oSIST prEN ISO 21676:2021

Water quality - Determination of the dissolved fraction of selected active pharmaceutical ingredients, transformation products and other organic substances in water and treated waste water - Method using high performance liquid chromatography and mass spectrometric detection (HPLC-MS/MS or -HRMS) after direct injection (ISO 21676:2018)

This document specifies a method for the determination of the dissolved fraction of selected active pharmaceutical ingredients and transformation products, as well as other organic substances (see Table 1 of the document) in drinking water, ground water, surface water and treated waste water.
The lower application range of this method can vary depending on the sensitivity of the equipment used and the matrix of the sample. For most compounds to which this document applies, the range is ≥ 0,025 μg/l for drinking water, ground water and surface water, and ≥ 0,050 μg/l for treated waste water.
The method can be used to determine further organic substances or in other types of water (e.g. process water) provided that accuracy has been tested and verified for each case, and that storage conditions of both samples and reference solutions have been validated.

Wasserbeschaffenheit - Bestimmung ausgewählter Arzneimittelwirkstoffe, Transformationsprodukte und weiterer organischer Stoffe gelöst in Wasser und gereinigtem Abwasser - Verfahren mittels Hochleistungs-Flüssigkeitschromatographie und massenspektrometrischer Detektion (HPLC-MS/MS oder -HRMS) nach Direktinjektion (ISO 21676:2018)

Dieses Dokument legt ein Verfahren zur Bestimmung des gelösten Anteils ausgewählter Arzneimittelwirkstoffe und Transformationsprodukte sowie weiterer organischer Substanzen (siehe Tabelle 1) in Trink-, Grund-, Oberflächen- und behandeltem Abwasser fest.
Der untere Anwendungsbereich des Verfahrens kann abhängig von der Empfindlichkeit der verwendeten Geräte und von der Matrix der Probe variieren. Für die meisten Verbindungen, für die dieses Dokument Anwendung findet, ist der Bereich ≥ 0,025 µg/l in Trink-, Grund- und Oberflächenwasser und ≥ 0,050 µg/l in behandeltem Abwasser.
Das Verfahren kann zur Bestimmung weiterer organischer Stoffe oder in anderen Wasserarten angewandt werden (z. B.: Brauchwasser), wenn die Richtigkeit im Einzelfall geprüft und nachgewiesen wurde und die Lagerbedingungen von Proben und Referenzlösungen validiert wurden. Tabelle 1 zeigt die Substanzen, für die eine Bestimmung nach der Methode getestet wurde. Tabelle E.1 enthält Beispiele für die Bestimmung anderer organischer Substanzen.
[Tabelle 1]

Qualité de l'eau - Détermination de la fraction dissoute des ingrédients pharmaceutiques actifs sélectionnés, des produits de la transformation et d'autres substances organiques dans l'eau et dans l'eau résiduaire - Méthode par chromatographie en phase liquide à haute performance et détection par spectrométrie de masse (CLHP-MS/MS ou -HRSM) après l'injection directe (ISO 21676:2018)

Kakovost vode - Določevanje raztopljenih frakcij izbranih aktivnih farmacevtskih učinkovin, produktov razgradnje in drugih organskih spojin v vodi in obdelani odpadni vodi - Metoda tekočinske kromatografije visoke ločljivosti in masne spektrometrije (HPLC-MS/MS ali -HRMS) po neposrednem injiciranju (ISO 21676:2018)

General Information

• Status: Not Published
• Public Enquiry End Date: 02-May-2021
• ICS: 13.060.50 - Examination of water for chemical substances
• Technical Committee: KAV - Water quality
• Current Stage: 4020 - Public enquire (PE) (Adopted Project)
• Start Date: 03-Feb-2021
• Due Date: 23-Jun-2021
• Completion Date: 31-May-2021
• Directive: 2000/60/EC - Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establishing a framework for Community action in the field of water policy (substitutes Directive Reference 01/991 - Common Position on WFD)
• Ref Project: EN ISO 21676:2021 - Water quality - Determination of the dissolved fraction of selected active pharmaceutical ingredients, transformation products and other organic substances in water and treated waste water - Method using high performance liquid chromatography and mass spectrometric detection (HPLC-MS/MS or -HRMS) after direct injection (ISO 21676:2018)

Standards Content (sample)

SLOVENSKI STANDARD
oSIST prEN ISO 21676:2021
01-april-2021
Kakovost vode - Določevanje raztopljenih frakcij izbranih aktivnih farmacevtskih
učinkovin, produktov razgradnje in drugih organskih spojin v vodi in obdelani
odpadni vodi - Metoda tekočinske kromatografije visoke ločljivosti in masne
spektrometrije (HPLC-MS/MS ali -HRMS) po neposrednem injiciranju (ISO
21676:2018)

