EN ISO 13646:2025

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 (ISO 13646:2025)

This document specifies methods for the determination of five selected estrogens in whole water samples listed in Table 1 (see Clause 4). The methods are based on solid-phase extraction (SPE; disk or cartridge) followed by liquid or gas chromatography-mass spectrometry detection (tandem mass spectrometry or high resolution mass spectrometry). Depending on the sample preparation chosen, the sample preparation can be applicable to the analysis of selected estrogens in drinking water, groundwater and surface water containing suspended particulate matter (SPM) up to 500 mg/l, dissolved organic carbon (DOC) content up to 14 mg/l (whole water samples).
The lower application range defined as verified limit of quantification can vary depending on the methods, the sensitivity of the equipment used and the matrix of the sample. The range is 0,006 ng/l to 1 ng/l for 17alpha-ethinylestradiol (EE2) and 0,038 ng/l to 1 ng/l for the other estrogens in drinking water, ground water and surface water. The upper limit of the working range is approximately tens of nanograms per litre.
For application that targets the measurements of very low level concentrations (between the lowest LOQ and 0,1 ng/l), every single step of the procedure becomes critical.
The methods can be used to determine further estrogens or hormones in other types of water, for example treated wastewater, if accuracy has been tested and verified for each case as well as storage conditions of both samples and reference solutions have been validated.

Wasserbeschaffenheit - Bestimmung ausgewählter Estrogene in Gesamtwasserproben - Verfahren mittels Festphasenextraktion (SPE) gefolgt von Flüssigkeitschromatographie (LC) oder Gaschromatographie (GC) gekoppelt mit massenspektrometrischer Detektion (MS) (ISO 13646:2025)

Dieses Dokument legt Verfahren für die Bestimmung von fünf ausgewählten Estrogenen in den in Tabelle 1 (siehe Abschnitt 4 aufgeführten Gesamtwasserproben fest. Die Verfahren beruht auf der Festphasenextraktion (SPE-Disk oder  Kartusche) und anschließendem Nachweis mittels Flüssigkeits- oder Gaschromatographie (Tandem-Massenspektrometrie oder Hochauflösungsmassenspektrometrie). Je nach gewählter Probenvorbereitung kann die Probenaufbereitung für die Analyse ausgewählter Estrogene in Trinkwasser, Grundwasser und Oberflächenwasser mit Schwebstoffen (SPM) bis zu 500 mg/l, einem Gehalt an gelösten organischen Kohlenstoff (DOC) bis zu 14 mg/l (Gesamtwasserproben) angewendet werden.
Der untere Anwendungsbereich, der als nachgewiesene Bestimmungsgrenze definiert ist, kann je nach Verfahren, Empfindlichkeit der verwendeten Geräte und Matrix der Probe variieren. Der Bereich liegt bei 0,006 ng/l bis 1 ng/l für 17α-Ethinylestradiol (EE2) und 0,038 ng/l bis 1 ng/l für die anderen Estrogene in Trinkwasser, Grundwasser und Oberflächenwasser. Die Obergrenze des Arbeitsbereichs liegt bei etwa 10 ng/l.
Für Anwendungen, die auf die Messung sehr niedriger Konzentrationen abzielen (zwischen der niedrigsten LOQ und 0,1 ng/l), ist jeder einzelne Schritt des Verfahrens kritisch.
Die Verfahren können zur Bestimmung weiterer Estrogene oder Hormone in anderen Wasserarten, z. B. in behandeltem Abwasser, verwendet werden, wenn die Genauigkeit in jedem Fall geprüft und verifiziert wurde und die Lagerungsbedingungen sowohl der Proben als auch der Referenzlösungen validiert wurden.
WARNUNG — Anwender dieses Dokuments sollten mit der üblichen Laborpraxis vertraut sein. Dieses Dokument gibt nicht vor, alle unter Umständen mit der Anwendung des Verfahrens verbundenen Sicherheitsaspekte anzusprechen. Es liegt in der Verantwortung des Arbeitgebers, angemessene Sicherheits- und Schutzmaßnahmen zu treffen.
WICHTIG — Es ist erforderlich, bei den Untersuchungen nach diesem Dokument Fachleute oder Facheinrichtungen einzuschalten.

