Water quality -- Determination of microcystins -- Method using liquid chromatography and tandem mass spectrometry (LC-MS/MS)

This document specifies a method for the quantification of twelve microcystin variants (microcystin-LR, -LA, -YR, -RR, -LY, -WR, -HtyR, -HilR, -LW, -LF, [Dha7]-microcystin-LR, and [Dha7]-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.

Qualité de l'eau -- Dosage des microcystines -- Méthode par chromatographie en phase liquide couplée à la spectrométrie de masse en tandem (CL-SM/SM)

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Publication Date
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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 22104:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO 22104:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

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
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 22104:2021(E)
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

© ISO 2021 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 22104:2021(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 2021 – All rights reserved
---------------------- Page: 4 ----------------------
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/
© ISO 2021 – All rights reserved 1
---------------------- Page: 5 ----------------------
ISO 22104:2021(E)
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
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
CAS-RN: Chemical Abstracts System Registration Number.

Nodularin can be naturally occurring in brackish water samples. Blank levels should be checked

before analysis for these samples. Alternatively, N-labelled microcystin surrogates should be used if

available.

NOTE Some microcystins (e.g. demethylated RR variants) have the same exact mass and a similar

3 7

chromatographic behaviour. While some can be distinguished by their fragmentation (e.g. [Asp , Mdha ] MC-

3 7 3 7 3

RR and [MeAsp , Dha )] MC-RR), others even show the same fragmentation (e.g. Asp , Mdha ] MC-RR and [Asp ,

Dhb ] MC-RR).

Water samples are homogenized to disperse cell aggregates. A 5 ml aliquot is transferred to a 15 ml

centrifuge tube, internal standard is added, and cells are lysed by three cycles of freeze/thaw. Solid

particles and cell debris are centrifuged and syringe filtered directly into an autosampler vial.

Quantification of microcystins is done by an internal standard method using LC-MS/MS or HRMS

(Annex A).

Alternatively, lysed and filtered samples can be injected using an on-line SPE instrumental configuration

(Annex B) or manual SPE (Annex C) for an increased sensitivity.
5 Interferences

This analysis was developed using liquid chromatography (LC) tandem mass spectrometry (MS/

MS) with electrospray ionization (ESI), on a triple quadrupole mass spectrometer. Acquisition mode

was based on multiple reaction monitoring (MRM). Isobaric interferences that are not resolved

by chromatography or the unit mass resolution of the tandem quadrupoles may be present in some

samples. These samples may require additional selectivity via additional sample clean-up and/or high-

resolution mass spectrometry (HRMS). Some microcystins with the same exact mass might not be able

to be distinguished by HRMS, but by their different fragmentation patterns, a few congeners cannot be

distinguished by either of these approaches.

Variable instrument response and/or inconsistent retention times may be observed in the first gradient

runs of the day. The column requires conditioning by running at least one gradient program prior to the

first sample injection of the day.
2 © ISO 2021 – All rights reserved
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ISO 22104:2021(E)
5.1 Biases

All labware that contacts microcystins should have relatively inert surfaces; otherwise, compound

losses may occur by adsorption onto the glass. Unscratched borosilicate glassware or polyethylene is

recommended. To further minimize this effect, sample preparation should be carried out in a timely

manner and quantification by matrix matched calibration standards is preferred.

Analytical results (method precision and accuracy) are calculated by internal standard quantitation

methods and may be affected by differences in the recovery of the internal standard relative to that of

the target compounds. When available, N-labelled microcystins should be used for this purpose.

The concentration of on-site samples will vary greatly depending on the density of algae at each

sampling point, and the concentration difference will also be large for each microcystins. Given this, the

multi-point calibration curves for the microcystins, using a fixed amount of internal standard, are non-

linear. Quantification is done by a second order (quadratic) curve-fitting procedure.

5.2 Limitations

The sample preparation method is restricted to water samples. Applicability of the method to samples

with very high organic content, such as water containing high concentrations of humic materials, is

unknown.

