SIST EN 16162:2012
(Main)Animal feeding stuffs - Determination of Decoquinate by HPLC with fluorescence detection
Animal feeding stuffs - Determination of Decoquinate by HPLC with fluorescence detection
This European Standard specifies a method for the determination of decoquinate. This high-performance liquid chromatographic (HPLC) method with a fluorescence detection is applicable to the quantification of decoquinate content in complete and complementary compound feeds, medicated feeds, semi-liquid feeds, premixtures and feed additives. The method was fully validated from LOQ to 60 000 mg/kg on different matrices during an international collaborative study , especially on complete compound feeds for poultry, at trace contamination level of 3 mg/kg and at European authorized level of 20 mg/kg to 40 mg/kg. The limit of detection is between 0,1 mg/kg and 0,3 mg/kg and the limit of quantification is around 0,5 mg/kg. These limits were validated during the collaborative study , from results on the blank feed. Lower limits of detection or quantification could be reached but a single laboratory validation is then requested.
Futtermittel - Bestimmung von Decoquinat mit Hochleistungs-Flüssigchromatographie (HPLC) und Fluoreszenzdetektion
Diese Europäische Norm beschreibt die Bestimmung von Decoquinat. Dieses hochleistungsflüssig-chromatographische (HPLC) Verfahren mit Fluoreszenzdetektion ist für die Quantifizierung des Decoquinat-gehaltes in Allein- und Ergänzungsfuttermitteln, Futtermittelergänzern, Vormischungen und Futterzusatzstoffen geeignet.
Die Nachweisgrenze liegt ungefähr bei 0,1 mg/kg oder 0,3 mg/kg und die Bestimmungsgrenze ungefähr bei 0,5 mg/kg. Diese Grenzen wurden während des Ringversuchs [11] anhand der mit dem Kontrollfuttermittel ohne Zusatz erzielten Ergebnisse validiert. Niedrigere Nachweis und Bestimmungsgrenzen könnten erreicht werden, jedoch ist dann eine eigene Validierung durch den Anwender erforderlich.
Aliments des animaux - Dosage du décoquinate par CLHP avec détection par fluorescence
La présente Norme européenne décrit le dosage du décoquinate. La présente méthode de chromatographie liquide à haute performance (CLHP) avec détection par fluorescence est applicable à la détermination de la teneur en décoquinate dans les aliments composés complets et complémentaires pour animaux, les suppléments, prémélanges et additifs utilisés en alimentation animale.
La limite de détection se situe à environ 0,1 mg/kg ou 0,3 mg/kg et la limite de quantification à environ 0,5 mg/kg. Ces limites ont été validées lors de l’étude interlaboratoires [11], à partir des résultats obtenus sur l’aliment témoin. Des limites de détection ou de quantification inférieures pourraient être atteintes à condition d’être validées en interne.
Krma - Določevanje dekokvinata s HPLC s fluorescentno detekcijo
Ta evropski standard določa metodo za določevanje dekokvinata. Ta metoda tekočinske kromatografije visoke ločljivosti (HPLC) s fluorescenčnim zaznavanjem se uporablja za kvantifikacijo vsebnosti dekokvinata v polnovrednih in dopolnilnih krmnih mešanicah, medicirani krmi, poltekoči krmi, premiksih in krmnih dodatkih. Metoda je bila z mednarodno medlaboratorijsko študijo v celoti validirana za razpon od meje kvantifikacije do 60.000 mg/kg na različnih matricah, zlasti na polnovrednih krmnih mešanicah za perutnino pri vrednosti onesnaženja v sledovih 3 mg/kg in evropski dovoljeni vrednosti 20 mg/kg do 40 mg/kg. Meja detekcije je med 0,1 mg/kg in 0,3 mg/kg, meja kvantifikacije pa približno 0,5 mg/kg. Ti meji sta bili validirani z medlaboratorijsko študijo na podlagi rezultatov iz slepe krme. Doseči je mogoče nižjo mejo detekcije ali kvantifikacije, vendar se za to zahteva ena sama laboratorijska validacija.
