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.

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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.

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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.
NOTE    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 may 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] [9]. For confirmatory purposes, LCMS is required.

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This method describes 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, 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 LCMS is recommended [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.

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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-1.
The limit of quantification of the method has been demonstrated to be better than 3 mg kg-1 for olaquindox and 4 mg kg-1 for carbadox.

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This draft European Standard defines predictive equations for the determination of 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.

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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.

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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.

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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.

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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.

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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 and T-2 toxin (T2) in the tested range of 7 µg/kg to 50 µg/kg in feed materials and 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 or better for each. This method is also applicable to the determination of 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.

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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.

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