The working range of this method is 0,05 µg/l to 1,6 µg/l. If samples with a higher microcystin

concentration than 1,5 µg/l are found or predicted, a smaller aliquot of sample should be taken, and a

dilution factor applied to the final result. Surface waters containing thick cyanobacterial blooms may

interfere with the instrumental analysis. In these cases, a smaller amount of sample can be diluted, and

volume should be recorded for the final calculation of microcystins concentration.

Standards of specific microcystin variants are not always available on a continuous basis. Foreign

suppliers are sometimes restricted by law and are not always able to export algal toxin standards to

different countries. Before being used, newly prepared standards shall be compared to standards in

current use. Purity of the different lots of standards should be checked against reference materials

when available. Alternatively, purity can also be confirmed using universal detector like HPLC-UV

(ISO 20179).
6 Reagents and standards
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 interferences shall be negligible. This shall

be checked regularly (see section for blank value measurements).

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

spectrometry.

Microcystins are potent hepatotoxins. Laboratory safety measures should be strictly followed

throughout the sample preparation (including lab gloves, labcoat, safety glasses) to prevent human

exposure to these toxins.

NOTE 1 High purity grades of solvent applicable for use are available commercially.

NOTE 2 Reagents listed as “prepare as required” have an expiry date of one year from the moment they were

prepared.

NOTE 3 Prepared standard solutions are stored at (5 ± 3) °C, with an expiry date of one year from the moment

they were prepared.

Stock and intermediate standard solutions should be used as a reference, other stock and intermediate

concentrations are acceptable to prepare the final working solutions.
© ISO 2021 – All rights reserved 3
---------------------- Page: 7 ----------------------
ISO 22104:2021(E)

6.1.1 Water, conforming with the requirements of ISO 3696, grade 1 or equivalent and without any

interfering blank values.
6.1.2 Methanol, CH OH, LC-MS grade.
6.1.3 Acetonitrile, CH CN, LC-MS grade.
6.1.4 Formic acid, CHCOOH, LC-MS grade, mass fraction ≥98 %.

6.1.5 Electrospray tuning mixture, in accordance with the specification of the instrument

manufacturer.
6.1.6 Sodium thiosulfate pentahydrate, Na S O ·5H 0, 99 % purity.
2 2 3 2
6.1.7 Concentrated phosphate-free detergent.

6.1.8 Internal standard substances like Nodularin, (CAS no 118399-22-7, ≥95 % purity determined

by HPLC) or isotope labelled compounds of reference substances.

6.1.9 Reference Substances as listed in Table 1, with known mass fraction or purity ≥95 %

determined by HPLC.
6.1.10 Microcystin-LR, 10 ng/µl certified reference standard.
6.2 Preparation of solutions

6.2.1 Tap water, quenched with sodium thiosulfate at 150 mg/l (for calibration standard solutions, QC

samples and sample dilutions).

The method blank, calibration standard solutions, QC samples and sample dilutions (if necessary) are

made with quenched laboratory tap water. This quenched water is made by taking 1 l of tap water and

adding 1,5 ml of sodium thiosulfate preservative solution (i.e. 150 mg sodium thiosulfate) (6.2.2). Cap

the bottle and shake vigorously to mix. This water is prepared as required before sample preparation

in order to quench any residual chlorine in the tap water which would oxidize the microcystins. Store

the reagent water at room temperature. Quenching is not necessary if it can be ensured that the used

tap water is provided without chlorination.

NOTE Depending on the application, method blank, calibration standard solutions, QC samples and sample

dilutions can be prepared with other matrices such as mineral water.
6.2.2 Sodium thiosulfate preservative solution, Na S O ,100 mg/ml.
2 2 3

Into a 1 l volumetric flask put 157 g of Na S O ·5H O (6.1.6), corresponding to 100 g of anhydrous

2 2 3 2

Na S O . Dissolve in water (6.1.1), and make up to 1 l with pure water. Prepare as required. Store the

2 2 3
preservative at room temperature.
6.2.3 Stock solution of internal standard substances
Prepare solutions with a mass concentration of, for example, 200 ng/μl.

