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Vo Nederlandse norm
NEN-EN 16158 (en)
Animal feeding stuffs - Determination of semduramicin content - Liquid chromatographic method using a "tree" analytical approach
Vervangt NEN-EN 16158:2010 Ontw.
ICS 65.120
maart 2012
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NEN-EN 16158
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Als Nederlandse norm is aanvaard: - EN 16158:2012,IDT
Normcommissie 370010 "Diervoeder"
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Apart from exceptions provided by the law, nothing from this publication may be duplicated and/or published by means of photocopy, microfilm, storage in computer files or otherwise, which also applies to full or partial processing, without the written consent of the Netherlands Standardization Institute.
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NEN-EN 16158
Nederlands voorwoord Voor de in deze norm vermelde normatieve verwijzingen bestaan in Nederland de volgende equivalenten: vermelde norm
Nederlandse norm
titel
prEN ISO 6498
NEN-EN-ISO 6498
Diervoeders - Richtlijnen voor de bereiding van analysemonsters
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NEN-EN 16158:2012
EN 16158
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
February 2012
ICS 65.120
English Version
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Animal feeding stuffs - Determination of semduramicin content Liquid chromatographic method using a "tree" analytical approach Futtermittel - Bestimmung des Semduramicingehalts Flüssigkeitschromatographisches Verfahren mit verzweigter analytischer Vorgehensweise
Aliments pour animaux - Dosage de la semduramicine Chromatographie liquide utilisant une approche analytique en arbre
This European Standard was approved by CEN on 30 December 2011.
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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.
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CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, 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.
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EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2012 CEN
All rights of exploitation in any form and by any means reserved worldwide for CEN national Members.
Ref. No. EN 16158:2012: E
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NEN-EN 16158:2012
EN 16158:2012 (E)
Contents
Page
Foreword ..............................................................................................................................................................3 Scope ......................................................................................................................................................4
2
Normative references ............................................................................................................................4
3
Principle ..................................................................................................................................................4
4
Reagents .................................................................................................................................................4
5
Apparatus ...............................................................................................................................................7
6
Sampling .................................................................................................................................................8
7 7.1 7.2 7.3 7.4
Preparation of test sample ....................................................................................................................8 General ....................................................................................................................................................8 Laboratory sample .................................................................................................................................9 Test sample ............................................................................................................................................9 Test portion ............................................................................................................................................9
8 8.1 8.2 8.3 8.4 8.4.1 8.4.2 8.5 8.5.1 8.5.2 8.5.3
Procedure ...............................................................................................................................................9 Preparation of positive and negative control samples ......................................................................9 Samples extraction ................................................................................................................................9 Filtration ..................................................................................................................................................9 HPLC analysis ........................................................................................................................................9 LC-MS ......................................................................................................................................................9 LC-PCD-UV .......................................................................................................................................... 11 HPLC determination ........................................................................................................................... 13 LC-MS method..................................................................................................................................... 13 LC-PCD-UV method ............................................................................................................................ 13 System suitability ............................................................................................................................... 13
9 9.1 9.2
Calculation ........................................................................................................................................... 14 LC-MS method..................................................................................................................................... 14 LC-PCD-UV method ............................................................................................................................ 14
10 10.1 10.2 10.3
Precision .............................................................................................................................................. 15 Collaborative study............................................................................................................................. 15 Repeatability........................................................................................................................................ 15 Reproducibility .................................................................................................................................... 15
11
Test report ........................................................................................................................................... 16
Annex A.1 A.2 A.3
A (informative) Results of collaborative study ................................................................................ 17 Procedure ............................................................................................................................................ 17 Statistical analysis of results ............................................................................................................ 18 Example chromatogram ..................................................................................................................... 22
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Bibliography ..................................................................................................................................................... 25
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NEN-EN 16158:2012
EN 16158:2012 (E)
Foreword This document (EN 16158:2012) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs”, the secretariat of which is held by NEN.
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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 2012, and conflicting national standards shall be withdrawn at the latest by August 2012. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] 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.
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According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, 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.
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NEN-EN 16158:2012
EN 16158:2012 (E)
1
Scope
This European standard specifies a high-performance liquid chromatographic (HPLC) method for the determination of the semduramicin content at authorized level in animal feeding stuffs [2], using mass spectrometry detection or post-column derivatization and (UV)-VIS detection (hereinafter UV detection). This method is applicable to poultry feed. The limit of quantitation is 1,0 mg/kg when mass spectrometry is used for detection and 3,0 mg/kg when the detection is performed by UV with post-column derivatization. Lower limits of quantitation are achievable but this is to be validated by the user.
