ISO 22283:2020

Traditional Chinese medicine — Determination of aflatoxins in natural products by LC-FLD

This document specifies the methods for the determination of aflatoxins (AFB1, AFB2, AFG1, AFG2) in natural products using LC-FLD. It is applicable to the analysis of aflatoxins in raw materials and manufactured products, including decoction pieces derived from plants and animals.

Médecine traditionnelle chinoise — Dosage des aflatoxines dans les produits naturels par CL-DF

General Information

• Status: Published
• Publication Date: 04-Aug-2020
• ICS: 11.120.10 - Medicaments
• Technical Committee: ISO/TC 249 - Traditional Chinese medicine
• Drafting Committee: ISO/TC 249/WG 2 - Quality and safety of manufactured TCM products
• Current Stage: 6060 - International Standard published
• Start Date: 05-Aug-2020
• Completion Date: 05-Aug-2020

Standards Content (sample)

INTERNATIONAL ISO
STANDARD 22283
First edition
2020-08
Traditional Chinese medicine —
Determination of aflatoxins in natural
products by LC-FLD
Médecine traditionnelle chinoise — Dosage des aflatoxines dans les
produits naturels par CL-DF
Reference number
ISO 22283:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 22283:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 22283:2020(E)
Contents Page

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

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

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

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

3 Terms and definition ........................................................................................................................................................................................ 1

4 Symbols and abbreviated terms ........................................................................................................................................................... 1

5 Reagents ........................................................................................................................................................................................................................ 2

6 Apparatus ..................................................................................................................................................................................................................... 2

6.1 LC-FLD ............................................................................................................................................................................................................ 2

6.2 Chromatographic column .............................................................................................................................................................. 2

6.3 Glass sample .............................................................................................................................................................................................. 2

6.4 Electronic balance ................................................................................................................................................................................ 2

6.5 Homogenizer ............................................................................................................................................................................................ 2

6.6 Centrifuge .................................................................................................................................................................................................... 2

6.7 Volumetric flask ..................................................................................................................................................................................... 2

7 Sample preparation ........................................................................................................................................................................................... 2

8 Test method ............................................................................................................................................................................................................... 3

8.1 Stock solution and working solution ................................................................................................................................... 3

8.2 LC-FLD conditions ................................................................................................................................................................................ 3

8.2.1 General...................................................................................................................................................................................... 3

8.2.2 LC-FLD conditions and system suitability ................................................................................................. 3

8.2.3 Post-column derivatization .................................................................................................................................... 4

8.2.4 Quantification of aflatoxins in the test sample using calibration curves......................... 4

8.3 Application of test method ........................................................................................................................................................... 4

9 Sampling and preservation ....................................................................................................................................................................... 5

9.1 Sampling ....................................................................................................................................................................................................... 5

9.2 Sample storage ........................................................................................................................................................................................ 5

Annex A (informative) LC-FLD method............................................................................................................................................................... 6

Annex B (informative) Chromatogram of AFG , AFG , AFB and AFB .............................................................................. 8

2 1 2 1

Annex C (informative) Reference of national, regional and organizational limits of

aflatoxins in natural products ................................................................................................................................................................ 9

Annex D (informative) Method validation ...................................................................................................................................................11

Bibliography .............................................................................................................................................................................................................................12

© ISO 2020 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 22283:2020(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 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 249, Traditional Chinese medicine.

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 2020 – All rights reserved
---------------------- Page: 4 ----------------------
ISO 22283:2020(E)
Introduction

Aflatoxins are naturally occurring mycotoxins produced by certain fungi, which can be found in a

variety of agriculture products, contaminated foods and natural medicines, including natural products,

decoction pieces and manufactured products. At least 14 different aflatoxins, mainly produced by

Aspergillus flavus and Aspergillus parasiticus, have been reported to be produced in nature. Among

these, aflatoxin B (AFB ) is considered the most toxic. Other important aflatoxins include aflatoxin B ,

1 1 2

M , M , G , G , Q Q and aflatoxicol. AFB , AFB , AFG and AFG are produced by Aspergillus flavus and

1 2 1 2 1, 2 1 2 1 2

Aspergillus parasiticus, while AFM and AFM are formed from AFB and AFB metabolism, respectively.