For this use, for example, transfer 10 mg of an internal standard (6.1.8) to a separate 50 ml volumetric

flask and dissolve it in methanol (6.1.2). Fill up to the 50 ml mark with methanol (6.1.2). The

concentration of this solution is 200 ng/μl.
4 © ISO 2021 – All rights reserved
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ISO 22104:2021(E)
6.2.4 Internal standard solution (IS1)

Prepare a working solution with internal standard mass concentrations of, for example, 8,0 ng/μl each.

For this use, for example, transfer 1,0 ml of each internal standard stock solution (6.2.3) to a 25 ml flask

and fill up to the mark with methanol (6.1.2).
6.2.5 Internal standard solution (IS2)

Prepare a working solution with internal standard mass concentrations of, for example, 80 pg/μl each.

For this use, for example, transfer 250 µl of each internal standard stock solution (6.2.4) to a 25 ml flask

and fill up to the mark with methanol (6.1.2).
6.2.6 MCYST mix solution (S1)

Prepare a solution with microcystin mass concentrations of, for example, 4 ng/μl.

7 7

For this use, for example, transfer 100 μg [Dha ] microcystin-LR (dmLR), 100 μg [Dha ] microcystin-RR

(dmRR), 100 μg microcystin-LF, 100 μg microcystin-LW, 100 μg microcystin-WR, 100 μg microcystin-

LY, 100 μg microcystin-HtyR, and 100 µg microcystin-HilR to a 25 ml volumetric flask and dissolve it

in methanol (6.1.2). Make up to 25 ml with methanol (6.1.2). The concentration of each microcystin is

4 ng/μl.
6.2.7 MCYST mix solution (S2)

Prepare a solution with microcystin mass concentrations of, for example, 400 pg/μl.

For this use, for example, transfer 2 500 µl of supplemental microcystin mix solution solution (6.2.6) to

a 25 ml flask and fill up to the mark with methanol (6.1.2).
6.2.8 MCYST mix solution (S3)

Prepare a solution with microcystin mass concentrations of, for example, 40 pg/μl.

For this use, for example, transfer 250 µl of supplemental microcystin mix solution (6.2.6) to a 25 ml

flask and fill up to the mark with methanol (6.1.2).
6.2.9 MCYST mix A solution

Prepare a solution with microcystin mass concentrations of, for example, 20 ng/μl.

For this use, for example, transfer 500 μg of microcystin-LR, 500 μg of microcystin-RR, 500 μg of

microcystin-YR and 500 μg of microcystin-LA to a 25 ml volumetric flask and dissolve it in methanol

(6.1.2). Make up to 25 ml with methanol (6.1.2). The concentration of each microcystin is 20 ng/μl.

6.2.10 MCYST mix B solution

Prepare a solution with microcystin mass concentrations of, for example, 2,0 ng/μl.

For this use, for example, dilute 2,5 ml of MCYST mix A solution (6.2.9) to 25 ml with methanol in a

25 ml volumetric flask. The concentration of each microcystin is 2,0 ng/μl.
6.2.11 MCYST mix C solution

Prepare a solution with microcystin mass concentrations of, for example, 200 pg/μl.

For this use, for example, dilute 250 µl of MCYST mix solution A (6.2.9) to 25 ml with methanol in a

25 ml volumetric flask. The concentration of each microcystin is 200 pg/μl.
© ISO 2021 – All rights reserved 5
---------------------- Page: 9 ----------------------
ISO 22104:2021(E)
6.2.12 MCYST mix D solution

Prepare a solution with microcystin mass concentrations of, for example, 20 pg/μl.

