SIST EN 16158:2012
(Main)Animal feeding stuffs - Determination of semduramicin content - Liquid chromatographic method using a "tree" analytical approach
Animal feeding stuffs - Determination of semduramicin content - Liquid chromatographic method using a "tree" analytical approach
This European standard specifies a high-performance liquid chromatographic (HPLC) method for the determination of the semduramicin content at authorized level in animal feeding stuffs [2], using mass spectrometry detection or post-column derivatization and (UV)-VIS detection (hereinafter UV detection). This method is applicable to poultry feed. The limit of quantitation is 1,0 mg/kg when mass spectrometry is used for detection and 3,0 mg/kg when the detection is performed by UV with post-column derivatization. Lower limits of quantitation are achievable but this is to be validated by the user. The method allows the discrimination of semduramicin from monensin, salinomycin, narasin, maduramicin and lasalocid.
Futtermittel - Bestimmung des Semduramicingehalts - Flüssigkeitschromatographisches Verfahren mit verzweigter analytischer Vorgehensweise
Die vorliegende Internationale Norm legt ein hochleistungsflüssigkeitschromatographisches (HPLC) Verfahren zur Bestimmung des Semduramicingehaltes bei einem zugelassenen Gehalt in Futtermitteln mittels massen-spektrometrischer Detektion oder Nachsäulenderivatisierung und UV Detektion fest. Das Verfahren gilt für Geflügelfutter. Bei der Anwendung von Massenspektrometrie zum Nachweis beträgt die Bestimmungsgrenze 1,0 mg kg–1 und beim Nachweis mittels UV mit Nachsäulenderivatisierung 3,0 mg kg 1. Niedrigere Bestimmungsgrenzen sind erreichbar, das ist jedoch vom Anwender zu validieren.
Das Verfahren ermöglicht es, Semduramicin von Monensin, Salinomycin, Narasin, Maduramicin und Lasalocid zu unterscheiden.
Aliments pour animaux - Dosage de la semduramicine - Chromatographie liquide utilisant une approche analytique en arbre
La présente Norme internationale décrit une méthode de dosage de la semduramicine au taux autorisé dans les aliments pour animaux, par chromatographie liquide haute performance (CLHP) avec détection par spectrométrie de masse ou dérivation post-colonne et détection UV. Cette méthode est applicable aux aliments pour volailles. La limite de quantification est de 1,0 mg kg-1 avec détection par spectrométrie de masse et de 3,0 mg kg-1 avec détection UV et dérivation post-colonne. Des limites de quantification inférieures peuvent être obtenues, à condition qu'elles soient validées par l'utilisateur.
La méthode permet de différencier la semduramicine du monensine, de la salinomycine, du narasine, de la maduramicine et du lasalocide.
Krma - Določevanje semduramicina - Metoda tekočinske kromatografije z "razvejanim" analitskim pristopom
Ta evropski standard določa metodo tekočinske kromatografije visoke ločljivosti (HPLC) za določevanje dovoljene vrednosti vsebnosti semduramicina v krmi [2] z masno spektrometrijo ali postkolonsko derivatizacijo in zaznavanjem (UV)-VIS (v nadaljnjem besedilu: zaznavanje UV). Ta metoda se uporablja za perutninsko krmo. Če se za zaznavanje uporablja masna spektrometrija, je meja kvantifikacije 1,0 mg/kg, če pa se zaznavanje izvaja z zaznavanjem UV s postkolonsko derivatizacijo, pa 3,0 mg/kg. Nižje meje kvantifikacije so dosegljive, vendar jih mora validirati uporabnik. Metoda omogoča razlikovanje semduramicina od monenzina, salinomicina, narazina, maduramicina in lasalocida.
