonachlor;
   non dioxin-like PCBs (ndl-PCBs), as the sum of PCB 28, 52, 101, 138, 153 and 180.
The method has been fully validated by a collaborative trial for the substances and corresponding ranges (ng/g) noted in Table 1.
The method has not been fully validated for oxychlordane, endrin ketone, cis- and trans-nonachlor and photo heptachlor in all matrices.
The method is not applicable to chlorocamphene (toxaphene), a complex mixture of polychlorinated camphenes. Chlorocamphene has a very distinctive chromatographic profile and is easily recognisable by GC/ECD. Positive identification of the toxaphene isomers can be performed by negative chemical ionisation mass spectrometry (NCI-MS), electron impact tandem mass spectrometry (EI MS × MS) or electron impact high resolution mass spectrometry (EI-HRMS), which is not within the scope of this method.
A limit of quantification (LOQ) for the mentioned organochlorine pesticides of 5 ng/g should normally be obtained. However, 10 ng/g applies for heptachlor aldrin, endrin, dieldrin, and endosulfan (α-, β- and sulphate). For the ndl-PCBs an LOQ of 0,5 to 1,0 ng/g should be obtained. The LOQs mentioned apply to the individual compounds (i.e. not the sum of two or more compounds). Individual laboratories are responsible for ensuring that the equipment that they used will achieve these LOQs. On customers' demand the standard may be applied to solely the analysis of PCBs or OCPs.

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This document is applicable to the determination of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), (together termed ‘dioxins’ (PCDD/Fs)) and dioxin-like PCBs and non-dioxin-like PCBs (dl-PCBs and ndl-PCBs) in animal feeding stuffs. Collaborative studies have been carried out. The method is suitable for the determination of dioxins, dl-PCBs and ndl-PCBs at the appropriate MRL in compound feed and ingredients e.g. oil, mineral clay. The method is applicable to samples containing trace level amounts of one or more dioxins, dioxin-like PCBs and non-dioxin-likePCBs. The limit of quantification (LOQ) is
-   0,05 pg/g (OCDD/F = 0,1 pg/g) for the relevant individual congeners of dioxins/furans,
-   0,05 pg/g for non-ortho PCBs,
-   10 pg/g for mono-ortho PCBs, and
-   100 pg/g for non-dioxin-like-PCBs.
For determination of dioxins and dioxin-like PCBs, the procedure can be used as confirmatory method as defined by Commission Regulation (EC) No 152/2009 for dioxins and dl-PCB in feed [1]. Confirmatory methods as described in this standard are high-resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS) methods. If only the analysis of non-dioxin-like PCBs is required, a GC-LRMS method can be used (e.g. EN 15741 [2]) provided that appropriate analytical performance criteria are met in the relevant range for the matrix of interest.
This document is split into four modules. Each module describes a part of the whole procedure (see Figure 1 and Figure 2) to be followed:
a)   Module A:   Description of standards which might be used;
b)   Module B:   Description of extraction procedures;
c)   Module C:   Description of clean-up procedures;
d)   Module D:    GC/HRMS determination.
Each module describes a part of the whole method as well as, when applicable, alternatives which should be equivalent. Each module has to be regarded as an example. Combining modules and/or alternatives gives a highly flexible, "performance based" procedure. It is permitted to modify the method if all performance criteria laid down in Commission Regulation (EC) No 152/2009 [1] are met.
Any deviation of the described method, combination of modules needs to be recorded as part of the QA/QC procedures of accredited laboratories and should be available on request.
Figure 1 - Flow scheme for the determination of dioxins, dl-PCBs and non-dioxin-like-PCBs in feed
Figure 2 - Flow scheme for the determination of dioxins, dl-PCBs and non-dioxin-like-PCBs in oil and fat

