EN 17299:2019
(Main)Animal feeding stuffs: Methods of sampling and analysis - Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination level, and of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatography - Tandem Mass Spectrometry detection (LC-MS/MS)
Animal feeding stuffs: Methods of sampling and analysis - Screening and determination of authorized coccidiostats at additive and 1 % and 3 % cross-contamination level, and of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound feed with High Performance Liquid Chromatography - Tandem Mass Spectrometry detection (LC-MS/MS)
This document specifies requirements for light fire storage units providing protection against fire.
The method of test is specified to determine the ability of light fire storage units to protect paper media from the effects of fire. Two levels of fire exposure periods (LFS 30 and LFS 60) are specified using the maximum temperature increase permitted within the storage space of the light fire storage unit.
Protection after the fire exposure of 30 min (LFS 30) or 60 min (LFS 60) is not ensured by this document, but by European Standard EN 1047-1. Requirements are also specified for the test specimen, the technical documentation for the test specimen, correlation of the test specimen with the technical documentation, preparation for type testing and test procedures.
A scheme to classify the light fire storage units from the test results is also given (see Table 1).
Futtermittel - Probenahme‑ und Untersuchungsverfahren - Screening und Bestimmung von zugelassenen Kokzidiostatika in Konzentrationen von Zusatzstoffen und deren Verschleppungen im Bereich von 1 % und 3 % sowie von nicht registrierten Kokzidiostatika und von einem Antibiotikum in Konzentrationen unterhalb von Zusatzstoffen in Mischfuttermitteln mittels Hochleistungs-Flüssigchromatographie mit Tandem-Massenspektrometrie-Nachweis (LC‑MS/MS)
Dieses Dokument legt ein Verfahren fest zum gleichzeitigen Screening und/oder zur Bestimmung des Ge-halts der elf zugelassenen Kokzidiostatika (Halofuginon, Hydrobromid, Robenidinhydrochlorid, Nicarbazin, Diclazuril, Decoquinat, Monensin Natrium, Salinomycin Natrium, Narasin, Lasalocid A Natrium, Semdura-micin Natrium und Maduramicin Ammonium Alpha) durch Hochleistungs Flüssigchromatographie in Kombination mit Tandem Massenspektrometrie (HPLC MS/MS) in Mischfuttermitteln und Einzelfuttermitteln pflanzlichen Ursprungs in Konzentrationen von Zusatzstoffen und deren Verschleppungen und von den fünf nicht registrierten Kokzidiostatika (Ethopabat, Clopidol, Ronidazol, Dimetridazol und Amprolium) in Konzentra¬tionen unterhalb von Zusatzstoffen und zum Screening auf das verbotene Antibiotikum Furazolidon in Konzentrationen unterhalb von Zusatzstoffen, in den gleichen Matrices. Das Verfahren ist nur für Geflügel , Rinder und Schweinemischfuttermittel vollständig validiert worden.
Der Anwendungsbereich des Verfahrens ist geeignet für das Screening auf und die Bestimmung von allen elf registrierten Kokzidiostatika bei den in der europäischen Gesetzgebung festgelegten Werten, für die nicht registrierten Kokzidiostatika und für das Screening auf das verbotene Antibiotikum.
Aliments des animaux: Méthodes d’échantillonnage et d’analyse - Recherche et dosage dans des aliments composés pour animaux des coccidiostatiques autorisés aux taux d’additif et de contamination croisée à 1 % et 3 %, de coccidiostatiques non enregistrés et d’un antibiotique aux taux sub-additifs, par chromatographie en phase liquide à haute performance couplée à une détection par spectrométrie de masse en tandem (CL-SM/SM)
Le présent document spécifie une méthode de chromatographie liquide à haute performance - spectrométrie de masse en tandem (CL-SM/SM) pour la recherche simultanée des onze coccidiostatiques autorisés (halofuginone, chlorhydrate de robénidine, nicarbazine, diclazuril, décoquinate, monensine sodium, salinomycine sodium, narasine, lasalocide sodium, semduramicine sodium et maduramicine ammonium alpha) et/ou leur dosage dans les aliments composés pour la volaille, le bétail et les porcs aux taux d’additif et de contamination croisée et de cinq coccidiostatiques non enregistrés (éthopabate, clopidol, ronidazole, dimétridazole et amprolium) à des taux sub-additifs, ainsi que pour la recherche de l’antibiotique interdit furazolidone au taux sub-additif, dans les mêmes matrices.
La plage d’application de la méthode est adéquate aux fins de recherche et de dosage de la totalité des onze coccidiostatiques aux valeurs définies par la législation européenne et des coccidiostatiques non enregistrés, ainsi que de recherche de l’antibiotique interdit.
Krma: metode vzorčenja in analize - Pregled in določevanje dovoljenih kokcidiostatikov v koncentracijah dodatkov in njihovih nosilcih v območju od 1 do 3 % v krmnih mešanicah s tekočinsko kromatografijo visoke ločljivosti - Tandemska masna spektrometrija (LC-MS/MS)
Ta dokument določa metodo tekočinske kromatografije visoke ločljivosti z masno spektrometrijo (LC-MS/MS) za hkratni pregled in/ali določevanje vsebnosti enajstih dovoljenih kokcidiostatikov (halofuginon, robenidin hidroklorid, nikarbazin, diklazuril, dekokvinat, natrijev monenzin, natrijev salinomicin, narazin, lasalocid natrija, natrijev semduramicin in alfa amonijev maduramicin) v krmi za perutnino, govedo in prašiče pri koncentracijah dodatkov in navzkrižnega onesnaženja ter petih neregistriranih kokcidiostatikov (etopabat, klopidol, ronidazol, dimetridazol in amprolium) pri koncentracijah, manjših od vsebnosti dodatkov, in za pregled prepovedanega antibiotika furazolidon pri koncentracijah, manjših od vsebnosti dodatkov, pri istih matrikah.