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) (ISO 13646:2025)

Le présent document spécifie des méthodes pour le dosage de cinq œstrogènes sélectionnés dans les échantillons d’eau totale, qui sont répertoriés dans le Tableau 1 (voir l’Article 4). Les méthodes reposent sur une extraction en phase solide (SPE sur disque et/ou cartouche) suivie d’une détection par chromatographie en phase liquide ou gazeuse couplée à la spectrométrie de masse (spectrométrie de masse en tandem et/ou spectrométrie de masse haute résolution). Selon la préparation des échantillons choisie, la préparation de l’échantillon peut être appliquée à l’analyse des œstrogènes sélectionnés dans l’eau potable, les eaux souterraines et les eaux de surface contenant une teneur en matières en suspension (MES) jusqu’à 500 mg/l et une teneur en carbone organique dissous (COD) jusqu’à 14 mg/l (échantillons d’eau totale).
La gamme d’application basse définie en tant que limite de quantification vérifiée peut varier selon les méthodes, la sensibilité de l’équipement utilisé et la matrice de l’échantillon. La gamme s’étend de 0,006 ng/l à 1 ng/l pour le 17alpha-éthinylestradiol (EE2) et de 0,038 ng/l à 1 ng/l pour les autres œstrogènes dans l’eau potable, les eaux souterraines et les eaux de surface. La limite supérieure du domaine d’application est de l’ordre de la dizaine de nanogrammes par litre.
Pour une application qui vise à mesurer de très faibles niveaux de concentration (entre la plus faible LQ et 0,1 ng/l), chaque étape individuelle des modes opératoires revêt une importance critique.
Les méthodes peuvent être utilisées pour doser d’autres œstrogènes ou hormones dans d’autres types d’eau, par exemple les eaux usées traitées, sous réserve que l’exactitude ait été soumise à essai et démontrée pour chaque cas et que les conditions de conservation des échantillons et des solutions de référence aient été validées.

Kakovost vode - Določanje izbranih estrogenov v celotnem vzorcu vode - Metoda tekočinske kromatografije (LC) ali plinske kromatografije (GC) z masno selektivnim detektorjem po ekstrakciji na trdni fazi (SPE) (ISO/FDIS 13646:2025)

General Information

• Status: Published
• Publication Date: 14-Oct-2025
• ICS: 13.060.50 - Examination of water for chemical substances
• Technical Committee: CEN/TC 230 - Water analysis
• Drafting Committee: CEN/TC 230/WG 1 - Physical and biochemical methods
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 15-Oct-2025
• Completion Date: 15-Oct-2025
• Ref Project: SIST EN ISO 13646:2026 - 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 (ISO/FDIS 13646:2025)

Overview

EN ISO 13646:2025 (CEN) specifies validated analytical methods for the determination of selected estrogens in whole water samples. The standard covers analysis of five target estrogens (see Table 1 in the standard) using solid-phase extraction (SPE) followed by liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometry (MS) detection - including tandem MS (MS/MS) and high-resolution MS (HRMS). Applicable matrices include drinking water, groundwater and surface water with suspended particulate matter (SPM) up to 500 mg/L and dissolved organic carbon (DOC) up to 14 mg/L.

Key topics and requirements

• Sample preparation: Whole water extraction using SPE (cartridge or disk), with protocols for clean-up and reconcentration.
• Chromatography and detection: Options for LC-MS/MS, LC-HRMS, GC-MS/MS, GC-HRMS; derivatization steps are addressed for GC where needed.
• Performance limits: Verified limits of quantification (LOQ) vary by analyte and method - e.g., EE2 (17α‑ethinylestradiol) LOQ range 0.006–1 ng/L; other estrogens 0.038–1 ng/L. Upper working range extends to tens of ng/L.
• Quality assurance / control: Identification criteria, blanks, calibration (including isotope-dilution calibration), recovery calculations and calibration checks are specified.
• Interferences and matrix effects: Guidance on interferences during sampling, extraction, chromatography and MS detection, and on managing complex matrices.
• Critical practices for ultra-trace analysis: For measurements between the lowest LOQ and 0.1 ng/L, the standard emphasizes that every procedural step (sampling, storage, extraction, instrument setup) is critical.
• Scope for extension: Methods can be applied to additional estrogens or other waters (e.g., treated wastewater) provided accuracy and storage conditions are validated.

