Animal feeding stuffs: Methods of sampling and analysis - Determination of carbadox and olaquindox by HPLC/UV

This European Standard specifies a high performance liquid chromatographic - UV detection (HPLC-UV) method for the simultaneous determination of two growth promoters Carbadox and Olaquindox contents in compound feeds and raw materials at levels ranging from the limit of quantification to 100 mg/kg.
The limit of quantification of the method has been demonstrated to be lower than 3 mg/kg for olaquindox and 4 mg/kg for carbadox.

Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von Carbadox und Olaquindox mittels HPLC/UV

Diese Europäische Norm legt ein Verfahren zur gleichzeitigen Bestimmung des jeweiligen Gehalts der beiden Wachstumsförderer Carbadox und Olaquindox in Mischfuttermitteln und Rohmaterialien bei Konzentrationen im Bereich der Bestimmungsgrenze (en: limit of quantification, LOQ) bis zu 100 mg/kg mit Hochleistungs¬flüssigkeitschromatographie und UV Detektion (HPLC UV) fest.
Die Bestimmungsgrenze des Verfahrens hat gezeigt, dass sie niedriger als 3 mg/kg bei Olaquindox und 4 mg/kg bei Carbadox ist.

Aliments des animaux - Méthodes d'échantillonnage et d'analyse - Détermination des teneurs en carbadox et olaquindox par CLHP/UV

La présente Norme européenne spécifie une méthode de chromatographie liquide à haute performance avec détection UV (CLHP-UV) pour la détermination simultanée des teneurs en carbadox et olaquindox, deux promoteurs de croissance, dans les aliments composés et les matières premières à des niveaux compris entre la limite de quantification et 100 mg/kg.
La limite de quantification de la méthode s’est révélée être inférieure à 3 mg/kg pour l’olaquindox et inférieure à 4 mg/kg pour le carbadox.

Krma: metode vzorčenja in analize - Določevanje karbadoksa in olakvindoksa s HPLC/UV

Ta evropski standard določa metodo tekočinske kromatografije visoke ločljivosti z UV-detekcijo (HPLC-UV) za hkratno določanje vsebnosti dveh pospeševalcev rasti karbadoksa in olakvindoksa v krmnih mešanicah in surovinah pri koncentracijah od mejne vrednosti kvantifikacije do 100 mg kg-1.
Mejna vrednost kvantifikacije metode je bila dokazano boljša od 3 mg kg-1 za olakvindoks in 4 mg kg-1 za karbadoks.

General Information

Status
Published
Publication Date
25-Jul-2017
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
26-Jul-2017
Completion Date
26-Jul-2017

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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.+3/&89Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung von Carbadox und Olaquindox mittels HPLC/UVAliments des animaux - Méthodes d'échantillonnage et d'analyse - Détermination des teneurs en carbadox et olaquindox par CLHP/UVAnimal feeding stuffs: Methods of sampling and analysis - Determination of carbadox and olaquindox by HPLC/UV71.040.50Fizikalnokemijske analitske metodePhysicochemical methods of analysis65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 16930:2017SIST EN 16930:2017en,fr,de01-september-2017SIST EN 16930:2017SLOVENSKI

STANDARD
SIST EN 16930:2017
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16930
July
t r s y ICS
x wä s t r English Version

Animal feeding stuffsã Methods of sampling and analysis æ Determination of carbadox aAliments des animaux æ Méthodes d 5échantillonnage et d 5analyse æ Détermination des teneurs en carbadox et

Futtermittel æ Probenahmeæ und Untersuchungsverfahren æ Bestimmung von Carbadox This European Standard was approved by CEN on

s v May
t r s yä

egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alterationä Upætoædate lists and bibliographical references concerning such national standards may be obtained on application to the CENæCENELEC Management Centre or to any CEN memberä

translation under the responsibility of a CEN member into its own language and notified to the CENæCENELEC Management Centre has the same status as the official versionsä

CEN members are the national standards bodies of Austriaá Belgiumá Bulgariaá Croatiaá Cyprusá Czech Republicá Denmarká Estoniaá Finlandá Former Yugoslav Republic of Macedoniaá Franceá Germanyá Greeceá Hungaryá Icelandá Irelandá Italyá Latviaá Lithuaniaá Luxembourgá Maltaá Netherlandsá Norwayá Polandá Portugalá Romaniaá Serbiaá Slovakiaá Sloveniaá Spainá Swedená Switzerlandá Turkey and United Kingdomä

