ISO 22104:2021

INTERNATIONAL ISO
STANDARD 22104
First edition
2021-07
Water quality — Determination of
microcystins — Method using liquid
chromatography and tandem mass
spectrometry (LC-MS/MS)
Qualité de l'eau — Dosage des microcystines — Méthode par
chromatographie en phase liquide couplée à la spectrométrie de
masse en tandem (CL-SM/SM)
Reference number
©
ISO 2021
© ISO 2021
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ii © ISO 2021 – All rights reserved

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Interferences . 2
5.1 Biases . 3
5.2 Limitations . 3
6 Reagents and standards . 3
6.1 General . 3
6.2 Preparation of solutions . 4
7 Apparatus . 6
8 Sampling . 7
9 Procedure. 8
9.1 Preparation of samples . 8
9.1.1 General. 8
9.1.2 Preparation of method blank sample . 8
9.1.3 Preparation of laboratory control spike sample . 8
9.1.4 Preparation of calibration control sample . 8
9.1.5 Preparation of calibration standard solutions . 8
9.1.6 Preparation of drinking water and freshwater sample . 9
9.1.7 Sample preparation procedure with freeze/thaw cycles . 9
9.2 Instrumental analysis by LC-MS/MS procedure .10
9.2.1 Instrument set-up parameters .10
9.3 Run processing and quality assurance .13
9.3.1 Run sequence .13
9.3.2 Run control operations / limits .13
10 Calibration .14
11 Evaluation and calculation of results .15
11.1 Identification and calculations .15
11.2 Calibration curve equation determination .15
11.3 Internal standard calculation .15
11.4 Internal standard recovery calculation .16
12 Expressing of results .16
13 Test report .16
Annex A (informative) Use of high resolution mass spectrometry detectors (HRMS).17
Annex B (informative) Use of online solid phase extraction coupled to liquid
chromatography for the automated analysis of microcystins .19
Annex C (informative) Use of manual solid phase extraction prior to instrumental analysis
for improved method detection limits .25
Bibliography .32
Foreword
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iv © ISO 2021 – All rights reserved

INTERNATIONAL STANDARD ISO 22104:2021(E)
Water quality — Determination of microcystins —
Method using liquid chromatography and tandem mass
spectrometry (LC-MS/MS)
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 quantification of twelve microcystin variants (microcystin-
7 7
LR, -LA, -YR, -RR, -LY, -WR, -HtyR, -HilR, -LW, -LF, [Dha ]-microcystin-LR, and [Dha ]-microcystin-RR)
in drinking water and freshwater samples between 0,05 µg/l to 1,6 µg/l. The method can be used to
determine further microcystins, provided that analytical conditions for chromatography and mass
spectrometric detection has been tested and validated for each microcystin. Samples are analysed by
LC-MS/MS using internal standard calibration.
This method is performance based. The laboratory is permitted to modify the method, e.g. increasing
direct flow injection volume for low interference samples or diluting the samples to increase the upper
working range limit, provided that all performance criteria in this method are met.
Detection of microcystins by high resolution mass spectrometry (HRMS) as an alternative for tandem
mass spectrometry (MS/MS) is described in Annex A.
An alternative automated sample preparation method based on on-line solid phase extraction coupled
to liquid chromatography is described in Annex B.
When instrumental sensitivity is not sufficient to reach the method detection limits by direct flow
injection, a solid phase extraction clean-up and concentration step is described in Annex C.
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 3696, Water for analytical laboratory use — Specification and test methods
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
This method is designed to identify and quantify total (free + intracellular) microcystins in water
by direct flow injection liquid chromatography and tandem mass spectrometry (LC-MS/MS) with
[1] [2]
electrospray ionization , . 12 microcystins (Table 1) are determined quantitatively by multi-point
calibration using nodularin as internal standard.
Table 1 — Microcystin variants included in the method
a
Microcystin variant CAS-RN Molecular formular
Microcystin-LR 101043-37-2 C H N O
49 74 10 12
Microcystin-RR 111755-374 C H N O
49 75 13 12
Microcystin-LA 96180-79-9 C H N O
46 67 7 12
Microcystin-YR 101064-48-6 C H N O
52 72 10 13
Microcystin-LY 123304-10-9 C H N O
52 71 7 13
Microcystin-WR 138234-58-9 C H N O
54 73 11 12
Microcystin-HtyR 913178-65-1 C H N O
52 72 10 13
Microcystin-HilR N/A C H N O
50 76 10 12
Microcystin-LW 157622-02-1 C H N O
54 72 8 12
Microcystin-LF 154037-70-4 C H N O
52 71 7 12
[Dha ]-Microcystin-LR (dmLR) 120011-66-7 C H N O
48 72 10 12
[Dha ]-Microcystin-RR (dmRR) 131022-02-1 C H N O
48 73 13 12
a
CAS-RN: Ch
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