Applications

• Regulatory and compliance monitoring of endocrine-disrupting compounds in water supplies and surface waters.
• Environmental surveillance for trace-level estrogens in river, lake and groundwater studies.
• Method development and validation in laboratories implementing SPE–LC/GC–MS workflows for ultra-trace organic contaminants.
• Research on partitioning of estrogens between dissolved phase and suspended particulate matter (SPM).

Who should use this standard

• Environmental laboratories and water quality testing facilities.
• Regulatory agencies and monitoring programs.
• Water utilities, wastewater treatment operators and environmental consultants.
• Researchers in environmental chemistry and ecotoxicology.

Related standards

• Other ISO/CEN water quality standards for trace organic analysis and sampling (refer to ISO/TC 147 - Water quality). Consult national standards bodies for cross-references and harmonized methods.

Standards Content (Sample)

SLOVENSKI STANDARD
oSIST prEN ISO 13646:2024
01-februar-2024
Kakovost vode - Določanje izbranih estrogenov v celotnem vzorcu vode - Metoda
tekočinske kromatografije (LC) ali plinske kromatografije (GC) z masno
selektivnim detektorjem po ekstrakciji na trdni fazi (SPE) (ISO/DIS 13646:2023)
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 (ISO/DIS
13646:2023)
Wasserbeschaffenheit - Bestimmung ausgewählter Estrogene in Gesamtwasserproben -
Verfahren mittels Festphasenextraktion (SPE) und anschließender
Flüssigkeitschromatographie (LC) oder Gaschromatographie (GC) gekoppelt mit
massenspektrometrischer Detektion (MS) (ISO/DIS 13646:2024)
Qualité des eaux - Dosage destrogènes sélectionnés dans des échantillons d'eau totale -
Méthode par extraction en phase solide (SPE), avec analyse par couplage
chromatographie-spectrométrie de masse (SM) (ISO/DIS 13646:2023)
Ta slovenski standard je istoveten z: prEN ISO 13646
ICS:
13.060.50 Preiskava vode na kemične Examination of water for
snovi chemical substances
oSIST prEN ISO 13646:2024 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN ISO 13646:2024
oSIST prEN ISO 13646:2024
DRAFT INTERNATIONAL STANDARD
ISO/DIS 13646
ISO/TC 147/SC 2 Secretariat: DIN
Voting begins on: Voting terminates on:
2024-01-01 2024-03-25
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
ICS: 13.060.50
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
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Reference number
NATIONAL REGULATIONS.
ISO/DIS 13646:2024(E)
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 2024

oSIST prEN ISO 13646:2024
ISO/DIS 13646:2024(E)
DRAFT INTERNATIONAL STANDARD
ISO/DIS 13646
ISO/TC 147/SC 2 Secretariat: DIN
Voting begins on: Voting terminates on:

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
ICS: 13.060.50
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
© ISO 2024
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY
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NOT BE REFERRED TO AS AN INTERNATIONAL
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NATIONAL REGULATIONS.
Website: www.iso.org ISO/DIS 13646:2023(E)
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ii
PROVIDE SUPPORTING DOCUMENTATION. © ISO 2023