Območje uporabe metode je primerno za namen pregleda in določevanja vseh enajstih kokcidiostatikov pri vrednostih, ki so določene z evropsko zakonodajo, neregistriranih kokcidiostatikov in pregleda prepovedanega antibiotika.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-oktober-2019
Krma: metode vzorčenja in analize - Pregled in določevanje dovoljenih
kokcidiostatikov v koncentracijah dodatkov in njihovih nosilcih v območju od 1 do
3 % v krmnih mešanicah s tekočinsko kromatografijo visoke ločljivosti -
Tandemska masna spektrometrija (LC-MS/MS)
Animal feeding stuffs: Methods of sampling and analysis - Screening and determination
of authorized coccidiostats at additive and 1 % and 3 % cross-contamination levels, and
of non-registered coccidiostats and of one antibiotic at sub-additive levels, in compound
feed with High Performance Liquid Chromatography - Tandem Mass Spectrometry
detection (LC-MS/MS)
Futtermittel - Probenahme- und Untersuchungsverfahren - Bestimmung zugelassener
Kokzidiostatika in Konzentrationen von Zusatzstoffen und deren Verschleppungen im
Bereich von 1 % bis 3 % in Mischfuttermitteln mittels Hochleistungs-LC-MS
Aliments des animaux: Méthodes d’échantillonnage et d’analyse - Recherche et dosage
dans des aliments composés pour animaux des coccidiostatiques autorisés au taux
d’additif et de contamination croisée à 1 % et 3 %, de coccidiostatiques non enregistrés
et d’un antibiotique aux taux sub-additifs, par chromatographie en phase liquide à haute
performance couplée à une détection par spectrométrie de masse en tandem (CL-
SM/SM)
Ta slovenski standard je istoveten z: EN 17299:2019
ICS:
65.120 Krmila Animal feeding stuffs
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 17299
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2019
EUROPÄISCHE NORM
ICS 65.120; 71.040.50
English Version
Animal feeding stuffs: Methods of sampling and analysis -
Screening and determination of authorized coccidiostats at
additive and 1 % and 3 % cross-contamination level, and
of non-registered coccidiostats and of one antibiotic at
sub-additive levels, in compound feed with High
Performance Liquid Chromatography - Tandem Mass
Spectrometry detection (LC-MS/MS)
Aliments des animaux: Méthodes d'échantillonnage et Futtermittel - Probenahme- und
d'analyse - Recherche et dosage dans des aliments Untersuchungsverfahren - Bestimmung zugelassener
composés pour animaux des coccidiostatiques Kokzidiostatika in Konzentrationen von Zusatzstoffen
autorisés au taux d'additif et de contamination croisée und deren Verschleppungen im Bereich von 1 % bis 3
à 1 % et 3 %, de coccidiostatiques non enregistrés et % in Mischfuttermitteln mittels Hochleistungs-LC-MS
d'un antibiotique aux taux sub-additifs, par
chromatographie en phase liquide à haute
performance couplée à une détection par
spectrométrie de masse en tandem (CL-SM/SM)
This European Standard was approved by CEN on 17 June 2019.
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, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17299:2019 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
1 Scope . 4
2 Normative references . 4
3 Terms and definitions . 4
4 Principle . 5
5 Reagents and materials . 5
6 Apparatus . 21
7 Sampling . 23
8 Preparation of test sample . 23
8.1 General . 23
8.2 Laboratory sample . 23
8.3 Test sample . 23
8.4 Test portion . 23
9 Procedure. 23
9.1 General . 23
9.2 Positive and negative control samples . 23
9.2.1 General . 23
9.2.2 Positive control sample PCS . 24
9.2.3 Negative control sample NCS . 24
9.3 Sample preparation . 24
9.3.1 Extraction . 24
9.3.2 Filtration . 24
9.3.3 Dilution . 24
9.4 Quantification through standard addition . 25
9.5 HPLC analysis . 25
9.5.1 Analytical conditions . 25
9.5.2 HPLC determination . 27
9.5.3 Criteria for acceptance of the analytical result . 28
10 Calculation . 28
11 Test report . 30
Annex A (informative) Complementary instrumental information. 31
European foreword
This document (EN 17299:2019) 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 February 2020, and conflicting national standards shall
be withdrawn at the latest by February 2020.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document has been prepared under a standardization request given to CEN by the European
Commission and the European Free Trade Association.
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, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United
Kingdom.
1 Scope
This document specifies a high performance liquid chromatographic – tandem mass spectrometry (HPLC-
MS/MS) method for the simultaneous screening and/or determination of the eleven authorized
coccidiostats (halofuginone hydrobromide, robenidine hydrochloride, nicarbazin, diclazuril,
decoquinate, monensin sodium, salinomycin sodium, narasin, lasalocid A sodium, semduramicin sodium
and maduramicin ammonium alpha) contents in compound feeds and feed materials of plant origin at
additive and cross-contamination levels and of the five non-registered coccidiostats (ethopabate,
clopidol, ronidazole, dimetridazole and amprolium) at sub-additive levels and for the screening of the
prohibited furazolidone antibiotic at sub-additive level, in the same matrices. It has been fully validated
only for poultry, cattle and pig compound feeds.