1 2 1 2

It has been well established that most aflatoxins are highly toxic and carcinogenic. Humans, in

particular young children, are less tolerant to aflatoxin toxicity. There are frequent reports of detection

of toxic aflatoxins in herbal medicines. Therefore, aflatoxins, in particular AFB and the total amount

of AFB , AFB , AFG and AFG , should be tested and limited as a quality and safety control measure

1 2 1 2

for natural products. There are two main methods to detect aflatoxins in natural products: the liquid

chromatography tandem mass spectrometry (LC-MS/MS) method and the liquid chromatography

coupled with fluorescence detector (LC-FLD) method. LC-FLD is preferentially chosen due to its high

sensitivity, high accuracy and reasonable operating cost (see Annex A, Table A.1).

© ISO 2020 – All rights reserved v
---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 22283:2020(E)
Traditional Chinese medicine — Determination of
aflatoxins in natural products by LC-FLD
1 Scope

This document specifies the methods for the determination of aflatoxins (AFB , AFB , AFG , AFG ) in

1 2 1 2
natural products using LC-FLD.

It is applicable to the analysis of aflatoxins in raw materials and manufactured products, including

decoction pieces derived from plants and animals.
2 Normative references
There are no normative references in this document.
3 Terms and definition
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
aflatoxin
mycotoxin produced mainly by Aspergillus flavus and Aspergillus parasiticus

Note 1 to entry: At least 13 different types of aflatoxin are produced in nature, and most of these are known to be

highly toxic and carcinogenic.

Note 2 to entry: Aflatoxin B and the sum of aflatoxins B , B , G and G shall be tested and limited.

1 1 2 1 2
4 Symbols and abbreviated terms
AFB aflatoxin B
1 1
AFB aflatoxin B
2 2
AFG aflatoxin G
1 1
AFG aflatoxin G
2 2
HPLC high-performance liquid chromatography
LC-FLD liquid chromatography coupled with fluorescence detector
LC-MS/MS liquid chromatography tandem mass spectrometry
© ISO 2020 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO 22283:2020(E)
5 Reagents

The purity of the reagents used shall be checked by running a blank determination. The chromatogram

obtained from the solvents shall have a baseline without noticeable peaks that would interfere with

targeted aflatoxins.

5.1 Water, of appropriate purity (the resistivity of water shall be at least 18,2 MΩ).

5.2 Methanol, CH OH, of HPLC grade.
5.3 Acetonitrile, CH CN, of HPLC grade.
5.4 Sodium chloride, NaCl, of AR (analytical) grade.
6 Apparatus
6.1 LC-FLD

The LC-FLD apparatus consists of a solvent pump system, a sample injector, a chromatographic column

(a column temperature controller may be used), a detector and a data acquisition system (or an

integrator or a chart recorder). The mobile phase is supplied from one or several reservoirs and flows

through the column and detector at a constant flow rate. The detector shall be a fluorescence detector.

6.2 Chromatographic column

A stainless-steel column sealed with octadecylsilyl silica gel for chromatography shall be used.

6.3 Glass sample

All glassware shall be thoroughly cleaned before use. The glassware used for aflatoxin analysis shall be

placed in a specific container filled with 0,5 % to 1,0 % sodium hypochlorite solution for more than 2 h

and then washed with an adequate amount of fresh running water. Finally, all glassware shall be rinsed

with distilled water and dried before use.
6.4 Electronic balance
The electronic balance shall be accurate to a minimum of 0,01 mg.
6.5 Homogenizer
The homogenizer shall have a rotation speed of up to 15 000 r/min.
6.6 Centrifuge
The centrifuge shall have a rotation speed of up to 5 000 r/min.
6.7 Volumetric flask
Volumetric flasks with a capacity of 2,0 ml and 50,0 ml shall be used.
7 Sample preparation

1) All natural products shall be crushed into powders and screened through a 24-mesh sieve.

