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)

This document specifies a high performance liquid chromatographic – tandem mass spectrometry (LC-MS/MS) method for the simultaneous screening and/or determination of the eleven authorised coccidiostats (halofuginone, robenidine hydrochloride, nicarbazin, diclazuril, decoquinate, monensin sodium, salinomycin sodium, narasin, lasalocid sodium, semduramicin sodium and maduramicin ammonium alpha) contents in poultry, cattle and pig feed at additive and cross-contamination levels and of 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.
The range of application of the method is fit for the purpose of the screening and determination of all eleven coccidiostats at the values set by European legislation, of the non-registered coccidiostats and of the screening of the banned antibiotic.

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

Dieses Dokument legt ein Verfahren fest zum gleichzeitigen Screening und/oder zur Bestimmung des Gehalts der elf zugelassenen Kokzidiostatika (Halofuginon, Robenidinhydrochlorid, Nicarbazin, Diclazuril, Decoquinat, Monensin-Natrium, Salinomycin Natrium, Narasin, Lasalocid Natrium, Semduramicin Natrium und Maduramicin Ammonium Alpha) durch Hochleistungsflüssigkeitschromatographie – Tandem Massenspektrometrie (LC MS/MS) in Geflügel , Rinder  und Schweinefutter in Konzentrationen von Zusatzstoffen und deren Verschleppungen und von fünf nicht registrierten Kokzidiostatika (Ethopabat, Clopidol, Ronidazol, Dimetridazol und Amprolium) in Konzentrationen unterhalb von Zusatzstoffen und zum Screening auf das verbotene Antibiotikum Furazolidon in Konzentrationen unterhalb von Zusatzstoffen, in den gleichen Matrices.
Der Anwendungsbereich des Verfahrens ist geeignet für das Screening auf und die Bestimmung von allen elf 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 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)

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

Status
Published
Public Enquiry End Date
24-Oct-2018
Publication Date
02-Sep-2019
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
19-Aug-2019
Due Date
24-Oct-2019
Completion Date
03-Sep-2019

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SLOVENSKI STANDARD
SIST EN 17299:2019
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
SIST EN 17299:2019 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 17299:2019
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SIST EN 17299:2019
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.
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SIST EN 17299:2019
EN 17299:2019 (E)
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

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EN 17299:2019 (E)
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.
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EN 17299:2019 (E)
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]
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SIST EN 17299:2019
EN 17299:2019 (E)
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.
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SIST EN 17299:2019
EN 17299:2019 (E)
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).
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EN 17299:2019 (E)

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).
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.
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EN 17299:2019 (E)
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).
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).
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).
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SIST EN 17299:2019
EN 17299:2019 (E)
(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).
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).
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.
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SIST EN 17299:2019
EN 17299:2019 (E)
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).
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).
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).
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SIST EN 17299:2019
EN 17299:2019 (E)
(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).
(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).
(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.
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SIST EN 17299:2019
EN 17299:2019 (E)
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).
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 cl
...

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