1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationAnimal feeding stuffs Determination of semduramicin content Liquid chromatographic method using a “tree” analytical approachBS EN 16158:2012National forewordThis British Standard
2、 is the UK implementation of EN 16158:2012.The UK participation in its preparation was entrusted to Technical CommitteeAW/10, Animal feeding stuffs.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the ne
3、cessary provisions of acontract. Users are responsible for its correct application. The British Standards Institution 2012Published by BSI Standards Limited 2012ISBN 978 0 580 67001 5ICS 65.120Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was p
4、ublished under the authority of the StandardsPolicy and Strategy Committee on 31 March 2012.Amendments issued since publicationAmd. No. Date Text affectedBRITISH STANDARDBS EN 16158:2012EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16158 February 2012 ICS 65.120 English Version Animal feeding
5、 stuffs - Determination of semduramicin content -Liquid chromatographic method using a “tree“ analytical approach Aliments pour animaux - Dosage de la semduramicine - Chromatographie liquide utilisant une approche analytique en arbre Futtermittel - Bestimmung des Semduramicingehalts - Flssigkeitschr
6、omatographisches Verfahren mit verzweigter analytischer Vorgehensweise This European Standard was approved by CEN on 30 December 2011. 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 s
7、tandard 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 i
8、n 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
9、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. EUROPEAN COMMITTEE FOR STANDARDIZATION COMI
10、T EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR 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: EBS EN 16158:2012EN 16158:2012 (E) 2 Contents Page Fore
11、word 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Apparatus .76 Sampling .87 Preparation of test sample 87.1 General 87.2 Laboratory sample .97.3 Test sample 97.4 Test portion 98 Procedure .98.1 Preparation of positive and negative control samples 98.2 Samples extraction 98.3 Filtra
12、tion 98.4 HPLC analysis 98.4.1 LC-MS 98.4.2 LC-PCD-UV 118.5 HPLC determination . 138.5.1 LC-MS method . 138.5.2 LC-PCD-UV method 138.5.3 System suitability . 139 Calculation . 149.1 LC-MS method . 149.2 LC-PCD-UV method 1410 Precision 1510.1 Collaborative study. 1510.2 Repeatability 1510.3 Reproduci
13、bility 1511 Test report . 16Annex A (informative) Results of collaborative study 17A.1 Procedure 17A.2 Statistical analysis of results 18A.3 Example chromatogram . 22Bibliography . 25BS EN 16158:2012EN 16158:2012 (E) 3 Foreword This document (EN 16158:2012) has been prepared by Technical Committee C
14、EN/TC 327 “Animal feeding stuffs”, 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 2012, and conflicting national standards shall be withdrawn at th
15、e 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
16、by the European Commission and the European Free Trade Association. 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,
17、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. BS EN 16158:2012EN 16158:2012 (E) 4 1 Scope This European
18、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
19、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. The method allo
20、ws the discrimination of semduramicin from monensin, salinomycin, narasin, maduramicin and lasalocid. 2 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 la
21、test edition of the referenced document (including any amendments) applies. prEN ISO 6498, Animal feeding stuffs Guidelines for sample preparation (ISO/DIS 6498) 3 Principle Semduramicin is extracted using acetonitrile with mechanical shaking during 30 min. The extracts are filtered through 0,2 m Ny
22、lon filters. Semduramicin is determined by reverse-phase liquid chromatography using electrospray (ESI) single quadrupole mass spectrometry detection in single ion monitoring (SIM) mode (LC-MS) 4 or using post-column derivatization with dimethylaminobenzaldehyde (DMAB) and spectrophotometric detecti
23、on 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. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise sp
24、ecified. 4.1 LC-MS. 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
25、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. BS EN 16158:2012EN 16158:2012 (E) 5 4.1.5.2 HPLC mobile phase. Mix methanol (4.
