ISO 13646

Water quality — Determination of selected estrogens in whole water samples — Method using solid phase extraction (SPE) followed by liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometry (MS) detection

This document specifies a method for the determination of 5 oestrogens, in whole water samples listed in Table 1. The method uses solid-phase extraction (SPE-disks and/or cartridges) followed by liquid or gas chromatography-mass spectrometry (tandem mass spectrometry and/or High resolution mass spectrometry). It is applicable to the analysis of selected estrogens in surface water containing suspended particulate matter (SPM) up to 50 mg/l , DOC content up to 7 mg/l (whole water samples), drinking water and groundwater.

Qualité de l’eau — Dosage d’œstrogènes sélectionnés dans des échantillons d’eau totale — Méthode par extraction en phase solide (SPE) suivie d’une détection par chromatographie en phase liquide (CL) ou en phase gazeuse (CG) couplée à la spectrométrie de masse (SM)

General Information

• Status: Not Published
• ICS: 13.060.50 - Examination of water for chemical substances
• Technical Committee: ISO/TC 147/SC 2 - Physical, chemical and biochemical methods
• Drafting Committee: ISO/TC 147/SC 2 - Physical, chemical and biochemical methods
• Current Stage: 6000 - International Standard under publication
• Start Date: 05-Sep-2025
• Completion Date: 13-Sep-2025

Relations

• Consolidates: ISO/IEC 21558-3:2022 - Telecommunications and information exchange between systems — Future network architecture — Part 3: Networking of everything
• Effective Date: 05-Aug-2023

Standards Content (Sample)

FINAL DRAFT
International
Standard
ISO/FDIS 13646
ISO/TC 147/SC 2
Water quality — Determination of
Secretariat: DIN
selected estrogens in whole water
Voting begins on:
samples — Method using solid
2025-07-10
phase extraction (SPE) followed by
Voting terminates on:
liquid chromatography (LC) or gas
2025-09-04
chromatography (GC) coupled to
mass spectrometry (MS) detection
Qualité de l’eau — Dosage d’œstrogènes sélectionnés dans des
échantillons d’eau totale — Méthode par extraction en phase
solide (SPE) suivie d’une détection par chromatographie en phase
liquide (CL) ou en phase gazeuse (CG) couplée à la spectrométrie
de masse (SM)
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
ISO/CEN PARALLEL PROCESSING 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 13646:2025(en) © ISO 2025

FINAL DRAFT
ISO/FDIS 13646:2025(en)
International
Standard
ISO/FDIS 13646
ISO/TC 147/SC 2
Water quality — Determination of
Secretariat: DIN
selected estrogens in whole water
Voting begins on:
samples — Method using solid
phase extraction (SPE) followed by
Voting terminates on:
liquid chromatography (LC) or gas
chromatography (GC) coupled to
mass spectrometry (MS) detection
Qualité de l’eau — Dosage d’œstrogènes sélectionnés dans des
échantillons d’eau totale — Méthode par extraction en phase
solide (SPE) suivie d’une détection par chromatographie en phase
liquide (CL) ou en phase gazeuse (CG) couplée à la spectrométrie
de masse (SM)
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
ISO/CEN PARALLEL PROCESSING
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: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland Reference number
ISO/FDIS 13646:2025(en) © ISO 2025

ii
ISO/FDIS 13646:2025(en)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms, definitions and subscripts . 2
3.1 Terms and definitions .2
3.2 Subscripts . .4
4 Principle . 4
5 Interferences . 5
5.1 General .5
5.2 Interferences with sampling, extraction and concentration . .5
5.3 Interferences during high performance liquid chromatography and mass spectrometry .6
5.4 Interferences during gas phase chromatography and mass spectrometry .7
5.5 Interferences from internal standards .8
6 Reagents . 8
7 Apparatus . 14
8 Sampling . 16
9 Procedure . 17
9.1 General .17
9.2 Sample preparation and extraction .18
9.2.1 General .18
9.2.2 Sample preparation .18
9.2.3 SPE cartridge extraction .19
9.2.4 SPE disk Extraction . 20
9.3 Sample clean-up (purification) . 20
9.3.1 General . 20
9.3.2 Principle .21
9.3.3 Procedure .21
9.4 Reconcentration .21
9.5 Liquid chromatography coupled to mass spectrometry . 22
9.5.1 High performance liquid chromatography (LC) . 22
9.5.2 Detection . 22
9.5.3 Derivatization . 23
9.6 Gas chromatography coupled to mass spectrometry .24
9.6.1 Derivatization .24
9.6.2 Gas chromatography (GC) . 25
9.6.3 Detection . 25
10 Calibration .26
10.1 General . 26
10.2 Calibration by isotope dilution . 26
10.3 Calibration check .27
11 Quality assurance and quality control (QA/QC) .27
11.1 Identification of the substances .27
11.2 Blanks .27
12 Limit of quantification (LOQ) .28
13 Calculation of recovery .28
13.1 General . 28
13.2 Calculation of analyte recovery using samples . 28
13.3 Recovery rates from internal standards . 29

