EPA Document #: EPA/600/R-14/474
METHOD 544. DETERMINATION OF MICROCYSTINS AND NODULARIN IN
DRINKING WATER BY SOLID PHASE EXTRACTION AND
LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY
(LC/MS/MS)
Version 1.0
February 2015
J.A. Shoemaker US EPA, Office of Research and Development, National Exposure
Research Laboratory
D.R. Tettenhorst US EPA, Office of Research and Development, National Exposure
Research Laboratory
A. de la Cruz US EPA, Office of Research and Development, National Exposure
Research Laboratory
NATIONAL EXPOSURE RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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METHOD 544
DETERMINATION OF MICROCYSTINS AND NODULARIN IN DRINKING WATER
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 drinking water. Accuracy and precision data have been generated in reagent water,
and finished ground and surface waters for compounds listed in the table below.
Chemical Abstract Services
Analvte Registry Number (CASRN)
microcystin-LA (MC-LA) 96180-79-9
microcystin-LF (MC-LF) 154037-70-4
microcystin-LR (MC-LR) 101043-37-2
microcystin-LY (MC-LY) 123304-10-9
microcystin-RR (MC-RR) 111755-37-4
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 2.9-22 ng/L, 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 meets the require-
ments described in Section 9.2.4.
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 1.2-4.6 ng/L, and are listed in Table 5.
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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 (Sect. 6.12, 9.1.1, 10.2, and 12.1). Changes may not
be made to sample collection and preservation (Sect. 8), sample extraction 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. Analvtes 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 that acceptable method performance can be verified in a real sample matrix
(Sect. 9.3.5).
NOTE: The above method flexibility section is intended as an abbreviated summation
of method flexibility. Sections 4-12 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 4-12, Sections 4-12 supersede Section 1.6.
2. SUMMARY OF METHOD
A 500-mL water sample (fortified with a surrogate) is filtered and both the filtrate and the
filter are collected. The filter is placed in a solution of methanol containing 20% reagent
water and held for at least one hour at -20 °C to release the intracellular toxins from
cyanobacteria cells captured on the filter. The liquid is drawn off the filter and added back to
the 500-mL aqueous filtrate. The 500-mL sample (plus the intracellular toxin solution) 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 methanol
containing 10% reagent water. The extract is concentrated to dryness by evaporation with
nitrogen in a heated water bath, and then adjusted to a 1-mL volume with methanol
containing 10% reagent water. A 10-|iL injection is made into an LC equipped with a Cs
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 external standard calibration.
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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 and/or
the number of field samples.
3.2. CALIBRATION STANDARD (CAL) - A solution prepared from the primary dilution
standard solution and/or stock standard solution, and the surrogate. 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, and surrogate(s). The CCC is analyzed periodically 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)
extracted together by the same person during a work day using the same lot of SPE
devices, solvents, surrogate, and fortifying solutions. Required QC samples include
Laboratory Reagent Blank, Laboratory Fortified Blank, Laboratory Fortified Sample
Matrix, and either a Field Duplicate or Laboratory Fortified Sample Matrix Duplicate.
3.7. FIELD DUPLICATES (FD1 and FD2) - Two separate samples collected at the same
time and place under identical circumstances, and treated exactly the same throughout
field and laboratory procedures. Analyses of FD1 and FD2 give a measure of the
precision associated with sample collection, preservation, and storage, as well as
laboratory procedures.
3.8. LABORATORY FORTIFIED BLANK (LFB) - A volume of reagent water or other
blank matrix to which known quantities of the method analytes and all the 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.
3.9. LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - A preserved field
sample to which known quantities of the method analytes are added in the laboratory.
The LFSM is processed and analyzed exactly like a sample, and its purpose is to
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determine whether the sample matrix contributes bias to the analytical results. The
background concentrations of the analytes in the sample matrix must be determined in
a separate sample extraction and the measured values in the LFSM corrected for
background concentrations.
3.10. 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.
3.11. 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, and surrogates 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, the reagents, or the apparatus.
