SIST EN ISO 13646:2026

SLOVENSKI STANDARD
01-april-2026
Kakovost vode - Določanje izbranih estrogenov v celotnem vzorcu vode - Metoda
tekočinske kromatografije (LC) ali plinske kromatografije (GC) z masno
selektivnim detektorjem (MS) po ekstrakciji na trdni fazi (SPE) (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)
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)
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)
Ta slovenski standard je istoveten z: EN ISO 13646:2025
ICS:
13.060.50 Preiskava vode na kemične Examination of water for
snovi chemical substances
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 13646
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2025
EUROPÄISCHE NORM
ICS 13.060.50
English Version
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)
Qualité de l'eau - Dosage d'œstrogènes sélectionnés Wasserbeschaffenheit - Bestimmung ausgewählter
dans des échantillons d'eau totale - Méthode par Estrogene in Gesamtwasserproben - Verfahren mittels
extraction en phase solide (SPE) suivie d'une détection Festphasenextraktion (SPE) gefolgt von
par chromatographie en phase liquide (CL) ou en Flüssigkeitschromatographie (LC) oder
phase gazeuse (CG) couplée à la spectrométrie de Gaschromatographie (GC) gekoppelt mit
masse (SM) (ISO 13646:2025) massenspektrometrischer Detektion (MS) (ISO
13646:2025)
This European Standard was approved by CEN on 5 October 2025.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2025 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 13646:2025 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 13646:2025) has been prepared by Technical Committee ISO/TC 147 "Water
quality" in collaboration with Technical Committee CEN/TC 230 “Water analysis” the secretariat of
which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by April 2026, and conflicting national standards shall be
withdrawn at the latest by April 2026.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 13646:2025 has been approved by CEN as EN ISO 13646:2025 without any modification.

International
Standard
ISO 13646
First edition
Water quality — Determination of
2025-10
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)
Reference number
ISO 13646:2025(en) © ISO 2025
ISO 13646:2025(en)
© ISO 2025
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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ISO copyright office
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Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 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 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 . 41
Annex F (informative) Example of LC-HRMS protocol .52
Annex G (informative) Examples of GC-MS/MS protocols .56
Annex H (informative) Example of GC-HRMS protocol . 61
Bibliography .62

iv
ISO 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,
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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,in collaboration with the European Committee for Standardization
(CEN) Technical Committee CEN/TC 230, Water analysis, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
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
ISO 13646:2025(en)
Introduction
Natural and synthetic estrogens 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 can
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).
Besides feminised fish and other endocrine disruptive effects in water ecosystems, they can also be a factor
[16]
in biodiversity loss. 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.

vi
International Standard ISO 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 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 (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.
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 8466-1:2021, Water quality — Calibration and evaluation of analytical methods — Part 1: Linear calibration
function
ISO 21253-1:2019, Water quality — Multi-compound class methods — Part 1: Criteria for the identification of
target compounds by gas and liquid chromatography and mass spectrometry
ISO 11352:2025, Water quality — Estimation of measurement uncertainty based on validation and quality
control data
ISO 13646:2025(en)
3 Terms, definitions and subscripts
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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/
3.1.1
accuracy
closeness of agreement between a measured quantity value and a true quantity value of a measurand
[SOURCE: ISO/IEC Guide 99:2007, 2.13, modified — the terms “measurement accuracy” and “accuracy of
measurement” and Notes 1, 2 and 3 to entry have been deleted.]
3.1.2
analyte
substance to be analyzed
[SOURCE: ISO 6107:2021, 3.31, modified — the domain and Notes 1 and 2 to entry have been deleted.]
3.1.3
blank
aliquot of reagent water (reagent blank) or of a matrix in which the analyte (3.1.2) 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 21253-2:2019, 3.2]
3.1.4
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.
[SOURCE: ISO/IEC Guide 99:2007, 2.39, modified — Note 1 to entry has been deleted.]
3.1.5
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, 5.14, modified — Notes 1, 2, 3, 4 and 5 to entry have been deleted.]

ISO 13646:2025(en)
3.1.6
integrity
property of the parameter(s) of interest, information or content of a sample stored in a container that has
not been altered or lost in an unauthorized manner or that has been subject to loss of representativeness
[SOURCE: ISO 5667-3:2024, 3.2]
3.1.7
isotope dilution quantification
process where isotopically labelled standards (e.g. deuterium- or carbon 13-labeled), which are chemically
similar isotopic analogs of the target analytes (3.1.2), are added to all environmental and quality control and
quality assurance samples before extraction and that follow the analytical procedure
Note 1 to entry: Isotope dilution quantification improves quantitative accuracy (3.1.1) by accounting for sample-
specific procedural losses in the determined analyte concentration
3.1.8
limit of quantification
LOQ
lowest value of a determinand that can be determined with an acceptable level of accuracy (3.1.1), which
could be estimated by different means and verified in the intended matrix
[SOURCE: ISO 21253-2:2019, 3.4]
3.1.9
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]
3.1.10
traceability
property of a measurement result whereby the result can be related to a reference through a documented
unbroken chain of calibrations (3.1.4), each contributing to the measurement uncertainty
[SOURCE: ISO/IEC Guide 99:2007, 2.41, modified — Notes 1, 2, 3, 4, 5, 6, 7 and 8 to entry have been deleted.]
3.1.11
yield
absolute recovery
amount of analyte (3.1.2) 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, 3.11]

ISO 13646:2025(en)
3.2 Subscripts
a measuring variables for addition
A addition
e measuring variables for the calibration
i identity of the substance
I internal standard
j consecutive number for value pairs
g found value
v default value
M measuring solution
P sample
R reference
4 Principle
A water sample is spiked with an appropriate amount of isotopic labelled standard analogous of each
targeted estrogen, before the sample is 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 by
implementing isotope dilution calibration.

