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

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 6,5Â mg/kg. LC-MS/MS is suitable for the determination of chloramphenicol content greater than 0,1Â ÎĽg/kg. Test samples which have deteriorated cannot be analysed with this method.

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

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Status
Published
Publication Date
26-Aug-2021
Current Stage
5060 - Close of voting Proof returned by Secretariat
Start Date
30-Jul-2021
Completion Date
30-Jul-2021
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INTERNATIONAL ISO
STANDARD 13493
Second edition
2021-08
Meat and meat products —
Determination of chloramphenicol
content — Reference method
Viande et produits à base de viande — Dosage du chloramphénicol —
Méthode de référence
Reference number
ISO 13493:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO 13493:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
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 Calculation ...............................................................................................................................................................................................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 and LC-

MS/MS chromatogram of chloramphenicol standard solution .........................................................................13

Bibliography .............................................................................................................................................................................................................................16

© ISO 2021 – All rights reserved iii
---------------------- Page: 3 ----------------------
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.
iv © ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
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

© ISO 2021 – All rights reserved 1
---------------------- Page: 5 ----------------------
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.
2 © ISO 2021 – All rights reserved
---------------------- Page: 6 ----------------------
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.
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.

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.

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

...

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
---------------------- Page: 1 ----------------------
ISO 13493:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
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

© ISO 2021 – All rights reserved PROOF/ÉPREUVE iii
---------------------- Page: 3 ----------------------
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.
iv PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
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

© ISO 2021 – All rights reserved PROOF/ÉPREUVE 1
---------------------- Page: 5 ----------------------
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.
2 PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 6 ----------------------
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.
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.

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.

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

...

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