ISO 23719:2025
(Main)Cereals and cereal products — Determination of 17 mycotoxins by ultra-high-performance liquid chromatography and tandem mass spectrometry method (UHPLC-MS/MS)
Cereals and cereal products — Determination of 17 mycotoxins by ultra-high-performance liquid chromatography and tandem mass spectrometry method (UHPLC-MS/MS)
This document specifies a method for the quantitative determination of 17 mycotoxins in cereals and cereal products (e.g. wheat, maize, husked rice, rice and their products) using ultra-high-performance liquid chromatography and tandem mass spectrometry method (UHPLC-MS/MS). The 17 mycotoxins are aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, deoxynivalenol, nivalenol, deoxynivalenol-3-glucoside, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, zearalenone, ochratoxin A, fumonisin B1, fumonisin B2, fumonisin B3, T-2 toxin, HT-2 toxin and sterigmatocystin. This document does not apply to foods for infants and young children. This document is applicable to other products (e.g. nuts) provided that the method is validated for each individual case. The calibration range of the method and the validated range during the interlaboratory study for each mycotoxin are listed in Table A.1.
Céréales et produits céréaliers — Détermination de 17 mycotoxines par chromatographie liquide à ultra haute performance et spectrométrie de masse en tandem (CLUHP-SM/SM)
General Information
Buy Standard
Standards Content (Sample)
International
Standard
ISO 23719
First edition
Cereals and cereal products —
2025-07
Determination of 17 mycotoxins
by ultra-high-performance liquid
chromatography and tandem
mass spectrometry method
(UHPLC-MS/MS)
Céréales et produits céréaliers — Détermination de 17 mycotoxines
par chromatographie liquide à ultra haute performance et
spectrométrie de masse en tandem (CLUHP-SM/SM)
Reference number
© ISO 2025
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
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 2
6 Apparatus and equipment . 4
7 Procedure . 5
7.1 Sampling and preparation of the laboratory sample . .5
7.2 Test portion weighing .5
7.3 Extraction .5
7.4 Preparation of the sample test solution .6
7.5 Preparation of control samples .6
7.6 Analysis .6
7.6.1 Operating conditions .6
7.6.2 Injection sequence .6
7.6.3 Data treatment .7
8 Identification . 7
9 Calculations . 7
10 Precision . 8
10.1 General .8
10.2 Repeatability .8
10.3 Reproducibility . .8
11 Test report . 8
Annex A (informative) Calibration range of the method and the validated range during the
interlaboratory study for each mycotoxin . 10
Annex B (informative) Information on 17 mycotoxins and ISTD standards .11
Annex C (informative) Example conditions for suitable LC-MS/MS systems .13
Annex D (informative) Example chromatogram of mycotoxins and ISTDs .20
Annex E (informative) Results of the interlaboratory test .22
Bibliography .40
iii
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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 4, Cereals
and pulses.
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
International Standard ISO 23719:2025(en)
Cereals and cereal products — Determination of
17 mycotoxins by ultra-high-performance liquid
chromatography and tandem mass spectrometry method
(UHPLC-MS/MS)
1 Scope
This document specifies a method for the quantitative determination of 17 mycotoxins in cereals and cereal
products (e.g. wheat, maize, husked rice, rice and their products) using ultra-high-performance liquid
chromatography and tandem mass spectrometry method (UHPLC-MS/MS).
The 17 mycotoxins are aflatoxin B , aflatoxin B , aflatoxin G , aflatoxin G , deoxynivalenol, nivalenol,
1 2 1 2
deoxynivalenol-3-glucoside, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, zearalenone, ochratoxin A,
fumonisin B , fumonisin B , fumonisin B , T-2 toxin, HT-2 toxin and sterigmatocystin.
1 2 3
This document does not apply to foods for infants and young children.
This document is applicable to other products (e.g. nuts) provided that the method is validated for each
individual case.
