SIST EN 16877:2017
(Main)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
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 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.
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von T-2- und HT-2-Toxinen, Deoxynivalenol und Zearalenon in Einzelfuttermitteln und Mischfuttermitteln mittels LC-MS
Dieses Analysenverfahren ist für die Bestimmung von HT-2-Toxin (HT2) im geprüften Bereich von 22 µg/kg bis 178 µg/kg, von T-2-Toxin (T2) im geprüften Bereich von 7 µg/kg bis 50 µg/kg, von Deoxynivalenol (DON) im geprüften Bereich von 88 µg/kg bis 559 µg/kg und von Zearalenon (ZON) im geprüften Bereich von 14 µg/kg bis 430 µg/kg in Getreide und in Mischfuttermitteln auf Getreidebasis anwendbar. Die tatsächlichen Arbeitsbereiche dürfen über die geprüften Bereiche hinausgehen. Es liegt in der Verantwortung des Laboratoriums nachzuweisen, dass die Bestimmungsgrenze (LOQ, en: limit of quantitation) für die HT-2- und T 2 Toxine 10 µg/kg, für DON 100 µg/kg und für ZON 20 µg/kg ist.
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
La présente méthode d'analyse s'applique au dosage de la toxine HT-2 (HT2) dans la gamme validée de 22 µg/kg à 178 µg/kg, de la toxine T-2 (T2) dans la gamme validée de 7 µg/kg à 50 µg/kg, du déoxynivalénol (DON) dans la gamme validée de 88 µg/kg à 559 µg/kg, et de la zéaralénone (ZON) dans la gamme validée de 14 µg/kg à 430 µg/kg, dans les céréales et les aliments composés pour animaux à base de céréales. Les gammes de travail réelles peuvent s'étendre au-delà des gammes validées. Il incombe au laboratoire de démontrer que la limite de quantification (LOQ) est ≤ 10 µg/kg pour les toxines HT-2 et T-2, ≤ 100 µg/kg pour DON et ≤ 20 µg/kg pour ZON.
Krma - Metode vzorčenja in analize - Določevanje toksinov T-2 in HT-2, deoksinivalenola in zearalenona v sestavinah krme in krmni mešanici z LC-MS
Ta metoda analize se uporablja za določevanje toksina HT-2 (HT2) v preskušanem obsegu od 22 µg/kg do 178 µg/kg in toksina T-2 (T2) v preskušanem obsegu od 7 µg/kg do 50 µg/kg v sestavinah krme in krmni mešanici. Dejanski delovni obsegi lahko presegajo preskušane obsege. Laboratorij mora dokazati, da je mejna vrednost za količinsko ovrednotenje (LOQ) za toksina HT-2 in T-2 10 µg/kg ali ustreznejša za posamezen toksin. Ta metoda se uporablja tudi za določevanje deoksinivalenola (DON) v preskušanem obsegu od 88 µg/kg do 559 µg/kg in zearalenona (ZON) v preskušanem obsegu od 14 µg/kg do 430 µg/kg.
General Information
Standards Content (Sample)
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
SIST EN 16877:2017
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
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre:
Avenue Marnix 17,
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
EN 16877:2016 (E) 2 Contents Page European foreword . 4 Introduction . 5 1 Scope . 6 2 Normative references . 6 3 Principle . 6 4 Reagents . 6 5 Apparatus . 8 6 Procedures. 9 6.1 Sample preparation . 9 6.2 Extraction . 9 6.3 Test solution . 10 6.4 Spiking procedure . 10 7 Measurements . 11 7.1 General . 11 7.2 LC conditions. 11 7.3 MS conditions . 11 7.4 Batch composition . 11 7.5 Peak identification . 11 7.6 Determination of DON, HT2, T2, and ZON in calibration or and test solutions . 11 7.7 Calibration . 11 8 Determination of mass fraction . 12 9 Precision . 13 9.1 Interlaboratory study . 13 9.2 Repeatability . 13 9.2.1 General . 13 9.2.2 HT-2 toxin . 13 9.2.3 T-2 toxin . 13 9.2.4 DON. 13 9.2.5 ZON . 13 9.3 Reproducibility . 13 9.3.1 General . 13 9.3.2 HT-2 toxin . 14 9.3.3 T-2 toxin . 14 9.3.4 DON. 14 9.3.5 ZON . 14 10 Test report . 14 Annex A (informative)
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
EN 16877:2016 (E) 3 B.2 Example 2 . 21 B.2.1 General . 21 B.2.2 LC conditions . 22 B.2.3 MS conditions . 22 B.3 Example 3 . 23 B.3.1 General . 23 B.3.2 LC conditions . 24 B.3.3 MS conditions . 24 Annex C (informative)
Examples of chromatograms according to the settings of the examples in Annex B . 26 Bibliography . 31
SIST EN 16877:2017
EN 16877:2016 (E) 4 European foreword This document (EN 16877:2016) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs - Methods of sampling and analysis”, the secretariat of which is held by NEN. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by May 2017, and conflicting national standards shall be withdrawn at the latest by May 2017. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN shall not be held responsible for identifying any or all such patent rights. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association.
