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

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.

Qualité de l'eau - Détermination 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

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

Ta dokument določa metodo za določevanje raztopljenih frakcij izbranih aktivnih farmacevtskih učinkovin, produktov razgradnje ter drugih organskih spojin (glej preglednico 1) v pitni vodi, podtalnici, površinski vodi in prečiščeni odpadni vodi.
Spodnje območje uporabe te metode se lahko razlikuje glede na občutljivost uporabljene opreme in matrico vzorca. Pri večini spojin, za katere se ta dokument uporablja, je območje ≥ 0,025 μg/l za pitno vodo, podtalnico in površinsko vodo ter ≥ 0,050 μg/l za prečiščeno odpadno vodo.
Metodo je mogoče uporabiti za določevanje nadaljnjih organskih spojin ali v drugih vrstah vode (npr. procesna voda) pod pogojem, da je bila natančnost preskušena in preverjena za vsak primer ter so bili pogoji skladiščenja obeh vzorcev in referenčnih raztopin potrjeni. V preglednici 1 so prikazane spojine, za katere je bilo preskušeno določevanje v skladu z metodo. V preglednici E.1 so podani primeri določevanja drugih organskih spojin.

General Information

Status
Published
Public Enquiry End Date
02-Jul-2018
Publication Date
11-Apr-2019
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
01-Feb-2019
Due Date
08-Apr-2019
Completion Date
12-Apr-2019

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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
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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
---------------------- Page: 1 ----------------------
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.
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Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
---------------------- Page: 2 ----------------------
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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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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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
---------------------- Page: 5 ----------------------
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

Common name Molecular Molar CAS-RN
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
IUPAC: International Union of Pure and Applied Chemistry.
CAS-RN: Chemical Abstracts System Registration Number.
© ISO 2018 – All rights reserved 1
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ISO 21676:2018(E)
Table 1 (continued)
Common name Molecular Molar CAS-RN
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
IUPAC: International Union of Pure and Applied Chemistry.
CAS-RN: Chemical Abstracts System Registration Number.
2 © ISO 2018 – All rights reserved
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ISO 21676:2018(E)
Table 1 (continued)
Common name Molecular Molar CAS-RN
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
IUPAC: International Union of Pure and Applied Chemistry.
CAS-RN: Chemical Abstracts System Registration Number.
© ISO 2018 – All rights reserved 3
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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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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 2018 – All rights reserved 5
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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
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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 t
...

SLOVENSKI STANDARD
SIST ISO 21676:2019
01-maj-2019
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XþLQNRYLQSURGXNWRYUD]JUDGQMHLQGUXJLKRUJDQVNLKVSRMLQYYRGLLQREGHODQL
RGSDGQLYRGL0HWRGDWHNRþLQVNHNURPDWRJUDILMHYLVRNHORþOMLYRVWLLQPDVQH
VSHNWURPHWULMH +3/&0606DOL+506 SRQHSRVUHGQHPLQMLFLUDQMX

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 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
Ta slovenski standard je istoveten z: ISO 21676:2018
ICS:
13.060.50 3UHLVNDYDYRGHQDNHPLþQH Examination of water for
VQRYL chemical substances
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
SIST ISO 21676:2019 en

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

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SIST ISO 21676:2019
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SIST ISO 21676:2019
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 ISO 21676:2019
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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SIST ISO 21676:2019
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 ISO 21676:2019
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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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 ISO 21676:2019
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SIST ISO 21676:2019
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

Common name Molecular Molar CAS-RN
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
IUPAC: International Union of Pure and Applied Chemistry.
CAS-RN: Chemical Abstracts System Registration Number.
© ISO 2018 – All rights reserved 1
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Table 1 (continued)
Common name Molecular Molar CAS-RN
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
IUPAC: International Union of Pure and Applied Chemistry.
CAS-RN: Chemical Abstracts System Registration Number.
2 © ISO 2018 – All rights reserved
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ISO 21676:2018(E)
Table 1 (continued)
Common name Molecular Molar CAS-RN
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
IUPAC: International Union of Pure and Applied Chemistry.
CAS-RN: Chemical Abstracts System Registration Number.
© ISO 2018 – All rights reserved 3
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SIST ISO 21676:2019
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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SIST ISO 21676:2019
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 2018 – All rights reserved 5
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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 tra
...