EUROPEAN COMMITTEE FOR STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre:
Avenue Marnix 17,
B-1000 Brussels

t r s y CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Membersä Refä Noä EN

s x { u rã t r s y ESIST EN 16930:2017

EN 16930:2017 (E) 2 Contents Page European foreword ....................................................................................................................................................... 4 1 Scope .................................................................................................................................................................... 5 2 Normative references .................................................................................................................................... 5 3 Principle ............................................................................................................................................................. 5 4 Reagents and materials ................................................................................................................................. 5 4.1 Water, demineralized or deionized or at least equivalent .............................................................. 6 4.2 Methanol ............................................................................................................................................................. 6 4.3 Acetonitrile ........................................................................................................................................................ 6 4.4 Glacial acetic acid, minimum purity 96 % .............................................................................................. 6 4.5 Ammonium acetate water free salt, CH3CO2NH4 .................................................................................. 6 4.6 Extraction solvent: methanol water mixture (1:1 v:v) ...................................................................... 6 4.7 Ammonium acetate buffer, 25 mM, pH = 4,35 ...................................................................................... 6 4.8 Mobile phase for HPLC: acetonitrile: buffer mixture (10:90; v:v) ................................................. 6 4.9 Carbadox dissolving solvent: methanol: acetonitrile mixture (1:1; v:v) .................................... 6 4.10 Reference standards ...................................................................................................................................... 6 4.11 Standard solutions .......................................................................................................................................... 6 4.12 Neutral aluminium oxide, Brockmann activity I .................................................................................. 8 4.13 Neutral aluminium oxide SPE cartridge, 2 cm3, 1850 mg ................................................................. 8 4.14 Technical methanol ........................................................................................................................................ 8 5 Apparatus ........................................................................................................................................................... 8 5.1 pH meter ............................................................................................................................................................. 8 5.2 Solvent filtration system, suitable for 0,45 µm membrane filters ................................................ 8 5.3 Ultrasonic bath ................................................................................................................................................. 8 5.4 Rotary on mechanical shaker ..................................................................................................................... 8 5.5 Centrifuge ........................................................................................................................................................... 8 5.6 Glass wool .......................................................................................................................................................... 8 5.7 Glass column for chromatography, length 200 mm to 400 mm; internal diameter 10 mm, restricted at the end and fitted with a wad of glass wool (5.6) or equivalent column ................................................................................................................................................................. 8 5.8 Filter papers or glass microfibre filter .................................................................................................... 8 5.9 Cellulose acetate membrane filters of pore size 0,45 µm ................................................................. 8 5.10 PVDF syringe filters of pore size 0,45 µm and adaptable syringes ............................................... 8 5.11 HPLC system ...................................................................................................................................................... 8 5.12 Refrigerator or refrigerated chamber at a temperature set between 0 °C and 8 °C ............... 9 6 Sampling ............................................................................................................................................................. 9 7 Preparation of test sample .......................................................................................................................... 9 7.1 General ................................................................................................................................................................ 9 7.2 Laboratory sample .......................................................................................................................................... 9 7.3 Test sample ....................................................................................................................................................... 9 7.4 Test portion ....................................................................................................................................................... 9 8 Procedure........................................................................................................................................................... 9 8.1 Extraction of feeding stuffs containing 0,5 mg/kg to 100 mg/kg of growth promoters ........ 9 8.2 Filtration ............................................................................................................................................................ 9 8.3 Purification ........................................................................................................................................................ 9 SIST EN 16930:2017

EN 16930:2017 (E) 3 8.4 HPLC analysis ................................................................................................................................................. 10 8.5 System suitability – Confirmation .......................................................................................................... 11 9 Calculation....................................................................................................................................................... 12 10 Precision .......................................................................................................................................................... 13 10.1 Collaborative study ...................................................................................................................................... 13 10.2 Repeatability .................................................................................................................................................. 13 10.3 Reproducibility .............................................................................................................................................. 13 11 Test report ...................................................................................................................................................... 14 Annex A (informative)