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.
SIST EN 16877:2017
EN 16877:2016 (E) 5 Introduction 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. SIST EN 16877:2017
EN 16877:2016 (E) 6 1 Scope 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). SIST EN 16877:2017
EN 16877:2016 (E) 7 4.11 T-2 toxin (T2). 4.12 Zearalenone (ZON). 4.13 13C15-Deoxynivalenol (13C15-DON). 4.14 13C22-HT-2 toxin (13C22-HT2). 4.15 13C24-T-2 toxin (13C24-T2). 4.16 13C18-Zearalenone (13C18-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 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
EN 16877:2016 (E) 8 Table 1 — Example calibration solutions Volume of Multitoxin working solution (4.18.) Total mass of analyte per vial [µl] [ng]
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
EN 16877:2016 (E) 9 5.11 Syringe filter: Small internal volume, Nylon, Pore size: 0,2 µm Nylon. 5.12 LC-MS: 5.12.1 Solvent delivery system capable of delivering a binary gradient at flow rates appropriate for the analytical column in use with sufficient accuracy. 5.12.2 Auto liquid sampler (ALS) capable of injecting an appropriate volume of injection solution with sufficient accuracy, cross-contamination below 0,1 %. 5.12.3 Analytical column capable of separating the four analytes with the following performance: Peak asymmetry factor at 10 % height: 0,9 < As < 1,4; minimum apparent retention factor for any of the four analytes: k
· 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
EN 16877:2016 (E) 10 — Vortex thoroughly until test portion is completely suspended. Do not let this suspension stand for more than 15 min to prevent effects due to enzymatic activities. — Add 16,0 ml of ethyl acetate (4.5). The accuracy of this volume is critical for the accuracy of the final result! — Extract for 27 to 33 min in a sonicator or by vigorously shaking (5.8). — Add between 7,2 and 8,8 g of sodium sulphate (4.8). — Instantly shake hard for 5 s. — Let stand for 10 to 20 min. — Centrifuge (5.10) at RCF 3 000 g for at least 1 min to aid settlement of particulate matter and phase separation. — If wanted for possible repeats: Transfer the extract (organic layer) into clean glass vial for storage of up to 7 d at 2 °C to 10 °C in the dark. — Transfer 500 µl of the extract (organic layer) into a deactivated glass vial (5.6) for further processing (the accuracy of this volume is critical for the accuracy of the final result!). 6.3 Test solution — Add 25 µl of the Multi ISTD stock solution (4.19) to the aliquot of the extract and/or the calibration solutions (4.20) (the accuracy of this volume is critical for the accuracy of the final result!). — Dry down the aliquot of the extract and/or the calibration solutions in an evaporator (5.9) with a gentle stream of dry nitrogen at 60 °C. — Add 250 µl of the organic mobile phase modifier used for LC-MS to the dry residue for reconstitution. — Vortex thoroughly for at least 10 s. — Add 250 µl deionized water (4.1) to the reconstituted extract. — Vortex thoroughly for at least 5 s. — Transfer the test solution into an ALS vial (5.7); if solution is turbid it may be filtered through a syringe filter (5.11). NOTE It has been shown that even very turbid samples can be injected without any negative effects to the life time of column and LC provided that appropriate in-line filters or guard columns are used. 6.4 Spiking procedure If recovery needs to be determined execute the following in duplicate: To three times 2 g of a material free of DON, HT2, T2, and ZON add three different volumes of the Multitoxin stock solution (4.17) such that 3 contamination levels across the calibration range result. Distribute the solutions evenly over the materials, mix to further distribute the spike, and leave for a minimum of 5 h to a maximum of 18 h. Proceed to 6.2 “Extraction” second step. NOTE Addition of 360, 980, and 1 600 4.17, Note 1 has been shown to work well. SIST EN 16877:2017
