ISO 22104:2021
(Main)Water quality — Determination of microcystins — Method using liquid chromatography and tandem mass spectrometry (LC-MS/MS)
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)
General Information
Standards Content (Sample)
INTERNATIONAL ISO
STANDARD 22104
First edition
2021-07
Water quality — Determination of
microcystins — Method using liquid
chromatography and tandem mass
spectrometry (LC-MS/MS)
Qualité de l'eau — Dosage des microcystines — Méthode par
chromatographie en phase liquide couplée à la spectrométrie de
masse en tandem (CL-SM/SM)
Reference number
©
ISO 2021
© ISO 2021
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ii © ISO 2021 – All rights reserved
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Interferences . 2
5.1 Biases . 3
5.2 Limitations . 3
6 Reagents and standards . 3
6.1 General . 3
6.2 Preparation of solutions . 4
7 Apparatus . 6
8 Sampling . 7
9 Procedure. 8
9.1 Preparation of samples . 8
9.1.1 General. 8
9.1.2 Preparation of method blank sample . 8
9.1.3 Preparation of laboratory control spike sample . 8
9.1.4 Preparation of calibration control sample . 8
9.1.5 Preparation of calibration standard solutions . 8
9.1.6 Preparation of drinking water and freshwater sample . 9
9.1.7 Sample preparation procedure with freeze/thaw cycles . 9
9.2 Instrumental analysis by LC-MS/MS procedure .10
9.2.1 Instrument set-up parameters .10
9.3 Run processing and quality assurance .13
9.3.1 Run sequence .13
9.3.2 Run control operations / limits .13
10 Calibration .14
11 Evaluation and calculation of results .15
11.1 Identification and calculations .15
11.2 Calibration curve equation determination .15
11.3 Internal standard calculation .15
11.4 Internal standard recovery calculation .16
12 Expressing of results .16
13 Test report .16
Annex A (informative) Use of high resolution mass spectrometry detectors (HRMS).17
Annex B (informative) Use of online solid phase extraction coupled to liquid
chromatography for the automated analysis of microcystins .19
Annex C (informative) Use of manual solid phase extraction prior to instrumental analysis
for improved method detection limits .25
Bibliography .32
Foreword
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electrotechnical standardization.
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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
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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
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iv © ISO 2021 – All rights reserved
INTERNATIONAL STANDARD ISO 22104:2021(E)
Water quality — Determination of microcystins —
Method using liquid chromatography and tandem mass
spectrometry (LC-MS/MS)
WARNING — Persons using this document should be familiar with normal laboratory practice.
This document does not purport to address all of the safety problems, if any, associated with its
use. It is the responsibility of the user to establish appropriate safety and health practices.
IMPORTANT — It is absolutely essential that tests conducted in accordance with this document
be carried out by suitably qualified staff.
1 Scope
This document specifies a method for the quantification of twelve microcystin variants (microcystin-
7 7
LR, -LA, -YR, -RR, -LY, -WR, -HtyR, -HilR, -LW, -LF, [Dha ]-microcystin-LR, and [Dha ]-microcystin-RR)
in drinking water and freshwater samples between 0,05 µg/l to 1,6 µg/l. The method can be used to
determine further microcystins, provided that analytical conditions for chromatography and mass
spectrometric detection has been tested and validated for each microcystin. Samples are analysed by
LC-MS/MS using internal standard calibration.
This method is performance based. The laboratory is permitted to modify the method, e.g. increasing
direct flow injection volume for low interference samples or diluting the samples to increase the upper
working range limit, provided that all performance criteria in this method are met.
Detection of microcystins by high resolution mass spectrometry (HRMS) as an alternative for tandem
mass spectrometry (MS/MS) is described in Annex A.
An alternative automated sample preparation method based on on-line solid phase extraction coupled
to liquid chromatography is described in Annex B.