2 © ISO 2020 – All rights reserved
---------------------- Page: 7 ----------------------
ISO 22283:2020(E)

2) A mixture of 15,0 g powders and 3,0 g sodium chloride shall be added into a 75,0 ml mixed solution

of methanol and water at 70:30 volume fraction.

3) The mixture shall be homogenized at a speed of higher than 11 000 rpm for 2 min and centrifuged

at 2 500 rpm for 5 min.

4) 15 ml of supernatant shall be added to a 50,0 ml volumetric flask and diluted with water, then

shaken and filtered through a 0,45 μm filter paper.

5) About 20,0 ml of the filtrate shall be passed through the immunoaffinity column at a flow rate of

3 ml/min. The column shall be washed with 20,0 ml of water and the eluent shall be abandoned

until the air has passed through the column to extrude the water.

6) The column shall be eluted with methanol and the eluent shall be collected and concentrated to

0,5 ml by nitrogen. The concentrated eluent shall be diluted with 0,5 ml of the mixed solution of

methanol and water at 50:50 volume fraction in the HPLC vial before use.
8 Test method
8.1 Stock solution and working solution

Stock solution shall be prepared by mixing a solution of aflatoxin standards (1,0 μg/ml, 0,3 μg/ml,

1,0 μg/ml and 0,3 μg/ml of AFB , AFB , AFG and AFG , respectively). A series of working solutions shall

1 2 1 2

be prepared by diluting the stock solution to 0,10 ng/ml to 100,00 ng/ml (AFB and AFG ) and 0,03 ng/

1 1

ml to 30,00 ng/ml (AFB and AFG ), respectively, with mobile phase of methanol and acetonitrile.

2 2
8.2 LC-FLD conditions
8.2.1 General

The LC-FLD method based on two different methods of derivatization, pre- and post-column

derivatization, shall be used for the simultaneous determination of aflatoxins. Commonly, post-

column derivatization methods, such as photochemical derivatization, iodine derivatization and

electrochemically generated bromine derivatization, have been applied in many countries, regions and

organizations including Europe, China, the United States, Japan and South Korea. The LC-FLD method

based on i
...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 22283
ISO/TC 249
Traditional Chinese medicine —
Secretariat: SAC
Determination of aflatoxins in natural
Voting begins on:
2020­04­29 products by LC-FLD
Voting terminates on:
2020­06­24
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/FDIS 22283:2020(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN­
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. ISO 2020
---------------------- Page: 1 ----------------------
ISO/FDIS 22283:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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
CH­1214 Vernier, Geneva
Phone: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/FDIS 22283:2020(E)
Contents Page

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

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

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

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

3 Terms and definition ........................................................................................................................................................................................ 1

4 Symbols and abbreviated terms ........................................................................................................................................................... 1

5 Reagents ........................................................................................................................................................................................................................ 2

6 Apparatus ..................................................................................................................................................................................................................... 2

6.1 LC­FLD ............................................................................................................................................................................................................ 2

6.2 Chromatographic column .............................................................................................................................................................. 2

6.3 Glass sample .............................................................................................................................................................................................. 2

6.4 Electronic balance ................................................................................................................................................................................ 2

6.5 Homogenizer ............................................................................................................................................................................................ 2

6.6 Centrifuge .................................................................................................................................................................................................... 2

6.7 Volumetric flask ..................................................................................................................................................................................... 2

7 Sample preparation ........................................................................................................................................................................................... 2

8 Test method ............................................................................................................................................................................................................... 3

8.1 Stock solution and working solution ................................................................................................................................... 3

8.2 LC­FLD conditions ................................................................................................................................................................................ 3

