SIST EN ISO 21676:2021
(Main)Water quality - Determination of the dissolved fraction of selected active pharmaceutical ingredients, transformation products and other organic substances in water and treated waste water - Method using high performance liquid chromatography and mass spectrometric detection (HPLC-MS/MS or -HRMS) after direct injection (ISO 21676:2018)
Water quality - Determination of the dissolved fraction of selected active pharmaceutical ingredients, transformation products and other organic substances in water and treated waste water - Method using high performance liquid chromatography and mass spectrometric detection (HPLC-MS/MS or -HRMS) after direct injection (ISO 21676:2018)
This document specifies a method for the determination of the dissolved fraction of selected active pharmaceutical ingredients and transformation products, as well as other organic substances (see Table 1 of the document) in drinking water, ground water, surface water and treated waste water.
The lower application range of this method can vary depending on the sensitivity of the equipment used and the matrix of the sample. For most compounds to which this document applies, the range is ≥ 0,025 μg/l for drinking water, ground water and surface water, and ≥ 0,050 μg/l for treated waste water.
The method can be used to determine further organic substances or in other types of water (e.g. process water) provided that accuracy has been tested and verified for each case, and that storage conditions of both samples and reference solutions have been validated.
Wasserbeschaffenheit - Bestimmung ausgewählter Arzneimittelwirkstoffe, Transformationsprodukte und weiterer organischer Stoffe gelöst in Wasser und gereinigtem Abwasser - Verfahren mittels Hochleistungs-Flüssigkeitschromatographie und massenspektrometrischer Detektion (HPLC-MS/MS oder -HRMS) nach Direktinjektion (ISO 21676:2018)
Dieses Dokument legt ein Verfahren zur Bestimmung des gelösten Anteils ausgewählter Arzneimittelwirkstoffe und Transformationsprodukte sowie weiterer organischer Substanzen (siehe Tabelle 1) in Trink-, Grund-, Oberflächen- und behandeltem Abwasser fest.
Der untere Anwendungsbereich des Verfahrens kann abhängig von der Empfindlichkeit der verwendeten Geräte und von der Matrix der Probe variieren. Für die meisten Verbindungen, für die dieses Dokument Anwendung findet, ist der Bereich ≥ 0,025 µg/l in Trink-, Grund- und Oberflächenwasser und ≥ 0,050 µg/l in behandeltem Abwasser.
Das Verfahren kann zur Bestimmung weiterer organischer Stoffe oder in anderen Wasserarten angewandt werden (z. B.: Brauchwasser), wenn die Richtigkeit im Einzelfall geprüft und nachgewiesen wurde und die Lagerbedingungen von Proben und Referenzlösungen validiert wurden. Tabelle 1 zeigt die Substanzen, für die eine Bestimmung nach dem Verfahren getestet wurde. Tabelle E.1 enthält Beispiele für die Bestimmung anderer organischer Substanzen.
[Tabelle 1]
Qualité de l'eau - Détermination de la fraction dissoute des ingrédients pharmaceutiques actifs sélectionnés, des produits de la transformation et d'autres substances organiques dans l'eau et dans l'eau résiduaire - Méthode par chromatographie en phase liquide à haute performance et détection par spectrométrie de masse (CLHP-MS/MS ou -HRSM) après l'injection directe (ISO 21676:2018)
Le présent document spécifie une méthode de détermination de la fraction dissoute des principes actifs pharmaceutiques sélectionnés et de leurs produits de transformation, ainsi que d'autres substances organiques (voir Tableau 1), dans l'eau potable, les eaux souterraines, les eaux de surface et les eaux usées traitées.
La gamme d'application basse de la présente méthode peut varier selon la sensibilité de l'équipement utilisé et la matrice de l'échantillon. Pour la plupart des composés concernés par le présent document, la gamme est ≥ 0,025 µg/l pour l'eau potable, les eaux souterraines et les eaux de surface, et ≥ 0,050 µg/l pour les eaux usées traitées.
La présente méthode peut être utilisée pour déterminer d'autres substances organiques ou pour d'autres types d'eaux (par exemple, l'eau de process), à condition que l’exactitude ait été testée et vérifiée dans chaque cas, et que les conditions de conservation des échantillons et des solutions de référence aient été validées. Le Tableau 1 indique les substances pour lesquelles la présente méthode a été appliquée. Le Tableau E.1 fournit d’autres exemples de substances organiques pour lesquelles la présente méthode peut être utilisée.