General Information
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Futtermittel - Bestimmung von Decoquinat mit Hochleistungs-Flüssigchromatographie (HPLC) und FluoreszenzdetektionAliments des animaux - Dosage du décoquinate par CLHP avec détection par fluorescenceAnimal feeding stuffs - Determination of Decoquinate by HPLC with fluorescence detection65.120KrmilaAnimal feeding stuffsICS:Ta slovenski standard je istoveten z:EN 16162:2012SIST EN 16162:2012en,fr,de01-september-2012SIST EN 16162:2012SLOVENSKI
STANDARD
SIST EN 16162:2012
EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 16162
March 2012 ICS 65.120 English Version
Animal feeding stuffs - Determination of decoquinate by HPLC with fluorescence detection
Aliments des animaux - Détermination du décoquinate par Chromatographie Liquide Haute Performance avec détection fluorimétrique
Futtermittel - Bestimmung von Decoquinat mit Hochleistungs-Flüssigchromatographie (HPLC) und Fluoreszenzdetektion This European Standard was approved by CEN on 4 February 2012.
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
Management Centre:
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 16162:2012: ESIST EN 16162:2012
EN 16162:2012 (E) 2 Contents Page Foreword .4Introduction .51Scope .62Normative references .63Principle .64Reagents .65Apparatus .86Sampling .87Sample preparation .88Procedure .88.1General .88.2Extraction of feeds (decoquinate content between 10 mg/kg to 500 mg/kg) .88.3Extraction of complementary feeds, premixtures and feed additives (decoquinate content higher than 500 mg/kg) .98.4Extraction of trace feeds (decoquinate content lower than 10 mg/kg) .98.5Quality control spiked feeds .98.5.1Blank Feed to spike at 30 mg/kg .98.5.2Blank feed to spike at 9 mg/kg .98.6HPLC parameters .98.7Standards’ injections and calibration curve . 108.8Sample extracts . 108.9Confirmation procedure . 109Calculations . 1010Precision . 1110.1Limit of Detection and Limit of Quantification . 1110.2Interlaboratory test . 1110.3Repeatability . 1110.4Reproducibility . 1111Test report . 12Annex A (informative)
Results inter-laboratory study . 13Annex B (informative)
Additional recovery results . 17B.1Recoveries from the familiarization phase . 17B.2Pre-trial; recovery test on a milk-replacer . 18B.3Recoveries from the single-laboratory validation . 18Annex C (informative)
Additional results for robustness purposes . 19C.1Robustness in terms of excitation wavelength, during the final collaborative study . 19C.2Comparison between ground and unground samples during the final collaborative study . 19C.3Robustness from one laboratory during the final collaborative study . 20Annex D (informative)
HPLC parameters and chromatogram examples . 22D.1General . 22D.2Chromatogram example: Calibration point at 0,6 µg/ml . 22D.3Chromatogram example: Commercial Lamb feed containing around 50 mg/kg of decoquinate22D.4Chromatogram example: same commercial Lamb feed, as in D.3, containing around 50 mg/kg of decoquinate . 23SIST EN 16162:2012
EN 16162:2012 (E) 3 Bibliography . 24 SIST EN 16162:2012
EN 16162:2012 (E) 4 Foreword This document (EN 16162:2012) 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 September 2012, and conflicting national standards shall be withdrawn at the latest by September 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 16162:2012
EN 16162:2012 (E) 5 Introduction This European Standard has been developed to quantify decoquinate in feeding stuffs to enable the European Commission to control the content of animal feed products. However, this method can also be used to evaluate the cross contamination from medicated feed to feedstuff. SIST EN 16162:2012
