Single Laboratory Validated Method for
Determination of Microcystins and Nodularin in
Ambient Freshwaters by Solid Phase Extraction and
Liquid Chromatography/Tandem Mass Spectrometry
(LC/MS/MS)
November 2017
U.S. Environmental Protection Agency
Office of Research and Development
1200 Pennsylvania Avenue, NW
EPA document # EPA/600/R-17/344
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Questions concerning this document should be addressed to:
Jody A. Shoemaker, Ph.D.
U.S. EPA, Office of Research and Development, National Exposure Research
Laboratory, 26 W. Martin Luther King Dr., Cincinnati, OH 45268
Phone: (513) 569-7298
shoemaker.j ody @epa. gov
Authors
Jody A. Shoemaker
Daniel R. Tettenhorst
Armah de la Cruz
U.S. EPA, Office of Research and Development, National Exposure Research
Laboratory
DISCLAIMER
This document has been reviewed by the U.S. Environmental Protection Agency,
Office of Research and Development, and approved for publication. Mention of
trade names or commercial products does not constitute endorsement or
recommendation for use.
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Table of Contents
1. SCOPE AND APPLICATION 3
2. SUMMARY OF METHOD 4
3. DEFINITIONS 5
4. INTERFERENCES 7
5. SAFETY 8
6. EQUIPMENT AND SUPPLIES 9
7. REAGENTS AND STANDARDS 12
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE 16
9. QUALITY CONTROL 17
10. CALIBRATION AND STANDARDIZATION 24
11. PROCEDURE 27
12. DATA ANALYSIS AND CALCULATION 32
13. SINGLE LABORATORY METHOD PERFORMANCE 33
14. POLLUTION PREVENTION 34
15. WASTE MANAGEMENT 34
16. REFERENCES 34
17. TABLES, DIAGRAMS, FLOWCHARTS AND VALIDATION DATA 36
FIGURE 1 46
FIGURE 2 47
FIGURE 3 48
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Single Laboratory Validated Method Determination of Microcystins and Nodularin in
Ambient Freshwaters by Solid Phase Extraction and Liquid Chromatography/Tandem
Mass Spectrometry (LC/MS/MS)
1. SCOPE AND APPLICATION
1.1 This is a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for
determination of microcystins and nodularin (combined intracellular and extracellular)
in ambient freshwater. Accuracy and precision data have been generated in reagent
water and ambient freshwaters for compounds listed in the table below.
Analyte
Chemical Abstract Services
Registry Number (CASRN)
3-desmethylated-microcystin-LR (3-dm-MC-LR)
120011-66-7
3-desmethylated-microcystin-RR (3-dm-MC-RR)
202120-08-9
7-desmethylated-microcystin-LR (7-dm-MC-LR)
134842-07-2
microcystin-HilR (MC-HilR)
Not assigned
microcystin-HtyR (MC-HtyR)
Not assigned
microcystin-LA (MC-LA)
96180-79-9
microcystin-LF (MC-LF)
154037-70-4
microcystin-LR (MC-LR)
101043-37-2
microcystin-LW (MC-LW)
157622-02-1
microcystin-LY (MC-LY)
123304-10-9
microcystin-RR (MC-RR)
111755-37-4
microcystin-WR (MC-WR)
138234-58-9
microcystin-YR (MC-YR)
101064-48-6
nodularin-R (NOD)
118399-22-7
1.2 The Minimum Reporting Level (MRL) is the lowest analyte concentration that meets
Data Quality Objectives (DQOs) that are developed based on the intended use of this
method. The single laboratory lowest concentration MRL (LCMRL) is the lowest true
concentration for which the future recovery is predicted to fall, with high confidence
(99%), between 50 and 150% recovery. Single laboratory LCMRLs for analytes in this
method range from 14-170 ng/L using Option A procedure (Sect. 11.4), and are listed
in Table 5. The procedure used to determine the LCMRL is described elsewhere.1
1.3 Laboratories using this method will not be required to determine the LCMRL for this
method, but will need to demonstrate that their laboratory MRL for this method meets
the requirements described in Section 9.2.4.
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1.4 Determining the Detection Limit (DL) for analytes in this method is optional
(Sect. 9.2.6). Detection limit is defined as the statistically calculated minimum
concentration that can be measured with 99% confidence that the reported value is
greater than zero.2 The DL is compound dependent and is dependent on extraction
efficiency, sample matrix, fortification concentration, and instrument performance.
DLs for analytes in this method range from 2.1-33 ng/L using Option A procedure
(Sect. 11.4), and are listed in Table 5.
1.5 This method is intended for use by analysts skilled in solid phase extractions,
operation of LC/MS/MS instruments, and the interpretation of associated data.
1.6 METHOD FLEXIBILITY - In recognition of technological advances in analytical
systems and techniques, the laboratory is permitted to modify the evaporation
technique, separation technique, LC column, mobile phase composition, LC conditions
and MS and MS/MS conditions. Changes may not be made to sample collection and
preservation (Sect. 8), sample extraction or intracellular toxin release steps (Sect. 11),
or to quality control requirements (Sect. 9). Method modifications should be
considered only to improve method performance. Modifications that are introduced in
the interest of reducing cost or sample processing time, but result in poorer method
performance, should not be used. Analytes must be adequately resolved
chromatographically in order to permit the mass spectrometer to dwell on a minimum
number of compounds eluting within a retention time window. Instrumental sensitivity
(or signal-to-noise) will decrease if too many compounds are permitted to elute within
a retention time window. In all cases where method modifications are proposed, the
analyst must perform the procedures outlined in the initial demonstration of capability
(IDC, Sect. 9.2), verify that all Quality Control (QC) acceptance criteria (Sect. 9) are
met, and ensure that acceptable method performance can be verified in a real sample
matrix (Sect. 9.3.6).
NOTE: The above section is intended as an abbreviated summation of method
flexibility. Sections 6-12 provide detailed information of specific portions of
the method that may be modified. If there is any perceived conflict between
the general method flexibility statement in Section 1.6 and specific
information in Sections 6-12, Sections 6-12 supersede Section 1.6.
2. SUMMARY OF METHOD
A water sample is filtered and intracellular toxins are released from cyanobacterial cells
following two possible procedures chosen by visual transparency or cell density of the
sample. Option A for clear to semi-transparent samples: A 100-mL water sample (fortified
with a surrogate) is filtered and both the filtrate and filter are collected. The filter is placed in
a solution of 80:20 methanol:reagent water (v/v)and held for at least one hour at -20 °C to
release intracellular toxins from cyanobacteria cells captured on the filter. The liquid is
drawn off the filter and added back to the 100-mL aqueous filtrate. Option B for semi-
transparent to opaque samples: A 10 mL water sample (fortified with a surrogate) is
combined with 30 mL of methanol in a centrifuge tube and held for at least two hours
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at -20 °C to release intracellular toxins from cyanobacteria cells. The sample is centrifuged
and the supernatant is filtered. The filtrate is diluted with reagent water to provide a sample
appropriate for extraction. The filtered sample, containing intracellular and extracellular
toxins (either Option A or B), is passed through a solid phase extraction (SPE) cartridge to
extract the method analytes and surrogate. Analytes are eluted from the solid phase with a
small amount of 90:10 methanol:reagent water (v/v). The extract is concentrated to dryness
by evaporation with nitrogen in a heated water bath, and then adjusted to a 1-mL volume
with 90:10 methanol:reagent water (v/v). A 10-|iL injection is made into an LC equipped
with a C8 column that is interfaced to an MS/MS. Analytes are separated and identified by
comparing the acquired mass spectra and retention times to reference spectra and retention
times for calibration standards acquired under identical LC/MS/MS conditions. The
concentration of each analyte is determined by internal standard calibration.
3. DEFINITIONS
3.1 ANALYSIS BATCH - A set of samples that is analyzed on the same instrument
during a 24-hour period, including no more than 20 field samples, that begins and ends
with the analysis of the appropriate Continuing Calibration Check (CCC) standards.
Additional CCCs may be required depending on the length of the analysis batch or the
number of field samples.
3.2 CALIBRATION STANDARD (CAL) - A solution prepared from the primary dilution
standard solution or stock standard solution, the surrogate, and the internal standard.
The CAL solutions are used to calibrate the instrument response with respect to
analyte concentration.
3.3 COLLISIONALLY ACTIVATED DISSOCIATION (CAD) - The process of
converting the translational energy of the precursor ion into internal energy by
collisions with neutral gas molecules to bring about dissociation into product ions.
3.4 CONTINUING CALIBRATION CHECK (CCC) - A calibration standard containing
the method analytes, surrogate(s) and internal standard(s). The CCC is analyzed to
verify the accuracy of the existing calibration for those analytes.
3.5 DETECTION LIMIT (DL) - The minimum concentration of an analyte that can be
identified, measured, and reported with 99% confidence that the analyte concentration
is greater than zero. This is a statistical determination of precision (Sect. 9.2.6), and
accurate quantitation is not expected at this level.2
3.6 EXTRACTION BATCH - A set of up to 20 field samples (not including QC samples)
processed together (filtration, toxin release, extraction and evaporation) by the same
person during a 24-hour work shift using the same lot of filters, SPE devices, solvents,
surrogate, and fortifying solutions. Required QC samples include Laboratory Reagent
Blank, Laboratory Fortified Blank, Laboratory Fortified Sample Matrix, and
Laboratory Fortified Sample Matrix Duplicate.
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3.7 FIELD DUPLICATES (FD) - Separate samples collected at the same time, shipped,
and stored under identical conditions as the field sample. Analyses of FDs give a
measure of the homogeneity of cyanotoxin concentrations within a cyanobacteria
bloom.
3.8 INTERNAL STANDARD (IS) - A pure compound that is added to all standard
solutions and samples in a known amount and used to measure the relative response of
other method analytes that are components of the same solution. The internal standard
must respond similarly to the method analytes, have no potential to be present in water
samples, and not be a method analyte.
3.9 ION SUPPRESSION/ENHANCEMENT - An observable decrease or increase in
analyte response in complex (field) samples as compared to the response obtained in
standard solutions.
3.10 LABORATORY FORTIFIED BLANK (LFB) - A volume of reagent water or other
blank matrix to which known quantities of method analytes and all preservation
compounds are added in the laboratory. The LFB is analyzed exactly like a sample,
and its purpose is to determine whether the methodology is in control, and whether the
laboratory is capable of making accurate and precise measurements.
3.11 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - A preserved field
sample to which known quantities of method analytes are added in the laboratory. The
LFSM is processed and analyzed exactly like a sample, and its purpose is to determine
whether the sample matrix contributes bias to the analytical results. Background
concentrations of the analytes in the sample matrix must be determined in a separate
sample extraction and measured values in the LFSM corrected for background
concentrations.
3.12 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) - A
duplicate of the Field sample used to prepare the LFSM. The LFSMD is fortified,
extracted, and analyzed identically to the LFSM. The LFSMD is used instead of the
Field Duplicate to assess method precision when the occurrence of method analytes is
infrequent.
3.13 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or other
blank matrix that is treated exactly as a sample including exposure to all glassware,
equipment, solvents and reagents, sample preservatives, surrogate(s) and internal
standard(s) that are used in the analysis batch. The LRB is used to determine if method
analytes or other interferences are present in the laboratory environment, reagents, or
apparatus.
3.14 LOWEST CONCENTRATION MINIMUM REPORTING LEVEL (LCMRL) - The
single laboratory LCMRL is the lowest true concentration for which a future recovery
is expected, with 99% confidence, to be between 50 and 150% recovery.1
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3.15 MINIMUM REPORTING LEVEL (MRL) - The minimum concentration that can be
reported as a quantitated value for a method analyte in a sample following analysis.
This defined concentration can be no lower than the concentration of the lowest
calibration standard for that analyte and can only be used if acceptable QC criteria for
this standard are met. A procedure for verifying a laboratory's MRL is provided in
Section 9.2.4.