oSIST prEN ISO 13646:2024
ISO/DIS 13646:2023(E)
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
2 Normative references . 2
3 Terms and definitions . 3
4 Principle . 5
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 coming from internal standards . 7
6 Reagents . 8
7 Apparatus .14
8 Sampling .16
9 Procedure .16
9.1 General . 16
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) . 21
9.3.1 General . 21
9.3.2 Principles . 21
9.3.3 Procedures . 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 . 27
10.3 Calibration check .28
11 QA/QC .28
11.1 Identification of the substances .28
11.2 Blanks .28
12 Limit of quantification (LOQ) .28
13 Calculation of recovery .29
13.1 General .29
13.2 Calculation of analyte recovery using samples .29
13.3 Recovery rates from internal standards .29
iii
oSIST prEN ISO 13646:2024
ISO/DIS 13646:2023(E)
14 Calculation of the concentration in the sample .30
15 Expression of results .31
16 Test reports .31
17 Performance Data .31
Annex A (informative) Performances data .32
Annex B (informative) Example of SPE extraction protocol .36
Annex C (informative) Examples of SPE disk extraction protocols .38
Annex D (informative) Examples of clean-up protocols .40
Annex E (informative) Examples of LC-MS/MS protocol .42
Annex F (informative) Example of LC-HRMS protocol .52
Annex G (informative) Examples of GC-MS/MS protocol .57
Annex H (informative) Examples of GC-HRMS protocol .61
Bibliography .62
iv
oSIST prEN ISO 13646:2024
ISO/DIS 13646:2023(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 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/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.
v
oSIST prEN ISO 13646:2024
ISO/DIS 13646:2023(E)
Introduction
Natural and synthetic oestrogens 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 may 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
[1]
water ecosystems also they may be a factor in biodiversity loss. Therefore, appropriate measurement
methods are necessary which allow estrogen levels below their ecotoxicological level (e.g. or predicted
no effect concentration (PNEC) or environmental quality standard (EQS) to be monitored and to
demonstrate if a water body is at risk.
This International Standard specifies validated methods for analysing whole water samples satisfying
future requirements in support of the European Water Framework Directive WFD and any others
regulation worldwide 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 www.iso.org/patents.
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.
vi
oSIST prEN ISO 13646:2024
DRAFT INTERNATIONAL STANDARD ISO/DIS 13646:2023(E)
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
use. It is the responsibility of the user to establish appropriate safety and health practices.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably qualified staff.
1 Scope
This document specifies methods for the determination of five selected estrogens in whole water
samples listed in Table 1. The methods are based on solid-phase extraction (SPE disk and/or cartridge)
followed by liquid or gas chromatography-mass spectrometry detection (tandem mass spectrometry
and/or High Resolution Mass Spectrometry). Depending on the sample preparation chosen, it is
applicable to the analysis of selected estrogens in drinking water, groundwater and surface water
containing suspended particulate matter (SPM) up to 500 mg/l, DOC content up to 14 mg/l (whole
water samples).
The lower application range defined as verified limit of quantification can vary depending on the
methods the sensitivity of the equipment used and the matrix of the sample. The range is 0,006 ng/l
to 1 ng/l for EE2 and 0,038 ng/l to 1 ng/l for the other estrogens in drinking water, ground water and
surface water. The upper limit of the working range is approximately tens ng/l.
For application that targets the measurements of very low level of concentrations (between the lowest
LOQ and 0,1 ng/l) every single step of the methods become critical and imply to fix some additional
requirements.
The method can be used to determine further estrogens or hormones in other types of water e.g.
treated wastewater if accuracy has been tested and verified for each case as well as storage conditions
of both samples and reference solutions have been validated.
Table 1 — The table summarizes names, abbreviations, structures, CAS numbers, formulas,
molecular weights, log Kow of the 5 selected estrogens
Molec-
CAS- Formu- ular
Names Structure Log K
a a ow
RN l weight
(g/mol)
17alpha- ethinylestradiol
(17αEE2)
57-63-6 C H O 296,40 4,52
20 24 2
IUPAC name: (13R,17S)-17-ethynyl-13-me-
thyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopen-
ta[a]phenanthrene-3,17-diol
a
CAS-RN: Chemical Abstracts System Registration Number
oSIST prEN ISO 13646:2024
ISO/DIS 13646:2023(E)