3.12. 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
3.13. MATERIAL SAFETY DATA SHEET (MSDS) - Written information provided by
vendors concerning a chemical's toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling precautions.
3.14. 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.
3.15. 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.
3.16. 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.
3.17. 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.
3.18. QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of known
concentrations that is obtained from a source external to the laboratory and different
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from the source of calibration standards. The second source stock standard solution is
used to fortify the QCS at a known concentration. The QCS is used to check
calibration standard integrity.
3.19. 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.
3.20. 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 must be meticulously cleaned. Wash glassware with detergent and tap
water, rinse with tap water, followed by a reagent water rinse. Non-volumetric
glassware can be heated in a muffle furnace for a minimum of 90 min at 400°C.
Volumetric glassware should be solvent rinsed and heated in an oven no hotter than
120 °C.
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 and/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. Total organic carbon
(TOC) is a good indicator of humic content of the sample.
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 The 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 the preservatives (Sect. 8.1.2) are added to sample
bottles. The potential exists for trace-level organic contaminants in these reagents.
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Interferences 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 field and 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
5.1. 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 MSDSs should be made available to all personnel involved
in the chemical analysis. Toxin decontamination/inactivation guidelines may be found
in Biosafety in Microbiological andBiomedical Laboratories, 5th edition.6
Additional references to laboratory safety are available.7"9
5.2. Pure standard materials and stock standard solutions of these method analytes should
be handled with suitable protection to skin and eyes, and care should be taken not to
breathe the vapors or ingest the materials.
6. EQUIPMENT AND SUPPLIES (Brand names and/or catalog numbers are included for
illustration only, and do not imply endorsement of the product.)
6.1 SAMPLE CONTAINERS - 500-mL amber glass bottles fitted with
polytetrafluoroethylene (PTFE)-lined screw caps.
6.2 SAMPLE FILTER APPARATUS (See Figure 1)
6.2.1 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).
6.2.2 FILTER BASE O-RING - PTFE/silicone sealing ring (Kimble Chase #410171-
4226 or equivalent).
6.2.3 BOTTLE CAP WITH HOLE - GL 45 bottle cap with hole for filter support base
(Kimble #410170-4534, or equivalent).
6.2.4 SUPPORT BASE - 47 mm fritted glass support base for filtration (Kimble Chase
#953752-5047 or equivalent).
6.2.5 HOSE BARB CONNECTOR - Barbed tubing adapter for filtration apparatus
(Kimble Chase #736400-1413 or equivalent).
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6.2.6 METAL CLAMP - 47 mm aluminum clamp (Kimble Chase #953753-0000 or
equivalent).
6.2.7 FUNNEL - 47 mm, 300 mL glass funnel (Kimble Chase #953751-0000 or
equivalent).
6.3 MEMBRANE FILTER - 47 mm Nuclepore polycarbonate filter membranes, pore size
0.4 um, (Whatman #111107 or equivalent).
6.4 ROUND BOTTOM CULTURE TUBES - 15-mL round bottom glass culture tubes
(Corning #9826-16X or equivalent) or other glassware suitable for use in releasing the
toxin from the filter.
6.5 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.
6.6 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).
6.7 MICRO SYRINGES - Suggested sizes include 5, 10, 25, 50, 100, 250, 500 and
1000-jiL syringes.
6.8 ANALYTICAL BALANCE - Capable of weighing to the nearest 0.0001 g.
6.9 SOLID PHASE EXTRACTION (SPE) APPARATUS FOR USING CARTRIDGES
6.9.1 SPE CARTRIDGES - Waters Oasis HLB, 150 mg, 6cc divinylbenzene N-
vinylpyrrolidone copolymer (Waters # 186003365).
6.9.2 VACUUM EXTRACTION MANIFOLD
6.9.2.1 Manual Extraction - A manual vacuum manifold with Visiprep™ large
volume sampler (Supelco #57030 and #57275 or equivalent) for cartridge
extractions.