iii
ISO/FDIS 13646:2025(en)
14 Calculation of the concentration in the sample .29
15 Expression of results .30
16 Test report .30
17 Performance data .31
Annex A (informative) Performance data .32
Annex B (informative) Examples of SPE cartridge extraction protocols .35
Annex C (informative) Examples of SPE disk extraction protocols .37
Annex D (informative) Examples of SPE clean-up protocols .39
Annex E (informative) Examples of LC-MS/MS protocols .
...

ISO/FDIS 13646:2025(E)
ISO/TC 147/SC 2/WG 84
Secretariat: DIN
Date: 2025-06-26
Water quality — Determination of selected estrogens in whole water
samples — Method using solid phase extraction (SPE) followed by
liquid chromatography (LC) or gas chromatography (GC) coupled to
mass spectrometry (MS) detection
Qualité de l’eau — Dosage d’œstrogènes sélectionnés dans des échantillons d’eau totale — Méthode par
extraction en phase solide (SPE) suivie d’une détection par chromatographie en phase liquide (CL) ou en phase
gazeuse (CG) couplée à la spectrométrie de masse (SM)
FDIS stage
Warning for WDs and CDs
This document is not an ISO International Standard. It is distributed for review and comment. It is subject to change
without notice and may not be referred to as an International Standard.
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 supporting documentation.

ISO/FDIS 13646:2025(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
EmailE-mail: [e-mail]
Website: www.iso.orgwww.iso.org
Published in Switzerland
ii
ISO/FDIS 13646:2025(en)
Contents
Foreword . vi
Introduction . vii
1 Scope . 1
2 Normative references . 1
3 Terms, definitions and subscripts . 2
3.1 Terms and definitions . 2
3.2 Subscripts . 4
4 Principle . 4
5 Interferences . 5
5.1 General . 5
5.2 Interferences with sampling, extraction and concentration . 6
5.3 Interferences during high performance liquid chromatography and mass spectrometry . 6
5.4 Interferences during gas phase chromatography and mass spectrometry . 8
5.5 Interferences from internal standards . 8
6 Reagents . 9
7 Apparatus . 17
8 Sampling . 19
9 Procedure . 19
9.1 General . 19
9.2 Sample preparation and extraction . 21
9.3 Sample clean-up (purification) . 24
9.4 Reconcentration . 25
9.5 Liquid chromatography coupled to mass spectrometry . 25
9.6 Gas chromatography coupled to mass spectrometry . 28
10 Calibration . 30
10.1 General . 30
10.2 Calibration by isotope dilution . 31
10.3 Calibration check . 32
11 Quality assurance and quality control (QA/QC) . 32
11.1 Identification of the substances . 32
11.2 Blanks . 32
12 Limit of quantification (LOQ) . 33
13 Calculation of recovery . 33
13.1 General . 33
13.2 Calculation of analyte recovery using samples . 33
13.3 Recovery rates from internal standards . 34
14 Calculation of the concentration in the sample . 35
15 Expression of results . 36
16 Test report . 36
17 Performance data . 36
Annex A (informative) Performance data . 37
Annex B (informative) Examples of SPE cartridge extraction protocols . 41
iii
ISO/FDIS 13646:2025(en)
Annex C (informative) Examples of SPE disk extraction protocols . 43
Annex D (informative) Examples of SPE clean-up protocols . 45
Annex E (informative) Examples of LC-MS/MS protocols . 47
Annex F (informative) Example of LC-HRMS protocol . 62
Annex G (informative) Examples of GC-MS/MS protocols . 67
Annex H (informative) Example of GC-HRMS protocol . 73
Bibliography . 74