Water quality - Determination of the dissolved fraction of selected active pharmaceutical

ingredients, transformation products and other organic substances in water and treated

waste water - Method using high performance liquid chromatography and mass
spectrometric detection (HPLC-MS/MS or -HRMS) after direct injection (ISO
21676:2018)
Wasserbeschaffenheit - Bestimmung ausgewählter Arzneimittelwirkstoffe,
Transformationsprodukte und weiterer organischer Stoffe gelöst in Wasser und

gereinigtem Abwasser - Verfahren mittels Hochleistungs-Flüssigkeitschromatographie

und massenspektrometrischer Detektion (HPLC-MS/MS oder -HRMS) nach
Direktinjektion (ISO 21676:2018)

Qualité de l'eau - Détermination de la fraction dissoute des ingrédients pharmaceutiques

actifs sélectionnés, des produits de la transformation et d'autres substances organiques

dans l'eau et dans l'eau résiduaire - Méthode par chromatographie en phase liquide à

haute performance et détection par spectrométrie de masse (CLHP-MS/MS ou -HRSM)
après l'injection directe (ISO 21676:2018)
Ta slovenski standard je istoveten z: prEN ISO 21676
ICS:
13.060.50 Preiskava vode na kemične Examination of water for
snovi chemical substances
oSIST prEN ISO 21676:2021 en,fr,de

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

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oSIST prEN ISO 21676:2021
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oSIST prEN ISO 21676:2021
INTERNATIONAL ISO
STANDARD 21676
First edition
2018-10
Water quality — Determination of
the dissolved fraction of selected
active pharmaceutical ingredients,
transformation products and
other organic substances in
water and treated waste water —
Method using high performance
liquid chromatography and mass
spectrometric detection (HPLC-MS/MS
or -HRMS) after direct injection
Qualité de l'eau — Détermination de la fraction dissoute des
ingrédients pharmaceutiques actifs sélectionnés, des produits de la
transformation et d'autres substances organiques dans l'eau et dans
l'eau résiduaire — Méthode par chromatographie en phase liquide à
haute performance et détection par spectrométrie de masse (CLHP-
MS/MS ou -HRSM) après l'injection directe
Reference number
ISO 21676:2018(E)
ISO 2018
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oSIST prEN ISO 21676:2021
ISO 21676:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
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oSIST prEN ISO 21676:2021
ISO 21676:2018(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

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

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 4

3 Terms and definitions ..................................................................................................................................................................................... 4

4 Principle ........................................................................................................................................................................................................................ 4

5 Interferences ............................................................................................................................................................................................................ 4

5.1 During sample preparation .......................................................................................................................................................... 4

5.2 During high performance liquid chromatography and mass spectrometry ...................................... 4

6 Reagents ........................................................................................................................................................................................................................ 5

6.1 General ........................................................................................................................................................................................................... 5

6.2 Preparation of solutions ................................................................................................................................................................. 5

7 Apparatus ..................................................................................................................................................................................................................... 7

8 Sampling ........................................................................................................................................................................................................................ 8

9 Procedure..................................................................................................................................................................................................................... 8

9.1 General ........................................................................................................................................................................................................... 8

9.2 Sample preparation ............................................................................................................................................................................ 8

9.3 High performance liquid chromatography (HPLC) ................................................................................................. 9

9.4 Detection ...................................................................................................................................................................................................... 9

9.4.1 General...................................................................................................................................................................................... 9

9.4.2 Tandem mass spectrometry (MS/MS) .......................................................................................................10

9.4.3 High-resolution mass spectrometry (HRMS) .......................................................................................10

9.5 Blank value measurements .......................................................................................................................................................10

10 Calibration ...............................................................................................................................................................................................................10

10.1 General ........................................................................................................................................................................................................10

10.2 Calibration with external standard ....................................................................................................................................12

10.3 Calibration with internal standard .....................................................................................................................................12

11 Calculation of recovery ...............................................................................................................................................................................13

11.1 General ........................................................................................................................................................................................................13

11.2 Calculation of analyte recovery using samples ........................................................................................................13

11.3 Recovery of internal standards ..............................................................................................................................................14

12 Evaluation .................................................................................................................................................................................................................14

12.1 Verification of individual substances ................................................................................................................................14

12.2 Calculation of the individual results using calibration with an external standard ...................15

12.3 Calculation of the individual results using calibration with an internal standard ....................15

13 Expression of results .....................................................................................................................................................................................16

14 Test report ................................................................................................................................................................................................................16

Annex A (informative) Performance data ....................................................................................................................................................17

Annex B (informative) Examples of recovery ...........................................................................................................................................22

Annex C (informative) Examples of HPLC columns and chromatograms ....................................................................24

Annex D (informative) Examples of detection .........................................................................................................................................30

Annex E (informative) Examples of extension of the method ..................................................................................................33

Bibliography .............................................................................................................................................................................................................................34

© ISO 2018 – All rights reserved iii
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oSIST prEN ISO 21676:2021
ISO 21676:2018(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO’s adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso

.org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2,

Physical, chemical and biochemical methods.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2018 – All rights reserved
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oSIST prEN ISO 21676:2021
ISO 21676:2018(E)
Introduction