3.16 PRECURSOR ION - For the purpose of this method, the precursor ion is the
protonated molecule ([M+H]+ or [M+2H]2+) of the method analyte. In MS/MS, the
precursor ion is mass selected and fragmented by CAD to produce distinctive product
ions of smaller m/z ratio.
3.17 PRIMARY DILUTION STANDARD (PDS) SOLUTION - A solution containing the
analytes prepared in the laboratory from stock standard solutions and diluted as needed
to prepare calibration solutions and other needed analyte solutions.
3.18 PRODUCT ION - For the purpose of this method, a product ion is one of the fragment
ions produced in MS/MS by CAD of the precursor ion.
3.19 QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of known
concentrations that is obtained from a source external to the laboratory and different
from the source of calibration standards. The QCS is used to check calibration
standard integrity.
3.20 REAGENT WATER - Purified water that does not contain any measurable quantity of
the method analytes or interfering compounds at or above 1/3 the MRL.
3.21 SAFETY DATA SHEET (SDS) - Written information provided by vendors
concerning a chemical's toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling precautions.
3.22 STOCK STANDARD SOLUTION (SSS) - A concentrated solution containing one or
more method analytes prepared in the laboratory using assayed reference materials or
purchased from a reputable commercial source.
3.23 SURROGATE ANALYTE (SUR) - A pure chemical which chemically resembles
method analytes and is extremely unlikely to be found in any sample. This chemical is
added to a sample aliquot in known amount(s) before processing and is measured with
the same procedures used to measure other method analytes. The purpose of the SUR
is to monitor method performance with each sample.
4. INTERFERENCES
4.1 All glassware and plasticware must be meticulously cleaned. Wash glassware and
plasticware with detergent and tap water, rinse with tap water, followed by a reagent
water rinse. Non-volumetric glassware must be heated in a muffle furnace for a
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minimum of 90 min at 400 °C. Volumetric glassware should be solvent rinsed and
allowed to air dry or heated in an oven no hotter than 120 °C. Plasticware should be
solvent rinsed and allowed to air dry.
4.2 Method interferences may be caused by contaminants in solvents, reagents (including
reagent water), sample bottles and caps, and other sample processing hardware that
lead to discrete artifacts and/or elevated baselines in chromatograms. All items must
be routinely demonstrated to be free from interferences (less than 1/3 the MRL for
each method analyte) under the conditions of the analysis by analyzing laboratory
reagent blanks as described in Section 9.3.1. Subtracting blank values from sample
results is not permitted.
4.3 Matrix interferences may be caused by contaminants that are co-extracted from the
sample. The extent of matrix interferences will vary considerably from source to
source, depending upon the nature of the water. Humic and/or fulvic material can be
co-extracted during SPE and high levels can cause signal enhancement or suppression
in the electrospray ionization source.3-4 Also, high levels of humic and/or fulvic
material can cause low recoveries on the SPE sorbent.
4.4 Although not observed during method development, suppression of analyte signals due
to electrolyte-induced ionization caused by dissolved salts in the mobile phase has
been reported in the literature.5 Addition of ammonium formate to the mobile phase in
this method aids in reducing the occurrence of this phenomenon.
4.5 Relatively large quantities of preservatives (Sect. 8.1.2) are added to sample bottles.
The potential exists for trace-level organic contaminants in these reagents. Interfer-
ences from these sources should be monitored by analysis of laboratory reagent blanks
(Sect. 9.3.1), particularly when new lots of reagents are acquired.
4.6 SPE cartridges can be a source of interferences. Analysis of laboratory reagent blanks
can provide important information regarding the presence or absence of such
interferences. Brands and lots of SPE devices should be tested to ensure that
contamination does not preclude analyte identification and quantitation.
5. SAFETY
Each chemical should be treated as a potential health hazard, and exposure to these chemicals
should be minimized. Each laboratory is responsible for maintaining an awareness of OSHA
regulations regarding safe handling of chemicals used in this method. A reference file of
SDSs should be made available to all personnel involved in the chemical analysis. Toxin
decontamination/inactivation guidelines may be found in Biosafety in Microbiological and
Biomedical Laboratories, 5th edition.6 Additional references to laboratory safety are
available.7"9
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6. EQUIPMENT AND SUPPLIES
(Brand names and catalog numbers are included for illustration only, and do not imply
endorsement of the product.)
6.1 SAMPLE CONTAINERS - 100-mL amber glass bottles fitted with
polytetrafluoroethylene (PTFE)-lined screw caps or 125-mL amber polyethylene
terephthalate glycol (PETG) bottles fitted with HDPE screw caps (Nalgene #322021-
0125).
6.2 STANDARD CONTAINERS - Amber-12mL glass screw thread sample vials
(Kimble #60815-1965 or equivalent) with black phenolic caps with PTFE-faced white
rubber liners (Kimble #73802-15425 or equivalent). If available, small volume amber
PETG bottles may also be used to prepare standards.
6.3 BULK COLLECTION CONTAINER - 500-mL (or larger) clear or amber PETG
media bottles (Nalgene #2019-0500). One bulk container per field sample (the LFSM
and LFSMD are drawn from the same bulk container as the field sample) is required.
6.4 SAMPLE FILTER APPARATUS (See Figure 1)
6.4.1 CONTAINERS FOR COLLECTING FILTRATE - 500-mL amber glass bottles
(Fisher #02-542-4C or equivalent) and GL 45 bottle cap (Fisher #13247GL45 or
equivalent; not shown in figure).
6.4.2 FILTER BASE O-RING - PTFE/silicone sealing ring (Kimble Chase
#410171-4226 or equivalent).
6.4.3 BOTTLE CAP WITH HOLE - GL 45 bottle cap with hole for filter support base
(Kimble #410170-4534, or equivalent).
6.4.4 SUPPORT BASE - 47 mm fritted glass support base for filtration (Kimble Chase
#953752-5047 or equivalent).
6.4.5 HOSE BARB CONNECTOR - Barbed tubing adapter for filtration apparatus
(Kimble Chase #736400-1413 or equivalent).
6.4.6 METAL CLAMP - 47 mm aluminum clamp (Kimble Chase #953753-0000 or
equivalent).
6.4.7 FUNNEL - 47 mm, 300 mL glass funnel (Kimble Chase #953751-0000 or
equivalent).
6.5 MEMBRANE FILTER - 47 mm Nuclepore polycarbonate filter membranes, pore size
0.8 [j,m, (Whatman #111109).
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6.6 ROUND BOTTOM CULTURE TUBES - 15-mL round bottom glass culture tubes
(Corning #9826-16X or equivalent) or other glassware suitable for use in releasing
toxins from the filter.
6.7 CONICAL CENTRIFUGE TUBES - 15-mL conical glass centrifuge tubes (Corning
#8082-15) or other glassware suitable for collection of the eluent from the solid phase
after extraction.
6.8 CONICAL CENTRIFUGE TUBES - 50-mL conical plastic centrifuge tubes (Fisher
#06-443-18) or other glassware suitable for freezing and centrifuging samples with
high cyanobacterial cell densities during the toxin release procedure.
6.9 AUTOSAMPLER VIALS - Amber glass 2.0-mL autosampler vials (National
Scientific #C4000-2W or equivalent) with caps containing PTFE-faced septa (National
Scientific #C4000-53 or equivalent).
6.10 MICRO SYRINGES - Suggested sizes include 5, 10, 25, 50, 100, 250, 500 and
1000-|iL syringes.
6.11 ANALYTICAL BALANCE - Capable of weighing to the nearest 0.0001 g.
6.12 CENTRIFUGE - Capable of centrifugation at 8,000 rpm and 4 °C.
6.13 SOLID PHASE EXTRACTION (SPE) APPARATUS FOR USING CARTRIDGES
6.13.1 SPE CARTRIDGES - Waters Oasis HLB, 150 mg, 6 cc divinylbenzene N-
vinylpyrrolidone copolymer (Waters # 186003365).
6.13 .2 VACUUM EXTRACTION MANIFOLD
6.13.2.1 Manual Extraction - A manual vacuum manifold with Visiprep™ large
volume sampler (Supelco #57030 and #57275 or equivalent) for cartridge
extractions.
6.13.2.2 SAMPLE DELIVERY SYSTEM - Use of a transfer tube system (Supelco
"Visiprep," #57275 or equivalent), which transfers sample directly from the
sample container to the SPE cartridge is recommended.
6.14 EXTRACT CONCENTRATION SYSTEM - Extracts are concentrated by
evaporation with nitrogen using a water bath set no higher than 60 °C (Meyer N-Evap,
Model 111, Organomation Associates, Inc. or equivalent).
6.15 LABORATORY OR ASPIRATOR VACUUM SYSTEM - Sufficient capacity to
maintain a vacuum of approximately 10 to 15 inches of mercury for extracting
cartridges.
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6.16 LIQUID CHROMATOGRAPHY (LC)/TANDEM MASS SPECTROMETER
(MS/MS) WITH DATA SYSTEM
6.16.1 LC SYSTEM - Instalment capable of reproducibly injecting up to 10-|aL aliquots,
and performing binary linear gradients at a constant flow rate near the flow rate
used for development of this method (0.3 mL/min). Usage of a column heater
capable of heating to 65 °C is required to achieve suitable peak shape for the IS. If
alternate ISs are used which meet the IS modification requirements (Sect. 7.2.1)
and do not need column heating to achieve suitable peak shape, then a column
heater is not required.
6.16.2 TANDEM MASS SPECTROMETER - The mass spectrometer must be capable
of positive ion electrospray ionization (ESI) near the suggested LC flow rate of
0.3 mL/min. The system must be capable of performing MS/MS to produce
unique product ions (Sect. 3.18) for method analytes within specified retention
time segments. A minimum of 10 scans across the chromatographic peak is
required to ensure adequate precision. Data demonstrated in Section 17 were
collected using a triple quadrupole mass spectrometer.
6.16.3 DATA SYSTEM - An interfaced data system is required to acquire, store, reduce,
and output mass spectral data. The computer software should have the capability
of processing stored LC/MS/MS data by recognizing an LC peak within any given
retention time window. The software must allow integration of the ion abundance
of any specific ion within specified time or scan number limits. The software must
be able to calculate relative response factors, construct linear regressions or
quadratic calibration curves, and calculate analyte concentrations.
6.16.4 ANALYTICAL COLUMN - Cs column (2.1 x 100 mm) packed with 2.6 |am Cs
solid phase particles (Phenomenex Kinetex #00D-4497-AN). Any equivalent
column that provides adequate resolution, peak shape, capacity, accuracy, and
precision (Sect. 1.6 and 9) may be used.
NOTE: This column has silanol groups which have the potential to impact
retention times and area counts of the microcystins. Retention times of the
microcystins have been found to slowly decrease with time on Cs and Ci8
columns both during method development and in the literature.10 In addition,
during method development, area counts of some microcystins (especially
arginine containing microcystins) increased as the number of ambient water
extract injections increased. A potential reason for these drifts may be that active
silanol sites on the column may be inactivated by the ambient water components;
thereby allowing less binding of the microcystins to the silanol sites. Thus,
sensitivity of the microcystins very slowly increased with time after repeated
injections of ambient water extracts during method development. In any given
batch, however, retention times and areas were very precise.
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6.16.5 ANALYTICAL GUARD COLUMN (optional) - Phenomenex SecurityGuard
Ultra Cartridges UHPLC C8 (#AJ0-8784, or equivalent).
7. REAGENTS AND STANDARDS
7.1 GASES, REAGENTS, AND SOLVENTS - Reagent grade or better chemicals should
be used. Unless otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used, provided it
is first determined that the reagent is of sufficiently high purity to permit its use
without lessening the quality of the determination.
7.1.1 REAGENT WATER - Purified water which does not contain any measurable
quantities of any method analytes or interfering compounds greater than 1/3 the
MRL for each method analyte of interest.
7.1.2 METHANOL (CH3OH, CASRN 67-56-1) - High purity, demonstrated to be free
of analytes and interferences (Fisher #A456-4, Optima LC/MS grade or
equivalent).