The range of application of the method is fit for the purpose of the screening and determination of all
eleven registered coccidiostats at the values set by European legislation, of the non-registered
coccidiostats and of the screening of the banned antibiotic.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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 (ISO 6498)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
Internal Standard
I.S.
compound of known concentration added to a sample to facilitate the qualitative identification and/or
quantitative determination of the sample components
[SOURCE: ISO 20752:2014, 3.2]
3.2
target feed
feed for target animal species or categories for which the additives are intended
[SOURCE: Commission Regulation (EC) No 124/2009]
3.3
non-target feed
feed for which the use of coccidiostats or histomonostats are not authorised, such as feed intended for
animal species or categories not provided for in the additive authorisation
[SOURCE: Commission Regulation (EC) No 124/2009]
4 Principle
All 11 authorized coccidiostats, non-registered coccidiostats and the banned antibiotic are extracted
using solid-liquid extraction. The extracts are centrifuged and supernatants filtered. After a first
screening analysis, the analytes are determined by reverse phase HPLC using electrospray ionization with
further tandem mass spectrometry detection. The quantitation of the detected target analytes is
performed using a multi-level standard additions approach.
5 Reagents and materials
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.
Use only reagents recognized as analytical grade at least unless otherwise stated.
5.1 Water, HPLC grade or equivalent (e.g. milli-Q purified water).
5.2 Acetonitrile, HPLC gradient grade or hypergrade LC-MS, minimum 99,9 % purity.
5.3 Methanol, HPLC grade or hypergrade LC-MS.
5.4 Formic acid, HPLC grade or hypergrade LC-MS, minimum purity 98 %.
5.5 Ethanol, absolute, minimum purity 99,9 %.
5.6 N, N-Dimethylformamide, minimum purity 99 %.
5.7 Dimethylsulfoxide, minimum purity 99,5 %.
5.8 Calcium chloride, anhydrous, minimum purity 93 %.
5.9 Acetonitrile solution in water 1:1, V:V.
Take 50 ml of acetonitrile (5.2) into a 100 ml graduated cylinder (6.11) and add 50 ml water (5.1).
Prepare freshly every 3 months.
5.10 1 % Calcium chloride solution in methanol
Accurately weigh to the nearest 0,1 g, 1,0 g of calcium chloride anhydrous (5.8) into a 100 ml volumetric
flask (6.9). Note down the exact weight of calcium chloride anhydrous (5.8). Dissolve in methanol (5.3)
and make up to 100 ml volume with methanol (5.3). Store at 4 °C in amber glassware (6.13). Prepare
freshly every 3 months.
5.11 Solvent mixture for feed extraction acetonitrile: methanol: water 80:10:10 V:V:V.
Take 800 ml of acetonitrile (5.2) into a 1 000 ml volumetric flask (6.9) or a 1 000 ml graduated cylinder
(6.11) and add 100 ml of methanol (5.3) and 100 ml of water (5.1). Mix thoroughly. Prepare freshly before
extraction and leave at room temperature.
5.12 Mobile phase for HPLC
5.12.1 Water, containing 0,5 % (V:V) formic acid – Phase A
Take 5 ml of formic acid (5.4) into a 1 000 ml volumetric flask (6.9) and make up to 1 000 ml of volume
with water (5.1). Prepare fresh solutions monthly.
5.12.2 Methanol, containing 0,5 % (V:V) formic acid – Phase B
Take 5 ml of formic acid (5.4) into a 1 000 ml volumetric flask (6.9) and make up to 1 000 ml of volume
with methanol (5.3). Prepare fresh solutions monthly.
5.13 Reference standards
Purity required for each lot of reference and internal standards.
NOTE In the event of non-availability of the standards described below, at the given purities, standard
substances with other purities can be used provided that the purity is duly taken into account in the calculation and
that the chromatographic behaviour e.g. peak shape, is not affected.
5.13.1 Halofuginone hydrobromide, CAS N° 64924-67-0, minimum purity 99 % expressed as
halofuginone hydrobromide.
5.13.2 Robenidine hydrochloride, CAS N° 25875-50-7, minimum purity 97 %.
5.13.3 Nicarbazin, CAS N° 330-95-0, minimum purity 97 %.
5.13.4 Diclazuril, CAS N° 101831-37-2, minimum purity 98 %.
5.13.5 Decoquinate, CAS N° 18507-89-6, minimum purity 98 %.
5.13.6 Semduramicin sodium, CAS N° 113378-31-7, minimum purity 93 % expressed as
semduramicin.
5.13.7 Monensin sodium, CAS N° 22373-78-0, minimum purity 86 % expressed as monensin A
sodium.
5.13.8 Maduramicin ammonium, CAS N° 84878-61-5, minimum purity 97 % expressed as
maduramicin ammonium alpha.
5.13.9 Salinomycin sodium, CAS N° 55721-31-8, minimum purity 93 %.
5.13.10 Lasalocid sodium, CAS N° 25999-20-6, minimum purity 95 % expressed as lasalocid A
sodium.
5.13.11 Narasin, CAS N° 55134-13-9, minimum purity 97 % expressed as narasin A.
5.13.12 Ethopabate, CAS N° 59-06-3, minimum purity 99 %.
5.13.13 Clopidol, CAS N° 2971-90-6, minimum purity 99 %.
5.13.14 Amprolium hydrochloride, CAS N° 137-88-2, minimum purity 99 %.
5.13.15 Furazolidone, CAS N° 67-45-8, minimum purity 99 %.
5.13.16 Ronidazole, CAS N° 7681-76-7, minimum purity 99 %.
5.13.17 Dimetridazole, CAS N° 551-92-8, minimum purity 99 %.
5.13.18 Robenidine-D , hydrochloride, CAS N° 1173097-77-2, minimum purity 98 %, to be used as
internal standard (I.S.) for robenidine (5.13.2).
