1、June 2012 Translation by DIN-Sprachendienst.English price group 13No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 6
2、5.120!$|Y8“1895421www.din.deDDIN EN 16162Animal feeding stuffs Determination of decoquinate by HPLC with fluorescence detectionEnglish translation of DIN EN 16162:2012-06Futtermittel Bestimmung von Decoquinat mit Hochleistungs-Flssigchromatographie (HPLC) undFluoreszenzdetektionEnglische bersetzung
3、von DIN EN 16162:2012-06Aliments des animaux Determination du dcoquinate par chromatographie liquide haute performance avecdtection fluorimtriqueTraduction anglaise de DIN EN 16162:2012-06www.beuth.deDocument comprises pagesIn case of doubt, the German-language original shall be considered authorita
4、tive.2605.12 DIN EN 16162:2012-06 2 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs Methods of sampling and analysis” (Secretariat: NEN, Netherlands). The responsible German body involved in its prepara
5、tion was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-03-03 AA Futtermittel. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 5725 (all p
6、arts) DIN ISO 5725 (all parts) National Annex NA (informative) Bibliography DIN ISO 5725, Accuracy (trueness and precision) of measurement methods and results Part 1 to Part 6 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16162 March 2012 ICS 65.120 English Version Animal feeding stuffs - Det
7、ermination of decoquinate by HPLC with fluorescence detection Aliments des animaux - Dtermination du dcoquinate par dtection fluorimtrique Futtermittel - Bestimmung von Decoquinat mit Hochleistungs-Flssigchromatographie (HPLC) und Fluoreszenzdetektion This European Standard was approved by CEN on 4
8、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 national standards may b
9、e 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
10、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, Latvia, Lithuania, Luxem
11、bourg, 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, B-1000 Brussels 2012 CEN
12、All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16162:2012: Echromatographie liquide haute performance avec EN 16162:2012 (E) 2 Contents Page Foreword 4Introduction .51 Scope 62 Normative references 63 Principle 64 Reagents .65 Apparat
13、us .86 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 (decoquina
14、te content 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 Precisio
15、n 1110.1 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 familiarizatio
16、n phase 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
17、ground 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 Chromatogra
18、m example: 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 . 23DIN EN 16162:2012-06 EN 16162:2012 (E) 3 Bibliography 24DIN EN 16162:2012-06 EN 16162:2012 (E) 4 Foreword This docu
19、ment (EN 16162:2012) 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
20、 2012, and conflicting 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
21、rights. This document 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: A
22、ustria, 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 Unite
23、d Kingdom. DIN EN 16162:2012-06 EN 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 contamina
24、tion from medicated feed to feedstuff. DIN EN 16162:2012-06 EN 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 deco
25、quinate content in complete 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 po
26、ultry, at trace contamination 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 th
27、e blank feed. Lower limits 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 refer
28、ences, 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) 3 Principle Decoquinate is extracted from samples with a solution of
29、1 % calcium chloride in methanol 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
30、HPLC analysis using an alternate 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
31、 use various regulations for 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. Dissolv
32、e a mass of calcium salt (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. DIN EN 16162:2012-06 EN 16162:2012 (E) 7 Dissolve a mass of calcium salt (4.3) equivalent to 10 g of calcium chloride anhydrous in technical me
33、thanol (4.2). Mix well and make 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
34、, approximately 300 g/ml. Accurately 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 pu
35、rity of the standard material (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 volumetri
36、c flask, dilute to volume with 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 absorba
37、nce range is between 0,67 and 0,73. 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). Tran
38、sfer by pipette the required volume 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 Fa
39、ctor g/ml A 5 200 40 0,15B 100 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 calciu
40、m salt (4.3) equivalent to 10 g 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
41、 calcium salt to flush the HPLC column and equipment at the end of each day of analysis. DIN EN 16162:2012-06 EN 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 f
42、ilter or equivalent. 5.3 Centrifuge 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 co
43、lumn like ACE or Luna or Symmetry or 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 shou
44、ld have been sent to the laboratory. 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 o
45、f premixtures and concentrates (feed 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 at
46、tention should be given to the addition 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 approximatel
47、y 5 min, is very helpful. Accurately 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
48、 supernatant extract with dilution solution (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, membrane filtration
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