Animal feeding stuffs - Determination of monensin, narasin and salinomycin contents - Liquid chromatographic method using post-column derivatization (ISO 14183:2005)

ISO 14183:2005 specifies a high-performance liquid chromatographic (HPLC) method for the determination of the monensin, narasin and salinomycin contents of animal feeding stuffs, supplements (dry and liquid) and mineral premixtures. The method is not applicable to drug premixes (pharmaceutical products). Lasalocid and semduramicin cannot be determined by this method.
The limit of quantitation is approximately 1 mg/kg, 2 mg/kg and 2 mg/kg for monensin, salinomycin and narasin, respectively. A lower limit of quantitation can be achievable but this is to be validated by the user.

Futtermittel - Bestimmung der Gehalte an Monensin, Narasin und Salinomycin - Flüssigkeitschromatographisches Verfahren mittels Nachsäulenderivatisierung (ISO 14183:2005)

Dieses Dokument legt ein Verfahren der Hochleistungsflüssigchromatographie (HPLC) zur Bestimmung der Monensin , Narasin  und Salinomycin Gehalte in Futtermitteln, Futtermittel Zusatzstoffen (trocken und flüssig) und mineralischen Futtermittel Vormischungen fest. Das Verfahren ist nicht anwendbar auf Gemische, die chemische Zusatzstoffe (pharmazeutische Produkte) enthalten. Lasalocid und Semduramicin können nach diesem Verfahren nicht bestimmt werden.
Die Bestimmungsgrenze liegt für Monensin etwa bei 1 mg/kg, für Salinomycin etwa bei 2 mg/kg und für Narasin etwa bei 2 mg/kg. Eine niedrigere Bestimmungsgrenze kann angewendet werden, ist dann aber vom Anwender zu validieren.

Aliments des animaux - Détermination des teneurs en monensine, narasine et salinomycine - Méthode par chromatographie liquide utilisant la dérivatisation post-colonne (ISO 14183:2005)

L'ISO 14183:2005 spécifie une méthode de détermination des teneurs en monensine, en narasine et en salinomycine dans les aliments pour animaux, les compléments (secs et liquides) et les prémélanges de minéraux, par chromatographie liquide à haute performance (CLHP). La méthode ne s'applique pas aux prémélanges médicamenteux (produits pharmaceutiques) et ne permet pas de déterminer le lasalocide et la semduramicine.
La limite de quantification est respectivement d'environ 1 mg/kg, 2 mg/kg et 2 mg/kg pour la monensine, la salinomycine et la narasine. Une limite de quantification inférieure peut être atteinte mais cela est sujet à validation par l'utilisateur.

Krma - Določevanje monezina, narazina in salinomicina - Metoda tekočinske kromatografije s postkolonsko derivatizacijo (ISO 14183:2005)

Ta evropski standard določa metodo tekočinske kromatografije visoke ločljivosti (HPLC) za določevanje vrednosti monezina, narazina in salinomicina v krmi, dodatkih (suhih in tekočih) ter mineralnih mešanicah. Ta metoda se ne uporablja za predmešanice za zdravilno krmno mešanico (farmacevtski izdelki). Vrednosti lasalocida in semduramicina ni mogoče določiti s to metodo. Za monezin, salinomicin in narazin so meje kvantifikacije približno 1 mg/kg, 2 mg/kg in 2 mg/kg. Nižja meja kvantifikacije je dosegljiva, vendar jo mora validirati uporabnik.

General Information

Status
Published
Publication Date
11-Nov-2008
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
12-Nov-2008
Completion Date
12-Nov-2008

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SLOVENSKI STANDARD
SIST EN ISO 14183:2009
01-april-2009
.UPD'RORþHYDQMHPRQH]LQDQDUD]LQDLQVDOLQRPLFLQD0HWRGDWHNRþLQVNH
NURPDWRJUDILMHVSRVWNRORQVNRGHULYDWL]DFLMR ,62

Animal feeding stuffs - Determination of monensin, narasin and salinomycin contents -