EN 16162:2012 (E) 6 1 Scope This European Standard specifies a method for the determination of decoquinate. This high-performance liquid chromatographic (HPLC) method with a fluorescence detection is applicable to the quantification of decoquinate content in complete and complementary compound feeds, medicated feeds, semi-liquid feeds, premixtures and feed additives. The method was fully validated from LOQ to 60 000 mg/kg on different matrices during an international collaborative study [11], especially on complete compound feeds for poultry, at trace contamination level of 3 mg/kg and at European authorized level of 20 mg/kg to 40 mg/kg [12]. The limit of detection is between 0,1 mg/kg and 0,3 mg/kg and the limit of quantification is around 0,5 mg/kg. These limits were validated during the collaborative study [11], from results on the blank feed. Lower limits of detection or quantification could be reached but a single laboratory validation is then requested. 2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. 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 Decoquinate is extracted from samples with a solution of 1 % calcium chloride in methanol using mechanical shaking or stirring for 60 min. After centrifugation or filtration, an aliquot is, if necessary, diluted with the extraction solvent and analysed by reversed phase HPLC with fluorescence detection. Positive trace level samples should be confirmed by HPLC analysis using an alternate excitation wavelength. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralised water or water of equivalent purity. WARNING — This method requires the handling of hazardous substances. It is recommended to use various regulations for potentially hazardous chemicals. Organisational, technical and personal safety has to be observed. 4.1 Methanol, HPLC grade. 4.2 Methanol, technical grade. 4.3 Calcium chloride anhydrous or Calcium chloride dihydrate, each > 99 % purity. 4.4 HPLC dilution solution. Dissolve a mass of calcium salt (4.3) equivalent to 10 g of calcium chloride anhydrous in methanol (4.1). Mix well and make up to 1 000 ml. 4.5 Extraction solvent. SIST EN 16162:2012
EN 16162:2012 (E) 7 Dissolve a mass of calcium salt (4.3) equivalent to 10 g of calcium chloride anhydrous in technical methanol (4.2). Mix well and make up to 1 000 ml. 4.6 Decoquinate standards. 4.6.1 Decoquinate powder reference standard with guaranteed purity. Purity shall be certified by a certificate of analysis. NOTE Pure reference standard is available at e.g. Alpharma®. 4.6.2 Decoquinate stock standard solution, approximately 300 µg/ml. Accurately weigh, to the nearest 0,1 mg, 30 mg of decoquinate reference standard (4.6.1) into a 100 ml volumetric flask and dissolve in dilution solution (4.4). Use ultrasonic bath if necessary to aid dissolution. Calculate the exact concentration taking into account the purity of the standard material (4.6.1), given in the certificate. Prepare fresh monthly. Store in the dark at 0 °C to 10 °C. 4.6.3 HPLC standard solutions. 4.6.3.1 Intermediate standard solution, approximately 6 µg/ml. Transfer by pipette 2,0 ml of stock standard solution (4.6.2) into a 100 ml volumetric flask, dilute to volume with dilution solution (4.4). Check that intermediate solution for each series of analysis. The absorbance density of intermediate solution can be evaluated at 265 nm, with dilution solvent (4.4) as reference for optical density measurement. In these conditions, the absorbance range is between 0,67 and 0,73. That intermediate solution is prepared fresh daily. 4.6.3.2 HPLC calibration standard solutions, approximately 0,15 µg/ml, 0,30 µg/ml, 0,60 µg/ml and 1,2 µg/ml. Prepare 4 concentrations of HPLC standard solutions as it is explained in Table 1 (standards A/B/C/D). Transfer by pipette the required volume of intermediate standard (4.6.3.1) into volumetric flasks, and make to volume with dilution solution (4.4). Mix well. Table 1 — preparation of calibration standard solutions Standard Parts of intermediate (4.6.3.1) solution, in ml Volumetric flask, in ml Dilution Factor ≅≅≅≅µg/ml A 5 200 40 0,15 B 5 100 20 0,30 C 10 100 10 0,60 D 10 50 5 1,20