Results of the collaborative study .......................................................................... 15 A.1 Procedure ........................................................................................................................................................ 15 A.2 Materials .......................................................................................................................................................... 15 A.3 Statistical analysis of results .................................................................................................................... 17 A.4 Results and interpretation ........................................................................................................................ 17 A.5 Example chromatogram ............................................................................................................................. 24 Bibliography ................................................................................................................................................................. 26

SIST EN 16930:2017

EN 16930:2017 (E) 4 European foreword This document (EN 16930:2017) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs - 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 January 2018, and conflicting national standards shall be withdrawn at the latest by January 2018. 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. WARNING — The use of this protocol involves hazardous materials, operations and equipment. This protocol does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this protocol to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. According to the CEN-CENELEC Internal Regulations, the national standards organisations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. SIST EN 16930:2017

EN 16930:2017 (E) 5 1 Scope This European Standard specifies a high performance liquid chromatographic – UV detection (HPLC-UV) method for the simultaneous determination of two growth promoters Carbadox and Olaquindox contents in compound feeds and raw materials at levels ranging from the limit of quantification to 100 mg/kg. The limit of quantification of the method has been demonstrated to be lower than 3 mg/kg for olaquindox and 4 mg/kg for carbadox. 2 Normative references The following documents, in whole or in part, is normatively referenced in this document and is indispensable for its application. For undated reference, the latest edition of the referenced document (including any amendments) applies. EN ISO 6498, Animal feeding stuffs - Guidelines for sample preparation (ISO 6498) 3 Principle The two growth promoters are extracted from the sample with a mixture of methanol and water 1:1, v:v. The extract of animal feeds is purified through a short open glass aluminium oxide column or by an alumina Solid Phase Extraction (SPE). The final extract is analysed by reversed phase HPLC with UV detection at a wavelength of 375 nm. Alternatively, for confirmation purposes, a Diode Array Detector (DAD) can be used. The presence of furazolidone can interfere with the determination of carbadox. If a DAD is used, many other veterinary drugs or feed additives can be detected (ronidazole, meticlorpindol, nitrofurazone, dimetridazol, furaltadon, sulphonamides...), but their signals do not interfere with the target ones. In some special feeds, matrix interfering peaks may be present, very close to the carbadox peak. 4 Reagents and materials WARNING — Persons using this European Standard should be familiar with normal laboratory practice. This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure compliance with any regulatory conditions. a) This method requires the use of solutions of Carbadox and Olaquindox. These substances were used as growth-promoting feed additives for piglets. Carbadox and Olaquindox are chemotherapeutics belonging to the quinoxaline group. They are suspected to be teratogen and mutagen. Avoid inhalation of and exposure to the toxic standard materials and solutions; b) These 2 growth promoters are subject to light degradation. Protect analytical work adequately from daylight, and keep standard solutions protected from light by using amber glassware, amber vials or aluminium foil. Use only reagents as analytical grade at least unless otherwise stated. SIST EN 16930:2017

EN 16930:2017 (E) 6 4.1 Water, demineralized or deionized or at least equivalent 4.2 Methanol 4.3 Acetonitrile 4.4 Glacial acetic acid, minimum purity 96 % 4.5 Ammonium acetate water free salt, CH3CO2NH4 4.6 Extraction solvent: methanol water mixture (1:1 v:v) Combine equal volumes of methanol (4.2) and water (4.1). Mix well. NOTE Only for 8.1 purposes, it is possible to combine equal volume of technical methanol (4.14) and water (4.1). It is essential to mix well. 4.7 Ammonium acetate buffer, 25 mM, pH = 4,35 Weigh 2,0 g of ammonium acetate (4.5) to the nearest 0,1 g, into a 1000 ml volumetric flask. Dissolve in 900 ml of water (4.1). Add 3,0 ml of glacial acetic acid (4.4). Adjust (5.1) the pH to pH = 4,35 with acetic acid (4.4), if necessary. Dilute to the mark with water (4.1) and mix well. 4.8 Mobile phase for HPLC: acetonitrile: buffer mixture (10:90; v:v) Transfer 100 ml of acetonitrile (4.3) into a 1000 ml volumetric flask. Dilute to the mark with ammonium acetate buffer (4.7). Filter the eluent through a 0,45 µm cellulose acetate membrane filter (5.9) using a solvent filtration system (5.2). If necessary, perform degassing for 5 min in an ultrasonic bath (5.3). NOTE This mobile phase is suitable for the three recommended columns in 5.11.8. If another column is used, optimization of the mobile phase composition may be necessary before performing real analysis. See 8.4.1. 4.9 Carbadox dissolving solvent: methanol: acetonitrile mixture (1:1; v:v) Combine equal volumes of methanol (4.2) and acetonitrile (4.3). Mix well. 4.10 Reference standards Guaranteed purity is required for each lot of reference standard. 4.10.1 Carbadox, 3- (2-quinoxalinyl methylene) carbazic acid methyl ester N,N’-dioxide 4.10.2 Olaquindox, (2-[N-2'-(hydroxyethyl) carbamoyl] 3–methyl quinoxaline) N,N’-dioxide 4.11 Standard solutions Protect all standard solutions from daily light 4.11.1 Carbadox stock standard solution ca. 100 µg/ml Weigh 25 mg of carbadox (4.10.1), to the nearest 0,1 mg, into a 250 ml amber volumetric flask. Dissolve in 200 ml of mixture (4.9). Mix well and place the flask in an ultrasonic bath (5.3) until total dissolution. Allow to cool down to room temperature, dilute to the mark with mixture (4.9) and mix well. Calculate the accurate concentration taking into account the purity of the reference standard (4.10.1). Prepare fresh every month. Store in the dark at 0 °C to 8 °C (5.12). SIST EN 16930:2017

