CEN/TC 327/WG 5 - Natural toxins

This document specifies a method for the determination of individual intact glucosinolates in feed materials including oilseeds and oilseed products and in compound feeds by high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS).
The method specified in this document has been successfully validated by collaborative trial in the following matrices: rape seed, camelina seed, Brassica oleracea seeds, mixed oilseeds, rape seed flakes, compound feed for bovine, porcine and poultry.
The method is applicable for the quantitative determination of epiprogroitrin, glucoalyssin, glucoarabin, glucobrassicanapin, glucobrassicin, glucocamelinin, glucoerucin, glucoiberin, gluconapin, gluconapoleiferin, gluconasturtiin, glucoraphanin, glucoraphenin, glucotropaeolin, homoglucocamelinin, 4-hydroxyglucobrassicin, 4-methoxyglucobrassicin, neoglucobrassicin, progoitrin, sinalbin and sinigrin.
The concentration ranges tested in the collaborative trial for each individual glucosinolate and for the total glucosinolate content are summarized in Table 1.
[table not represented]

This document specifies a method for the quantitative determination of pyrrolizidine alkaloids (PA) in the concentration ranges shown in Table 1 in complete and supplementary feed and in forages by liquid chromatography tandem mass spectrometry (LC-MS/MS) after solid phase extraction (SPE) clean-up.
Table 1 - Summary of concentration ranges per PA tested in the collaborative trial
NOTE 1   A second method was part of the method validation collaborative main trial. For this method PA-N-Oxides are reduced by adding zinc powder to the extract of the feed material. The following steps correspond to the first and main method. Quantitative results for each PA except the otonecine type PA senkirkine represent the sum of the free PA base and its corresponding N-oxide.
NOTE 2   Due to insufficient numbers of data for some analyte-matrix combinations statistical evaluation was not valid for standardization. Received data indicated the methods applicability in experienced laboratories with appropriate quality assurance measures. Therefore, the method description is included as an informative annex (Annex D).

This document specifies a method for the determination of free gossypol, extractable by acidified acetonitrile/water, in cottonseed, cottonseed products and compound feeds by liquid chromatography with tandem mass spectrometry (LC-MS/MS).
The method described in this document has been successfully validated in the range of 69 mg/kg to 5 950 mg/kg by collaborative trial in the following matrices: cottonseed, cottonseed products (cake/meal, hulls) and compound feeds for bovine, porcine and poultry.
NOTE   It is possible to reach quantification limits of approximatively 5 mg/kg in compound feeds. The method might be applicable at lower and at higher concentrations than the concentration range validated in the collaborative trial. However, this needs to be assessed by in-house validation.

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.

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.

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.

This document specifies performance criteria for the selection of single-laboratory validated or collaborative study validated methods of analysis of mycotoxins in feed. The terms and definition of the relevant parameters for method validation are included. The performance requirements and characteristics are provided. This document could 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 mycotoxins.

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.