EN ISO 7012-3:2025

SLOVENSKI STANDARD
01-junij-2025
Barve in laki - Določanje konzervansov v premazih, topnih v vodi - 3. del:
Določanje izotiazolinonov v pločevinki/posodi s tekočinsko kromatografijo LC-UV
in LC-MS (ISO 7012-3:2025)
Paints and varnishes - Determination of preservatives in water-dilutable coating
materials - Part 3: Determination of in-can isothiazolinones with LC-UV and LC-MS (ISO
7012-3:2025)
Beschichtungsstoffe - Bestimmung von Konservierungsmitteln in wasserverdünnbaren
Beschichtungsstoffen - Teil 3: Bestimmung von Isothiazolinonen im Gebinde mit LC-UV
und LC‑MS‑MS (ISO 7012-3:2025)
Peintures et vernis - Dosage des agents de préservation dans les produits de peinture
diluables à l’eau - Partie 3: Dosage des isothiazolinones en pot par CL-UV et CL-SM
(ISO 7012-3:2025)
Ta slovenski standard je istoveten z: EN ISO 7012-3:2025
ICS:
87.040 Barve in laki Paints and varnishes
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 7012-3
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2025
EUROPÄISCHE NORM
ICS 87.040
English Version
Paints and varnishes - Determination of preservatives in
water-dilutable coating materials - Part 3: Determination
of in-can isothiazolinones with LC-UV and LC-MS (ISO
7012-3:2025)
Peintures et vernis - Dosage des agents de préservation Beschichtungsstoffe - Bestimmung von
dans les produits de peinture diluables à l'eau - Partie Konservierungsmitteln in wasserverdünnbaren
3: Dosage des isothiazolinones en pot par CL-UV et CL- Beschichtungsstoffen - Teil 3: Bestimmung von
SM (ISO 7012-3:2025) Isothiazolinonen im Gebinde mit LC-UV und LC-MS-MS
(ISO 7012-3:2025)
This European Standard was approved by CEN on 27 March 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 7012-3:2025 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 7012-3:2025) has been prepared by Technical Committee ISO/TC 35 "Paints
and varnishes" in collaboration with Technical Committee CEN/TC 139 “Paints and varnishes” 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 October 2025, and conflicting national standards shall
be withdrawn at the latest by October 2025.
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 7012-3:2025 has been approved by CEN as EN ISO 7012-3:2025 without any
modification.
International
Standard
ISO 7012-3
First edition
Paints and varnishes —
2025-03
Determination of preservatives in
water-dilutable coating materials —
Part 3:
Determination of in-can
isothiazolinones with LC-UV and LC-
MS
Peintures et vernis — Dosage des agents de préservation dans les
produits de peinture diluables à l’eau —
Partie 3: Dosage des isothiazolinones en pot par CL-UV et CL-SM
Reference number
ISO 7012-3:2025(en) © ISO 2025

ISO 7012-3:2025(en)
© ISO 2025
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
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Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 7012-3:2025(en)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Apparatus . 2
5.1 General .2
5.2 Equipment for extraction and sample preparation .2
5.3 LC-UV/VIS system . .3
5.4 LC-MS/MS or LC-MS system .3
6 Reagents and materials . 3
7 Sampling . 5
8 Procedure . 5
8.1 General .5
8.2 LC-UV/VIS method .5
8.2.1 Extraction of LC-UV/VIS .5
8.2.2 Operation conditions of LC-UV/VIS .6
8.2.3 Blank .6
8.2.4 Interferences of LC-UV/VIS .6
8.2.5 Calibration of LC-UV/VIS . .6
8.2.6 Determination by LC-UV/VIS .7
8.3 LC-MS method .7
8.3.1 Extraction of LC-MS .7
8.3.2 Further dilution . .7
8.3.3 Filtration .8
8.3.4 Operation conditions of LC-MS .8
8.3.5 Blank .8
8.3.6 Interferences of LC-MS .8
8.3.7 Calibration of LC-MS .8
8.3.8 Determination by LC-MS .9
9 Calculation . 9
10 Precision . 10
10.1 General .10
10.2 Repeatability limit,r .10
10.3 Reproducibility limit,R .10
11 Test report . 10
Annex A (informative) Example of LC conditions .12
Annex B (informative) Example of multiple reaction monitoring LC-MS/MS conditions .15
Annex C (normative) Calculation of preservative content with standard addition for LC-MS
method .18
Annex D (informative) Mechanical method for homogenization and extraction.20
Annex E (informative) Results of the interlaboratory test .21
Bibliography .23

iii
ISO 7012-3:2025(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 35, Paints and varnishes, Subcommittee SC 16,
Chemical analysis, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 139, Paints and varnishes, in accordance with the Agreement on technical cooperation
between ISO and CEN (Vienna Agreement).
A list of all parts in the ISO 7012 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
International Standard ISO 7012-3:2025(en)
Paints and varnishes — Determination of preservatives in
water-dilutable coating materials —
Part 3:
Determination of in-can isothiazolinones with LC-UV and LC-MS
1 Scope
This document specifies the apparatus and the analytical methods for determining the content of in-can
isothiazolinone preservatives in water-dilutable coating materials or related products.
This document is also applicable to polymer dispersions.
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 1513, Paints and varnishes — Examination and preparation of test samples
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 15528, Paints, varnishes and raw materials for paints and varnishes — Sampling
3 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
ready for use
state of a product when it is mixed in accordance with the manufacturer’s instructions in the correct
proportions and thinned if required using the correct thinners so that it is ready for application by the
approved method
[SOURCE: ISO 11890-2:2020, 3.7]