The calibration range of the method and the validated range during the interlaboratory study for each
mycotoxin are listed in Table A.1.
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
No terms and definitions are listed in this document.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 Principle
Mycotoxins are extracted using an acetonitrile-water-acetic acid (70:29:1, v/v/v) solution. After
centrifugation, the extract is diluted with water, centrifuged in low temperature and filtered. The final
solution is mixed with isotope-labelled internal standard solution and subjected to UHPLC-MS/MS analysis.
Quantification is performed by the isotopic dilution approach using C isotopically labelled mycotoxins as
internal standards (ISTDs).
WARNING — Mycotoxins are generally considered to be carcinogenic, neurotoxic and
[1]
immunosuppressive. Observe appropriate safety precautions for handling such compounds
and avoid handling in dry form as the electrostatic nature can result in dispersion and inhalation.
Glassware can be decontaminated with 4 % sodium hypochlorite solution. Attention is drawn to the
[2][3]
statement made by the International Agency for Research on Cancer (WHO).
5 Reagents
WARNING — This document requires handling of hazardous substances. Technical, organizational
and personal safety measures shall be followed.
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only
distilled water or water of grade 1 in accordance with ISO 3696. Solvents shall be of quality for LC analysis.
5.1 Acetonitrile, LC grade or equivalent.
5.2 Methanol, LC grade or equivalent.
5.3 Ammonium acetate, American Chemical Society (ACS) grade, > 99 %.
5.4 Acetic acid, ACS grade.
5.5 Formic acid, ACS grade.
5.6 Extraction solvent: Mix 70 volume parts of acetonitrile (5.1) and 29 volume parts of water and 1
volume part of acetic acid (5.4).
5.7 Solvent of calibration solutions: Mix 50 volume parts of extraction solvent (5.6) and 50 volume parts
of water.
5.8 Individual stock solutions, either prepared by dissolving neat (solid) certified standards in an
appropriate solvent or from individual stock solutions purchased as such.
The mycotoxins covered in this document dissolve well in acetonitrile, except for fumonisins which
are soluble in a mixture of acetonitrile and water (50:50, v/v). The information and concentration of 17
mycotoxins and the isotope-labelled internal standards is shown in Table B.1.
5.9 Mixed stock solution.
Prepare a mixed stock solution containing all individual mycotoxins at the concentration given in Table 1,
using the appropriate pipettes (6.1) and dilute with deionized water. This solution can be used for six months
if stored in the dark at −20 °C.
This mixed stock solution may be used for the preparation of positive control samples (see 7.5).
Table 1 — Concentration of mixed stock solution for 17 mycotoxins
Compounds Concentration Compounds Concentration
...
FINAL DRAFT
International
Standard
ISO/FDIS 23719
ISO/TC 34/SC 4
Cereals and cereal products —
Secretariat: SAC
Determination of 17 mycotoxins
Voting begins on:
by ultra-high-performance liquid
2025-04-04
chromatography and tandem mass
Voting terminates on:
spectrometry method (UHPLC-MS/
2025-05-30
MS)
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
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO
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.
Reference number
ISO/FDIS 23719:2025(en) © ISO 2025
FINAL DRAFT
ISO/FDIS 23719:2025(en)
International
Standard
ISO/FDIS 23719
ISO/TC 34/SC 4
Cereals and cereal products —
Secretariat: SAC
Determination of 17 mycotoxins
Voting begins on:
by ultra-high-performance liquid
chromatography and tandem
Voting terminates on:
mass spectrometry method
(UHPLC-MS/MS)
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.
© ISO 2025
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
or ISO’s member body in the country of the requester.