EN 16930:2017 (E) 7 4.11.2 Olaquindox stock standard solution ca. 250 µg/ml Weigh 50 mg of olaquindox (4.10.2), to the nearest 0,1 mg, into a 200 ml amber volumetric flask. Dissolve in about 190 ml of water (4.1). Mix well and place the flask in an ultrasonic bath (5.3) until total dissolution. Allow to cool down to room temperature. Dilute to the mark with water (4.1) and mix well. Calculate the accurate concentration taking into account the purity of the reference standard (4.10.2). Prepare fresh every month. Store in the dark at 0 °C to 8 °C (5.12). 4.11.3 Calibrations solutions 4.11.3.1 Carbadox/olaquindox calibration solution ca. 10 µg/ml Pipette 5 ml of the carbadox stock standard solution (4.11.1) and 2 ml of the olaquindox stock standard solution (4.11.2) in a 50 ml volumetric flask. Dilute to the mark with the extraction solvent (4.6) and mix well. Prepare fresh for each series of samples. 4.11.3.2 Carbadox/olaquindox calibration solution ca. 5 µg/ml Pipette 5 ml of the carbadox stock standard solution (4.11.1) and 2 ml of the olaquindox stock standard solution (4.11.2) in a 100 ml volumetric flask. Dilute to the mark with the extraction solvent (4.6) and mix well. Prepare fresh for each series of samples. 4.11.3.3 Carbadox/olaquindox calibration solution ca. 2,5 µg/ml Pipette 5 ml of the carbadox stock standard solution (4.11.1) and 2 ml of the olaquindox stock standard solution (4.11.2) in a 200 ml volumetric flask. Dilute to the mark with the extraction solvent (4.6) and mix well. Prepare fresh for each series of samples. 4.11.3.4 Carbadox/olaquindox calibration solution ca. 1 µg/ml Pipette 5 ml of the Carbadox/olaquindox calibration solution ca. 10 µg/ml (4.11.3.1) in a 50 ml volumetric flask. Dilute to the mark with the extraction solvent (4.6) and mix well. Prepare fresh for each series of samples. 4.11.3.5 Carbadox/olaquindox calibration solution ca. 0,5 µg/ml Pipette 5 ml of the Carbadox/olaquindox calibration solution ca. 5 µg/ml (4.11.3.2) in a 50 ml volumetric flask. Dilute to the mark with the extraction solvent (4.6) and mix well. Prepare fresh for each series of samples. 4.11.3.6 Carbadox/olaquindox calibration solution ca. 0,25 µg/ml Pipette 5 ml of the Carbadox/olaquindox calibration solution ca. 2,5 µg/ml (4.11.3.3) in a 50 ml volumetric flask. Dilute to the mark with the extraction solvent (4.6) and mix well. Prepare fresh for each series of samples. SIST EN 16930:2017

