SVENSK STANDARD SS-EN 17049:2018

Transkript

1 SVENSK STANDARD SS-EN 17049:2018 Fastställd/Approved: Publicerad/Published: Utgåva/Edition: 1 Språk/Language: engelska/english ICS: Identifiering av tylosin, spiramycin, virginiamycin, carbadox och olaquindox i låga halter (under tillsatsnivå) Konfirmerande analys med LC-MS Animal feeding stuffs: Methods of sampling and analysis Identification of tylosin, spiramycin, virginiamycin, carbadox and olaquindox at sub-additive levels in compound feed Confirmatory analysis by LC-MS

2 Standarder får världen att fungera SIS (Swedish Standards Institute) är en fristående ideell förening med medlemmar från både privat och offentlig sektor. Vi är en del av det europeiska och globala nätverk som utarbetar internationella standarder. Standarder är dokumenterad kunskap utvecklad av framstående aktörer inom industri, näringsliv och samhälle och befrämjar handel över gränser, bidrar till att processer och produkter blir säkrare samt effektiviserar din verksamhet. Delta och påverka Som medlem i SIS har du möjlighet att påverka framtida standarder inom ditt område på nationell, europeisk och global nivå. Du får samtidigt tillgång till tidig information om utvecklingen inom din bransch. Ta del av det färdiga arbetet Vi erbjuder våra kunder allt som rör standarder och deras tillämpning. Hos oss kan du köpa alla publikationer du behöver allt från enskilda standarder, tekniska rapporter och standardpaket till handböcker och onlinetjänster. Genom vår webbtjänst e-nav får du tillgång till ett lättnavigerat bibliotek där alla standarder som är aktuella för ditt företag finns tillgängliga. Standarder och handböcker är källor till kunskap. Vi säljer dem. Utveckla din kompetens och lyckas bättre i ditt arbete Hos SIS kan du gå öppna eller företagsinterna utbildningar kring innehåll och tillämpning av standarder. Genom vår närhet till den internationella utvecklingen och ISO får du rätt kunskap i rätt tid, direkt från källan. Med vår kunskap om standarders möjligheter hjälper vi våra kunder att skapa verklig nytta och lönsamhet i sina verksamheter. Vill du veta mer om SIS eller hur standarder kan effektivisera din verksamhet är du välkommen in på eller ta kontakt med oss på tel Standards make the world go round SIS (Swedish Standards Institute) is an independent non-profit organisation with members from both the private and public sectors. We are part of the European and global network that draws up international standards. Standards consist of documented knowledge developed by prominent actors within the industry, business world and society. They promote cross-border trade, they help to make processes and products safer and they streamline your organisation. Take part and have influence As a member of SIS you will have the possibility to participate in standardization activities on national, European and global level. The membership in SIS will give you the opportunity to influence future standards and gain access to early stage information about developments within your field. Get to know the finished work We offer our customers everything in connection with standards and their application. You can purchase all the publications you need from us - everything from individual standards, technical reports and standard packages through to manuals and online services. Our web service e-nav gives you access to an easy-to-navigate library where all standards that are relevant to your company are available. Standards and manuals are sources of knowledge. We sell them. Increase understanding and improve perception With SIS you can undergo either shared or in-house training in the content and application of standards. Thanks to our proximity to international development and ISO you receive the right knowledge at the right time, direct from the source. With our knowledge about the potential of standards, we assist our customers in creating tangible benefit and profitability in their organisations. If you want to know more about SIS, or how standards can streamline your organisation, please visit or contact us on phone +46 (0)