iv
ISO/FDIS 13646:2025(en)
Contents
v
ISO/FDIS 13646:2025(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.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2, Physical,
chemical and biochemical methods.
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.
vi
ISO/FDIS 13646:2025(en)
Introduction
Natural and synthetic oestrogensestrogens are widely used worldwide, e.g. for contraception. Through
application or improper disposal, these estrogens can enter the water cycle unchanged or transformed. They
can therefore be detected in surface and groundwater, as well as in treated wastewater. It is known that
estrogens maycan end up in surface waters via wastewater, and due to their physicochemical properties, they
can partition in the different compartments ([water and suspended particulate matter (SPM)))] of water
systems. They are of rising concern, due to their high estrogenic activity even at the measured ultra-trace
levels (far below ng/l). Beside feminised fish and other endocrine disruptive effects in water ecosystems, they
[ ]
maycan also be a factor in biodiversity loss. 0 [1]. Therefore, appropriate measurement methods are required
to monitor estrogen levels below their ecotoxicological level ([e.g. predicted no effect concentration (PNEC)
or environmental quality standard (EQS)))] and accordingly demonstrate if a water body is at risk.
This document specifies validated methods for analysing water samples in monitoring programs aiming at
qualifying the quality of the water environment with respects to the selected estrogens.
The International Organization for Standardization (ISO) draws attention to the fact that it is claimed that
compliance with this document may involve the use of a patent.
ISO takes no position concerning the evidence, validity and scope of this patent right.
The holder of this patent right has assured ISO that he/she is willing to negotiate licences under reasonable
and non-discriminatory terms and conditions with applicants throughout the world. In this respect, the
statement of the holder of this patent right is registered with ISO. Information may be obtained from the patent
database available at .
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights other than those in the patent database. ISO shall not be held responsible for identifying any or all such
patent rights.
vii
ISO/FDIS 13646:2025(en)
Water quality — Determination of selected estrogens in whole water
samples — Method using solid phase extraction (SPE) followed by
liquid chromatography (LC) or gas chromatography (GC) coupled to
mass spectrometry (MS) detection
WARNING — Persons using this document should be familiar with normal laboratory practice. This
document does not purport to address all of the safety problems, if any, associated with its u
...

PROJET FINAL
Norme
internationale
ISO/FDIS 13646
ISO/TC 147/SC 2
Qualité de l’eau — Dosage
Secrétariat: DIN
d’œstrogènes sélectionnés dans
Début de vote:
des échantillons d’eau totale —
2025-07-10
Méthode par extraction en phase
Vote clos le:
solide (SPE) suivie d’une détection
2025-09-04
par chromatographie en phase
liquide (CL) ou en phase gazeuse
(CG) couplée à la spectrométrie de
masse (SM)
Water quality — Determination of selected estrogens in whole
water samples — Method using solid phase extraction (SPE)
followed by liquid chromatography (LC) or gas chromatography
(GC) coupled to mass spectrometry (MS) detection
LES DESTINATAIRES DU PRÉSENT PROJET SONT
INVITÉS À PRÉSENTER, AVEC LEURS OBSERVATIONS,
NOTIFICATION DES DROITS DE PROPRIÉTÉ DONT ILS
AURAIENT ÉVENTUELLEMENT CONNAISSANCE ET À
FOURNIR UNE DOCUMENTATION EXPLICATIVE.
OUTRE LE FAIT D’ÊTRE EXAMINÉS POUR
ÉTABLIR S’ILS SONT ACCEPTABLES À DES FINS
INDUSTRIELLES, TECHNOLOGIQUES ET COM-MERCIALES,
AINSI QUE DU POINT DE VUE DES UTILISATEURS, LES
PROJETS DE NORMES
TRAITEMENT PARALLÈLE ISO/CEN
INTERNATIONALES DOIVENT PARFOIS ÊTRE CONSIDÉRÉS
DU POINT DE VUE DE LEUR POSSI BILITÉ DE DEVENIR DES
NORMES POUVANT
SERVIR DE RÉFÉRENCE DANS LA RÉGLEMENTATION
NATIONALE.
Numéro de référence
ISO/FDIS 13646:2025(fr) © ISO 2025

