ISO 20179:2005

Water quality — Determination of microcystins — Method using solid phase extraction (SPE) and high performance liquid chromatography (HPLC) with ultraviolet (UV) detection

ISO 20179:2005 specifies a method for the determination and quantification of microcystins in raw water (containing biomass) and treated water, such as tap water. The method described is validated for MCYST-RR, MCYST-YR, and MCYST-LR. It is also applicable for the determination of several structure variants of these microcystins, but an unambiguous identification cannot be made due to the lack of commercially available standards and due to co-elution.

Qualité de l'eau — Dosage des microcystines — Méthode utilisant l'extraction en phase solide (SPE) et la chromatographie en phase liquide à haute performance (CLHP) avec détection dans l'ultraviolet (UV)

Kakovost vode - Določevanje mikrocistinov - Metoda z ekstrakcijo na trdni fazi (SPE) in s tekočinsko kromatografijo visoke ločljivosti (HPLC) z ultravijolično (UV) detekcijo

Ta mednarodni standard določa metodo določevanja in količinskega določanja mikrocistinov v neobdelani vodi (ki vsebuje biomaso) in obdelani vodi, kot je voda iz pipe. Opisana metoda je potrjena za MCYST-RR, MCYST-YR in MCYST-LR. Prav tako velja za določevanje več strukturnih različic [1] teh mikrocistinov, vendar nedvomnega prepoznavanja ni možno izvesti zaradi pomanjkanja standardov, ki bi bili komercialno razpoložljivi in zaradi skupnega spiranja. Pragovna vrednost 1 µg/l MCYST-LR v vodi, kot jo predlaga Svetovna zdravstvena organizacija, lahko sledi obogatitvi z mikrocistini z ekstrakcijo na trdni fazi (SPE).

General Information

• Status: Published
• Publication Date: 10-Oct-2005
• ICS: 13.060.50 - Examination of water for chemical substances
• Technical Committee: ISO/TC 147/SC 2 - Physical, chemical and biochemical methods
• Drafting Committee: ISO/TC 147/SC 2 - Physical, chemical and biochemical methods
• Current Stage: 9093 - International Standard confirmed
• Start Date: 07-Apr-2026
• Completion Date: 11-Apr-2026
• Ref Project: SIST ISO 20179:2010 - Water quality - Determination of microcystins - Method using solid phase extraction (SPE) and high performance liquid chromatography (HPLC) with ultraviolet (UV) detection

Overview

ISO 20179:2005 specifies a laboratory method for the determination and quantification of microcystins in raw water (containing cyanobacterial biomass) and treated water (e.g., tap water). The method combines solid phase extraction (SPE) for sample enrichment and cleanup with reversed‑phase high performance liquid chromatography (RP‑HPLC) and UV/diode‑array detection (238 nm) for quantification. It is validated for MCYST‑LR, MCYST‑YR and MCYST‑RR and can be applied to related structural variants (though unambiguous identification may be limited by co‑elution and lack of standards). The standard supports following the World Health Organization guidance value (1 µg/L MCYST‑LR) after SPE enrichment.

Safety note: Microcystins are highly hepatotoxic. ISO 20179 warns that laboratory wastes must be handled and disposed of as highly toxic chemical waste and recommends appropriate safety procedures.

Key Topics and Requirements

• Scope: Analysis of microcystins in raw and treated waters; validated for MCYST‑LR, ‑YR, ‑RR.
• Principle: Filter biomass, solvent extraction (methanol/water), SPE clean‑up, SPE elution (methanol/water 90:10 + 0.1% TFA), RP‑HPLC-UV at 238 nm.
• Reagents & standards: HPLC‑grade methanol, acetonitrile, trifluoroacetic acid (TFA); preparation and spectrophotometric verification of microcystin stock solutions (use of molar absorptivity and molar mass data provided).
• SPE performance: Cartridges must retain ≥100 µg analyte capacity and deliver recoveries of ≥80% for MCYST‑LR and ≥70% for MCYST‑RR/‑YR under specified test conditions. Cartridge material/brand affects recovery.
• HPLC system requirements: C‑18 type column (e.g., 250 mm length, 2–4.6 mm ID, 3–5 µm particles), column oven at ~35 °C, flow 0.3–1.0 mL/min, injection 5–20 µL, PDA/UV detector (200–300 nm). System LOD ≤ 0.1 ng/µL and LOQ ≤ 0.2 ng/µL (signal‑to‑noise criteria).
• Quality control: Preparation of mixed standards, spiking solution (1 µg/L) for method control, and performance data (precision, typical chromatograms, and optional MS detection in informative annex).