EN 16877:2016 (E) 11 7 Measurements 7.1 General The LC-MS system shall meet the requirements laid out in 5.12 and its sub-entries. 7.2 LC conditions Choose an analytical column, mobile phase, gradient settings, and injection volume that let you meet the requirements in 5.12.3 (for examples see Annex B). 7.3 MS conditions Choose an ion source with sufficient ionization yield for the four analytes and ion source settings such that a stable spray is achieved. Choose for each analyte an appropriate precursor ion (adducts of the molecule with a Proton, Sodium, Ammonia, etc. in positive mode, or deprotonation, etc. in negative mode). If more than one precursor ion per analyte is detectable choosing the strongest is a good starting point. But one shall be aware that the choice of precursor ion will affect repeatability and, by that, LOD and LOQ. For SRM select two product ions in the MS/MS spectrum for each selected precursor ion of each analyte. Set up SRM transitions with these precursor/ product ion combinations (for SRM examples see Annex B). The selected MS settings shall be such that for a relevant feed material with a contamination of ca. 90 µg/kg DON, 10 µg/kg HT-2 toxin, 10 µg/kg T-2 toxin, and 10 µg/kg ZON, prepared according to Clause 6, signal-to-noise ratios (peak-to-peak) of larger than 10 are obtained (see Annex C). 7.4 Batch composition Always start a batch of measurements with a reagent blank run to prove non-contamination of the system. Then inject the calibration solutions once again followed by a reagent blank to check for possible carry-over. Subsequently inject the test solutions. At the end of the batch reinject the calibration solutions for a second run. 7.5 Peak identification Identify an analyte peak in the test solution by plotting the extracted ion currents of the analyte and its respective labelled analogue and then A) comparing the retention time of the analyte with the retention time of the respective labelled analogue (difference shall be smaller than 0,25 times peak width (FWHM)), and B) comparing the ratio of the two measured transitions of the analyte with that of a calibration solution of comparable signal intensity. For example chromatograms see Annex C. 7.6 Determination of DON, HT2, T2, and ZON in calibration or and test solutions Inject aliquots of the calibration and test solutions (6.3) onto the column using identical conditions. For each injection calculate the ratio of the peak area of the analyte divided by the peak area of the respective labelled analogue. These peak area ratios will be used in all subsequent calculations. For peak area determination integrate the extracted ion current of the transition with the least interferences (best signal-to-noise ratio). If both transitions of a given analyte are equally well suited integrate the sum of both. 7.7 Calibration Plot the peak area ratios of all the measured calibration solutions against the corresponding total masses in the calibration solution of DON, HT2, T2, and ZON separately. Do not use means of the SIST EN 16877:2017
EN 16877:2016 (E) 12 multiple injections! With weighted least-square regression over all data estimate slope and possible intercept of each of the four calibration functions (DON, HT2, T2, ZON). Check for significance of the intercept (95 % confidence level) and test for linearity (use e.g. a residuals vs fitted-values plot, a lack-of-fit test (95 % confidence), Mandel's fitting test (95 % confidence)). If a nonlinearity is indicated identify the cause and, if necessary, rerun the analyses. 8 Determination of mass fraction To calculate the mass fractions (An,Sw) of a specific analyte in the test portion use the following model formula: −×××011ISTD,SEtOAcAn,SISTD,CAliqSmRw= (1) where An,Sw is the mass fraction of analyte in the test portion [ng/g]; R is the mean of the peak area ratios of replicate injections; 1 is the slope, estimated with weighted least-square regression from calibration data (7.7) [area ratio/ng]; 0 is the intercept, estimated with weighted least-square regression from calibration data (becomes zero if not significant (see 7.7)) [area ratio]; ISTD,Sm is the mass of the labelled analogue in the test solution [g]; ISTD,Cm is the mass of the labelled analogue in the calibration solution [g]; AliqV is t
...