PROJET FINAL
ISO/FDIS 13646:2025(fr)
Norme
internationale
ISO/FDIS 13646
ISO/TC 147/SC 2
Qualité de l’eau — Dosage
Secrétariat: DIN
d’œstrogènes sélectionnés dans
Début de vote:
des échantillons d’eau totale —
2025-07-10
Méthode par extraction en phase
Vote clos le:
solide (SPE) suivie d’une détection
2025-09-04
par chromatographie en phase
liquide (CL) ou en phase gazeuse
(CG) couplée à la spectrométrie de
masse (SM)
Water quality — Determination of selected estrogens in whole
water samples — Method using solid phase extraction (SPE)
followed by liquid chromatography (LC) or gas chromatography
(GC) coupled to mass spectrometry (MS) detection
LES DESTINATAIRES DU PRÉSENT PROJET SONT
INVITÉS À PRÉSENTER, AVEC LEURS OBSERVATIONS,
NOTIFICATION DES DROITS DE PROPRIÉTÉ DONT ILS
AURAIENT ÉVENTUELLEMENT CONNAISSANCE ET À
FOURNIR UNE DOCUMENTATION EXPLICATIVE.
DOCUMENT PROTÉGÉ PAR COPYRIGHT
OUTRE LE FAIT D’ÊTRE EXAMINÉS POUR
ÉTABLIR S’ILS SONT ACCEPTABLES À DES FINS
© ISO 2025 INDUSTRIELLES, TECHNOLOGIQUES ET COM-MERCIALES,
AINSI QUE DU POINT DE VUE DES UTILISATEURS, LES
Tous droits réservés. Sauf prescription différente ou nécessité dans le contexte de sa mise en œuvre, aucune partie de cette
PROJETS DE NORMES
TRAITEMENT PARALLÈLE ISO/CEN
INTERNATIONALES DOIVENT PARFOIS ÊTRE CONSIDÉRÉS
publication ne peut être reproduite ni utilisée sous quelque forme que ce soit et par aucun procédé, électronique ou mécanique,
DU POINT DE VUE DE LEUR POSSI BILITÉ DE DEVENIR DES
y compris la photocopie, ou la diffusion sur l’internet ou sur un intranet, sans autorisation écrite préalable. Une autorisation peut
NORMES POUVANT
être demandée à l’ISO à l’adresse ci-après ou au comité membre de l’ISO dans le pays du demandeur.
SERVIR DE RÉFÉRENCE DANS LA RÉGLEMENTATION
NATIONALE.
ISO copyright office
Case postale 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Genève
Tél.: [phone]
E-mail: [e-mail]
Web: www.iso.org
Publié en Suisse Numéro de référence
ISO/FDIS 13646:2025(fr) © ISO 2025

ii
ISO/FDIS 13646:2025(fr)
Sommaire Page
Avant-propos .v
Introduction .vi
1 Domaine d’application . 1
2 Références normatives . 1
3 Termes, définitions et indices . 2
3.1 Termes et définitions .2
3.2 Indices .4
4 Principe. 4
5 Interférences . 5
5.1 Généralités .5
5.2 Interférences liées à l’échantillonnage, l’extraction et la concentration .6
5.3 Interférences lors de l’analyse par chromatographie en phase liquide haute
performance et spectrométrie de masse .6
5.4 Interférences lors de l’analyse par chromatographie en phase gazeuse et spectrométrie
de masse .7
5.5 Interférences des étalons internes .8
6 Réactifs . 8
7 Appareillage . 14
8 Échantillonnage .16
9 Mode opératoire . 17
9.1 Généralités .17
9.2 Préparation de l’échantillon et extraction .18
9.2.1 Généralités .18
9.2.2 Préparation de l’échantillon .18
9.2.3 Extraction SPE sur cartouche .19
9.2.4 Extraction SPE sur disque . 20
9.3 Purification de l’échantillon . .21
9.3.1 Généralités .21
9.3.2 Principe.21
9.3.3 Mode opératoire . .21
9.4 Reconcentration . 22
9.5 Chromatographie en phase liquide couplée à la spectrométrie de masse . 22
9.5.1 Chromatographie en phase liquide haute performance (CL) . 22
9.5.2 Détection . 22
9.5.3 Dérivation . 23
9.6 Chromatographie en phase gazeuse couplée à la spectrométrie de masse . 25
9.6.1 Dérivation . 25
9.6.2 Chromatographie en phase gazeuse (CG) . 25
9.6.3 Détection . 25
10 Étalonnage .26
10.1 Généralités . 26
10.2 Étalonnage par dilution isotopique .27
10.3 Contrôle de l’étalonnage .27
11 Programme d’assurance qualité et de contrôle qualité (AQ/CQ) .28
11.1 Identification des composés . 28
11.2 Blancs . 28
12 Limite de quantification (LQ) .28
13 Calcul du taux de récupération .29
13.1 Généralités . 29

iii
ISO/FDIS 13646:2025(fr)
13.2 Calcul du taux de récupération des analytes à l’aide d’échantillons . 29
13.3 Taux de récupération des étalons internes . 30
14 Calcul de la concentration dans l’échantillon .30
15 Expression des résultats .31
16 Rapport d’essai .31
17 Données de performance .31
Annexe A (informative) Données de performance .32
Annexe B (informative) Exemples de protocoles d’extraction SPE sur cartouche .36
Annexe C (informative) Exemples de protocoles d’extraction SPE sur disque .38
Annexe D (info
...