Applications and Users

ISO 20179 is intended for:

• Municipal and private drinking water laboratories monitoring cyanotoxins.
• Environmental monitoring of lakes, reservoirs and raw water sources.
• Public health agencies assessing compliance with WHO guidance and local regulations.
• Research laboratories developing or comparing microcystin occurrence data.
• Water utilities conducting routine or investigative testing after algal bloom events.

Practical applications include routine screening, method validation, and supporting regulatory compliance decisions where reliable quantification of MCYST‑LR, ‑YR and ‑RR is required.

Related Standards

• ISO 3696:1987 - Water for analytical laboratory use (quality of reagents/water).
• ISO 5667‑4 and ISO 5667‑5 - Guidance on sampling from lakes and drinking water sampling.
• ISO 20179 Annex A - Mass spectrometry (MS) as alternative detection (informative).

Keywords: ISO 20179, microcystins, SPE, HPLC‑UV, water quality testing, cyanotoxins, MCYST‑LR, MCYST‑YR, MCYST‑RR.

Standards Content (Sample)

SLOVENSKI STANDARD
01-september-2010
.DNRYRVWYRGH'RORþHYDQMHPLNURFLVWLQRY0HWRGD]HNVWUDNFLMRQDWUGQLID]L
63(LQVWHNRþLQVNRNURPDWRJUDILMRYLVRNHORþOMLYRVWL+3/&]XOWUDYLMROLþQR89
GHWHNFLMR
Water quality - Determination of microcystins - Method using solid phase extraction
(SPE) and high performance liquid chromatography (HPLC) with ultraviolet (UV)
detection
Qualité de l'eau - Dosage des microcystines - Méthode utilisant l'extraction en phase
solide (SPE) et la chromatographie en phase liquide à haute performance (CLHP) avec
détection dans l'ultraviolet (UV)
Ta slovenski standard je istoveten z: ISO 20179:2005
ICS:
13.060.50 3UHLVNDYDYRGHQDNHPLþQH Examination of water for
VQRYL chemical substances
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

INTERNATIONAL ISO
STANDARD 20179
First edition
2005-10-01
Water quality — Determination of
microcystins — Method using solid
phase extraction (SPE) and high
performance liquid chromatography
(HPLC) with ultraviolet (UV) detection
Qualité de l'eau — Dosage des microcystines — Méthode utilisant
l'extraction en phase solide (SPE) et la chromatographie en phase
liquide à haute performance (CLHP) avec détection dans l'ultraviolet
(UV)
Reference number
©
ISO 2005
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.

©  ISO 2005
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail [e-mail]
Web www.iso.org
Published in Switzerland
ii © ISO 2005 – All rights reserved

Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Abbreviated terms . 1
4 Principle. 2
5 Reagents. 2
6 Apparatus . 5
7 Procedure . 6
8 Method performance characteristics and data. 10
9 Test report . 10
Annex A (informative) Mass spectrometry (MS) as an alternative detection . 11
Annex B (informative) Typical chromatogram and absorption spectra . 14
Annex C (informative) Precision data. 16
Bibliography . 17

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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 20179 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2, Physical,
chemical and biochemical methods.
iv © ISO 2005 – All rights reserved

Introduction
The user should be aware that particular problems could require the specification of additional conditions.

INTERNATIONAL STANDARD ISO 20179:2005(E)