When instrumental sensitivity is not sufficient to reach the method detection limits by direct flow
injection, a solid phase extraction clean-up and concentration step is described in Annex C.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 3696, Water for analytical laboratory use — Specification and test methods
3 Terms and definitions
No terms and definitions are listed in this document.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
4 Principle
This method is designed to identify and quantify total (free + intracellular) microcystins in water
by direct flow injection liquid chromatography and tandem mass spectrometry (LC-MS/MS) with
[1] [2]
electrospray ionization , . 12 microcystins (Table 1) are determined quantitatively by multi-point
calibration using nodularin as internal standard.
Table 1 — Microcystin variants included in the method
a
Microcystin variant CAS-RN Molecular formular
Microcystin-LR 101043-37-2 C H N O
49 74 10 12
Microcystin-RR 111755-374 C H N O
49 75 13 12
Microcystin-LA 96180-79-9 C H N O
46 67 7 12
Microcystin-YR 101064-48-6 C H N O
52 72 10 13
Microcystin-LY 123304-10-9 C H N O
52 71 7 13
Microcystin-WR 138234-58-9 C H N O
54 73 11 12
Microcystin-HtyR 913178-65-1 C H N O
52 72 10 13
Microcystin-HilR N/A C H N O
50 76 10 12
Microcystin-LW 157622-02-1 C H N O
54 72 8 12
Microcystin-LF 154037-70-4 C H N O
52 71 7 12
[Dha ]-Microcystin-LR (dmLR) 120011-66-7 C H N O
48 72 10 12
[Dha ]-Microcystin-RR (dmRR) 131022-02-1 C H N O
48 73 13 12
a
CAS-RN: 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
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.
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
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.
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
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 constant flow of 0,35 ml/min using the analytical
column described in 7.16. The instrument should also be equipped with an autosampler capable to
accommodate enough samples to process an entire batch.
7.16 Analytical column, C , 2,1 mm ID × 150 mm length, 1,8 μm particle size, suitable for
chromatography of the selected substances. Other columns showing similar performance can be used
alternatively.
7.17 Mass spectrometer (MS)
The mass spectrometer should have a triple quadrupole (tandem MS/MS) configuration capable of
performing collision induced dissociation (CID) experiments at different collision energies (CE) and
acquire in multiple reaction monitoring (MRM) mode. The instrument should also be equipped with
an ionization interface such as an electrospray ionization probe (ESI) with adjustable capillary voltage
and a combination f pumps capable to provide sufficient vacuum for the correct operation of the system.
The instrument shall also be supplied with the corresponding gases for the correct operation of the
electrospray source (cone and desolvation gases, usually nitrogen) and collision cell (usually argon or
nitrogen). Alternatively, a high-resolution mass spectrometer (HRMS) can be used instead of MS/MS, as
described in Annex A.
8 Sampling
Collect the samples in 500 ml amber glass bottles (7.1). A minimum of 500 ml should be submitted
for testing. Samples should be preserved with 150 mg/l of sodium thiosulfate (6.1.6) as a neutralizing
additive to remove chlorine: add 0,75 ml of sodium thiosulfate preservative solution (6.2.2) to
500 ml sample. This is particularly important for treated drinking waters or those waters suspected
of containing residual chlorine. Higher concentration of preservative can be employed in samples
containing large amount of chlorine (ISO 5667-1, ISO 5667-3; ISO 5667-4, ISO 5667-5, ISO 5667-6).
For a sample preparation procedure with manual SPE (Annex C) a minimum volume of 1 l should be
taken in amber glass bottles. Samples should be preserved by adding 1,5 ml of sodium thiosulfate
preservative solution (6.2.2) to 1 000 ml sample.
Samples should be stored in the dark at (5 ± 3) °C, and should be extracted within 21 d of sampling.
9 Procedure
9.1 Preparation of samples
IMPORTANT — Allow all working standard solutions to warm to room temperature before
opening the vials. Rinse syringes with methanol.
9.1.1 General
All glassware should be cleaned prior to the analysis (6.1.7).
9.1.2 Preparation of method blank sample
For each batch of samples, prepare a method blank sample by transferring 5 ml of quenched tap
water (6.2.1) into a 15 ml centrifuge tube (7.4).