TTabablele 1 1 ((ccoonnttiinnueuedd))
Molec-
CAS- Formu- ular
Names Structure Log K
a a ow
RN l weight
(g/mol)
17alpha-estradiol
(17αE2)
57-91-0 C H O 272,38 4,13
18 24 2
IUPAC name: (8R,9S,13S,14S,17R)-13-me-
thyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopen-
ta[a]phenanthrene-3,17-diol
17beta-estradiol
(17βE2)
50-28-2 C H O 272,38 4,13
18 24 2
IUPAC name: (8R,9S,13S,14S,17S)-13-me-
thyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopen-
ta[a]phenanthrene-3,17-diol
Estriol
(E3)
50-27-1 C H O 288,38 2,94
18 24 3
IUPAC name: (8R,9S,13S,14S,16R,17R)-13-me-
thyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopen-
ta[a]phenanthrene-3,16,17-triol
Estrone
(E1)
53-16-7 C H O 270,37 3,69
18 22 2
IUPAC name: (8R,9S,13S,14S)-3-hydroxy-13-me-
thyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopen-
ta[a]phenanthren-17-one
a
CAS-RN: Chemical Abstracts System Registration Number
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 4796-2, Laboratory glassware — Bottles — Part 2: Conical neck bottles
ISO 5667:3, Water quality — Sampling — Part 3: Preservation and handling of water samples
ISO 5667-4, Water quality — Sampling — Part 4: Guidance on sampling from lakes, natural and man-made
ISO 5667-5, Water quality — Sampling — Part 5: Guidance on sampling of drinking water from treatment
works and piped distribution systems
ISO 5667-6, Water quality — Sampling — Part 6: Guidance on sampling of rivers and streams
ISO 5667-11, Water quality — Sampling — Part 11: Guidance on sampling of groundwaters
ISO 8466-1, Water quality — Calibration and evaluation of analytical methods and estimation of
performance characteristics — Part 1: Linear calibration function
ISO 21253-1, Water quality — Multi-compound class methods — Part 1: Criteria for the identification of
target compounds by gas and liquid chromatography and mass spectrometry
ISO 21253-2, Water quality — Multi-compound class methods — Part 2: Criteria for the quantitative
determination of organic substances using a multi-compound class analytical method
oSIST prEN ISO 13646:2024
ISO/DIS 13646:2023(E)
ISO/TS 13530:2009, Water quality — Guidance on analytical quality control for chemical and
physicochemical water analysis
ISO 11352:2012, Water quality — Estimation of measurement uncertainty based on validation and quality
control data
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 https:// www .electropedia .org/
3.1
accuracy
closeness of agreement between a measured quantity value and a true quantity value of a measurand
Note 1 to entry: The concept "measurement accuracy" is not a quantity and is not given a numerical quantity
value. A measurement is said to be more accurate when it offers a smaller measurement error.
Note 2 to entry: The term "measurement accuracy" should not be used for measurement trueness and the term
measurement precision should not be used for ‘measurement accuracy’, which, however, is related to both these
concepts.
Note 3 to entry: "Measurement accuracy" is sometimes understood as closeness of agreement between measured
quantity values that are being attributed to the measurand.
[SOURCE: ISO/IEC Guide 99:2007, 2.13]
3.2
analyte
substance to be analyzed
[SOURCE: ISO 21253-2:2019, definition 3.1]
3.3
blank
aliquot of reagent water (reagent blank) or of a matrix in which the analyte (3.2) is absent (matrix blank)
that is treated exactly as a sample through the complete analytical procedure including extraction,
clean-up, identification and quantification including all the relevant reagents and materials
Note 1 to entry: It is crucial that the laboratory specifies which blank is considered.
[SOURCE: ISO 21253-1: 2019, definition 3.2]
3.4
calibration
operation that, under specified conditions, in a first step, establishes a relation between the quantity
values with measurement uncertainties provided by measurement standards and corresponding
indications with associated measurement uncertainties and, in a second step, uses this information to
establish a relation for obtaining a measurement result from an indication
Note 1 to entry: A calibration may be expressed by a statement, calibration function, calibration diagram,
calibration curve, or calibration table. In some cases, it may consist of an additive or multiplicative correction of
the indication with associated measurement uncertainty.
Note 2 to entry: Calibration should not be confused with adjustment of a measuring system, often mistakenly
called “self-calibration”, nor with verification of calibration.
oSIST prEN ISO 13646:2024
ISO/DIS 13646:2023(E)
[SOURCE: ISO/IEC Guide 99:2007, definition 2.39]
3.5
certified reference material
CRM
reference material, accompanied by documentation issued by an authoritative body and providing
one or more specified property values with associated uncertainties and traceabilities, using valid