  • Standard
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This document's method of analysis is applicable for the determination of:
-   deoxynivalenol (DON) in the tested range of 100 µg/kg to 3 300 µg/kg,
-   aflatoxin B1 (AfB1) in the tested range of 2,5 µg/kg to 440 µg/kg,
-   fumonisin B1 (FB1) in the tested range of 690 µg/kg to 7 500 µg/kg,
-   fumonisin B2 (FB2) in the tested range of 200 µg/kg to 2 500 µg/kg,
-   T-2 toxin in the tested range of 7,5 µg/kg to 360 µg/kg,
-   HT-2 toxin in the tested range of 14 µg/kg to 1 800 µg/kg,
-   zearalenone (ZEN) in the tested range of 30 µg/kg to 600 µg/kg, and
-   ochratoxin A (OTA) in the tested range of 10 µg/kg to 230 µg/kg
in cereals and cereal-based compound feed by liquid-chromatography tandem mass spectrometry (LC-MS/MS). The actual working ranges could extend beyond the tested ranges.

  • Standard
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This document method is applicable for the determination of theobromine in compound feed by liquid chromatography with UV detection in the tested range of 27 to 307 mg/kg. This method has been validated using complementary compound feed for adult dogs and complementary compound feedstuff for horses. The actual working range may extend beyond the tested range. Alternative chromatography conditions using liquid chromatography tandem mass spectrometry (LC-MS/MS) are also provided for the validated range of 49 to 307 mg/kg. This method has also been shown to be fit for purpose for the determination of theobromine in baking chocolate by both HPLC-UV and LC-MS/MS.

  • Standard
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This document describes a method for the determination of individual ergot alkaloids and tropane alkaloids in unprocessed cereals and cereal-based compound feeds by high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS).
This method has been successfully validated by collaborative trial in the following matrices: rye, barley, wheat, complete feed for bovine, porcine and poultry. Validation in buckwheat produced acceptable results, but the relative standard reproducibility was higher for most analytes in comparison with the other matrices. This may be related to the matrix. The validated range of the method is approximately 10 to 250 µg/kg for individual alkaloids. Determination of concentrations above 250 µg/kg is possible by applying a higher spiking level and dilution of the sample extract, but this has not been validated in the collaborative trial.
The method is applicable for the determination, by means of one-point standard addition to the sample, of ergocornine in the tested range of 12 µg/kg to 221 µg/kg, ergocorninine in the tested range of 9 µg/kg to 196 µg/kg, ergocristine in the tested range of 14 µg/kg to 312 µg/kg, ergocristinine in the tested range of 12 µg/kg to 258 µg/kg, α-ergocryptine in the tested range of 10 µg/kg to 184 µg/kg, α-ergocryptinine in the tested range of 8 µg/kg to 171 µg/kg, ergometrine in the tested range of 12 µg/kg to 174 µg/kg, ergometrinine in the tested range of 3 µg/kg to 172 µg/kg, ergosine in the tested range of 12 µg/kg to 226 µg/kg, ergosinine in the tested range of 9 µg/kg to 273 µg/kg, ergotamine in the tested range of 11 µg/kg to 443 µg/kg, ergotaminine in the tested range of 10 µg/kg to 273 µg/kg, atropine in the tested range of 16 µg/kg to 252 µg/kg and scopolamine in the tested range of 15 µg/kg to 246 µg/kg.

  • Standard
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This document gives guidance to those involved in designing, executing and evaluating interlaboratory comparison studies for multi-analyte methods of analysis, developed by CEN/TC 327 “Animal feeding stuffs: Methods of sampling and analysis” and its working groups.
For the validation of multi-analyte methods their particularities must be considered which might necessitate deviations from the prescribed validation protocols. This study provides information whether the method is fit for its purpose and which performance can be expected in practical work while at the same time keeping the necessary effort for the study organizer and the participating laboratories minimal.
Next to the abovementioned aspects regarding interlaboratory comparison studies, this document also gives guidance on the preceding steps, viz. in-house validation and preparation of the method protocol. Guidance is also given on the transferability of the method protocol and the familiarization with the method protocol through a training study, elements that – depending on the specific method – could be included in the design of the study.