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
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 Reference number
ISO/FDIS 23719:2025(en) © ISO 2025
ii
ISO/FDIS 23719:2025(en)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 2
6 Apparatus and equipment . 4
7 Procedure . 5
7.1 Sampling and preparation of the laboratory sample . .5
7.2 Test portion weighing .5
7.3 Extraction .5
7.4 Preparation of the sample test solution .6
7.5 Preparation of control samples .6
7.6 Analysis .6
7.6.1 Operating conditions .6
7.6.2 Injection sequence .6
7.6.3 Data treatment .7
8 Identification . 7
9 Calculations . 7
10 Precision . 8
10.1 General .8
10.2 Repeatability .8
10.3 Reproducibility . .8
11 Test report . 8
Annex A (informative) Calibration range of the method and the validated range during the
interlaboratory study for each mycotoxin . 9
Annex B (informative) Information on 17 mycotoxins and ISTD standards .10
Annex C (informative) Example conditions for suitable LC-MS/MS systems .12
Annex D (informative) Example chromatogram of mycotoxins and ISTDs . 19
Annex E (informative) Results of the interlaboratory test .21
Bibliography .39
iii
ISO/FDIS 23719:2025(en)
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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.
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 4, Cereals and pulses.
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
FINAL DRAFT International Standard ISO/FDIS 23719:2025(en)
Cereals and cereal products — Determination of
17 mycotoxins by ultra-high-performance liquid
chromatography and tandem mass spectrometry method
(UHPLC-MS/MS)
1 Scope
This document specifies a method for the quantitative determination of 17 mycotoxins in cereals and cereal
products (e.g. wheat, maize, husked rice, rice and their products) using ultra-high-performance liquid
chromatography and tandem mass spectrometry method (UHPLC-MS/MS).
The 17 mycotoxins are aflatoxin B , aflatoxin B , aflatoxin G , aflatoxin G , deoxynivalenol, nivalenol,
1 2 1 2
deoxynivalenol-3-Glucoside, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, zearalenone, ochratoxin A,
fumonisin B , fumonisin B , fumonisin B , T-2 toxin, HT-2 toxin and sterigmatocystin.
1 2 3
This document does not apply to foods for infants and young children.
This document is applicable to other products (e.g. nuts) provided that the method is validated for each
individual case.
The calibration range of the method and the validated range during the interlaboratory study for each
mycotoxin are listed in Table A.1.
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
No terms and definitions are listed in this document.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 Principle
Mycotoxins are extracted using an acetonitrile-water-acetic acid (70:29:1, v/v/v) solution. After
centrifugation, the extract is diluted with water, centrifuged in low temperature and filtered. The final
solution is mixed with isotope-labelled internal standard solution and subjected to UHPLC-MS/MS analysis.
Quantification is performed by the isotopic dilution approach using C isotopically labelled mycotoxins as
internal standards (ISTDs).
WARNING — Mycotoxins are generally considered to be carcinogenic, neurotoxic and
[1]
immunosuppressive. Observe appropriate safety precautions for handling such compounds
and avoid handling in dry form as the electrostatic nature can result in dispersion and inhalation.
ISO/FDIS 23719:2025(en)
Glassware can be decontaminated with 4 % sodium hypochlorite solution. Attention is drawn to the
[2][3]
statement made by the International Agency for Research on Cancer (WHO).
5 Reagents
WARNING — This document requires handling of hazardous substances. Technical, organizational
and personal safety measures shall be followed.
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only
distilled water or water of grade 1 in accordance with ISO 3696. Solvents shall be of quality for LC analysis.
5.1 Acetonitrile, LC grade or equivalent.
5.2 Methanol, LC grade or equivalent.
5.3 Ammonium acetate, American Chemical Society (ACS) grade, > 99
...
