1、February 2009DEUTSCHE NORM English price group 13No 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!$Uvf“1508367ww
2、w.din.deDDIN EN ISO 14183Animal feeding stuffs Determination of monensin, narasin and salinomycin contents Liquid chromatographic method using post-column derivatization(ISO 14183:2005)English version of DIN EN ISO 14183:2009-02Futtermittel Bestimmung der Gehalte an Monensin, Narasin und Salinomycin
3、 Flssigkeitschromatographisches Verfahren mittels Nachsulenderivatisierung(ISO 14183:2005)Englische Fassung DIN EN ISO 14183:2009-02www.beuth.deDocument comprises 26 pagesDIN EN ISO 14183:2009-02 2 National foreword This standard has been prepared by Technical Committee ISO/TC 34 “Food products” (Se
4、cretariat: AFNOR, France), Subcommittee SC 10 “Animal feeding stuffs” (Secretariat: NEN, Netherlands) in collaboration with Technical Committee CEN/TC 327 “Animal feeding stuffs Methods of sampling and analysis” (Secretariat: NEN, Netherlands). The responsible German body involved in its preparation
5、 was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-03-03 AA Futtermittel. This European standard has been prepared under a mandate given to CEN by the European Commission and the European Free Trade
6、Association, and supports essential requirements of EU Directive(s). The DIN Standard corresponding to the International Standard referred to in this document is as follows: ISO 6497 DIN EN ISO 6497 National Annex NA (informative) Bibliography DIN EN ISO 6497, Animal feeding stuffs Sampling EUROPEAN
7、 STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 14183November 2008ICS 65.120English VersionAnimal feeding stuffs - Determination of monensin, narasin andsalinomycin contents - Liquid chromatographic method usingpost-column derivatization (ISO 14183:2005)Aliments des animaux - Dtermination des teneurs
8、enmonensine, narasine et salinomycine - Mthode parchromatographie liquide utilisant la drivatisation post-colonne (ISO 14183:2005)Futtermittel - Bestimmung der Gehalte an Monensin,Narasin und Salinomycin -Flssigkeitschromatographisches Verfahren mittelsNachsulenderivatisierung (ISO 14183:2005)This E
9、uropean Standard was approved by CEN on 25 October 2008.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
10、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
11、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, Lithuan
12、ia, 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: rue de Stassart, 36 B-1050 Brussels 2008 CEN Al
13、l rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 14183:2008: EContents Page 1 2 3 4 5 6 7 8 8.1 8.2 8.3 8.4 9 9.1 9.2 9.3 10 10.1 10.2 10.3 10.4 10.5 11 11.1 11.2 11.3 11.4 12 EN ISO 14183:2008 (E) 2DIN EN ISO 14183:2009-02 Scope . 4 No
14、rmative references . 4 Principle. 4 Reagents 4 Sampling 8 Preparation of test sample. 8 Procedure 9 Extraction 9 Calculation of results . 13 Monensin . 13 Narasin. 15 Precision 17 Repeatability 17 Limit of quantitation . 18 Test report . 18 Annex A (informative) Results of interlaboratory test 19 Bi
15、bliography . 24 Reproducibility 17 Interlaboratory test . 17 Interpretation of confirmation data. 16 Salinomycin. 14 General. 13 Hexane extraction. 13 Preparation of quality control sample 9 Post-column derivatization with DMAB 12 HPLC confirmation . 12 HPLC analysis . 10 Foreword. 3 Determination 1
16、1 General. 12 Apparatus .7 Foreword The text of ISO 14183:2005 has been prepared by Technical Committee ISO/TC 34 “Agricultural food products” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 14183:2008 by Technical Committee CEN/TC 327 “Animal feeding st
17、uffs - Methods of sampling and analysis” 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 May 2009, and conflicting national standards shall be withdrawn at
18、 the latest by May 2009. 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 nation
19、al 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, Po
20、land, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 14183:2005 has been approved by CEN as a EN ISO 14183:2008 without any modification. EN ISO 14183:2008 (E) 3DIN EN ISO 14183:2009-02 1 Scope This International Standard
21、specifies a high-performance liquid chromatographic (HPLC) method for the determination of the monensin, narasin and salinomycin contents of animal feeding stuffs, supplements (dry and liquid) and mineral premixtures. The method is not applicable to drug premixes (pharmaceutical products). Lasalocid
22、 and semduramicin cannot be determined by this method. The limit of quantitation is approximately 1 mg/kg, 2 mg/kg and 2 mg/kg for monensin, salinomycin and narasin, respectively. A lower limit of quantitation can be achievable but this is to be validated by the user. 2 Normative references The foll
23、owing 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. ISO 6498:1998, Animal feeding stuffs Preparation of test
24、samples 3 Principle The ionophores monensin, narasin and salinomycin are extracted using methanol/water (90 + 10) with mechanical shaking for 1 h, then the extracts are filtered. The ionophores are determined by reverse-phase HPLC using post-column derivatization with vanillin and detection at 520 n
25、m. Suspect positive trace-level samples and medicated feed samples containing unexpected ionophores are confirmed using a hexane extraction or post-column derivatization with dimethylaminobenzaldehyde (DMAB). 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified. 4.