Kakovost vode - Določevanje raztopljenih frakcij izbranih aktivnih farmacevtskih učinkovin, produktov razgradnje in drugih organskih spojin v vodi in obdelani odpadni vodi - Metoda tekočinske kromatografije visoke ločljivosti in masne spektrometrije (HPLC-MS/MS ali -HRMS) po neposrednem injiciranju (ISO 21676:2018)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 21676:2021
01-oktober-2021
Kakovost vode - Določevanje raztopljenih frakcij izbranih aktivnih farmacevtskih
učinkovin, produktov razgradnje in drugih organskih spojin v vodi in obdelani
odpadni vodi - Metoda tekočinske kromatografije visoke ločljivosti in masne
spektrometrije (HPLC-MS/MS ali -HRMS) po neposrednem injiciranju (ISO
21676:2018)
Water quality - Determination of the dissolved fraction of selected active pharmaceutical
ingredients, transformation products and other organic substances in water and treated
waste water - Method using high performance liquid chromatography and mass
spectrometric detection (HPLC-MS/MS or -HRMS) after direct injection (ISO
21676:2018)
Wasserbeschaffenheit - Bestimmung ausgewählter Arzneimittelwirkstoffe,
Transformationsprodukte und weiterer organischer Stoffe gelöst in Wasser und
gereinigtem Abwasser - Verfahren mittels Hochleistungs-Flüssigkeitschromatographie
und massenspektrometrischer Detektion (HPLC-MS/MS oder -HRMS) nach
Direktinjektion (ISO 21676:2018)
Qualité de l'eau - Détermination de la fraction dissoute des ingrédients pharmaceutiques
actifs sélectionnés, des produits de la transformation et d'autres substances organiques
dans l'eau et dans l'eau résiduaire - Méthode par chromatographie en phase liquide à
haute performance et détection par spectrométrie de masse (CLHP-MS/MS ou -HRSM)
après l'injection directe (ISO 21676:2018)
Ta slovenski standard je istoveten z: EN ISO 21676:2021
ICS:
13.060.50 Preiskava vode na kemične Examination of water for
snovi chemical substances
SIST EN ISO 21676:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 21676:2021
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SIST EN ISO 21676:2021
EN ISO 21676
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2021
EUROPÄISCHE NORM
ICS 13.060.50
English Version
Water quality - Determination of the dissolved fraction of
selected active pharmaceutical ingredients, transformation
products and other organic substances in water and
treated waste water - Method using high performance
liquid chromatography and mass spectrometric detection
(HPLC-MS/MS or -HRMS) after direct injection (ISO
21676:2018)
Qualité de l'eau - Détermination de la fraction dissoute Wasserbeschaffenheit - Bestimmung ausgewählter
des ingrédients pharmaceutiques actifs sélectionnés, Arzneimittelwirkstoffe, Transformationsprodukte und
des produits de la transformation et d'autres weiterer organischer Stoffe gelöst in Wasser und
substances organiques dans l'eau et dans l'eau gereinigtem Abwasser - Verfahren mittels
résiduaire - Méthode par chromatographie en phase Hochleistungs-Flüssigkeitschromatographie und
liquide à haute performance et détection par massenspektrometrischer Detektion (HPLC-MS/MS
spectrométrie de masse (CLHP-MS/MS ou -HRSM) oder -HRMS) nach Direktinjektion (ISO 21676:2018)
après l'injection directe (ISO 21676:2018)
This European Standard was approved by CEN on 18 July 2021.
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, Turkey 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
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21676:2021 E
worldwide for CEN national Members.
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SIST EN ISO 21676:2021
EN ISO 21676:2021 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 21676:2021
EN ISO 21676:2021 (E)
European foreword
The text of ISO 21676:2018 has been prepared by Technical Committee ISO/TC 147 "Water quality” of
the International Organization for Standardization (ISO) and has been taken over as EN ISO 21676:2021
by 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 February 2022, and conflicting national standards
shall be withdrawn at the latest by February 2022.
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.
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, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 21676:2018 has been approved by CEN as EN ISO 21676:2021 without any modification.
3
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SIST EN ISO 21676:2021
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SIST EN ISO 21676:2021
INTERNATIONAL ISO
STANDARD 21676
First edition
2018-10
Water quality — Determination of
the dissolved fraction of selected
active pharmaceutical ingredients,
transformation products and
other organic substances in
water and treated waste water —
Method using high performance
liquid chromatography and mass
spectrometric detection (HPLC-MS/MS
or -HRMS) after direct injection
Qualité de l'eau — Détermination de la fraction dissoute des
ingrédients pharmaceutiques actifs sélectionnés, des produits de la
transformation et d'autres substances organiques dans l'eau et dans
l'eau résiduaire — Méthode par chromatographie en phase liquide à
haute performance et détection par spectrométrie de masse (CLHP-
MS/MS ou -HRSM) après l'injection directe
Reference number
ISO 21676:2018(E)
©
ISO 2018
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SIST EN ISO 21676:2021
ISO 21676:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018
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
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Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
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SIST EN ISO 21676:2021
ISO 21676:2018(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 4
3 Terms and definitions . 4
4 Principle . 4
5 Interferences . 4
5.1 During sample preparation . 4
5.2 During high performance liquid chromatography and mass spectrometry . 4
6 Reagents . 5
6.1 General . 5
6.2 Preparation of solutions . 5
7 Apparatus . 7
8 Sampling . 8
9 Procedure. 8
9.1 General . 8
9.2 Sample preparation . 8
9.3 High performance liquid chromatography (HPLC) . 9
9.4 Detection . 9
9.4.1 General. 9
9.4.2 Tandem mass spectrometry (MS/MS) .10
9.4.3 High-resolution mass spectrometry (HRMS) .10
9.5 Blank value measurements .10
10 Calibration .10
10.1 General .10
10.2 Calibration with external standard .12
10.3 Calibration with internal standard .12
11 Calculation of recovery .13
11.1 General .13
11.2 Calculation of analyte recovery using samples .13
11.3 Recovery of internal standards .14
12 Evaluation .14
12.1 Verification of individual substances .14
12.2 Calculation of the individual results using calibration with an external standard .15
12.3 Calculation of the individual results using calibration with an internal standard .15
13 Expression of results .16
14 Test report .16
Annex A (informative) Performance data .17
Annex B (informative) Examples of recovery .22
Annex C (informative) Examples of HPLC columns and chromatograms .24
Annex D (informative) Examples of detection .30
Annex E (informative) Examples of extension of the method .33
Bibliography .34
© ISO 2018 – All rights reserved iii
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SIST EN ISO 21676:2021
ISO 21676:2018(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 documents 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).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
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.