SLOVENSKI STANDARD
oSIST prEN 16877:2015
01-september-2015
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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
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von T-2- und HT
-2-Toxinen, Deoxynivalenol und Zearalenon in Einzelfuttermitteln und Mischfuttermitteln
mittels LC-MS
Aliments des animaux - Méthodes d'échantillonnage et d'analyse - Dosage par LC-MS
des toxines T-2 et HT-2, du déoxynivalénol et de la zéaralénone dans les matières
premières pour aliments et les aliments composés
Ta slovenski standard je istoveten z: prEN 16877
ICS:
65.120 Krmila Animal feeding stuffs
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
oSIST prEN 16877:2015 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
oSIST prEN 16877:2015
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oSIST prEN 16877:2015
EUROPEAN STANDARD
DRAFT
prEN 16877
NORME EUROPÉENNE
EUROPÄISCHE NORM
May 2015
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 Futtermittel - Probenahme- und Untersuchungsverfahren -
d'analyse - Dosage par LC-MS des toxines T-2 et HT-2, du Bestimmung von T-2- und HT-2-Toxinen, Deoxynivalenol
déoxynivalénol et de la zéaralénone dans les matières und Zearalenon in Einzelfuttermitteln und Mischfuttermitteln
premières pour aliments et les aliments composés mittels LC-MS
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee CEN/TC 327.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to
provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and
shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16877:2015 E
worldwide for CEN national Members.
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Contents Page
Foreword .4
1 Scope .5
2 Normative references .5
3 Principle .5
4 Reagents .5
4.1 Water (deionized) .5
4.2 Water (LC-MS grade, double-distilled water or water of grade 1 as defined in EN ISO 3696) .5
4.3 Methanol (LC-MS grade) .5
4.4 Methanol (p.a.) .5
4.5 Ethyl acetate (p.a.) .5
4.6 Formic acid (98 %–100 %, LC-MS grade) .5
4.7 Acetonitrile (LC-MS grade) .6
4.8 Sodium sulphate .6
4.9 Deoxynivalenol (DON) .6
4.10 HT-2 toxin (HT2) .6
4.11 T-2 toxin (T2) .6
4.12 Zearalenone (ZON) .6
13 13
4.13 C15-Deoxynivalenol ( C -DON) .6
15
13 13
4.14 C22-HT-2 toxin ( C -HT2) .6
22
13 13
4.15 C24-T-2 toxin ( C -T2) .6
24
13 13
4.16 C -Zearalenone ( C -ZON) .6
18 18
4.17 Multitoxin stock solution .6
4.18 Multitoxin working solution .7
4.19 Multi internal standard (ISTD) stock solution .7
4.20 Calibration .7
4.21 Quality control material .8
5 Apparatus .8
5.1 Mill .8
5.2 Mixer .8
5.3 Conical polypropylen screw-cap centrifuge tubes 50 mL with caps .8
5.4 Volumetric flasks .8
5.5 Pipettors .8
5.6 Deactivated glass vials .8
5.7 Auto Liquid Sampler (ALS) vials .8
5.8 Shaker or Sonicator .8
5.9 Evaporator .8
5.10 Centrifuge .8
5.11 Syringe filter .8
5.12 LC-MS .8
5.12.1 Solvent delivery system .8
5.12.2 Auto liquid sampler (ALS) .9
5.12.3 Analytical column .9
5.12.4 Mass spectrometer .9
5.13 Balance .9
6 Procedures .9
6.1 Sample preparation .9
6.2 Extraction .9
6.3 Test solution . 10
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6.4 Spiking procedure . 10
7 Measurements . 11
7.1 General . 11
7.2 LC conditions . 11
7.3 MS conditions . 11
7.4 Batch composition . 11
7.5 Peak identification . 11
7.6 Determination of DON, HT2, T2, and ZON in calibration or and test solutions . 11
7.7 Calibration . 12
8 Determination of mass fraction . 12
9 Precision. 13
9.1 Interlaboratory study . 13
9.2 Repeatability . 13
9.2.1 HT-2 toxin . 13
9.2.2 T-2 toxin . 13
9.2.3 DON . 13
9.2.4 ZON . 13
9.3 Reproducibility . 13
9.3.1 HT-2 toxin . 14
9.3.2 T-2 toxin . 14
9.3.3 DON . 14
9.3.4 ZON . 14
10 Test report . 14
Annex A (informative) Precision data . 15
Annex B (informative) Examples . 20
B.1 Example 1 . 20
B.2 Example 2 . 21
B.3 Example 3 . 23
Annex C (informative) Example chromatograms . 25
Bibliography . 30
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Foreword
This document (prEN 16877:2015) has been prepared by Technical Committee CEN/TC 327 “Animal feeding
stuffs - Methods of sampling and analysis”, the secretariat of which is held by NEN.