Pharmaceutical ingredients are essential for human and animal health. Through application or improper

disposal, active pharmaceutical ingredients enter the water cycle unchanged or transformed. This can

happen via municipal waste water, treated at treatment plants. There, some active pharmaceutical

ingredients and transformation products cannot be removed completely from the waste water by

conventional treatment techniques. Active pharmaceutical ingredients and their transformation

products also travel through sludge to the soil and subsequently enter water bodies via leachate,

depending on the nature of the ground and the active ingredients. Active pharmaceutical ingredients

and their transformation products are therefore found in treated waste water, as well as in surface

and ground water. This document specifies a liquid chromatography method with mass spectrometric

detection for the determination of selected active pharmaceutical ingredients and their transformation

products in the dissolved fraction.
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oSIST prEN ISO 21676:2021
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oSIST prEN ISO 21676:2021
INTERNATIONAL STANDARD ISO 21676:2018(E)
Water quality — Determination of the dissolved fraction of
selected active pharmaceutical ingredients, transformation
products and other organic substances in water and
treated waste water — Method using high performance
liquid chromatography and mass spectrometric detection
(HPLC-MS/MS or -HRMS) after direct injection

WARNING — Persons using this document should be familiar with normal laboratory practice.

This document does not purport to address all of the safety problems, if any, associated with its

use. It is the responsibility of the user to establish appropriate safety and health practices.

IMPORTANT — It is absolutely essential that tests conducted in accordance with this document

be carried out by suitably qualified staff.
1 Scope

This document specifies a method for the determination of the dissolved fraction of selected active

pharmaceutical ingredients and transformation products, as well as other organic substances

(see Table 1) in drinking water, ground water, surface water and treated waste water.

The lower application range of this method can vary depending on the sensitivity of the equipment used

and the matrix of the sample. For most compounds to which this document applies, the range is ≥ 0,025 µg/l

for drinking water, ground water and surface water, and ≥ 0,050 µg/l for treated waste water.

The method can be used to determine further organic substances or in other types of water (e.g.

process water) provided that accuracy has been tested and verified for each case, and that storage

conditions of both samples and reference solutions have been validated. Table 1 shows the substances

for which a determination was tested in accordance with the method. Table E.1 provides examples of

the determination of other organic substances.

Table 1 — Substances for which a determination was tested in accordance with this method

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.
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oSIST prEN ISO 21676:2021
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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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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.
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2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 1042, Laboratory glassware — One-mark volumetric flasks
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 4796-2, Laboratory glassware — Bottles — Part 2: Conical neck bottles

ISO 5667-4, Water quality — Sampling — Part 4: Guidance on sampling from lakes, natural and man-made

ISO 5667-5, Water quality — Sampling — Part 5: Guidance on sampling of drinking water from treatment

works and piped distribution systems

ISO 5667-6, Water quality — Sampling — Part 6: Guidance on sampling of rivers and streams

ISO 5667-10, Water quality — Sampling — Part 10: Guidance on sampling of waste waters

ISO 5667-11, Water quality — Sampling — Part 11: Guidance on sampling of groundwaters

ISO 8466-1, Water quality — Calibration and evaluation of analytical methods and estimation of

performance characteristics — Part 1: Statistical evaluation of the linear calibration function

3 Terms and definitions
No terms and definitions are listed in this document.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
4 Principle

The water sample is injected directly into the analysis system. The identification and quantitative

determination is performed using high performance liquid chromatography coupled with mass

spectrometric detection (HPLC-MS/MS, HPLC-HRMS).
5 Interferences
5.1 During sample preparation

Loss of analytes can occur during filtration of the sample as a result of sorption.

5.2 During high performance liquid chromatography and mass spectrometry

Peak tailing, peak fronting and/or wide peaks are indications of a malfunctioning of HPLC and/or

interferences occurring during chromatography. However, some compounds tend to show more signal

tailing than others depending on the chromatographic conditions.

Interferences from accompanying substances (matrix) can occur in both positive and negative

ionization modes depending on the measured compound (e.g. diclofenac in negative ESI mode).

Accompanying substances (matrix) can affect the ionization of the target substances (e.g. ion suppression

or signal enhancement). This can result in underestimation or overestimation of concentration during

4 © ISO 2018 – All rights reserved
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oSIST prEN ISO 21676:2021
ISO 21676:2018(E)

quantification. These interferences can be detected and corrected for as needed using analyte recovery

(11.2 and Annex B) and/or internal standardization (10.3 and Table D.3).
6 Reagents
6.1 General

If available, reagents of purity grade “for analysis” or “for residue analysis” are used. The amount of

impurities contributing to the blank value or causing signal interference shall be negligible. This shall

be checked regularly (see 9.5).

Solvents, water and reagents intended for use as elution agents shall be compatible with HPLC and mass

spectrometry.

NOTE High purity grades of solvent applicable for use are available commercially.

6.1.1 Water, complying with the requirements of ISO 3696, grade 1 or equivalent without any interfering

blank values.
6.1.2 Methanol, CH OH.
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.

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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

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