General Information
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Futtermittel - Bestimmung des Semduramicingehalts - Flüssigkeitschromatographisches Verfahren mit verzweigter analytischer VorgehensweiseAliments pour animaux - Dosage de la semduramicine - Chromatographie liquide utilisant une approche analytique en arbreAnimal feeding stuffs - Determination of semduramicin content - Liquid chromatographic method using a "tree" analytical approach65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 16158:2012SIST EN 16158:2012en,fr,de01-april-2012SIST EN 16158:2012SLOVENSKI
STANDARD
SIST EN 16158:2012
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16158
February 2012 ICS 65.120 English Version
Animal feeding stuffs - Determination of semduramicin content -Liquid chromatographic method using a "tree" analytical approach
Aliments pour animaux - Dosage de la semduramicine - Chromatographie liquide utilisant une approche analytique en arbre
Futtermittel - Bestimmung des Semduramicingehalts - Flüssigkeitschromatographisches Verfahren mit verzweigter analytischer Vorgehensweise This European Standard was approved by CEN on 30 December 2011.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC 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 translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, 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, Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
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Avenue Marnix 17,
B-1000 Brussels © 2012 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 16158:2012: ESIST EN 16158:2012
EN 16158:2012 (E) 2 Contents Page Foreword .31Scope .42Normative references .43Principle .44Reagents .45Apparatus .76Sampling .87Preparation of test sample .87.1General .87.2Laboratory sample .97.3Test sample .97.4Test portion .98Procedure .98.1Preparation of positive and negative control samples .98.2Samples extraction .98.3Filtration .98.4HPLC analysis .98.4.1LC-MS .98.4.2LC-PCD-UV . 118.5HPLC determination . 138.5.1LC-MS method . 138.5.2LC-PCD-UV method . 138.5.3System suitability . 139Calculation . 149.1LC-MS method . 149.2LC-PCD-UV method . 1410Precision . 1510.1Collaborative study. 1510.2Repeatability . 1510.3Reproducibility . 1511Test report . 16Annex A (informative)
Results of collaborative study . 17A.1Procedure . 17A.2Statistical analysis of results . 18A.3Example chromatogram . 22Bibliography . 25
SIST EN 16158:2012
EN 16158:2012 (E) 3 Foreword This document (EN 16158:2012) 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 August 2012, and conflicting national standards shall be withdrawn at the latest by August 2012. 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. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. 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, Croatia, 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, Turkey and the United Kingdom. SIST EN 16158:2012
EN 16158:2012 (E) 4 1 Scope This European standard specifies a high-performance liquid chromatographic (HPLC) method for the determination of the semduramicin content at authorized level in animal feeding stuffs [2], using mass spectrometry detection or post-column derivatization and (UV)-VIS detection (hereinafter UV detection). This method is applicable to poultry feed. The limit of quantitation is 1,0 mg/kg when mass spectrometry is used for detection and 3,0 mg/kg when the detection is performed by UV with post-column derivatization. Lower limits of quantitation are achievable but this is to be validated by the user. The method allows the discrimination of semduramicin from monensin, salinomycin, narasin, maduramicin and lasalocid. 2 Normative references The following referenced documents are indispensable for the application of this protocol. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. prEN ISO 6498, Animal feeding stuffs Guidelines for sample preparation (ISO/DIS 6498) 3 Principle Semduramicin is extracted using acetonitrile with mechanical shaking during 30 min. The extracts are filtered through 0,2 µm Nylon filters. Semduramicin is determined by reverse-phase liquid chromatography using electrospray (ESI) single quadrupole mass spectrometry detection in single ion monitoring (SIM) mode (LC-MS) [4] or using post-column derivatization with dimethylaminobenzaldehyde (DMAB) and spectrophotometric detection at 598 nm (LC-PCD-UV) [5]. If the detection used is ESI-MS the quantitation is performed through a standard addition approach. When LC-PCD-UV is used the quantitation is performed through external standard calibration.
4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified.