Water quality — Determination of microcystins — Method using
solid phase extraction (SPE) and high performance liquid
chromatography (HPLC) with ultraviolet (UV) detection
WARNING — The method requires use of microcystin-containing solutions. Microcystins are highly
hepatotoxic to humans. Laboratory wastes of microcystins shall be collected separately and disposed
as highly toxic chemical waste. Long-term decontamination with concentrated sodium hypochlorite
(NaClO) solution is also possible.
Persons using this International Standard should be familiar with normal laboratory practice. This
standard 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 and to ensure
compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted according to this standard be carried
out by suitably trained staff.
1 Scope
This International Standard specifies a method for the determination and quantification of microcystins in raw
water (containing biomass) and treated water, such as tap water. The method described is validated for
MCYST-RR, MCYST-YR, and MCYST-LR. It is also applicable for the determination of several structure
[1]
variants of these microcystins, but an unambiguous identification cannot be made due to the lack of
commercially available standards and due to co-elution.
The threshold value of 1 µg/l of MCYST-LR in water, proposed by the World Health Organization, can be
followed after microcystin enrichment using solid phase extraction (SPE).
2 Normative references
The following referenced documents are indispensable for the application 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 3696:1987, Water for analytical laboratory use — Specification and test methods
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
3 Abbreviated terms
For the purposes of this document, the following abbreviated terms apply.
APCI atmospheric pressure chemical ionization
MCYST microcystin
MCYST-LR microcystin containing leucine (L) and arginine (R)
MCYST-RR microcystin containing two arginine (R) units
MCYST-YR microcystin containing tyrosine (Y) and arginine (R)
SIM selected ion monitoring
SEC size exclusion chromatography
4 Principle
Water samples containing cyanobacterial material (biomass) shall be filtered first. The biomass is extracted
separately with a solvent (methanol/water). The extract is filtered, diluted and a solid phase extraction (SPE) is
applied for sample clean-up. The filtrate is treated as a pure water sample (see below).
Pure water samples such as tap water are enriched using SPE. The microcystins are eluted from the SPE
cartridges with methanol/water [90/10 by volume] containing 0,1 % by volume of trifluoroacetic acid (TFA).
Microcystins are quantified by reversed-phase high performance liquid chromatography (RP-HPLC) with
ultraviolet/diode array detection at 238 nm.
5 Reagents
Use only reagents of recognized analytical grade and water complying with grade 3 as specified in
ISO 3696:1987, unless otherwise specified.
5.1 Methanol, CH OH, HPLC grade.
5.2 Acetonitrile, CH CN, HPLC grade.
5.3 Trifluoroacetic acid, TFA, CF COOH.
5.4 Standard dilution solution, SPE rinsing solvent, and re-dissolving solvent.
Methanol/water [20/80 by volume].
5.5 Extraction solution
Methanol/water [75/25 by volume].
5.6 SPE elution solution
Methanol/water [90/10 by volume] containing 0,1 % by volume TFA.
5.7 Sodium thiosulfate, solution.
Dissolve 1 g of sodium thiosulfate Na S O (anhydrous or with 5 H O) in 100 ml of water. The final
2 2 3 2
concentration is ρ = 10 g/l (63 mmolar in case of anhydrous Na SO ).
2 3
5.8 Ammonium hydroxide solution
Commercially available ∼ 1 mol/l of ammonium hydroxide solution, NH OH.
5.9 Solid phase extraction cartridges (SPE) for microcystin enrichment
The column shall have a minimum capacity (amount of analyte to be retained by the column) of not less than
100 µg of each microcystin and shall give a recovery of not less than 80 % for MCYST-LR and not less than
70 % for MCYST-RR and MCYST-YR when applied as a standard solution in water containing 0,05 µg of
each microcystin.
2 © ISO 2005 – All rights reserved