7.1.3 AMMONIUM FORMATE (CH5O2N, CASRN 540-69-2) - High purity,
demonstrated to be free of analytes and interferences (LC/MS grade (Fluka
#55674) or equivalent).
7.1.4 20 mM FORMATE BUFFER - To prepare 1 L, add 1.26 g ammonium formate to
1 L of reagent water. This solution is prone to volatility losses and should be
replaced at least every 48 hours.
7.1.5 SAMPLE PRESERVATION REAGENTS - The following preservatives are
solids at room temperature and may be added to the sample bottle before
shipment to the field.
7.1.5.1 TRIZMA PRESET CRYSTALS, pH 7.0 (Sigma-Aldrich #T-7193 or
equivalent) - Reagent grade. A premixed blend of Tris [Tris(hydroxy-
methyl)aminomethane] and Tris HCL [Tris(hydroxymethyl)aminomethane
hydrochloride]. Alternatively, a mix of the two components with a weight
ratio of 15.5/1 Tris HCL/Tris may be used. These blends are targeted to
produce a pH near 7.0 at 25 °C in reagent water. Trizma functions as a buffer
(Sect. 8.1.2).
7.1.5.2 2-CHLOROACETAMIDE (CASRN 79-07-2) - Inhibits microbial growth and
analyte degradation (Sigma-Aldrich #C0267 or equivalent).11
7.1.5.3 ETHYLENEDIAMINETETRAACETIC ACID, TRISODIUM SALT
HYDRATE (Trisodium EDTA, CASRN 85715-60-2) - Inhibits metal-
catalyzed hydrolysis of analytes. Trisodium salt is used instead of the
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disodium salt because the trisodium salt solution pH is closer to the desired
pH of 7 (Sigma #ED3SS or equivalent).
7.1.6 NITROGEN - DESOLVATION GAS - High purity compressed gas (e.g.,
nitrogen or zero-air) used for desolvation in the mass spectrometer. The specific
type of gas, purity, and pressure requirements will depend on the instrument
manufacturer's specifications. Nitrogen was used to generate the data in
Section 17.
7.1.7 COLLISION GAS - High purity compressed gas (e.g., nitrogen or argon) used
for CAD in the mass spectrometer. The specific type of gas, purity, and pressure
requirements will depend on the instrument manufacturer's specifications. Argon
was used to generate the data in Section 17.
7.2 STANDARD SOLUTIONS - When the purity of a compound is assayed to be 95% or
greater, the weight can be used without correction to calculate concentration of the
stock standard. The suggested concentrations are a description of concentrations used
during method development, and may be modified to conform to instrument
sensitivity. Standards for sample fortification generally should be prepared in the
smallest volume that can be accurately measured to minimize addition of excess
organic solvent to aqueous samples. Stock standards, PDSs and calibration standards
were found to be stable for a minimum of three months during method development.
Laboratories should use standard QC practices to determine when standards need to be
replaced. The target analyte manufacturer's guidelines may be helpful when making
the determination.
7.2.1 INTERNAL STANDARD (IS) SOLUTIONS - Cyclosporin-A, 13C2, d4„ obtained
from Toronto Research Chemicals as neat material, (Cat # C988901) is used as
the IS. Although alternate IS standards may be used provided they are isotopically
labeled compounds with similar functional groups as method analytes, the analyst
must have documented reasons for using alternate IS standards (e.g., isotopically
labeled microcystins become commercially available). In addition, alternate IS
standards must meet QC requirements in Section 9.3.4.
7.2.1.1 IS STOCK SOLUTION (500 ng/|iL) -The IS stock standard solution is
prepared by diluting 0.5 mg of the IS in one mL of methanol. This IS stock
standard is stored at -15 °C or less in amber glass screw cap vials.
7.2.1.2 IS PRIMARY DILUTION STANDARD - (IS PDS; 1.0 ng/^L) - The IS PDS
is prepared at 1.0 ng/|iL by diluting 20 [j,L of the IS stock standard in 10 mL
of methanol. Ten [j,L of this 1.0 ng/(j,L solution is used to fortify the final
1-mL extracts (Sect. 11.6.5). This will yield an IS concentration of 10 [j.g/L in
the 1-mL extracts. This IS PDS is stored at -15 °C or less in amber glass
screw cap vials. The IS concentration may be adjusted to accommodate
instrument sensitivity.
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.2 SURROGATE (SUR) ANALYTE STANDARD SOLUTIONS - Ethylated MC-
LR, ds (C2D5-MC-LR), obtained from Tamarack Environmental Laboratories
(now available through Cambridge Isotopes) as neat material, is used as the SUR.
This isotopically labeled SUR standard was carefully chosen during method
development because it contains similar functional groups as the method analytes.
Although alternate SUR standards may be used provided they are isotopically
labeled compounds with similar functional groups as method analytes, the analyst
must have documented reasons for using alternate SUR standards. In addition,
alternate SUR standards must meet QC requirements in Section 9.3.5.
.2.2.1 SUR STOCK STANDARD; 100 |ig/mL) - The SUR stock standard solution
is prepared by diluting 0.1 mg of the SUR in one mL of methanol. This SUR
stock standard is stored at -15 °C or less in amber glass screw cap vials.
.2.2.2 SUR PRIMARY DILUTION STANDARD (SUR PDS; 5.0 ng/|iL) - The
SUR PDS was prepared at 5.0 ng/|iL by diluting 500 |iL of the SUR stock
standard with 10 mL of methanol. This solution is used to fortify all QC and
field samples. The PDS has been shown to be stable for at least one month
when stored at -15 °C or less. Use 20 |iL of this 5 ng/|iL SUR PDS to fortify
the 100 mL aqueous QC and field samples prior to extraction (Sect. 11.3.4).
This will yield a concentration of 1000 ng/L of the SUR in 100-mL aqueous
QC and field samples. The SUR concentration may be adjusted to
accommodate instrument sensitivity.
.3 ANALYTE STANDARD SOLUTIONS - Analyte standards may be purchased
commercially as ampulized solutions or prepared from neat materials (see Table 3
for analyte sources used during method development).
.2.3.1 ANALYTE STOCK STANDARD SOLUTION (10-100 |ig/mL) - Neat
cyanotoxins are typically purchased in quantities of 10-100 |ig. Due to the
small quantity and toxicity of these analytes, weighing the cyanotoxins is not
feasible. If preparing from neat material, simply add 1 mL of methanol to the
purchased neat material (10-100 |ig) for a final concentration of 10-
100 |ig/mL, Repeat for each method analyte prepared from neat material.
Alternatively, purchase commercially available stock standard solutions of the
analytes, preferably in methanol, if available. These stock standards were stored
at -15 °C or less in amber glass screw cap vials.
ANALYTE PRIMARY DILUTION STANDARD (PDS) SOLUTION (0.33-
6.0 ng/|aL) - The Analyte PDS contains all, or a portion, of method analytes at
various concentrations in methanol. ESI and MS/MS response varies by
compound; therefore, a mix of concentrations may be needed in the Analyte
PDS. During method development, Analyte PDS solutions were prepared
such that approximately the same instrument response was obtained for all
analytes. The Analyte PDS was prepared in 10 mL of methanol at
concentrations of 0.33-6.0 ng/|aL as shown in the following table. The Analyte
14
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PDS is prepared by dilution of the combined Analyte Stock Standard
Solutions (Sect. 7.2.3.1) and is used to prepare CAL standards, and fortify
LFBs, LFSMs, and LFSMDs with method analytes. The Analyte PDS was
stored at -15 °C or less in amber glass screw cap vials.
Analyte
Cone, of Analyte
Stock Standard
Solution (ng/jiL)
Vol. of Analyte
Stock Standard
Solution (jiL)
Final Cone, of
Analyte in 10-mL
PDS (ng/jiL)
Nodularin
10.3
480
0.49
MC-YR
100
200
2.0
MC-HtyR
100
200
2.0
MC-RR
10.3
320
0.33
3 -de smethylated-MC -RR
100
100
1.0
MC-LR
10.1
1890
1.9
MC-WR
100
600
6.0
7 -de smethylated-MC-LR
9.4
2000
1.9
MC-HilR
25
800
2.0
3 -de smethylated-MC -LR
100
400
4.0
MC-LA
100
200
2.0
MC-LY
100
200
2.0
MC-LW
100
200
2.0
MC-LF
100
200
2.0
7.2.4 CALIBRATION STANDARDS (CAL) - Prepare a series of at least five
concentrations of calibration solutions in 90:10 methanol:reagent water (v/v),
from dilutions of the Analyte PDS (Sect 7.2.3.2). The suggested concentrations in
this section are a description of concentrations used during method development,
and may be modified to conform with instrument sensitivity. Typical calibration
standard concentration ranges are depicted in the table below. Larger
concentration ranges will require more calibration points. The IS and SUR are
typically added to CAL standards at constant concentration (see Note below).
During method development, the concentration of the IS was 10 |ig/L in each
standard and the concentration of the SUR was 100 |ig/L in each standard
(1000 ng/L in the aqueous sample). The lowest concentration CAL standard must
be at or below the MRL, which may depend on system sensitivity. CAL standards
may also be used as CCCs (Sect. 9.3.2). During method development, CAL
standards were stored at -4 °C or less.
NOTE: Alternatively, a calibration curve may be used for quantitation of the
SUR. Analysts may be hesitant to analyze undiluted extracts obtained from a
water sample with high cyanobacterial cell densities. Dilution of extracts may be
required or preferred causing the inability to accurately calculate SUR recoveries
using average response factors. Generation of a calibration curve using a
concentration range for the SUR will enable calculation of the SUR in diluted
extracts.
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Cal standard Concentration
Concentration Range in 100-mL
Analyte
Range, ug/L
aqueous sample, ug/L
Nodularin
1.5-247
0.015-2.47
MC-YR
6.0-1000
0.060- 10.00
MC-HtyR
6.0-1000
0.060- 10.00
MC-RR
0.99-165
0.0099- 1.65
3-dm-MC-RR
3.0-500
0.030-5.00
MC-LR
5.7-945
0.057-9.45
MC-WR
18-3000
0.18-30.00
7-dm-MC-LR
5.6-940
0.056-9.40
MC-HilR
6.0-1000
0.060- 10.00
3-dm-MC-LR
12-2000
0.12-20.00
MC-LA
6.0-1000
0.060- 10.00
MC-LY
6.0-1000
0.060- 10.00
MC-LW
6.0-1000
0.060- 10.00
MC-LF
6.0-1000
0.060- 10.00
8. SAMPLE COLLECTION. PRESERVATION. AND STORAGE
8.1 SAMPLE BOTTLE PREPARATION
8.1.1 Collect 100-mL samples in amber glass bottles or 125-mL amber PETG bottles
(Sect. 6.1). It is recommended that more samples be collected than are needed to
meet QC requirements in Section 9. This will allow laboratories some flexibility
to analyze samples by the Option B procedure if samples fail to filter in the 8 h
time period required in Option A.
8.1.2 Preservation reagents, listed in the table below, are added to each sample bottle as
a solid prior to shipment to the field (or prior to sample collection).
Compound
Amount
Purpose
Trizma
7.75 g/L
buffering reagent (pH 7)
2-Chloroacetamide
2.0 g/L
antimicrobial
Ethylenediaminetetraacetic acid
trisodium salt
0.35 g/L
inhibit binding of the targets
to metals
8.1.3 Optional: If 125 mL amber bottles do not have visible 100-mL demarcations, a
marker may be added to outside of the bottle to serve as a fill line and to prevent
over-filling the bottle.
8.2 SAMPLE COLLECTION (The sample collection procedure in Section 8.2 is
recommended but program requirements may involve alternate sample collection
procedures, and it is incumbent upon the laboratory and field samplers to verify such
requirements. Changes to preservation agents are not permitted.)
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8.2.1 Collect approximately 500 mL of sample water in a 500-mL PETG container
(Sect. 6.3).
8.2.2 Gently shake the 500-mL PETG container at least 25 times between sample draws
to aid in homogenizing the sample. Immediately pour 100 mL of the sample water
into the bottle containing preservatives. Do not completely fill the bottle as
preservatives have been added at quantities appropriate for 100 mL samples.