26、1 Water, HPLC grade, or equivalent (e.g. Milli-Q purified water). 4.2 Methanol (CH3OH), HPLC grade. 4.3 Sulfuric acid (H2SO4), 97 % to 98 %. 4.4 Acetic acid (CH2CH3CO2H), glacial, 97 % to 98 %. 4.5 Sodium hydrogen carbonate (NaHCO3), minimum 99 % purity. 4.6 Vanillin (4-hydroxy-3-methoxybenzaldehyde
27、), minimum 99 % purity. EN ISO 14183:2008 (E) 4DIN EN ISO 14183:2009-02 4.7 Dimethylaminobenzaldehyde (DMAB), minimum 99 % purity. 4.8 Hexane CH3(CH2)4CH3, distilled in glass. 4.9 Extraction solvent, methanol/water (90 + 10). Combine 1 800 ml of methanol (4.2) and 200 ml of water (4.1) in a 2 l flas
28、k. Mix well. 4.10 Mobile phases 4.10.1 Post-column reaction system While stirring gently, slowly add by pipette 20 ml of sulfuric acid (4.3) to 950 ml of methanol (4.2). Allow to cool, then add 30 g of vanillin (4.6) while stirring. Protect from light. Prepare fresh daily. 4.10.2 HPLC column Use met
29、hanol (4.2)/water (4.1)/acetic acid (4.4) (940/60/1). Filter under vacuum using the equipment in 5.7. 4.11 Neutralized methanol Add 1,0 g of sodium hydrogen carbonate (4.5) into 4 l of methanol (4.2). Mix well and filter if necessary through an 11 m filter paper (e.g. Whatman No. 1)1). See Note to 4
30、.13. 4.12 Reference standards Composition or potency is required for each lot of reference standard. 4.12.1 Monensin sodium2)4.12.2 Narasin2)4.12.3 Sodium salinomycin3)WARNING Avoid inhalation of and exposure to the toxic standard materials and solutions thereof. Work in a fume-hood when handling th
31、e solvents and solutions. Wear safety glasses and protective clothing. 4.13 Ionophore stock standards, ca. 0,50 mg/ml. Accurately weigh, to the nearest 0,1 mg, 25 mg of each standard (4.12.1 to 4.12.3) into separate 50 ml volumetric flasks. Dissolve in neutralized methanol (4.11) and dilute to volum
32、e. Prepare freshly every month. Store in a refrigerator. Protect all standard solutions from light or prepare them in low actinic flasks. NOTE The requirement for neutralized methanol has not been verified for salinomycin. It is not required if analysing monensin only, but is required for analysis o
33、f narasin. 1) This is an example of a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of this product. 2) Available from Lilly Research Laboratories, Lilly Corporate Center, I
34、ndianapolis, Indiana 46285, USA. 3) Available from Alpharma Inc., Animal Health Division, 1 Duggar Drive, Willow Island, WV, USA 26134-97111, and Hoechst Roussel Vet, D-65926 Frankfurt am Main, Gebaude H 790, Germany. EN ISO 14183:2008 (E) 5DIN EN ISO 14183:2009-02 4.13.1 Monensin stock standard Pre
35、pare as described in 4.13. Calculate the concentration of stock standard based on the principle component, monensin A. The minor component, monensin B, which elutes just before monensin A 4is determined in test samples based on monensin A. Use the component composition identified on the reference st
36、andard profile sheet: MM0,5100S = where 0,5 is the concentration of the stock standard (4.13), in milligrams per millilitre, recorded to three significant figures; Misthe concentration of the given component monensin A in the stock standard, in milligrams per millilitre; SMis the proportion of the g
37、iven component monensin A in the reference standard according to the profile sheet, in percent. EXAMPLE Reference standard lot RS0234 contains 93,71 % of monensin A on an “as-is” basis. 4.13.2 Salinomycin stock standard Prepare as described in 4.13. Determine the concentration using the reference st