iv © ISO 2018 – All rights reserved
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SIST EN ISO 21676:2021
ISO 21676:2018(E)
Introduction
Pharmaceutical ingredients are essential for human and animal health. Through application or improper
disposal, active pharmaceutical ingredients enter the water cycle unchanged or transformed. This can
happen via municipal waste water, treated at treatment plants. There, some active pharmaceutical
ingredients and transformation products cannot be removed completely from the waste water by
conventional treatment techniques. Active pharmaceutical ingredients and their transformation
products also travel through sludge to the soil and subsequently enter water bodies via leachate,
depending on the nature of the ground and the active ingredients. Active pharmaceutical ingredients
and their transformation products are therefore found in treated waste water, as well as in surface
and ground water. This document specifies a liquid chromatography method with mass spectrometric
detection for the determination of selected active pharmaceutical ingredients and their transformation
products in the dissolved fraction.
© ISO 2018 – All rights reserved v
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SIST EN ISO 21676:2021
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SIST EN ISO 21676:2021
INTERNATIONAL STANDARD ISO 21676:2018(E)
Water quality — Determination of the dissolved fraction of
selected active pharmaceutical ingredients, transformation
products and other organic substances in water and
treated waste water — Method using high performance
liquid chromatography and mass spectrometric detection
(HPLC-MS/MS or -HRMS) after direct injection
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 a method for the determination of the dissolved fraction of selected active
pharmaceutical ingredients and transformation products, as well as other organic substances
(see Table 1) in drinking water, ground water, surface water and treated waste water.
The lower application range of this method can vary depending on the sensitivity of the equipment used
and the matrix of the sample. For most compounds to which this document applies, the range is ≥ 0,025 µg/l
for drinking water, ground water and surface water, and ≥ 0,050 µg/l for treated waste water.
The method can be used to determine further organic substances or in other types of water (e.g.
process water) provided that accuracy has been tested and verified for each case, and that storage
conditions of both samples and reference solutions have been validated. Table 1 shows the substances
for which a determination was tested in accordance with the method. Table E.1 provides examples of
the determination of other organic substances.
Table 1 — Substances for which a determination was tested in accordance with this method
b
Common name Molecular Molar CAS-RN
a
Chemical name (IUPAC ) formula mass
g/mol
4-Acetylaminoantipyrine
C H N O 245,28 83-15-8
13 15 3 2
N-(2,3-Dimethyl-5-oxo-1-phenyl-3-pyrazolin-4-yl)acetamide
N4-Acetyl sulfamethoxazole
C H N O S 295,32 21312-10-7
12 13 3 4
N-{4-[(5-Methyl-1,2-oxazol-3-yl)sulfamoyl]phenyl}-acetamide
Diatrizoic acid (amidotricoic acid)
C H I N O 613,91 117-96-4
11 9 3 2 4
3,5-Bis(acetamido)-2,4,6-triiodobenzoic acid
Atenolol
C H N O 266,34 29122-68-7
14 22 2 3
(RS)-2-[4-[2-Hydroxy-3-(1-methylethylamino) propoxy]phenyl]
ethanamide
a
IUPAC: International Union of Pure and Applied Chemistry.
b
CAS-RN: Chemical Abstracts System Registration Number.
© ISO 2018 – All rights reserved 1
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SIST EN ISO 21676:2021
ISO 21676:2018(E)
Table 1 (continued)
b
Common name Molecular Molar CAS-RN
a
Chemical name (IUPAC ) formula mass
g/mol
Bezafibrate
C H ClNO 361,80 41859-67-0
19 20 4
2-{4-[2-(4-Chlorbenzamido)ethyl]phenoxyl}-2-
methylpropanoic acid
Bisoprolol
C H NO 325,45 66722-44-9
18 31 4
(RS)-1-[4-(2-Isopropoxyethoxymethyl)phenoxy]-3-
isopropylamino-2-propanol
Carbamazepine
C H N O 236,27 298-46-4
15 12 2