Evaluate precisely each exact concentration by using the exact concentration of the stock solution (4.6.2). All solutions described here (standards A/B/C/D) are prepared fresh daily. 4.7 HPLC Mobile Phase. Dissolve a mass of calcium salt (4.3) equivalent to 10 g calcium chloride anhydrous into 1 l of a solvent mixture of methanol (4.1) / water in proportion by volume of 825/175. Filter under vacuum (5.2) before use. NOTE HPLC shutdown solution. Prepare a methanol (4.1) / water solution in proportion by volume of 85/15 without calcium salt to flush the HPLC column and equipment at the end of each day of analysis. SIST EN 16162:2012
EN 16162:2012 (E) 8 5 Apparatus Usual laboratory apparatus, in particular, the following: 5.1 Mechanical shaker or magnetic stirrer. 5.2 Solvent filtration system, suitable for 0,45 µm PTFE filter or equivalent. 5.3 Centrifuge and centrifuge tube (50 ml). 5.4 HPLC system consisting of the following: 5.4.1 Pump, pulse free, flow capacity of 0,2 ml/min to 5 ml/min. 5.4.2 Injection system, manual or autosampler, with loop suitable for 10 µl to 50 µl injection volumes. 5.4.3 Analytical C18 column like ACE® or Luna® or Symmetry® or Restek Ultra®; 5 µm; 4.6 mm x 250 mm or equivalent. 5.4.4 Fluorescence detector suitable for measurement using 330 nm and 260 nm excitation wavelengths and 390 nm emission wavelength. 5.4.5 Integrator or computer data system. 6 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this European Standard. A recommended sampling procedure is given in EN ISO 6497 [1]. 7 Sample preparation All feed samples, with the exception of premixtures and concentrates (feed additives), are ground extraction as recommended in the guidelines prEN ISO 6498. 8 Procedure 8.1 General Preferably, duplicate analysis is performed. 8.2 Extraction of feeds (decoquinate content between 10 mg/kg to 500 mg/kg) NOTE For milk-replacer particular attention should be given to the addition of extraction solvent. Small or large lumps could be formed and quantitative results will be compromised. The extraction solvent addition should be done slowly under rotate shaking of the conical flask. To dissolve lumps, ultrasonic system, during approximately 5 min, is very helpful. Accurately weigh, to the nearest 0,1 g, 10,0 g of feed in a 250 ml amber conical flask. Add 100 ml of the extraction solution (4.5). Shake or stir for 1 h on apparatus (5.1). Allow contents of flask to settle particles for about 10 min. Dilute an aliquot of the almost clear supernatant extract with dilution solution (4.4) to obtain a concentration between 0,3 µg/ml and 0,6 µg/ml. Filter the diluted solution on membrane filter (5.2) and inject it onto HPLC system (5.4). For cloudy extracts, if necessary, transfer at least 40 ml of the extract into 50 ml centrifuge tube (5.3) and centrifuge before dilution, membrane filtration and HPLC injection. SIST EN 16162:2012
EN 16162:2012 (E) 9 8.3 Extraction of complementary feeds, premixtures and feed additives (decoquinate content higher than 500 mg/kg) Accurately weigh, to the nearest 0,01 g, depending on the declared concentration of decoquinate in the sample, 0,50 g to 2,00 g of sample into a 250 ml amber conical flask. Add 100 ml of the extraction solution (4.5). Shake or stir for 1 h on equipment (5.1). Allow contents of flask to settle particles for 10 min. Dilute an aliquot of the almost clear supernatant extract with dilution solution (4.4) to obtain a concentration between 0,3 µg/ml and 0,6 µg/ml. Filter the diluted solution on membrane filter (5.2) and inject it onto HPLC system (5.4). For cloudy extracts, if necessary, transfer at least 40 ml of the extract into 50 ml centrifuge tube (5.3) and centrifuge before dilution, membrane filtration and HPLC injection. NOTE For a decoquinate declaration of 1 000 mg/kg, weigh 2 g of test portion and dilute 1 ml of clear extract in a 100 ml volumetric flask. For feed additives declared at 6 %, weigh 0,5 g and dilute by a factor of 1/400. 