1、Animal feeding stuffs Methods of sampling and analysis Determination of carbadox and olaquindox by HPLC/UVBS EN 16930:2017BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16930 July 2017 ICS 65.120 English Version A
2、nimal feeding stuffs: Methods of sampling and analysis - Determination of carbadox and olaquindox by HPLC/UV Aliments des animaux - Mthodes dchantillonnage et danalyse - Dtermination des teneurs en carbadox et olaquindox par CLHP/UV Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung
3、von Carbadox und Olaquindox mittels HPLC/UV This European Standard was approved by CEN on 14 May 2017. 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.
4、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 tra
5、nslation 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, Finl
6、and, 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 STAND
7、ARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16930:2017 ENational forewordThis Britis
8、h Standard is the UK implementation of EN 16930:2017.The UK participation in its preparation was entrusted to Technical Committee AW/10, Animal feeding stuffs.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to includ
9、e all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2017 Published by BSI Standards Limited 2017ISBN 978 0 580 91485 0ICS 65.120Compliance with a British Standard cannot confer immunity from legal obligations.This British
10、 Standard was published under the authority of the Standards Policy and Strategy Committee on 31 August 2017.Amendments/corrigenda issued since publicationDate Text affectedBRITISH STANDARDBS EN 16930:2017EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16930 July 2017 ICS 65.120 English Version
11、 Animal feeding stuffs: Methods of sampling and analysis - Determination of carbadox and olaquindox by HPLC/UV Aliments des animaux - Mthodes dchantillonnage et danalyse - Dtermination des teneurs en carbadox et olaquindox par CLHP/UV Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmun
12、g von Carbadox und Olaquindox mittels HPLC/UV This European Standard was approved by CEN on 14 May 2017. 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
13、. 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 t
14、ranslation 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, Fi
15、nland, 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 STA
16、NDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16930:2017 EBS EN 16930:2017EN 16930:2
17、017 (E) 2 Contents Page European foreword . 4 1 Scope 5 2 Normative references 5 3 Principle . 5 4 Reagents and materials . 5 4.1 Water, demineralized or deionized or at least equivalent 6 4.2 Methanol . 6 4.3 Acetonitrile 6 4.4 Glacial acetic acid, minimum purity 96 % 6 4.5 Ammonium acetate water f
18、ree salt, CH3CO2NH46 4.6 Extraction solvent: methanol water mixture (1:1 v:v) 6 4.7 Ammonium acetate buffer, 25 mM, pH = 4,35 6 4.8 Mobile phase for HPLC: acetonitrile: buffer mixture (10:90; v:v) . 6 4.9 Carbadox dissolving solvent: methanol: acetonitrile mixture (1:1; v:v) 6 4.10 Reference standar
19、ds 6 4.11 Standard solutions 6 4.12 Neutral aluminium oxide, Brockmann activity I 8 4.13 Neutral aluminium oxide SPE cartridge, 2 cm3, 1850 mg . 8 4.14 Technical methanol 8 5 Apparatus . 8 5.1 pH meter . 8 5.2 Solvent filtration system, suitable for 0,45 m membrane filters 8 5.3 Ultrasonic bath . 8
20、5.4 Rotary on mechanical shaker . 8 5.5 Centrifuge . 8 5.6 Glass wool 8 5.7 Glass column for chromatography, length 200 mm to 400 mm; internal diameter 10 mm, restricted at the end and fitted with a wad of glass wool (5.6) or equivalent column . 8 5.8 Filter papers or glass microfibre filter 8 5.9 C
21、ellulose acetate membrane filters of pore size 0,45 m . 8 5.10 PVDF syringe filters of pore size 0,45 m and adaptable syringes . 8 5.11 HPLC system 8 5.12 Refrigerator or refrigerated chamber at a temperature set between 0 C and 8 C . 9 6 Sampling . 9 7 Preparation of test sample 9 7.1 General 9 7.2