4.1 LC-MS. 4.1.1 Water, HPLC grade, or equivalent (e.g. Milli-Q purified water). 4.1.2 Acetonitrile, HPLC gradient grade, minimum 99,9 % purity. 4.1.3 Methanol, HPLC grade or hypergrade LC-MS. 4.1.4 Ammonium formate, HPLC grade. 4.1.5 Mobile phase. 4.1.5.1 Ammonium formate solution, c = 20 mmol/l. Accurately weigh 1,25 g to the nearest 0,01 g of ammonium formate (4.1.4) into a 1 000 ml volumetric flask. Dissolve in water (4.1.1) and make up to 1 000 ml of volume with water. Prepare fresh solutions monthly. 4.1.5.2 HPLC mobile phase. SIST EN 16158:2012
EN 16158:2012 (E) 5 Mix methanol (4.1.3) and ammonium formate solution (4.1.5.1) in proportion of 90+10 (v+v). Filter under vacuum using a solvent filtration system (5.11) and Nylon filters (5.13). 4.2 LC-PCD-UV. In addition to the reagents 4.1.1, 4.1.2, 4.1.3 and 4.1.4: 4.2.1 Sulphuric acid, minimum 98 % purity. 4.2.2 Dimethylaminobenzaldehyde (DMAB), minimum 99 % purity. 4.2.3 Formic acid, minimum 98 % purity. 4.2.4 Mobile phase. 4.2.4.1 Post-column reaction reagent. In a 500 ml volumetric flask (5.7) add first about 250 ml cold methanol (4.1.3) then 15 ml sulphuric acid (4.2.1). Dissolve 15 g DMAB (4.2.2) in the mixture. Cool down and make up to 500 ml with methanol (4.1.3). Filter under vacuum using the equipment in (5.11) and a membrane filter (5.12). Store in a refrigerator (from +2 °C to + 8 °C). This reagent is stable for 28 days. NOTE The methanol used for preparing the post-column reaction reagent should be kept refrigerated (from +2 °C to +8 °C).
4.2.4.2 Ammonium formate solution, c = 100 mmol/l at pH = 3. Accurately weigh 6,30 g to the nearest 0,01 g of ammonium formate (4.1.4) into a 1 000 ml volumetric flask (5.7). Dissolve in 900 ml water (4.1.1). Adjust the pH to 3,0 using formic acid (4.2.3) and make up to 1 000 ml with purified water. Prepare fresh monthly. 4.2.4.3 HPLC mobile phase (solvent blank). Mix methanol (4.1.3) and ammonium formate solution (4.2.4.2) in proportion of 90+10 (v+v). Filter under a vacuum using a solvent filtration system (5.11) and Nylon filters (5.13). 4.3 Reference standards LC-PCD-UV method. 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.
Declaration of purity is required for each lot of reference standard.
4.3.1 Semduramicin sodium standard, minimum 93 % purity expressed as semduramicin.
NOTE Available from Phibro Animal Health Corporation, Third Floor 65 Challenger Road Ridgefield Park, NJ 07660-2103 USA. This information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results. 4.4 Reference standards LC-MS method. In addition to the reference standard 4.3.1: 4.4.1 Nigericin sodium standard, minimum 98 % purity to be used as internal standard (I.S.). NOTE Available from Calbiochem, A Brand of EMD Biosciences, Inc. 10394 Pacific Center Court, San Diego, CA 92121 USA. This information is given for the convenience of users of this European Standard and does not constitute an SIST EN 16158:2012
EN 16158:2012 (E) 6 endorsement by CEN of the product named. Equivalent products may be used if they can be shown to lead to the same results.” 4.5 Standard solutions. Protect all standard solutions from daily light. 4.5.1 Semduramicin stock standard solution, ca. 1 mg/ml. Accurately weigh 10 mg to the nearest 0,1 mg of semduramicin sodium standard (4.3.1) into a 10 ml volumetric flask (5.7). Note down the exact weight of semduramicin sodium. Dissolve in methanol (4.1.3) and make up to 10 ml with methanol. Store at -20 °C and protected from light. Prepare freshly every 3 months. Determine the experimental concentration of the semduramicin stock solution using the reference standard purity value provided by the supplier expressed as semduramicin using Equation (1). PmCs×=10 (1) where Cs
is the experimental concentration of semduramicin in the stock standard in mg/ml; P is the purity of the semduramicin standard expressed as semduramicin given by the supplier in percent e.g. 0,934; m
is the weighed mass of semduramicin sodium standard (4.3.1) in mg. 4.5.2 Nigericin sodium stock standard solution (for the LC-MS method), ca. 1 mg/ml. Accurately weigh 10 mg to the nearest 0,1 mg of nigericin sodium standard (4.4.1) into a 10 ml volumetric flask (5.7). Note down the exact weight of nigericin sodium. Dissolve in methanol (4.1.3) and make up to 10 ml with methanol. Store at -20 °C and protected from light. Prepare freshly every 3 months.