ISO/DIS FDIS 23719:2025(en)
ISO/TC 34/SC 4/WG 8
Secretariat: SAC
Date: 2025-03-1018
Cereals and cereal products — Determination of 17 mycotoxins by
ultra-high-performance liquid chromatography and tandem mass
spectrometry method (UHPLC-MS/MS)
FDIS stage
ISO/FDIS 23719:2025(en)
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
E-mail: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO/FDIS 23719:2025(en)
Contents
Foreword . iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 1
5 Reagents . 2
6 Apparatus and equipment . 4
7 Procedure . 5
7.1 Sampling and preparation of the laboratory sample . 5
7.2 Test portion weighing . 5
7.3 Extraction . 6
7.4 Preparation of the sample test solution . 6
7.5 Preparation of control samples . 6
7.6 Analysis . 6
8 Identification . 7
9 Calculations . 7
10 Precision . 8
10.1 General . 8
10.2 Repeatability . 8
10.3 Reproducibility . 8
11 Test report . 8
Annex A (informative) Calibration range of the method and the validated range during the
interlaboratory study for each mycotoxin . 10
Annex B (informative) Information on 17 mycotoxins and ISTD standards . 11
Annex C (informative) Example conditions for suitable LC-MS/MS systems . 13
Annex D (informative) Example chromatogram of mycotoxins and ISTDs . 20
Annex E (informative) Results of the interlaboratory test . 24
Bibliography . 42
iii
ISO/FDIS 23719:2025(en)
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights
in respect thereof. As of the date of publication of this document, ISO had not received notice of (a) patent(s)
which may be required to implement this document. However, implementers are cautioned that this may not
represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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.
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 4, Cereals and
pulses.
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/FDIS 23719:2025(en)
Cereals and cereal products — Determination of 17 mycotoxins by
ultra-high-performance liquid chromatography and tandem mass
spectrometry method (UHPLC-MS/MS)
1 Scope
This document specifies a method for the quantitative determination of 17 mycotoxins in cereals and cereal
products (e.g. wheat, maize, husked rice, rice and their products) using ultra-high-performance liquid
chromatography and tandem mass spectrometry method (UHPLC-MS/MS).
The 17 mycotoxins are aflatoxin B , aflatoxin B , aflatoxin G , aflatoxin G , deoxynivalenol, nivalenol,
1 2 1 2
deoxynivalenol-3-Glucoside, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, zearalenone, ochratoxin A,
fumonisin B , fumonisin B , fumonisin B , T-2 toxin, HT-2 toxin and sterigmatocystin.
1 2 3
This document does not apply to foods for infants and young children.
This document is applicable to other products (e.g. nuts) provided that the method is validated for each
individual case.
The calibration range of the method and the validated range during the interlaboratory study for each
mycotoxin are listed in Table A.1.
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
No terms and definitions are listed in this document.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
4 Principle
Mycotoxins are extracted using an acetonitrile-water-acetic acid (70:29:1, v/v/v) solution. After
centrifugation, the extract is diluted with water, centrifuged in low temperature and filtered. The final solution
is mixed with isotope-labelled internal standard solution and subjected to UHPLC-MS/MS analysis.
Quantification is performed by the isotopic dilution approach using C isotopically labelled mycotoxins as
internal standards (ISTDs).
WARNING — Mycotoxins are generally considered to be carcinogenic, neurotoxic and
[1]
immunosuppressive. Observe appropriate safety precautions for handling such compounds and
avoid handling in dry form as the electrostatic nature can result in dispersion and inhalation.
Glassware can be decontaminated with 4 % sodium hypochlorite solution. Attention is drawn to the
[2][3]
statement made by the International Agency for Research on Cancer (WHO).
ISO/FDIS 23719:2025(en)
5 Reagents
WARNING — This document requires handling of hazardous substances. Technical, organizational and
personal safety measures shall be followed.
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only
distilled water or water of grade 1 in accordance with ISO 3696. Solvents shall be of quality for LC analysis.
5.1 Acetonitrile, LC grade or equivalent.
5.2 Methanol, LC grade or equivalent.
5.3 Ammonium acetate, American Chemical Society (ACS) grade, > 99 %.
5.4 Acetic acid, ACS grade.
5.5 Formic acid, ACS grade.
5.6 Extraction solvent: Mix 70 volume parts of acetonitrile (5.1) and 29 volume parts of water and 1
volume part of acetic acid (5.4).