NOTE The recovery strongly depends on the SPE cartridge material/brand, material specifications such as carbon
load, particle size etc. The recovery data are based on C-18 cartridges determined by a single measurement. The material
should have the following material specifications: carbon load (16,9 %), particle diameter (54 µm), surface coverage
(333 µg/m based on % C) cartridge volume (3 ml), material per cartridge (500 mg). If the above required recovery values
can not be reached, changing the brand of the SPE cartridge is recommended.
Disk-type SPE cartridges may also be used for the microcystin enrichment from water samples [2].
5.10 HPLC mobile phase solution (A)
To a 1 000 ml volumetric flask, add 800 ml of acetonitrile (5.2) and 500 µl of TFA (5.3) and bring to volume
with acetonitrile. Transfer this solution in a HPLC-eluent bottle. Degas the solution before use.
This solution is stable at room temperature for about 3 weeks.
5.11 HPLC mobile phase solution (B)
To a 1 000 ml volumetric flask, add 800 ml of water and 500 µl of TFA (5.3) and bring to volume with water.
Transfer this solution in a HPLC-eluent bottle. Degas this solution before use.
This solution is stable at room temperature for about 2 weeks.
5.12 HPLC mobile phase gradient (an example)
Table 1 — HPLC mobile phase gradient
Time HPLC mobile phase solution (A) HPLC mobile phase solution (B); Total volume flow rate,
Acetonitrile with 0,05 % TFA (5.10) water with 0,05 % TFA (5.11) depending on the column
min % % ml/min
0 30 70 0,3 to 1,0
10 35 65 0,3 to 1,0
40 70 30 0,3 to 1,0
42 100 0 0,3 to 1,0
44 100 0 0,3 to 1,0
46 30 70 0,3 to 1,0
55 30 70 0,3 to 1,0
5.13 Microcystins, commercially available film in ampoules.
NOTE The quality of commercially available microcystins is very variable. Thus, it is important to follow the procedure
given in 5.14.
5.14 Microcystin stock solutions
To determine the exact concentration of microcystins, dissolve in each stock solution the individual
microcystin delivered from the supplier in pure methanol (5.1). Record the absorption curve between 220 nm
and 250 nm in 1 cm quartz glass cells in a spectrophotometer with methanol (5.1) in the reference cell.
Calculate the mass concentration of each microcystin, ρ, in micrograms per millilitre, µg/ml, using
i
Equation (1):
AM⋅⋅1000
max i
ρ = (1)
i
ε ⋅ d
i
where
A is the absorbance determined at the maximum of the absorption curve;
max
M is the molar mass of each microcystin, in grams per mol; g/mol;
i
ε is the molar absorptivity of each microcystin in methanol (5.1), in litres per (mole × centimetre),
i
l/(mol × cm);
d is the optical path length of the cell, in centimetres, cm;
1 000 is a calculation factor to achieve the final unit micrograms per millilitre, µg/ml.
M and ε are tabulated in Table 2.
i i
Table 2 — Molar mass and molar absorptivity of MCYST-LR, -YR, and -RR (in methanol, at 238 nm)
Microcystin M ε
i i
−1 −1 −1
g mol l mol cm
-LR 994 39 800
-YR 1 044 39 800
-RR 1 037 39 800
NOTE Data taken from Reference [1]. For further details
refer to this reference.
For further HPLC analysis, the solvent methanol/water ratio for the MCYST-LR, -YR, and -RR standards can
be adjusted to 20/80 by volume (i.e. to the standard dilution solution described in 5.4), by adding water and
allowing a concentration of 10 µg/ml for each microcystin.
5.15 Mixed microcystin stock solutions
Prepare a standard solution containing 2,5 µg/ml each of MCYST-LR, -YR, and -RR in the standard dilution
solution (5.4). Store it below −16 °C. To avoid incorporation of water by condensation, do not open the vial
until its contents have reached room temperature.
If the solution is to be stored for a long period, use a hermetic vial. In case of doubt, weigh the vial and record
any changes in mass during storage.
5.16 Mixed microcystin standard solutions
Pipette the volumes of microcystin stock solutions (5.14) given in Table 3 into 1 ml vials.
To each vial, add the volume of the standard dilution solution (5.4) given in Table 3 to achieve a final volume
of 1 000 µl, and shake well.
4 © ISO 2005 – All rights reserved