Determine the experimental concentration of the nigericin sodium stock solutions using the reference standard purity value provided by the supplier using Equation (2). PmCn×=10 (2) where Cn is the experimental concentration of nigericin sodium in the stock standard in mg/ml; P is the purity of the nigericin sodium standard given by the supplier in % e.g. 0,98; m is the weighed mass of nigericin sodium standard (4.4.1) in mg. 4.5.3 Semduramicin intermediate standard solution (for the LC-MS method), ca. 100 µg/ml. Accurately pipette 1,0 ml of the semduramicin stock standard solution (4.5.1) into a 10 ml volumetric flask (5.7). Make up to 10 ml with acetonitrile (4.1.2). Store at -20 °C and protected from light. Prepare fresh intermediate solutions monthly. 4.5.4 Nigericin sodium intermediate standard solution (for the LC-MS method), ca. 100 µg/ml. SIST EN 16158:2012
EN 16158:2012 (E) 7 Accurately pipette 1,0 ml of the nigericin sodium stock standard solution (4.5.2) into a 10 ml volumetric flask (5.7). Make up to 10 ml of with acetonitrile (4.1.2). Store at -20 °C and protected from light. Prepare fresh intermediate solutions monthly. 4.5.5 Semduramicin spiking solution (for the LC-MS method), ca. 2 µg/ml. Accurately pipette 200 µl of the semduramicin intermediate solution (4.5.3) with an appropriate automatic pipette (5.6) into a 10 ml volumetric flask (5.7). Make up to 10 ml with acetonitrile (4.1.2). Store at -20 °C and protected from light. Prepare fresh spiking solutions weekly. 4.5.6 Nigericin sodium spiking solution (for the LC-MS method), ca. 2 µg/ml. Accurately pipette 200 µl of the nigericin sodium intermediate solution (4.5.4) with an appropriate automatic pipette (5.6) into a 10 ml volumetric flask. Make up to 10 ml with acetonitrile (4.1.2). Store at -20 °C and protected from light. Prepare fresh spiking solutions weekly. 5 Apparatus Usual laboratory apparatus and, in particular, the following:
5.1 LC-MS method 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 a loop suitable for 10 µl injections. 5.1.3 Single quadrupole mass spectrometer or triple quadrupole mass spectrometer used in the MS configuration able to operate in a mass range at least between 200 m/z to 1 000 m/z. NOTE For the MS detector the vacuum should be as stable as possible to ensure satisfactory repeatability. The system should therefore be pumped at least 48 h before starting the measurements. 5.1.4 Computer data system. 5.1.5 Analytical column, Alltima HP C18, 5 µm ,150 mm x 2,1 mm, 190 Å, 12 % C load and 200 m2/g or equivalent. 5.1.6 Guard column, Alltima HP C18, 5 µm, 7,5 mm x 2,1 mm, 190 Å, 12% C load and 200 m2/g or equivalent. 5.2 LC-PCD-UV method HPLC system consisting of the following: 5.2.1 Pump, pulse free, flow capacity 0,1 ml/min to 2,0 ml/min. 5.2.2 Injection system, manual or autosampler, with a loop suitable for 100 µl injections. 5.2.3 UV/VIS detector, variable wavelength, suitable for reliable measurements at 598 nm, or UV/VIS photodiode array detector (DAD). 5.2.4 Computer data system. 5.2.5 Post-column reactor, with a 1,5 ml to 2,0 ml reaction coil, for operation at 92 °C. The coil may be a commercially available coil or it may be made using 1/16” 316 SS tubing, 0,020" <> 0,5 mm ID (between 8 m and 10 m length) folded to fit the reactor heating chamber. To ensure effective mixing of reagent and column effluent, use a vortex or static mixing tee (not a regular tee) before the reaction coil. SIST EN 16158:2012