8.2.1 General...................................................................................................................................................................................... 3

8.2.2 LC-FLD conditions and system suitability ................................................................................................. 3

8.2.3 Post­column derivatization .................................................................................................................................... 4

8.2.4 Quantification of aflatoxins in the test sample using calibration curves......................... 4

8.3 Application of test method ........................................................................................................................................................... 4

9 Sampling and preservation ....................................................................................................................................................................... 5

9.1 Sampling ....................................................................................................................................................................................................... 5

9.2 Sample storage ........................................................................................................................................................................................ 5

Annex A (informative) LC-FLD method............................................................................................................................................................... 6

Annex B (informative) Chromatogram of AFG , AFG , AFB and AFB .............................................................................. 8

2 1 2 1

Annex C (informative) Reference of national, regional and organizational limits of

aflatoxins in natural products ................................................................................................................................................................ 9

Annex D (informative) Method validation ...................................................................................................................................................11

Bibliography .............................................................................................................................................................................................................................12

© ISO 2020 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO/FDIS 22283:2020(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 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 249, Traditional Chinese medicine.

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 2020 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/FDIS 22283:2020(E)
Introduction

Aflatoxins are naturally occurring mycotoxins produced by certain fungi, which can be found in a

variety of agriculture products, contaminated foods and natural medicines, including natural products,

decoction pieces and manufactured products. At least 14 different aflatoxins, mainly produced by

Aspergillus flavus and Aspergillus parasiticus, have been reported to be produced in nature. Among

these, aflatoxin B (AFB ) is considered the most toxic. Other important aflatoxins include aflatoxin B ,

1 1 2

M , M , G , G , Q Q and aflatoxicol. AFB , AFB , AFG and AFG are produced by Aspergillus flavus and

1 2 1 2 1, 2 1 2 1 2

Aspergillus parasiticus, while AFM and AFM are formed from AFB and AFB metabolism, respectively.

1 2 1 2

It has been well established that most aflatoxins are highly toxic and carcinogenic. Humans, in

particular young children, are less tolerant of aflatoxin toxicity. There are frequent reports of detection

of toxic aflatoxins in herbal medicines. Therefore, AFB and the total amount of AFB , AFB , AFG and

1 1 2 1

AFG should be tested and limited as a quality and safety control measure for natural products. There

are two main methods to detect aflatoxins in natural products: the liquid chromatography tandem

mass spectrometry (LC-MS/MS) method and the liquid chromatography coupled with fluorescence

detector (LC-FLD) method. LC-FLD is preferentially chosen due to its high sensitivity, high accuracy and

reasonable operating cost (see Annex A, Table A.1).
© ISO 2020 – All rights reserved v
---------------------- Page: 5 ----------------------
FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 22283:2020(E)
Traditional Chinese medicine — Determination of
aflatoxins in natural products by LC-FLD
1 Scope

This document specifies the determination of aflatoxins (AFB , AFB , AFG , AFG ) in natural products

1 2 1 2
using LC­FLD.

It is applicable to the analysis of aflatoxins in raw materials and manufactured products, including

decoction pieces derived from plants and animals.
2 Normative references
There are no normative references in this document.
3 Terms and definition
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
aflatoxin
mycotoxin produced mainly by Aspergillus flavus and Aspergillus parasiticus

Note 1 to entry: At least 13 different types of aflatoxin are produced in nature, and most of these are known to be

highly toxic and carcinogenic.

Note 2 to entry: Aflatoxin B and the sum of aflatoxins B , B , G and G shall be tested and limited.

1 1 2 1 2
4 Symbols and abbreviated terms
AFB aflatoxin B
1 1
AFB aflatoxin B
2 2
AFG aflatoxin G
1 1
AFG aflatoxin G
2 2
HPLC high-performance liquid chromatography
LC­FLD liquid chromatography coupled with fluorescence detector
LC­MS/MS liquid chromatography tandem mass spectrometry
© ISO 2020 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO/FDIS 22283:2020(E)
5 Reagents

The purity of the reagents used shall be checked by running a blank determination. The chromatogram

obtained from the solvents shall have a baseline without noticeable peaks that would interfere with

targeted aflatoxins.