5.13.19 Dinitrocarbanilide-D , CAS N° 1156508-87-0, minimum purity 99 %, to be used as internal
standard (I.S.) for dinitrocarbanilide (nicarbazin (5.13.3) marker).
5.13.20 Bis-Diclazuril, CAS N° 103337-71-9, minimum purity 97 %, to be used as internal standard (I.S.)
for diclazuril (5.13.4).
5.13.21 Decoquinate-D , CAS N° 1228182-55-5, minimum purity 98 %, to be used as internal standard
(I.S.) for decoquinate (5.13.5).
5.13.22 Nigericin sodium, CAS N° 28643-80-3, minimum purity 98 % to be used as internal standard
(I.S.) for ionophore coccidiostats (5.13.6; 5.13.7; 5.13.8; 5.13.9; 5.13.10; 5.13.11).
5.13.23 Dimetridazole-D , CAS N° 64678-69-9, minimum purity 99 % to be used as internal standard
(I.S.) for dimetridazole (5.13.17).
5.13.24 Ethopabate-D , CAS N° 59-06-03 - unlabelled, minimum purity 99 % to be used as internal
standard (I.S.) for ethopabate (5.13.12).
5.13.25 Furazolidone-D , CAS N° 1217222-76-8, minimum purity 99 % to be used as internal standard
(I.S.) for furazolidone (5.13.15).
5.13.26 Ronidazole-D , CAS N° 1015855-87-4, minimum purity 99 % to be used as internal standard
(I.S.) for ronidazole (5.13.16) and clopidol (5.13.13).
5.14 Standard solutions
5.14.1 Stock standard solutions, ca. 1 mg/ml.
5.14.1.1 General
5.14.1.2 Halofuginone hydrobromide
Accurately weigh to the nearest 0,1 mg 10 mg of halofuginone hydrobromide standard (5.13.1) into a
10 ml volumetric flask (6.9). Note down the exact weight of halofuginone hydrobromide. Dissolve in the
acetonitrile solution in water (5.9) and make up to 10 ml volume with the acetonitrile solution in water
(5.9). Store at −20 °C in amber vials (6.13). Prepare freshly every 3 months.
Determine the accurate concentration of the halofuginone hydrobromide stock standard solution using
the reference standard purity value provided by the supplier using Formula (1).
m
C × P (1)
HAL
where
C is the concentration of halofuginone hydrobromide in the stock standard solution, in mg/ml;
HAL
P is the purity of the halofuginone hydrobromide standard given by the supplier, in %;
NOTE For example 0,99.
m is the weighed mass of halofuginone hydrobromide standard (5.13.1), in mg.
=
5.14.1.3 Robenidine hydrochloride
Accurately weigh to the nearest 0,1 mg 10 mg of robenidine hydrochloride standard (5.13.2) into a 10 ml
volumetric flask (6.9). Note down the exact weight of robenidine hydrochloride. Dissolve in ethanol (5.5)
and make up to 10 ml volume with ethanol (5.5). Store at −20 °C in amber vials (6.13). Prepare freshly
every 3 months.
Determine the accurate concentration of the robenidine hydrochloride stock standard solution using the
reference standard purity value provided by the supplier using Formula (2).
m
C × P (2)
ROB
where
C is the concentration of robenidine hydrochloride in the stock standard solution, in mg/ml;
ROB
P is the purity of the robenidine hydrochloride standard given by the supplier, in %;
NOTE For example 0,97.
m is the weighed mass of robenidine hydrochloride standard (5.13.2), in mg.
5.14.1.4 Nicarbazin
Accurately weigh to the nearest 0,1 mg 10 mg of nicarbazin standard (5.13.3) into a 10 ml volumetric
flask (6.9). Note down the exact weight of nicarbazin. Dissolve in dimethylsulfoxide (5.7) and make up to
10 ml volume with dimethylsulfoxide (5.7). Store at −20 °C in amber vials (6.13). Prepare freshly every
3 months.
Determine the accurate concentration of the nicarbazin stock standard solution using the reference
standard purity value provided by the supplier using Formula (3).
m
C × P (3)
NIC
where
C is the concentration of nicarbazin in the stock standard solution, in mg/ml;
NIC
P is the purity of the nicarbazin standard given by the supplier, in %;
NOTE For example 0,97.
m is the weighed mass of nicarbazin standard (5.13.3), in mg.
5.14.1.5 Diclazuril
Accurately weigh to the nearest 0,1 mg 10 mg of diclazuril standard (5.13.4) into a 10 ml volumetric flask
(6.9). Note down the exact weight of diclazuril. Dissolve in N,N-dimethylformamide (5.6) and make up to
10 ml volume with N,N-dimethylformamide (5.6). Store at −20 °C in amber vials (6.13). Prepare freshly
every 3 months.
Determine the accurate concentration of the diclazuril stock standard solution using the reference
standard purity value provided by the supplier using Formula (4).
=
=
m
(4)
C × P
DICL
where
C is the concentration of diclazuril in the stock standard solution, in mg/ml;
DICL
P is the purity of the diclazuril standard given by the supplier, in %;
NOTE For example 0,98.
m is the weighed mass of diclazuril standard (5.13.4), in mg.