Table 3 — Pipetting scheme for the mixed microcystin standard solutions
Standard Withdrawal volume from Volume to add from the Mass concentration of standard solution
solution each microcystin stock standard dilution solution
solution [MCYST- (5.4) to achieve a final
LR, -YR, -RR (5.14)] volume of 1 000 µl
µl µl µg/ml
MCYST-LR MCYST-YR MCYST-RR
1 20 940 0,2 0,2 0,2
2 40 880 0,4 0,4 0,4
3 100 700 1,0 1,0 1,0
4 200 400 2,0 2,0 2,0
5 300 100 3,0 3,0 3,0
5.17 Spiking solution for method control
Prepare a spiking solution by pipetting 200 µl of the mixed microcystin stock solution (prepared according to
5.15) into a 500 ml volumetric flask. Dilute it to the mark with water (tap-water or blank water from a natural
lake), and shake well.
The concentration of this spiking solution is 1 µg/l for MCYST-LR, -YR, and -RR.
6 Apparatus
6.1 General
The laboratory glassware and equipment to be used is not specified in this International Standard, as the
choice of apparatus will depend on the specific applications and circumstances.
Avoid the use of plastics whenever possible. This is necessary because the use of plastics (e.g. plastic
pipettes, plastic tubing or plastic cartridges) may cause losses of microcystins through absorption on the
surface walls.
6.2 Adjustable horizontal shaker, needed only for the analysis of samples containing phytoplankton.
6.3 Glass microfibre filter paper, retention size 1 µm to 2 µm.
The maximum diameter of the filter should be 47 mm.
Filtration is needed only for the analysis of samples containing phytoplankton.
6.4 SPE reservoir, 500 ml with connector for cartridges.
6.5 Vacuum pump for SPE
−1
6.6 Laboratory centrifuge, W 4 000 min , relative centrifugal force (RCF) W 10 000 g.
The use of an explosion-proof centrifuge is strongly advised due to the use of inflammable extraction solvents.
6.7 Ultrasonic probe, with characteristics of ∼ 60 W, ∼ 20 kHz.
6.8 Ultrasonic bath
6.9 Heating block with temperature control and nitrogen gas delivery unit, with the following
characteristics: block-temperature 30 °C to 50 °C; gas temperature: ∼ 20 °C; and gas-purity W 99,996 %.
6.10 Disposable filter unit, pore size < 0,45 µm.
Prior to use, verify that no microcystin losses occur during filtration (recovery testing).
NOTE There is a possibility that various filter materials may retain microcystins. Optionally a microfuge may be used
in order to avoid losses.
6.11 Sampling bottles, dark glassware, sterile and pre-cleaned.
6.12 HPLC system, consisting of the following.
6.12.1 Binary HPLC pump, suitable for volume flow rates between 0,3 ml/min and 1,0 ml/min.
6.12.2 HPLC column oven, with temperature control unit (35 °C).
6.12.3 Injection system, with injection volume range from 5 µl to 20 µl.
6.12.4 HPLC column (e.g. C column), packed with material of particle size 3 µm to 5 µm and inner
diameter 2 mm to 4,6 mm, length 250 mm, to ensure baseline resolution of MCYST-LR, -YR, and -RR
standards.
A suitable guard-column should be used. Pressure range should be 70,000 hPa to 200,000 hPa (1 015 psi to
2 900 psi).
6.12.5 UV/photo diode array (PDA) detector, with a wavelength of λ = 238 nm including background
correction.
The wavelength range of the PDA shall be 200 nm to 300 nm.
The limit of detection (LOD) for the system should be u 0,1 ng/µl (signal-to-noise-ratio = 3), and the limit of
quantification (LOQ) should be u 0,2 ng/µl (signal-to-noise-ratio = 6) for each microcystin (using a standard
solution).
7 Procedure
7.1 Sampling and conditioning
Collect water samples as specified in ISO 5667-4 and ISO 5667-5 and store the samples (not longer than
48 h) in a cool (4 °C to 8 °C) and dark place.
Prior to conditioning, adjust the SPE cartridge (5.9) to room temperature.
For conditioning, refer to the suppliers´ recommendation. If this is not indicated, first pass 4 ml of methanol
(5.1) through the cartridge. Then pass 4 ml of water through the cartridge. Let the solvents pass at a rate
< 10 ml/min through the column, and make sure that a small portion of the solvent remains on top of the
column until the sample solution is applied.
7.2 Sample preparation
7.2.1 Treated water/tap water
Concentrate microcystins in water samples using solid phase extraction (7.4).
6 © ISO 2005 – All rights reserved

7.2.2 Raw water containing phytoplankton
First pass the sample (recommended volume: 50 ml to 100 ml) through a filter (6.3) to separate the biomass
from the liquid fraction. If floatin
...

INTERNATIONAL ISO
STANDARD 20179
First edition
2005-10-01
Water quality — Determination of
microcystins — Method using solid
phase extraction (SPE) and high
performance liquid chromatography
(HPLC) with ultraviolet (UV) detection
Qualité de l'eau — Dosage des microcystines — Méthode utilisant
l'extraction en phase solide (SPE) et la chromatographie en phase
liquide à haute performance (CLHP) avec détection dans l'ultraviolet
(UV)
Reference number
©
ISO 2005
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.

©  ISO 2005
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail [e-mail]
Web www.iso.org
Published in Switzerland
ii © ISO 2005 – All rights reserved

Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Abbreviated terms . 1
4 Principle. 2
5 Reagents. 2
6 Apparatus . 5
7 Procedure . 6
8 Method performance characteristics and data. 10
9 Test report . 10
Annex A (informative) Mass spectrometry (MS) as an alternative detection . 11
Annex B (informative) Typical chromatogram and absorption spectra . 14
Annex C (informative) Precision data. 16
Bibliography . 17

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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 20179 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2, Physical,
chemical and biochemical methods.
iv © ISO 2005 – All rights reserved

Introduction
The user should be aware that particular problems could require the specification of additional conditions.

