1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN 16162:2012Animal feeding stuffs Determination of decoquinateby HPLC with fluorescencedetectionBS EN 16162:2012 BRITISH STANDARDNational forewordThis British Standard is the
2、 UK implementation of EN 16162:2012.The UK participation in its preparation was entrusted to TechnicalCommittee AW/10, Animal feeding stuffs.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessary
3、provisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2012. Published by BSI StandardsLimited 2012ISBN 978 0 580 67002 2ICS 65.120Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was publishe
4、d under the authority of theStandards Policy and Strategy Committee on 31 March 2012.Amendments issued since publicationDate Text affectedBS EN 16162:2012EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16162 March 2012 ICS 65.120 English Version Animal feeding stuffs - Determination of decoquin
5、ate by HPLC with fluorescence detection Aliments des animaux - Dtermination du dcoquinate par Chromatographie Liquide Haute Performance avec dtection fluorimtrique Futtermittel - Bestimmung von Decoquinat mit Hochleistungs-Flssigchromatographie (HPLC) und Fluoreszenzdetektion This European Standard
6、was approved by CEN on 4 February 2012. 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
7、 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
8、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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy,
9、 Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17,
10、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 16162:2012: EBS EN 16162:2012EN 16162:2012 (E) 2 Contents Page Foreword 4Introduction .51 Scope 62 Normative references 63 Principle 64 Reagents .65 Apparatus .86
11、 Sampling .87 Sample preparation .88 Procedure .88.1 General 88.2 Extraction of feeds (decoquinate content between 10 mg/kg to 500 mg/kg) 88.3 Extraction of complementary feeds, premixtures and feed additives (decoquinate content higher than 500 mg/kg) 98.4 Extraction of trace feeds (decoquinate con
12、tent lower than 10 mg/kg) 98.5 Quality control spiked feeds .98.5.1 Blank Feed to spike at 30 mg/kg 98.5.2 Blank feed to spike at 9 mg/kg .98.6 HPLC parameters .98.7 Standards injections and calibration curve 108.8 Sample extracts 108.9 Confirmation procedure . 109 Calculations . 1010 Precision 1110
13、.1 Limit of Detection and Limit of Quantification . 1110.2 Interlaboratory test . 1110.3 Repeatability 1110.4 Reproducibility 1111 Test report . 12Annex A (informative) Results inter-laboratory study 13Annex B (informative) Additional recovery results . 17B.1 Recoveries from the familiarization phas
14、e 17B.2 Pre-trial; recovery test on a milk-replacer 18B.3 Recoveries from the single-laboratory validation . 18Annex C (informative) Additional results for robustness purposes . 19C.1 Robustness in terms of excitation wavelength, during the final collaborative study . 19C.2 Comparison between ground
15、 and unground samples during the final collaborative study 19C.3 Robustness from one laboratory during the final collaborative study . 20Annex D (informative) HPLC parameters and chromatogram examples 22D.1 General . 22D.2 Chromatogram example: Calibration point at 0,6 g/ml . 22D.3 Chromatogram exam
16、ple: Commercial Lamb feed containing around 50 mg/kg of decoquinate22D.4 Chromatogram example: same commercial Lamb feed, as in D.3, containing around 50 mg/kg of decoquinate . 23BS EN 16162:2012EN 16162:2012 (E) 3 Bibliography 24BS EN 16162:2012EN 16162:2012 (E) 4 Foreword This document (EN 16162:2
17、012) has been prepared by Technical Committee CEN/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 September 2012, and confl
18、icting national standards shall be withdrawn at the latest by September 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 doc
19、ument 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 organizations of the following countries are bound to implement this European Standard: Austria, Belgium,
20、 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. BS EN