Liquid chromatographic method using post-column derivatization (ISO 14183:2005)
Futtermittel - Bestimmung der Gehalte an Monensin, Narasin und Salinomycin -
Flüssigkeitschromatographisches Verfahren mittels Nachsäulenderivatisierung (ISO
14183:2005)
Aliments des animaux - Détermination des teneurs en monensine, narasine et

salinomycine - Méthode par chromatographie liquide utilisant la dérivatisation post-

colonne (ISO 14183:2005)
Ta slovenski standard je istoveten z: EN ISO 14183:2008
ICS:
65.120 Krmila Animal feeding stuffs
SIST EN ISO 14183:2009 en,fr,de

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

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SIST EN ISO 14183:2009
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SIST EN ISO 14183:2009
EUROPEAN STANDARD
EN ISO 14183
NORME EUROPÉENNE
EUROPÄISCHE NORM
November 2008
ICS 65.120
English Version
Animal feeding stuffs - Determination of monensin, narasin and
salinomycin contents - Liquid chromatographic method using
post-column derivatization (ISO 14183:2005)

Aliments des animaux - Détermination des teneurs en Futtermittel - Bestimmung der Gehalte an Monensin,

monensine, narasine et salinomycine - Méthode par Narasin und Salinomycin -

chromatographie liquide utilisant la dérivatisation post- Flüssigkeitschromatographisches Verfahren mittels

colonne (ISO 14183:2005) Nachsäulenderivatisierung (ISO 14183:2005)
This European Standard was approved by CEN on 25 October 2008.

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 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 Management Centre has the same status as the

official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, 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.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36 B-1050 Brussels

© 2008 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 14183:2008: E

worldwide for CEN national Members.
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SIST EN ISO 14183:2009
EN ISO 14183:2008 (E)
Contents Page

Foreword..............................................................................................................................................................3

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SIST EN ISO 14183:2009
EN ISO 14183:2008 (E)
Foreword

The text of ISO 14183:2005 has been prepared by Technical Committee ISO/TC 34 “Agricultural food

products” of the International Organization for Standardization (ISO) and has been taken over as EN ISO

14183:2008 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 May 2009, and conflicting national standards shall be withdrawn at the

latest by May 2009.

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.

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, 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 the United Kingdom.
Endorsement notice

The text of ISO 14183:2005 has been approved by CEN as a EN ISO 14183:2008 without any modification.

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SIST EN ISO 14183:2009
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SIST EN ISO 14183:2009
INTERNATIONAL ISO
STANDARD 14183
First edition
2005-11-01
Animal feeding stuffs — Determination of
monensin, narasin and salinomycin
contents — Liquid chromatographic
method using post-column derivatization
Aliments des animaux — Détermination des teneurs en monensine,
narasine et salinomycine — Méthode par chromatographie liquide
utilisant la dérivatisation post-colonne
Reference number
ISO 14183:2005(E)
ISO 2005
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SIST EN ISO 14183:2009
ISO 14183:2005(E)
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© ISO 2005

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,

electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or

ISO's member body in the country of the requester.
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Published in Switzerland
ii © ISO 2005 – All rights reserved
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SIST EN ISO 14183:2009
ISO 14183:2005(E)
Contents Page

Foreword............................................................................................................................................................ iv

1 Scope ..................................................................................................................................................... 1

2 Normative references ........................................................................................................................... 1

3 Principle................................................................................................................................................. 1

4 Reagents................................................................................................................................................ 1

5 Apparatus .............................................................................................................................................. 4

6 Sampling................................................................................................................................................ 5

7 Preparation of test sample................................................................................................................... 5

8 Procedure .............................................................................................................................................. 6

8.1 Preparation of quality control sample ................................................................................................ 6

8.2 Extraction .............................................................................................................................................. 6

8.3 HPLC analysis ....................................................................................................................................... 7

8.4 Determination........................................................................................................................................ 8

9 HPLC confirmation ............................................................................................................................... 9

9.1 General................................................................................................................................................... 9

9.2 Post-column derivatization with DMAB.............................................................................................. 9

9.3 Hexane extraction............................................................................................................................... 10

10 Calculation of results ......................................................................................................................... 10

10.1 General................................................................................................................................................. 10

10.2 Monensin ............................................................................................................................................. 10

10.3 Salinomycin......................................................................................................................................... 11

10.4 Narasin................................................................................................................................................. 12

10.5 Interpretation of confirmation data................................................................................................... 13

11 Precision.............................................................................................................................................. 14

11.1 Interlaboratory test ............................................................................................................................. 14

11.2 Repeatability........................................................................................................................................ 14

11.3 Reproducibility.................................................................................................................................... 14