INTERNATIONAL STANDARD ISO 20179:2005(E)

Water quality — Determination of microcystins — Method using
solid phase extraction (SPE) and high performance liquid
chromatography (HPLC) with ultraviolet (UV) detection
WARNING — The method requires use of microcystin-containing solutions. Microcystins are highly
hepatotoxic to humans. Laboratory wastes of microcystins shall be collected separately and disposed
as highly toxic chemical waste. Long-term decontamination with concentrated sodium hypochlorite
(NaClO) solution is also possible.
Persons using this International Standard should be familiar with normal laboratory practice. This
standard 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 and to ensure
compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted according to this standard be carried
out by suitably trained staff.
1 Scope
This International Standard specifies a method for the determination and quantification of microcystins in raw
water (containing biomass) and treated water, such as tap water. The method described is validated for
MCYST-RR, MCYST-YR, and MCYST-LR. It is also applicable for the determination of several structure
[1]
variants of these microcystins, but an unambiguous identification cannot be made due to the lack of
commercially available standards and due to co-elution.
The threshold value of 1 µg/l of MCYST-LR in water, proposed by the World Health Organization, can be
followed after microcystin enrichment using solid phase extraction (SPE).
2 Normative references
The following referenced documents are indispensable for the application 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 3696:1987, Water for analytical laboratory use — Specification and test methods
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
3 Abbreviated terms
For the purposes of this document, the following abbreviated terms apply.
APCI atmospheric pressure chemical ionization
MCYST microcystin
MCYST-LR microcystin containing leucine (L) and arginine (R)
MCYST-RR microcystin containing two arginine (R) units
MCYST-YR microcystin containing tyrosine (Y) and arginine (R)
SIM selected ion monitoring
SEC size exclusion chromatography
4 Principle
Water samples containing cyanobacterial material (biomass) shall be filtered first. The biomass is extracted
separately with a solvent (methanol/water). The extract is filtered, diluted and a solid phase extraction (SPE) is
applied for sample clean-up. The filtrate is treated as a pure water sample (see below).
Pure water samples such as tap water are enriched using SPE. The microcystins are eluted from the SPE
cartridges with methanol/water [90/10 by volume] containing 0,1 % by volume of trifluoroacetic acid (TFA).
Microcystins are quantified by reversed-phase high performance liquid chromatography (RP-HPLC) with
ultraviolet/diode array detection at 238 nm.
5 Reagents
Use only reagents of recognized analytical grade and water complying with grade 3 as specified in
ISO 3696:1987, unless otherwise specified.
5.1 Methanol, CH OH, HPLC grade.
5.2 Acetonitrile, CH CN, HPLC grade.
5.3 Trifluoroacetic acid, TFA, CF COOH.
5.4 Standard dilution solution, SPE rinsing solvent, and re-dissolving solvent.
Methanol/water [20/80 by volume].
5.5 Extraction solution
Methanol/water [75/25 by volume].
5.6 SPE elution solution
Methanol/water [90/10 by volume] containing 0,1 % by volume TFA.
5.7 Sodium thiosulfate, solution.
Dissolve 1 g of sodium thiosulfate Na S O (anhydrous or with 5 H O) in 100 ml of water. The final
2 2 3 2
concentration is ρ = 10 g/l (63 mmolar in case of anhydrous Na SO ).
2 3
5.8 Ammonium hydroxide solution
Commercially available ∼ 1 mol/l of ammonium hydroxide solution, NH OH.
5.9 Solid phase extraction cartridges (SPE) for microcystin enrichment
The column shall have a minimum capacity (amount of analyte to be retained by the column) of not less than
100 µg of each microcystin and shall give a recovery of not less than 80 % for MCYST-LR and not less than
70 % for MCYST-RR and MCYST-YR when applied as a standard solution in water containing 0,05 µg of
each microcystin.
2 © ISO 2005 – All rights reserved

