1、BRITISH STANDARDAnimal feeding stuffs Determination of monensin, narasin and salinomycin contents Liquid chromatographic method using post-column derivatizationICS 65.120g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g4
2、0g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN ISO 14183:2008This British Standard waspublished under the authorityof the Standards Policy andStrategy Committee on 24 January 2006 BSI 2009National forewordAmendments/corrigenda issued since publicationBS EN ISO 14183:2008
3、This British Standard is the UK implementation of EN ISO 14183:2008. It is identical with ISO 14183:2005. It supersedes BS ISO 14183:2005, which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/10, Animal feeding stuffs.A list of organizations represented
4、on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for it correct application.Compliance with a British Standard cannot confer immunity from legal obligations. ISBN 978 0 580 629
5、55 6Date Comments 30 September 2009 This corrigendum renumbers BS ISO 14183:2005 as BS EN ISO 14183:2008EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 14183November 2008ICS 65.120English VersionAnimal feeding stuffs - Determination of monensin, narasin andsalinomycin contents - Liquid chromat
6、ographic method usingpost-column derivatization (ISO 14183:2005)Aliments des animaux - Dtermination des teneurs enmonensine, narasine et salinomycine - Mthode parchromatographie liquide utilisant la drivatisation post-colonne (ISO 14183:2005)Futtermittel - Bestimmung der Gehalte an Monensin,Narasin
7、und Salinomycin -Flssigkeitschromatographisches Verfahren mittelsNachsulenderivatisierung (ISO 14183:2005)This European 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 EuropeanSta
8、ndard 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
9、, 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, Czec
10、h 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 STANDARDIZATIONCOMIT EUROPEN
11、DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2008 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 14183:2008: EEN ISO 14183:2008 (E) Foreword The text of ISO 14183:2005 has
12、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 stuffs - Methods of sampling and analysis” the secretariat of which i
13、s 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 the latest by May 2009. Attention is drawn to the possibility that
14、 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
15、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, Switzer
16、land 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. ii EN ISO 14183:2008BSiiiContents Page 1 Scope . 1 2 Normative references . 1 3 Principle. 1 4 Reagents 1 5 Apparatus 4 6 Sampling 5 7 Preparation of t
17、est sample. 5 8 Procedure 6 8.1 Preparation of quality control sample 6 8.2 Extraction 6 8.3 HPLC analysis . 7 8.4 Determination 8 9 HPLC confirmation . 9 9.1 General. 9 9.2 Post-column derivatization with DMAB 9 9.3 Hexane extraction . 10 10 Calculation of results . 10 10.1 General. 10 10.2 Monensi
18、n . 10 10.3 Salinomycin. 11 10.4 Narasin. 12 10.5 Interpretation of confirmation data. 13 11 Precision 14 11.1 Interlaboratory test . 14 11.2 Repeatability 14 11.3 Reproducibility 14 11.4 Limit of quantitation . 15 12 Test report . 15 Annex A (informative) Results of interlaboratory test 16 Bibliogr
19、aphy . 21 EN ISO 14183:2008 (E) EN ISO 14183:2008BS1Animal feeding stuffs Determination of monensin, narasin and salinomycin contents Liquid chromatographic method using post-column derivatization 1 Scope This International Standard specifies a high-performance liquid chromatographic (HPLC) method f
20、or 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 and semduramicin cannot be determined by this method. The limit of
21、 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 following referenced documents are indispensable for the application of
22、 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 samples 3 Principle The ionophores monensin, narasin and salinomyci
23、n 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 nm. Suspect positive trace-level samples and medicated feed samples
24、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. 4.1 Water, HPLC grade, or equivalent (e.g. Milli-Q purified water). 4
25、.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), minimum 99 % purity. EN ISO 14183:2008 (E) EN ISO 14183:2008BS2
26、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 flask. Mix well. 4.10 Mobile phases 4.10.1 Post-column reaction system While
27、 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 methanol (4.2)/water (4.1)/acetic acid (4.4) (940/60/1). Filter under vacuu
28、m 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 4.13. 4.12 Reference standards Composition or potency is required for eac
29、h 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 the solvents and solutions. Wear safety glasses and protective clothing. 4
30、.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 volume. Prepare freshly every month. Store in a refrigerator. Protect all sta
31、ndard 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 of narasin. 1) This is an example of a suitable product available commerc
32、ially. 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, Indianapolis, Indiana 46285, USA. 3) Available from Alpharma Inc., Animal
33、 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) EN ISO 14183:2008BS34.13.1 Monensin stock standard Prepare as described in 4.13. Calculate the concentration of stock standard based
34、 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 standard profile sheet: MM0,5100S = where 0,5 is the concentration of the stock
35、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 given component monensin A in the reference standard according to the profile s
36、heet, 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 standard concentration value provided by the supplier 2: S0,51000w = where Sis t
37、he 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 concentration is 986 g/mg. 4.13.3 Narasin stock standard Prepare as described
38、 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 narasin A. Use the component composition identified on the reference standard profil
39、e 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 the profile sheet, in percent. EXAMPLE For reference standard lot RS0302, the pe
40、rcentage 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) EN ISO 14183:2008BS4 4.14 Intermediate mixed standard solution, ca. 20 g/ml, 40 g/ml and 40 g/ml monensin, salinomycin and narasin, respectively. Transfer by pipette 10,
41、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 month. 4.15 Mixed HPLC standards Prepare five mixed HPLC standard solutions by pipettin
42、g 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. Table 1 Approximate HPLC standard concentration g/ml Mixed HPLC standard identifi
43、cation 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 1,0 ml of monensin stock standard (4.13.1) into a 100 ml low-actinic volumetric fla
44、sk. 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) into a 100 ml low-actinic volumetric flask. Dilute to volume with extraction solvent (
45、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 volume with extraction solvent (4.9). Mix well. Prepare freshly every month. Store in
46、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 system, manual or autosampler, with loop suitable for 100 l injections. EN ISO 14183:
47、2008 (E) EN ISO 14183:2008BS55.1.3 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 2,0 ml reaction coil, for operation at 98 C. The coil may be a commercially available kn
48、itted 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
49、. 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-column reagent pump, pulse free, with flow capacity 0,5 ml/min to 2,0 ml/min. 5.1.7 Analytical column. NOTE A 5 m C18, 25 0,46 cm Nucleosil 120A, Partisil 5 ODS-3, or Waters Nova Pak (4 m), or equivalent, has been found to be suitable.4)5.1.8 Guard column, C18. 5.2 Nitrogen evaporator, for evaporation of solvents under a stream of nitrogen. 5.3 Shaker, rotary or wrist-action. 5.4 Bala