22、 Laboratory sample 9 7.3 Test sample . 9 7.4 Test portion . 9 8 Procedure. 9 8.1 Extraction of feeding stuffs containing 0,5 mg/kg to 100 mg/kg of growth promoters 9 8.2 Filtration 9 8.3 Purification 9 BS EN 16930:2017EN 16930:2017 (E) 3 8.4 HPLC analysis . 10 8.5 System suitability Confirmation 11
23、9 Calculation. 12 10 Precision 13 10.1 Collaborative study 13 10.2 Repeatability 13 10.3 Reproducibility 13 11 Test report 14 Annex A (informative) Results of the collaborative study 15 A.1 Procedure 15 A.2 Materials 15 A.3 Statistical analysis of results 17 A.4 Results and interpretation 17 A.5 Exa
24、mple chromatogram . 24 Bibliography . 26 BS EN 16930:2017EN 16930:2017 (E) 4 European foreword This document (EN 16930:2017) 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
25、 shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by January 2018, and conflicting national standards shall be withdrawn at the latest by January 2018. Attention is drawn to the possibility that some of the elements of this
26、 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. WARNING The use of this prot
27、ocol involves hazardous materials, operations and equipment. This protocol does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this protocol to establish appropriate safety and health practices and determine the applicability of regula
28、tory limitations prior to use. 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 Rep
29、ublic 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. BS EN 16930:2017EN 16930:2017 (E) 5 1 Scope This
30、 European Standard specifies a high performance liquid chromatographic UV detection (HPLC-UV) method for the simultaneous determination of two growth promoters Carbadox and Olaquindox contents in compound feeds and raw materials at levels ranging from the limit of quantification to 100 mg/kg. The li
31、mit of quantification of the method has been demonstrated to be lower than 3 mg/kg for olaquindox and 4 mg/kg for carbadox. 2 Normative references The following documents, in whole or in part, is normatively referenced in this document and is indispensable for its application. For undated reference,
32、 the latest edition of the referenced document (including any amendments) applies. EN ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498) 3 Principle The two growth promoters are extracted from the sample with a mixture of methanol and water 1:1, v:v. The extract of animal
33、 feeds is purified through a short open glass aluminium oxide column or by an alumina Solid Phase Extraction (SPE). The final extract is analysed by reversed phase HPLC with UV detection at a wavelength of 375 nm. Alternatively, for confirmation purposes, a Diode Array Detector (DAD) can be used. Th
34、e presence of furazolidone can interfere with the determination of carbadox. If a DAD is used, many other veterinary drugs or feed additives can be detected (ronidazole, meticlorpindol, nitrofurazone, dimetridazol, furaltadon, sulphonamides.), but their signals do not interfere with the target ones.
35、 In some special feeds, matrix interfering peaks may be present, very close to the carbadox peak. 4 Reagents and materials WARNING Persons using this European Standard should be familiar with normal laboratory practice. This standard does not purport to address all of the safety problems, if any, as
36、sociated with its use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure compliance with any regulatory conditions. a) This method requires the use of solutions of Carbadox and Olaquindox. These substances were used as growth-promoting feed addit
37、ives for piglets. Carbadox and Olaquindox are chemotherapeutics belonging to the quinoxaline group. They are suspected to be teratogen and mutagen. Avoid inhalation of and exposure to the toxic standard materials and solutions; b) These 2 growth promoters are subject to light degradation. Protect an