Samples do not need to be collected headspace free. Pour duplicate samples for
the LFSM and LFSMD (if necessary to meet minimum QC requirements in
Sections 9.3.6 and 9.3.7) from the same draw of water.
8.2.3 If a FD is desired, collect the FD from a second draw of water from the water
body. Cyanobacterial blooms typically display heterogeneity in water bodies and
collection of the first water sample will also disturb the bloom even further. Thus,
the FD cannot be used as a measure of sample collection precision. However, if
desired, the FD can be used as a measure of heterogeneity of the cyanobacterial
bloom in the water body.
8.2.4 After pouring the sample, cap the sample bottle and agitate by hand until
preservative is dissolved. Note that 2-chloroacetamide is slow to dissolve
especially in cold water. Keep the sample sealed from time of collection until
extraction.
8.3 SAMPLE SHIPMENT AND STORAGE - Samples must be chilled during shipment
and must not exceed 10 °C during the first 48 hours after collection. Sample
temperature must be confirmed to be at or below 10 °C when samples are received at
the laboratory. Samples stored in the lab must be held at or below 6 °C until
extraction, but should not be frozen.
NOTE: Samples that are significantly above 10 °C, at the time of collection, may need
to be iced or refrigerated for a period of time, in order to chill them prior to
shipping. This will allow them to be shipped with sufficient ice to meet the
above requirements.
8.4 SAMPLE AND EXTRACT HOLDING TIMES - Water samples should be extracted
as soon as possible after collection but must be extracted within 28 days of collection.
Extracts must be stored at < -4 °C and analyzed within 28 days after extraction.
Sample and extract holding time data are presented in Tables 10 and 11.
9. QUALITY CONTROL
9.1 QC requirements include the Initial Demonstration of Capability (IDC) and ongoing
QC requirements that must be met when preparing and analyzing field samples. This
section describes QC parameters, their required frequencies, and performance criteria
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that must be met in order to meet EPA quality objectives. These QC requirements are
considered the minimum acceptable QC criteria. Laboratories are encouraged to
institute additional QC practices to meet their specific needs.
9.1.1 METHOD MODIFICATIONS - The analyst is permitted to modify LC columns,
LC conditions, evaporation techniques, internal standards, surrogate standards,
and MS and MS/MS conditions. Each time such method modifications are made,
the analyst must repeat the procedures of the IDC. Modifications to LC
conditions should still minimize co-elution of method analytes to reduce the
probability of suppression/enhancement effects.
9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) - The IDC must be
successfully performed for the Option A (Sect. 11.4) and Option B (Sect. 11.5)
procedures prior to analyzing any field samples. Prior to conducting the IDC, the
analyst must first generate an acceptable Initial Calibration following the procedure
outlined in Section 10.2.
9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND - Any time
a new lot of filters, SPE cartridges, solvents, centrifuge tubes, disposable pipets,
and autosampler vials are used, it must be demonstrated that an LRB is reasonably
free of contamination and that criteria in Section 9.3.1 are met. If an automated
extraction system is used, an LRB should be extracted on each port to ensure that
all valves and tubing are free from potential contamination.
9.2.2 INITIAL DEMONSTRATION OF PRECISION (IDP) - Prepare, extract, and
analyze four to seven replicate LFBs fortified near the midrange of the initial
calibration curve according to the procedure described in Section 11. Sample
preservatives, as described in Section 8.1.2, must be added to these samples. The
relative standard deviation (RSD) of the results of replicate analyses must be less
than 30%.
9.2.3 INITIAL DEMONSTRATION OF ACCURACY (IDA) - Using the same set of
replicate data generated for Section 9.2.2, calculate average recovery. The average
recovery of replicate values must be within ± 30% of the true value except for
MC-WR and MC-LW which must be 50-130%) of the true value.
9.2.4 MINIMUM REPORTING LEVEL (MRL) CONFIRMATION - Establish target
concentrations for the MRL based on the intended use of the method for the
Option A and Option B procedures. The MRL may be established by a laboratory
for their specific purpose or may be set by a regulatory agency. Establish an
Initial Calibration following the procedure outlined in Section 10.2. The lowest
CAL standard used to establish Initial Calibration (as well as the low-level CCC,
Section 10.3) must be at or below the concentration of the MRL. Establishing the
MRL concentration too low may cause repeated failure of ongoing QC
requirements. Confirm MRLs following the procedure outlined below using the
Option A and Option B procedures.
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9.2.4.1 Fortify, extract, and analyze seven replicate LFBs at the proposed MRL
concentration. These LFBs must contain all method preservatives described in
Section 8.1.2. Calculate the mean measured concentration (Mean) and
standard deviation for these replicates. Determine the Half Range for the
prediction interval of results (HRpir) using the equation below
HRPm = 3.963 s
where
5 = standard deviation
3.963 = a constant value for seven replicates.1
9.2.4.2 Confirm that the upper and lower limits for the Prediction Interval of Result
(PIR = Mean + HRpir) meet the upper and lower recovery limits as shown
below
The Upper PIR Limit must be < 150% recovery.
Mean + HRpir
Fortified Concentration
The Lower PIR Limit must be > 50% recovery.
Mean — HRpir
x 100% <150%
Fortified Concentration
x 100% >50%
9.2.4.3 The MRL is validated if both the Upper and Lower PIR Limits meet the
criteria described above. If these criteria are not met, the MRL has been set
too low and must be determined again at a higher concentration.
9.2.5 CALIBRATION CONFIRMATION - Analyze a QCS (if available) as described
in Section 9.3.9 to confirm the accuracy of the standards/calibration curve.
9.2.6 DETECTION LIMIT DETERMINATION (optional) - While DL determination
is not a specific requirement of this method, it may be required by various
regulatory bodies associated with compliance monitoring. It is the responsibility
of the laboratory to determine if DL determination is required based upon the
intended use of the data.
Replicate analyses for this procedure should be done over at least three days (i.e.,
both the sample extraction and the LC/MS/MS analyses should be done over at
least three days). Prepare at least seven replicate LFBs at a concentration
estimated to be near the DL. This concentration may be estimated by selecting a
concentration at two to five times the noise level. DLs in Table 5 were calculated
from LFBs fortified at various concentrations as indicated in the table.
Appropriate fortification concentrations will be dependent upon the sensitivity of
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the LC/MS/MS system used. All preservation reagents listed in Section 8.1.2 must
also be added to these samples. Analyze the seven replicates through all steps of
Section 11. This procedure must be conducted for Option A and Option B
procedures.
NOTE: If an MRL confirmation data set meets these requirements, a DL may be
calculated from the MRL confirmation data, and no additional analyses
are necessary.
Calculate the DL using the following equation
DL = sx t^n_^ 1_a=0 99)
where
5 = standard deviation of replicate analyses
i o-i, i-a=o.99) = Student's t value for the 99% confidence level with
n-1 degrees of freedom
n = number of replicates.
NOTE: Do not subtract blank values when performing DL calculations.
ONGOING QC REQUIREMENTS - This section summarizes ongoing QC criteria
that must be followed when processing and analyzing field samples.
.3.1 LABORATORY REAGENT BLANK (LRB) - An LRB is required with each
extraction batch (Sect. 3.6) to confirm that potential background contaminants are
not interfering with identification or quantitation of method analytes. If more than
20 field samples are included in a batch, analyze an LRB for every 20 samples. If
the LRB produces a peak within the retention time window of any analyte that
would prevent determination of that analyte, determine the source of
contamination and eliminate the interference before processing samples.
Background contamination must be reduced to an acceptable level before
proceeding. Background from method analytes or other contaminants that
interfere with the measurement of method analytes must be below 1/3 of the
MRL. If method analytes are detected in the LRB at concentrations equal to or
greater than this level, then all data for the problem analyte(s) must be considered
invalid for all samples in the extraction batch. Blank contamination is estimated
by extrapolation, if the concentration is below the lowest CAL standard. This
extrapolation procedure is not allowed for sample results as it may not meet data
quality objectives.
NOTE: Although quantitative data below the MRL may not be accurate enough
for data reporting, such data are useful in determining the magnitude of
background interference. Therefore, blank contamination levels may be
estimated by extrapolation when the concentration is below the MRL.
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9.3.2 CONTINUING CALIBRATION CHECK (CCC) - CCC standards are analyzed
at the beginning of each analysis batch (Sect. 3.1), after every 10 field samples,
and at the end of the analysis batch. See Section 10.3 for concentration
requirements and acceptance criteria.
9.3.3 LABORATORY FORTIFIED BLANK (LFB) - An LFB is required with each
extraction batch (Sect. 3.6). The fortified concentration of the LFB must be
rotated between low, medium, and high concentrations from batch to batch. The
low concentration LFB must be as near as practical to, but no more than two
times, the MRL. Similarly, the high concentration LFB should be near the high
end of the calibration range established during the initial calibration (Sect. 10.2).
Results of low-level LFB analyses must be 50-150% of the true value. Results of
medium and high-level LFB analyses must be 70-130% of the true value except
for MC-WR and MC-LW which must be 50-130%). If LFB results do not meet
these criteria for method analytes, then all data for the problem analyte(s) must be
considered invalid for all samples in the extraction batch.
9.3.4 INTERNAL STAND ARD(S) (IS) - The analyst must monitor peak areas of the
IS(s) in all injections during each analysis day. Internal standard responses (as
indicated by peak areas) for any chromatographic run must not deviate by more
than ± 50% from average areas measured during the initial calibration for the
internal standards. If IS areas in a chromatographic run do not meet these criteria,
inject a second aliquot of that standard or extract.
9.3.4.1 If the reinjected aliquot produces an acceptable IS response, report results for
that aliquot.
9.3.4.2 If the reinjected extract fails again, the analyst should check the calibration by
reanalyzing the most recently acceptable CAL standard. If the CAL standard
fails the criteria of Section 10.3, recalibration is in order per Section 10.2. If
the CAL standard is acceptable, report results obtained from the reinjected
extract, but annotate as suspect. Alternatively, if a duplicate sample was
collected at the time of sample collection and is still within the holding time,
extract the duplicate sample and re-analyze.
9.3.5 SURROGATE RECOVERY - The SUR standard is fortified into all samples,
CCCs, LRBs, LFBs, LFSMs, LFSMDs, and FDs prior to extraction. It is also
added to CAL standards. The SUR is a means of assessing method performance
from extraction to final chromatographic measurement. Calculate recovery (%>R)
for the SUR using the following equation
where
A = measured SUR concentration for the QC or Field sample
B = fortified concentration of the SUR.
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SUR recovery in extracts must be in the range of 60-130%. SUR recovery in
CCCs must be 70-130%. A wider recovery range is allowed for the SUR in
extracts due to SPE recoveries typically being 15-20% lower for this SUR
during method development. When SUR recovery does not meet these
criteria, check 1) calculations to locate possible errors, 2) standard solutions
for degradation, 3) contamination, 4) instrument performance and
5) extraction procedure. Correct the problem and reanalyze the extract.
If the extract reanalysis meets the SUR recovery criterion, report only data for
the reanalyzed extract.
If the extract reanalysis fails the 60-130%) recovery criterion, the analyst
should check the calibration by injecting the last CAL standard that passed. If
the CAL standard fails the criteria of Section 10.3, recalibration is in order per
Section 10.2. If the CAL standard is acceptable, report all data for that sample
as suspect/SUR recovery to inform the data user that the results are suspect
due to SUR recovery. Alternatively, if a duplicate sample was collected at the
time of sample collection and is still within the holding time, extract the
duplicate sample and re-analyze.
9.3 .6 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - Analysis of an
LFSM is required in each extraction batch and is used to determine that the
sample matrix does not adversely affect method accuracy. Assessment of method
precision is accomplished by analysis of a LFSMD (Sect. 9.3.7). If a variety of
different sample matrices are analyzed regularly method performance should be
established for each. Over time, LFSM data should be documented by the
laboratory for all routine sample sources.
