SIST EN 15781:2009

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.UDILMRFuttermittel - Bestimmung von Maduramicin-Ammonium durch Umkehrphasen HPLC-Verfahren mittels NachsäulenderivatisierungAliments des animaux - Détermination de la maduramicine ammonium par HPLC en phase inverse à l'aide de la dérivation post-colonneAnimal feeding stuffs - Determination of maduramicin-ammonium by reversed-phase HPLC using post-column derivatisation65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 15781:2009SIST EN 15781:2009en,fr,de01-december-2009SIST EN 15781:2009SLOVENSKI
STANDARD



SIST EN 15781:2009



EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 15781August 2009ICS 65.120 English VersionAnimal feeding stuffs - Determination of maduramicin-ammonium by reversed-phase HPLC using post-columnderivatisationAliments des animaux - Détermination de la maduramicineammonium par HPLC en phase inverse à l'aide de ladérivation post-colonneFuttermittel - Bestimmung von Maduramicin-Ammoniumdurch Umkehrphasen HPLC-Verfahren mittelsNachsäulenderivatisierungThis 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 and 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 responsibility 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, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre:
Avenue Marnix 17,
B-1000 Brussels© 2009 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15781:2009: ESIST EN 15781:2009



EN 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 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
SIST EN 15781:2009



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 identical 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 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, 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. SIST EN 15781:2009



EN 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 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 0,5 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 latest 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 derivatization 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 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 with water (4.1). 4.6 Potassium dihydrogen phosphate (KH2PO4) SIST EN 15781:2009



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-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 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 vacuum 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 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 µµµµg/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 4°C. Prepare fresh every month. 4.11.2 Standard solution, 10 µµµµg/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 4°C. Prepare fresh every week. 4.11.3 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) and mix. These solutions correspond to 0,2 µg, 0,4 µg, 0,6 µg, 0,8 µg, 1,0 µg, 1,2 µg, 1,6 µg, and 2,0 µg of maduramicin per ml respectively. Alternatively, you may use 5 calibration solutions with maduramicin concentrations of 0,4 µg, 0,7 µg, 1,0 µg, 1,5 µg and 2,0 µg 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 Centrifuge SIST EN 15781:2009



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 system, manual or autosampler, with loop suitable for 50 µl injection 5.3.3 UV/VIS detector, suitable for measurements at 520 nm NOTE Noise preferably should be < 1⋅ 10-5 AU (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 reaction coil, 1,5 ml to 2,0 ml reaction coil, for operating at 95°C 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 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 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 optimized if there are problems with bubbles. NOTE 2 A temperature of 92°C to 98°C is possible, high stability (1°C) should be guaranteed. 5.3.5.3 Reactor oven or water bath for the PTFE-reaction coil, suitable for operating at 95°C 5.3.6 Post column reagent pump, pulse free, flow cap
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