8.4 Extraction of trace feeds (decoquinate content lower than 10 mg/kg) Accurately weigh, to the nearest 0,1 g, 10,0 g of feed in a 250 ml amber conical flask. Add 100 ml of the extraction solution (4.5). Shake or stir for 1 h on apparatus (5.1). Allow contents of flask to settle particles for 10 min. Dilute an aliquot of the almost clear supernatant extract with dilution solution (4.4) to obtain a concentration between 0,3 µg/ml and 0,6 µg/ml. Filter the diluted solution on membrane filter (5.2) and inject it onto HPLC system (5.4). For cloudy extracts, if necessary, transfer at least 40 ml of the extract into 50 ml centrifuge tube (5.3) and centrifuge before dilution, membrane filtration and HPLC injection. 8.5 Quality control spiked feeds 8.5.1 Blank Feed to spike at 30 mg/kg Accurately weigh, to the nearest 0,1 g, 10,0 g of quality control blank feed in a 250 ml amber conical flask. Add 1 ml of stock solution (4.6.2). Wait for 15 min. Then add 100 ml of the extraction solution (4.5). Proceed as described in 8.2. The recovery shall be from 80 % to 110 %. 8.5.2
Blank feed to spike at 9 mg/kg Accurately weigh, to the nearest 0,1 g, 10,0 g of quality control blank feed in a 250 ml amber conical flask. Add 15 ml of intermediate solution (4.6.3.1). Wait for 15 min. Then add 85 ml of the extraction solution (4.5). Proceed as described in 8.4. The recovery shall be from 80 % to 120 %. 8.6 HPLC parameters These HPLC parameters are given for guidance. Other parameters should be applicable (column, flow rate adapted to HPLC columns and optimum response of the fluorescence detector…). Analytical column: C18 column; 5 µm; 4.6 mm x 250 mm as described in 5.4.3; Column Temperature: ambient or 30 °C; Mobile phase (4.7): MeOH/water in proportion by volume of 825/
...
SLOVENSKI STANDARD
oSIST prEN 16162:2011
01-februar-2011
.UPD'RORþHYDQMHGHNRNYLQDWDV+3/&VIOXRUHVFHQWQRGHWHNFLMR
Animal feeding stuffs - Determination of Decoquinate by HPLC with fluorescence
detection
Futtermittel - Bestimmung von Decoquinat mit Hochleistungs-Flüssigchromatographie
(HPLC) und Fluoreszenzdetektion
Aliments des animaux - Dosage du décoquinate par CLHP avec détection par
fluorescence
Ta slovenski standard je istoveten z: prEN 16162
ICS:
65.120 Krmila Animal feeding stuffs
oSIST prEN 16162:2011 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 16162:2011
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oSIST prEN 16162:2011
EUROPEAN STANDARD
DRAFT
prEN 16162
NORME EUROPÉENNE
EUROPÄISCHE NORM
October 2010
ICS 65.120
English Version
Animal feeding stuffs - Determination of Decoquinate by HPLC
with fluorescence detection
Aliments des animaux - Dosage du décoquinate par CLHP Futtermittel - Bestimmung von Decoquinat mit
avec détection par fluorescence Hochleistungs-Flüssigchromatographie (HPLC) und
Fluoreszenzdetektion
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee CEN/TC 327.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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 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 and United Kingdom.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without notice and
shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2010 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16162:2010: E
worldwide for CEN national Members.
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oSIST prEN 16162:2011
prEN 16162:2010 (E)
Contents Page
Foreword .3
Introduction .3
1 Scope .4
2 Normative references .4
3 Principle .4
4 Reagents .4
5 Apparatus .5
6 Sampling .6
7 Sample preparation .6
8 Procedure .6
9 Calculations .8
10 Precision .8
11 Test report .9
Annex A (informative) Results interlaboratory study . 11
Annex B (informative) Additional recovery results . 14
Annex C (informative) Additional results for robustness purposes . 16
Annex D (informative) HPLC parameters and chromatogram examples . 19
Bibliography . 21
2
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oSIST prEN 16162:2011
prEN 16162:2010 (E)
Foreword
This document (prEN 16162:2010) has been prepared by Technical Committee CEN/TC 327 “Animal feeding stuffs”,
the secretariat of which is held by NEN.
This document is currently submitted to the CEN Enquiry.