5.7 Solvent of calibration solutions: Mix 50 volume parts of extraction solvent (5.6) and 50 volume
parts of water.
5.8 Individual stock solutions, either prepared by dissolving neat (solid) certified standards in an
appropriate solvent or from individual stock solutions purchased as such.
The mycotoxins covered in this document dissolve well in acetonitrile, except for fumonisins which are soluble
in a mixture of acetonitrile and water (50:50, v/v). The information and concentration of 17 mycotoxins and
the isotope-labelled internal standards is shown in Table B.1.
5.9 Mixed stock solution.
Prepare a mixed stock solution containing all individual mycotoxins at the concentration given in Table 1,
using the appropriate pipettes (6.1) and dilute with deioniseddeionized water. This solution can be used for
six months if stored in the dark at -−20 °C.
This mixed stock solution may be used for the preparation of positive control samples (see 7.5).
Table 1 — Concentration of stock solution for 17 mixed mycotoxins
Compounds Concentration Compounds Concentration
μg/ml μg/ml
NIV 20 FB 12,5
DON 15 FB 7,5
DON-3G 2,5 FB3 9,0
3-AcDON 4 T-2 0,2
15-AcDON 2 HT-2 1
AFB 0,1 ZEN 2
AFB 0,1 OTA 0,2
AFG1 0,1 ST 0,1
AFG2 0,1 - -
ISO/FDIS 23719:2025(en)
5.10 Mixed isotope-labelled internal standard (ISTD) solution.
Isotopically labelled mycotoxins are generally available as certified standard solutions. Prepare a mixed ISTD
solution in a mixture of acetonitrile and water (30:70, v/v), containing all isotopically labelled mycotoxins at
the concentration given in Table 2. This solution can be used for six months if stored in the dark at -−20 °C.
Table 2 — Concentration of isotope-labelled internal standard stock solution for 17 mixed
mycotoxins
Compounds Concentration Compounds Concentration
µg/ml µg/ml
13 13
[ C15]-NIV 2,5 [ C34]-FB1 0,5
13 13
[ C ]-DON 2 [ C ]-FB 0,3
15 34 2
13 13
[ C ]-DON-3G 1 [ C ]-FB 0,3
21 34 3
13 13
[ C17]-3-AcDON 1 [ C24]-T-2 0,05
13 13
[ C17]-15-AcDON 1 [ C22]-HT-2 0,125
13 13
[ C17]-AFB1 0,01 [ C18]-ZEN 0,2
13 13
[ C ]-AFB 0,01 [ C ]-OTA 0,04
17 2 20
13 13
[ C ]-AFG 0,01 [ C ]-ST 0,025
17 1 18
[ C17]-AFG2 0,01 - -
5.11 Calibration solutions.
Transfer appropriate volumes of the mixed stock solutions (5.9) into six separate 1,5 ml injection vials as
described in Table 3. The concentrations for the calibration solutions are shown in Table 4. Store them at –
20 °C for no longer than two weeks before use.
Transfer a 180 µl aliquot of the calibration solutions into an injection vial (6.3), individually. Add 20 µl mixed
ISTD solution (5.10) into all the vials. The same ISTD solution as used in sample preparation shall be used. Cap
and mix.
Table 3 — Example of calibration solutions
No. Calibration Calibration standard Volume of standard Volume of acetonitrile-
standard solution solution solution water-acetic acid
Nono. µl (35:64.5:0.5, v/v/v) (5.7)
µl
mixed stock solution
Step 1 SS1 50 950
(5.9)
mixed stock solution
Step 2 SS2 20 980
(5.9)
Step 3 SS3 SS1 200 800
Step 4 SS4 SS2 200 600
Step 5 SS5 SS3 200 600
Step 6 SS6 SS4 200 600
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.