For this use, for example, dilute 25 µl of MCYST mix solution A (6.2.9) to 25 ml with methanol in a 25 ml

volumetric flask. The concentration of each microcystin is 20 pg/μl.
6.2.13 Instrument check mix (high) solution

Into a 25 ml volumetric flask put 25 µl of MCYST mix solution A (6.2.9) and 250 μl of supplemental

microcystin mix solution (6.2.6). Make up to 25 ml with pure water (6.1.1). The concentrations of

microcystins -LR (6.2.10), -RR, -LA, -YR are 20 pg/μl. The remaining supplemental microcystins are at a

concentration of 40 pg/μl.
6.2.14 Calibration control standard (CS1)

The calibration control standard is a reference substance solution produced independently of the other

stock solutions (6.2.5 to 6.2.13), e.g. a solution from an alternative batch or manufacturer.

For this use, for example, a microcystin-LR, 10 ng/µl certified concentration standard can be purchased

or prepared.

Other microcystins with certified concentration should also be used to validate standard mixture

concentration when available.
6.2.15 Calibration control standard (CS2)

Prepare a solution with microcystin mass concentrations of, for example, 100 pg/μl.

For this use, for example, dilute 100 µl of calibration control standard CS1 (6.2.14) to 10 ml with

methanol in a 10 ml volumetric flask. The concentration of each microcystin is 100 pg/μl.

6.2.16 Mobile phase A, water with 0,1 % formic acid.

Measure 1 l of pure water (6.1.1) using a graduated cylinder and pour into a 1 l amber bottle. Transfer

1 ml of formic acid (6.1.4) using a 1 ml pipette into the water. Cap the bottle and shake vigorously to

mix. Store the reagent at room temperature. Prepare as required. Mobile phase A should be replaced at

least on a weekly basis and should be degassed before the chromatographic run.
6.2.17 Mobile phase B, acetonitrile with 0,1 % formic acid.

Measure 1 l of acetonitrile (6.1.3) using a graduated cylinder and pour into a 1 l amber bottle. Transfer

1 ml of formic acid (6.1.4) using a 1 ml pipette into the acetonitrile. Cap the bottle and shake vigorously

to mix. Store the reagent at room temperature. Prepare as required. Mobile phase B should be degassed

before the chromatographic run.
7 Apparatus

NOTE Labware, reagents and equipment equivalent to those listed in this document are acceptable.

7.1 Bottles 500 ml, 1 l, amber glass, with polytetrafluoroethylene (PTFE) screw caps.

7.2 Cylinders, graduated, glass, 25 ml, 50 ml, 100 ml, 250 ml, 500 ml, 1 000 ml, 2 000 ml.

7.3 Microsyringes.
7.4 Centrifuge tubes, polypropylene, 15 ml, 17 mm diameter.
6 © ISO 2021 – All rights reserved
---------------------- Page: 10 ----------------------
ISO 22104:2021(E)
7.5 Centrifuge, suitable for 15 ml centrifuge tubes (7.4)
7.6 Pipette, 1 ml to 5 ml, adjustable.
7.7 Pipette tips, polypropylene, flextips, 1 ml to 5 ml.
7.8 Syringe, polypropylene, 5 ml.
7.9 Syringe filters, with low dead volume, GHP membrane, 13 mm, 0,2 μm.

7.10 Sample vials, appropriate for automated sample injection and with low adsorption, nominal

volume 1,5 ml, clear glass, screw or crimp cap with PTFE/silicone septa with slit.

7.11 Freezer, capable of reaching −28 °C.
7.12 Temperature controlled water bath, capable of reaching 50 °C.
7.13 Ultrasonic bath
7.14 Homogenizer, capable of reaching 10 000 RPM.
7.15 Liquid chromatograph (LC)

The LC shall include a binary pump capable to run gradients from 95 % aqueous mobile phase A

(6.2.16) and 5 % organic mobile phase B (6.2.17) to 5 % aqueous mobile phase A and 95 % organic

mobile phase B, providing enough pressure to run at a constan
...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 22104
ISO/TC 147/SC 2
Water quality — Determination of
Secretariat: DIN
microcystins — Method using liquid
Voting begins on:
2021­03­04 chromatography and tandem mass
spectrometry (LC-MS/MS)
Voting terminates on:
2021­04­29
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/FDIS 22104:2021(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN­
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NATIONAL REGULATIONS. ISO 2021
---------------------- Page: 1 ----------------------
ISO/FDIS 22104:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

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
CH­1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/FDIS 22104:2021(E)
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 set­up 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

© ISO 2021 – All rights reserved iii
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ISO/FDIS 22104:2021(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

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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.