11.4 Limit of quantitation ........................................................................................................................... 15

12 Test report ........................................................................................................................................... 15

Annex A (informative) Results of interlaboratory test.................................................................................. 16

Bibliography ..................................................................................................................................................... 21

© ISO 2005 – All rights reserved iii
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SIST EN ISO 14183:2009
ISO 14183:2005(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies

(ISO member bodies). The work of preparing International Standards is normally carried out through ISO

technical committees. Each member body interested in a subject for which a technical committee has been

established has the right to be represented on that committee. International organizations, governmental and

non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the

International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards

adopted by the technical committees are circulated to the member bodies for voting. Publication as an

International Standard requires approval by at least 75 % of the member bodies casting a vote.

Attention is drawn to the possibility that some of the elements of this document may be the subject of patent

rights. ISO shall not be held responsible for identifying any or all such patent rights.

ISO 14183 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 10, Animal

feeding stuffs.
iv © ISO 2005 – All rights reserved
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SIST EN ISO 14183:2009
INTERNATIONAL STANDARD ISO 14183:2005(E)
Animal feeding stuffs — Determination of monensin, narasin
and salinomycin contents — Liquid chromatographic method
using post-column derivatization
1 Scope

This International Standard specifies a high-performance liquid chromatographic (HPLC) method for the

determination of the monensin, narasin and salinomycin contents of animal feeding stuffs, supplements (dry

and liquid) and mineral premixtures. The method is not applicable to drug premixes (pharmaceutical products).

Lasalocid and semduramicin cannot be determined by this method.

The limit of quantitation is approximately 1 mg/kg, 2 mg/kg and 2 mg/kg for monensin, salinomycin and

narasin, respectively. A lower limit of quantitation can be achievable but this is to be validated by the user.

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.
ISO 6498:1998, Animal feeding stuffs — Preparation of test samples
3 Principle

The ionophores monensin, narasin and salinomycin are extracted using methanol/water (90 + 10) with

mechanical shaking for 1 h, then the extracts are filtered. The ionophores are determined by reverse-phase

HPLC using post-column derivatization with vanillin and detection at 520 nm. Suspect positive trace-level

samples and medicated feed samples containing unexpected ionophores are confirmed using a hexane

extraction or post-column derivatization with dimethylaminobenzaldehyde (DMAB).
4 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
4.1 Water, HPLC grade, or equivalent (e.g. Milli-Q purified water).
4.2 Methanol (CH OH), HPLC grade.
4.3 Sulfuric acid (H SO ), 97 % to 98 %.
2 4
4.4 Acetic acid (CH CH CO H), glacial, 97 % to 98 %.
2 3 2
4.5 Sodium hydrogen carbonate (NaHCO ), minimum 99 % purity.
4.6 Vanillin (4-hydroxy-3-methoxybenzaldehyde), minimum 99 % purity.
© ISO 2005 – All rights reserved 1
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SIST EN ISO 14183:2009
ISO 14183:2005(E)
4.7 Dimethylaminobenzaldehyde (DMAB), minimum 99 % purity.
4.8 Hexane [CH (CH ) CH ], distilled in glass.
3 2 4 3
4.9 Extraction solvent, methanol/water (90 + 10).

Combine 1 800 ml of methanol (4.2) and 200 ml of water (4.1) in a 2 l flask. Mix well.

4.10 Mobile phases
4.10.1 Post-column reaction system

While stirring gently, slowly add by pipette 20 ml of sulfuric acid (4.3) to 950 ml of methanol (4.2). Allow to cool,

then add 30 g of vanillin (4.6) while stirring. Protect from light. Prepare fresh daily.

4.10.2 HPLC column

Use methanol (4.2)/water (4.1)/acetic acid (4.4) (940/60/1). Filter under vacuum using the equipment in 5.7.

4.11 Neutralized methanol

Add 1,0 g of sodium hydrogen carbonate (4.5) into 4 l of methanol (4.2). Mix well and filter if necessary

through an 11 µm filter paper (e.g. Whatman No. 1) . See Note to 4.13.
4.12 Reference standards
Composition or potency is required for each lot of reference standard.
4.12.1 Monensin sodium
4.12.2 Narasin
4.12.3 Sodium salinomycin

WARNING — Avoid inhalation of and exposure to the toxic standard materials and solutions thereof.