  • Technical report
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  • Technical report
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This document specifies a high-performance liquid chromatographic (HPLC) mass spectrometric (MS) method for screening and quantification of melamine and cyanuric acid in the concentration range between 1 mg/kg and 100 mg/kg feed.
The method is validated in an international collaborative trial for melamine in complete feed, complementary feed, feed material, milk replacer and pet food including canned pet food in the range between 1 mg/kg and 80 mg/kg with particular regard to the maximum level of 2,5 mg/kg as established by the European Commission.
Laboratory experiences have shown that the method is also applicable for cyanuric acid in the same concentration range in complete feed (n = 7), complementary feed (n = 6), feed material (n = 7, resp. 9), milk replacer (n = 7) and pet food (n = 7) including canned pet food.
Since the LC-MS/MS sensitivity for cyanuric acid is lower than for melamine, it has to be ensured that the LC-MS/MS system is in excellent working order. The method is applicable to feeding stuffs but not tested for pre-mixtures and feed additives.
Quantification of concentrations above 100 mg/kg is possible, but the method has to be validated by the operator.

  • Standard
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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.

  • Standard
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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.

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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).

  • Standard
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This document specifies performance criteria for the selection of single-laboratory validated or collaborative-trial validated methods of analysis of elements and their chemical species in feed. The terms and definition of the relevant parameters for method validation are included. The performance requirements and characteristics are provided. This document may serve as a guide:
-   to assess the quality of new European Standard methods under validation;
-   to review the quality of previous collaborative trials;
-   to confirm the extension of the scope of an already published European Standard applied to other analyte concentrations or matrices; or
-   to evaluate the fitness-for-purpose of single-validated methods.
The performance criteria can apply to methods dedicated to the determination of heavy metals, trace elements, major elements and minerals.

  • Technical specification
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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.

  • Standard
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This European Standard specifies a method for the determination of trace elements, heavy metals and other elements in animal feed by inductively coupled plasma mass spectrometry (ICP-MS). The method is used to determine As, Cd, Co, Cu, Fe, Hg, Mn, Mo, Pb, Se, Tl, U and Zn in the extraction solution after pressurized digestion. For the determination of extractable lead in minerals and feeds containing phyllosilicates (e.g. kaolinite clay) wet digestion with nitric acid should be used. The method described is suitable for use in quadrupole instruments equipped either with or without additional technology to reduce molecular ion interferences (e.g. collision or reaction technologies) as well as in high-resolution sector-field systems.
The method was fully statistically tested and evaluated in a collaborative trial comprising eight animal feeding stuff samples for the elements As, Cd, Co, Cu, Fe, Hg, Mn, Mo, Pb, Se, Tl, U and Zn. For elements with a HORRAT value higher than 2 (e.g. mercury, see Annex A) the method is more applicable as a screening method and not for confirmatory purposes. High-resolution sector-field ICP-MS was not tested in the validation ring trial.
The limit of quantification for each element is dependent on the sample matrix as well as the instrument. For the elements Co, Mn, Mo, Pb, Tl, U a limit of quantification of 0,10 mg/kg should normally be obtained, for the elements Fe and Zn 5,0 mg/kg, while for Cd 0,03 mg/kg, Hg 0,04 mg/kg and As 0,05 mg/kg should normally be quantifiable.
Details on the successfully tested working range for each element are described in this standard.

  • Standard
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ISO 12099:2017 gives guidelines for the determination by near infrared spectroscopy of constituents such as moisture, fat, protein, starch and crude fibre and parameters such as digestibility in animal feeding stuffs, cereals and milled cereal products.
The determinations are based on spectrometric measurement in the near infrared spectral region.

  • Standard
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This European standard specifies a method for the determination of iodine in animal feeding stuffs by inductively coupled plasma mass spectrometry (ICP-MS) following extraction with an alkaline solution.
This method was successfully tested in the range of 0,70 to 631 mg/kg in following animal feeds: seaweed meal, mineral premixture, fish meal, plant based ingredient, marine based compound feed and a synthetic iodine solution.