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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 2021 – All rights reserved
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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 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

LC­MS/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/
© ISO 2021 – All rights reserved 1
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ISO/FDIS 22104:2021(E)
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
Microcystin variant CAS-RN Molecular formular
Microcystin-LR 101043­37­2 C H N O
49 74 10 12
Microcystin-RR 111755­374 C H N O
49 75 13 12
Microcystin-LA 96180­79­9 C H N O
46 67 7 12
Microcystin-YR 101064­48­6 C H N O
52 72 10 13
Microcystin-LY 123304­10­9 C H N O
52 71 7 13
Microcystin-WR 138234­58­9 C H N O
54 73 11 12
Microcystin-HtyR 913178­65­1 C H N O
52 72 10 13
Microcystin-HilR N/A C H N O
50 76 10 12
Microcystin-LW 157622­02­1 C H N O
54 72 8 12
Microcystin-LF 154037­70­4 C H N O
52 71 7 12
[Dha ]-Microcystin-LR (dmLR) 120011­66­7 C H N O
48 72 10 12
[Dha ]-Microcystin-RR (dmRR) 131022­02­1 C H N O
48 73 13 12
CAS-RN: Chemical Abstracts System Registration Number.

Nodularin can be naturally occurring in brackish water samples. Blank levels should be checked

before analysis for these samples. Alternatively, N-labelled microcystin surrogates should be used if

available.

NOTE Some microcystins (e.g. demethylated RR variants) have the same exact mass and a similar

3 7

chromatographic behaviour. While some can be distinguished by their fragmentation (e.g. [Asp , Mdha ] MC­

3 7 3 7 3

RR and [MeAsp , Dha )] MC­RR), others even show the same fragmentation (e.g. Asp , Mdha ] MC-RR and [Asp ,

Dhb ] MC­RR).

Water samples are homogenized to disperse cell aggregates. A 5 ml aliquot is transferred to a 15 ml

centrifuge tube, internal standard is added, and cells are lysed by three cycles of freeze/thaw. Solid

particles and cell debris are centrifuged and syringe filtered directly into an autosampler vial.

Quantification of microcystins is done by an internal standard method using LC-MS/MS or HRMS

(Annex A).

Alternatively, lysed and filtered samples can be injected using an on-line SPE instrumental configuration

(Annex B) or manual SPE (Annex C) for an increased sensitivity.
5 Interferences

This analysis was developed using liquid chromatography (LC) tandem mass spectrometry (MS/

MS) with electrospray ionization (ESI), on a triple quadrupole mass spectrometer. Acquisition mode

was based on multiple reaction monitoring (MRM). Isobaric interferences that are not resolved

by chromatography or the unit mass resolution of the tandem quadrupoles may be present in some

samples. These samples may require additional selectivity via additional sample clean-up and/or high-

resolution mass spectrometry (HRMS). Some microcystins with the same exact mass might not be able

to be distinguished by HRMS, but by their different fragmentation patterns, a few congeners cannot be

distinguished by either of these approaches.

Variable instrument response and/or inconsistent retention times may be observed in the first gradient

runs of the day. The column requires conditioning by running at least one gradient program prior to the

first sample injection of the day.
2 © ISO 2021 – All rights reserved
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ISO/FDIS 22104:2021(E)
5.1 Biases

All labware that contacts microcystins should have relatively inert surfaces; otherwise, compound

losses may occur by adsorption onto the glass. Unscratched borosilicate glassware or polyethylene is

recommended. To further minimize this effect, sample preparation should be carried out in a timely

manner and quantification by matrix matched calibration standards is preferred.