ISO 7012-3:2025(en)
3.2
water-dilutable coating material
water-reducible coating material
water-based coating material
water-borne coating material
water-thinnable coating material
DEPRECATED: water paint
coating material whose viscosity is reduced by the addition of water
[SOURCE: ISO 4618:2023, 3.272]
3.3
detection limit of instrument
three times the standard deviation of the result obtained in the blank test using a specific instrument
[SOURCE: ISO 8124-5:2015, 3.5]
3.4
in-can preservative
biocide used to prevent growth of microorganisms during storage of a stock solution of a coating material or
water-based coating material (3.2)
[SOURCE: ISO 4618:2023, 3.141]
4 Principle
The sample is extracted with suitable dilution solvent using an appropriate homogenization method, such as
ultrasonic water bath. The extract is purified by centrifugation, after which the isothiazolinones are identified
and quantified by liquid chromatography (LC) in combination with ultraviolet/visible (UV/VIS) detection
or different mass spectrometry detectors. This includes liquid chromatography-tandem quadrupole mass
spectrometry (LC-MS/MS) and liquid chromatography-single quadrupole mass spectrometry (LC-MS).
For the purposes of this document, LC also designates high-performance liquid chromatography (HPLC) or
ultra-performance liquid chromatography (e.g. UPLC or UHPLC).
Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) have been found suitable.
Other ionization techniques and mass analysers with sufficient performance can also be used.
NOTE The difference between the results is expected to be smaller than the reproducibility limits of the methods
for a standard water-borne coating sample. In the interlaboratory test according to Annex E, the determination by
the LC-UV/VIS method and the LC-MS or LC-MS/MS method turned out to deliver comparable results within the
reproducibility limits of the methods, validating the above statement.
This document is generally applicable for n-octyl-4-isothiazolin-3-one (OIT) and 4,5-dichloro-n-octyl-
4-isothiazolin-3-one (DCOIT). However, the document was not validated for OIT and DCOIT in the
interlaboratory test. There are national standards covering these compounds.
5 Apparatus
5.1 General
The usual laboratory apparatus and, in particular, the following apparatus stated in 5.2 to 5.4 shall be used.
5.2 Equipment for extraction and sample preparation
5.2.1 High speed centrifuge, with an acceleration at a suitable speed.
NOTE A rotation speed of (5 000 - 30 000) revolutions per minute (r/min) has been found suitable.

ISO 7012-3:2025(en)
5.2.2 Ultrasonic water bath, with a frequency from 35 kHz to 45 kHz.
5.2.3 Analytical balance, capable of weighing to an accuracy of 0,1 mg.
5.2.4 One-mark volumetric flask, of 10 ml, 25 ml, 50 ml and 100 ml nominal capacity.
5.2.5 Pipettes, suitable single-volume pipettes, graduated pipettes and Pasteur pipettes.
5.2.6 Centrifuge tubes, with a suitable snap/screw cap.
5.2.7 Glass autosampler vials, with a suitable snap/screw pre-slit cap, 2 ml.
5.2.8 Syringe, minimum volume 2 ml.
5.2.9 Filtration membrane, about 0,22 μm pore size.
5.3 LC-UV/VIS system
5.3.1 LC system, equipped with a sample injection system, a solvent pumping system capable of mixing
solvents, a sample compartment capable of maintaining required temperature and a temperature-controlled
column compartment, a degassing system and data processing software. An LC system that can perform
at the flows, pressures, controlled temperatures, sample volumes and other requirements of the document
shall be used.
5.3.2 Analytical column; reverse phase C18 particle columns were used to develop this test method. Any
column that achieves adequate resolution may be used. The retention times and order of elution may change
depending on the column used and shall be monitored.
5.3.3 UV/VIS detector, or optionally a diode array detector (DAD).
5.4 LC-MS/MS or LC-MS system
5.4.1 LC system, equipped with a sample injection system, a solvent pumping system capable of mixing
solvents, a sample compartment capable of maintaining required temperature and a temperature-controlled
column compartment, a degassing system and data processing software. An LC system that can perform at
the flows, pressures, controlled temperatures, sample volumes, and other requirements of the document
shall be used.
5.4.2 Analytical column, as specified in 5.3.2.
5.4.3 MS/MS or MS-system, equipped with APCI or ESI. Other ionization techniques and mass analysers
with sufficient performance can also be used.
6 Reagents and materials
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only
distilled water or water of grade 1 in accordance with ISO 3696. Solvents shall be of a suitable quality for LC-
UV/VIS and LC-MS, i.e. LC-MS grade for LC-MS analysis.