9.1.3 Preparation of laboratory control spike sample
For each batch of samples processed, prepare a laboratory control spike sample consisting of
5 ml of quenched tap water (6.2.1) in a 15 ml centrifuge tube (7.4) spiked with 8,0 μl of MCYST mix
C solution (6.2.11) and 40 μl of supplemental microcystin mix solution S3 (6.2.8) using the appropriate
volume microsyringes (7.3) to give a concentration of 0,32 μg/l for each target microcystin. This target
concentration should be used as an example, the laboratory can modify the control spike concentration
depending on the analysis expectations.
9.1.4 Preparation of calibration control sample
For each batch of samples processed, prepare a calibration control sample consisting of 5 ml of quenched
tap water (6.2.1) in a 15 ml centrifuge tube (7.4) spiked with 16 μl of the control standard solution
solution CS2 (6.2.15) to give a concentration of 0,32 μg/l for microcystin-LR, for example.
NOTE Other microcystins with certified concentration can also be used to validate standard mixture
concentration when available.
9.1.5 Preparation of calibration standard solutions
The calibration standard solutions are used for calibration of the method including the whole sample
preparation procedure.
Two different microcystin standard stock solutions are prepared: MCYST mix C and D solutions (6.2.11,
6.2.12) include those commercially available variants that are more commonly found in freshwater
[3] [4] [5]
environments or regulated by guidelines (LR, LA, RR and YR , , ).
Supplemental microcystins solutions S2 and S3 (6.2.7, 6.2.8) include those variants that are also
commercially available, but are usually not detected at high concentrations in freshwater (dmLR,
dmRR, LF, LW, WR, LY, HtyR, HilR), even though some of them might be present in higher concentrations
depending on the nature of the algal bloom (e.g. dmRR in Planktothrix blooms). Depending on standard
availability or sample expectations, some laboratories might decide to spike the supplemental
microcystins only for the lower range of the calibration curve, for example MR1 to MR5.
NOTE 1 Alternative mixtures of stock solutions including other microcystins can be prepared depending on
expected local variants.
8 © ISO 2021 – All rights reserved
For each batch of samples processed, prepare the following method recovery (MR) samples consisting
of 5 ml of quenched tap water (6.2.1) in 15 ml centrifuge tube (7.4). Table 2 gives the volume (µl) of each
MCYST mix solutions and supplement mix solutions that is added to each 5 ml calibration standard
solution to produce the required target compound concentrations.
NOTE 2 For freshwater samples, blank lake water can be used alternatively for the preparation of calibration
standard solutions.
Table 2 — Calibration standard solutions preparation
MCYST MCYST Supplemental Supplemental
Compound
MR No mix D mix C mix S3 mix S2
concentration
(6.2.12) (6.2.11) (6.2.8) (6.2.7)
µg/l µl µl µl µl
MR1 0 0 0 0 0
MR2 0,05 12,5 0 6,25 0
MR3 0,08 20 0 10 0
MR4 0,16 40 0 20 0
MR5 0,32 0 8 40 0
MR6 0,80 0 20 0 10
MR7 1,2 0 30 0 15
MR8 1,6 0 40 0 20
Not all MRs need to be prepared and processed. If sufficient information is known about the expected
concentrations, fewer MRs are needed. The MRs selected are based on the concentrations that would
sufficiently bracket the algal toxin concentrations in the samples. The working range of MR solutions
can be modified to meet different guidelines values.
9.1.6 Preparation of drinking water and freshwater sample
Allow the sample bottles to warm to room temperature.
Shake samples well right before taking a 5 ml aliquot of the homogenized sample with a 5 ml
pipette (7.6). Samples of fresh water samples having a high density of cyanobacteria (algal blooms)
are homogenized using homogenizer (7.14) at 10 000 RPM for 5 min before aliquoting. For fresh
water samples the preparation and analysis shall be performed in three replicates due to a possible
inhomogeneous distribution of cyanobacteria with intracellular microcystin.