  • Standard
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This European Standard specifies a method for the determination of the elements cadmium and lead in animal feeding stuffs by graphite furnace atomic absorption spectrometry (GF-AAS) after pressure digestion.
The method was successfully tested in the range of 0,015 to 5,65 mg/kg for Cd and 0,18 to 40,3 mg/kg for lead in 11 animal feeds: 2 complete feeds (pig feed, sheep feed), 2 complementary feeds (2 mineral feeds), 1 mineral premixture, 4 feed materials (MgO, 2 phosphates, CaCO3) and 2 feed additives (CuSO4, bentonite).
For the determination of extractable lead in minerals and feeds, containing phyllosilicates (e.g. kaolinite clay) wet digestion with nitric acid should be used.
The method limit of quantification for each element is dependent on the sample matrix as well as the instrument. For cadmium a limit of quantification of 0,05 mg/kg should normally be obtained while for lead, a limit of quantification of 0,5 mg/kg should be obtained.

  • Standard
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This European Standard specifies a method for the determination of the elements calcium, sodium, phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt in animal feeding stuffs by inductively coupled plasma atomic emission spectrometry (ICP-AES) after pressure digestion.
The method was fully statistically tested and evaluated for the elements calcium, sodium, phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt within the following 11 animal feeds: 2 complete feeds (pig feed, sheep feed), 3 complementary feeds (3 mineral feeds), 1 mineral premixture, 3 feed materials (MgO, phosphate, CaCO3) and 2 feed additives (CuSO4, bentonite).
For potassium and sulphur the HORRAT values were mostly higher than 2. Therefore, for these elements the method is more applicable as a screening method and not for confirmatory purposes.
Other elements like molybdenum, lead, cadmium, arsenic were not fully statistically tested and evaluated within 11 animal feeding stuff samples because these elements did not occur in concentrations higher than the limit of quantification in most of these samples. A single laboratory validation is therefore necessary for the use of this multi element method for these elements.
For the determination of extractable lead in minerals and feeds, containing phyllosilicates (e.g. kaolinite clay) wet digestion with nitric acid should be used.
The method limit of quantification for each element is dependent on the sample matrix as well as on the instrument. The method is not applicable for determination of low concentrations of elements. A limit of quantification of 1 mg/kg should normally be obtained.
NOTE 1   This method can also be used for the determination in products with high content (> 5 %) of the element to be measured, but for this purpose the accuracy of the method has to be checked individually.
NOTE 2   Results of this European Standard EN 15621 may be higher than of EN 15510 because EN 15621 is using pressure digestion mode.

  • Standard
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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.

  • Standard
    26 pages
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This European Standard specifies the inductively coupled plasma atomic emission spectroscopy (ICP-AES) method for the determination of the elements calcium, sodium, phosphorus, magnesium, potassium, iron, zinc, copper, manganese, cobalt, molybdenum and lead.
The elements calcium, sodium, phosphorus, magnesium, potassium, iron, zinc, copper, manganese, cobalt, molybdenum and lead are extracted either in feeds mainly consisting of organic matter after dry ashing and dissolving in hydrochloric acid or in feeds mainly consisting of inorganic matter after wet digestion with hydrochloric acid.
For the determination of extractable lead in minerals and feeds containing phyllosilicates (e.g. kaolinite clay) wet digestion with nitric acid should be used.
The method was successfully tested for:
-   calcium, sodium, phosphorus, magnesium, potassium, iron, zinc, copper, manganese, cobalt and molybdenum in the following animal feeding stuffs: 2 complete feeds (pig feed, sheep feed), 1 feed material  (phosphate), 1 mineral premixture and 2 complementary feeds (2 mineral feeds),
-   lead in 2 feed materials (phosphate, CaCO3), 2 feed additives (Bentonite, CuSO4), 1 complementary feed (mineral feed)
The method detection limit for each element is dependent on the sample matrix and the instrument. The method is not applicable for the determination of a low concentration of elements. The limit of quantification should be 3 mg/kg or lower.
This method also applies for the determination in products with high element content (>5 %). For this purpose the accuracy of the method has to be checked individually.
NOTE 1   EN 15621 uses the pressure digestion mode, therefore lower results may be obtained with the described method in this standard.