This document is currently submitted to the CEN Enquiry.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
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1 Scope
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, Water for analytical laboratory use ― Specification and test methods (ISO 3696)
3 Principle
Two gram of finely ground and homogeneous test material is suspended in water. After addition of 16,0 mL
ethyl acetate the sample is agitated for 30 min. Then sodium sulphate is added to facilitate phase separation
and after 10 min to 20 min 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. 500 µL of the organic phase, an
equivalent of 1/16th of a gram of the test material, are mixed with stable-isotope labelled analogues of the
analytes and evaporated to dryness in deactivated glass vials. After reconstitution of the dry extract with
250 µL of organic mobile phase modifier, addition of 250 µL of water, and thorough mixing the analytes are
quantified with a Liquid Chromatography-Mass Spectrometry (LC-MS) system.
4 Reagents
4.1 Water (deionized)
4.2 Water (LC-MS grade, double-distilled water or water of grade 1 as defined in
EN ISO 3696)
4.3 Methanol (LC-MS grade)
WARNING — Methanol is hazardous and handling shall be carried out inside a fume cupboard. Appropriate
safety equipment (lab coat, goggles, gloves) shall be worn.
4.4 Methanol (p.a.)
WARNING — Methanol is hazardous and handling shall be carried out inside a fume cupboard. Appropriate
safety equipment (lab coat, goggles, gloves) shall be worn.
4.5 Ethyl acetate (p.a.)
WARNING — Ethyl acetate is hazardous and handling shall be carried out inside a fume cupboard.
Appropriate safety equipment (lab coat, goggles, gloves) shall be worn.
4.6 Formic acid (98 % – 100 %, LC-MS grade)
WARNING — Formic acid is hazardous and handling shall be carried out inside a fume cupboard. Appropriate
safety equipment (lab coat, goggles, gloves) shall be worn.
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4.7 Acetonitrile (LC-MS grade)
WARNING — Acetonitrile is hazardous and handling shall be carried out inside a fume cupboard. Appropriate
safety equipment (lab coat, goggles, gloves) shall be worn.
4.8 Sodium sulphate
anhydrous, granulated
4.9 Deoxynivalenol (DON)
WARNING — Deoxynivalenol is highly toxic. Gloves and safety glasses shall be worn at all times and all
standard and sample preparation stages shall be carried out in a fume cupboard.
4.10 HT-2 toxin (HT2)
WARNING — HT-2 toxin is highly toxic. Gloves and safety glasses shall be worn at all times and all standard
and sample preparation stages shall be carried out in a fume cupboard.
4.11 T-2 toxin (T2)
WARNING — T-2 toxin is highly toxic. Gloves and safety glasses shall be worn at all times and all standard
and sample preparation stages shall be carried out in a fume cupboard.
4.12 Zearalenone (ZON)
WARNING — Zearalenone is highly toxic. Gloves and safety glasses shall be worn at all times and all
standard and sample preparation stages shall be carried out in a fume cupboard.
13 13
4.13 C15-Deoxynivalenol ( C -DON)
15
13
WARNING — C -Deoxynivalenol is highly toxic. Gloves and safety glasses shall be worn at all times and all
15
standard and sample preparation stages shall be carried out in a fume cupboard.
13 13
4.14 C22-HT-2 toxin ( C -HT2)
22
13
WARNING — C -HT-2 toxin is highly toxic. Gloves and safety glasses shall be worn at all times and all
22
standard and sample preparation stages shall be carried out in a fume cupboard.
