BS EN-16877-2016 pdf free download.Animal feeding stuffs: Methods of sampling and analysis — Determination of T-2 and HT-2 toxins, Deoxynivalenol and Zearalenone, in feed materials and compound feed by LC-MS
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.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use – Specification and test methods (ISO 3696:1987)
3 Principle
Finely ground and homogeneous test material is suspended in water. After addition of ethyl acetate the sample is agitated. Then sodium sulphate is added to facilitate phase separation and after a delay the sample is centrifuged to pellet particulate matter at the bottom of the extraction tube. The organic phase is transferred to a clean vial for possible storage. An aliquote of the organic phase is mixed with stable-isotope labelled analogues of the analytes and evaporated to dryness in deactivated glass vials. After reconstitution of the dry extract with organic mobile phase modifier and water, and thorough mixing, the analytes are quantified with a Liquid Chromatography-Mass Spectrometry (LC-MS) system.
4 Reagents
WARNING The method described in this standard implies the use of reagents that pose a hazard to health. The standard does not claim to address all associated safety problems. It is the responsibility of the user of this standard to take appropriate measures for the health and safety protection of the personnel prior to use of the standard and to ensure that regulatory and legal requirements are complied with.
4.1 Water (deionized).
4.2 Water (LC-MS grade, double-distilled or water of grade 1 as defined in EN ISO 3696:1995).
4.3 Methanol (LC-MS grade).
4.4 Methanol (p.a.).
4.5 Ethyl acetate (p.a.).
4.6 Formic acid (98-100 %, LC-MS grade).
4.7 Acetonitrile (LC-MS grade).
4.8 Sodium sulfate, anhydrous, granulated.
4.9 Deoxynivalenol (DON).
4.10 HT-2 toxin (HT2).
4.11 T-2 toxin (T2).
4.12 Zearalenone (ZON).
4.13 13 C 15 -Deoxynivalenol ( 13 C 15 -DON).
4.14 13 C 22 -HT-2 toxin ( 13 C 22 -HT2).
4.15 13 C 24 -T-2 toxin ( 13 C 24 -T2).
4.16 13 C 18 -Zearalenone ( 13 C 18 -ZON).
4.17 Multitoxin stock solution:
A mixture containing Deoxynivalenol (4.9), HT-2 toxin (4.10), T-2 toxin (4.11), and Zearalenone (4.12)in neat acetonitrile (4.7) at relevant concentrations.
When preparing this solution the certified purities of the mycotoxin reference materials need to be properly accounted for. In any case the purities shall be ≥ 95 %.
NOTE 1 3,2 µg/ml DON, 0,5 µg/ml HT-2 toxin, 0,3 µg/ml T-2 toxin, and 0,3 µg/ml ZON in neat acetonitrile have been used during the collaborative study. This solution is stable for three months in the dark at 2–8 °C.
To compare a new stock solution against an old one add 25 µl of each into separate deactivated vials (5.6) and proceed as described in “Test solution” (6.3).
NOTE 2 If 6.4“Spiking procedure” is executed at least 6 ml of the stock solution are needed.
4.18 Multitoxin working solution: Dilute Multitoxin stock solution (4.17) with Methanol (4.3) such that the resulting concentration in the working solution is applicable to the calibration range of the different compounds. Only prepare enough volume for one full calibration. NOTE Adding 188 µl of the Multitoxin stock solution described in 4.17, Note 1 to a 3 ml volumetric flask and making up to the mark with methanol will result in a solution containing 0,2 µg/ml DON, 0,031 µg/ml HT-2 toxin, 0,019 µg/ml T-2 toxin, and 0,019 µg/ml ZON in methanol/acetonitrile (94/6, v/v).
4.19 Multi internal standard (ISTD) stock solution: A mixture containing 13 C 15 -DON (4.13), 13 C 22 -HT-2 toxin (4.14), 13 C 24 -T-2 toxin (4.15), and 13 C 18 -ZON (4.16) in neat acetonitrile (4.7) at the same concentrations as the respective native compounds in the Multitoxin stock solution (4.17). NOTE This solution is stable for three months in the dark at (2–8) °C. 4.20 Calibration: To six deactivated glass vials (5.6) add different volumes of the Multitoxin working solution (4.18) such that six equidistant calibration levels across the calibration range result. Proceed as described in 6.3, “Test solution”. Table 1 below shows example calibration levels using the solution described in the Note to 4.18 above. Once it has been shown that there is linearity the number of levels may be adjusted to local needs and requirements.BS EN-16877 pdf download.