5H-Dibenzo[b,f]azepine-5-carbamide
Clarithromycin
(2R,3R,4S,5R,8R,9S,10S,11R,12R,14R)-11-[(2S,3R,4S,6R)-4-
(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy-5-ethyl-
C H NO 747,95 81103-11-9
38 69 13
3,4-dihydroxy-9-[(2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-
dimethyl-oxan-2-yl]oxy-12-methoxy-2,4,8,10,12,14-hexa-
methyl-6-oxacyclotetradecane-1,7-dione
Clofibric acid
C H ClO 214,70 882-09-7
10 11 3
2-(4-Chlorophenoxy)-2-methylpropanoic acid
Dehydrato-Erythromycin (anhydro-erythromycin)
(2R,3R,4S,5S,8R,9S,10S,11R,12R)-11-{[4-(dimethylamino)-3-hy-
C H NO 715,91 23893-13-2
37 65 12
droxy-6-methyloxan-2-yl]oxy}-5-ethyl-3-hydroxy-9-[(5-hydroxy-
4-methoxy-4,6-dimethyloxan-2-yl)oxy]-2,4,8,10,12,14-hexame-
thyl-6,15,16-trioxatricyclo[10.2.1.1{1,4}]hexadecane-7-one
Diazepam
C H ClN O 284,74 439-14-5
16 13 2
(RS)-7-Chlor-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-
benzodiazepine-2-on
Diclofenac
C H Cl NO 296,15 15307-86-5
14 11 2 2
2-[2-[(2,6-Dichlorphenyl)amino]phenyl]acetic acid
10,11-Dihydro-10,11-dihydroxy carbamazepine
C H N O 270,29 58955-93-4
15 14 2 3
(5S,6S)-5,6-Dihydroxy-5,6-dihydrobenzo[b][1]benzazepie-
11-carboxamide
Erythromycin
6-(4-Dimethylamino-3-hydroxy-6-methyl-oxan-2-yl)oxy-
C H NO 733,93 114-07-8
37 67 13
14-ethyl-7,12,13-trihydroxy-4-(5-hydroxy-4-methoxy-4,6-
dimethyl-oxan-2-yl)-oxy-3,5,7,9,11,13-hexamethyl-1-oxacyclo-
tetradecane-2,10-dione
4-Formylaminoantipyrine
C H N O 231,25 1672-58-8
12 13 3 2
N-(2,3-Dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl)
formamide
Gemfibrozil
C H O 250,34 25812-30-0
15 22 3
5-(2,5-Chlorophenoxy)-2,2-methylpropanoic acid
Ibuprofen
C H O 206,28 15687-27-1
13 18 2
(RS)-2-[4-(2-Methylpropyl)phenyl]propanoic acid
a
IUPAC: International Union of Pure and Applied Chemistry.
b
CAS-RN: Chemical Abstracts System Registration Number.
2 © ISO 2018 – All rights reserved
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SIST EN ISO 21676:2021
ISO 21676:2018(E)
Table 1 (continued)
b
Common name Molecular Molar CAS-RN
a
Chemical name (IUPAC ) formula mass
g/mol
Iomeprol
C H I N O 777,09 78649-41-9
17 22 3 3 8
(±)-N,N′-Bis-(2,3-dihydroxypropyl)-5-[(2-hydroxy-acetyl)
methylamino]-2,4,6-triiodo isophthalamide
Iopamidol
C H I N O 777,08 60166-93-0
17 22 3 3 3
(S)-N,N′-Bis[2-hydroxy-1-(hydroxymethyl)ethyl]-5-[(2-hy-
droxypropanoyl)amino]-2,4,6-triiodobenzene-1,3-dicarbamide
Iopromide
C H I N O 791,12 73334-07-3
18 24 3 3 8
(±)-N,N′-Bis(2,3-dihydroxypropyl)-2,4,6-triiodo-5-
(2-methoxyacetamido)-N-methylisophthalamide
Metoprolol
C H NO 267,36 37350-58-6
15 25 3
(RS)-1-(Isopropylamino)-3-[4-(2-methoxyethyl) phenoxy]
propan-2-ol
Naproxen
C H O 230,26 22204-53-1
14 14 3
(S)-2-(6-Methoxy-2-naphthyl)propanoic acid
Oxazepam
C H ClN O 286,71 604-75-1
15 11 2 2
(RS)-7-Chloro-3-hydroxy-5-phenyl-1,3-dihydro-2H-1,4-
benzodiazepin-2-on
Phenazone
C H N O 188,23 60-80-0
11 12 2
1,5-Dimethyl-2-phenyl-2,3-dihydro-1H-pyrazol-3-on
Primidone
C H N O 218,25 125-33-7
12 14 2 2
5-Ethyl-5-phenylhexahydropyrimidin-4,6-dione
Propyphenazone
C H N O 230,31 479-92-5
14 18 2
1,5-Dimethyl-4-(1-methylethyl)-2-phenyl-1,2-dihydro-3H-
pyrazol-3-one
Roxithromycin
(3R,4S,5S,6R,7R,9R,11S,12R,13S,14R)-6-{[(2S,3R,4S,6R)-
4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]
C H N O 837,05 80214-83-1
41 76 2 15
oxy}-14-ethyl-7,12,13-trihydroxy-4-{[(2R,4R,5S,6S)-5-hy-
droxy-4-methoxy-4,6-dimethyloxan-2-yl]oxy}-3,5,7,9,11,13-
hexamethyl-10-(2,4,7-trioxa-1-azaoctan-1-ylidene)-1-
oxacyclotetradecane-2-one
Sotalol
C H N O S 272,36 3930-20-9
12 20 2 3
(RS)-4′-(1-Hydroxy-2-isopropylaminoethyl)
methanesulfonanilide
Sulfamethoxazole
C H N O S 253,28 723-46-6
10 11 3 3
4-Amino-N-(5-methyl-1,2-oxazol-3-yl)benzene-sulfonamide
Temazepam
C H ClN O 300,74 846-50-4
16 13 2 2
(RS)-7-Chloro-3-hydroxy-1-methyl-5-phenyl-1,3-dihydro-2H-
1,4-benzodiazepin-2-one
Trimethoprim
C H N O 290,32 738-70-5
14 18 4 3
2,4-Diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine
a
IUPAC: International Union of Pure and Applied Chemistry.
b
CAS-RN: Chemical Abstracts System Registration Number.