EN 16930:2017 (E) 8 4.12 Neutral aluminium oxide, Brockmann activity I 4.13 Neutral aluminium oxide SPE cartridge, 2 cm3, 1850 mg 4.14 Technical methanol 5 Apparatus Usual laboratory apparatus and, in particular, the following: 5.1 pH meter 5.2 Solvent filtration system, suitable for 0,45 µm membrane filters 5.3 Ultrasonic bath 5.4 Rotary on mechanical shaker 5.5 Centrifuge 5.6 Glass wool 5.7 Glass column for chromatography, length 200 mm to 400 mm; internal diameter 10 mm, restricted at the end and fitted with a wad of glass wool (5.6) or equivalent column 5.8 Filter papers or glass microfibre filter 5.9 Cellulose acetate membrane filters of pore size 0,45 µm 5.10 PVDF syringe filters of pore size 0,45 µm and adaptable syringes 5.11 HPLC system 5.11.1 pump, capable of maintaining a volume flow rate of 0,4 ml to 1,5 ml min 5.11.2 Column heater set at 30 °C 5.11.3 Injection system, with a loop suitable for 10 µl to 50 µl injections 5.11.4 UV detector, suitable for measurements at a wavelength of 375 nm 5.11.5 Diode array detector for confirmation purposes 5.11.6 Recorder or data acquisition system 5.11.7 Guard column, silica bounded C8 5.11.8 LC analytical column, 250 × 3 mm, 5 µm particle size, packed with Lichrosorb C8 or Lichrospher C8 or Lichrospher C18 or equivalent column. NOTE During the method validation, these recommended LC columns have proved to be fit for purpose, especially with samples containing interfering peaks close to the carbadox zone. SIST EN 16930:2017

EN 16930:2017 (E) 9 5.12 Refrigerator or refrigerated chamber at a temperature set between 0 °C and 8 °C 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. Recommended sampling methods are given in: — Annex I of the Commission Regulation (EU) No152/2009 amended by Commission Regulation (EU) No 691/2013 [1] — EN ISO 6497 [12]. 7 Preparation of test sample 7.1 General Prepare the test sample in accordance with EN ISO 6498. 7.2 Laboratory sample Grind the laboratory sample (usually 50 g) so that is passes completely through a sieve with 1 mm apertures. Mix thoroughly. 7.3 Test sample The test sample consists of a representative and homogenized aliquot of the ground laboratory sample of at least 20 g. 7.4 Test portion Accurately weigh 10,0 g to the nearest 0,1 g of the thoroughly mixed test sample into a 250 ml conical flask. Note down the mass expressed in g. Submit it to the analysis procedure (8). 8 Procedure 8.1 Extraction of feeding stuffs containing 0,5 mg/kg to 100 mg/kg of growth promoters Add 100,0 ml of extraction solvent (4.6) to the test portion (7.4), stopper and shake vigorously for 1 h or 2 h on the rotary shaker (5.4). 8.2 Filtration Filter the solution obtained in 8.1 through a paper or glass micro fibre filter (5.8). Alternatively centrifugation (5.5) is possible, if filtration is too long, for example. Use the filtrate or the supernatant for the purification step. 8.3 Purification 8.3.1 General Apply 8.3.2 or 8.3.3. SIST EN 16930:2017

EN 16930:2017 (E) 10 8.3.2 Open glass clean-up For each sample extract, dry-pack a glass column (5.7), fitted at the end with a plug of glass wool (5.6) with 5 g of aluminium oxide (4.12). Load 20 ml of extract (8.2) on the column and discard the first 5 ml of eluate. Collect the next 5 ml. Filtrate these 5 ml through a PVDF syringe filter (5.10). Use this filtrate for HPLC determination. 8.3.3 SPE clean-up Load 8 ml of extract (8.2), on a SPE cartridge (4.13) and discard the first 1,5 ml of eluate. Collect the next 2 ml. Filtrate these 2 ml through a PVDF syringe filter (5.10). Use this filtrate for HPLC determination. 8.4 HPLC analysis 8.4.1 Analytical conditions 8.4.1.1 General The following conditions are provided for guidance. Other conditions may be used provided they yield to equivalent results. 8.4.1.2 HPLC column: as in 5.11.8 8.4.1.3 guard column: as in 5.11.7 8.4.1.4 mobile phases: as in 4.8, flow rate: 0,5 ml min 8.4.1.5 injection volume: 20-50 µl 8.4.1.6 column temperature: 30 °C 8.4.1.7 detection wavelength: 375 nm Using these conditions the retention times should approximately be 4,0 min to 6,0 min and 18,0 min to 22,0 min for olaquindox and carbadox respectively with respective capacity factors of 3 and 13. 8.4.1.8 System set-up Check the stability of the HPLC system by injecting several times one of the calibration solutions (4.11.3.1; 4.11.3.2; 4.11.3.3) until constant peak heights and retention times are achieved. NOTE 1 Furazolidone has approximately the same retention time than carbadox. In case of doubt, the result needs to be confirmed. See 8.5.2 or 8.5.3. NOTE 2 In few particular feeds, a matrix interfering peak is present and has approximately the same retention time than carbadox. Using the described LC parameters, the interfering peak is eluted just before the carbadox peak. It is advised that Apex be however sufficiently separated. In case of doubt, the result needs to be confirmed. See 8.5.2 or 8.5.3. 8.4.2 External calibration curve Inject each calibration solution (4.11.3) at the beginning and at the end of a sample series. Plot each of the individual measured peak height or peak area (y-axis) versus the respective concentration of carbadox or olaquindox (x-axis) in µg/ml. SIST EN 16930:2017