21、 16162:2012EN 16162:2012 (E) 5 Introduction This European Standard has been developed to quantify decoquinate in feeding stuffs to enable the European Commission to control the content of animal feed products. However, this method can also be used to evaluate the cross contamination from medicated f
22、eed to feedstuff. BS EN 16162:2012EN 16162:2012 (E) 6 1 Scope This European Standard specifies a method for the determination of decoquinate. This high-performance liquid chromatographic (HPLC) method with a fluorescence detection is applicable to the quantification of decoquinate content in complet
23、e and complementary compound feeds, medicated feeds, semi-liquid feeds, premixtures and feed additives. The method was fully validated from LOQ to 60 000 mg/kg on different matrices during an international collaborative study 11, especially on complete compound feeds for poultry, at trace contaminat
24、ion level of 3 mg/kg and at European authorized level of 20 mg/kg to 40 mg/kg 12. The limit of detection is between 0,1 mg/kg and 0,3 mg/kg and the limit of quantification is around 0,5 mg/kg. These limits were validated during the collaborative study 11, from results on the blank feed. Lower limits
25、 of detection or quantification could be reached but a single laboratory validation is then requested. 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 ci
26、ted 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) 3 Principle Decoquinate is extracted from samples with a solution of 1 % calcium chloride in me
27、thanol using mechanical shaking or stirring for 60 min. After centrifugation or filtration, an aliquot is, if necessary, diluted with the extraction solvent and analysed by reversed phase HPLC with fluorescence detection. Positive trace level samples should be confirmed by HPLC analysis using an alt
28、ernate excitation wavelength. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralised water or water of equivalent purity. WARNING This method requires the handling of hazardous substances. It is recommended to use various regulations f
29、or potentially hazardous chemicals. Organisational, technical and personal safety has to be observed. 4.1 Methanol, HPLC grade. 4.2 Methanol, technical grade. 4.3 Calcium chloride anhydrous or Calcium chloride dihydrate, each 99 % purity. 4.4 HPLC dilution solution. Dissolve a mass of calcium salt (
30、4.3) equivalent to 10 g of calcium chloride anhydrous in methanol (4.1). Mix well and make up to 1 000 ml. 4.5 Extraction solvent. BS EN 16162:2012EN 16162:2012 (E) 7 Dissolve a mass of calcium salt (4.3) equivalent to 10 g of calcium chloride anhydrous in technical methanol (4.2). Mix well and make
31、 up to 1 000 ml. 4.6 Decoquinate standards. 4.6.1 Decoquinate powder reference standard with guaranteed purity. Purity shall be certified by a certificate of analysis. NOTE Pure reference standard is available at e.g. Alpharma. 4.6.2 Decoquinate stock standard solution, approximately 300 g/ml. Accur
32、ately weigh, to the nearest 0,1 mg, 30 mg of decoquinate reference standard (4.6.1) into a 100 ml volumetric flask and dissolve in dilution solution (4.4). Use ultrasonic bath if necessary to aid dissolution. Calculate the exact concentration taking into account the purity of the standard material (
33、4.6.1), given in the certificate. Prepare fresh monthly. Store in the dark at 0 C to 10 C. 4.6.3 HPLC standard solutions. 4.6.3.1 Intermediate standard solution, approximately 6 g/ml. Transfer by pipette 2,0 ml of stock standard solution (4.6.2) into a 100 ml volumetric flask, dilute to volume with
34、dilution solution (4.4). Check that intermediate solution for each series of analysis. The absorbance density of intermediate solution can be evaluated at 265 nm, with dilution solvent (4.4) as reference for optical density measurement. In these conditions, the absorbance range is between 0,67 and 0
35、,73. That intermediate solution is prepared fresh daily. 4.6.3.2 HPLC calibration standard solutions, approximately 0,15 g/ml, 0,30 g/ml, 0,60 g/ml and 1,2 g/ml. Prepare 4 concentrations of HPLC standard solutions as it is explained in Table 1 (standards A/B/C/D). Transfer by pipette the required vo