  • Standard
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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.

  • Standard
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This European Standard specifies predictive formulae for the determination of metabolizable energy (ME) in
-   Products of vegetable or animal origin, in their natural state, fresh or preserved, such as meat, offal, milk products, cooked starch sources; highly digestible special products such as milk substitutes or diets for enteral nutrition;
-   Complete or complementary products derived from the industrial processing for cats and dogs.

  • Standard
    32 pages
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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.

  • Standard
    16 pages
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This method of analysis is applicable to the determination of HT-2 toxin (HT2) in the tested range of 22 µg/kg to 178 µg/kg, T-2 toxin (T2) in the tested range of 7 µg/kg to 50 µg/kg, Deoxynivalenol (DON) in the tested range of 88 µg/kg to 559 µg/kg, and Zearalenone (ZON) in the tested range of 14 µg/kg to 430 µg/kg in cereals and cereal-based compound animal feed. The actual working ranges may extend beyond the tested ranges. It is the responsibility of the laboratory to prove that the limit of quantitation (LOQ) for HT-2 and T-2 toxin is ≤ 10 µg/kg, for DON ≤ 100 µg/kg, and for ZON ≤ 20µg/kg.

  • Standard
    31 pages
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ISO 13904:2016 specifies a method for determination of the total and free tryptophan (Trp) content in feeding stuffs (e.g. complete and complementary feeds, supplementary feeds, raw materials, ingredients, and concentrates) and determination of free tryptophan in commercial pure substances and premixtures containing more than 2 % of tryptophan.
It does not distinguish between D- and L-forms.

  • Standard
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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.

  • Standard
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This European Standard specifies an Ion-Selective Electrode method (ISE) after hydrochloric acid treatment for the determination of fluoride from animal feeding stuffs. The content of fluoride (F-) corresponds to that of fluorine (F) specified in Commission Regulation (EU) 574/2011[3].
This European Standard is strictly based on several conventions such as those contained in the following example:
EXAMPLE   0,5 g test portion for extraction of fluoride from animal feeds by means of an acid treatment with 20 ml of 1 mol/l hydrochloric acid solution at ambient temperature (20 °C to 25 ºC) for 20 min. The pH is controlled and adjusted to 5,5 in the buffered test solution before determination of fluoride by ISE using standard addition technique.
The method was successfully tested in an interlaboratory study in concentrations between 100 mg/kg up to 500 mg/kg. If this method is followed strictly, then theoretically all concentrations from 40 mg/kg up to 4 000 mg/kg can be analysed within the linear calibration function.
Only for concentrations lower than 40 mg/kg is the use of an interpolation technique required instead of standard addition Annex C.
The quantification limit for fluoride using the conventions of the method including the standard addition technique is 40 mg/kg or lower than 2,5 mg/kg when using interpolation Annex C.

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This European Standard specifies a method for the determination of mercury in animal feeding stuffs by Cold-Vapour Atomic Absorption Spectrometry (CVAAS) after microwave pressure digestion. The limit of quantification in the test solution should be 0,25 µg/l or lower. Using a test portion of 0,5 g and a volume of the test solution of 25 ml a limit of quantification of 0,0125 mg/kg or lower should be obtained.

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This European Standard describes a procedure for the determination of inorganic arsenic in animal feeding stuffs of marine origin by Solid Phase Extraction (SPE) and Hydride Generation Atomic Absorption Spectrometry (HG-AAS). The method has been successfully tested in a collaborative trial with a working range from 0,19 mg/kg to 2,7 mg/kg (HORRATvalues < 2). The LOQ of the method is usually approximately 0,1 mg/kg or lower.

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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.

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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.