NORME ISO
INTERNATIONALE 21676
Première édition
2018-10
Qualité de l'eau — Détermination
de la fraction dissoute des principes
actifs pharmaceutiques sélectionnés,
de leurs produits de transformation
et d'autres substances organiques
dans les eaux et les eaux résiduaires
— Méthode par chromatographie
en phase liquide haute performance
et détection par spectrométrie de
masse (HPLC-MS/MS ou -HRMS) après
injection directe
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
Numéro de référence
ISO 21676:2018(F)
ISO 2018
---------------------- Page: 1 ----------------------
ISO 21676:2018(F)
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© ISO 2018

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ii © ISO 2018 – Tous droits réservés
---------------------- Page: 2 ----------------------
ISO 21676:2018(F)
Sommaire Page

Avant-propos ..............................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Domaine d'application ................................................................................................................................................................................... 1

2 Références normatives ................................................................................................................................................................................... 4

3 Termes et définitions ....................................................................................................................................................................................... 4

4 Principe .......................................................................................................................................................................................................................... 5

5 Interférences ............................................................................................................................................................................................................ 5

5.1 Pendant la préparation de l'échantillon ............................................................................................................................ 5

5.2 Pendant la chromatographie en phase liquide haute performance et la

spectrométrie de masse .................................................................................................................................................................. 5

6 Réactifs ........................................................................................................................................................................................................................... 5

6.1 Généralités .................................................................................................................................................................................................. 5

6.2 Préparation des solutions .............................................................................................................................................................. 6

7 Appareillage .............................................................................................................................................................................................................. 8

8 Échantillonnage ..................................................................................................................................................................................................... 9

9 Mode opératoire.................................................................................................................................................................................................... 9

9.1 Généralités .................................................................................................................................................................................................. 9

9.2 Préparation des échantillons ...................................................................................................................................................... 9

9.3 Chromatographie en phase liquide haute performance (HPLC) ...............................................................10

9.4 Détection ...................................................................................................................................................................................................10

9.4.1 Généralités .........................................................................................................................................................................10

9.4.2 Spectrométrie de masse en tandem (MS/MS) ....................................................................................11

9.4.3 Spectrométrie de masse à haute résolution (HRMS) ....................................................................11

9.5 Détermination des valeurs de blanc ..................................................................................................................................11

10 Étalonnage ...............................................................................................................................................................................................................11

10.1 Généralités ...............................................................................................................................................................................................11

10.2 Étalonnage avec un étalon externe .....................................................................................................................................13

10.3 Étalonnage avec un étalon interne ......................................................................................................................................13

11 Calcul du taux de récupération ...........................................................................................................................................................14

11.1 Généralités ...............................................................................................................................................................................................14

11.2 Calcul du taux de récupération avec des échantillons .......................................................................................15

11.3 Rendement d'extraction des étalons internes ..........................................................................................................15

12 Évaluation .................................................................................................................................................................................................................15

12.1 Vérification des substances individuelles .....................................................................................................................15

12.2 Calcul des résultats individuels en utilisant l'étalonnage avec un étalon externe.....................17

12.3 Calcul des résultats individuels en utilisant l'étalonnage avec un étalon interne .....................17

13 Expression des résultats............................................................................................................................................................................17

14 Rapport d'essai ...................................................................................................................................................................................................18

Annexe A (informative) Données de performance ..............................................................................................................................19

Annexe B (informative) Exemples de taux de récupération ......................................................................................................25

Annexe C (informative) Exemples de colonnes HPLC et de chromatogrammes ...................................................27

Annexe D (informative) Exemples de détection .....................................................................................................................................33

Annexe E (informative) Exemples d'extension de la méthode ................................................................................................36

Bibliographie ...........................................................................................................................................................................................................................37

© ISO 2018 – Tous droits réservés iii
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ISO 21676:2018(F)
Avant-propos

L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes

nationaux de normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est

en général confiée aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude

a le droit de faire partie du comité technique créé à cet effet. Les organisations internationales,

gouvernementales et non gouvernementales, en liaison avec l'ISO participent également aux travaux.