EN 16158:2012 (E) 8 5.2.6 Post column reagent pump, pulse free, flow capacity from 0,5 ml/min to 2,0 ml/min. 5.2.7 Analytical column, Inertsil ODS-3 C18, 5 µm, 150 x 4,6 mm 100 Å 15% carbon load and 450m2/g or equivalent. 5.2.8 Guard column, Prevail C18, 5 µm, 7,5 x 4,6 mm or equivalent. 5.3 Glass vials, 1,5 ml HPLC glass vials. 5.4 Shaker, head-over-head or equivalent. 5.5 Balances, one analytical, of 10 g capacity or greater with 0,1 mg readability. 5.6 Variable-volume positive displacement piston pipettes, suitable for pipetting volumes ranged from 20 µl to 1 000 µl. 5.7 Volumetric flasks, 10 ml, 20 ml, 500 ml and 1 000 ml. 5.8 Glass graduated cylinders, 100 ml and 1 000 ml. 5.9 Polypropylene centrifuge tubes, 50 ml capacity, stoppered (Falcon® or equivalent). 5.10 Disposable syringes, 20 ml. 5.11 Solvent filtration system, all glass filter apparatus suitable for 47 mm filters. 5.12 Membrane filters, Durapore® membrane filters, PVDF, hydrophobic, of 47 mm diameter and pore size 0,45 µm or equivalent. 5.13 Nylon filters, 47 mm diameter and pore size 0,22 µm or equivalent. 5.14 Syringe filters Nylon 0,2 µm or equivalent i.e. full chemical compatibility with acetonitrile. 5.15 pH meter. 5.16 Grinding instrument. 5.17 Sieve, with 1 mm apertures. 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. Sampling is not part of the method specified in this European standard. A recommended sampling method is given in EN ISO 6497 [1]. 7 Preparation of test sample 7.1 General Prepare the test sample in accordance with prEN ISO 6498. SIST EN 16158:2012
EN 16158:2012 (E) 9 7.2 Laboratory sample Grind the laboratory sample (usually 50 g) so that it passes completely through a sieve with 1 mm apertures. Mix thoroughly.
7.3 Test sample The test sample consists of a representative and homogenised aliquot of the ground laboratory sample of at least 20 g. 7.4 Test portion Accurately weigh 5,0 g ± 0,1 g of the thoroughly mixed test sample into a 50 ml polypropylene stoppered tube (Falcon® tube or equivalent). Note down the mass expressed in mg (Wtp). Submit it to the analysis procedure (8). 8 Procedure 8.1 Preparation of positive and negative control samples The use of quality control samples and quality control charts is recommended. With each set of samples, include a positive control sample (PCS) and a negative control sample (NCS) on a daily basis. For the PCS, weigh 5,0 g ± 0,1 g of a poultry blank feed. Accurately pipette, with an appropriate automatic pipette (5.6), 130 µl of the semduramicin stock standard solution (4.5.1) and add to the poultry blank. Mix thoroughly to ensure maximum contact with the feed. Allow penetration for at least 1 hour before extraction. The NCS is constituted by 5,0 g ± 0,1 g of non-spiked poultry blank feed. Both PCS and NCS will be submitted to the same treatment as unknown samples. 8.2 Samples extraction
Accurately weigh 5,0 g ± 0,1 g test portion into a polypropylene centrifuge tube (5.9). Add 15 ml acetonitrile (4.1.2), close tightly the polypropylene centrifuge tube (5.9) and check that there is no solvent leak through the cap. Shake vigorously to ensure appropriate suspension of the feed into the extraction solvent. Perform a solid-liquid extraction by shaking the mixture for 30 min on the shaker (5.4). 8.3 Filtration Filter the supernatants (8.2) through a 0,2 µm Nylon syringe filter (5.14) into a clean polypropylene tube (5.9).