3 Europastandarden EN 17049:2018 gäller som svensk standard. Detta dokument innehåller den officiella engelska versionen av EN 17049:2018. The European Standard EN 17049:2018 has the status of a Swedish Standard. This document contains the official version of EN 17049:2018. Copyright / Upphovsrätten till denna produkt tillhör SIS, Swedish Standards Institute, Stockholm, Sverige. Användningen av denna produkt regleras av slutanvändarlicensen som återfinns i denna produkt, se standardens sista sidor. Copyright SIS, Swedish Standards Institute, Stockholm, Sweden. All rights reserved. The use of this product is governed by the end-user licence for this product. You will find the licence in the end of this document. Upplysningar om sakinnehållet i standarden lämnas av SIS, Swedish Standards Institute, telefon Standarder kan beställas hos SIS som även lämnar allmänna upplysningar om svensk och utländsk standard. Information about the content of the standard is available from the Swedish Standards Institute (SIS), telephone Standards may be ordered from SIS, who can also provide general information about Swedish and foreign standards. Denna standard är framtagen av kommittén för Djurfoder, SIS / TK 435 / AG 02. Har du synpunkter på innehållet i den här standarden, vill du delta i ett kommande revideringsarbete eller vara med och ta fram andra standarder inom området? Gå in på - där hittar du mer information.

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5 EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM EN February 2018 ICS English Version Animal feeding stuffs: Methods of sampling and analysis - Identification of tylosin, spiramycin, virginiamycin, carbadox and olaquindox at sub-additive levels in compound feed - Confirmatory analysis by LC-MS Aliments des animaux: Méthodes d'échantillonnage et d'analyse - Identification de la tylosine, spiramycine, virginiamycine, du carbadox et de l'olaquindix dans les aliments composés pour animaux à des concentrations inférieures à celles des additifs - Analyse de confirmation par CL-SM Futtermittel: Probenahme- und Untersuchungsverfahren - Identifizierung von Tylosin, Spiramycin, Virginiamycin, Carbadox und Olaquindox in Konzentrationen unterhalb von Zusatzstoffen in Mischfuttermitteln - Bestätigungsanalyse mittels LC- MS This European Standard was approved by CEN on 8 January 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, Serbia, 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: Rue de la Science 23, B-1040 Brussels 2018 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 17049:2018 E

6 Contents Page European foreword Scope Normative references Principle Reagents and materials General Reagents and materials Acetonitrile (LC-MS grade) Methanol (LC-MS grade) Formic acid (LC-MS grade) Tylosin Spiramycin Virginiamycin Carbadox Olaquindox Solutions HPLC Mobile phase A: Formic acid 5mM HPLC Mobile phase B: Formic acid 50 mm/ acetonitrile (10/90, v/v) Standard solutions Stock solution tylosin (500 μg/ml) Stock solution spiramycin (500 μg/ml) Stock solution virginiamycin (500 μg/ml) Stock solution carbadox (500 μg/ml) Stock solution olaquindox (500 μg/ml) Mixed stock solution Mixed stock solution Calibration standard Apparatus Sampling Sample preparation Sample pre-treatment Quality control samples Sample extraction

7 7.4 Sample purification Sample preparation for LC-MS/MS Confirmation control LC-MS/MS analysis General LC-MS/MS experimental conditions Initial test Analysis of samples Data processing and interpretation of results Data processing Recording and calculation of identification parameters Criteria for acceptance of the analytical results General Run acceptance Identification of the analyte General Retention time criterion Ion ratio criterion Test report Annex A (informative) Results of the interlaboratory study A.1 Procedure A.2 Materials A.3 Statistical analysis of results A.4 Results and interpretation - Precision Annex B (informative) Run and sample acceptance form Annex C (informative) Quantitative analysis C.1 General C.2 Procedure quantitative analysis C.2.1 Sample pre-treatment quantitative analysis C.2.2 Quality control samples C.2.3 Sample extraction C.2.4 Sample purification C.2.5 Sample preparation for LC-MS/MS C.2.6 Recovery control C.2.7 Confirmation control C.2.8 LC-MS/MS analysis