© ISO 2018 – All rights reserved 3
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SIST EN ISO 21676:2021
ISO 21676:2018(E)
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 1042, Laboratory glassware — One-mark volumetric flasks
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 4796-2, Laboratory glassware — Bottles — Part 2: Conical neck bottles
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 5667-10, Water quality — Sampling — Part 10: Guidance on sampling of waste waters
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: Statistical evaluation of the linear calibration function
3 Terms and definitions
No terms and definitions are listed in this document.
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 http: //www .electropedia .org/
4 Principle
The water sample is injected directly into the analysis system. The identification and quantitative
determination is performed using high performance liquid chromatography coupled with mass
spectrometric detection (HPLC-MS/MS, HPLC-HRMS).
5 Interferences
5.1 During sample preparation
Loss of analytes can occur during filtration of the sample as a result of sorption.
5.2 During high performance liquid chromatography and mass spectrometry
Peak tailing, peak fronting and/or wide peaks are indications of a malfunctioning of HPLC and/or
interferences occurring during chromatography. However, some compounds tend to show more signa
...
SLOVENSKI STANDARD
oSIST prEN ISO 21676:2021
01-april-2021
Kakovost vode - Določevanje raztopljenih frakcij izbranih aktivnih farmacevtskih
učinkovin, produktov razgradnje in drugih organskih spojin v vodi in obdelani
odpadni vodi - Metoda tekočinske kromatografije visoke ločljivosti in masne
spektrometrije (HPLC-MS/MS ali -HRMS) po neposrednem injiciranju (ISO
21676:2018)
Water quality - Determination of the dissolved fraction of selected active pharmaceutical
ingredients, transformation products and other organic substances in water and treated
waste water - Method using high performance liquid chromatography and mass
spectrometric detection (HPLC-MS/MS or -HRMS) after direct injection (ISO
21676:2018)
Wasserbeschaffenheit - Bestimmung ausgewählter Arzneimittelwirkstoffe,
Transformationsprodukte und weiterer organischer Stoffe gelöst in Wasser und
gereinigtem Abwasser - Verfahren mittels Hochleistungs-Flüssigkeitschromatographie
und massenspektrometrischer Detektion (HPLC-MS/MS oder -HRMS) nach
Direktinjektion (ISO 21676:2018)
Qualité de l'eau - Détermination de la fraction dissoute des ingrédients pharmaceutiques
actifs sélectionnés, des produits de la transformation et d'autres substances organiques
dans l'eau et dans l'eau résiduaire - Méthode par chromatographie en phase liquide à
haute performance et détection par spectrométrie de masse (CLHP-MS/MS ou -HRSM)
après l'injection directe (ISO 21676:2018)
Ta slovenski standard je istoveten z: prEN ISO 21676
ICS:
13.060.50 Preiskava vode na kemične Examination of water for
snovi chemical substances
oSIST prEN ISO 21676:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 21676:2021
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oSIST prEN ISO 21676:2021
INTERNATIONAL ISO
STANDARD 21676
First edition
2018-10
Water quality — Determination of
the dissolved fraction of selected
active pharmaceutical ingredients,
transformation products and
other organic substances in
water and treated waste water —
Method using high performance
liquid chromatography and mass
spectrometric detection (HPLC-MS/MS
or -HRMS) after direct injection
Qualité de l'eau — Détermination de la fraction dissoute des
ingrédients pharmaceutiques actifs sélectionnés, des produits de la
transformation et d'autres substances organiques dans l'eau et dans
l'eau résiduaire — Méthode par chromatographie en phase liquide à
haute performance et détection par spectrométrie de masse (CLHP-
MS/MS ou -HRSM) après l'injection directe
Reference number
ISO 21676:2018(E)
©
ISO 2018
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oSIST prEN ISO 21676:2021
ISO 21676:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
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oSIST prEN ISO 21676:2021
ISO 21676:2018(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 4
3 Terms and definitions . 4
4 Principle . 4
5 Interferences . 4
5.1 During sample preparation . 4
5.2 During high performance liquid chromatography and mass spectrometry . 4
6 Reagents . 5
6.1 General . 5
6.2 Preparation of solutions . 5
7 Apparatus . 7
8 Sampling . 8
9 Procedure. 8
9.1 General . 8
9.2 Sample preparation . 8
9.3 High performance liquid chromatography (HPLC) . 9
9.4 Detection . 9
9.4.1 General. 9
9.4.2 Tandem mass spectrometry (MS/MS) .10
9.4.3 High-resolution mass spectrometry (HRMS) .10
9.5 Blank value measurements .10
10 Calibration .10
10.1 General .10
10.2 Calibration with external standard .12
10.3 Calibration with internal standard .12
11 Calculation of recovery .13
11.1 General .13
11.2 Calculation of analyte recovery using samples .13
11.3 Recovery of internal standards .14
12 Evaluation .14
12.1 Verification of individual substances .14
12.2 Calculation of the individual results using calibration with an external standard .15
12.3 Calculation of the individual results using calibration with an internal standard .15
13 Expression of results .16
14 Test report .16
Annex A (informative) Performance data .17
Annex B (informative) Examples of recovery .22
Annex C (informative) Examples of HPLC columns and chromatograms .24
Annex D (informative) Examples of detection .30
Annex E (informative) Examples of extension of the method .33
Bibliography .34
© ISO 2018 – All rights reserved iii
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oSIST prEN ISO 21676:2021
ISO 21676:2018(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 documents 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).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
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.