5.14.1.6 Semduramicin sodium
Accurately weigh to the nearest 0,1 mg 10 mg of semduramicin sodium standard (5.13.6) into a 10 ml
volumetric flask (6.9). Note down the exact weight of semduramicin sodium. Dissolve in 3 ml methanol
(5.3) and make up to 10 ml volume with acetonitrile (5.2). Store at −20 °C in amber vials (6.13). Prepare
freshly every 3 months.
Determine the accurate concentration of the semduramicin sodium stock standard solution using the
reference standard purity value provided by the supplier using Formula (5).
m
C × P (5)
SEM
where
C is the concentration of semduramicin sodium in the stock standard solution, in mg/ml;
SEM
P is the purity of the semduramicin sodium standard given by the supplier, in %;
NOTE For example 0,93.
m is the weighed mass of semduramicin sodium standard (5.13.6), in mg.
5.14.1.7 Monensin sodium
Accurately weigh to the nearest 0,1 mg 10 mg of monensin sodium standard (5.13.7) into a 10 ml
volumetric flask (6.9). Note down the exact weight of monensin sodium. Dissolve in 3 ml methanol (5.3)
and make up to 10 ml volume with acetonitrile (5.2). Store at −20 °C in amber vials (6.13). Prepare freshly
every 3 months.
Determine the accurate concentration of the monensin sodium stock standard solution using the
reference standard purity value provided by the supplier using Formula (6).
m
C × P (6)
MON
where
C is the concentration of monensin sodium in the stock standard solution, in mg/ml;
MON
P is the purity of the monensin sodium standard given by the supplier, in %;
NOTE For example 0,86.
m is the weighed mass of monensin sodium standard (5.13.7), in mg.
=
=
=
5.14.1.8 Maduramicin ammonium
Accurately weigh to the nearest 0,1 mg 10 mg of maduramicin ammonium standard (5.13.8) into a 10 ml
volumetric flask (6.9). Note down the exact weight of maduramicin ammonium. Dissolve in 3 ml methanol
(5.3) and make up to 10 ml volume with acetonitrile (5.2). Store at −20 °C in amber vials (6.13). Prepare
freshly every 3 months.
Determine the accurate concentration of the maduramicin ammonium stock standard solution using the
reference standard purity value provided by the supplier using Formula (7).
m
CP× (7)
MAD
where
C is the concentration of maduramicin ammonium in the stock standard solution, in mg/ml;
MAD
P is the purity of the maduramicin ammonium alpha standard given by the supplier, in %;
NOTE For example 0,97.
m is the weighed mass of maduramicin ammonium alpha standard (5.13.8), in mg.
5.14.1.9 Salinomycin sodium
Accurately weigh to the nearest 0,1 mg 10 mg of salinomycin sodium standard (5.13.9) into a 10 ml
volumetric flask (6.9). Note down the exact weight of salinomycin sodium. Dissolve in 3 ml methanol (5.3)
and make up to 10 ml volume with acetonitrile (5.2). Store at −20 °C in amber vials (6.13). Prepare freshly
every 3 months.
Determine the accurate concentration of the salinomycin sodium stock standard solution using the
reference standard purity value provided by the supplier using Formula (8).
m
C × P (8)
SAL
where
C is the concentration of salinomycin sodium in the stock standard solution, in mg/ml;
SAL
P is the purity of the salinomycin sodium standard given by the supplier, in %;
NOTE For example 0,93.
m is the weighed mass of salinomycin sodium standard (5.13.9), in mg.
5.14.1.10 Lasalocid A sodium
Accurately weigh to the nearest 0,1 mg 10 mg of lasalocid sodium standard (5.13.10) into a 10 ml
volumetric flask (6.9). Note down the exact weight of lasalocid sodium. Dissolve in 3 ml methanol (5.3)
and make up to 10 ml volume with acetonitrile (5.2). Store at −20 °C in amber vials (6.13). Prepare freshly
every 3 months.
Determine the accurate concentration of the lasalocid A sodium stock standard solution using the
reference standard purity value provided by the supplier using Formula (9).
=
=
m
(9)
CP ×
LAS
where
C is the concentration of lasalocid A sodium in the stock standard solution, in mg/ml;
LAS
P is the purity of the lasalocid sodium standard given by the supplier, in %;
NOTE For example 0,95.
m is the weighed mass of lasalocid sodium standard (5.13.10), in mg.
5.14.1.11 Narasin
Accurately weigh to the nearest 0,1 mg 10 mg of narasin standard (5.13.11) into a 10 ml volumetric flask
(6.9). Note down the exact weight of narasin. Dissolve in 3 ml methanol (5.3) and make up to 10 ml
volume with acetonitrile (5.2). Store at −20 °C in amber vials (6.13). Prepare freshly every 3 months.
Determine the accurate concentration of the narasin stock standard solution using the reference standard
purity value provided by the supplier using Formula (10).
m
(10)
CP ×
NAR
where
C is the concentration of narasin in the stock standard solution, in mg/ml;
NAR
P is the purity of the narasin standard given by the supplier, in %;
NOTE For example 0,97.
m is the weighed mass of narasin standard (5.13.11), in mg.
5.14.1.12 Amprolium
Accurately weigh to the nearest 0,1 mg 10 mg of amprolium hydrochloride standard (5.13.14) into a
10 ml volumetric flask (6.9). Note down the exact weight of amprolium. Dissolve in 5 ml methanol (5.3)
and make up to 10 ml volume with methanol (5.2). Store at −20 °C in amber vials (6.13). Prepare freshly
every 3 months.