Introduction
This European Standard has been developed to quantify Decoquinate in feedingstuff to enable the European
Commission to control their content of animal feed products. However, that method can be used to evaluate the cross
contamination from medicated feed to feedstuff.
3
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oSIST prEN 16162:2011
prEN 16162:2010 (E)
1 Scope
This European Standard describes the determination of decoquinate. This high-performance liquid chromatographic
(HPLC) method with a fluorescence detection is applicable to the quantification of decoquinate content in complete -,
complementary compound feeds, supplements, premixture and feed additives.
The limit of detection is around 0,1 or 0,3 mg/kg and the limit of quantification is around 0,5 mg/kg. These limits were
validated during the collaborative study [11], from results on the blank feed. Lower limits of detection or quantification
could be reached but an in-house validation is then requested.
2 Normative references
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 6498, Animal feeding stuffs — Guidelines for sample preparation
3 Principle
Decoquinate is extracted from samples with a solution of 1% calcium chloride in methanol using mechanical shaking
or stirring for 60 minutes. After centrifugation or filtration, an aliquot is, if necessary, diluted with the extraction solvent
and analysed by reversed phase HPLC with fluorescence detection. Positive trace level samples should be confirmed
by HPLC analysis using an alternate excitation wavelength.
4 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralised water or
water of equivalent purity.
WARNING — This method requires the handling of hazardous substances. It is recommended to use various
regulations for potentially hazardous chemicals. Organisational, technical and personal safety has to be
observed.
4.1 Methanol, HPLC grade
4.2 Methanol, technical grade
4.3 Calcium chloride anhydrous or Calcium chloride dehydrate, each >99% purity
4.4 HPLC dilution solution
Dissolve a mass of calcium salt (4.3) equivalent to 10 g of calcium chloride anhydrous in methanol (4.1). Mix well and
make up to 1000 ml.
4.5 Extraction solvent
Dissolve a mass of calcium salt (4.3) equivalent to 10 g of calcium chloride anhydrous in technical methanol (4.2). Mix
well and make up to 1 000 ml.
4.6 Decoquinate reference standard with guaranteed purity
Pure reference standard is available by Alpharma® supplier
4
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oSIST prEN 16162:2011
prEN 16162:2010 (E)
Purity should be certified by a certificate of analysis.
4.6.1 Decoquinate stock standard solution, approximately 300 µg/ml
Accurately weigh, to the nearest 0,1 mg, 30 mg of decoquinate reference standard (4.6) into a 100 ml volumetric flask
and dissolve in dilution solution (4.4). Use ultrasonic bath if necessary to aid dissolution. Calculate the exact
concentration taking into account the purity of the standard material (4.6), given in the certificate.
Prepare fresh monthly. Store in the dark at 0°C to 10°C.
4.6.2 HPLC standard solutions
All solutions described under that clause, are prepared fresh daily.
4.6.2.1 Intermediate standard solution, approximately 6 µg/ml
Transfer by pipette, 2,0 ml of stock standard (4.6.1) into a 100 ml volumetric flask, dilute to volume with dilution
solution (4.4). Check that intermediate solution for each series of analysis. The absorbance density of intermediate
solution (4.6.2.1) can be evaluated at 265 nm, with dilution solvent (4.4) as reference for optical density measurement.
In these conditions, the absorbance range is between 0,67 and 0,73.
4.6.2.2 HPLC calibration standard solutions, approximately 0,15, 0,30, 0,60 and 1,2 µg/ml
Prepare 4 concentrations of HPLC standard solutions as it is explained in table 1.
Transfer by pipette the required volume of intermediate standard (4.6.2.1) into volumetric flasks, and make to volume
with dilution solution (4.4). Mix well.
Table 1 — preparation of calibration standard solutions
Parts of intermediate Volumetric
Standard Dilution Factor
≅µg/ml
(4.6.2.1) solution, in ml flask, in ml
A 5 200 40 0,15
B 5 100 20 0,30
C 10 100 10 0,60
D 10 50 5 1,20
Evaluate precisely each exact concentration by using the exact concentration of the stock solution (4.6.1)
4.7 HPLC Mobile Phase
Dissolve a mass of calcium salt (4.3) equivalent to 10 g calcium chloride anhydrous into 1l of a solvent mixture of
methanol (4.1)/water in proportion of 825/175 (v/v). Filter under vacuum (5.2) before use.
