1、November 2009DEUTSCHE NORM English price group 10No part of this standard 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 65.120!$ZaE“1556234ww
2、w.din.deDDIN EN 15781Animal feeding stuffs Determination of maduramicin-ammonium by reversed-phase HPLCusing post-column derivatisationEnglish version of DIN EN 15781:2009-11Futtermittel Bestimmung von Maduramicin-Ammonium durch Umkehrphasen HPLC-Verfahren mittelsNachsulenderivatisierungEnglische Fa
3、ssung DIN EN 15781:2009-11www.beuth.deDocument comprises pages15DIN EN 15781:2009-11 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 i
4、ts preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-03-03 AA Futtermittel. The DIN Standards corresponding to the International Standards referred to in this document are as follows: EN
5、ISO 3696 DIN ISO 3696 EN ISO 6497 DIN EN ISO 6497 ISO 5725-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO 5725-2 ISO 5725-3 DIN ISO 5725-3 ISO 5725-4 DIN ISO 5725-4 ISO 5725-5 DIN ISO 5725-5 ISO 5725-6 DIN ISO 5725-6 ISO 6498 E DIN EN ISO 6498 (to be published) National Annex NA (informative) Bibliography DIN
6、EN ISO 6497, Animal feeding stuffs Sampling DIN ISO 3696, Water for analytical laboratory use Specification and test methods DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions DIN ISO 5725-2, Accuracy (trueness and precisio
7、n) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method DIN ISO 5725-3, Accuracy (trueness and precision) of measurement methods and results Part 3: Intermediate measures of the precision of a standard mea
8、surement method DIN ISO 5725-4, Accuracy (trueness and precision) of measurement methods and results Part 4: Basic methods for the determination of the trueness of a standard measurement method DIN ISO 5725-5, Accuracy (trueness and precision) of measurement methods and results Part 5: Alternative m
9、ethods for the determination of the precision of a standard measurement method DIN ISO 5725-5, Corrigendum 1, Accuracy (trueness and precision) of measurement methods and results Part 5: Alternative methods for the determination of the precision of a standard measurement method DIN ISO 5725-6, Accur
10、acy (trueness and precision) of measurement methods and results Part 6: Use in practice of accuracy values E DIN EN ISO 6498, Animal feeding stuffs Guidelines for sample preparation 2 EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15781August 2009ICS 65.120English VersionAnimal feeding stuffs - D
11、etermination of maduramicin-ammonium by reversed-phase HPLC using post-columnderivatisationAliments des animaux - Dtermination de la maduramicineammonium par HPLC en phase inverse laide de ladrivation post-colonneFuttermittel - Bestimmung von Maduramicin-Ammoniumdurch Umkehrphasen HPLC-Verfahren mit
12、telsNachsulenderivatisierungThis European Standard was approved by CEN on 3 July 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists a
13、nd bibliographical references concerning such nationalstandards may be obtained on application to the CEN 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 translationunder the responsibi
14、lity of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, I
15、reland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17,
16、 B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15781:2009: EEN 15781:2009 (E) 2 Contents Page Foreword 3 1 Scope 4 2 Normative reference(s) 4 3 Principle 4 4 Reagents .4 5 Apparatus .5 6 Sampling .6 7 Preparati
17、on of test sample 7 8 Procedure .7 9 Calculation of results 8 10 Interpretation of confirmation data 9 11 Precision .9 12 Test report 9 Annex A (informative) Results of the interlaboratory study 10 Annex B (informative) Conditions for post-column derivatisation with DMAB . 12 Bibliography . 13 DIN E