Analytical results (method precision and accuracy) are calculated by internal standard quantitation

methods and may be affected by differences in the recovery of the internal standard relative to that of

the target compounds. When available, N-labelled microcystins should be used for this purpose.

The concentration of on-site samples will vary greatly depending on the density of algae at each

sampling point, and the concentration difference will also be large for each microcystins. Given this, the

multi-point calibration curves for the microcystins, using a fixed amount of internal standard, are non-

linear. Quantification is done by a second order (quadratic) curve-fitting procedure.

5.2 Limitations

The sample preparation method is restricted to water samples. Applicability of the method to samples

with very high organic content, such as water containing high concentrations of humic materials, is

unknown.

The working range of this method is 0,05 µg/l to 1,6 µg/l. If samples with a higher microcystin

concentration than 1,5 µg/l are found or predicted, a smaller aliquot of sample should be taken, and a

dilution factor applied to the final result. Surface waters containing thick cyanobacterial blooms may

interfere with the instrumental analysis. In these cases, a smaller amount of sample can be diluted, and

volume should be recorded for the final calculation of microcystins concentration.

Standards of specific microcystin variants are not always available on a continuous basis. Foreign

suppliers are sometimes restricted by law and are not always able to export algal toxin standards to

different countries. Before being used, newly prepared standards shall be compared to standards in

current use. Purity of the different lots of standards should be checked against reference materials

when available. Alternatively, purity can also be confirmed using universal detector like HPLC-UV

(ISO 20179).
6 Reagents and standards
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 interferences shall be negligible. This shall

be checked regularly (see section for blank value measurements).

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

spectrometry.

Microcystins are potent hepatotoxins. Laboratory safety measures should be strictly followed

throughout the sample preparation (including lab gloves, labcoat, safety glasses) to prevent human

exposure to these toxins.

NOTE 1 High purity grades of solvent applicable for use are available commercially.

NOTE 2 Reagents listed as “prepare as required” have an expiry date of one year from the moment they were

prepared.

NOTE 3 Prepared standard solutions are stored at (5 ± 3) °C, with an expiry date of one year from the moment

they were prepared.

Stock and intermediate standard solutions should be used as a reference, other stock and intermediate

concentrations are acceptable to prepare the final working solutions.
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ISO/FDIS 22104:2021(E)

6.1.1 Water, conforming with the requirements of ISO 3696, grade 1 or equivalent and without any

interfering blank values.
6.1.2 Methanol, CH OH, LC­MS grade.
6.1.3 Acetonitrile, CH CN, LC­MS grade.
6.1.4 Formic acid, CHCOOH, LC-MS grade, mass fraction ≥98 %.

6.1.5 Electrospray tuning mixture, in accordance with the specification of the instrument

manufacturer.
6.1.6 Sodium thiosulfate pentahydrate, Na S O ·5H 0, 99 % purity.
2 2 3 2
6.1.7 Concentrated phosphate-free detergent.

6.1.8 Internal standard substances like Nodularin, (CAS no 118399-22-7, ≥95 % purity determined

by HPLC) or isotope labelled coumpounds of reference substances.

6.1.9 Reference Substances as listed in Table 1, with known mass fraction or purity ≥95 %

determined by HPLC.
6.1.10 Microcystin-LR, 10 ng/µl certified reference standard.
6.2 Preparation of solutions

6.2.1 Tap water, quenched with sodium thiosulfate at 150 mg/l (for calibration standard solutions, QC

samples and sample dilutions).