9.3.6.1 Within each extraction batch (Sect. 3.6), a minimum of one Field sample is
fortified as an LFSM for every 20 field samples analyzed. The LFSM is
prepared by spiking a sample with an appropriate amount of the Analyte PDS
(Sect. 0). Select a spiking concentration that is greater than or equal to the
matrix background concentration, if known. Use historical data and rotate
through low, mid and high concentrations when selecting a fortifying
concentration. If high levels of method analytes are suspected, it may not be
possible to spike the LFSM above the native amount. In this case, spike with
the highest concentration within the calibration curve.
9.3.6.2 Calculate percent recovery (%R) for each analyte using the equation
%r = (a~bK\oo
C
where A = measured concentration in the fortified sample
B = measured concentration in the unfortified sample
C = fortification concentration.
9.3.5.2
9.3.5.3
22
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9.3.6.3 Analyte recoveries may exhibit matrix bias. For samples fortified at or above
their native concentration, recoveries should range between 60-140%, except
for low-level fortification near or at the MRL (within a factor of two-times the
MRL concentration) where 50-150% recoveries are acceptable. If the
accuracy of any analyte falls outside the designated range, and laboratory
performance for that analyte is shown to be in control in CCCs, the recovery
is judged to be matrix biased. The result for that analyte in the unfortified
sample is labeled suspect/matrix to inform the data user that results are
suspect due to matrix effects.
9.3.7 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) -
Within each extraction batch (not to exceed 20 field samples, Sect. 3.6), a
minimum of one LFSMD must be analyzed. Duplicates check the precision
associated with sample collection, preservation, storage, and laboratory
procedures.
9.3.7.1 Calculate relative percent difference (RPD) for duplicate LFSMs (LFSM and
LFSMD) using the equation
\LFSM-LFSMD\
RPD = y-! r-! xlOO
(LFSM + LFSMD)/2
9.3.7.2 RPDs for duplicate LFSMs should be < 50% for samples fortified at or above
their native concentration. If the RPD of any analyte falls outside the
designated range, and laboratory performance for that analyte is shown to be
in control in the CCC, the recovery is judged to be matrix biased. The result
for that analyte in the unfortified sample is labeled suspect/matrix to inform
the data user that results are suspect due to matrix effects.
9.3.8 FIELD DUPLICATES (FD) - FDs may be collected and analyzed as a part of a
sample batch, if desired. No QC criteria are being mandated as a part of this
method because FDs can only be used as a measure of heterogeneity of the
cyanobacterial bloom and not as a measure of sample collection or laboratory
precision.
9.3.9 QUALITY CONTROL SAMPLES (QCS) - As part of the IDC (Sect. 9.2 ), each
time a new Analyte PDS (Sect. 0) is prepared, and at least quarterly, analyze a
QCS sample from a source different from the source of the CAL standards. If a
second vendor is not available, then a different lot of the standard should be used.
The QCS should be prepared and analyzed just like a CCC. Fortify the QCS near
the midpoint of the calibration range. The expectation is that the calculated value for
each analyte should be within ±30% of the expected value, but due to the lack of
certified standards, calculated values within ±40% of the expected values are
acceptable.
23
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OPTIONAL: If available, certified reference materials are suggested for use in the
QCS if not already being used in the Analyte PDS.
10. CALIBRATION AND STANDARDIZATION
10.1 Demonstration and documentation of acceptable initial calibration is required before
any samples are analyzed. The MS tune check and initial calibration must be repeated
each time a major instrument modification is made, or maintenance is performed.
10.2 INITIAL CALIBRATION
10.2.1 ESI-MS/MS TUNE
10.2.1.1 Calibrate the mass scale of the MS with the calibration compounds and
procedures prescribed by the manufacturer.
10.2.1.2 Optimize the precursor ion (Sect. 3.16, [M+H]+ or [M+2H]2+) for each method
analyte by infusing approximately 1-5 ng/|iL of each analyte (prepared in the
initial mobile phase conditions) directly into the MS at the chosen LC mobile
phase flow rate (approximately 0.3 mL/min). This tune can be done on a mix
of method analytes, provided analytes have different MS/MS transitions. MS
parameters (voltages, temperatures, gas flows, etc.) are varied until optimal
analyte responses are determined (see caution below). Method analytes may
have different optima requiring some compromise between the optima. See
Table 2 for ESI-MS conditions used in method development. Conditions may
vary on different instruments, including whether the precursor ion is doubly
charged or not. Precursor ions other than those listed may be selected.
CAUTION: Desolvation temperature was identified as a parameter that
can affect the degree of analyte suppression observed in
matrices. Desolvation temperature is applied in different
ways to different instruments; a heated gas or a heated
stainless steel capillary is used in ESI source designs. Thus, it
is highly recommended that desolvation temperature be
minimized and that temperatures of <400 °C be used for the
heated gas source designs and <275 °C for heated capillary
source designs (these recommended temperatures are based
on LC conditions employed during development of this
method).
10.2.1.3 Optimize the product ion (Sect. 3.18) for each analyte by infusing
approximately 1-5 ng/|iL of each analyte (prepared as PDS in methanol)
directly into the MS at the chosen LC mobile phase flow rate (approximately
0.3 mL/min). This tune can be done on a mix of method analytes. MS/MS
parameters (collision gas pressure, collision energy, etc.) are varied until
optimal analyte responses are determined. See Table 4 for MS/MS conditions
used in method development.
24
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10.2.2 Establish LC operating parameters that optimize resolution and peak shape.
Suggested LC conditions can be found in Table 1. LC conditions listed in Table 1
may not be optimum for all LC systems and may need to be optimized by the
analyst.
10.2.3 Inject a mid-level CAL standard under LC/MS conditions to obtain retention
times of each method analyte. Divide the chromatogram into retention time
windows (segments) each of which contains one or more chromatographic peaks.
During MS/MS analysis, fragment a small number of selected precursor ions
([M+H]+ or [M+2H]2+; Sect. 3.16) for the analytes in each window and choose the
most abundant product ion. Product ions (also quantitation ions) chosen during
method development are in Table 4. Product ions other than those listed may be
selected. For maximum sensitivity in subsequent MS/MS analyses, minimize the
number of transitions that are simultaneously monitored within each segment.
10.2.4 Inject a mid-level CAL standard under optimized LC/MS/MS conditions to ensure
that each method analyte is observed in its MS/MS window and that there are at
least 10 scans across the peak for optimum precision.
10.2.5 Prepare a set of at least five CAL standards as described in Section 7.2.4. The
lowest concentration CAL standard must be at or below the MRL, which may
depend on system sensitivity. It is recommended that at least four of the CAL
standards are at a concentration greater than or equal to the MRL.
10.2.6 The LC/MS/MS system is calibrated using the internal standard technique. Use
the LC/MS/MS data system software to generate a linear regression or quadratic
calibration curve for each of the analytes. Curves may be concentration weighted,
if necessary. Forcing zero as part of the calibration is not permitted.
10.2.7 CALIBRATION ACCEPTANCE CRITERIA - Validate the initial calibration by
calculating the concentration of each analyte as an unknown against its regression
equation. For calibration levels that are < MRL, the result for each analyte should
be within ± 50% of the true value. All other calibration points must calculate to be
within ± 30% of their true value. If these criteria cannot be met, the analyst will
have difficulty meeting ongoing QC criteria. It is recommended that corrective
action is taken to reanalyze the CAL standards, restrict the range of calibration, or
select an alternate method of calibration.
CAUTION: When acquiring MS/MS data, LC operating conditions must be
carefully reproduced for each analysis to provide reproducible
retention times. If this is not done, the correct ions will not be
monitored at appropriate times. As a precautionary measure,
chromatographic peaks in each window must not elute too close to
the edge of the segment time window.
25
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10.3 CONTINUING CALIBRATION CHECK (CCC) - Analyze a CCC to verify the initial
calibration at the beginning of each analysis batch, after every tenth Field sample, and
at the end of each analysis batch. LRBs, CCCs, LFBs, LFSMs, LFSMDs and FDs are
not counted as samples. The beginning CCC of each analysis batch must be at or
below the MRL in order to verify instrument sensitivity prior to any analyses. If
standards have been prepared such that all low CAL points are not in the same CAL
solution, it may be necessary to analyze two CAL standards to meet this requirement.
Alternatively, analyte concentrations in the Analyte PDS may be customized to meet
these criteria. Subsequent CCCs should alternate between a medium and high
concentration CAL standard.
10.3.1 Inject an aliquot of the appropriate concentration CAL standard and analyze with
the same conditions used during the initial calibration.
10.3.2 Determine that the absolute areas of the quantitation ions of the IS(s) are within
50-150% of the average areas measured during initial calibration. If any of the IS
areas has changed by more than these amounts, adjustments must be made to
restore system sensitivity. These adjustments may include cleaning of the MS ion
source, or other maintenance as indicated in Section 10.3.4. Major instrument
maintenance requires recalibration (Sect 10.2) and verification of sensitivity by
analyzing a CCC at or below the MRL (Sect 10.3). Control charts are useful aids
in documenting system sensitivity changes.
10.3.3 Calculate the concentration of each analyte and SUR in the CCC. The calculated
amount for the SUR must be within ± 30% of the true value. Each analyte
fortified at a level < MRL must calculate to be within ± 50% of the true value.
The calculated concentration of method analytes in CCCs fortified at all other
levels must be within ± 30%. If these conditions do not exist, then all data for the
problem analyte must be considered invalid, and remedial action must be taken
(Sect. 10.3.4) which may require recalibration. Any Field or QC Samples that
have been analyzed since the last acceptable calibration verification should be
reanalyzed after adequate calibration has been restored, with the following
exception. If the CCC fails because the calculated concentration is greater
than 130% (150% for the low-level CCC) for a particular method analyte,
and Field sample extracts show no detection for that method analyte, non-
detects may be reported without re-analysis.
10.3.4 REMEDIAL ACTION - Failure to meet CCC QC performance criteria may
require remedial action. Major maintenance, such as cleaning the electrospray
probe, atmospheric pressure ionization source, mass analyzer, replacing the LC
column, LC maintenance, etc., requires recalibration (Sect 10.2) and verification
of sensitivity by analyzing a CCC at or below the MRL (Sect 10.3).
26
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11. PROCEDURE
11.1 Ambient water samples may contain cyanobacterial cells at widely different densities.
Thus, two procedural options are offered below: Option A - for samples with cell
densities that can be filtered within 8 hours, and Option B - for samples with cell
densities so high that direct filtration of the water sample is not practical. In most
cases, the Option A procedure will be the procedure used. Visual inspection of the
water sample and analyst experience will weigh heavily in determining the appropriate
procedure to apply to samples. Note that Option B decreases the sensitivity of analysis
and will require adjustment of the reported MRL for samples processed using this
option.
11.2 This procedure may be performed manually or in an automated mode using a robotic
or automatic sample preparation device. Data presented in Tables 5-11 demonstrate
data collected by manual extraction. An automatic/robotic sample preparation system,
designed for use with SPE cartridges, may be used if all QC requirements discussed in
Section 9 are met. If an automated system is used to prepare samples, follow
manufacturer's operating instructions, but all extraction and elution steps must be the
same as in the manual procedure. Extraction and elution steps may not be changed or
omitted to accommodate use of an automated system. If an automated system is used,
LRBs should be rotated among the ports to ensure that all valves and tubing meet LRB
requirements (Sect. 9.3.1).
NOTE: SPE cartridges described in this section are designed as single use items and
must be discarded after use. They may not be reconditioned for reuse in subsequent
analyses.
11.3 SAMPLE PREPARATION
11.3.1 Samples are preserved, collected and stored as presented in Section 8. All Field
and QC Samples, including the LRB, and LFB, must contain the preservatives
listed in Section 8.1.2. Before processing, gently shake the sample at least twenty-
five times to homogenize, then verify that the sample pH is 7 ± 0.5. If the sample
pH does not meet this requirement, discard the sample. If the sample pH is
acceptable, proceed with the analysis. Visually inspect the sample cell density
during pH verification and make a judgment as to whether the sample can be
filtered following Option A (Sect 11.4; filterable within 8 h from the start of
filtration) or Option B (Sect. 11.5). Samples that are observed during pH
measurement to be semitransparent can typically be processed following the
Option A procedure. If samples are not semi-transparent, but rather are thick and
viscous, the samples are unlikely to filter within the required 8 h time period,
therefore these samples must be processed with the Option B procedure.