L'ISO collabore étroitement avec la Commission électrotechnique internationale (IEC) en ce qui

concerne la normalisation électrotechnique.

Les procédures utilisées pour élaborer le présent document et celles destinées à sa mise à jour sont

décrites dans les Directives ISO/IEC, Partie 1. Il convient, en particulier de prendre note des différents

critères d'approbation requis pour les différents types de documents ISO. Le présent document a été

rédigé conformément aux règles de rédaction données dans les Directives ISO/IEC, Partie 2 (voir www

.iso .org/ directives).

L'attention est attirée sur le fait que certains des éléments du présent document peuvent faire l'objet de

droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour responsable

de ne pas avoir identifié de tels droits de propriété et averti de leur existence. Les détails concernant

les références aux droits de propriété intellectuelle ou autres droits analogues identifiés lors de

l'élaboration du document sont indiqués dans l'Introduction et/ou dans la liste des déclarations de

brevets reçues par l'ISO (voir www .iso .org/ brevets).

Les appellations commerciales éventuellement mentionnées dans le présent document sont données

pour information, par souci de commodité, à l'intention des utilisateurs et ne sauraient constituer un

engagement.

Pour une explication de la nature volontaire des normes, la signification des termes et expressions

spécifiques de l'ISO liés à l'évaluation de la conformité, ou pour toute information au sujet de l'adhésion

de l'ISO aux principes de l'Organisation mondiale du commerce (OMC) concernant les obstacles

techniques au commerce (OTC), voir www .iso .org/ avant -propos.

Le présent document a été élaboré par le comité technique ISO/TC 147, Qualité de l'eau, sous-comité

SC 2, Méthodes physiques, chimiques et biochimiques.

Il convient que l'utilisateur adresse tout retour d'information ou toute question concernant le présent

document à l'organisme national de normalisation de son pays. Une liste exhaustive desdits organismes

se trouve à l'adresse www .iso .org/ fr/ members .html.
iv © ISO 2018 – Tous droits réservés
---------------------- Page: 4 ----------------------
ISO 21676:2018(F)
Introduction

Les composés pharmaceutiques sont essentiels pour la santé humaine et animale. Au moment de

l'application ou du fait d'une élimination inappropriée, les principes actifs pharmaceutiques pénètrent le

cycle de l'eau, avec ou sans transformation. Cela peut se produire par le biais des eaux usées municipales,

traitées dans les stations de traitement. En effet, certains principes actifs pharmaceutiques et leurs

produits de transformation ne sont pas complètement éliminés des eaux usées par les traitements

classiques. Les principes actifs pharmaceutiques et leurs produits de transformation sont ainsi

transférés dans le sol par les boues, et pénètrent ensuite les masses d'eau par lessivage, en fonction

de la nature du sol et des principes actifs. Des principes actifs pharmaceutiques et leurs produits de

transformation se trouvent ainsi dans les eaux usées traitées, ainsi que dans les eaux de surface et

souterraines. Le présent document spécifie une méthode par chromatographie en phase liquide avec

détection par spectrométrie de masse pour la détermination de la fraction dissoute des principes actifs

pharmaceutiques sélectionnés et de leurs produits de transformation.
© ISO 2018 – Tous droits réservés v
---------------------- Page: 5 ----------------------
NORME INTERNATIONALE ISO 21676:2018(F)
Qualité de l'eau — Détermination de la fraction dissoute
des principes actifs pharmaceutiques sélectionnés, de
leurs produits de transformation et d'autres substances
organiques dans les eaux et les eaux résiduaires —
Méthode par chromatographie en phase liquide haute
performance et détection par spectrométrie de masse
(HPLC-MS/MS ou -HRMS) après injection directe

AVERTISSEMENT — Il convient que l'utilisateur du présent document connaisse bien les

pratiques courantes de laboratoire. Le présent document n'a pas pour but de traiter tous les

problèmes de sécurité qui sont, le cas échéant, liés à son utilisation. Il est de la responsabilité de

l'utilisateur de mettre en place des mesures de sécurité et d'hygiène appropriées.