The method blank, calibration standard solutions, QC samples and sample dilutions (if necessary) are

made with quenched laboratory tap water. This quenched water is made by taking 1 l of tap water and

adding 1,5 ml of sodium thiosulfate preservative solution (i.e. 150 mg sodium thiosulfate) (6.2.2). Cap

the bottle and shake vigorously to mix. This water is prepared as required before sample preparation

in order to quench any residual chlorine in the tap water which would oxidize the microcystins. Store

the reagent water at room temperature. Quenching is not necessary if it can be ensured that the used

tap water is provided without chlorination.

NOTE Depending on the application, method blank, calibration standard solutions, QC samples and sample

dilutions can be prepared with other matrices such as mineral water.
6.2.2 Sodium thiosulfate preservative solution, Na S O ,100 mg/ml.
2 2 3

Into a 1 l volumetric flask put 157 g of Na S O ·5H O (6.1.6), corresponding to 100 g of anhydrous

2 2 3 2

Na S O . Dissolve in water (6.1.1), and make up to 1 l with pure water. Prepare as required. Store the

2 2 3
preservative at room temperature.
6.2.3 Stock solution of internal standard substances
Prepare solutions with a mass concentration of, for example, 200 ng/μl.

For this use, for example, transfer 10 mg of an internal standard (6.1.8) to a separate 50 ml volumetric

flask and dissolve it in methanol (6.1.2). Fill up to the 50 ml mark with methanol (6.1.2). The

concentration of this solution is 200 ng/μl.
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ISO/FDIS 22104:2021(E)
6.2.4 Internal standard solution (IS1)

Prepare a working solution with internal standard mass concentrations of, for example, 8,0 ng/μl each.

For this use, for example, transfer 1,0 ml of each internal standard stock solution (6.2.3) to a 25 ml flask

and fill up to the mark with methanol (6.1.2).
6.2.5 Internal standard solution (IS2)

Prepare a working solution with internal standard mass concentrations of, for example, 80 pg/μl each.

For this use, for example, transfer 250 µl of each internal standard stock solution (6.2.4) to a 25 ml flask

and fill up to the mark with methanol (6.1.2).
6.2.6 MCYST mix solution (S1)

Prepare a solution with microcystin mass concentrations of, for example, 4 ng/μl.

7 7

For this use, for example, transfer 100 μg [Dha ] microcystin-LR (dmLR), 100 μg [Dha ] microcystin-RR

(dmRR), 100 μg microcystin-LF, 100 μg microcystin-LW, 100 μg microcystin-WR, 100 μg microcystin-

LY, 100 μg microcystin-HtyR, and 100 µg microcystin-HilR to a 25 ml volumetric flask and dissolve it

in methanol (6.1.2). Make up to 25 ml with methanol (6.1.2). The concentration of each microcystin is

4 ng/μl.
6.2.7 MCYST mix solution (S2)

Prepare a solution with microcystin mass concentrations of, for example, 400 pg/μl.

For this use, for example, transfer 2 500 µl of supplemental microcystin mix solution solution (6.2.6) to

a 25 ml flask and fill up to the mark with methanol (6.1.2).
6.2.8 MCYST mix solution (S3)

Prepare a solution with microcystin mass concentrations of, for example, 40 pg/μl.

For this use, for example, transfer 250 µl of supplemental microcystin mix solution solution (6.2.5) to a

25 ml flask and fill up to the mark with methanol (6.1.2).
6.2.9 MCYST mix A solution

Prepare a solution with microcystin mass concentrations of, for example, 20 ng/μl.

For this use, for example, transfer 500 μg of microcystin-LR, 500 μg of microcystin-RR, 500 μg of

microcystin-YR and 500 μg of microcystin-LA to a 25 ml volumetric flask and dissolve it in methanol

(6.1.2). Make up to 25 ml with methanol (6.1.2). The concentration of each microcystin is 20 ng/μl.

6.2.10 MCYST mix B solution

Prepare a solution with microcystin mass concentrations of, for example, 2,0 ng/μl.