11.3.2 If using the Option A procedure, weigh the sample bottle with collected sample to
the nearest 1 g, after pH measurement, but before filtration. Measurement by
volume will be used for the Option B procedure.
27
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11.3.3 If using Option A, a 100-mL volume is used for the LRB, LFB, LFSM and
LFSMD. If using Option B, a 10-mL volume is used for the LRB, LFB, LFSM
and LFSMD.
11.3.4 Add an aliquot of the SUR PDS (Sect. 7.2.2.2) to each sample to be extracted, cap
and invert to mix. During method development, a 20-|aL aliquot of the 5.0 ng/|jL
SUR PDS (Sect. 7.2.2.2) was added to 100 mL for a final concentration of
1,000 ng/L in the aqueous sample using Option A. For Option B, a 20-|aL aliquot
of the 5.0 ng/|jL SUR PDS (Sect. 7.2.2.2) was added to a 10-mL sample aliquot
for a final concentration of 10,000 ng/L in the aqueous sample.
11.3.5 In addition to SUR and preservatives, if the sample is an LFB, LFSM, or LFSMD,
add the necessary amount of Analyte PDS (Sect. 0). Cap and invert each sample
to mix.
11.3.6 Proceed to the Option A (Sect. 11.4) or Option B (Sect. 11.5) procedure.
11.4 OPTION A PROCEDURE
11.4.1 INTRACELLULAR TOXIN RELEASE PROCEDURE
11.4.1.1 Filter the 100-mL water sample using a Nuclepore filter (Sect. 6.5) with the
shiny side up; collect the filtrate into a 500 mL amber glass bottle (Sect. 6.4.1)
for extraction in Sect. 11.6. During the filtration of the samples, turn off the
hood lights to protect the sample from potential photodegradation.
NOTE: The entire 100-mL sample must be filtered within an 8 h period. If the
sample cannot be filtered within an 8 h period and another replicate
sample is not available to analyze by the Option B procedure, then
allow the sample to finish filtering, if possible. However, the results
from this sample must be flagged as suspect due to slow filtration.
11.4.1.2 Rinse sample bottle with 5 mL of 90:10 methanol:reagent water (v/v). Pour
bottle rinsate into filter apparatus and combine the rinsate with the filtered
water sample in Sect. 11.4.1.1.
11.4.1.3 Rinse the sides of the funnel with another 2.5 mL of 90:10 methanol: reagent
water (v/v) and combine with the filtered water sample in Sect. 11.4.1.1.
NOTE: If a different type of filtration apparatus is used (than what is
described in Section 6.4) that requires transfer of the filtrate from the
receiving container to another container, additional solvent washes (90:10
methanol:water) will be necessary to prevent loss of analytes. Do not exceed
25% methanol in the final 100-mL sample to be extracted.
28
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11.4.1.4 Using metal forceps remove the filter from the filter apparatus and fold the
filter in half (top of the filter inward) while only touching the edges of the
filter. Continue to fold the filter until it is small enough to fit into a glass test
tube (Sect.6.6). Push the filter to the bottom of the glass test tube using a glass
pipet.
11.4.1.5 Add 2 mL of 80:20 methanol:reagent water (v/v) to the test tube containing
the filter (ensure that the filter is covered with liquid) and manually swirl the
tube gently a few times.
11.4.1.6 Place the test tube containing the 2 mL filter solution and the filter in a freezer
at -20 °C for a minimum 1 hour. Do not exceed 24 hours in the freezer. If the
filter is kept frozen for more than 2 hours, the 100-mL aqueous filtrate from
Section 11.4.1.1 must be kept refrigerated at <6 °C until completion of the
toxin release procedure.
11.4.1.7 Remove the test tube from the freezer, swirl gently a few times, then draw off
the 2 mL of liquid using a glass pipet. Transfer the 2 mL of liquid to the
filtered 100 mL water sample collected in Section 11.4.1.1.
11.4.1.8 Rinse the filter and test tube by adding another 2 mL of 80:20
methanol: reagent water (v/v) to the test tube and swirl gently. Draw off the
2 mL of liquid using a glass pipet and transfer the 2 mL of liquid to the
filtered 100 mL water sample collected in Section 11.4.1.1.
11.4.1.9 Rinse the filter a second time by adding another 1 mL of 80:20
methanol: reagent water (v/v) to the test tube and swirl gently. Draw off the
1 mL of liquid using a glass pipet and transfer the 1 mL of liquid to the
filtered 100 mL water sample collected in Section 11.4.1.1. Swirl the 100 mL
sample several times to homogenize the sample. Perform SPE as directed in
Sect. 11.6.
11.4.2 SAMPLE VOLUME DETERMINATION - Weigh the empty sample bottle to the
nearest 1 g and determine the sample weight by subtraction of the empty bottle
weight from the original sample weight (Sect. 11.3.2). Assume a sample density of
1.0 g/mL. The sample volume for the Option A procedure must be 75-115 mL. If
the sample volume is not within this range, the sample results must be flagged as
incorrect sample volume collected. The sample volume will be used in the final
calculations of the analyte concentration (Sect. 12.2).
11.5 OPTION B PROCEDURE - Option B is a semi-quantitative procedure for samples
difficult to filter due to high cell densities. Because of the need to aliquot, use, and
analyze only a portion of the sample volume, quantitation of the method analytes will
be affected. These effects arise from a lack of sample homogeneity as well as potential
sample bottle adsorption losses. Results from this Option B procedure are expected to
29
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be biased low; however, identity of the major congeners present in the sample can be
determined.
11.5.1 INTRACELLULAR TOXIN RELEASE PROCEDURE
11.5.1.1 Using a glass graduated cylinder, place a 10 mL sample aliquot in a plastic
centrifuge tube (Sect. 6.8), add 30 mL of methanol using the same graduated
cylinder, cap and shake the tube gently, and freeze at -20 °C for a minimum of
2 hours and maximum of 24 hours.
11.5.1.2 Remove sample from the freezer and centrifuge at 8,000 rpm and 4 °C for
10 min. NOTE: Cyanobacterial cells may contain gas vesicles and depending
on the age of the cells, some cells may remain buoyant even after the toxin
release procedure and centrifugation.
11.5.1.3 Decant the supernatant into the filter apparatus and filter the supernatant using
a Nuclepore filter (Sect. 6.5) with the shiny side up; collect the filtrate into a
500 mL amber glass bottle (Sect. 6.4.1) for extraction in Sect. 11.6. Try not to
dislodge the pellet if possible. During filtration of the samples, turn off the
hood lights to protect the sample from potential photodegradation.
NOTE: The entire supernatant must be filtered within an 8 h period. If the
sample cannot be filtered within an 8 h period then allow the sample
to finish filtering, if possible. However, the results from this sample
must be flagged as suspect due to slow filtration.
11.5.1.4 Rinse the centrifuge tube with two 2-mL aliquots of 90:10 methanol:reagent
water (v/v). Pour the centrifuge tube rinsate into filter apparatus (while not
dislodging the pellet) and pass the rinsate through the filter into the water
sample filtered in Sect. 11.5.1.3.
11.5.1.5 Add enough reagent water to the filtrate in the 500-mL amber bottle to bring
the total volume up to approximately 150 mL (dilutes the methanol to levels
that do not affect the SPE).
11.5.1.6 Proceed with SPE of the 150 mL sample in Section 11.6.
11.6 CARTRIDGE SPE PROCEDURE
11.6.1 CARTRIDGE CLEAN-UP AND CONDITIONING - DO NOT allow cartridge
packing material to go dry during any of the conditioning steps. Rinse each
cartridge with 15 mL of methanol. Next, rinse each cartridge with 15 mL of
reagent water, without allowing the water to drop below the top edge of the
packing. If the cartridge goes dry during the conditioning phase, the conditioning
must be started over. Add 4-5 mL of reagent water to each cartridge, attach
30
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sample transfer tubes (Sect. 6.13.2.2), turn on the vacuum, and begin adding
filtered sample (containing the released intracellular toxins) to the cartridge.
11.6.2 SAMPLE EXTRACTION - Adjust the vacuum so that the approximate flow rate
is 10-15 mL/min. Do not allow the cartridge to go dry before all the sample has
passed through.
11.6.3 SAMPLE BOTTLE AND CARTRIDGE RINSE - After the entire sample has
passed through the cartridge, rinse the sample bottles with 10 mL of reagent water
and draw the rinse through the sample transfer tubes and the cartridges. Remove
the sample transfer tubes from the cartridges, but keep the tubes in their respective
bottles for the elution step in Section 11.6.4. Rinse the cartridges with another
5 mL of reagent water. Draw air or nitrogen through the cartridge for 10 min at
high vacuum (10-15 in Hg).
11.6.4 SAMPLE BOTTLE AND CARTRIDGE ELUTION - Turn off and release the
vacuum. Lift the extraction manifold top and insert a rack with collection tubes
into the extraction tank to collect the extracts as they are eluted from the
cartridges. Turn the vacuum back on, but ensure the vacuum does not exceed
10 in Hg during elution. Rinse the sample bottles with 5 mL of 90:10
methanol:reagent water (v/v) and elute the analytes from the cartridges by pulling
the 5 mL of solution (used to rinse the bottles) through the sample transfer tubes
and the cartridges. A soak time (draw a small amount of the elution solvent
through the cartridge, release the vacuum), up to 5 min, is permitted, but not
required, during the elution step to aid in recovery. Use a low vacuum such that
the solvent exits the cartridge in a dropwise fashion. Repeat sample bottle rinse
and cartridge elution with a second 5-mL aliquot of 90:10 methanol:reagent water
(v/v).
11.6.5 EXTRACT CONCENTRATION - Concentrate the extract to dryness under a
gentle stream of nitrogen in a heated water bath (60 °C). Care should be taken, as
the extract approached dryness, to keep the nitrogen flow low to prevent blowing
dried material out of the tube. Add 990 |iL of 90:10 methanol: reagent water (v/v)
and 10 |iL of the IS PDS (Sect. 7.2.1.2) to the collection vial and vortex. Transfer
an aliquot to an autosampler vial.
11.7 EXTRACT ANALYSIS
11.7.1 Establish operating conditions equivalent to those summarized in Tables 1-4 of
Section 17. Instrument conditions and columns should be optimized prior to
initiation of the IDC.
CAUTION: Diverting the first 6-8 minutes of the LC flow to waste is highly
recommended. These extracts will contain small quantities of some of the
preservatives which elute early in the chromatogram. Thus, diverting the early
portion of the analysis will minimize fouling of the MS source.
31
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11.7.2 Establish an appropriate retention time window for each analyte. This should be
based on measurements of actual retention time variation for each method analyte
in CAL standard solutions analyzed on the LC over the course of time. A value of
plus or minus three times the standard deviation of the retention time obtained for
each method analyte while establishing the initial calibration and completing the
IDC can be used to calculate a suggested window size. However, the experience
of the analyst should weigh heavily on the determination of the appropriate
retention window size.
11.7.3 Establish a valid initial calibration following the procedures outlined in Sect. 10.2
or confirm that the calibration is still valid by running a CCC as described in
Sect. 10.3. If establishing an initial calibration, complete the IDC as described in
Section 9.2.
11.7.4 Begin analyzing field samples, including QC samples, at their appropriate
frequency by injecting the same size aliquots (10 |aL was used in method
development), under the same conditions used to analyze the CAL standards.
11.7.5 At the conclusion of data acquisition, use the same software that was used in the
calibration procedure to identify peaks of interest in predetermined retention time
windows. Use the data system software to examine the ion abundances of the
peaks in the chromatogram. Identify an analyte by comparison of its retention
time with that of the corresponding method analyte peak in a reference standard.
