ISO 13493:1998

INTERNATIONAL ISO
STANDARD 13493
First edition
1998-09-01
Meat and meat products — Determination
of chloramphenicol content — Method
using liquid chromatography
Viande et produits à base de viande — Dosage du chloramphénicol —
Méthode par chromatographie en phase liquide
A
Reference number
ISO 13493:1998(E)

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ISO 13493:1998(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.
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.
International Standard ISO 13493 was prepared by Technical Committee
ISO/TC 34, Agricultural food products, Subcommittee SC 6, Meat and meat
products.
Annex A of this International Standard is for information only.
©  ISO 1998
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 the publisher.
International Organization for Standardization
Case postale 56 • CH-1211 Genève 20 • Switzerland
Internet iso@iso.ch
Printed in Switzerland
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INTERNATIONAL STANDARD  ISO ISO 13493:1998(E)
Meat and meat products — Determination of chloramphenicol
content — Method using liquid chromatography
1  Scope
This International Standard specifies a liquid chromatographic method for the determination of the chloramphenicol
content of the muscle tissue of meat, including poultry.
The method is suitable for the determination of chloramphenicol contents greater than 6,5 μg/kg.
Test samples which have deteriorated cannot be analysed with this method.
NOTE  This International Standard may be applicable for the determination of the chloramphenicol content of all kinds of meat
and meat products. However, materials other than muscle tissue were not included in the collaborative testing of the method.
2  Normative reference
The following standard contains provisions which, through reference in this text, constitute provisions of this
International Standard. At the time of publication, the edition indicated was valid. All standards are subject to
revision, and parties to agreements based on this International Standard are encouraged to investigate the
possibility of applying the most recent edition of the standard indicated below. Members of IEC and ISO maintain
registers of currently valid International Standards.
ISO 3696:1987, Water for analytical laboratory use — Specification and test methods.
3  Definition
For the purposes of this International Standard, the following definition applies.
3.1
chloramphenicol content of meat and meat products
mass fraction of chloramphenicol residue determined according to the procedure specified in this International
Standard.
NOTE  The chloramphenicol content is expressed in micrograms per kilogram.
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ISO 13493:1998(E) ISO
4  Principle
A test portion is extracted with water. Filtration and solid-phase extraction are used to isolate the lipophilic
components from the aqueous solution. The chloramphenicol is eluted from the cartridge with dichloromethane. The
organic phase is evaporated and purified by liquid-liquid extraction with water and toluene. The chloramphenicol is
measured with reverse-phase chromatography by ultraviolet (UV) detection.
5  Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
5.1  Water, complying with at least grade 3 in accordance with ISO 3696. The water shall be free of organic
compounds.
5.2  Nitrogen, suitable for evaporating solvents.
5.3  Dichloromethane.
5.4  Toluene.
5.5  Acetate buffer, c(CH CO Na) = 0,01 mol/l, pH = 4,3.
3 2
Dissolve 0,82 g of anhydrous sodium acetate in about 970 ml of water. Adjust the pH to 4,3 with 50 % (m/m) dilute
acetic acid (CH CO H) using the pH-meter (6.1). Transfer the solution to a 1000 ml one-mark volumetric flask.
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Dilute to the mark with water and mix.
5.6  Acetonitrile, suitable for UV spectroscopy.
5.7  Mobile phase.
Add 750 ml of acetate buffer (5.5) to 250 ml of acetonitrile (5.6) and mix thoroughly.
Before use, filter the eluent through a 0,22 mm filter (6.2) and degas.
5.8  Chloramphenicol stock solution, 100 mg/ml.
Weigh, to the nearest 0,1 mg, 10 mg of chloramphenicol and transfer it to a 100 ml one-mark volumetric flask. Dilute
to the mark with methanol and mix.
This stock solution is stable for 1 month when stored in the dark.
5.9  Chloramphenicol standard solutions.
Pipette 5,0 ml of the stock solution (5.8) into a 100 ml one-mark volumetric flask. Dilute to the mark with water and
mix.
Prepare four standard solutions by diluting 1,0 ml, 2,0 ml, 5,0 ml and 15,0 ml of this solution to 100 ml with water to
obtain solutions with a chloramphenicol content of 0,05 mg/ml, 0,10 mg/ml, 0,25 mg/ml and 0,75 mg/ml respectively.
These standard solutions are stable for 1 week when stored in the dark.
6  Apparatus
Usual laboratory apparatus and, in particular, the following.
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ISO 13493:1998(E)
6.1  pH-meter.
6.2  Membrane filter, of low dead volume and pore size 0,22 mm.
6.3  Mechanical or electrical equipment capable of homogenizing the laboratory sample.
This includes a high-speed rotational cutter, or a mincer fitted with a plate with apertures not exceeding 4,0 mm in
diameter.
1)
6.4  Laboratory blender (e.g. Stomacher blender or vortex type).
6.5  Filter paper, quantitative, fast filtration rate, of diameter about 15 cm.
1)
NOTE For example, Whatman 41 proved to be suitable .
6.6  Extraction cartridges, of capacity 20 ml, containing diatomaceous earth that extracts lipophilic components
from aqueous solutions.
1)
NOTE Extrelut®, manufactured by Merck, Darmstadt, Germany (No. 11737), proved to be suitable .
6.7  Water bath or heating block, capable of being maintained at (40 ± 1) °C, with equipment for drying with
nitrogen (5.2); or rotary vacuum evaporator.
6.8  Centrifuge tubes, of capacity 25 ml.
-1
6.9  Vortex mixer, operating at a rotation frequency of about 700 min .
6.10  Centrifuge, operating at a radial acceleration of about 1 000 g.
6.11  Micropipettes, of capacity 300 ml.
6.12  Liquid chromatograph, equipped with:
— a constant-flow pump;
— an injector;
— a reverse-phase C or C column with an internal diameter of 3 mm, length of 20 cm, and particle size of
8 18
5 μm, or a column of equivalent quality;
— a UV/VIS detector suitable for measurements at a wavelength of 285 nm; if available, a diode array detector
(for confirmation purposes);
— a recorder with variable measuring range or an integrator.
7  Sampling
Sampling is not part of the method specified in this International Standard. A recommended sampling method is
given in ISO 3100-1 [1].
It is important that the laboratory receive a sample which is truly representative and has not been damaged or
changed during transport or storage.
Proceed from a representative sample of at least 200 g. Store the sample in such a way that deterioration and
change in composition are prevented.

1) 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.
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