18、N 15781:2009-11 EN 15781:2009 (E) 3 Foreword This document (EN 15781:2009) 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 identi
19、cal text or by endorsement, at the latest by February 2010, and conflicting national standards shall be withdrawn at the latest by February 2010. 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
20、 responsible for identifying any or all such patent rights. 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, Cyprus, Czech Republic, Denmark, Estonia, Finland,
21、 France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. DIN EN 15781:2009-11 EN 15781:2009 (E) 4 1 Scope This European Standard specifies a
22、 high performance liquid chromatography (HPLC) method for the determination of the content of maduramicin in feeding stuffs and premixtures. The usual concentration of maduramicin in feedstuffs is 5 mg/kg, in premixtures 500 mg/kg. The limit of quantification is 2 mg/kg. The limit of detection is 0,
23、5 mg/kg. NOTE A lower limit of quantification may be achievable but shall be validated by the user. 2 Normative reference(s) The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the la
24、test edition of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987) 3 Principle The sample is extracted with methanol. Maduramicin is determined by reversed-phase HPLC using post-column derivati
25、zation with vanillin and detection at 520 nm. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified. WARNING Use all solvents and solutions in a fume hood. Wear safety glasses, protective clothing and avoid skin contact. 4.1 Water, complying with EN-ISO 3696, grade
26、1 4.2 Methanol (CH3OH), HPLC grade 4.3 1,5-Dimethylhexylamine (CH3(CH2)4CH2N(CH3)2)4.4 Sulfuric acid (H2SO4), purity 95% to 97% by volume 4.5 Ortho-phosphoric acid, (H3PO4), purity approximately 85% by volume 4.5.1 Diluted o-phosphoric acid Dissolve 10 ml of ortho-phosphoric acid (4.5) to 100 ml wit
27、h water (4.1). 4.6 Potassium dihydrogen phosphate (KH2PO4) DIN EN 15781:2009-11 EN 15781:2009 (E) 5 4.7 Phosphate buffer solution, (KH2PO4) = 10 mmol/l, pH 4,0 Dissolve 1,36 g of potassium dihydrogen phosphate (4.6) in 500 ml of water (4.1). Add 3,0 ml of ortho-phosphoric acid (4.5) and 10 ml of 1,5
28、-dimethylhexylamine (4.3). Adjust the pH to 4,0 with diluted ortho-phosphoric acid (4.5.1) and fill with demineralised water to 1 000 ml. The solution can be stored for some weeks, but in case of fungal growth, prepare a new one. 4.8 Mobile phase Dilute 100 ml of phosphate buffer solution (4.7) with
29、 methanol (4.2) to 1 000 ml. 4.9 Vanillin, 4-hydroxy-3-methoxybenzaldehyde, minimum 98% purity by volume (HPLC grade) 4.9.1 Vanillin reagent Dissolve 10 g of vanillin (4.9) in a mixture of 250 ml of methanol (4.2) and 5,0 ml of sulphuric acid (4.4). Mix well and sonicate for some minutes under vacuu
30、m at room temperature. This solution has to be prepared daily prior to use and has to be cooled with ice water during use. NOTE Dimethylaminobenzaldehyde (DMAB) is also suitable as a reagent for post-column derivatisation (details are given in Annex B) although a full validation of this reagent has
31、not been performed. 4.10 Maduramicin WARNING Maduramicin is very toxic. Avoid inhalation and exposure to the toxic standard material and solutions thereof. 4.11 Standard solutions 4.11.1 Stock-standard-solution, 100 g/ml Accurately weigh 10 mg to the nearest 0,1 mg maduramicin (4.10) into a 100 ml v
32、olumetric flask. Dissolve in methanol (4.2) and dilute to volume. Store below 4C. Prepare fresh every month. 4.11.2 Standard solution, 10 g/ml Dilute 10 ml of the stock-standard-solution (4.11.1) to 100 ml with methanol (4.2) in a 100 ml volumetric flask. Store below 4C. Prepare fresh every week. 4.
