ISO 13493:2021

INTERNATIONAL ISO
STANDARD 13493
Second edition
Meat and meat products —
Determination of chloramphenicol
content — Reference method
Viande et produits à base de viande — Dosage du chloramphénicol —
Méthode de référence
PROOF/ÉPREUVE
Reference number
ISO 13493:2021(E)
©
ISO 2021

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ISO 13493:2021(E)

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© ISO 2021
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Published in Switzerland
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ISO 13493:2021(E)

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
4.1 Liquid chromatographic method. 1
4.2 Liquid chromatography tandem mass spectrometry method . 2
5 Sampling . 2
6 Preparation of test sample . 2
7 Test method of liquid chromatography . 2
7.1 Reagents. 2
7.2 Apparatus . 3
7.3 Procedure . 4
7.3.1 General. 4
7.3.2 Test portion . 4
7.3.3 Preparation of extract . . 4
7.3.4 Solid-phase extraction . 4
7.3.5 Liquid-liquid extraction . 4
7.3.6 Chromatographic analysis . 5
7.4 Calculation . 5
7.5 Precision . 6
7.5.1 Interlaboratory test . 6
7.5.2 Repeatability . 6
7.5.3 Reproducibility . 6
8 Test method of liquid chromatography tandem mass spectrometry method
(reference method). 6
8.1 Reagents and materials . 6
8.2 Apparatus . 8
8.3 Procedure . 8
8.3.1 General. 8
8.3.2 Test portion . 8
8.3.3 Preparation of extract . . 8
8.3.4 Solid-phase extraction . 9
8.3.5 LC-MS/MS analysis . 9
8.4 Calculations .11
8.5 Precision .11
8.5.1 Interlaboratory test .11
8.5.2 Repeatability .11
8.5.3 Reproducibility .11
9 Test report .11
Annex A (informative) HPLC chromatogram of chloramphenicol standard solution .13
Bibliography .15
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ISO 13493:2021(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 6,
Meat, poultry, fish, eggs and their products.
This second edition cancels and replaces the first edition (ISO 13493:1998), which has been technically
revised. The main changes compared with the previous edition are as follows:
— A new test method, the liquid chromatography tandem mass spectrometry method (LC-MS/MS) has
been added. The reference test method has been changed from liquid chromatography to LC-MS/
MS.
— The Scope has been expanded to include muscle, casing and the liver of meat and meat products,
including livestock, poultry and seafood.
— The title of the document has been modified.
— The document structure has been rearranged.
— The introductory texts for the Foreword, Clause 2 and Clause 3 have been modified.
— Clause 2 has been updated.
— In 7.3.6.1, “Chromatographic conditions”, “detector range”, “recorder ranger” and “paper speed”
have been deleted.
— Clause 8 “Test method of liquid chromatography tandem mass spectrometry method (reference
method)” has been added.
— The Bibliography has been updated.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
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INTERNATIONAL STANDARD ISO 13493:2021(E)
Meat and meat products — Determination of
chloramphenicol content — Reference method
1 Scope
This document specifies the liquid chromatographic (LC) method for the determination of
chloramphenicol content of muscle tissue of meat, including livestock and poultry.
This document specifies the liquid chromatography tandem mass spectrometry method (LC-MS/
MS) for the determination of chloramphenicol content of muscle tissue, casing, liver of meat and meat
products, including livestock and poultry.
This document specifies LC-MS/MS as the reference method.
The LC method is suitable for the determination of chloramphenicol content greater than 6,5 mg/kg.
LC-MS/MS is suitable for the determination of chloramphenicol content greater than 0,1 μg/kg.
Test samples which have deteriorated cannot be analysed with this method.
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.
ISO 3696, Water for analytical laboratory use — Specification and test methods
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:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
chloramphenicol content
mass fraction of chloramphenicol residue in meat and meat products
Note 1 to entry: The chloramphenicol content is expressed in micrograms per kilogram.
Note 2 to entry: The chloramphenicol content is determined according to the procedure specified in this
document.
4 Principle
4.1 Liquid chromatographic method
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
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ISO 13493:2021(E)

and toluene. The chloramphenicol is measured with reverse-phase chromatography by ultraviolet (UV)
detection.
4.2 Liquid chromatography tandem mass spectrometry method
The test portion is extracted with ethyl acetate, defatted with n-hexane and cleaned up with hydrophile-
lipophile balance (HLB) solid phase extraction. The chloramphenicol is determined and confirmed by
LC-MS/MS in multiple reaction monitoring (MRM) mode, operating in negative ionization.
5 Sampling
Sampling is not part of the method specified in this document. A recommended sampling method is
given in ISO 17604.
It is important that the laboratory receive a sample which is truly representative and has not been
damaged or changed during transport or storage.
Start from a representative sample of at least 200 g. Store the sample in such a way that deterioration
and change in composition are prevented.
6 Preparation of test sample
Allow the sample to reach room temperature. Remove excess of fat and inedible parts.
Homogenize the laboratory sample with the appropriate equipment (7.2.3). Take care that the
temperature of the sample material does not rise above 25 °C. If a mincer is used, pass the sample at
least twice through the equipment.
Fill a suitable airtight container with the prepared sample. Close the container and store in such a way
that deterioration and change in composition are prevented. Analyse the sample as soon as practicable,
but always within 24 h of homogenization.
7 Test method of liquid chromatography
7.1 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
7.1.1 Water, conforming to at least grade 3 in accordance with ISO 3696. The water shall be free of
organic compounds.
7.1.2 Nitrogen, suitable for evaporating solvents.
7.1.3 Dichloromethane.
7.1.4 Toluene.
7.1.5 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 a mass
fraction of 50 %dilute acetic acid (CH CO H) using the pH-meter (7.2.1). Transfer the solution to a
3 2
1 000 ml one-mark volumetric flask. Dilute to the mark with water and mix.
7.1.6 Acetonitrile, HPLC grade.
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ISO 13493:2021(E)

7.1.7 Mobile phase.
Add 750 ml of acetate buffer (7.1.5) to 250 ml of acetonitrile (7.1.6) and mix thoroughly.
Before use, filter the eluent through a 0,22 µm filter (7.2.2) and degas.
7.1.8 Chloramphenicol stock solution, 100 µg/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 one month when stored in the dark.
7.1.9 Chloramphenicol standard solutions.
Pipette 5,0 ml of the stock solution (7.1.8) 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 µg/ml, 0,10 µg/ml, 0,25 µg/ml
and 0,75 µg/ml, respectively.
These standard solutions are stable for one week when stored in the dark.
7.2 Apparatus
The usual laboratory apparatus and, in particular, the following shall be used.
7.2.1 pH-meter.
7.2.2 Membrane filter, of low dead volume and pore size 0,22 µm.
7.2.3 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)
7.2.4 Laboratory blender (e.g. Stomacher blender or vortex type).
7.2.5 Filter paper, quantitative, fast filtration rate, of diameter about 15 cm.
1)
NOTE For example, Whatman 41, 1441-047-GE Whatman Grade 41, proved to be suitable .
7.2.6 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 .
7.2.7 Water bath or heating block, capable of being maintained at (40 ± 1) °C, with equipment for
drying with nitrogen (7.1.2), or rotary vacuum evaporator.
7.2.8 Centrifuge tubes, of capacity 25 ml.
7.2.9 Vortex mixer, operating at a rotation frequency of about 700 r/min.
1) These are examples of suitable products available commercially. This information is given for the convenience
of
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