9.1.7 Sample preparation procedure with freeze/thaw cycles
9.1.7.1 Add 15,0 μl of the working internal standard solution IS2 (6.2.5) to the method blank (9.1.2),
laboratory control spike sample (9.1.3), calibration control sample (9.1.4), calibration standard
solutions (9.1.5) and samples (9.1.6) to give a internal standard concentration of 0,24 μg/l. Cap the
centrifuge tubes and shake well to mix the samples.
9.1.7.2 Place all the 15 ml centrifuge tubes containing the samples (9.1.7.1) in tube rack(s) and put the
rack(s) in the freezer (7.11) at −28 °C for 50 min. Make sure all samples are completely frozen by visual
inspection.
9.1.7.3 Place the centrifuge tubes rack(s) with the frozen samples in the water bath (7.12) at 50 °C for
15 min. Make sure all samples are completely thawed by visual inspection.
9.1.7.4 Repeat steps 9.1.7.2 and 9.1.7.3 two more times to complete three freeze/thaw cycles.
9.1.7.5 Centrifuge all the 15 ml centrifuge tubes containing the samples (9.1.7.1) at 4 000 r/min for
5 min using a centrifuge (7.5).
9.1.7.6 Pour 4 ml of the supernatant into an opened plastic syringe (7.8) connected to a 0,2 µm GHP
syringe filter (7.9). Discard the first 2 ml filtered to condition the membrane and collect around 1,5 ml
filtered in a glass sample vial (7.10). Store the samples at (3 ± 2) °C until ready for analysis.
9.2 Instrumental analysis by LC-MS/MS procedure
9.2.1 Instrument set-up parameters
IMPORTANT — 9.2.1.1 and 9.2.1.2 require basic proficiency and familiarization of the analyst
with the vendor’s LC-MS/MS hardware and software. Set up conditions may vary depending on
the different instrument models and the values detailed in the section below should be used as
an example.
9.2.1.1 Typical liquid chromatograph (LC) parameters
Table 3 — Typical LC-parameters
Parameter Value
Mobile phase - Reservoir A: Water with 0,1 % formic acid
Mobile phase - Reservoir B: Acetonitrile with 0,1 % formic acid
Gradient program: Start at 95 % A until minute 3,75
linear ramp to 5 % A until minute 8,25
hold at 5 % A until minute 10
linear ramp to 95 % A until minute 10,10
hold at 95 % A until minute 13
Flowrate: Constant flow, 0,35 ml/min
Back pressure: 6,8 MPa (typical at initial conditions)
Column compartment: 45 °C
Autosampler compartment: 5 °C
Injection volume: 50 μl
Filling speed: 10 μl/s
Injection speed: 5 μl/s
Stop time: 13 min
Maximum pressure: 11 MPa
Minimum pressure: 0 MPa
NOTE Changes in the instrumental conditions can be accepted as long as they offer similar chromatographic
performance. Injection volume can be modified depending on instrumental sensitivity.
Operate the HPLC instrumentation in accordance with the instruction provided by the manufacturer.
Use a suitable HPLC column (7.16) for chromatographic separation and select the chromatograhic
conditions in Table 3, for example.
Complete separation of the substances is not necessary provided that interferences of the quantitative
determination does not occur during peak overlapping. Optimize the separation of the analytes with
gradient elution if necessary.