HPLC shutdown solution
Prepare a methanol (4.1)/water solution in proportion of 85/15 (v/v) to flush the HPLC column and equipment at the
end of each day of analysis.
5 Apparatus
Usual laboratory apparatus, in particular, the following.
5
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oSIST prEN 16162:2011
prEN 16162:2010 (E)
5.1 Mechanical shaker or magnetic stirrer
5.2 Solvent filtration system, suitable for 0,45 µm PTFE filter or equivalent
5.3 Centrifuge and centrifuge tube (50 ml)
5.4 HPLC system consisting of the following
5.4.1 Pump, pulse free, flow capacity of 0,2 ml/min to 5 mL/min
5.4.2 Injection system, manual or autosampler, with loop suitable for 10 to 50 µl injection volumes
5.4.3 Analytical C18 column like ACE® or Luna® or Symmetry® ; 5µm ; 4.6 * 250 mm or equivalent
5.4.4 Fluorescence detector suitable for measurement using 330 nm and 260 nm excitation wavelengths and
390 nm emission wavelength
5.4.5 Integrator or computer data system
6 Sampling
A representative sample should have been sent to the laboratory. It should not have been damaged or changed
during transport or storage. Sampling is not part of the method specified in this European Standard. A recommended
sampling procedure is given in ISO 6497 [1].
7 Sample preparation
All sample feeds are grinded before extraction as recommended in guideline EN ISO 6498. Premixtures and
concentrates (feed additives) are not obligatory grinded.
8 Procedure
Preferably, duplicate analysis is performed. Portions of samples are weighed into amber conical flasks.
8.1 Extraction of feeds (decoquinate content between 10 to 500 mg/kg)
NOTE For milk-replacer give a particular attention in adding extraction solvent. Small or large lumps could be formed and
quantitative results will be compromised. The extraction solvent addition should be done slowly under rotative shaking of the
conical flask. To dissolve lumps, ultrasonic system, during approximately five minutes, is very helpful.
Accurately weigh, to the nearest 0,1 g , 10,0 g of feed in a 250 ml conical flask. Add 100 ml of the extraction solution
(4.5). Shake or stir for 1 hour on apparatus (5.1). Let settle down particules about 10 minutes. Transfer an aliquot of
the nearly clear overstanding extraction solution. For uncleared extracts, if necessary, transfer at least 40 ml of the
extract into 50 ml centrifuge tube (5.3) and centrifuge. Dilute the clear extract with dilution solution (4.4) to obtain a
concentration between 0,3 and 0,6 µg/ml. Filter on membrane filter (5.2) the diluted solution. Inject onto HPLC system
(5.4).
8.2 Extraction of complementary feeds, premixtures and feed additives (decoquinate content higher than
500 mg/kg)
Accurately weigh, to the nearest 0,01 g, in function of guarantees, 0,50 to 2,00 g of sample into a 250 ml conical flask.
Add 100 ml of the extraction solution (4.5). Shake or stir for 1 hour on equipment (5.1). Let settle down particules
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about 10 minutes. Transfer an aliquot of the nearly clear overstanding extraction solution. For uncleared extracts, if
necessary, transfer at least 40 ml of the extract into 50 ml centrifuge tube (5.3) and centrifuge. Dilute with dilution
solution (4.4) to obtain a concentration between 0,30 and 0,6 µg/ml. Filter on membrane filter and inject onto HPLC
system (5.4).
NOTE For a decoquinate guarantee of 1 000 mg/kg, weigh 2 g of test portion and dilute 1 ml of clear extract in a 100 ml
volumetric flask. For feed additives guaranteed at 6%, weigh 0,5g and dilute of a factor 1/400.