Determine the accurate concentration of the amprolium stock standard solution using the reference
standard purity value provided by the supplier using Formula (11).
m
(11)
CP×
AMP
where
C is the concentration of amprolium in the stock standard solution, in mg/ml;
AMP
P is the purity of the amprolium standard given by the supplier, in %;
NOTE For example 0,99.
m is the weighed mass of amprolium standard (5.13.14), in mg.
=
=
=
5.14.1.13 Clopidol
Accurately weigh to the nearest 0,1 mg 10 mg of clopidol standard (5.13.13) into a 10 ml volumetric flask
(6.9). Note down the exact weight of clopidol. Dissolve in 5 ml dimethylsulfoxide (5.7) and make up to
10 ml volume with dimethylsulfoxide (5.7). Store at −20 °C in amber vials (6.13). Prepare freshly every
3 months.
Determine the accurate concentration of the clopidol stock standard solution using the reference
standard purity value provided by the supplier using Formula (12).
m
CP × (12)
CLOP
where
C is the concentration of clopidol in the stock standard solution, in mg/ml;
CLOP
P is the purity of the clopidol standard given by the supplier, in %;
NOTE For example 0,99.
m is the weighed mass of clopidol standard (5.13.13), in mg.
5.14.1.14 Dimetridazole
Accurately weigh to the nearest 0,1 mg 10 mg of dimetridazole standard (5.13.17) into a 10 ml volumetric
flask (6.9). Note down the exact weight of dimetridazole. Dissolve in 5 ml ethanol (5.5) and make up to
10 ml volume with ethanol (5.5). Store at −20 °C in amber vials (6.13). Prepare freshly every 3 months.
Determine the accurate concentration of the dimetridazole stock standard solution using the reference
standard purity value provided by the supplier using Formula (13).
m
CP × (13)
DIM
where
C is the concentration of dimetridazole in the stock standard solution, in mg/ml;
DIM
P is the purity of the dimetridazole standard given by the supplier, in %;
NOTE For example 0,98.
m is the weighed mass of dimetridazole standard (5.13.17), in mg.
5.14.1.15 Ethopabate
Accurately weigh to the nearest 0,1 mg 10 mg of ethopabate standard (5.13.12) into a 10 ml volumetric
flask (6.9). Note down the exact weight of ethopabate. Dissolve in 5 ml ethanol (5.5) and make up to 10 ml
volume with ethanol (5.5). Store at −20 °C in amber vials (6.13). Prepare freshly every 3 months.
Determine the accurate concentration of the ethopabate stock standard solution using the reference
standard purity value provided by the supplier using Formula (14).
=
=
m
(14)
C × P
ETHO
where
C is the concentration of ethopabate in the stock standard solution, in mg/ml;
ETHO
P is the purity of the ethopabate standard given by the supplier, in %;
NOTE For example 0,99.
m is the weighed mass of ethopabate standard (5.13.12), in mg.
5.14.1.16 Furazolidone
Accurately weigh to the nearest 0,1 mg 10 mg of furazolidone standard (5.13.15) into a 10 ml volumetric
flask (6.9). Note down the exact weight of furazolidone. Dissolve in 5 ml dimethylsulfoxide (5.7) and make
up to 10 ml volume with dimethylsulfoxide (5.7). Store at −20 °C in amber vials (6.13). Prepare freshly
every 3 months.
Determine the accurate concentration of the furazolidone stock standard solution using the reference
standard purity value provided by the supplier using Formula (15).
m
CP × (15)
FURA
where
C is the concentration of furazolidone in the stock standard solution, in mg/ml;
FURA
P is the purity of the furazolidone standard given by the supplier, in %;
NOTE For example 0,99.
m is the weighed mass of furazolidone standard (5.13.15), in mg.
5.14.1.17 Ronidazole
Accurately weigh to the nearest 0,1 mg 10 mg of ronidazole standard (5.13.16) into a 10 ml volumetric
flask (6.9). Note down the exact weight of ronidazole. Dissolve in 5 ml ethanol (5.5) and make up to 10 ml
volume with ethanol (5.5). Store at −20 °C in amber vials (6.13). Prepare freshly every 3 months.
Determine the accurate concentration of the ronidazole stock standard solution using the reference
standard purity value provided by the supplier using Formula (16).
m
(16)
C × P
RON
where
C is the concentration of ronidazole in the stock standard solution, in mg/ml;
RON
P is the purity of the ronidazole standard given by the supplier, in %;
NOTE For example 0,99.
m is the weighed mass of ronidazole standard (5.13.16), in mg.
=
=
=
5.14.1.18 Nigericin (I.S.)
Accurately weigh to the nearest 0,1 mg 10 mg of nigericin sodium standard (5.13.22) into a 10 ml
volumetric flask (6.9). Note down the exact weight of nigericin sodium. Dissolve in methanol (5.3) and
make up to 10 ml volume with methanol (5.3). Store at −20 °C in amber vials (6.13). Prepare freshly every
3 months.
Determine the accurate concentration of the nigericin stock standard solution using the reference
standard purity value provided by the supplier using Formula (17).
m
CP × (17)
NIG
where
C is the concentration of nigericin in the stock standard solution, in mg/ml;
NIG
P is the purity of the nigericin sodium standard given by the supplier, in %;
NOTE For example 0,98.
m is the weighed mass of nigericin sodium standard (5.13.22), in mg.