38、alytical work adequately from daylight, and keep standard solutions protected from light by using amber glassware, amber vials or aluminium foil. Use only reagents as analytical grade at least unless otherwise stated. BS EN 16930:2017EN 16930:2017 (E) 6 4.1 Water, demineralized or deionized or at le
39、ast equivalent 4.2 Methanol 4.3 Acetonitrile 4.4 Glacial acetic acid, minimum purity 96 % 4.5 Ammonium acetate water free salt, CH3CO2NH4 4.6 Extraction solvent: methanol water mixture (1:1 v:v) Combine equal volumes of methanol (4.2) and water (4.1). Mix well. NOTE Only for 8.1 purposes, it is poss
40、ible to combine equal volume of technical methanol (4.14) and water (4.1). It is essential to mix well. 4.7 Ammonium acetate buffer, 25 mM, pH = 4,35 Weigh 2,0 g of ammonium acetate (4.5) to the nearest 0,1 g, into a 1000 ml volumetric flask. Dissolve in 900 ml of water (4.1). Add 3,0 ml of glacial
41、acetic acid (4.4). Adjust (5.1) the pH to pH = 4,35 with acetic acid (4.4), if necessary. Dilute to the mark with water (4.1) and mix well. 4.8 Mobile phase for HPLC: acetonitrile: buffer mixture (10:90; v:v) Transfer 100 ml of acetonitrile (4.3) into a 1000 ml volumetric flask. Dilute to the mark w
42、ith ammonium acetate buffer (4.7). Filter the eluent through a 0,45 m cellulose acetate membrane filter (5.9) using a solvent filtration system (5.2). If necessary, perform degassing for 5 min in an ultrasonic bath (5.3). NOTE This mobile phase is suitable for the three recommended columns in 5.11.8
43、. If another column is used, optimization of the mobile phase composition may be necessary before performing real analysis. See 8.4.1. 4.9 Carbadox dissolving solvent: methanol: acetonitrile mixture (1:1; v:v) Combine equal volumes of methanol (4.2) and acetonitrile (4.3). Mix well. 4.10 Reference s
44、tandards Guaranteed purity is required for each lot of reference standard. 4.10.1 Carbadox, 3- (2-quinoxalinyl methylene) carbazic acid methyl ester N,N-dioxide 4.10.2 Olaquindox, (2-N-2-(hydroxyethyl) carbamoyl 3methyl quinoxaline) N,N-dioxide 4.11 Standard solutions Protect all standard solutions
45、from daily light 4.11.1 Carbadox stock standard solution ca. 100 g/ml Weigh 25 mg of carbadox (4.10.1), to the nearest 0,1 mg, into a 250 ml amber volumetric flask. Dissolve in 200 ml of mixture (4.9). Mix well and place the flask in an ultrasonic bath (5.3) until total dissolution. Allow to cool do
46、wn to room temperature, dilute to the mark with mixture (4.9) and mix well. Calculate the accurate concentration taking into account the purity of the reference standard (4.10.1). Prepare fresh every month. Store in the dark at 0 C to 8 C (5.12). BS EN 16930:2017EN 16930:2017 (E) 7 4.11.2 Olaquindox
47、 stock standard solution ca. 250 g/ml Weigh 50 mg of olaquindox (4.10.2), to the nearest 0,1 mg, into a 200 ml amber volumetric flask. Dissolve in about 190 ml of water (4.1). Mix well and place the flask in an ultrasonic bath (5.3) until total dissolution. Allow to cool down to room temperature. Di
48、lute to the mark with water (4.1) and mix well. Calculate the accurate concentration taking into account the purity of the reference standard (4.10.2). Prepare fresh every month. Store in the dark at 0 C to 8 C (5.12). 4.11.3 Calibrations solutions 4.11.3.1 Carbadox/olaquindox calibration solution c
49、a. 10 g/ml Pipette 5 ml of the carbadox stock standard solution (4.11.1) and 2 ml of the olaquindox stock standard solution (4.11.2) in a 50 ml volumetric flask. Dilute to the mark with the extraction solvent (4.6) and mix well. Prepare fresh for each series of samples. 4.11.3.2 Carbadox/olaquindox calibration solution ca. 5 g/ml Pipette 5 ml of the carbadox stock standard solution (4.11.1) and 2 ml of the olaquindox stock standard solution (4.11.2) in a 100 ml volumetric flask. Dilute to the mark with the extraction solvent (4.6)
