1、BS EN 15781:2009ICS 65.120NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDAnimal feeding stuffs Determinationof maduramicin-ammonium byreversed-phase HPLCusing post-columnderivatisationThis British Standardwas published underthe authority of theStandards Policy
2、andStrategy Committee on 30September 2009 BSI 2009ISBN 978 0 580 61797 3Amendments/corrigenda issued since publicationDate CommentsBS EN 15781:2009National forewordThis British Standard is the UK implementation of EN 15781:2009.The UK participation in its preparation was entrusted to TechnicalCommit
3、tee AW/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 necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a British Standa
4、rd cannot confer immunityfrom legal obligations.BS EN 15781:2009EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15781August 2009ICS 65.120English VersionAnimal feeding stuffs - Determination of maduramicin-ammonium by reversed-phase HPLC using post-columnderivatisationAliments des animaux - Dtermi
5、nation de la maduramicineammonium par HPLC en phase inverse laide de ladrivation post-colonneFuttermittel - Bestimmung von Maduramicin-Ammoniumdurch Umkehrphasen HPLC-Verfahren mittelsNachsulenderivatisierungThis European Standard was approved by CEN on 3 July 2009.CEN members are bound to comply wi
6、th 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 and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Cen
7、tre 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 responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial
8、versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia,
9、Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Mem
10、bers.Ref. No. EN 15781:2009: EBS EN 15781:2009EN 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 Preparation of test sample 7 8 Procedure .7 9 Calculation of results 8 10 Interpretation of confirmation data 9 1
11、1 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 BS EN 15781:2009EN 15781:2009 (E) 3 Foreword This document (EN 15781:2009) has been prepared by Technical Com
12、mittee 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 February 2010, and conflicting national standards shall be withd
13、rawn 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 responsible for identifying any or all such patent rights. According to the CEN/CENELEC Internal Regulations
14、, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands
15、, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 15781:2009EN 15781:2009 (E) 4 1 Scope This European Standard specifies a high performance liquid chromatography (HPLC) method for the determination of the content of maduramicin in feedi
16、ng 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 0,5 mg/kg. NOTE A lower limit of quantification may be achievable but shall be validated by the user. 2 Normative re
17、ference(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 latest edition of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical labo
18、ratory 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 derivatization with vanillin and detection at 520 nm. 4 Reagents Use only reagents of recognized analytical grade, unless
19、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 1 4.2 Methanol (CH3OH), HPLC grade 4.3 1,5-Dimethylhexylamine (CH3(CH2)4CH2N(CH3)2)4.4 Sulfuric acid (H2SO4), puri
20、ty 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 with water (4.1). 4.6 Potassium dihydrogen phosphate (KH2PO4) BS EN 15781:2009EN 15781:2009 (E) 5 4.7 Phosphate buffe
21、r 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-dimethylhexylamine (4.3). Adjust the pH to 4,0 with diluted ortho-phosphoric acid (4.5.1) and fill with demineralised
22、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 methanol (4.2) to 1 000 ml. 4.9 Vanillin, 4-hydroxy-3-methoxybenzaldehyde, minimum 98% purity by volume (HPLC grade) 4
23、.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 vacuum at room temperature. This solution has to be prepared daily prior to use and has to be cooled with ice water during u
24、se. 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 not been performed. 4.10 Maduramicin WARNING Maduramicin is very toxic. Avoid inhalation and exposure to the toxic stan
25、dard 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 volumetric flask. Dissolve in methanol (4.2) and dilute to volume. Store below 4C. Prepare fresh every month. 4.11.2 Sta
26、ndard 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. 4.11.3 Calibration solutions The interlaboratory study was performed with 8 calibration solutions. Into a series of 50 ml
27、 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) and 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
28、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 should be prepared on the day of analysis. 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1
29、 Centrifuge BS EN 15781:2009EN 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 system, manual or autosampler, with loop suitable for 50 l injection 5.3.3 UV/VIS detector, suitable for measurements at 52
30、0 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 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 r
31、eaction 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, 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
32、 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, use a vortex or static mixing tee (not a regular tee) before the reaction coil. NOTE 1 The length of the polytetrafluoroethyle
33、ne (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 optimized if there are problems with bubbles. NOTE 2 A temperature of 92C to 98C is possible, high stability (1C) should be guar
34、anteed. 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 Liquid chromatographic column, 250 mm x 4,6 mm, 5 m material, Hypersil BDS C18 or equivalent 5.3.8 Column oven, suitable for
35、 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 the laboratory receives a sample that is truly representative and has not been damaged or changed during transport or storage
36、. BS EN 15781:2009EN 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 Preparation of test sample Sample preparation is not part of the method specified in this European Standard. A recommended method tha
37、t 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 (8.1.2) a blank feed should be analyzed to check that neither maduramicin nor interfering substances are present. The blank feed
38、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 by analyzing the blank feed which has been fortified by the addition of a quantity of maduramicin, similar to that present in th
39、e 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, mixing again several times before proceeding with the extraction step (8.2) Alternatively, if a blank feed similar in type to that
40、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 with a quantity of maduramicin similar to that already present in the sample. This sample is analyzed together with the unfortified
41、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 nearest 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 f
42、lask 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 15 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 fo
43、r 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
44、suitable method, and place in an ultrasonic bath (5.2) at 50C for 20 min. Cool down to room temperature, shake vigorously (5.4), store some min and dilute an aliquot of the clear supernatant 1:10 with methanol and place in a freezer (5.5) for 2 h to 3 h to settle down fat. Then centrifuge an aliquot
45、 for 1 min to 2 min. After membrane filtration (5.6), 50 l of this solution is injected into the HPLC-apparatus. NOTE A larger sample amount may be used, but shall be validated by the user. In the case a larger sample amount is used the volume of the extraction solvent has to be adjusted accordingly
46、. BS EN 15781:2009EN 15781:2009 (E) 8 WARNING Maduramicin is very toxic. Avoid inhalation and exposure to the toxic material when grinding the sample. 8.3 HPLC determination 8.3.1 HPLC conditions The following conditions are offered for guidance, other conditions may be used provided that they give
47、equivalent results. analytical column as in 5.3.7 mobile phase as in 4.8 column oven 40C flow rate 0,4 ml/min detection wavelength 520 nm injection volume 50 l post-column reagent vanillin reagent (4.9.1) flow rate reagent pump 0,4 ml/min reactor temperature 95C retention time approx. 25 min run tim
48、e 30 min to 35 min Check the stability of the chromatographic system, injecting several times the calibration solution (4.11.3) containing 1,0 g/ml, until constant peak areas and retention times are achieved. Working with the described conditions there is baseline separation from other ionophores li
49、ke salinomycin, narasin, monensin and semduramycin. 8.3.2 Calibration graph Inject each calibration solution (4.11.3) several times and determine the mean peak areas for each concentration. Plot a calibration graph using the mean peak areas of the calibration solutions as the ordinate and the corresponding concentrations in g/ml as the abscissae. 8.3.3 Sample solution Inject the sample extract (8.2) at least 2 times using the same volume as taken for the calibration solutions and determine the mean peak area of the ma