13 13
4.15 C24-T-2 toxin ( C -T2)
24
13
WARNING — C -T-2 toxin is highly toxic. Gloves and safety glasses shall be worn at all times and all
24
standard and sample preparation stages shall be carried out in a fume cupboard.
13 13
4.16 C -Zearalenone ( C -ZON)
18 18
13
WARNING — C -Zearalenone is highly toxic. Gloves and safety glasses shall be worn at all times and all
18
standard and sample preparation stages shall be carried out in a fume cupboard.
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 %.
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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
shown to work well. This solution is stable for three months in the dark at 2 °C –8 °C.
NOTE 2 Compare a new stock solution against the old one by adding 25 µL of each into separate deactivated vials
(5.6) and proceeding as described in “Test solution” (6.3).
NOTE 3 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 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
13 13 13 13
A mixture containing C -DON (4.13), C -HT-2 toxin (4.14), C -T-2 toxin (4.15), and C -ZON (4.16) in
15 22 24 18
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 °C – 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”.
NOTE Table 1 below shows example calibration levels using the solutions described in the notes above.
Once it has been shown that there is linearity the number of levels may be adjusted to local needs and
requirements.
Table 1— Calibration solutions
Volume of Multitoxin working Total mass of analyte
solution (4.18) per vial
[µL] [ng]
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
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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 mL, 5 mL, and 10 mL
5.5 Pipettors
Adjustable 10 µL - 100 µL and adjustable 100 µL – 1 000 µL, properly calibrated.
5.6 Deactivated glass vials
Silanized glass vials, e.g. 4 mL 45 × 14,7 mm.
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 °C - 60 °C with a constant flow of dry nitrogen.
5.10 Centrifuge
Capable of generating a relative centrifugal force (RCF) of 3 000 g.
5.11 Syringe filter
Small internal volume, Nylon, Pore size: 0,2 µm Nylon.
5.12 LC-MS
5.12.1 Solvent delivery system
Capable of delivering a binary gradient at flow rates appropriate for the analytical column in use with sufficient
accuracy.
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5.12.2 Auto liquid sampler (ALS)
Capable of injecting an appropriate volume of injection solution with sufficient accuracy, cross-contamination
below 0,1 %.
5.12.3 Analytical column
Capable of separating the four analytes with the following performance:
Peak asymmetry factor at 10 % height: 0,9 < As < 1,4; minimum apparent retention factor for any of the four
analytes: k ≥ 2; minimum plate number for any of the four analytes: N ≥ 1 200; 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
Balance with readability d = 0,001 g or better.
6 Procedures
6.1 Sample preparation
It is important that the laboratory receives a laboratory sample which is truly representative and has not been
damaged or altered during transport or storage. Laboratory samples should be taken and prepared in
accordance with European legislation where applicable [1] [2]. 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, for instance, 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 g 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 mL to 8,8 mL of deionized water (4.1).
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— Vortex thoroughly until test portion is completely suspended. Do not let this suspension stand for more
than 15 min to prevent effects due to enzymatic activities.
— Add 16,0 mL of ethyl acetate (4.5, the accuracy of this volume is critical for the accuracy of the final
result!).
— Extract for 27 min to 33 min in a sonicator or by vigorously shaking (5.8).
— Add between 7,2 g and 8,8 g of sodium sulphate (4.8).
— Instantly shake hard for 5 s.
— Let stand for 10 min to 20 min.
— Centrifuge (5.10) at RCF 3 000 g for at least 1 min to aid settlement of particulate matter and phase
separation.
— If wanted for possible repeats: Transfer the extract (organic layer) into clean glass vial for storage of up to
7 days at 2 °C to 10 °C in the dark.
— Transfer 500 µL of the extract (organic layer) into a deactivated glass vial (5.6) for further processing (the
accuracy of this volume is critical for the accuracy of the final result!).
6.3 Test solution
— Add 25 µL of the Multi ISTD stock solution (4.19) to the aliquot of the extract and/or the calibration
solutions (4.20) (the accuracy of this volume is critical for the accuracy of the final result).
— Dry down the
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