IMPORTANT — Il est absolument essentiel que les essais effectués conformément au présent

document soient réalisés par du personnel ayant reçu une qualification appropriée.

1 Domaine d'application

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.
© ISO 2018 – Tous droits réservés 1
---------------------- Page: 6 ----------------------
ISO 21676:2018(F)
Tableau 1 — Substances pour lesquelles la présente méthode a été appliquée
Nom commun Formule Masse Numéro CAS
Nom chimique (IUPAC ) brute molaire
g/mol
4-Acétylaminoantipyrine
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-Acétyl sulfaméthoxazole
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
Acide diatrizoïque (acide amidotrizoïque)
C H I N O 613,91 117-96-4
11 9 3 2 4
3,5-Bis(acetamido)-2,4,6-triiodobenzoic acid
Aténolol
C H N O 266,34 29122-68-7
(RS)-2-[4-[2-Hydroxy-3-(1-methylethylamino) propoxy]phenyl] 14 22 2 3
ethanamide
Bézafibrate
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
(RS)-1-[4-(2-Isopropoxyethoxymethyl)phenoxy]-3- 18 31 4
isopropylamino-2-propanol
Carbamazépine
C H N O 236,27 298-46-4
15 12 2
5H-Dibenzo[b,f]azepine-5-carbamide
Clarithromycine
(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-di-
methyl-oxan-2-yl]oxy-12-methoxy-2,4,8,10,12,14-hexa-
methyl-6-oxacyclotetradecane-1,7-dione
Acide clofibrique
C H ClO 214,70 882-09-7
10 11 3
2-(4-Chlorophenoxy)-2-methylpropanoic acid
Déhydro-érythromycine (anhydro-érythromycine) (érythro-
mycine-H2O)
(2R,3R,4S,5S,8R,9S,10S,11R,12R)-11-{[4-(dimethylamino)-3-
C H NO 715,91 23893-13-2
hydroxy-6-methyloxan-2-yl]oxy}-5-ethyl-3-hydroxy-9-[(5-hy- 37 65 12
droxy-4-methoxy-4,6-dimethyloxan-2-yl)oxy]-2,4,8,10,12,14-
hexamethyl-6,15,16-trioxatricyclo[10.2.1.1{1,4}]hexadecane-
7-one
Diazépam
C H ClN O 284,74 439-14-5
(RS)-7-Chlor-1-methyl-5-phenyl-1,3-dihydro-2H-1,4- 16 13 2
benzodiazepine-2-on
Diclofénac
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 carbamazépine
C H N O 270,29 58955-93-4
(5S,6S)-5,6-Dihydroxy-5,6-dihydrobenzo[b][1]benzazepie- 15 14 2 3
11-carboxamide
IUPAC: Union internationale de chimie pure et appliquée.
Numéro CAS: Numéro de registre du Chemical Abstracts Service.
2 © ISO 2018 – Tous droits réservés
---------------------- Page: 7 ----------------------
ISO 21676:2018(F)
Tableau 1 (suite)
Nom commun Formule Masse Numéro CAS
Nom chimique (IUPAC ) brute molaire
g/mol
Érythromycine
6-(4-Dimethylamino-3-hydroxy-6-methyl-oxan-2-yl)oxy-
C H NO 733,93 114-07-8