11.7.6 The analyst must not extrapolate beyond the established calibration range. If an
analyte peak area exceeds the range of the initial calibration curve, the extract
may be diluted with 90:10 methanol:reagent water (v/v) and the appropriate
amount of internal standard added to match the original level. Re-inject the
diluted extract. Incorporate the dilution factor into the final concentration
calculations. Acceptable SUR performance (Sect. 9.3.5) should be determined
from the undiluted sample extract if analyzed. If the undiluted sample extract is
not analyzed, the SUR recovery should be calculated from a standard calibration
curve generated from a calibration curve containing a SUR concentration range.
The resulting data should be documented as a dilution and MRLs should be
adjusted accordingly. It is recommended that samples processed by Option B be
analyzed after a 10-fold or 100-fold dilution as the undiluted sample is likely to
foul the instrumentation and results are likely to be beyond the established
calibration range.
12. DATA ANALYSIS AND CALCULATION
12.1 Complete chromatographic resolution is not necessary for accurate and precise
measurements of analyte concentrations using MS/MS. Figure 2 demonstrates the
chromatogram achieved using the method conditions. In validating this method,
32
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concentrations were calculated by measuring the product ions listed in Table 4. Other
ions may be selected at the discretion of the analyst.
12.2 Calculate analyte and SUR concentrations using the multipoint calibration established
in Section 10.2. Do not use daily calibration verification data to quantitate analytes in
samples. Adjust final analyte concentrations to reflect the actual sample volume
determined in Section 11.4.2 (Option A) or the volume aliquoted in Section 11.5.1.1
(Option B).
12.3 Prior to reporting data, the chromatogram should be reviewed for any incorrect peak
identification or poor integration.
12.4 Calculations must utilize all available digits of precision, but final reported
concentrations should be rounded to an appropriate number of significant figures (one
digit of uncertainty), typically two, and not more than three significant figures.
13. SINGLE LABORATORY METHOD PERFORMANCE
13.1 PRECISION, ACCURACY, AND MINIMUM REPORTING LEVELS - Tables for
these data are presented in Section 17. LCMRLs and DLs for each method analyte are
presented in Table 5. Precision and accuracy are presented for two water matrices:
reagent water (Tables 6 and 8) and lake water (Table 7 and 9) using either Option A or
Option B.
13.2 AQUEOUS SAMPLE STORAGE STABILITY STUDIES - An analyte storage
stability study was conducted by fortifying the analytes into lake water samples that
were collected, preserved, and stored as described in Section 8. Precision and mean
recovery (n=4) of analyses, conducted on Days 0, 7, 14, 21 and 28 are presented in
Table 10.
13.3 EXTRACT STORAGE STABILITY STUDIES - Extract storage stability studies
were conducted on extracts obtained from lake water fortified with method analytes.
Precision and mean recovery (n=4) of injections conducted on Days 0, 7, 14, 21, and
28 are reported in Table 11.
13.4 Performance of the method was evaluated in 14 different ambient water sources across
the U.S. The box plots in Figure 3 show that QC criteria (dashed lines) were
consistently met for 882 analyte measurements in 63 LFSMs except for the 33 analyte
failures (96.3% QC pass rate) shown as outliers (green triangles) in the box plots.
LFSM failures were due to matrix effects observed in the fortified matrices. Some of
the matrices collected contained significant cyanobacterial blooms, including a few
cyanobacterial scum samples for which the Option B Procedure was followed.
33
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14. POLLUTION PREVENTION
14.1 This method utilizes SPE to extract analytes from water. It requires the use of very
small volumes of organic solvent and very small quantities of pure analytes, thereby
minimizing potential hazards to both the analyst and the environment as compared to
the use of large volumes of organic solvents in conventional liquid-liquid extractions.
14.2 For information about pollution prevention applicable to laboratory operations
described in this method, consult: Less is Better, Guide to Minimizing Waste in
Laboratories, a web-based resource available from the American Chemical Society
website.
15. WASTE MANAGEMENT
Analytical procedures described in this method generate relatively small amounts of waste
since only small amounts of reagents and solvents are used. However, laboratory waste
management practices must be conducted consistent with all applicable rules and regulations,
and that laboratories protect the air, water, and land by minimizing and controlling all
releases from fume hoods and bench operations. Also, compliance is required with any
sewage discharge permits and regulations, particularly the hazardous waste identification
rules and land disposal restrictions.
16. REFERENCES
1. Winslow, S.D., Pepich, B.V., Martin, J.J., Hallberg, G.R., Munch, D.J., Frebis, C.P.,
Hedrick, E. J., Krop, R. A. "Statistical Procedures for Determination and Verification of
Minimum Reporting Levels for Drinking Water Methods." Environ. Sci. Technol. 2004, 40,
281-288.
2. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, W.L. Budde, "Trace Analyses for
Wastewaters." Environ. Sci. Technol. 1981, 15, 1426-1435.
3. Leenheer, J.A., Rostad, C.E., Gates, P.M., Furlong, E.T., Ferrer, I. "Molecular Resolution
and Fragmentation of Fulvic Acid by Electrospray Ionization/Multistage Tandem Mass
Spectrometry." Anal. Chem. 2001, 73, 1461-1471.
4. Cahill, J.D., Furlong E.T., Burkhardt, M.R., Kolpin, D., Anderson, L.G. "Determination of
Pharmaceutical Compounds in Surface- and Ground-Water Samples by Solid-Phase
Extraction and High-Performance Liquid Chromatography Electrospray Ionization Mass
Spectrometry." J. Chromatogr. A, 2004, 1041, 171-180.
5. Draper, W.M., Xu, D., Perera, S.K. "Electrolyte-Induced Ionization Suppression and
Microcystin Toxins: Ammonium Formate Suppresses Sodium Replacement Ions and
Enhances Protiated and Ammoniated Ions for Improved Specificity in Quantitative LC-MS-
MS." Anal. Chem. 2009, 81, 4153-4160.
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6. "Biosafety in Microbiological and Biomedical Laboratories", 5th edition, Appendix I—
Guidelines for Work with Toxins of Biological Origin. A web-based resource available from
U.S. Department of Health and Human Services, Public Health Service Centers for Disease
Control and Prevention, National Institutes of Health.
7. "Prudent Practices in the Laboratory: Handling and Disposal of Chemicals," a web-based
resource available from National Academies Press (1995).
8. "OSHA Safety and Health Standards, General Industry," (29CFR1910), Occupational Safety
and Health Administration, OSHA 2206, (Revised, July 2001).
9. "Safety in Academic Chemistry Laboratories," a web-based resource available from
American Chemical Society Publication, Committee on Chemical Safety, 7th Edition.
10. Draper W.M., Xu, D., Behniwal, P., McKinney, M.J., Jayalath, P., Dhoot, J.S.,Wijekoon, D.
"Optimizing LC-MS-MS Determination of Microcystin Toxins in Natural Water and
Drinking Water Supplies." Anal. Methods, 2013, 5, 6796-6806.
11. Winslow, S. D. , Pepich, S. D. , Bassett, M. V., Wendelken, S. C., Munch, D. J., Sinclair, J.
L. "Microbial Inhibitors for U.S. EPA Drinking Water Methods for the Determination of
Organic Compounds."Environ. Sci. Technol., 2001, 35, 4103-4110
35
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17. TABLES. DIAGRAMS. FLOWCHARTS AND VALIDATION DATA
TABLE 1. LC METHOD CONDITIONS
Time (min)
% 20 mM Ammonium Formate
% Methanol
Initial
90
10
2.0
90
10
16.0
20
80
16.1
10
90
22.0
10
90
22.1
90
10
26.0
90
10
Phenomenex Kinetex Cs column, 2.6 |am, 2.1 x 100 mm
Flow rate of 0.3 mL/min
Column temperature of 65 °C
10 |oL partial loop injection into a 20 |iL loop
TABLE 2. ESI-MS/MS METHOD CONDITIONS
ESI Parameter
Settings
Polarity
Positive ion
Capillary needle voltage
4 kV
Cone gas flow
50 L/h
Nitrogen desolvation gas
1000 L/h
Desolvation gas temp.
350 °C
36
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TABLE 3. METHOD ANALYTE SOURCE AND RETENTION TIMES (RTs)
Peak ID
Analyte
Method Analyte Source3
RT (min)
1
Nodularin
National Research Council Canada
10.06
2
MC-YR
Enzo Life Sciences
10.15
3
MC-HtyR
Enzo Life Sciences
10.18
4
MC-RR
National Research Council Canada
10.55
5
3 -desm ethyl ated-MC -RR
Enzo Life Sciences
10.58
6
MC-LR
National Research Council Canada
10.67
7
MC-WR
Enzo Life Sciences
10.98
8
7-desmethyl ated-MC -LR
National Research Council Canada
11.08
9
MC-HilR
Enzo Life Sciences
11.19
10
3 -desmethyl ated-MC -LR
Enzo Life Sciences
11.35
11
MC-LA
GreenWater Laboratories
11.47
12
MC-LY
Enzo Life Sciences
11.49
13
MC-LW
Enzo Life Sciences
12.45
14
MC-LF
Enzo Life Sciences
13.07
15
C2D5-MC-LR (SUR)
Tamarack Environmental
13.55
16
Cyclosporin-A, 13C2, d4 (IS)
Toronto Research Chemicals
17.19
a Data presented in this method were obtained using analytes purchased from these vendors.
Other vendors' materials can be used provided the QC requirements in Section 9 can be met.
37
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TABLE 4. M
[S/MS METHOD CONDITIONS3'
Segment0
Analyte
Precursor Iond
(m/z)
Product
Iond'e (m/z)
Cone
Voltage (v)
Collision
Energyf (v)
1
Nodularin
825.4 [M+H]+
134.9
45
55
1
MC-YR
523.4 [M+2H]2+
134.9
20
20
1
MC-HtyR
1059.6 [M+H]+
134.9
60
75
2
MC-RR
519.9 [M+2H]2+
134.9
35
30
2
3 -desm ethyl ated-MC -RR
512.9 [M+2H]2+
134.9
40
30
2
MC-LR
995.6 [M+H]+
134.9
60
70
3
MC-WR
1068.6 [M+H]+
134.9
60
75
3
7-desmethyl ated-MC -LR
981.5 [M+H]+
134.9
75
65
3
MC-HilR
1009.6 [M+H]+
134.9
70
65
3
3 -desmethyl ated-MC -LR
981.5 [M+H]+
134.9
70
65
3
MC-LA
910.5 [M+H]+
134.9
40
50
3
MC-LY
1002.5 [M+H]+
134.9
40
60
4
MC-LW
1025.5 [M+H]+
134.9
45
65
4
MC-LF
986.5 [M+H]+
134.9
40
60
4
C2D5-MC-LR (SUR)
1028.6 [M+H]+
134.9
55
70
5
Cyclosporin-A, 13C2, d4 (IS)
1208.9 [M+H]+
99.9
65
90
a An LC/MS/MS chromatogram of the analytes is shown in Figure 2.
b Conditions may vary on different instruments, including whether the precursor ion is doubly
charged or not. The conditions in this table are suggested conditions. Other conditions and MS/MS
transitions are permitted.
c Segments are time durations in which single or multiple scan events occur.
d During MS and MS/MS optimization, the analyst should determine the precursor and product ion
masses to one decimal place by locating the apex of the mass spectral peak place (e.g., m/z
523.4^134.9 for MC-YR). These precursor and product ion masses (with one decimal place)
should be used in the MS/MS method for all analyses.
e Ions used for quantitation purposes.
f Argon used as collision gas at a flow rate of 0.3 mL/min.