procedures
[SOURCE: ISO/IEC Guide 99:2007, definition 5.14]
3.6
integrity
property that the parameter(s) of interest, information or content of the sample container has not been
altered or lost in an unauthorized manner or subject to loss of representativeness
[SOURCE: ISO 5667-3, definition 3.1]
3.7
isotope-dilution quantification
process where isotopically labelled standards (eg.deuterium- or carbon 13-labeled) chemically
similar isotopic analogs of the target analytes, are added to all environmental and quality-control
and quality-assurance samples before extraction and follow all the analytical procedure. It improve
quantitative accuracy by accounting for sample-specific procedural losses in the determined analyte
concentration
3.8
limit of quantification
LOQ
lowest value of a determinand that can be determined with an acceptable level of accuracy, which could
be estimated by different means and shall be verified in the intended matrix
[SOURCE: ISO 21253-2: 2019, definition 3.4]
3.9
recovery
relative recovery
extent to which a known, added quantity of determinant in a sample can be measured by an analytical
system
Note 1 to entry: Recovery is calculated from the difference between results obtained from a spiked and an
unspiked aliquot of sample and is usually expressed as a percentage.
[SOURCE: ISO 5667-14:2014, 3.8]
3.10
traceability
property of a measurement result whereby the result can be related to a reference through a
documented unbroken chain of calibrations, each contributing to the measurement uncertainty
[SOURCE: ISO/IEC Guide 99:2007, definition 2.41]
3.11
yield
absolute recovery
amount of analyte (3.2) added in the test sample corrected by the relative recovery of the internal
standard (analyte-to-internal standard ratio)
Note 1 to entry: Yield is a value that accounts for both sample matrix effect and compound recovery.
[SOURCE: ISO 21253-2: 2019, definition 3.11]
oSIST prEN ISO 13646:2024
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4 Principle
The water sample is spiked with an appropriate amount of isotopic labelled standard analogous of each
targeted estrogens, before the sample to be extracted by solid phase extraction (SPE) cartridge or disk
and then cleaned up by SPE. The separation of the substances is achieved by liquid chromatography
(LC) or gas chromatography (GC) with an identification and quantification based on mass spectrometry
(tandem mass spectrometry (MS/MS) or High Resolution Mass spectrometry (HRMS)). The result is
calculated implementing isotope dilution calibration.
5 Interferences
5.1 General
Solvents, reagents, glassware, and other sample processing hardware may yield artefacts, elevated
baselines, and/or lock-mass suppression causing misinterpretation of chromatograms.
Proper cleaning of glassware is extremely important, because glassware may not only contaminate the
samples but may also remove the analytes of interest by adsorption on the glass surface.
Glassware should be rinsed with solvent and washed with a detergent solution as soon after use as is
practical. Sonication of glassware containing a detergent solution for approximately 30 s may aid in
cleaning. After detergent washing, glassware should be rinsed first with solvent (e.g. methanol) and
then with ultrapure water (6.1). Baking of glassware in an oven, programmable and capable of heating
to at least 450 °C for 2 h may be warranted.
Sample contamination is a concern because estrogenic substances are biogenic and can be present on
human skin or might be used as pharmaceuticals or personal care products. It is important that field
and laboratory personnel exercise care to avoid contamination of the samples by avoiding consumption
or contact with such materials immediately before and during sample collection and processing
procedures. Exercising care is important for both the acquisition and subsequent handling of samples
and sample extracts to avoid contamination.
5.2 Interferences with sampling, extraction and concentration
Use sampling containers of materials (7.1) that do not affect the analyte content during the contact time,
preferably glass. Avoid plastics and organic materials during sampling, sample storage at (5 ± 3) °C
or extraction especially if very low level of concentrations are targeted. High-density polyethylene
(HDPE) or polytetrafluoroethylene (PTFE) may be used but are not recommended if very low level of
concentrations (< 0,1 ng/l) is to be measured.
If automatic samplers are used, avoid the use of silicone or rubber material for the tubes. If these
materials are present, ensure that the contact time is minimized. Rinse the sampling line with the
water to be sampled before taking the test sample. ISO 5667-1 and ISO 5667-3 provide guidance.
Storage temperature is at (5 ± 3) °C. For sampling and sample preservation see Clause 8.
