SIST EN ISO 21676:2021

SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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.

Contents Page
European foreword . 3

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.

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
ISO 21676:2018(E)
© ISO 2018
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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Published in Switzerland
ii © ISO 2018 – All rights reserved

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 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

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.
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 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

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 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

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.
6.1.3 Acetonitrile, CH CN.
6.1.4 Acetic acid, w(CH COOH) = 100 % mass fraction.
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.
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 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
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.
6.2.6 Intermediate dilution D (internal standards)
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 internal standard 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.
6.2.7 Intermediate dilution E (internal standards)
Prepare an intermediate solution with substance mass concentrations of, for example, 50 ng/ml each.
6 © ISO 2018 – All rights reserved

ISO 21676:2018(E)
This involves transferring, for example, 0,5 ml of the intermediate dilution D (see 6.2.6) 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 for generating calibration samples and for spiked samples.
6.2.8 Calibration samples
Prepare calibration samples from the corresponding dilutions of the intermediate dilution C (see 6.2.5).
For calibration with an internal standard (see 10.3), apply the same amount of internal standards to
each calibration sample.
Prepare calibration samples, e.g. solutions in which the mass concentrations of the substances to be
determined correspond to 0,025 µg/l and those of the internal standard substances correspond to
0,250 µg/l (see 10.1).
This involves transferring, for example, 50 µl of the intermediate dilution C (see 6.2.5) to a 10 ml
volumetric flask, mixing 50 µl of the intermediate dilution E (see 6.2.7) and then making the solution up
to the mark with, for example, water (6.1.1).
If possible, the composition of the calibration samples should be similar to that of the samples to be
examined and shall not result in interfering peak broadening. When using calibration samples in
drinking, ground or surface water, ensure that the substances to be determined are not present.
NOTE When calibration samples are prepared in ultrapure water, this can lead to lower findings of
macrolides. In these cases, the use of ultrapure water is not preferred, but matrix matched calibration instead.
Prepare new calibration samples for each new measurement sequence if their stability cannot be
verified.
6.2.9 Control standard
The control standard is a reference substance solution produced independently of the stock solutions,
e.g. a solution from an alternative batch or manufacturer. The solution should contain all of the
substances to be determined.
7 Apparatus
Equipment or parts of equipment that come into contact with the water sample shall have no blank
values for the compounds measured within this method. All equipment used should preferably be made
of glass, stainless steel or polytetrafluoroethylene (PTFE).
7.1 Narrow-neck flat-bottomed bottles, preferably of brown glass conical joint with glass stoppers,
e.g. laboratory bottles, volume of 250 ml, as per ISO 4796-2 — NS 250.
7.2 Volumetric flasks, nominal volume 10 ml, 25 ml, 50 ml, e.g. volumetric flasks, as per
ISO 1042 — A50-C.
7.3 Microsyringes.
7.4 Syringe filters, with low dead volume, e.g. diameter of 13 mm with a regenerated cellulose
membrane.
Filtration should not lead to significant losses of individual substances, and the type of filter used shall
be selected by testing this. Verify no contamination or significant loss from filtering by passing blanks
and reference substance solutions through the same filters.
ISO 21676:2018(E)
7.5 Sample vials, appropriate for the automated sample injector, e.g. crimp-top vial, nominal volume
1,5 ml with crimp cap and septum of rubber/PTFE.
NOTE Sample vials of polyethylene (PE) can be used in an additional run to minimize losses of macrolides.
7.6 HPLC column, preferably with precolumn, suitable for chromatography of the selected substances.
See Annex C for examples.
7.7 High performance liquid chromatograph, coupled to mass spectrometer, consisting of the
following.
7.7.1 Degasser unit, e.g. vacuum degasser.
7.7.2 Analytical pumping systems, low-pulsation, suitable for binary gradient elution.
7.7.3 Manual or automated sample injector.
7.7.4 Apparatus for thermostat control of the separation column, e.g. column thermostat.