8.4 HPLC analysis 8.4.1 LC-MS 8.4.1.1 Dilution
Accurately pipette with an appropriate automatic pipette (5.6), 75 µl of the filtered extract (8.3) into a 10 ml volumetric flask (5.7) Make up to 10 ml volume with acetonitrile (4.1.2).
8.4.1.2 Standard additions
Accurately pipette with an appropriate automatic pipette (5.6), four 900 µl aliquots of the diluted extract (8.4.1.1) into 1,5 ml glass vials (5.3) and label as S0, S1, S2 and S3 respectively. SIST EN 16158:2012
EN 16158:2012 (E) 10 Accurately pipette with an appropriate automatic pipette (5.6), 25 µl of the nigericin sodium spiking solution (4.5.6) and add to each of the vials labelled as S0, S1, S2 and S3 respectively. Accurately pipette with an appropriate automatic pipette (5.6), 0 µl, 15 µl, 45 µl and 75 µl of the semduramicin spiking solution (4.5.5) and add to the vials labelled as S0, S1, S2 and S3 respectively.
Accurately pipette with an appropriate automatic pipette (5.6), 75 µl, 60 µl, 30 µl and 0 µl of acetonitrile (4.1.2) and add to the vials labelled as S0, S1, S2 and S3 respectively.
Cap all vials and shake vigorously.
NOTE When using highly sensitive mass spectrometers an appropriate further dilution of all the solutions (e.g. 1:10) from the vials labelled as S0, S1, S2 and S3 is recommended in order to prevent source saturation. 8.4.1.3 Analytical conditions a) HPLC separation parameters:
1) column: as in (5.1.5); 2) guard column: as in (5.1.6); 3) mobile phase: as in (4.1.5.2)
4) flow rate: 0,25 ml/min; 5) injection volume: 10 µl; 6) injection mode: full loop; 7) column temperature: 25 °C; 8) autosampler temperature: 4 °C. In these conditions the retention times are approximately 4,0 min and 7,0 min for semduramicin and nigericin respectively. The total run time is 15 min. NOTE 1 If longer retention times are observed, increase appropriately the total run time.
b) MS parameters 1) ionisation mode: ESI+ ; 2) acquisition mode: single ion monitoring (SIM); 3) probe temperature: 250 °C; 4) cone voltage: 60 V; 5) needle voltage: 2,7 kV; 6) ammonium adducts (used for identification and for quantification): i) semduramicin m/z > 890,5; ii) nigericin (I.S.) m/z > 742,5; NOTE 2 All conditions mentioned were optimised for the LC-MS used at EC-JRC-IRMM for the optimisation and validation of this protocol being therefore only indicative values and have to be optimised for the used equipment. SIST EN 16158:2012
EN 16158:2012 (E) 11 7) sodium adducts (used only for identification): i) semduramicin m/z > 895,5; ii) nigericin (I.S.) m/z > 747,5;
8) mass span: ± 0,25; 9) dwell time: 0,50 s. NOTE 3 The quantification is based on the signal of the correspondent ammonium adducts of the molecular ions. If a MS/MS instrumentation is used, it shall be used in MS configuration. NOTE 4 Optimize the instrumental parameters in order to have a similar absolute signal of the semduramicin and nigericin ammonium adducts in a solution containing both substances at the same concentration level, e.g. prepare a sample containing 25 µl solution (4.5.5) + 25 µl solution (4.5.6) + 950 µl acetonitrile and check that in the optimized conditions the obtained absolute signal for the respective ammonium adducts are of the same order of magnitude. NOTE 5 After a maximum of 50 injections a cleaning step is strongly recommended. This cleaning step involves flushing the column with pure methanol (4.1.3) at 0,25 ml/min during 30 min followed by re-conditioning of the column with an acetonitrile (4.1.2) injection with the mobile phase (4.1.5.2) used for the analysis for the HPLC column. It is advised to include the cleaning step always after the full standard addition of one sample. 8.4.2 LC-PCD-UV 8.4.2.1 System set up Start up the system as follows. Switch on the oven of the post column derivatization system. Turn on the HPLC pump with a low flow (e.g. 0,1 ml/min) before starting the post-column reagent flow to prevent back flow of reagent into the analytical column. Start then the post-column reagent flow with a low flow (e.g. 0,1 ml/min). Slowly increase alternatively the two flows until reaching 0,7 ml/min and 0,9 ml/min respectively for the HPLC flow and the post-column reagent flow. Wait until the temperature is stabilized at 92 °C. 8.4.2.2 External calibration curve 8.4.2.2.1 General Accurately pipette with an appropriate automatic pipette (5.6), one aliquot of the filtered extract (8.3) into
1,5 ml glass vials (5.3) and label it as S0. Cap the vial and shake vigorously.