NOTE The recovery strongly depends on the SPE cartridge material/brand, material specifications such as carbon
load, particle size etc. The recovery data are based on C-18 cartridges determined by a single measurement. The material
should have the following material specifications: carbon load (16,9 %), particle diameter (54 µm), surface coverage
(333 µg/m based on % C) cartridge volume (3 ml), material per cartridge (500 mg). If the above required recovery values
can not be reached, changing the brand of the SPE cartridge is recommended.
Disk-type SPE cartridges may also be used for the microcystin enrichment from water samples [2].
5.10 HPLC mobile phase solution (A)
To a 1 000 ml volumetric flask, add 800 ml of acetonitrile (5.2) and 500 µl of TFA (5.3) and bring to volume
with acetonitrile. Transfer this solution in a HPLC-eluent bottle. Degas the solution before use.
This solution is stable at room temperature for about 3 weeks.
5.11 HPLC mobile phase solution (B)
To a 1 000 ml volumetric flask, add 800 ml of water and 500 µl of TFA (5.3) and bring to volume with water.
Transfer this solution in a HPLC-eluent bottle. Degas this solution before use.
This solution is stable at room temperature for about 2 weeks.
5.12 HPLC mobile phase gradient (an example)
Table 1 — HPLC mobile phase gradient
Time HPLC mobile phase solution (A) HPLC mobile phase solution (B); Total volume flow rate,
Acetonitrile with 0,05 % TFA (5.10) water with 0,05 % TFA (5.11) depending on the column
min % % ml/min
0 30 70 0,3 to 1,0
10 35 65 0,3 to 1,0
40 70 30 0,3 to 1,0
42 100 0 0,3 to 1,0
44 100 0 0,3 to 1,0
46 30 70 0,3 to 1,0
55 30 70 0,3 to 1,0
5.13 Microcystins, commercially available film in ampoules.
NOTE The quality of commercially available microcystins is very variable. Thus, it is important to follow the procedure
given in 5.14.
5.14 Microcystin stock solutions
To determine the exact concentration of microcystins, dissolve in each stock solution the individual
microcystin delivered from the supplier in pure methanol (5.1). Record the absorption curve between 220 nm
and 250 nm in 1 cm quartz glass cells in a spectrophotometer with methanol (5.1) in the reference cell.
Calculate the mass concentration of each microcystin, ρ, in micrograms per millilitre, µg/ml, using
i
Equation (1):
AM⋅⋅1000
max i
ρ = (1)
i
ε ⋅ d
i
where
A is the absorbance determined at the maximum of the absorption curve;
max
M is the molar mass of each microcystin, in grams per mol; g/mol;
i
ε is the molar absorptivity of each microcystin in methanol (5.1), in litres per (mole × centimetre),
i
l/(mol × cm);
d is the optical path length of the cell, in centimetres, cm;
1 000 is a calculation factor to achieve the final unit micrograms per millilitre, µg/ml.
M and ε are tabulated in Table 2.
i i
Table 2 — Molar mass and molar absorptivity of MCYST-LR, -YR, and -RR (in methanol, at 238 nm)
Microcystin M ε
i i
−1 −1 −1
g mol l mol cm
-LR 994 39 800
-YR 1 044 39 800
-RR 1 037 39 800
NOTE Data taken from Reference [1]. For further details
refer to this reference.
For further HPLC analysis, the solvent methanol/water ratio for the MCYST-LR, -YR, and -RR standards can
be adjusted to 20/80 by volume (i.e. to the standard dilution solution described in 5.4), by adding water and
allowing a concentration of 10 µg/ml for each microcystin.
5.15 Mixed microcystin stock solutions
Prepare a standard solution containing 2,5 µg/ml each of MCYST-LR, -YR, and -RR in the standard dilution
solution (5.4). Store it below −16 °C. To avoid incorporation of water by condensation, do not open the vial
until its contents have reached room temperature.
If the solution is to be stored for a long period, use a hermetic vial. In case of doubt, weigh the vial and record
any changes in mass during storage.
5.16 Mixed microcystin standard solutions
Pipette the volumes of microcystin stock solutions (5.14) given in Table 3 into 1 ml vials.
To each vial, add the volume of the standard dilution solution (5.4) given in Table 3 to achieve a final volume
of 1 000 µl, and shake well.
4 © ISO 2005 – All rights reserved