38、andard concentration value provided by the supplier 2: S0,51000w = where Sis the concentration of salinomycin in the stock standard, in milligrams per millilitre; wis theconcentration of the salinomycin standard given by the supplier, in micrograms per milligram. EXAMPLE For lot WS-19B, the standard
39、 concentration is 986 g/mg. 4.13.3 Narasin stock standard Prepare as described in 4.13. Calculate the concentration of the stock standard based on the principle component, narasin A. The minor components (narasin D and l), which elute after narasin A 5, are determined in test samples based on narasi
40、n A. Use the component composition identified on the reference standard profile sheet: NN0,5100S = where Nisthe concentration of the component narasin A in the stock standard, in milligrams per millilitre; SNis the proportion of the given component narasin A in the reference standard according to th
41、e profile sheet, in percent. EXAMPLE For reference standard lot RS0302, the percentage of each component on an “as-is” basis is: narasin A = 85,4 %, narasin D = 1,9 %, narasin I = 0,7 %. EN ISO 14183:2008 (E) 6DIN EN ISO 14183:2009-02 4.14 Intermediate mixed standard solution, ca. 20 g/ml, 40 g/ml a
42、nd 40 g/ml monensin, salinomycin and narasin, respectively. Transfer by pipette 10,0 ml, 20,0 ml and 20,0 ml of monensin, salinomycin and narasin stock standards (4.13), respectively, into a 250 ml volumetric flask. Dilute to volume with extraction solvent (4.9). Mix well. Prepare freshly every mont
43、h. 4.15 Mixed HPLC standards Prepare five mixed HPLC standard solutions by pipetting an aliquot of the mixed intermediate standard (4.14) into 100 ml low-actinic volumetric flasks and diluting to volume with extraction solvent (4.9), as specified in the Table 1. Mix well. Prepare freshly every month
44、. Table 1 Approximate HPLC standard concentration g/ml Mixed HPLC standard identification Amount of intermediate standard (4.14) ml Monensin Salinomycin Narasin A 1 0,2 0,4 0,4 B 5 1 2 2 C 10 2 4 4 D 25 5 10 10 E 50 10 20 20 4.16 Single HPLC standards 4.16.1 Monensin, ca. 5 g/ml. Accurately pipette
45、1,0 ml of monensin stock standard (4.13.1) into a 100 ml low-actinic volumetric flask. Dilute to volume with extraction solvent (4.9). Mix well. Prepare freshly every month. Store in a refrigerator. 4.16.2 Salinomycin, ca. 10 g/ml. Accurately pipette 2,0 ml of salinomycin stock standard (4.13.2) int
46、o a 100 ml low-actinic volumetric flask. Dilute to volume with extraction solvent (4.9). Mix well. Prepare freshly every month. Store in a refrigerator. 4.16.3 Narasin, ca. 10 g/ml. Accurately pipette 2,0 ml of narasin stock standard (4.13.3) into a 100 ml low-actinic volumetric flask. Dilute to vol
47、ume with extraction solvent (4.9). Mix well. Prepare freshly every month. Store in a refrigerator. 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 HPLC system consisting of the following. 5.1.1 Pump, pulse free, flow capacity 0,1 ml/min to 2,0 ml/min. 5.1.2 Injection sy
48、stem, manual or autosampler, with loop suitable for 100 l injections. EN ISO 14183:2008 (E) 7DIN EN ISO 14183:2009-02 5.1.3 UV/VIS detector, with variable wavelength, suitable for measurements at 520 nm and 592 nm. 5.1.4 Integrator or computer data system. 5.1.5 Post-column reactor, with a 1,5 ml to
49、 2,0 ml reaction coil, for operation at 98 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. For example, wrap the coil in sufficient 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. 5.1.6 Post-