8.3 Extraction of trace feeds (decoquinate content lower than 10 mg/kg)
Accurately weigh, to the nearest 0,1 g, 10,0 g of feed in a 250 ml conical flask. Add 100 ml of the extraction solution
(4.5). Shake or stir for 1 hour on apparatus (5.1). Let settle down particules about 10 minutes. Transfer an aliquot of
the nearly clear overstanding extraction solution. For uncleared extracts, if necessary, transfer at least 40 ml of the
extract into 50 ml centrifuge tube (5.3) and centrifuge. That extract, without dilution, is filtered on membrane filter and
injected onto HPLC system (5.4).
8.4 Quality control spiked feeds
Accurately weigh, to the nearest 0,1 g, 10,0 g of quality control blank feed in a 250 ml amber conical flask.
8.4.1 Blank Feed to spike at 30 mg/kg
Add 1 ml of stock solution (4.6.1). Wait for 15 minutes. Then add 100 ml of the extraction solution (4.5). Proceed as
described in (8.1). The recovery must be from 80 to 110%.
8.4.2 Blank feed to spike at 9 mg/kg
Add 15 ml of intermediate solution (4.6.2.1). Wait for 15 minutes. Then add 85 ml of the extraction solution (4.5).
Proceed as described in (8.3). The recovery must be from 80 to 120%.
8.5 HPLC parameters
This HPLC parameters are given for guidance. Other parameters should be applicable (column, flow rate adapted to
HPLC columns and optimum response of the fluorescence detector, …)
Column: analytical ACE C18 column; 5µm; 4.6 * 250 mm
Column Temperature: ambient or 30°C
Mobile phase (4.7): MeOH/water 825/175 (v/v) with 1% of CaCl2
Injection Volume: 20 µl
Flow rate: 0,5 ml/min
Excitation wavelength: 330 nm
Emission wavelength: 390 nm
Equilibrate the system by running mobile phase before beginning the sequence. Check the stability of the HPLC
system by injecting several times one of the calibration solutions (4.6.2.2), until constant peak heights (areas) and
retention times are achieved.
With these HPLC conditions, the decoquinate retention time is within 14 minutes and 18 minutes (capacity factor = 5).
After each day of analysis, flush all the HPLC system with shutdown solution.
8.6 Standards’ injections and calibration curve
The linearity system has been tested between 0,015 µg/ml to 1,5µg/ml
Inject the HPLC standard solutions on HPLC system at the beginning and the end of a samples’ sequence.
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Plot calibration graphs using the peak areas of the calibration solutions as the ordinates and the corresponding
concentrations, in µg/ml, of decoquinate, as the abscissas.
Use a linear regression as mathematic model (y=ax+b).
8.7 Sample extracts
Inject several times, if necessary, the sample extract obtained in (8.1), (8.2), (8.3) or (8.4), using the same injection
volume as taken with calibration solutions. Determine the mean area of decoquinate peaks.
8.8 Confirmation procedure
In cases of doubts on the decoquinate peak identification, in sample solution or for trace levels, a confirmatory
excitation wavelength at 260 nm must be applied. The HPLC standards solutions and sample solutions will be re-
injected in these confirmatory conditions at 260 nm. Others LC parameters stay identical as described in (8.5).
9 Calculations
Report the area of unknown sample on the calibration curve and evaluate the concentration C of the injected solution.
The decoquinate amount A, in the sample, in mg/kg, is obtained by application of the formula:
∗ ∗
Α=(C DF V)/m (1)
where
C is the decoquinate concentration of the sample extract, in µg/ml;
DF is the dilution factor;
V is the total volume, in ml, of extraction solvent added to the test portion (100 ml);
m is the mass, in g, of the test portion (10 g for feeds).
Round the result to the nearest:
0,1 mg/kg for feedingstuffs containing 0,5 to 10 mg/kg of decoquinate
1 mg/kg for feedingstuffs containing 10 to 100 mg/kg of decoquinate
10 mg/kg for feedingstuffs containing 100 to 1 000 mg/kg of decoquinate
100 mg/kg for premixtures containing 1 000 to 10 000 mg/kg (= 1%) of decoquinate
0,1% for feed additives cont
...
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