iv © ISO 2018 – All rights reserved
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oSIST prEN ISO 21676:2021
ISO 21676:2018(E)
Introduction
Pharmaceutical ingredients are essential for human and animal health. Through application or improper
disposal, active pharmaceutical ingredients enter the water cycle unchanged or transformed. This can
happen via municipal waste water, treated at treatment plants. There, some active pharmaceutical
ingredients and transformation products cannot be removed completely from the waste water by
conventional treatment techniques. Active pharmaceutical ingredients and their transformation
products also travel through sludge to the soil and subsequently enter water bodies via leachate,
depending on the nature of the ground and the active ingredients. Active pharmaceutical ingredients
and their transformation products are therefore found in treated waste water, as well as in surface
and ground water. This document specifies a liquid chromatography method with mass spectrometric
detection for the determination of selected active pharmaceutical ingredients and their transformation
products in the dissolved fraction.
© ISO 2018 – All rights reserved v
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oSIST prEN ISO 21676:2021
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oSIST prEN ISO 21676:2021
INTERNATIONAL STANDARD ISO 21676:2018(E)
Water quality — Determination of the dissolved fraction of
selected active pharmaceutical ingredients, transformation
products and other organic substances in water and
treated waste water — Method using high performance
liquid chromatography and mass spectrometric detection
(HPLC-MS/MS or -HRMS) after direct injection
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 a method for the determination of the dissolved fraction of selected active
pharmaceutical ingredients and transformation products, as well as other organic substances
(see Table 1) in drinking water, ground water, surface water and treated waste water.
The lower application range of this method can vary depending on the sensitivity of the equipment used
and the matrix of the sample. For most compounds to which this document applies, the range is ≥ 0,025 µg/l
for drinking water, ground water and surface water, and ≥ 0,050 µg/l for treated waste water.
The method can be used to determine further organic substances or in other types of water (e.g.
process water) provided that accuracy has been tested and verified for each case, and that storage
conditions of both samples and reference solutions have been validated. Table 1 shows the substances
for which a determination was tested in accordance with the method. Table E.1 provides examples of
the determination of other organic substances.
Table 1 — Substances for which a determination was tested in accordance with this method
b
Common name Molecular Molar CAS-RN
a
Chemical name (IUPAC ) formula mass
g/mol
4-Acetylaminoantipyrine
C H N O 245,28 83-15-8
13 15 3 2
N-(2,3-Dimethyl-5-oxo-1-phenyl-3-pyrazolin-4-yl)acetamide
N4-Acetyl sulfamethoxazole
C H N O S 295,32 21312-10-7
12 13 3 4
N-{4-[(5-Methyl-1,2-oxazol-3-yl)sulfamoyl]phenyl}-acetamide
Diatrizoic acid (amidotricoic acid)
C H I N O 613,91 117-96-4
11 9 3 2 4
3,5-Bis(acetamido)-2,4,6-triiodobenzoic acid
Atenolol
C H N O 266,34 29122-68-7
14 22 2 3
(RS)-2-[4-[2-Hydroxy-3-(1-methylethylamino) propoxy]phenyl]
ethanamide
a
IUPAC: International Union of Pure and Applied Chemistry.
b
CAS-RN: Chemical Abstracts System Registration Number.
© ISO 2018 – All rights reserved 1
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oSIST prEN ISO 21676:2021
ISO 21676:2018(E)
Table 1 (continued)
b
Common name Molecular Molar CAS-RN
a
Chemical name (IUPAC ) formula mass
g/mol
Bezafibrate
C H ClNO 361,80 41859-67-0
19 20 4
2-{4-[2-(4-Chlorbenzamido)ethyl]phenoxyl}-2-
methylpropanoic acid
Bisoprolol
C H NO 325,45 66722-44-9
18 31 4
(RS)-1-[4-(2-Isopropoxyethoxymethyl)phenoxy]-3-
isopropylamino-2-propanol
Carbamazepine
C H N O 236,27 298-46-4
15 12 2