During storage of the test samples, losses of components may occur due to adsorption on the walls of
the containers. The extent of the losses may depend on the storage time.
Commercially available solid-phase extraction (SPE cartridge or disk) may differ in quality. Variations
in the selectivity of the materials also frequently occur from batch to batch, thus possibly causing
significant deviations in extraction yield. This does not basically impair their suitability, apart from a
resulting higher quantification limit for individual substances.
Avoid major fluctuations in the extraction times and elution procedures within one sample sequence
when analyzing the samples.
Water samples containing high content of SPM or DOC can lead to clogging in case of SPE cartridge
(see subclause 9.2.2) that could be prejudicial to extraction recovery. To overpass, reduction of sample
oSIST prEN ISO 13646:2024
ISO/DIS 13646:2023(E)
volume or switching to SPE disk (see subclause 9.2.3) extraction may be implemented. Another solution
is the application of glass wool or sand to the cartridge (filling height 1 cm to 2 cm) to achieve higher
sample volumes.
Repeated uses and cleaning procedures of glassware may cause active sites on the glass surface that
may irreversibly adsorb the selected estrogens and also be responsible of cross contamination.
To prevent cross contamination between series, SPE apparatus, tubing and pieces in contact with
samples shall be dismantled and cleaned with successively e.g. (hot) water with detergents, solvents
(e.g. methanol, acetone, ethanol) then rinsed with water and dried.
Interferences co-extracted from samples will vary considerably from source to source, depending on
the diversity of the site being sampled. Interfering substances may be present at concentrations several
orders of magnitude higher than the selected estrogens. The most frequently encountered interferences
are humic and other acids. Because very low levels of estrogens shall be measured by this method,
elimination of interferences is essential (implementation of clean-up (see subclause 9.3).
5.3 Interferences during high performance liquid chromatography and mass
spectrometry
Substances with similar retention times and masses as the target estrogens may lead to interferences
and overlapping or incompletely resolved peaks in the chromatogram. Depending on their intensity
those co-eluents can affect the trueness of the analysis.
17αE2 and 17βE2 are epi-isomers and as consequence have the same transitions in MS. It is critical to
separate both substances. A minimal chromatographic resolution (R) ≥ 1,2 is suitable. If the criterion
cannot be reached, a suitable column shall be chosen to meet the required resolution [see Annex E
and Annex F for examples].
The chromatographic resolution is calculated according to Figure 1 and Formula (1).
Key
X time
Y intensity
t , t retention time of eluting substances 1 and 2 in seconds (s)
R1 R2
w , w peak width at the base of each peak in seconds (s)
b1 b2
Figure 1 — Resolution of chromatographic peaks
oSIST prEN ISO 13646:2024
ISO/DIS 13646:2023(E)
tt−
()
RR21
R=2 (1)
ww+
bb12
where
R is the resolution;
t , t Retention time of eluting substances 1 and 2 in seconds (s);
R1 R2
w , w Peak width at the base of each peak in seconds (s).
b1 b2
For some targeted estrogens some sensitive transitions have shown a lack of selectivity/specificity
leading to possibly false positive and overestimation of results.
Accompanying substances (matrix) can affect the ionization of the target substances (e.g. ion suppression
or signal enhancement). This can result in underestimation or overestimation of concentration during
quantification and significantly affect the sensitivity of the method. These interferences can be detected
and corrected by implementating a clean-up step (see subclause 9.3) and isotope dilution quantification
(3.7).
5.4 Interferences during gas phase chromatography and mass spectrometry
Substances with similar retention times and masses as the target estrogens may lead to interferences
and overlapping or incompletely resolved peaks in the chromatogram. Depending on their intensity
those co-eluents can affect the trueness of the analysis.
17αE2 and 17βE2 are epi-isomers and as consequence have the same transitions in MS. It is critical to
separate both substances. A minimal chromatographic resolution (R) ≥ 1,2 is suitable. If the criterion
cannot be reached, a suitable column shall be chosen to meet the required resolution [see Annex E and
Annex D for example].
Interferences from accompanying substances (matrix) can occur depending on methods applied.
5.5 Interferences coming from internal standards
For the mass spectrometric detection, it is recommended to use a stable isotopically labelled analogue