For this use, for example, dilute 2,5 ml of MCYST mix A solution (6.2.9) to 25 ml with methanol in a

25 ml volumetric flask. The concentration of each microcystin is 2,0 ng/μl.
6.2.11 MCYST mix C solution

Prepare a solution with microcystin mass concentrations of, for example, 200 pg/μl.

For this use, for example, dilute 250 µl of MCYST mix solution A (6.2.9) to 25 ml with methanol in a

25 ml volumetric flask. The concentration of each microcystin is 200 pg/μl.
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ISO/FDIS 22104:2021(E)
6.2.12 MCYST mix D solution

Prepare a solution with microcystin mass concentrations of, for example, 20 pg/μl.

For this use, for example, dilute 25 µl of MCYST mix solution A (6.2.9) to 25 ml with methanol in a 25 ml

volumetric flask. The concentration of each microcystin is 20 pg/μl.
6.2.13 Instrument check mix (high) solution

Into a 25 ml volumetric flask put 25 µl of MCYST mix solution A (6.2.9) and 250 μl of supplemental

microcystin mix solution (6.2.6). Make up to 25 ml with pure water (6.1.1). The concentrations of

microcystins -LR, -RR, -LA, -YR are 20 pg/μl. The remaining supplemental microcystins are at a

concentration of 40 pg/μl.
6.2.14 Calibration control standard (CS1)

The calibration control standard is a reference substance solution produced independently of the other

stock solutions (6.2.5 to 6.2.13), e.g. a solution from an alternative batch or manufacturer.

For this use, for example, a microcystin-LR, 10 ng/µl certified concentration standard can be purchased

or prepared.

Other microcystins with certified concentration should also be used to validate standard mixture

concentration when available.
6.2.15 Calibration control standard (CS2)

Prepare a solution with microcystin mass concentrations of, for example, 100 pg/μl.

For this use, for example, dilute 100 µl of calibration control standard CS1 (6.2.14) to 10 ml with

methanol in a 10 ml volumetric flask. The concentration of each microcystin is 100 pg/μl.

6.2.16 Mobile phase A, water with 0,1 % formic acid.

Measure 1 l of pure water (6.1.1) using a graduated cylinder and pour into a 1 l amber bottle. Transfer

1 ml of formic acid (6.1.4) using a 1 ml pipette into the water. Cap the bottle and shake vigorously to

mix. Store the reagent at room temperature. Prepare as required. Mobile phase A should be replaced at

least on a weekly basis and should be degassed before the chromatographic run.
6.2.17 Mobile phase B, acetonitrile with 0,1 % formic acid.

Measure 1 l of acetonitrile (6.1.3) using a graduated cylinder and pour into a 1 l amber bottle. Transfer

1 ml of formic acid (6.1.4) using a 1 ml pipette into the acetonitrile. Cap the bottle and shake vigorously

to mix. Store the reagent at room temperature. Prepare as required. Mobile phase B should be degassed

before the chromatographic run.
7 Apparatus

NOTE Labware, reagents and equipment equivalent to those listed in this document are acceptable.

7.1 Bottles 500 ml, 1 l, amber glass, with polytetrafluoroethylene (PTFE) screw caps.

7.2 Cylinders, graduated, glass, 25 ml, 50 ml, 100 ml, 250 ml, 500 ml, 1 000 ml, 2 000 ml.

7.3 Microsyringes.
7.4 Centrifuge tubes, polypropylene, 15 ml, 17 mm diameter.
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ISO/FDIS 22104:2021(E)
7.5 Centrifuge, suitable for 15 ml centrifuge tubes (7.4)
7.6 Pipette, 1 ml to 5 ml, adjustable.
7.7 Pipette tips, polypropylene, flextips, 1 ml to 5 ml.
7.8 Syringe, polypropylene, 5 ml.
7.9 Syringe filters, with low dead volume, GHP membrane, 13 mm, 0,2 μm.

7.10 Sample vials, appropriate for automated sample injection and with low adsorption, nominal

volume 1,5 ml, clear glass, screw or crimp cap with PTFE
...

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