5.14.1.19 Robenidine-D hydrochloride (I.S.)
Accurately weigh to the nearest 0,1 mg 10 mg of robenidine-D hydrochloride standard (5.13.18) into a
10 ml volumetric flask (6.9). Note down the exact weight of robenidine-D hydrochloride. Dissolve in
ethanol (5.5) and make up to 10 ml volume with ethanol (5.5). Store at −20 °C in amber vials (6.13).
Prepare freshly every 3 months.
Determine the accurate concentration of the robenidine-D stock standard solution using the reference
standard purity value provided by the supplier using Formula (18).
m
C × P (18)
ROB−D
Where
is the concentration of robenidine-D in the stock standard solution, in mg/ml;
C
ROB−D
P is the purity of the robenidine-D hydrochloride standard given by the supplier, in %;
NOTE For example 0,97.
m is the weighed mass of robenidine-D hydrochloride standard (5.13.18), in mg.
5.14.1.20 Decoquinate-D (I.S.)
Accurately weigh to the nearest 0,1 mg 10 mg of decoquinate-D standard (5.13.21) into a 10 ml
volumetric flask (6.9). Note down the exact weight of decoquinate-D . Dissolve in 1 % calcium chloride
solution in methanol (5.10) and make up to 10 ml volume with 1 % calcium chloride solution in methanol
(5.10). Store at −20 °C in amber vials (6.13). Prepare freshly every 3 months.
Determine the accurate concentration of the decoquinate-D stock standard solution using the reference
standard purity value provided by the supplier using Formula (19).
=
=
m
(19)
CP ×
DEC−D
where
is the concentration of decoquinate-D in the stock standard solution, in mg/ml;
C
DEC−D
P is the purity of the decoquinate-D standard given by the supplier, in %;
NOTE For example 0,98.
m is the weighed mass of decoquinate-D standard (5.13.21), in mg.
5.14.1.21 Dimetridazole-D (I.S.)
Accurately weigh to the nearest 0,1 mg 10 mg of dimetridazole-D standard (5.13.23) into a 10 ml
volumetric flask (6.9). Note down the exact weight of dimetridazole-D . Dissolve in ethanol (5.5) and
make up to 10 ml volume with ethanol (5.5). Store at −20 °C in amber vials (6.13). Prepare freshly every
3 months.
Determine the accurate concentration of the dimetridazole-D stock standard solution using the
reference standard purity value provided by the supplier using Formula (20).
m
C × P (20)
DIM−D
where
is the concentration of dimetridazole-D in the stock standard solution, in mg/ml;
C
DIM−D
P is the purity of the dimetridazole-D standard given by the supplier, in %;
NOTE For example 0,99.
m is the weighed mass of dimetridazole-D standard (5.13.23), in mg.
5.14.1.22 Ethopabate-D (I.S.)
Accurately weigh to the nearest 0,1 mg 10 mg of ethopabate-D standard (5.13.24) into a 10 ml
volumetric flask (6.9). Note down the exact weight of ethopabate-D . Dissolve in ethanol (5.5) and make
up to 10 ml volume with ethanol (5.5). Store at −20 °C in amber vials (6.13). Prepare freshly every
3 months.
Determine the accurate concentration of the ethopabate-D stock standard solution using the reference
standard purity value provided by the supplier using Formula (21).
=
=
m
(21)
C × P
ETHO−D
where
is the concentration of ethopabate-D in the stock standard solution, in mg/ml;
C
ETHO−D
P is the purity of the ethopabate-D standard given by the supplier, in %;
NOTE For example 0,99.
m is the weighed mass of ethopabate-D standard (5.13.24), in mg.
5.14.1.23 Furazolidone-D (I.S.)
Accurately weigh to the nearest 0,1 mg 10 mg of furazolidone-D standard (5.13.25) into a 10 ml
volumetric flask (6.9). Note down the exact weight of furazolidone-D . Dissolve in dimethylsulfoxide (5.7)
and make up to 10 ml volume with dimethylsulfoxide (5.7). Store at −20 °C in amber vials (6.13). Prepare
freshly every 3 months.
Determine the accurate concentration of the furazolidone-D stock standard solution using the reference
standard purity value provided by the supplier using Formula (22).
m
CP × (22)
FURA−D
where
is the concentration of furazolidone-D in the stock standard solution, in mg/ml;
C
FURA−D
P is the purity of the furazolidone-D standard given by the supplier, in %;
NOTE For example 0,99.
m is the weighed mass of furazolidone-D standard (5.13.25), in mg.
5.14.1.24 Ronidazole-D (I.S.)
Accurately weigh to the nearest 0,1 mg 10 mg of ronidazole-D standard (5.13.26) into a 10 ml
volumetric flask (6.9). Note down the exact weight of ronidazole-D . Dissolve in ethanol (5.5) and make
up to 10 ml volume with ethanol (5.5). Store at −20 °C in amber vials (6.13). Prepare freshly every 3
months.
Determine the accurate concentration of the ronidazole-D stock standard solution using the reference
standard purity value provided by the supplier using Formula (23).
=
=
m
(23)
C × P
RON−D
Where
is the concentration of ronidazole-D in the stock standard solution, in mg/ml;
C 3
RON−D
P is the purity of the ronidazole-D standard given by the supplier, in %;
NOTE For example 0,99.
m is the weighed mass of ronidazole-D standard (5.13.26), in mg.
5.14.2 Stock standard solutions, ca. 0,3 mg/ml
5.14.2.1 Decoquinate
Accurately weigh to the nearest 0,1 mg 15 mg of decoquinate standard (5.13.5) into a 50 ml volumetric
flask (6.9). Note down the exact weight of decoquinate. Dissolve in 1 % calcium chloride in methanol
(5.10) and make up to 50 ml volume with 1 % calcium chloride in methanol (5.10). Store at −20 °C in
amber vials (6.13). Prepare freshly every 3 months.