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The method allows the discrimination of semduramicin from monensin, salinomycin, narasin, maduramicin and lasalocid.
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Normative references
The following referenced documents are indispensable for the application of this protocol. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. prEN ISO 6498, Animal feeding stuffs Guidelines for sample preparation (ISO/DIS 6498)
Principle
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Semduramicin is extracted using acetonitrile with mechanical shaking during 30 min. The extracts are filtered through 0,2 µm Nylon filters. Semduramicin is determined by reverse-phase liquid chromatography using electrospray (ESI) single quadrupole mass spectrometry detection in single ion monitoring (SIM) mode (LCMS) [4] or using post-column derivatization with dimethylaminobenzaldehyde (DMAB) and spectrophotometric detection at 598 nm (LC-PCD-UV) [5]. If the detection used is ESI-MS the quantitation is performed through a standard addition approach. When LC-PCD-UV is used the quantitation is performed through external standard calibration.
Reagents
Use only reagents of recognized analytical grade, unless otherwise specified. LC-MS.
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4.1 4.1.1
Water, HPLC grade, or equivalent (e.g. Milli-Q purified water).
4.1.2
Acetonitrile, HPLC gradient grade, minimum 99,9 % purity.
4.1.3
Methanol, HPLC grade or hypergrade LC-MS.
4.1.4
Ammonium formate, HPLC grade.
4.1.5
Mobile phase.
4.1.5.1
Ammonium formate solution, c = 20 mmol/l.
Accurately weigh 1,25 g to the nearest 0,01 g of ammonium formate (4.1.4) into a 1 000 ml volumetric flask. Dissolve in water (4.1.1) and make up to 1 000 ml of volume with water. Prepare fresh solutions monthly.
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NEN-EN 16158:2012
EN 16158:2012 (E)
4.1.5.2
HPLC mobile phase.
Mix methanol (4.1.3) and ammonium formate solution (4.1.5.1) in proportion of 90+10 (v+v). Filter under vacuum using a solvent filtration system (5.11) and Nylon filters (5.13). 4.2
LC-PCD-UV.
In addition to the reagents 4.1.1, 4.1.2, 4.1.3 and 4.1.4: Sulphuric acid, minimum 98 % purity.
4.2.2
Dimethylaminobenzaldehyde (DMAB), minimum 99 % purity.
4.2.3
Formic acid, minimum 98 % purity.
4.2.4
Mobile phase.
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4.2.1
4.2.4.1
Post-column reaction reagent.
NOTE +8 °C).
4.2.4.2
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In a 500 ml volumetric flask (5.7) add first about 250 ml cold methanol (4.1.3) then 15 ml sulphuric acid (4.2.1). Dissolve 15 g DMAB (4.2.2) in the mixture. Cool down and make up to 500 ml with methanol (4.1.3). Filter under vacuum using the equipment in (5.11) and a membrane filter (5.12). Store in a refrigerator (from +2 °C to + 8 °C). This reagent is stable for 28 days. The methanol used for preparing the post-column reaction reagent should be kept refrigerated (from +2 °C to
Ammonium formate solution, c = 100 mmol/l at pH = 3.
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HPLC mobile phase (solvent blank).
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4.2.4.3
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Accurately weigh 6,30 g to the nearest 0,01 g of ammonium formate (4.1.4) into a 1 000 ml volumetric flask (5.7). Dissolve in 900 ml water (4.1.1). Adjust the pH to 3,0 using formic acid (4.2.3) and make up to 1 000 ml with purified water. Prepare fresh monthly.
Mix methanol (4.1.3) and ammonium formate solution (4.2.4.2) in proportion of 90+10 (v+v). Filter under a vacuum using a solvent filtration system (5.11) and Nylon filters (5.13). 4.3
Reference standards LC-PCD-UV method.
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WARNING — Avoid inhalation of and exposure to the toxic standard materials and solutions thereof. Work in a fume-hood when handling the solvents and solutions. Wear safety glasses and protective clothing. Declaration of purity is required for each lot of reference standard. 4.3.1
Semduramicin sodium standard, minimum 93 % purity expressed as semduramicin.
NOTE Available from Phibro Animal Health Corporation, Third Floor 65 Challenger Road Ridgefield Park, NJ 076602103 USA. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results.
4.4
Reference standards LC-MS method.
In addition to the reference standard 4.3.1:
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