Table 3 — Pipetting scheme for the mixed microcystin standard solutions
Standard Withdrawal volume from Volume to add from the Mass concentration of standard solution
solution each microcystin stock standard dilution solution
solution [MCYST- (5.4) to achieve a final
LR, -YR, -RR (5.14)] volume of 1 000 µl
µl µl µg/ml
MCYST-LR MCYST-YR MCYST-RR
1 20 940 0,2 0,2 0,2
2 40 880 0,4 0,4 0,4
3 100 700 1,0 1,0 1,0
4 200 400 2,0 2,0 2,0
5 300 100 3,0 3,0 3,0
5.17 Spiking solution for method control
Prepare a spiking solution by pipetting 200 µl of the mixed microcystin stock solution (prepared according to
5.15) into a 500 ml volumetric flask. Dilute it to the mark with water (tap-water or blank water from a natural
lake), and shake well.
The concentration of this spiking solution is 1 µg/l for MCYST-LR, -YR, and -RR.
6 Apparatus
6.1 General
The laboratory glassware and equipment to be used is not specified in this International Standard, as the
choice of apparatus will depend on the specific applications and circumstances.
Avoid the use of plastics whenever possible. This is necessary because the use of plastics (e.g. plastic
pipettes, plastic tubing or plastic cartridges) may cause losses of microcystins through absorption on the
surface walls.
6.2 Adjustable horizontal shaker, needed only for the analysis of samples containing phytoplankton.
6.3 Glass microfibre filter paper, retention size 1 µm to 2 µm.
The maximum diameter of the filter should be 47 mm.
Filtration is needed only for the analysis of samples containing phytoplankton.
6.4 SPE reservoir, 500 ml with connector for cartridges.
6.5 Vacuum pump for SPE
−1
6.6 Laboratory centrifuge, W 4 000 min , relative centrifugal force (RCF) W 10 000 g.
The use of an explosion-proof centrifuge is strongly advised due to the use of inflammable extraction solvents.
6.7 Ultrasonic probe, with characteristics of ∼ 60 W, ∼ 20 kHz.
6.8 Ultrasonic bath
6.9 Heating block with temperature control and nitrogen gas delivery unit, with the following
characteristics: block-temperature 30 °C to 50 °C; gas temperature: ∼ 20 °C; and gas-purity W 99,996 %.
6.10 Disposable filter unit, pore size < 0,45 µm.
Prior to use, verify that no microcystin losses occur during filtration (recovery testing).
NOTE There is a possibility that various filter materials may retain microcystins. Optionally a microfuge may be used
in order to avoid losses.
6.11 Sampling bottles, dark glassware, sterile and pre-cleaned.
6.12 HPLC system, consisting of the following.
6.12.1 Binary HPLC pump, suitable for volume flow rates between 0,3 ml/min and 1,0 ml/min.
6.12.2 HPLC column oven, with temperature control unit (35 °C).
6.12.3 Injection system, with injection volume range from 5 µl to 20 µl.
6.12.4 HPLC column (e.g. C column), packed with material of particle size 3 µm to 5 µm and inner
diameter 2 mm to 4,6 mm, length 250 mm, to ensure baseline resolution of MCYST-LR, -YR, and -RR
standards.
A suitable guard-column should be used. Pressure range should be 70,000 hPa to 200,000 hPa (1 015 psi to
2 900 psi).
6.12.5 UV/photo diode array (PDA) detector, with a wavelength of λ = 238 nm including background
correction.
The wavelength range of the PDA shall be 200 nm to 300 nm.
The limit of detection (LOD) for the system should be u 0,1 ng/µl (signal-to-noise-ratio = 3), and the limit of
quantification (LOQ) should be u 0,2 ng/µl (signal-to-noise-ratio = 6) for each microcystin (using a standard
solution).
7 Procedure
7.1 Sampling and conditioning
Collect water samples as specified in ISO 5667-4 and ISO 5667-5 and store the samples (not longer than
48 h) in a cool (4 °C to 8 °C) and dark place.
Prior to conditioning, adjust the SPE cartridge (5.9) to room temperature.
For conditioning, refer to the suppliers´ recommendation. If this is not indicated, first pass 4 ml of methanol
(5.1) through the cartridge. Then pass 4 ml of water through the cartridge. Let the solvents pass at a rate
< 10 ml/min through the column, and make sure that a small portion of the solvent remains on top of the
column until the sample solution is applied.
7.2 Sample preparation
7.2.1 Treated water/tap water
Concentrate microcystins in water samples using solid phase extraction (7.4).
6 © ISO 2005 – All rights reserved

7.2.2 Raw water containing phytoplankton
First pass the sample (recommended volume: 50 ml to 100 ml) through a filter (6.3) to separate the biomass
from the liquid fraction. If floating layers of algae are present, one filter may be insufficient for the filtration of
even 50 ml of water. In this case, immediately replace the clogged filter with a fresh one. Concentrate the
microcystins in the filtrate by solid phase extraction (see 7.4).
Extract the biomass on the filter separately (see 7.3) followed by clean-up (see 7.5) of the extract prior to
HPLC analysis (see 7.6).
NOTE If a gravimetric filter is used, the mass of the biomass may be determined and the microcystin content can be
expressed in micrograms per gram (µg/g).
7.3 Extraction of microcystins from the cells on the filter
To extract the cells on the filter (if
...