33、11.3 Calibration solutions The interlaboratory study was performed with 8 calibration solutions. Into a series of 50 ml volumetric flasks transfer 1,0 ml, 2,0 ml, 3,0 ml, 4,0 ml, 5,0 ml, 6,0 ml, 8,0 ml and 10,0 ml of the intermediate standard solution (4.11.2). Dilute to volume with methanol (4.2) a
34、nd mix. These solutions correspond to 0,2 g, 0,4 g, 0,6 g, 0,8 g, 1,0 g, 1,2 g, 1,6 g, and 2,0 g of maduramicin per ml respectively. Alternatively, you may use 5 calibration solutions with maduramicin concentrations of 0,4 g, 0,7 g, 1,0 g, 1,5 g and 2,0 g per ml respectively. Calibration solutions s
35、hould be prepared on the day of analysis. 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 Centrifuge DIN EN 15781:2009-11 EN 15781:2009 (E) 6 5.2 Ultrasonic bath 5.3 HPLC system consisting of the following 5.3.1 Pump, pulse free, flow capacity 0,4 ml/min 5.3.2 Injection
36、 system, manual or autosampler, with loop suitable for 50 l injection 5.3.3 UV/VIS detector, suitable for measurements at 520 nm NOTE Noise preferably should be 1 10-5AU (250 nm, 600 nm) 5.3.4 Integrator, or computer data system. 5.3.5 Post column reactor consisting of the following NOTE The use of
37、stainless steel tubing in the post-column reactor and detector should be avoided. 5.3.5.1 PEEK mixing chamber 5.3.5.2 PTFE reaction coil, 1,5 ml to 2,0 ml reaction coil, for operating at 95C The coil may be a commercially available knitted coil or it may be made using 7,5 m to 10 m of 316 SS tubing,
38、 0,5 mm ID, coiled in a format to fit the reactor heating chamber (a suggestion is to wrap the coil in enough aluminium foil to make it fit snugly in the heater and to provide good heat transfer to the coil). A knitted coil is preferable. To ensure effective mixing of reagent and column effluent, us
39、e a vortex or static mixing tee (not a regular tee) before the reaction coil. NOTE 1 The length of the polytetrafluoroethylene (PTFE) tube (e.g. 1 m ID 0,25 mm) between reagent-pump and mixing chamber and the length of the Teflon tube (e.g. 3 m ID 0,17 mm) between reactor and detector should be opti
40、mized if there are problems with bubbles. NOTE 2 A temperature of 92C to 98C is possible, high stability (1C) should be guaranteed. 5.3.5.3 Reactor oven or water bath for the PTFE-reaction coil, suitable for operating at 95C 5.3.6 Post column reagent pump, pulse free, flow capacity 0,4 ml/min 5.3.7
41、Liquid chromatographic column, 250 mm x 4,6 mm, 5 m material, Hypersil BDS C18 or equivalent 5.3.8 Column oven, suitable for operating at 40C 5.4 Shaker, rotary or wrist-action shaker 5.5 Freezer 5.6 Membrane-filter, PTFE, pore size within the range of 0,20 m 0,45 m 6 Sampling It is important that t
42、he laboratory receives a sample that is truly representative and has not been damaged or changed during transport or storage. DIN EN 15781:2009-11 EN 15781:2009 (E) 7 Sampling is not part of the method specified in this European Standard. A recommended sampling method is given in EN ISO 6497. 7 Prep
43、aration of test sample Sample preparation is not part of the method specified in this European Standard. A recommended method that can be used for sample preparation is given in ISO 6498. 8 Procedure 8.1 Preparation of quality control sample 8.1.1 Blank feed For the performance of the recovery test
44、(8.1.2) a blank feed should be analyzed to check that neither maduramicin nor interfering substances are present. The blank feed should be similar in type to that of the sample and maduramicin or interfering substances should not be detected. 8.1.2 Recovery test A recovery test should be carried out
45、 by analyzing the blank feed which has been fortified by the addition of a quantity of maduramicin, similar to that present in the sample. To fortify at a level of 5 mg/kg, transfer 500 l stock-standard solution (4.11.1) to a flask. Add 10 g of the blank feed, mix thoroughly and leave for 10 min, mi
46、xing again several times before proceeding with the extraction step (8.2) Alternatively, if a blank feed similar in type to that of the sample is not available (8.1.1), a recovery test can be performed by means of the standard addition method. In this case, the sample to be analyzed is fortified wit
47、h a quantity of maduramicin similar to that already present in the sample. This sample is analyzed together with the unfortified sample and the recovery can be calculated by subtraction. Acceptable recovery is between 90% and 110%. 8.2 Extraction 8.2.1 Feeding stuffs Accurately weigh 10 g to the nea
48、rest 0,01 g of the ground sample (with particles of 1mm) into a 250 ml volumetric flask and add 50 ml methanol (4.2). Close the flask with a suitable method, and place it in an ultrasonic bath (5.2) at 50C for 20 min. Shake vigorously (5.4), store and cool down to room temperature in approximately 1
49、5 min, decant the clear supernatant and place in a freezer (5.5) for 2 h to 3 h to settle down fat. Then centrifuge an aliquot for 1 min to 2 min. After membrane (5.6) filtration, 50 l of this solution is injected into the HPLC-apparatus. 8.2.2 Premixes Accurately weigh 1 g to the nearest 0,01 g of the ground sample (with particles of 0,5 mm) into a 250 ml volumetric flask and add 50 ml methanol (4.2). Close the flask with a suitable method, and place in an ultrasonic bath (5.2) at 50C for 20 min. Coo