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9.2.1.2 Typical mass spectrometer (MS) conditions
Table 4 — Typical MS-conditions
Parameter Value
Scan type: Multiple reaction monitoring (MRM)
Ion mode: Positive ESI
Resolution Q1: Unit
Resolution Q3: Unit
MS/MS transitions: see Table 5
Acquisition window: ± 30 s from retention time
Table 5 — Microcystins MS/MS acquisition conditions
Microcystin Retention time Precursor ion Quantifier Qualifier prod- Collision energy
(min) product ion uct ion (eV)
Microcystin-dmRR 6,85 512,8 135,1 199,1 45
Microcystin-RR 6,90 519,8 135,1 213,1 45
Nodularin (IS) 7,14 825,5 135,1 227,1 70
Microcystin-YR 7,22 1 045,5 135,1 213,1 102
Microcystin-dmLR 7,23 981,5 135,1 199,1 98
Microcystin-HtyR 7,24 1 059,6 135,1 213,1 105
Microcystin-LR 7,27 995,6 135,1 213,1 98
Microcystin-HilR 7,33 1 009,6 135,1 213,1 97
Microcystin-WR 7,36 1 068,6 135,1 213,1 105
Microcystin-LA 8,17 910,5 135,1 213,1 87
Microcystin-LY 8,20 1 002,5 135,1 213,1 97
Microcystin-LW 8,57 1 025,5 135,1 213,1 98
Microcystin-LF 8,70 986,5 135,1 213,1 92
NOTE Optimum collision energies to maximize the abundance of qualifying and quantifying ions can vary
depending on the instrument
Identify the optimal settings for ionization under the specified chromatographic conditions and set
the method-specific settings for the source and MS parameters (Tables 4 and 5), for example. Figure 1
shows a typical chromatogram of the twelve microcystins and nodularin as internal standard under
the instrumental conditions described in 9.2.1 (Tables 3, 4 and 5).
Key
Y1 microcystin-dmRR Y8 microcystin-HilR
Y2 microcystin-RR Y9 microcystin-WR
Y3 nodularin Y10 microcystin-LA
12 © ISO 2021 – All rights reserved
Y4 microcystin-Yr Y11 microcystin-LY
Y5 microcystin-dmLR Y12 microcystinLW
Y6 microcystins-HtyR Y13 microcystin-LF
Y7 microcystin-LR
Figure 1 — LC-MS/MS chromatogram of the 12 microcystins and nodularin as internal standard
(IS)
9.3 Run processing and quality assurance
9.3.1 Run sequence
A possible run sequence includes analysing, for example:
1) Instrument check mix (high) solution (6.2.13); minimum of two injections;
2) Method blank (9.1.2);
3) Calibration standard solutions (9.1.5);
4) Laboratory control spike sample (9.1.3);
5) Calibration control sample (9.1.4);
6) Samples, including replicates (9.1.6);
7) Calibration standard solution (MR5) every 20 samples to check for instrument drift during long
sequences and at the end of the samples;
8) Instrument check mix (high) solution (6.2.13).
9.3.2 Run control operations / limits
9.3.2.1 Instrument check mix (high) solution
The instrument check mix (high) solution (6.2.13) is injected at least twice at the beginning of
each instrument run. The two sample runs are used to stabilize the instrument system plus check
chromatographic performance (peak shape, retention time, sensitivity) and general mass spectrometer
sensitivity. Good chromatography is exemplified by Gaussian peaks with peak widths < 15 s at the
baseline. If the chromatography is not adequate, the analytical run is stopped, and system maintenance
is done in accordance with the instructions of the instrument manufacturer.
9.3.2.2 Low level method recovery (MR2) sample
A typical way to evaluate the chromatographic performance would be measuring the 0,05 µg/l method
recovery (MR2) sample to ensure adequate instrument sensitivity and chromatographic peak shape.
The microcystin-LR MRM transition 995,6 -> 135,1 is used to assess sensitivity and chromatographic
peak shape. The microcystin-LR chromatographic peak should be less than 0,25 min (15 s) at the
baseline. If the peak does not meet peak shape and sensitivity requirements the analytical run is
stopped, and instrument maintenance is done in accordance with the instructions of the instrument
manufacturer.
9.3.2.3 Method blank measurements
The method blank (9.1.2) is used to assess cleanliness of the method labwares, reagents and equipment.
The method blank should not contain target compounds or interference peaks ≥0,025 μg/l. If interfering
blank values occur, identify the cause using systematic examination and eliminate the source of
contamination.
9.3.2.4 Laboratory control spike sample measurements
The laboratory control spike sample (9.1.3) is a method spike used to assess method performance and
check the validity of the calibration curve generated for the batch of samples analysed. The sample
contains microcystins at 0,32 µg/l, for example.
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