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This European Standard is applicable to the quantitative analysis of (bound and free) hydrocyanic acid (HCN) in feed materials of plant origin and compound feed by High Performance Liquid Chromatography (HPLC).
The method is validated from 10 mg HCN/kg to 350 mg HCN/kg. When the method is used outside this range it should be validated at least within the laboratory. A limit of quantification of 2 mg HCN/kg should normally be obtained.

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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.

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This European Standard specifies a method for the determination of total arsenic in animal feeding stuffs by hydride generation atomic absorption spectrometry (HGAAS) after microwave pressure digestion. The limit of quantification is 0,5 µg/l of the test solution. Using a test portion of 0,5 g, a volume of the test solution of 25 ml and an aliquot of 5 ml for pre-reduction the limit of quantification is 0,125 mg/kg in the feed material.
NOTE   For feed materials containing organic arsenic species from compounds of marine origin (i.e. arsenobetaine and tetramethylarsine oxide) a higher digestion temperature of the microwave system up to 300 °C may be necessary in order to enable the hydridisation of these arsenic compounds and in order to determine all different kinds of arsenic species in the corresponding feeding stuffs. Alternatively, the digestion procedure of Annex D can be used if the microwave system does not reach higher temperatures up to 300 °C to ensure complete mineralization for HGAAS determination.

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This European Standard specifies a method for the determination of selenium in animal feeding stuffs by hydride generation atomic absorption spectrometry (HGAAS) after microwave pressure digestion.
The method was successfully tested by an inter-laboratory study of CEN/TC 327/WG 4 in the range of 0,25 mg/kg to 74 mg/kg.
The limit of quantification is 0,5 µg/l of the test solution which corresponds to the calibration standard 2. Using a test portion of 0,5 g and a volume of the test solution of 25 ml after pressure digestion the limit of quantification is calculated as 0,125 mg/kg in the feed material.
NOTE   A lower limit of quantification could be achieved – each laboratory has to prove it.

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This European Standard is applicable to the determination of Fumonisin B1 & B2 (FB1 & FB2) in compound animal feed at levels starting from 3 mg/kg up to 16 mg/kg.

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This European Standard specifies a method for the determination of Ochratoxin A (OTA) in cereal based animal feed using immunoaffinity for clean-up followed by liquid-chromatography with fluorescence detection.
NOTE   The validated mass fraction range was 39 µg/kg to 338 µg/kg OTA.

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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.

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This International Standard is applicable to the determination of zearalenone in animal feed at concentrations from 30 µg/kg to 3 000 µg/kg.

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This Standard is applicable to the determination of deoxynivalenol (DON) in animal compound feed at concentrations of 150 μg/kg up to at least 4 000 μg/kg.

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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.

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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.

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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.

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ISO 5983-2:2009 specifies a method for the determination of nitrogen content of animal feeding stuffs according to the Kjeldahl method, and a method for the calculation of the crude protein content.
It is suitable for use as a semi-micro rapid routine method using block digestion, copper catalyst, and steam distillation into boric acid.
The method is applicable to the determination of greater than 0,5 % mass fraction Kjeldahl nitrogen in animal feeding stuffs, pet foods, and their raw materials.
The method does not measure oxidized forms of nitrogen nor heterocyclic nitrogen compounds.
The method does not distinguish between protein nitrogen and non-protein nitrogen.

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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.

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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.

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ISO 16634-1:2008 specifies a method for the determination of the total nitrogen content and the calculation of crude protein content of oilseeds and animal feeding stuffs.
This method, like the Kjeldahl method, does not distinguish between protein nitrogen and non-protein nitrogen. For the calculation of protein content, various conversion factors are used.
This method is not applicable to milk and milk products.

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This Technical Specification describes the quantitative determination of specific sugars (glucose, fructose, galactose, sucrose, maltose, and lactose) in dry animal feeding stuffs at the g/kg level by a sophisticated high performance anion exchange chromatography in combination with pulsed amperometric detection (HPAEC-PAD).

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