14-ethyl-7,12,13-trihydroxy-4-(5-hydroxy-4-methoxy-4,6- 37 67 13
dimethyl-oxan-2-yl)-oxy-3,5,7,9,11,13-hexamethyl-1-oxacyclote-
tradecane-2,10-dione
4-Formylaminoantipyrine
C H N O 231,25 1672-58-8
N-(2,3-Dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl) 12 13 3 2
formamide
Gemfibrozil
C H O 250,34 25812-30-0
15 22 3
5-(2,5-Chlorophenoxy)-2,2-methylpropanoic acid
Ibuprofène
C H O 206,28 15687-27-1
13 18 2
(RS)-2-[4-(2-Methylpropyl)phenyl]propanoic acid
Ioméprol
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 isophtalamide
Iopamidol
C H I N O 777,08 60166-93-0
(S)-N,N′-Bis[2-hydroxy-1-(hydroxymethyl)ethyl]-5-[(2-hy- 17 22 3 3 3
droxypropanoyl)amino]-2,4,6-triiodobenzene-1,3-dicarbamide
Iopromide
C H I N O 791,12 73334-07-3
(±)-N,N′-Bis(2,3-dihydroxypropyl)-2,4,6-triiodo-5- 18 24 3 3 8
(2-methoxyacetamido)-N-methylisophtalamide
Métoprolol
C H NO 267,36 37350-58-6
(RS)-1-(Isopropylamino)-3-[4-(2-methoxyethyl) phenoxy] 15 25 3
propan-2-ol
Naproxène
C H O 230,26 22204-53-1
14 14 3
(S)-2-(6-Methoxy-2-naphtyl)propanoic acid
Oxazépam
C H ClN O 286,71 604-75-1
(RS)-7-Chloro-3-hydroxy-5-phenyl-1,3-dihydro-2H-1,4- 15 11 2 2
benzodiazepin-2-on
Phénazone
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
Propyphénazone
C H N O 230,31 479-92-5
1,5-Dimethyl-4-(1-methylethyl)-2-phenyl-1,2-dihydro-3H- 14 18 2
pyrazol-3-one
Roxithromycine
(3R,4S,5S,6R,7R,9R,11S,12R,13S,14R)-6-{[(2S,3R,4S,6R)-4-
(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy}-14-ethyl-
C H N O 837,05 80214-83-1
7,12,13-trihydroxy-4-{[(2R,4R,5S,6S)-5-hydroxy-4-methoxy- 41 76 2 15
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
IUPAC: Union internationale de chimie pure et appliquée.
Numéro CAS: Numéro de registre du Chemical Abstracts Service.
© ISO 2018 – Tous droits réservés 3
---------------------- Page: 8 ----------------------
ISO 21676:2018(F)
Tableau 1 (suite)
Nom commun Formule Masse Numéro CAS
Nom chimique (IUPAC ) brute molaire
g/mol
Sotalol
C H N O S 272,36 3930-20-9
(RS)-4′-(1-Hydroxy-2-isopropylaminoethyl) 12 20 2 3
methanesulfonanilide
Sulfaméthoxazole
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
Témazépam
C H ClN O 300,74 846-50-4
(RS)-7-Chloro-3-hydroxy-1-methyl-5-phenyl-1,3-dihydro-2H- 16 13 2 2
1,4-benzodiazepin-2-one
Triméthoprime
C H N O 290,32 738-70-5
14 18 4 3
2,4-Diamino-5-(3,4,5-trimethoxybenzyl)pyrimidine
IUPAC: Union internationale de chimie pure et appliquée.
Numéro CAS: Numéro de registre du Chemical Abstracts Service.
2 Références normatives