38
-------
TABLE 5. DLs AND LCMRLs IN B
JCAGENT WA1
"ER (Option A)
Analyte
Fortified
Cone. (ng/L)a
DLb
(ng/L)
LCMRLC
(ng/L)
Nodularin
4.94
2.3
14
MC-YR
20.0
7.0
54
MC-HtyR
20.0
11
36
MC-RR
3.30
2.1
17
3 -desm ethyl ated-MC -RR
10.0
4.4
28
MC-LR
19.1
10
79
MC-WR
60.0
33
170
7-desmethyl ated-MC -LR
18.8
7.6
49
MC-HilR
20.0
13
62
3 -desmethyl ated-MC -LR
40.0
13
89
MC-LA
20.0
6.9
22
MC-LY
20.0
10
50
MC-LW
20.0
7.6
39
MC-LF
20.0
6.5
23
a Spiking concentration used to determine DL.
b Detection limits were determined by analyzing seven replicates over three days
according to Section 9.2.6.
c LCMRLs were calculated according to the procedure in reference 2.
39
-------
TABLE 6. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
Analyte
Fortified
Cone. (ng/L)
Mean %
Recovery
%
RSD
Fortified
Cone. (ng/L)
Mean %
Recovery
%
RSD
Nodularin
247
108
2.9
49.4
107
1.2
MC-YR
1000
92.6
3.0
200
108
6.4
MC-HtyR
1000
99.7
2.1
200
105
6.2
MC-RR
165
95.2
4.1
33.0
106
5.3
3 -desmethyl ated-MC-RR
500
102
3.8
100
107
3.9
MC-LR
954
105
1.9
191
100
6.9
MC-WR
3000
99.0
1.8
600
93.7
5.4
7-desmethyl ated-MC-LR
940
97.4
2.8
188
96.0
2.9
MC-HilR
1000
105
2.2
200
98.1
3.3
3 -desmethyl ated-MC-LR
2000
99.6
2.0
400
94.8
6.3
MC-LA
1000
93.1
4.0
200
102
3.2
MC-LY
1000
93.4
2.8
200
99.5
4.3
MC-LW
1000
83.3
3.5
200
89.5
6.3
MC-LF
1000
92.2
2.9
200
97.4
2.1
C2D5-MC-LR (SUR)
1000
93.8
2.0
1000
91.7
2.1
Cyclosporin-A, 13C2, d4 (IS)
10000
87.7
6.9
10000
81.0
5.4
40
-------
TABLE 7. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
FORTIFIED IN LAKE WATER (n=4; Option A)
Analyte
Fortified
Cone. (ng/L)
Mean %
Recovery
%
RSD
Fortified
Cone. (ng/L)
Mean %
Recovery
%
RSD
Nodularin
247
93.0
1.2
49.4
95.6
3.0
MC-YR
1000
88.7
2.2
200
87.8
6.0
MC-HtyR
1000
112
1.2
200
109
12
MC-RR
165
91.8
1.4
33.0
84.9
2.7
3 -desmethyl ated-MC-RR
500
94.1
1.7
100
95.7
3.4
MC-LR
954
110
2.4
191
105
5.1
MC-WR
3000
96.7
1.5
600
97.2
11
7-desmethyl ated-MC-LR
940
105
3.1
188
104
4.7
MC-HilR
1000
104
4.7
200
105
5.9
3 -desmethyl ated-MC-LR
2000
106
3.5
400
110
5.3
MC-LA
1000
95.9
1.3
200
87.1
6.3
MC-LY
1000
95.6
2.0
200
92.4
4.8
MC-LW
1000
67.0
5.7
200
68.1
17
MC-LF
1000
88.9
2.0
200
86.8
7.0
C2D5-MC-LR (SUR)
1000
85.7
2.4
1000
83.5
6.0
Cyclosporin-A, 13C2, d4 (IS)
10000
89.9
5.0
10000
91.6
3.5
41
-------
TABLE 8. PRECISION AND ACCURACY DATA FOR METHOD
ANALYTES FORTIFIED IN REAGENT WATER
(n=4; Option B)
Analyte
Fortified
Cone. (jig/L)
Mean %
Recovery
%
RSD
Nodularin
2.50
95.1
6.6
MC-YR
10.0
94.6
7.2
MC-HtyR
10.0
86.8
14
MC-RR
1.80
87.4
12
3 -desmethyl ated-MC-RR
5.00
83.4
13
MC-LR
10.0
90.5
10
MC-WR
30.0
90.4
11
7-desmethyl ated-MC-LR
9.40
89.8
6.9
MC-HilR
10.0
89.1
5.6
3 -desmethyl ated-MC-LR
20.0
96.5
7.8
MC-LA
10.0
98.1
5.9
MC-LY
10.0
92.8
11
MC-LW
10.0
88.5
7.3
MC-LF
10.0
89.9
6.9
C2D5-MC-LR (SUR)
10.0
87.0
5.3
Cyclosporin-A, 13C2, d4 (IS)
10.0
94.2
12
42
-------
TABLE 9. PRECISION AND ACCURACY DATA FOR METHOD
Analyte
Fortified
Cone. (jig/L)
Mean %
Recovery
%
RSD
Nodularin
2.50
92.6
7.7
MC-YR
10.0
94.4
6.9
MC-HtyR
10.0
85.4
3.9
MC-RR
1.80
89.7
6.2
3 -desmethyl ated-MC-RR
5.00
91.8
8.3
MC-LR
10.0
89.7
5.7
MC-WR
30.0
86.3
6.5
7-desmethyl ated-MC-LR
9.40
87.7
6.3
MC-HilR
10.0
88.1
6.5
3 -desmethyl ated-MC-LR
20.0
92.0
6.8
MC-LA
10.0
95.8
3.7
MC-LY
10.0
91.1
5.1
MC-LW
10.0
86.2
4.4
MC-LF
10.0
86.2
5.0
C2D5-MC-LR (SUR)
10.0
85.3
6.1
Cyclosporin-A, 13C2, d4 (IS)
10.0
91.3
13
43
-------
TABLE 10. AQUEOUS SAMPLE HOLDING TIME DATA FOR LAKE WATER SAMPLES FORTIFIED WITH
METHOD ANALYTES AND PRESERVED AND STORED ACCORDING TO SI
Analyte
Fortified
Cone. (ng/L)
Day 0
Day 0
Day 7
Day 7
Day 14
Day 14
Day 21
Day 21
Day 28
Day 28
Mean
%
Mean
%
Mean
%
Mean
%
Mean
%
%Rec
RSD
%Rec
RSD
%Rec
RSD
%Rec
RSD
%Rec
RSD
Nodularin
494.4
87.4
3.3
91.1
3.3
80.8
3.8
82.6
4.7
83.9
2.1
MC-YR
2000
92.6
1.8
100.4
1.6
87.5
2.0
88.6
2.9
86.6
4.8
MC-HtyR
2000
90.3
3.7
97.7
3.0
86.9
1.8
90.4
2.8
87.8
2.7
MC-RR
329.6
83.4
5.2
88.9
2.5
78.4
1.9
83.3
2.7
82.5
1.6
3 -desmethyl ated-MC-RR
1000
91.8
2.3
100.7
2.5
88.9
3.0
93.8
3.7
91.9
1.7
MC-LR
2000
89.5
5.8
92.4
1.2
86.0
3.1
91.1
4.0
91.5
2.7
MC-WR
6000
91.9
6.1
99.4
1.0
91.0
1.9
95.5
5.0
91.4
1.9
7-desmethyl ated-MC-LR
1880
93.2
3.6
97.8
0.6
89.8
1.3
93.0
4.2
91.0
2.7
MC-HilR
2000
91.1
3.9
97.8
4.6
88.9
1.5
92.6
5.9
89.3
2.5
3 -desmethyl ated-MC-LR
4000
98.0
3.4
105.5
2.4
97.2
1.1
96.2
3.1
96.2
2.1
MC-LA
2000
83.3
4.0
96.4
4.0
83.2
6.8
84.7
4.4
86.3
4.1
MC-LY
2000
92.1
3.2
95.7
3.8
80.2
4.0
89.4
6.4
91.3
1.4
MC-LW
2000
80.3
3.7
91.7
3.9
81.3
1.2
83.0
2.7
80.5
3.3
MC-LF
2000
85.4
1.4
92.2
0.8
85.4
0.6
89.1
1.8
88.5
2.1
C2D5-MC-LR (SUR)a
1664
82.8
4.7
89.7
3.7
84.9
2.3
85.6
4.2
87.4
2.4
Surrogate was not added to samples until the day of extraction.
44
-------
TABLE 11. EXTRACT HOLDING TIME DATA FOR SAMPLES FROM A LAKE WATER SOURCE, FORTIFIED WITH
Analyte
Fortified
Cone. (ng/L)
Day 0
Day 0
Day 7
Day 7
Day 14
Day 14
Day 21
Day 21
Day 28
Day 28
Mean
%
Mean
%
Mean
%
Mean
%
Mean
%
%Rec
RSD
%Rec
RSD
%Rec
RSD
%Rec
RSD
%Rec
RSD
Nodularin
494.4
87.4
3.3
86.1
2.7
81.5
2.2
82.5
2.5
77.7
2.7
MC-YR
2000
92.6
1.8
98.0
4.1
84.9
2.0
89.1
2.0
81.4
1.1
MC-HtyR
2000
90.3
3.7
94.9
2.8
87.1
3.2
92.6
2.2
82.1
3.3
MC-RR
329.6
83.4
5.2
84.6
3.3
77.1
0.6
82.3
2.5
73.4
2.3
3 -desmethyl ated-MC-RR
1000
91.8
2.3
98.8
3.5
90.5
1.8
91.6
2.6
85.2
2.6
MC-LR
2000
89.5
5.8
90.0
3.6
87.0
1.3
92.7
1.4
82.8
2.7
MC-WR
6000
91.9
6.1
98.5
4.0
92.6
1.4
99.2
3.6
89.5
0.5
7-desmethyl ated-MC-LR
1880
93.2
3.6
95.9
2.6
89.9
3.3
97.8
3.2
86.6
1.6
MC-HilR
2000
91.1
3.9
98.0
3.2
88.5
3.4
98.7
1.7
83.7
1.8
3 -desmethyl ated-MC-LR
4000
98.0
3.4
102.7
4.2
97.8
0.9
101.5
2.2
92.5
2.1
MC-LA
2000
83.3
4.0
92.5
5.0
81.5
5.9
87.1
2.1
91.2
0.8
MC-LY
2000
92.1
3.2
90.1
4.9
82.2
3.3
91.7
4.6
83.0
2.6
MC-LW
2000
80.3
3.7
81.6
3.5
78.0
6.2
83.3
1.6
88.1
1.9
MC-LF
2000
85.4
1.4
86.9
5.9
83.9
3.3
86.1
3.0
82.6
3.2
C2D5-MC-LR (SUR)
1664
82.8
4.7
79.8
4.0
80.8
2.7
86.1
1.2
86.9
2.9
45
-------
FIGURE 1.
DIAGRAM OF FILTER APPARATUS WITH PART NUMBERS (SECT. 6.4)
953751-0000
953753-0000
953752-5047
\\\
tHC
% 736400-1413
410170-4534 (jTjT [Tj
410171-4226 f;:: •• '
02-542-4C (Fisher) '
46
-------
FIGURE 2.
EXAMPLE CHROMATOGRAMS (OVERLAID MS/MS SEGMENTS) OF A CALIBRATION STANDARD WITH
ANALYTES AT MID-LEVEL CALIBRATION CONCENTRATIONS. SEE TABLE 3 FOR PEAK IDs.
100-,
¦
14
12
9.50
10.00
10.50
11.00
11.50
12.00
12.50
13.00
13.50
14.00
14.50
15.00
15.50
16.00
16.50
17.00
17.50
18.00
47
-------
FIGURE 3.
BOX PLOTS SHOWING DISTRIBUTION OF LFSM RECOVERIES OBTAINED IN AMBIENT
WATERS FROM 14 DIFFERENT WATER BODIES ACROSS THE U.S. (SECT. 13.4). GREEN
TRAINGLES REPRESENT THE 33 ANALYTE QC FAILURES OUT OF 882 ANALYTE
MEASUREMENTS.
400 q
350 ;
300 j
&250 -
8 200
/
af
$
v
"5
48
------- |