SIST EN 16877:2017

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.+7Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von T-2- und HT-2-Toxinen, Deoxynivalenol und Zearalenon in Einzelfuttermitteln und Mischfuttermitteln mittels LC-MSAliments des animaux - Méthodes d'échantillonnage et d'analyse - Dosage par CL-SM des toxines T-2 et HT-2, du déoxynivalénol et de la zéaralénone dans les matières premières pour aliments et les aliments composésAnimal 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-MS71.040.50Fizikalnokemijske analitske metodePhysicochemical methods of analysis65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 16877:2016SIST EN 16877:2017en,fr,de01-februar-2017SIST EN 16877:2017SLOVENSKI
STANDARD
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16877
November 2016 ICS 65.120; 71.040.50
English Version
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
Aliments des animaux - Méthodes d'échantillonnage et d'analyse - Dosage par CL-SM des toxines T-2 et HT-2, du déoxynivalénol et de la zéaralénone dans les matières premières pour aliments et les aliments composés
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von T-2- und HT-2-Toxinen, Deoxynivalenol und Zearalenon in Einzelfuttermitteln und Mischfuttermitteln mittels LC-MS This European Standard was approved by CEN on 26 September 2016.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
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B-1000 Brussels © 2016 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16877:2016 E SIST EN 16877:2017

Precision data . 15 Annex B (informative)
Examples . 20 B.1 Example 1 . 20 B.1.1 General . 20 B.1.2 LC conditions. 20 B.1.3 MS conditions . 21 SIST EN 16877:2017

Examples of chromatograms according to the settings of the examples in Annex B . 26 Bibliography . 31
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
¶ 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). SIST EN 16877:2017

· 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 13C15-DON (4.13), 13C22-HT-2 toxin (4.14), 13C24-T-2 toxin (4.15), and 13C18-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. SIST EN 16877:2017

DON HT-2 T-2 ZON 25 5 0,78 0,48 0,48 180 36 5,6 3,4 3,4 335 67 10 6,4 6,4 490 98 15 9,3 9,3 645 129 20 12 12 800 160 25 15 15 4.21 Quality control material: An appropriate material with natural contamination or fortification of the tested mycotoxins which is sufficiently stable. 5 Apparatus 5.1 Mill: Single mill or multiple mills capable of comminuting test materials to particle sizes of < 500 µm. 5.2 Mixer capable of sufficiently homogenizing the comminuted test materials. NOTE A tumble mixer that uses a folding action either through moving paddles or fins, or an end-over-end movement has shown to work well. 5.3 Conical polypropylen screw-cap centrifuge tubes, 50 ml with caps. 5.4 Volumetric flasks: 3, 5, and 10 ml. 5.5 Pipettors: adjustable (10-100) µl and adjustable (100-1 000) µl. 5.6 Deactivated glass vials: Silanized glass vials, minimum volume 1,5 ml. 5.7 Auto Liquid Sampler (ALS) vials of appropriate size for the Auto Liquid Sampler in use. 5.8 Shaker or Sonicator. 5.9 Evaporator capable of maintaining a stable temperature in the range of 30 - 60 °C with a constant flow of dry nitrogen. 5.10 Centrifuge capable of generating a relative centrifugal force (RCF) of 3 000 g. SIST EN 16877:2017

· 2; minimum plate number for any of the four analytes: N
·
s t r râ minimum resolution between two adjacent analyte peaks: Rs
· 4. 5.12.4 Mass spectrometer: An instrument capable of performing selected reaction monitoring (SRM) with a sufficiently wide dynamic range. Any ionization source giving sufficient yield may be employed. 5.13 Balance with readability d = 0,001 g or better. 6 Procedures 6.1 Sample preparation Laboratory samples should be taken and prepared in accordance with European legislation ([1], [2]) where applicable or, in any other case, with EN ISO 6498. The laboratory sample should be finely ground and thoroughly mixed using a mill (5.1) and a mixer (5.2) or another process for which complete homogenization has been demonstrated before a test portion is removed for analysis. The recommended way is to comminute the laboratory sample in several steps. Beginning with the totality of the laboratory sample each step consists of taking a representative aliquot of the previous step after sufficient homogenization. This aliquot is then comminuted to the next smaller particle size until a subsample of ca. 50 g of the final particle size is obtained. It is of utmost importance that the test portion is taken from a subsample which is sufficiently homogenous with a particle size of
¶ 500 µm. Care should be taken to not overheat the sample during this process. In all instances everything should be at room temperature before any kind of manipulation takes place. 6.2 Extraction Some of the steps described below are more critical for the accuracy of the results than others. These steps are marked as such and should be carried out with the necessary attention. A scale-up of the test portion size is deemed to be acceptable if such a need is assumed. In that case the amounts of added water, ethyl acetate, and sodium sulphate need to be increased at the same rate, f.i. scale-up by factor of 2: 4 g test portion, 16 ml water, 32 ml ethyl acetate, 16 g sodium sulphate. In no way shall a scale-up be seen as replacement for proper sample preparation (6.1). — For the test portion weigh 1,9 to 2,1 g of the homogeneous sample into a conical polypropylene screw-cap tube (5.3), round and record the weight to the second decimal (the accuracy of this weight is critical for the accuracy of the final result!). — Add 7,2 to 8,8 ml of deionized water (4.1). SIST EN 16877:2017
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