ISO 7012-3:2025(en)
®1)
6.1 Reference substances: methylisothiazolinone (MIT, CAS RN 2682-20-4), 5-chloro-2-methyl-4-
isothiazolin-3-one (CMIT, CAS RN 26172-55-4), 1,2-benzisothiazolin-3-one (BIT, CAS RN 2634-33-5).
6.2 Methanol (CAS RN 67-56-1).
6.3 Acetonitrile (CAS RN 75-05-8).
6.4 Formic acid (CAS RN 64-18-6).
6.5 Acetic acid (CAS RN 64-19-7), ≥ 99 % mass fraction, optional for better recovery rates.
NOTE The acetic acid is found useful to improve the recovery rate by loosening up the structure of the emulsion paint.
6.6 Ammonium acetate (CAS RN 631-61-8).
6.7 Precipitating agent (optional for better precipitation when using LC-UV/VIS), e.g. aluminium
sulfate ≥ 98 %, Carrez reagent.
A precipitating agent (e.g. aluminium sulfate, Carrez reagent) can be used to clean up the sample solution
after the extraction. In this case, tests shall be carried out to ensure that the use of the precipitating agent
does not reduce the recovery rate as compared to the analysis without precipitation agent.
6.8 Dilution solvent, suitable for diluting the sample. It shall have a purity of at least 99 % by mass or
shall be of known purity, and it shall not contain any substances which interfere with the determination,
e.g. causing overlapping peaks in the chromatogram. Methanol and mixture of water and methanol (1:1 by
volume) have been found suitable.
Alternatively, other suitable solvents may also be used. However, it should be ensured that the sample does
not agglomerate, which can lead to the inclusion of preservatives and thus to analytical errors.
6.9 Internal standard (ISTD) for LC-MS method, which should be added at the extraction step and to
standard working solutions.
Isotope labelled internal standards should be used as much as possible.
6.10 Calibration standard solutions
6.10.1 General
The calibration standard solutions should be prepared in the same dilution solvent as the final sample
solution. All standard solutions used in this method shall be prepared as described in 6.10.
Commercially available standard stock solutions with known content may also be used. If necessary, the
content of these solutions shall be determined using certified/traceable standards (see 6.1).
NOTE If commercially available standard stock solution is used, standard working solutions are prepared in one-
mark volumetric flask (5.2.4) by diluting the stock solution with dilution solvent (6.8).
6.10.2 Standard stock solutions
Weigh each of the target reference substances (6.1) into a 100 ml one-mark volumetric flask (5.2.4) at
concentrations that are sufficiently high to allow the preparation of standard working solutions. Fill up
to the mark with the dilution solvent (6.8) and let them dissolve completely. Transfer the standard stock
solutions to amber-glass flask and seal with glass cap or PTFE-coated cap. Seal the bottle neck again with
1) 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.

ISO 7012-3:2025(en)
flexible laboratory sealing film. The preparation of standard stock solutions shall be repeated after a time
period that shall be specified by the laboratory, after which a degradation can occur.
NOTE Normally, a stock solution can be kept in the refrigerator at 2 °C to 6 °C in the dark for up to 1 month, when
stability has been proven, or according to the expiry date of the producer.
Given the broad applicability of this method and the partly divergent pH-stability of preservatives, analyte
mixtures of different composition can be necessary.
6.10.3 Standard working solutions
Prepare standard working solutions of a minimum of five concentration levels from the standard stock
solution (6.10.2) by diluting with dilution solvent (6.8). It is possible that they are stable for only a few days.
The preparation of standard working solutions shall be repeated after a time period that shall be specified
by the laboratory, after which a degradation can occur.
If internal standard is used, a fixed volume of internal standard solution shall be added to the standard
working solutions before dilution.
7 Sampling
Take a representative sample of the product to be tested (or of each product in the case of a multi-coat
system) as specified in ISO 15528.
Examine and prepare each sample for testing, as specified in ISO 1513, preparing the final sample for testing
in the “ready for use” state.
8 Procedure
8.1 General
Carry out all tests by taking duplicate coating samples as described in Clause 7.
8.2 LC-UV/VIS method
8.2.1 Extraction of LC-UV/VIS
Weigh, to the nearest 0,1 mg, 2 g ± 0,2 g of the test sample into a 25 ml one-mark volumetric flask (5.2.4).
Add about 10 ml dilution solvent (6.8) to the one-mark volumetric flasks, shake it in the ultrasonic water
bath (5.2.2) until it is homogenized (typically 3 min of manual shaking is sufficient), fill up to the mark with
dilution solvent (6.8) and shake again until it is homogenized (typically 30 s of manual shaking is sufficient).
Another sample mass may also be used, as long as the mass of the sample and the volume of the one-mark
...