5H-Dibenzo[b,f]azepine-5-carbamide
Clarithromycin
(2R,3R,4S,5R,8R,9S,10S,11R,12R,14R)-11-[(2S,3R,4S,6R)-4-
(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy-5-ethyl-
C H NO 747,95 81103-11-9
38 69 13
3,4-dihydroxy-9-[(2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-
dimethyl-oxan-2-yl]oxy-12-methoxy-2,4,8,10,12,14-hexa-
methyl-6-oxacyclotetradecane-1,7-dione
Clofibric acid
C H ClO 214,70 882-09-7
10 11 3
2-(4-Chlorophenoxy)-2-methylpropanoic acid
Dehydrato-Erythromycin (anhydro-erythromycin)
(2R,3R,4S,5S,8R,9S,10S,11R,12R)-11-{[4-(dimethylamino)-3-hy-
C H NO 715,91 23893-13-2
37 65 12
droxy-6-methyloxan-2-yl]oxy}-5-ethyl-3-hydroxy-9-[(5-hydroxy-
4-methoxy-4,6-dimethyloxan-2-yl)oxy]-2,4,8,10,12,14-hexame-
thyl-6,15,16-trioxatricyclo[10.2.1.1{1,4}]hexadecane-7-one
Diazepam
C H ClN O 284,74 439-14-5
16 13 2
(RS)-7-Chlor-1-methyl-5-phenyl-1,3-dihydro-2H-1,4-
benzodiazepine-2-on
Diclofenac
C H Cl NO 296,15 15307-86-5
14 11 2 2
2-[2-[(2,6-Dichlorphenyl)amino]phenyl]acetic acid
10,11-Dihydro-10,11-dihydroxy carbamazepine
C H N O 270,29 58955-93-4
15 14 2 3
(5S,6S)-5,6-Dihydroxy-5,6-dihydrobenzo[b][1]benzazepie-
11-carboxamide
Erythromycin
6-(4-Dimethylamino-3-hydroxy-6-methyl-oxan-2-yl)oxy-
C H NO 733,93 114-07-8
37 67 13
14-ethyl-7,12,13-trihydroxy-4-(5-hydroxy-4-methoxy-4,6-
dimethyl-oxan-2-yl)-oxy-3,5,7,9,11,13-hexamethyl-1-oxacyclo-
tetradecane-2,10-dione
4-Formylaminoantipyrine
C H N O 231,25 1672-58-8
12 13 3 2
N-(2,3-Dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl)
formamide
Gemfibrozil
C H O 250,34 25812-30-0
15 22 3
5-(2,5-Chlorophenoxy)-2,2-methylpropanoic acid
Ibuprofen
C H O 206,28 15687-27-1
13 18 2
(RS)-2-[4-(2-Methylpropyl)phenyl]propanoic acid
a
IUPAC: International Union of Pure and Applied Chemistry.
b
CAS-RN: Chemical Abstracts System Registration Number.
2 © ISO 2018 – All rights reserved
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oSIST prEN ISO 21676:2021
ISO 21676:2018(E)
Table 1 (continued)
b
Common name Molecular Molar CAS-RN
a
Chemical name (IUPAC ) formula mass
g/mol
Iomeprol
C H I N O 777,09 78649-41-9
17 22 3 3 8
(±)-N,N′-Bis-(2,3-dihydroxypropyl)-5-[(2-hydroxy-acetyl)
methylamino]-2,4,6-triiodo isophthalamide
Iopamidol
C H I N O 777,08 60166-93-0
17 22 3 3 3
(S)-N,N′-Bis[2-hydroxy-1-(hydroxymethyl)ethyl]-5-[(2-hy-
droxypropanoyl)amino]-2,4,6-triiodobenzene-1,3-dicarbamide
Iopromide
C H I N O 791,12 73334-07-3
18 24 3 3 8
(±)-N,N′-Bis(2,3-dihydroxypropyl)-2,4,6-triiodo-5-
(2-methoxyacetamido)-N-methylisophthalamide
Metoprolol
C H NO 267,36 37350-58-6
15 25 3
(RS)-1-(Isopropylamino)-3-[4-(2-methoxyethyl) phenoxy]
propan-2-ol
Naproxen
C H O 230,26 22204-53-1
14 14 3
(S)-2-(6-Methoxy-2-naphthyl)propanoic acid
Oxazepam
C H ClN O 286,71 604-75-1
15 11 2 2
(RS)-7-Chloro-3-hydroxy-5-phenyl-1,3-dihydro-2H-1,4-
benzodiazepin-2-on
Phenazone
C H N O 188,23 60-80-0
11 12 2
1,5-Dimethyl-2-phenyl-2,3-dihydro-1H-pyrazol-3-on
Primidone
C H N O 218,25 125-33-7
12 14 2 2
5-Ethyl-5-phenylhexahydropyrimidin-4,6-dione
Propyphenazone
C H N O 230,31 479-92-5
14 18 2
1,5-Dimethyl-4-(1-methylethyl)-2-phenyl-1,2-dihydro-3H-
pyrazol-3-one
Roxithromycin
(3R,4S,5S,6R,7R,9R,11S,12R,13S,14R)-6-{[(2S,3R,4S,6R)-
4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]
C H N O 837,05 80214-83-1
41 76 2 15
oxy}-14-ethyl-7,12,13-trihydroxy-4-{[(2R,4R,5S,6S)-5-hy-
droxy-4-methoxy-4,6-dimethyloxan-2-yl]oxy}-3,5,7,9,11,13-
hexamethyl-10-(2,4,7-trioxa-1-azaoctan-1-ylidene)-1-
oxacyclotetradecane-2-one
Sotalol
C H N O S 272,36 3930-20-9
12 20 2 3
(RS)-4′-(1-Hydroxy-2-isopropylaminoethyl)
methanesulfonanilide
Sulfamethoxazole
C H N O S 253,28 723-46-6
10 11 3 3
4-Amino-N-(5-methyl-1,2-oxazol-3-yl)benzene-sulfonamide
Temazepam
C H ClN O 300,74 846-50-4
16 13 2 2
(RS)-7-Chloro-3-hydroxy-1-methyl-5-phenyl-1,3-dihydro-2H-
1,4-benzodiazepin-2-one
Trimethoprim
C H N O 290,32 738-70-5
14 18 4 3
2,4-Diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine
a
IUPAC: International Union of Pure and Applied Chemistry.
b
CAS-RN: Chemical Abstracts System Registration Number.