EN 16930:2017 (E) 11 8.4.3 Sample extracts Inject several times (the determined area or peak height should be stable) the sample extract obtained in (8.3). The injection volume should be the same than the one selected for the calibration solutions. Determine the mean peak height (or peak area) for the carbadox and/or olaquindox signals. 8.5 System suitability – Confirmation 8.5.1 General The identity of the target analyte(s) can be confirmed by co-chromatography or by using a DAD. 8.5.2 Co-chromatography Prepare a spiked sample extract adding an appropriate amount of one of the carbadox/olaquindox calibration solutions (4.11.3) to the sample extract. The amount of carbadox or olaquindox added shall be approximately equal to the estimated amount of the corresponding growth promoter detected in the sample extract. Inject the spiked sample extract. If the signal detected in the chromatogram obtained in 8.4.3 was linked to the detection of carbadox, only this signal intensity should increase. The related peak height increase should be proportional to the spiking level of carbadox and the related peak width at half height should increase by no more than ± 10 % of the original width. If the signal detected in the chromatogram obtained in 8.4.3 was linked to the detection of olaquindox, only this signal intensity should increase. The related peak height increase should be proportional to the spiking level of olaquindox and the related peak width at half height should increase by no more than ± 10 % of the original width. If the signals detected in the chromatogram obtained in 8.4.3 were linked to the detection of carbadox and olaquindox, only these signals intensities should increase. The respective related peak heights increase should be proportional to the spiking level of carbadox and olaquindox respectively and the respective related peak widths at half height should increase by no more than ± 10 % of the respective original widths. 8.5.3 DAD 8.5.3.1 Analytical parameters If a DAD is used instead of a UV detector, conditions to be used are: a) Wavelength: 375 nm; b) Bandwidth: 4 nm; c) Spectrum range: 220 to 400 nm; d) Spectrum: baseline, apex, up-slope and down slope inflection points. 8.5.3.2 Evaluation Plot the normalized spectra differences (sample – baseline) of the sample peak, recorded both at the apex and at the up-slope and down-slope inflection points. Plot the normalized spectra of the sample peak and of the carbadox or olaquindox corresponding calibration solution peak, both recorded at the apex. SIST EN 16930:2017

EN 16930:2017 (E) 12 8.5.3.3 Confirmation criteria The identity of the analyte is confirmed if the following three criteria are satisfied: a) The retention time of the sample peak shall be equal (±5 %) to the retention time of the standard peak. In case of doubt, standard addition (reference standard added to the sample) shall be performed. b) Assess the purity of the sample peak on the basis of the conformity of the spectra differences, recorded at the apex and at up-slope and down-slope inflection points. For those parts of the spectra with a relative absorption of at least 10 % (between 10 % and 100 % in a normalized spectrum), at each wavelength the relative absorption shall be equal (within 15 % for contents higher than 20 mg/kg and 25 % for contents lower than 20 mg/kg) for all spectra. c) The spectrum differences of the sample and standard peaks recorded at the peak apex shall not be visually different for those parts of the spectra with a relative absorption of at least 10 % (between 10 % and 100 % in a normalized spectrum). This criterion is met when: — the same maxima wavelengths are present within a margin determined by the resolution of the detection system (4 nm); — at th

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