ISO 13646:2025(en)
Table 1 — Characteristics of the five selected estrogens
a
Name Structure CAS RN Formula Molecular log K
ow
weight
g/mol
17alpha-ethinylestradiol (EE2)
IUPAC name: (13R,17S)-17-ethynyl-13-me-
57-63-6 C H O 296,40 4,52
20 24 2
thyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]
phenanthrene-3,17-diol
17alpha-estradiol (17αE2)
IUPAC name: (8R,9S,13S,14S,17R)-13-me-
57-91-0 C H O 272,38 4,13
18 24 2
thyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]
phenanthrene-3,17-diol
17beta-estradiol (17βE2)
IUPAC name: (8R,9S,13S,14S,17S)-13-me-
50-28-2 C H O 272,38 4,13
18 24 2
thyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]
phenanthrene-3,17-diol
Estriol (E3)
IUPAC name: (8R,9S,13S,14S,16R,17R)-13-me-
50-27-1 C H O 288,38 2,94
18 24 3
thyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]
phenanthrene-3,16,17-triol
Estrone (E1)
IUPAC name: (8R,9S,13S,14S)-3-hydroxy-13-methyl-
53-16-7 C H O 270,37 3,69
18 22 2
7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenan-
threne-17-one
a
CAS Registry Number® is a trademark of the American Chemical Society (ACS). This information is given for the convenience of users of
this document and does not constitute an endorsement by ISO of the product named. Equivalent products may be used if they can be shown to
lead to the same results.
5 Interferences
5.1 General
Solvents, reagents, glassware and other sample processing hardware can yield artefacts, elevated baselines
or lock-mass suppression causing misinterpretation of chromatograms.
Proper cleaning of glassware is extremely important, because glassware cannot only contaminate the
samples but can 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 30 s can 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 can be
warranted.
Sample contamination is a concern because estrogenic substances are biogenic and can be present on human
skin or can 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

ISO 13646:2025(en)
polytetrafluoroethylene (PTFE) may be used but are not recommended if very low concentration levels
(< 0,1 ng/l) are 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.
The storage temperature is (5 ± 3) °C. For sampling and sample preservation, see Clause 8.
During storage of the test samples, losses of components can occur due to adsorption on the walls of the
containers. The extent of the losses depends on the storage time.
Commercially available solid-phase extraction (SPE cartridge or disk) can 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 use
(see 9.2.2) that can be prejudicial to extraction recovery. To overcome this, a reduction of sample volume or
switching to SPE disk (see 9.2.3) extraction can be implemented. Another solution is the application of glass
wool or sand to the cartridge (filling height 1 cm to 2 cm) to achieve the extraction of higher sample volumes.
Repeated uses and cleaning procedures of glassware can cause active sites on the glass surface that can
irreversibly adsorb the selected estrogens and also be responsible of cross contamination.
To prevent cross contamination between series, SPE apparatus, tubing and items in contact with samples
shall be cleaned with successively, for example, (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 can be present at concentrations several orders
of magnitude higher than the selected estrogens. The most frequently encountered interferences are humic
and other acids. Given that very low levels of estrogens can be measured by this method, elimination of
interferences is essential (implementation of clean-up, see 9.3).
5.3 Interferences during high performance liquid chromatography and mass spectrometry
Substances with similar retention times and masses as the target estrogens can 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/MS. It is critical
to separate both substances. A minimal chromatographic resolution of R ≥ 1,2 must be achieved. If the
criterion cannot be achieved, 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).

ISO 13646:2025(en)
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
()tt−
RR21
R=2 (1)
ww+
bb12
where
R is the resolution;
t , t is the retention time of eluting substances 1 and 2 in seconds (s);
R1 R2
w , w is the peak width at the base of each peak in seconds (s).
b1 b2
NOTE Alternative calculation methods for R are also possible.
For some targeted estrogens, some sensitive transitions have shown a lack of selectivity or 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 implementing a clean-up step (see 9.3) and isotope dilution quantification.
5.4 Interferences during gas phase chromatography and mass spectrometry
Substances with similar retention times and masses as the target estrogens can 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 of R ≥ 1,2 is suitable. If the criterion cannot
be reached, a suitable column shall be chosen to meet the required resolution (see Annex G and Annex H for
an example).
Interferences from accompanying substances (matrix) can occur depending on methods applied.

ISO 13646:2025(en)
5.5 Interferences from internal standards
For mass spectrometric detection, it is recommended to use a stable isotopically labelled analogue as an
13 15
internal standard; C or N-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 can 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 applications, the labelled analogue should preferably have a mass difference of at least 3 Da
to the native target substance.
The isotopic purity of the internal standards shall be checked. It is not a critical factor provided that
its impurities, including unlabelled analogue, have preferably zero contribution on target compounds of
the analytical method. In the event that this is not possible, the contribution of the labelled analogue to the
unlabelled analogue should be negligible at LOQ level.
If internal standards with less than 3 Da are implemented, the above criteria shall be checked and
documented to prevent from false positive and misquantification.
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
as the sample to be measured.
Commercially available mineral water in glass bottles can also be used to determine method blank when
ultrapure water has significant background interference.
6.2 Sodium thiosulfate pentahydrate,w(Na S O ·5H O) of at least 99 %.
2 2 3 2
6.3 Disodium salt of ethylenediaminetetraacetic acid(EDTA), (ρ ≈ 0,025 g/ml), (C H N Na O ⋅2H O),
10 14 2 2 8 2
at least 99 %.
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 acet
...