7.7.5 Mass spectrometry detector (MS/MS, HRMS), preferably with electrospray ionization (ESI).
8 Sampling
Take the samples in accordance with the specifications given in ISO 5667-4, ISO 5667-5, ISO 5667-6,
ISO 5667-10 and ISO 5667-11.
Use flat-bottomed bottles (7.1) for sampling and fill the bottles with the water to be examined.
When taking drinking water that may contain oxidants, also add approximately 50 mg of sodium
thiosulfate pentahydrate (6.1.8) per litre.
Analyse the water sample as soon as possible after it is sampled.
Store the sample at temperatures of (3 ± 2) °C, protected from light, for a maximum of three weeks.
NOTE If longer storage times are necessary and/or in case of presumed or validated instability of individual
substances, suitable measures can be implemented (e.g. preservation by freezing of samples).
9 Procedure
9.1 General
The implementation of the method depends on the type of calibration and the measures planned to
recognize and, if necessary, correct for matrix effects.
9.2 Sample preparation
If the sample is not visibly absent of particles, filter the sample through a syringe filter (7.4).
During filtration of the sample, sorption may cause loss of analytes, particularly hydrophobic substances
(e.g. macrolides). In this case, do not filter the sample before chromatography, but use an in-line filter as
an alternative to protect the separation column from particles.
When calibrating with an external standard (see 10.2), obtain an aliquot of the sample to determine the
recovery if necessary (see 11.2).
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ISO 21676:2018(E)
When calibrating with an internal standard (see 10.3), add the internal standards so that the mass
concentrations of the internal standards in the sample are equal to those in the calibration samples
(see 6.2.8).
Take into account the dilution of the sample caused by the addition of reagents when calculating the
individual results (see 12.2) if it amounts to more than 1 %.
Monitor an early eluter, e.g. metformin (see Table E.1), in terms of retention time and peak shape. The
level of organic solvents in a prepared sample shall not result in any additional peak broadening.
9.3 High performance liquid chromatography (HPLC)
Operate the HPLC instrumentation in accordance with the instructions provided by the manufacturer.
Use a suitable HPLC column (7.6) for chromatographic separation and optimize the separation of the
analytes with gradient elution.
Select the chromatographic conditions to achieve optimal sensitivity for mass spectrometric detection
(see Annex C for examples).
NOTE 1 The use of gradient programmes with acetonitrile/water/acetic acid is advantageous in terms of
sensitivity for most substances.
NOTE 2 At the same linear flow rate of the eluent, columns with a lower internal diameter exhibit better
sensitivities than those with wider diameter.
Complete separation of the substances is not necessary provided that interference of the quantitative
determination does not occur during peak overlapping.
The shortest retention time shall correspond to a minimum of three times the HPLC column dead
volume time.
Use chromatography to separate substances that cannot be completely separated from each other using
spectrometry. In these cases, chromatographic resolution R should be a minimum of R = 1,2.
Choose an appropriate injection volume so that no interfering peak widening or interference of the
quantitative determination occur.
NOTE 3 For larger injection volumes, e.g. 1 ml, column switching techniques with suitable enrichment columns
is possible but is outside the scope of this method.
Check retention time standard deviation once during initial assessment. It shall not exceed 0,03 min for
six consecutive chromatograms.
9.4 Detection
9.4.1 General
Operate the mass spectrometer in accordance with the manufacturer’s instructions and select the
correct settings for the device.
The ESI mode is typically preferred when ionizing substances. This usually produces quasi-molecular
+ ⎺ + +
ions of the type [M+H] or [M-H] . In isolated cases, adduct ions, e.g. [M+NH ] or [M+Na] , can also be
formed under certain chromatographic conditions.
Most substances listed in Table 1 can be detected using the positive ESI mode. Substances that are to be
detected in the negative ESI mode can be analysed in a single run by switching polarity or tested in a
separate run (see Table D.1).
When performing simultaneous detection of negative and positive ions, choose switch times for
changing polarity that are short enough to preserve a sufficient number of data points.
ISO 21676:2018(E)
Each peak shall be registered with a minimum of eight data points.
Identify and set the method-specific settings for the source parameters and the MS parameters using
less sensitive substances, e.g. ibuprofen.
NOTE The signal is usually smoothed prior to peak integration. Depending on the algorithm used this
can result in a disproportionately high loss of signal intensity when the number of data points is too low.
Reproducibility of peak int
...