ISO/DIS 13493
(Main)Meat and meat products -- Determination of chloramphenicol content -- Reference method
Meat and meat products -- Determination of chloramphenicol content -- Reference method
Viande et produits à base de viande -- Dosage du chloramphénicol -- Méthode de référence
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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: 67.120.10THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
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ISO/DIS 13493:2020(E)
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PROVIDE SUPPORTING DOCUMENTATION. ISO 2020
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ISO/DIS 13493:2020(E)
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© ISO 2020
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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
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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.
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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 methodsISO 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 SamplingSampling 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 2one-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
...
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