Prepare the respective HPLC calibrants as follows:
8.4.2.2.2 HPLC standard 1, ca. 2,5 µg/ml Accurately pipette 50 µl of semduramicin stock standard solution (4.5.1) with an automatic pipette (5.6) into a 20 ml volumetric flask. Make up to 20 ml with acetonitrile (4.1.2). Mix well. Store at -20 °C. Prepare fresh weekly. 8.4.2.2.3 HPLC standard 2, ca. 6,5 µg/ml Accurately pipette 130 µl of semduramicin stock standard solution (4.5.1) with an automatic pipette (5.6) into a 20 ml volumetric flask. Make up to 20 ml with acetonitrile (4.1.2). Mix well. Store at -20 °C. Prepare fresh weekly. SIST EN 16158:2012
EN 16158:2012 (E) 12 8.4.2.2.4 HPLC standard 3, ca. 10,5 µg/ml Accurately pipette 210 µl of semduramicin stock standard solution (4.5.1) with an automatic pipette (5.6) into a 20 ml volumetric flask. Make up to 20 ml with acetonitrile (4.1.2). Mix well. Store at -20 °C. Prepare fresh weekly. 8.4.2.2.5 HPLC standard 4, ca. 14,5 µg/ml Accurately pipette 290 µl of semduramicin stock standard solution (4.5.1) with an automatic pipette (5.6) into a 20 ml volumetric flask. Make up to 20 ml with acetonitrile (4.1.2). Mix well. Store at -20 °C. Prepare fresh weekly. 8.4.2.2.6 HPLC standard 5, ca. 18,5 µg/ml Accurately pipette 370 µl of semduramicin stock standard solution (4.5.1) with an automatic pipette (5.6) into a 20 ml volumetric flask. Make up to 20 ml with acetonitrile (4.1.2). Mix well. Store at -20 °C. Prepare fresh weekly. 8.4.2.3 Analytical conditions a) HPLC separation parameters: 1) analytical column (5.2.7); 2) guard column (5.2.8); 3) column temperature: 38 °C; 4) mobile phase: as in (4.2.4.3); 5) flow rate: 0,7 ml/min; 6) wavelength: 598 nm; b) Post-column reaction parameters: 1) post-column reactor: as in (5.2.5); 2) mobile phase: as in (4.2.4.1); 3) flow rate: 0,9 ml/min; 4) reactor temperature: 92 °C. NOTE During the series of measurements, the post column reaction reagent (4.2.4.1) shall be kept cool and protected from the light by means of ice blocks or in an ice bath. Using the analytical column (5.2.7) guard column (5.2.8) combination, with the above conditions, the retention time for semduramicin is approximately 10 min. The total run is set at e.g. 25 min to allow the elution of all the other coccidiostats, if present.
8.4.2.4 System shutdown Shut down the system as follows. Turn off the post-column reagent flow before stopping HPLC pump to prevent back flow
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Questions, Comments and Discussion
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