ISO/DIS 13493

Meat and meat products -- Determination of chloramphenicol content -- Reference method

Viande et produits à base de viande -- Dosage du chloramphénicol -- Méthode de référence

Standards Content (sample)

DRAFT INTERNATIONAL STANDARD
ISO/DIS 13493
ISO/TC 34/SC 6 Secretariat: SAC
Voting begins on: Voting terminates on:
2020-11-25 2021-02-17
Meat and meat products — Determination of
chloramphenicol content — Reference method

Viande et produits à base de viande — Dosage du chloramphénicol — Méthode de référence

ICS: 67.120.10
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ISO/DIS 13493:2020(E)
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ISO/DIS 13493:2020(E)
Contents Page

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

Introduction ..................................................................................................................................................................................................................................v

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

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

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

4.1 Liquid chromatographic method............................................................................................................................................. 2

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 ............................................................................................................................................. 5

7.3.6 Chromatographic analysis ....................................................................................................................................... 5

7.4 Calculation .................................................................................................................................................................................................. 6

7.5 Precision ....................................................................................................................................................................................................... 6

7.5.1 Inter laboratory test ...................................................................................................................................................... 6

7.5.2 Repeatability ....................................................................................................................................................................... 6

7.5.3 Reproducibility .................................................................................................................................................................. 6

8 Test method of liquid chromatography tandem mass spectrometry method

(reference method)............................................................................................................................................................................................ 7

8.1 Reagents and materials ................................................................................................................................................................... 7

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 ........................................................................................................................................... ....... 9

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 Inter laboratory test ...................................................................................................................................................11

8.5.2 Repeatability ....................................................................................................................................................................11

8.5.3 Reproducibility ...............................................................................................................................................................12

9 Test report ................................................................................................................................................................................................................12

Annex A (informative) LC-MS/MS chromatogram of Chloramphenicol standard solution .......................13

Bibliography .............................................................................................................................................................................................................................14

© ISO 2020 – All rights reserved iii
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ISO/DIS 13493:2020(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 on 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 the following URL: www .iso .org/ iso/ foreword .htmL.

The committee responsible for this document is 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 to the previous edition are as follows:

— A new test method-liquid chromatography tandem mass spectrometry method(LC-MS/MS) method

is added, and the provision that the test method of liquid chromatography asreference method is

cancelled, accordingly, the test method of liquid chromatography tandem mass spectrometry

method(LC-MS/MS) is stipulated as reference method (namely referee method).

— The determination scope is expanded to muscle, casing and liver of meat and meat products including

livestock, poultry and sea food.
— The clauses order of the document has been rearranged.

— The introductory texts for “Foreword”, “Normative references” and “Terms and definitions” have

been modified in accordance with the requirements of ISO/IEC Directives, Part 2.
— The title of the document has been modified.
— The scope of the document has been modified.
— The documents list of “Normative references” has been updated.

— In the “Chromatographic conditions” of the test method of liquid chromatography, “detector range”,

“Recorder ranger” and “paper speed” have been deleted.

— The clauses of “Test method of liquid chromatography tandem mass spectrometry method (reference

method)” have been added.
— The documents list of “Bibliography” has been updated.
iv © ISO 2020 – All rights reserved
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ISO/DIS 13493:2020(E)
Introduction
This document does not involve the use of patents.
© ISO 2020 – All rights reserved v
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DRAFT INTERNATIONAL STANDARD ISO/DIS 13493:2020(E)
Meat and meat products — Determination of
chloramphenicol content — Reference method
1 Scope

This document specifies liquid chromatographic (LC) method for the determination of Chloramphenicol

content of muscle tissue of meat, including livestock and poultry.

This document specifies 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 liquid chromatography tandem mass spectrometry method as the

reference method.

Liquid chromatographic (LC) method is suitable for the determination of Chloramphenicol contents

great than 6.5 mg/kg.

Liquid chromatography tandem mass spectrometry method (LC-MS/MS) is suitable for the

determination of chloramphenicol contents great than 0.1 μg/kg.
Test samples which have deteriorated cannot be analyzed 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
ISO 648, Laboratory glassware — Single-volume pipettes
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 of meat and meat products

Mass fraction of chloramphenicol residue determined according to the procedure specified in this

International Standard.

Note 1 to entry: The chloramphenicol content is expressed in micrograms per kilogram.

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ISO/DIS 13493:2020(E)
4 Principle
4.1 Liquid chromatographic method

A test portion is extracted with water. Filltration and solid-phase extraction are used to isolate the

lipophillic components from the aquesous 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.
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 International Standard. A recommended sampling

[1]
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 after homogenization.
7 Test method of liquid chromatography
7.1 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.

7.1.1 Water, complying with 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.
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ISO/DIS 13493:2020(E)
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

50%(m/m) dilute acetic acid (CH CO H) using the pH-meter (7.2.1). Transfer the solution to a 1000 mL

3 2
one-mark volumetric flask. Dilute to the mark with water and mix.
7.1.6 Acetonitrile, HPLC grade.
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 0,22µm filter (7.2.2) and degas.
7.1.8 Chloramphenicol stock solution, 100 µg/mL.

Weigh, to the nearest 0.1mg, 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.
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 1 week when stored in the dark.
7.2 Apparatus
The usual laboratory apparatus and, in particular
...