Work in a fume-hood when handling the solvents and solutions. Wear safety glasses and protective

clothing.
4.13 Ionophore stock standards, ca. 0,50 mg/ml.

Accurately weigh, to the nearest 0,1 mg, 25 mg of each standard (4.12.1 to 4.12.3) into separate 50 ml

volumetric flasks. Dissolve in neutralized methanol (4.11) and dilute to volume. Prepare freshly every month.

Store in a refrigerator.
Protect all standard solutions from light or prepare them in low actinic flasks.

NOTE The requirement for neutralized methanol has not been verified for salinomycin. It is not required if analysing

monensin only, but is required for analysis of narasin.

1) This is an example of a suitable product available commercially. This information is given for the convenience of users

of this International Standard and does not constitute an endorsement by ISO of this product.

2) Available from Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana 46285, USA.

3) Available from Alpharma Inc., Animal Health Division, 1 Duggar Drive, Willow Island, WV, USA 26134-97111, and

Hoechst Roussel Vet, D-65926 Frankfurt am Main, Gebaude H 790, Germany.
2 © ISO 2005 – All rights reserved
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SIST EN ISO 14183:2009
ISO 14183:2005(E)
4.13.1 Monensin stock standard

Prepare as described in 4.13. Calculate the concentration of stock standard based on the principle component,

[4]

monensin A. The minor component, monensin B, which elutes just before monensin A is determined in test

samples based on monensin A. Use the component composition identified on the reference standard profile

sheet:
0,5 S
ρ =
100
where

0,5 is the concentration of the stock standard (4.13), in milligrams per millilitre, recorded to three

significant figures;

ρ is the concentration of the given component monensin A in the stock standard, in milligrams per

millilitre;

S is the proportion of the given component monensin A in the reference standard according to the

profile sheet, in percent.

EXAMPLE Reference standard lot RS0234 contains 93,71 % of monensin A on an “as-is” basis.

4.13.2 Salinomycin stock standard

Prepare as described in 4.13. Determine the concentration using the reference standard concentration value

[2]
provided by the supplier :
0,5 w
ρ =
1000
where

ρ is the concentration of salinomycin in the stock standard, in milligrams per millilitre;

w is the concentration of the salinomycin standard given by the supplier, in micrograms per milligram.

EXAMPLE For lot WS-19B, the standard concentration is 986 µg/mg.
4.13.3 Narasin stock standard

Prepare as described in 4.13. Calculate the concentration of the stock standard based on the principle

[5]

component, narasin A. The minor components (narasin D and l), which elute after narasin A , are

determined in test samples based on narasin A. Use the component composition identified on the reference

standard profile sheet:
0,5 S
ρ =
100
where

ρ is the concentration of the component narasin A in the stock standard, in milligrams per millilitre;

S is the proportion of the given component narasin A in the reference standard according to the profile

sheet, in percent.

EXAMPLE For reference standard lot RS0302, the percentage of each component on an “as-is” basis is:

narasin A = 85,4 %,
narasin D = 1,9 %,
narasin I = 0,7 %.
© ISO 2005 – All rights reserved 3
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SIST EN ISO 14183:2009
ISO 14183:2005(E)

4.14 Intermediate mixed standard solution, ca. 20 µg/ml, 40 µg/ml and 40 µg/ml monensin, salinomycin

and narasin, respectively.

Transfer by pipette 10,0 ml, 20,0 ml and 20,0 ml of monensin, salinomycin and narasin stock standards (4.13),

respectively, into a 250 ml volumetric flask. Dilute to volume with extraction solvent (4.9). Mix well. Prepare

freshly every month.
4.15 Mixed HPLC standards

Prepare five mixed HPLC standard solutions by pipetting an aliquot of the mixed intermediate standard (4.14)

into 100 ml low-actinic volumetric flasks and diluting to volume with extraction solvent (4.9), as specified in the

Table 1. Mix well. Prepare freshly every month.
Table 1
Mixed HPLC Amount of Approximate HPLC standard concentration
standard intermediate
µg/ml
identification standard (4.14)
Monensin Salinomycin Narasin
A 1 0,2 0,4 0,4
B 5 1 2 2
C 10 2 4 4
D 25 5 10 10
E 50 10 20 20
4.16 Single HPLC standards
4.16.1 Monensin, ca. 5 µg/ml.