Les documents suivants sont cités dans le texte de sorte qu'ils constituent, pour tout ou partie de leur

contenu, des exigences du présent document. Pour les références datées, seule l'édition citée s'applique.

Pour les références non datées, la dernière édition du document de référence s'applique (y compris les

éventuels amendements).
ISO 1042, Verrerie de laboratoire — Fioles jaugées à un trait

ISO 3696, Eau pour laboratoire à usage analytique — Spécification et méthodes d'essai

ISO 4796-2, Verrerie de laboratoire — Flacons — Partie 2: Flacons à col conique

ISO 5667-4, Qualité de l'eau — Échantillonnage — Partie 4: Lignes directrices pour l'échantillonnage des

eaux des lacs naturels et des lacs artificiels

ISO 5667-5, Qualité de l'eau — Échantillonnage — Partie 5: Lignes directrices pour l'échantillonnage de

l'eau potable des usines de traitement et du réseau de distribution

ISO 5667-6, Qualité de l'eau — Échantillonnage — Partie 6: Lignes directrices pour l'échantillonnage des

rivières et des cours d'eau

ISO 5667-10, Qualité de l'eau — Échantillonnage — Partie 10: Lignes directrices pour l'échantillonnage des

eaux résiduaires

ISO 5667-11, Qualité de l'eau — Échantillonnage — Partie 11: Lignes directrices pour l'échantillonnage des

eaux souterraines

ISO 8466-1, Qualité de l’eau — Étalonnage et évaluation des méthodes d’analyse et estimation des

caractères de performance — Partie 1: Évaluation statistique de la fonction linéaire d’étalonnage

3 Termes et définitions
Aucun terme n'est défini dans le présent document.

L'ISO et l'IEC tiennent à jour des bases de données terminologiques destinées à être utilisées en

normalisation, consultables aux adresses suivantes:

— ISO Online browsing platform: disponible à l'adresse https:// www .iso .org/ obp

4 © ISO 2018 – Tous droits réservés
---------------------- Page: 9 ----------------------
ISO 21676:2018(F)
— IEC Electropedia: disponible à l'adresse https:// www .electropedia .org/
4 Principe

L'échantillon d'eau est injecté directement dans le système d'analyse. L'identification et la détermination

quantitative sont réalisées en associant la chromatographie en phase liquide haute performance et la

détection par spectrométrie de masse (HPLC-MS/MS ou HPLC-HRMS).
5 Interférences
5.1 Pendant la préparation de l'échantillon

Des pertes d'analytes peuvent avoir lieu pendant la filtration de l'échantillon à cause de la sorption.

5.2 Pendant la chromatographie en phase liquide haute performance et la
spectrométrie de masse

Les traînées de pics, les diffusions frontales et/ou les pics larges indiquent un dysfonctionnement du

système HPLC et/ou la présence d'interférences pendant l'analyse chromatographique. Toutefois,

certains composés ont tendance à présenter davantage de traînées de pics que d'autres, en fonction des

conditions chromatographiques.

Des interférences dues aux substances concomitantes (matrice) peuvent se produire à la fois avec

l'ionisation positive et négative, selon le composé analysé (par exemple, le diclofénac en mode ESI

négatif).

Les substances concomitantes (matrice) peuvent affecter l'ionisation des substances cibles (par

exemple, suppression d'ions ou exaltation du signal). Cela peut donner lieu à une sous-estimation ou à

une surestimation de la concentration lors de la quantification. Ces interférences peuvent être détectées

et corrigées de manière adéquate en utilisant le taux de récupération de l'analyte (11.2 et Annexe B) et/

ou un étalonnage interne (10.3 et Tableau D.3).
6 Réactifs
6.1 Généralités

S'ils sont disponibles, des réactifs de qualité «pour analyse» ou «pour analyse de résidus» sont utilisés.

La quantité d'impuretés contribuant à la valeur de blanc ou à l'origine d'une interférence dans le signal

doit être négligeable. Cela doit être vérifié régulièrement (voir 9.5).

Les solvants, l'eau et les réactifs prévus comme agents d'élution doivent être compatibles avec la HPLC

et la spectrométrie de masse.

NOTE Les qualités de solvants ultra purs applicables pour cette utilisation sont disponibles dans le

commerce.

6.1.1 Eau, respectant les exigences de l'ISO 3696, de qualité 1 ou équivalente, exempte d'interférence

dans le blanc.
6.1.2 Méthanol, CH OH.
6.1.3 Acétonitrile, CH CN.
6.1.4 Acide acétique, w(CH COOH) = 100 % de la fraction massique.
© ISO 2018 – Tous droits réservés 5
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ISO 21676:2018(F)
6.1.5 Acide formique, w(HCOOH) au moins 98 % de la fraction massique.
6.1.6 Acétate d'ammonium, w(CH COONH ) au moins 99 % de la fraction massique.
3 4
6.1.7 Formiate d'ammonium, w(HCOONH ) au moins 99 % de la fraction massique.
6.1.8 Thiosulfate de sodium pentahydraté, Na S O ·5H O.
2 2 3 2

6.1.9 Gaz pour le spectromètre de masse, conformes aux spécifications du fabricant de l'instrument.

6.1.10 Étalons de référence, selon les indications du Tableau 1, avec une fraction massique connue.