8 C LC-MS/MS experimental conditions C Initial test C Analysis of samples C.3 Data processing and interpretation of results C.3.1 Data processing C.3.2 Recording and calculation of identification parameters C.3.3 Calculation of the amount of analyte in the sample C.3.4 Calculation of recovery percentage C.4 Criteria for the acceptance of the analytical result C.4.1 General C.4.2 Run acceptance C.4.3 Identification of the analyte C General C Retention time criterion C Ion ratio criterion C.5 Notes on the procedure C.5.1 Influence of ionization suppression C.5.2 Comments on the quantitive accuracy C.5.3 Comments on the relevance of recovery percentage Annex D (informative) Run and sample acceptance form Bibliography

9 European foreword This document (EN 17049:2018) 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 August 2018, and conflicting national standards shall be withdrawn at the latest by August 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. 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. 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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. 5

10 1 Scope 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. 2 Normative references There are no normative references in this document. 3 Principle The compounds are extracted from the feed with a mixture of water and methanol. An aliquot of the liquid phase is diluted and applied to a pre-conditioned SPE column. After washing of the SPE column, compounds of interest are eluted with methanol. The obtained extract is evaporated and re-dissolved in dilute formic acid. The resulting extract is analysed by LC-MS/MS. Separation is carried out on a silicabased C18 bonded phase column and detection is performed by mass spectrometry in multiple reaction monitoring mode. The validation of this method was performed at concentration levels that were calculated on a weight (w/w) basis. Expression of working ranges in terms of w/w concentration is common practice in residue analysis of veterinary drugs, in fact Maximum Residue Limits (MRL) are exclusively expressed on a w/w basis. For feed additives however, tolerances have been expressed traditionally as microbiological activity. To translate the validation experiments concerning the level at which they were performed, to units expressed as microbiological activity, the w/w concentrations should be corrected for the microbiological potency of the preparation used for spiking experiments. 4 Reagents and materials WARNING Use all solvents and solutions in a fume hood. Wear safety glasses, protective clothing and avoid skin contact. 4.1 General All reagents are of 'Analytical reagent' grade or better unless otherwise stated. Throughout this method, water means demineralized water with a conductivity of at least 10 MΩ.cm 1. Guaranteed purity is required for each lot of reference standard. 6

11 4.2 Reagents and materials Acetonitrile (LC-MS grade) Methanol (LC-MS grade) Formic acid (LC-MS grade) Tylosin Spiramycin Virginiamycin Carbadox Olaquindox 4.3 Solutions HPLC Mobile phase A: Formic acid 5mM Measure 200 μl formic acid (4.2.3) and transfer to a volumetric flask of ml, make up to the mark with water. Filter and degas before use HPLC Mobile phase B: Formic acid 50 mm/ acetonitrile (10/90, v/v) Measure 200 μl formic acid (4.2.3) and transfer to a volumetric flask of ml, add 100 ml water and make up to the mark with acetonitrile (4.2.1). Filter and degas before use. 4.4 Standard solutions Stock solution tylosin (500 μg/ml) Weigh between 10 and 50 mg of tylosin standard substance (4.2.4) and transfer to a brown glass bottle. Calculate the required amount of methanol (4.2.2) and add that amount (on a weight basis) to obtain a standard solution of 500 μg/ml. Store this stock solution in the dark at 4-8 C. Under these conditions it is stable for at least one month Stock solution spiramycin (500 μg/ml) Weigh between 10 and 50 mg of spiramycin standard substance (4.2.5) and transfer to a brown glass bottle. Calculate the required amount of methanol (4.2.2) and add that amount (on a weight basis) to obtain a standard solution of 500 μg/ml. Store this stock solution in the dark at 4-8 C. Under these conditions it is stable for at least one month Stock solution virginiamycin (500 μg/ml) Weigh between 10 and 50 mg of virginiamycin standard substance (4.2.6) and transfer to a brown glass bottle. Calculate the required amount of methanol (4.2.2) and add that amount (on a weight basis) to obtain a standard solution of 500 μg/ml. Store this stock solution in the dark at 4-8 C. Under these conditions it is stable for at least one month. 7