26、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: 4.2.1 Sulphuric acid, minimum 98 % purity. 4.2.2 Dimethylaminobe
27、nzaldehyde (DMAB), minimum 99 % purity. 4.2.3 Formic acid, minimum 98 % purity. 4.2.4 Mobile phase. 4.2.4.1 Post-column reaction reagent. 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
28、 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. NOTE The methanol used for preparing the post-column reaction reagent should be kept re
29、frigerated (from +2 C to +8 C). 4.2.4.2 Ammonium formate solution, c = 100 mmol/l at pH = 3. 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
30、to 1 000 ml with purified water. Prepare fresh monthly. 4.2.4.3 HPLC mobile phase (solvent blank). 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 standar
31、ds LC-PCD-UV method. 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
32、Semduramicin sodium standard, minimum 93 % purity expressed as semduramicin. NOTE Available from Phibro Animal Health Corporation, Third Floor 65 Challenger Road Ridgefield Park, NJ 07660-2103 USA. This information is given for the convenience of users of this European Standard and does not constitu
33、te 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: BS EN 16158:2012EN 16158:2012 (E) 6 4.4.1 Nigericin sodium standard, minimum 98 % purity
34、 to be used as internal standard (I.S.). NOTE Available from Calbiochem, A Brand of EMD Biosciences, Inc. 10394 Pacific Center Court, San Diego, CA 92121 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
35、 named. Equivalent products may be used if they can be shown to lead to the same results.” 4.5 Standard solutions. Protect all standard solutions from daily light. 4.5.1 Semduramicin stock standard solution, ca. 1 mg/ml. Accurately weigh 10 mg to the nearest 0,1 mg of semduramicin sodium standard (4
36、3.1) into a 10 ml volumetric flask (5.7). Note down the exact weight of semduramicin sodium. Dissolve in methanol (4.1.3) and make up to 10 ml with methanol. Store at -20 C and protected from light. Prepare freshly every 3 months. Determine the experimental concentration of the semduramicin stock s
37、olution using the reference standard purity value provided by the supplier expressed as semduramicin using Equation (1). PmCs=10(1) where Csis the experimental concentration of semduramicin in the stock standard in mg/ml; P is the purity of the semduramicin standard expressed as semduramicin given b
38、y the supplier in percent e.g. 0,934; m is the weighed mass of semduramicin sodium standard (4.3.1) in mg. 4.5.2 Nigericin sodium stock standard solution (for the LC-MS method), ca. 1 mg/ml. Accurately weigh 10 mg to the nearest 0,1 mg of nigericin sodium standard (4.4.1) into a 10 ml volumetric fla
39、sk (5.7). Note down the exact weight of nigericin sodium. Dissolve in methanol (4.1.3) and make up to 10 ml with methanol. Store at -20 C and protected from light. Prepare freshly every 3 months. Determine the experimental concentration of the nigericin sodium stock solutions using the reference sta
40、ndard purity value provided by the supplier using Equation (2). PmCn=10(2) where Cn is the experimental concentration of nigericin sodium in the stock standard in mg/ml; P is the purity of the nigericin sodium standard given by the supplier in % e.g. 0,98; m is the weighed mass of nigericin sodium s
41、tandard (4.4.1) in mg. 4.5.3 Semduramicin intermediate standard solution (for the LC-MS method), ca. 100 g/ml. Accurately pipette 1,0 ml of the semduramicin stock standard solution (4.5.1) into a 10 ml volumetric flask (5.7). Make up to 10 ml with acetonitrile (4.1.2). Store at -20 C and protected f
42、rom light. Prepare fresh intermediate solutions monthly. BS EN 16158:2012EN 16158:2012 (E) 7 4.5.4 Nigericin sodium intermediate standard solution (for the LC-MS method), ca. 100 g/ml. Accurately pipette 1,0 ml of the nigericin sodium stock standard solution (4.5.2) into a 10 ml volumetric flask (5.
43、7). Make up to 10 ml of with acetonitrile (4.1.2). Store at -20 C and protected from light. Prepare fresh intermediate solutions monthly. 4.5.5 Semduramicin spiking solution (for the LC-MS method), ca. 2 g/ml. Accurately pipette 200 l of the semduramicin intermediate solution (4.5.3) with an appropr
44、iate automatic pipette (5.6) into a 10 ml volumetric flask (5.7). Make up to 10 ml with acetonitrile (4.1.2). Store at -20 C and protected from light. Prepare fresh spiking solutions weekly. 4.5.6 Nigericin sodium spiking solution (for the LC-MS method), ca. 2 g/ml. Accurately pipette 200 l of the n
45、igericin sodium intermediate solution (4.5.4) with an appropriate automatic pipette (5.6) into a 10 ml volumetric flask. Make up to 10 ml with acetonitrile (4.1.2). Store at -20 C and protected from light. Prepare fresh spiking solutions weekly. 5 Apparatus Usual laboratory apparatus and, in particu
46、lar, the following: 5.1 LC-MS method HPLC system consisting of the following: 5.1.1 Pump, pulse free, flow capacity 0,1 ml/min to 2,0 ml/min. 5.1.2 Injection system, manual or autosampler, with a loop suitable for 10 l injections. 5.1.3 Single quadrupole mass spectrometer or triple quadrupole mass s
47、pectrometer used in the MS configuration able to operate in a mass range at least between 200 m/z to 1 000 m/z. NOTE For the MS detector the vacuum should be as stable as possible to ensure satisfactory repeatability. The system should therefore be pumped at least 48 h before starting the measuremen
48、ts. 5.1.4 Computer data system. 5.1.5 Analytical column, Alltima HP C18, 5 m ,150 mm x 2,1 mm, 190 , 12 % C load and 200 m2/g or equivalent. 5.1.6 Guard column, Alltima HP C18, 5 m, 7,5 mm x 2,1 mm, 190 , 12% C load and 200 m2/g or equivalent. 5.2 LC-PCD-UV method HPLC system consisting of the follo
49、wing: 5.2.1 Pump, pulse free, flow capacity 0,1 ml/min to 2,0 ml/min. 5.2.2 Injection system, manual or autosampler, with a loop suitable for 100 l injections. 5.2.3 UV/VIS detector, variable wavelength, suitable for reliable measurements at 598 nm, or UV/VIS photodiode array detector (DAD). 5.2.4 Computer data system. 5.2.5 Post-column reactor, with a 1,5 ml to 2,0 ml reaction coil, for op