Accurately pipette 1,0 ml of monensin stock standard (4.13.1) into a 100 ml low-actinic volumetric flask. Dilute

to volume with extraction solvent (4.9). Mix well. Prepare freshly every month. Store in a refrigerator.

4.16.2 Salinomycin, ca. 10 µg/ml.

Accurately pipette 2,0 ml of salinomycin stock standard (4.13.2) into a 100 ml low-actinic volumetric flask.

Dilute to volume with extraction solvent (4.9). Mix well. Prepare freshly every month. Store in a refrigerator.

4.16.3 Narasin, ca. 10 µg/ml.

Accurately pipette 2,0 ml of narasin stock standard (4.13.3) into a 100 ml low-actinic volumetric flask. Dilute to

volume with extraction solvent (4.9). Mix well. Prepare freshly every month. Store in a refrigerator.

5 Apparatus
Usual laboratory apparatus and, in particular, the following.
5.1 HPLC system consisting of the following.
5.1.1 Pump, pulse free, flow capacity 0,1 ml/min to 2,0 ml/min.

5.1.2 Injection system, manual or autosampler, with loop suitable for 100 µl injections.

4 © ISO 2005 – All rights reserved
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SIST EN ISO 14183:2009
ISO 14183:2005(E)

5.1.3 UV/VIS detector, with variable wavelength, suitable for measurements at 520 nm and 592 nm.

5.1.4 Integrator or computer data system.

5.1.5 Post-column reactor, with a 1,5 ml to 2,0 ml reaction coil, for operation at 98 °C.

The coil may be a commercially available knitted coil or it may be made using 7,5 m to 10 m of 316 SS tubing,

0,5 mm ID, coiled in a format to fit the reactor heating chamber. For example, wrap the coil in sufficient

aluminium foil to make it fit snugly in the heater and to provide good heat transfer to the coil. A knitted coil is

preferable. To ensure effective mixing of reagent and column effluent, use a vortex or static mixing tee (not a

regular tee) before the reaction coil.

5.1.6 Post-column reagent pump, pulse free, with flow capacity 0,5 ml/min to 2,0 ml/min.

5.1.7 Analytical column.

NOTE A 5 µm C , 25 × 0,46 cm Nucleosil 120A, Partisil 5 ODS-3, or Waters Nova Pak (4 µm), or equivalent, has

been found to be suitable.
5.1.8 Guard column, C .
5.2 Nitrogen evaporator, for evaporation of solvents under a stream of nitrogen.
5.3 Shaker, rotary or wrist-action.

5.4 Balances: analytical balance of 10 g capacity or greater with 0,1 mg readability, and another balance of

100 g capacity or greater with 0,01 g readability.

5.5 Erlenmeyer flasks, of capacities 125 ml, 250 ml and 500 ml, with glass stoppers.

5.6 Filter papers, Whatman No. 41 (15 cm), Whatman No. 42 (15 cm), and Whatman No. 1 (15 cm), or

equivalent.

5.7 Solvent filtration system, all glass filter apparatus, suitable for 47 mm filter, and 47 mm diameter

nylon filter of pore size 0,45 µm.

5.8 Sample filtration system, equipped with nylon or PTFE filter of pore size 0,45 µm.

5.9 Sieve, with 1 mm apertures.
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 International Standard. A recommended sampling method

is given in ISO 6497.
7 Preparation of test sample
Prepare the test sample in accordance with ISO 6498.

Grind the laboratory sample (> 200 g) so that it passes completely through a sieve with 1 mm apertures. For

trace-level samples, grind the entire laboratory sample. Mix thoroughly.

4) These are examples of suitable products available commercially. This information is given for the convenience of

users of this International Standard and does not constitute an endorsement by ISO of these products.

© ISO 2005 – All rights reserved 5
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SIST EN ISO 14183:2009
ISO 14183:2005(E)
8 Procedure
8.1 Preparation of quality control sample
The use of quality control samples and quality control charts is recommended.

With each set, include a sample spiked at approx. 100 mg/kg, 50 mg/kg, 50 mg/kg (for medication levels) or at

4 mg/kg, 6 mg/kg, and 6 mg/kg (for trace levels) for monensin, salinomycin and narasin, respectively.