as an internal standard; 13C or 15N-labelled compounds are preferred rather than deuterated
compounds. There is generally an observable drift in retention time compared to the natural compound
when working with isotopically labelled standards (isotope effect).
Only a stable part of the molecule shall be labelled, and degree of labelling shall not change over the
course of analysis. Exchanges may take place, via tautomerization processes for example, and so this
type of mechanism shall be experimentally verified in the event that the abundance of internal standard
is shown to vary.
Spectral overlapping between labelled analogue and native substances shall be avoided. Consequently,
for the majority of small-molecule applications, the labelled analogue should preferably have a
monoisotopic mass greater than at least 3 Da. The isotopic purity of the internal standards shall be
checked. It is not a critical factor provided that its impurities, including unlabeled analogue, have
preferably zero contribution on target compounds of the analytical method. In case it is not possible,
the contribution of the labelled analogue to the unlabeled analogue should be negligible at LOQ level.
If internal standards with less than 3Da are implemented, the above criteria shall be checked and
documented to prevent from false positive and misquantification.
oSIST prEN ISO 13646:2024
ISO/DIS 13646:2023(E)
6 Reagents
As far as available, analytical grade or residue-analytical grade reagents shall be used. The
content of impurities contributing to blank values or causing interfering signals shall be negligible
(below 1/3 LOQ). This shall be checked in regular intervals.
Reagents, solvents and water used as eluents shall be suitable for LC or respectively GC and mass
spectrometry.
When very low level of estrogens are to be measured (below 0,1 ng/l) and the lowest LOQ shall be
reached the quality of reagents and materials become highly critical and shall be routinely monitored
to prevent cross contamination and false positive results.
6.1 Water, H O
Ultrapure water without any interfering blank values. The quality of water is checked by the same
procedure than the sample to be measured.
Commercially available mineral water in glass bottle can also be used to determine method blank when
ultrapure water has significant background.
6.2 Sodium thiosulfate pentahydrate, w (Na S O ·5H2O) at least 99 %
2 2 3
6.3 Disodium salt of ethylenediaminetetraacetic acid, EDTA (ρ ≈ 0,025 g/ml),
(C H N Na O ⋅2H O), at least 99 %
10 14 2 2 8 2
Dissolve 25 g EDTA in 1 000 ml of water.
6.4 Methanol, MeOH, (CH OH), for extraction, purification, mobile phase and preparation of
reference solutions
6.5 Acetonitrile, ACN, (CH CN), for extraction, purification, mobile phase and preparation of
reference solutions
6.6 Ethyl acetate, EA, (C H O , for extraction and purification
4 8 2)
6.7 Acetone, (C H O), for extraction and purification and derivatization (GC)
3 6
6.8 n-Hexane, (C H ), for extraction and purification
6 14
6.9 Methyl tert-butyl ether, MTBE, (C H O), for extraction and purification
5 12
6.10 Acetone extra dry, (C H O), for derivatization
3 6
6.11 Acetic acid, w (CH COOH) at least 99 %, for mobile phase (LC)
6.12 Formic acid, w (HCOOH) at least 98 %, for mobile phase (LC)
6.13 Hydrochloric acid, w HCl at least 36 %, for pH adjustment of water sample
6.14 Sulphuric acid, w H SO at least 98 %, for pH adjustment of water sample
2 4
6.15 Sodium Chlorid, w NaCl at least 98 %, for preservation of analytes in water sample
6.16 Ascorbic acid, w C H O at least 98 %, for preservation of analytes in water sample
6 8 6
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6.17 Ammonium fluoride, NH F at least 98 %, for mobile phase (LC)
6.18 Pyridine, C H N, for derivatization (GC)
5 5
6.19 N-Trimethylsilylimidazole, TMSI, C H N Si for derivatization (GC)
6 12 2
6.20 5-(Dimethylamino)naphthalene-1-sulfonyl chloride, dansyl chloride, DNSCl,(C H ClNO S),
12 12 2
LC grade for dansylation (LC)
6.21 Pyridine-3-sulfonyl chloride, Pyridine-3-sulfonyl chloride, PS-Cl, (C H ClNO S), HPLC grade
5 4 2
for derivatization (LC)
6.22 Sodium carbonate concentrate (0,1 M, pH = 10,5), Na CO in water, for derivatization (LC)
2 3
6.23 Sodium bicarbonate concentrate, 0,1 mol/l, for derivatization (LC)
6.24 Sodium hydroxide solution, 10 mol/l, for derivatization (LC) and pH adjument of water sample
6.25 Dansyl reagent solution, for dansylation (LC)
The volume of dansyl reagent to be prepared shall be calculated beforehand because it depends on the
number of samples to be dansyled. To do this, weigh 0,783 mg of dansyl chloride (6.20) for 1 ml of extra
dry acetone (6.10) in an amber glass bottle of suitable volume, then mix well. The dansyl reagent thus
prepared cannot be stored for more than
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