Determine the accurate concentration of the decoquinate stock standard solution using the reference
standard purity value provided by the supplier using Formula (24).
m
CP × (24)
DEC
where
C
is the concentration of decoquinate in the stock standard solution, in mg/ml;
DEC
P is the purity of the decoquinate standard given by the supplier, in %;
NOTE For example 0,98.
m is the weighed mass of decoquinate standard (5.13.5), in mg.
5.14.3 Stock standard solutions, ca. 0,2 mg/ml
5.14.3.1 Bis-diclazuril (I.S.)
Accurately weigh to the nearest 0,1 mg 10 mg of bis-diclazuril standard (5.13.20) into a 50 ml volumetric
flask (6.9). Note down the exact weight of bis-diclazuril. Dissolve in N,N-dimethylformamide (5.6) and
make up to 50 ml volume with N,N-dimethylformamide (5.6). Store at −20 °C in amber vials (6.13).
Prepare freshly every 3 months.
Determine the accurate concentration of the bis-diclazuril stock standard solution using the reference
standard purity value provided by the supplier using Formula (25).
=
=
m
(25)
C × P
bis−DICL
where
C is the concentration of bis-diclazuril in the stock standard solution, in mg/ml;
bis-DICL
P is the purity of the bis-diclazuril standard given by the supplier, in %;
NOTE For example 0,97.
m is the weighed mass of bis-diclazuril standard (5.13.20), in mg.
5.14.3.2 Dinitrocarbanilide-D (I.S.)
Accurately weigh to the nearest 0,1 mg 10 mg of dinitrocarbanilide-D standard (5.13.19) into a 50 ml
volumetric flask (6.9). Note down the exact weight of dinitrocarbanilide-D . Dissolve in N,N-
dimethylformamide (5.6) and make up to 50 ml volume with N,N-dimethylformamide (5.6). Store at
−20 °C in amber vials (6.13). Prepare freshly every 3 months.
Determine the accurate concentration of the dinitrocarbanilide-D stock standard solution using the
reference standard purity value provided by the supplier using Formula (26).
m
CP × (26)
DNC−D
Where
is the concentration of dinitrocarbanilide-D in the stock standard solution, in mg/ml;
C
DNC−D
P is the purity of the dinitrocarbanilide-D standard given by the supplier, in %;
NOTE For example 0,99.
m is the weighed mass of dinitrocarbanilide-D standard (5.13.19), in mg.
5.14.4 Intermediate standard solutions, ca. 0,1 mg/ml.
5.14.4.1 Maduramicin ammonium
Accurately pipette (6.8) 1,0 ml of the maduramicin ammonium stock standard solution (5.14.1.8) into a
10 ml volumetric flask (6.9). Make up to 10 ml volume with acetonitrile (5.2). Store at −20 °C in amber
vials (6.13). Prepare freshly every month.
5.14.4.2 Halofuginone hydrobromide
Accurately pipette (6.8) 1,0 ml of the halofuginone hydrobromide stock standard solution (5.14.1.2) into
a 10 ml volumetric flask (6.9). Make up to 10 ml volume with acetonitrile (5.2). Store at −20 °C in amber
vials (6.13). Prepare freshly every month.
5.14.4.3 Diclazuril
Accurately pipette (6.8) 1,0 ml of the diclazuril stock standard solution (5.14.1.5) into a 10 ml volumetric
flask (6.9). Make up to 10 ml volume with acetonitrile (5.2). Store at −20 °C in amber vials (6.13). Prepare
freshly every month.
=
=
5.15 Standard addition spiking solutions
5.15.1 Target feed – coccidiostats spiking solution
Accurately pipette(6.8) into a 20 ml volumetric flask (6.9):
— 315 µl of lasalocid sodium stock standard solution (5.14.1.10);
— 175 µl of narasin stock standard solution (5.14.1.11);
— 175 µl of salinomycin sodium stock standard solution (5.14.1.9);
— 315 µl of monensin sodium stock standard solution (5.14.1.7);
— 65 µl of semduramicin sodium stock standard solution (5.14.1.6);
— 175 µl of robenidine hydrochloride stock standard solution (5.14.1.3);
— 310 µl of nicarbazin stock standard solution (5.14.1.4);
— 330 µl of decoquinate stock standard solution (5.14.2.1);
— 125 µl of maduramicin ammonium intermediate standard solution (5.14.4.1);
— 75 µl of halofuginone hydrobromide intermediate standard solution (5.14.4.2);
— 25 µl of diclazuril intermediate standard solution (5.14.4.3).
If the protocol is to be applied simultaneously to the determination of the non-registered coccidiostats
and of the furazolidone antibiotic at sub-additive levels, add:
— 100 µl of amprolium stock standard solution (5.14.1.12);
— 100 µl of clopidol stock standard solution (5.14.1.13);
— 100 µl of dimetridazole stock standard solution (5.14.1.14);
— 100 µl of ethopabate stock standard solution (5.14.1.15);
— 100 µl of furazolidone stock standard solution (5.14.1.16);
— 100 µl of ronidazole stock standard solution (5.14.1.17);
before making up to 20 ml volume with acetonitrile (5.2).
Store at −20 °C in amber vials (6.13). Prepare freshly every 3 months.
5.15.2 Target feed – coccidiost
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.
Loading comments...