EXAMPLE 1 4,0 ml of monensin stock standard added to 20 g of sample gives 100 mg/kg, and 2,0 ml each of

salinomycin and narasin stock standard gives 50 mg/kg. All stock standards have approximately equal concentrations

(0,50 mg/ml, see 4.13).

EXAMPLE 2 3,0 ml of mixed intermediate standard (4.14) added to 20 g of sample gives 3 mg/kg monensin and

6 mg/kg salinomycin and narasin.

Acceptable recovery for medication level samples (> 10 mg/kg) is between 95 % and 108 %. Acceptable

recovery for trace-level samples (< 10 mg/kg) is between 90 % and 110 %.
8.2 Extraction
8.2.1 Dry feeds and premixes containing < 5 000 mg/kg

Accurately weigh a 20 g test portion into a 250 ml Erlenmeyer flask. For mineral premixes, add 5 g of sodium

hydrogen carbonate. Add 100 ml of extraction solvent (4.9). Stopper the flask and shake vigorously for 1 h on

the shaker (5.3).
8.2.2 Dry feeds and premixes containing > 5 000 mg/kg

Accurately weigh a 5 g test portion into a 500 ml Erlenmeyer flask. For mineral premixes, add 2 g of sodium

hydrogen carbonate. Add 200 ml of extraction solvent (4.9). Stopper the flask and shake vigorously for 1 h on

the shaker (5.3).
8.2.3 Liquid samples

Homogenize the sample by stirring the sample bottle contents on a magnetic stirrer or with a propeller mixer.

Measure a 20 ml liquid sample into a tared 25 ml graduated cylinder. Weigh and transfer the sample to a

500 ml Erlenmeyer flask. Add 180 ml of methanol (4.2) (using some to rinse the graduated cylinder). Stopper

the flask and shake vigorously for 1 h on the shaker (5.3).
8.2.4 Filter extract

Filter extracts through a No. 41 Whatman filter paper (5.6) into a 125 ml Erlenmeyer flask.

For extracts containing a high level of ionophores, dilute to the approximate concentration of HPLC

standard D (4.15). The dilution required, D, can be calculated using the following formula:

D=×
ρ V
std e
where
w is the sample target level, in milligrams per kilogram;
ρ is the concentration of the HPLC standard, in micrograms per millilitre;
std
m is the mass of the test portion, in grams;
V is the volume of extractant, in millilitres.

Pass the above extract or eluate through a 0,45 µm filter before proceeding to HPLC analysis.

6 © ISO 2005 – All rights reserved
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SIST EN ISO 14183:2009
ISO 14183:2005(E)
8.3 HPLC analysis
8.3.1 HPLC conditions
a) HPLC separation parameters:
⎯ column: as in 5.1.7
⎯ mobile phase: as in 4.10.2
⎯ flow rate: 0,7 ml/min
⎯ wavelength: 520 nm
⎯ chart speed: 0,5 cm/min
⎯ injection volume: 100 µl

⎯ guard column: as in 5.1.8 (change or repack the guard column frequently, especially when

analysing trace level samples)

⎯ attenuation: adjust to give 50 % to 60 % full-scale deflection for HPLC standard B (4.15)

for low level sample, and HPLC standard D for samples containing medication
levels.
b) Post-column reaction parameters:
⎯ post-column reactor: as in 5.1.5
⎯ mobile phase: as in 4.10.1
⎯ flow rate: 0,9 ml/min
⎯ reactor temperature: 98 °C

The system suitability criteria in 8.3.2 shall be met. The three ionophores and minor components should be

baseline resolved, however, a minor component of salinomycin may appear as a shoulder peak on the front

side of the narasin peak. Using the Nucleosil column (5.1.7), with the above conditions, retention times for

monensin B, monensin A, salinomycin, narasin A and narasin (D + I) should be approximately 8,7 min,

9,8 min, 11,2 min, 12,8 min and 14,6 min, respectively.

The flow rates and mobile phase for the analytical column may be varied slightly, however, the total flow rate

shall be between 1,5 ml/min and 1,6 ml/min to allow at least a 1 min reaction time. Sensitivity is determined by

the reaction conditions and detector signal/noise.

Maduramicin is a potential interference when analysing trace levels of salinomycin; maduramicin elutes about

0,3 min before salinomycin. Semduramicin is a potential interference when analysing monensin; it elutes

about 0,4 min before monensin B. Both maduramicin and semduramicin exhibit low sensitivity and can be

...

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