© ISO 2018 – All rights reserved 3
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oSIST prEN ISO 21676:2021
ISO 21676:2018(E)
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 1042, Laboratory glassware — One-mark volumetric flasks
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 4796-2, Laboratory glassware — Bottles — Part 2: Conical neck bottles
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 5667-10, Water quality — Sampling — Part 10: Guidance on sampling of waste waters
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: Statistical evaluation of the linear calibration function
3 Terms and definitions
No terms and definitions are listed in this document.
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 http: //www .electropedia .org/
4 Principle
The water sample is injected directly into the analysis system. The identification and quantitative
determination is performed using high performance liquid chromatography coupled with mass
spectrometric detection (HPLC-MS/MS, HPLC-HRMS).
5 Interferences
5.1 During sample preparation
Loss of analytes can occur during filtration of the sample as a result of sorption.
5.2 During high performance liquid chromatography and mass spectrometry
Peak tailing, peak fronting and/or wide peaks are indications of a malfunctioning of HPLC and/or
interferences occurring during chromatography. However, some compounds tend to show more signal
tailing than others depending on the chromatographic conditions.
Interferences from accompanying substances (matrix) can occur in both positive and negative
ionization modes depending on the measured compound (e.g. diclofenac in negative ESI mode).
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
4 © ISO 2018 – All rights reserved
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oSIST prEN ISO 21676:2021
ISO 21676:2018(E)
quantification. These interferences can be detected and corrected for as needed using analyte recovery
(11.2 and Annex B) and/or internal standardization (10.3 and Table D.3).
6 Reagents
6.1 General
If available, reagents of purity grade “for analysis” or “for residue analysis” are used. The amount of
impurities contributing to the blank value or causing signal interference shall be negligible. This shall
be checked regularly (see 9.5).
Solvents, water and reagents intended for use as elution agents shall be compatible with HPLC and mass
spectrometry.
NOTE High purity grades of solvent applicable for use are available commercially.
6.1.1 Water, complying with the requirements of ISO 3696, grade 1 or equivalent without any interfering
blank values.
6.1.2 Methanol, CH OH.
3
6.1.3 Acetonitrile, CH CN.
3
6.1.4 Acetic acid, w(CH COOH) = 100 % mass fraction.
3
6.1.5 Formic acid, w(HCOOH) not less than 98 % mass fraction.
6.1.6 Ammonium acetate, w(CH COONH ) not less than 99 % mass fraction.
3 4
6.1.7 Ammonium formate, w(HCOONH ) not less than 99 % mass fraction.
4
6.1.8 Sodium thiosulfate pentahydrate, Na S O ·5H O.
2 2 3 2
6.1.9 Operating gases for the mass spectrometer, in accordance with the specifications of the
instrument manufacturer.
6.1.10 Reference substances, as listed in Table 1, with known mass fraction.
6.1.11 Internal standard substances, preferably isotope-labelled compounds of reference substances
(see Table D.3).
The internal standards shall not lead to analyte interferences (see 9.5).
6.2 Preparation of solutions
6.2.1 General
Solutions of internal standard substances are needed only once calibration and evaluation have been
performed in accordance with 10.3 and 12.3.
Test the accuracy of the reference substance solutions against a control standard (see 6.2.9), e.g. during
calibration (see 10.1).
NOTE Reference substance solutions and internal standard substances are available commercially.
© ISO 2018 – All rights reserved 5
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oSIST prEN ISO 21676:2021
ISO 21676:2018(E)
6.2.2 Stock solutions (reference substances/internal standard substances)
Prepare solutions with a mass concentration of, for example, 0,1 mg/ml of each substance.
For this, use, for example, a 5 mg amount of a substance (6.1.10) in separate 50 ml volumetric flasks
(7.2), dissolve them in acetonitrile (6.1.3) or methanol (6.1.2), and then add solvent to solution until it
reaches the mark.
NOTE Alternatively, commercially available (or custom made) stock solutions of individual reference
substances (or internal standard substances) in organic solvent can be used for preparing further dilutions.
Store the solutions at temperatures below −15 °C and protected from light and evaporation. Under these
conditions they are stable for one year.
6.2.3 Intermediate dilution A (reference substances)
Prepare an intermediate solution with substance mass concentrations of, for example, 1 µg/ml each.
This involves transferring, for example, 0,5 ml of each reference substance stock solution (see 6.2.2) to
a 50 ml volumetric flask (7.2) and then making the solution up to the mark with acetonitrile (6.1.3) to
the mark.
Store the solution at temperatures below −15 °C and protected from light and evaporation. Under these
conditions it is stable for one year.
6.2.4 Intermediate dilution B (reference substances)
Prepare an intermediate solution with substance mass concentrations of, for example, 50 ng/ml each.
This involves transferring, for example, 0,5 ml of the intermediate dilution A (see 6.2.3) to a 10 ml
volumetric flask (7.2) and then making the solution up to the mark with water (6.1.1) to the mark.
Store the solution at between 2 °C and 8 °C and protected from light and evaporation. Under these
conditions it is stable for one month.
Use the solution to spike analytes to the samples to determine the recovery (see 11.2).
6.2.5 Intermediate dilution C (reference substances)
Prepare an intermediate solution with substance mass concentrations of, for example, 5 ng/ml each.
This involves transferring, for example, 0,25 ml of the intermediate dilution A (see 6.2.3) to a 50 ml
...
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