5.1 Water, of appropriate purity (the resistivity of water shall be at least 18,2 MΩ).

5.2 Methanol, CH OH, of HPLC grade.
5.3 Acetonitrile, CH CN, of HPLC grade.
5.4 Sodium chloride, NaCl, of AR (analytical) grade.
6 Apparatus
6.1 LC-FLD

The LC-FLD apparatus consists of a solvent pump system, a sample injector, a chromatographic column

(a column temperature controller may be used), a detector and a data acquisition system (or an

integrator or a chart recorder). The mobile phase is supplied from one or several reservoirs and flows

through the column and detector at a constant flow rate. The detector shall be a fluorescence detector.

6.2 Chromatographic column

A stainless-steel column sealed with octadecylsilyl silica gel for chromatography shall be used.

6.3 Glass sample

All glassware shall be thoroughly cleaned before use. The glassware used for aflatoxin analysis shall be

placed in a specific container filled with 0,5 % to 1,0 % sodium hypochlorite solution for more than 2 h

and then washed with an adequate amount of fresh running water. Finally, all glassware shall be rinsed

with distilled water and dried before use.
6.4 Electronic balance
The electronic balance shall be accurate to a minimum of 0,01 mg.
6.5 Homogenizer
The homogenizer shall have a rotation speed of up to 15,000 r/min.
6.6 Centrifuge
The centrifuge shall have a rotation speed of up to 5,000 r/min.
6.7 Volumetric flask
Volumetric flasks with a capacity of 2,0 ml and 50,0 ml shall be used.
7 Sample preparation

1) All natural products shall be crushed into powders and screened through a [24] mesh sieve.

2 © ISO 2020 – All rights reserved
---------------------- Page: 7 ----------------------
ISO/FDIS 22283:2020(E)

2) A mixture of [15,0 g] powders and [3,0 g] sodium chloride shall be added into a [75,0 ml] mixed

solution of methanol and water at [70:30 volume fraction].

3) All the mixture shall be homogenized at a speed of higher than [11,000 rpm] for [2 min] and

centrifuged at [2,500 rpm] for [5 min].

4) [15 ml] of supernatant shall be moved to a [50,0 ml] volumetric flask and diluted with water, then

shaken and filtered through a 0,45 μm filter paper.

5) About [20,0 ml] of the filtrate shall be passed through the immunoaffinity column at a flow rate of

3 ml/min. The column shall be washed with [20,0 ml] of water and the eluent shall be abandoned

until the air has passed through the column to extrude the water.

6) The column shall be eluted with methanol and the eluent shall be collected and concentrated to

0,5 ml by nitrogen. The concentrated eluent shall be diluted with 0,5 ml of the mixed solution of

methanol and water at [50:50 volume fraction] in the HPLC vial before use.
8 Test method
8.1 Stock solution and working solution

Stock solution shall be prepared by mixing a solution of aflatoxin standards (1,0 μg/ml, 0,3 μg/ml,

1,0 μg/ml and 0,3 μg/ml of AFB , AFB , AFG and AFG , respectively). A series of working solutions shall

1 2 1 2

be prepared by diluting the stock solution to 0,10 ng/ml to 100,00 ng/ml (AFB and AFG ) and 0,03 ng/

1 1

ml to 30,00 ng/ml (AFB and AFG ), respectively, with mobile phase of methanol and acetonitrile.

2 2
8.2 LC-FLD conditions
8.2.1 General

The LC­FLD method based on two different methods of derivatization, pre­ and post­column

derivatization, shall be used for the simultaneous determination of aflatoxins. Commonly, post-

column derivatization methods, such as photochemical derivatization, iodine derivatization and

electrochemical
...