36、lume of intermediate standard (4.6.3.1) into volumetric flasks, and make to volume with dilution solution (4.4). Mix well. Table 1 preparation of calibration standard solutions Standard Parts of intermediate (4.6.3.1) solution, in ml Volumetric flask, in ml Dilution Factor g/ml A 5 200 40 0,15B 100
37、20 0,30 C 10 100 10 0,60D 10 50 5 1,20Evaluate precisely each exact concentration by using the exact concentration of the stock solution (4.6.2). All solutions described here (standards A/B/C/D) are prepared fresh daily. 4.7 HPLC Mobile Phase. Dissolve a mass of calcium salt (4.3) equivalent to 10 g
38、 calcium chloride anhydrous into 1 l of a solvent mixture of methanol (4.1) / water in proportion by volume of 825/175. Filter under vacuum (5.2) before use. NOTE HPLC shutdown solution. Prepare a methanol (4.1) / water solution in proportion by volume of 85/15 without calcium salt to flush the HPLC
39、 column and equipment at the end of each day of analysis. BS EN 16162:2012EN 16162:2012 (E) 8 5 Apparatus Usual laboratory apparatus, in particular, the following: 5.1 Mechanical shaker or magnetic stirrer. 5.2 Solvent filtration system, suitable for 0,45 m PTFE filter or equivalent. 5.3 Centrifuge
40、and centrifuge tube (50 ml). 5.4 HPLC system consisting of the following: 5.4.1 Pump, pulse free, flow capacity of 0,2 ml/min to 5 ml/min. 5.4.2 Injection system, manual or autosampler, with loop suitable for 10 l to 50 l injection volumes. 5.4.3 Analytical C18 column like ACE or Luna or Symmetry or
41、 Restek Ultra; 5 m; 4.6 mm x 250 mm or equivalent. 5.4.4 Fluorescence detector suitable for measurement using 330 nm and 260 nm excitation wavelengths and 390 nm emission wavelength. 5.4.5 Integrator or computer data system. 6 Sampling A representative sample should have been sent to the laboratory.
42、 It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this European Standard. A recommended sampling procedure is given in EN ISO 6497 1. 7 Sample preparation All feed samples, with the exception of premixtures and concentrates (feed
43、 additives), are ground extraction as recommended in the guidelines prEN ISO 6498. 8 Procedure 8.1 General Preferably, duplicate analysis is performed. 8.2 Extraction of feeds (decoquinate content between 10 mg/kg to 500 mg/kg) NOTE For milk-replacer particular attention should be given to the addit
44、ion of extraction solvent. Small or large lumps could be formed and quantitative results will be compromised. The extraction solvent addition should be done slowly under rotate shaking of the conical flask. To dissolve lumps, ultrasonic system, during approximately 5 min, is very helpful. Accurately
45、 weigh, to the nearest 0,1 g, 10,0 g of feed in a 250 ml amber conical flask. Add 100 ml of the extraction solution (4.5). Shake or stir for 1 h on apparatus (5.1). Allow contents of flask to settle particles for about 10 min. Dilute an aliquot of the almost clear supernatant extract with dilution s
46、olution (4.4) to obtain a concentration between 0,3 g/ml and 0,6 g/ml. Filter the diluted solution on membrane filter (5.2) and inject it onto HPLC system (5.4). For cloudy extracts, if necessary, transfer at least 40 ml of the extract into 50 ml centrifuge tube (5.3) and centrifuge before dilution,
47、 membrane filtration and HPLC injection. BS EN 16162:2012EN 16162:2012 (E) 9 8.3 Extraction of complementary feeds, premixtures and feed additives (decoquinate content higher than 500 mg/kg) Accurately weigh, to the nearest 0,01 g, depending on the declared concentration of decoquinate in the sample
48、, 0,50 g to 2,00 g of sample into a 250 ml amber conical flask. Add 100 ml of the extraction solution (4.5). Shake or stir for 1 h on equipment (5.1). Allow contents of flask to settle particles for 10 min. Dilute an aliquot of the almost clear supernatant extract with dilution solution (4.4) to obt
49、ain a concentration between 0,3 g/ml and 0,6 g/ml. Filter the diluted solution on membrane filter (5.2) and inject it onto HPLC system (5.4). For cloudy extracts, if necessary, transfer at least 40 ml of the extract into 50 ml centrifuge tube (5.3) and centrifuge before dilution, membrane filtration and HPLC injection. NOTE For a decoquinate declaration of 1 000 mg/kg, weigh 2 g of
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