6.1.11 Étalons internes, de préférence les composés marqués avec un isotope (voir Tableau D.3) des

étalons de référence.

Les étalons internes ne doivent pas provoquer d'interférences avec les analytes (voir 9.5).

6.2 Préparation des solutions
6.2.1 Généralités

Les solutions des étalons internes sont nécessaires seulement une fois que l'étalonnage et l'évaluation

ont été réalisés conformément au 10.3 et au 12.3.

Tester la justesse des solutions d’étalon de référence en les comparant à un étalon de contrôle (voir

6.2.9), par exemple pendant l'étalonnage (voir 10.1).

NOTE Des solutions d’étalon de référence et des étalons internes sont disponibles dans le commerce.

6.2.2 Solutions mères (étalons de référence/étalons internes)

Préparer les solutions avec une concentration massique de 0,1 mg/ml de chaque substance, par exemple.

Pour cela, utiliser, par exemple, 5 mg d'une substance (6.1.10) dans différentes fioles jaugées

de 50 ml (7.2), les dissoudre dans l'acétonitrile (6.1.3) ou le méthanol (6.1.2), puis ajouter du solvant

jusqu'à la marque.

NOTE Sinon, il est possible d'utiliser des solutions mères des étalons de référence (ou des étalons internes)

individuelles du commerce (ou sur mesure) dans un solvant organique, pour préparer les dilutions nécessaires.

Stocker les solutions à une température inférieure à −15 °C et les protéger de la lumière et de

l'évaporation. Dans ces conditions, elles sont stables pendant un an.
6.2.3 Dilution intermédiaire A (étalons de référence)

Préparer une solution intermédiaire avec des concentrations massiques de 1 µg/ml de chaque

substance, par exemple.

Cela implique de transférer, par exemple, 0,5 ml de chaque solution mère d’étalon de référence (voir

6.2.2) dans une fiole jaugée de 50 ml (7.2), puis de remplir la fiole d'acétonitrile (6.1.3) jusqu'à la marque.

Stocker la solution à une température inférieure à −15 °C et la protéger de la lumière et de l'évaporation.

Dans ces conditions, elle reste stable pendant un an.
6.2.4 Dilution intermédiaire B (étalons de référence)

Préparer une solution intermédiaire avec des concentrations massiques de 50 ng/ml de chaque

substance, par exemple.
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ISO 21676:2018(F)

Cela implique de transférer, par exemple, 0,5 ml de la dilution intermédiaire A (voir 6.2.3) dans une fiole

jaugée de 10 ml (7.2), puis de remplir la fiole d'eau (6.1.1) jusqu'à la marque.

Stocker la solution à une température entre 2 °C et 8 °C et la protéger de la lumière et de l'évaporation.

Dans ces conditions, elle est stable pendant un mois.

Utiliser cette solution pour doper les échantillons avec les analytes, pour déterminer les taux de

récupération (voir 11.2).
6.2.5 Dilution intermédiaire C (étalons de référence)

Préparer une solution intermédiaire avec des concentrations massiques de 5 ng/ml de chaque

substance, par exemple.

Cela implique de transférer, par exemple, 0,25 ml de la dilution intermédiaire A (voir 6.2.3) dans une

fiole jaugée de 50 ml (7.2), puis de remplir la fiole d'eau (6.1.1) jusqu'à la marque.

Stocker la solution à une température entre 2 °C et 8 °C et la protéger de la lumière et de l'évaporation.

Dans ces conditions, elle est stable pendant un mois.
6.2.6 Dilution intermédiaire D (étalons
...

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