6.9.2.2 SAMPLE DELIVERY SYSTEM - Use of a transfer tube system (Supelco
"Visiprep," #57275 or equivalent), which transfers the sample directly from
the sample container to the SPE cartridge is recommended.
6.10 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).
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6.11 LABORATORY OR ASPIRATOR VACUUM SYSTEM - Sufficient capacity to
maintain a vacuum of approximately 10 to 15 inches of mercury for extracting
cartridges.
6.12 LIQUID CHROMATOGRAPHY (LC)/TANDEM MASS SPECTROMETER
(MS/MS) WITH DATA SYSTEM
6.12.1 LC SYSTEM - Instrument 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 is optional.
6.12.2 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. 3.17) 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.
6.12.3 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.
6.12.4 ANALYTICAL COLUMN - An LC C8 column (2.1 x 100 mm) packed
with 2.6 jam Cs solid phase particles (Phenomenex Kinetex #OOD-4497-AN)
was used. Any equivalent column that provides adequate resolution, peak
shape, capacity, accuracy, and precision (Sect. 1.6 and 9) may be used.
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.
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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, CAS#: 67-56-1) - High purity, demonstrated to be free of
analytes and interferences (Fisher Optima LC/MS grade or equivalent).
7.1.3 AMMONIUM FORMATE (CH5O2N, CAS# 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 L-ASCORBIC ACID (CAS# 50-81-7) - Reduces free chlorine at the time of
sample collection (Sigma-Aldrich #255564 or equivalent).10
7.1.5.3 2-CHLOROACETAMIDE (CAS# 79-07-2) - Inhibits microbial growth and
analyte degradation (Sigma-Aldrich #C0267 or equivalent).10
7.1.5.4 ETHYLENEDIAMINETETRAACETIC ACID, TRISODIUM SALT
(Trisodium EDTA, CAS# 10378-22-0) - Inhibits metal-catalyzed hydrolysis
of analytes. The trisodium salt is used instead of the disodium salt because
the trisodium salt solution pH is closer to the desired pH of 7 (Sigma
#ED3SS or equivalent).
7.1.6 NITROGEN - Aids in aerosol generation and desolvation of the ESI liquid spray
and is used as collision gas in some MS/MS instruments. Nitrogen used should
meet or exceed instrument manufacturer's specifications.
7.1.7 ARGON - Used as collision gas during MS/MS experiments. Argon should meet
or exceed instrument manufacturer's specifications. Nitrogen gas may be used as
collision gas provided sufficient sensitivity (product ion formation) is achieved.
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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 Even though stability times for standard
solutions are suggested in the following sections, laboratories should use standard
QC practices to determine when their standards need to be replaced.
NOTE: Pipets using polypropylene tips must not be used for dispensing solutions
containing method analytes as adsorption of microcystins to polypropylene
has been reported.11
7.2.1 SURROGATE (SUR) ANALYTE STANDARD SOLUTIONS - The SUR for
this method is ethylated MC-LR, ds (C2Ds-MC-LR; see Table 3). 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 the QC requirements in Section 9.3.4.
7.2.1.1 SURROGATE PRIMARY DILUTION STANDARD (SUR PDS; 6.49
ng/|iL) - The SUR PDS was prepared by diluting 64.9 jig of neat material
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 6.49 ng/|iL SUR PDS to fortify
the 500 mL aqueous QC and field samples prior to extraction (Sect. 11.2.2).
This will yield a concentration of 259.6 ng/L of the SUR in aqueous QC and
field samples. The SUR concentration may be adjusted to accommodate
instrument sensitivity.
7.2.2 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).
7.2.2.1 ANALYTE STOCK STANDARD SOLUTION (10-500 |ig/mL) - Neat
cyanotoxins are typically purchased in quantities of 10-500 jig. 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 (10-500 jig) for a final concentration of 10-
500 |ig/mL. Repeat for each method analyte prepared from neat material.
Alternatively, purchase commercially available stock standards of the analytes,
preferably in methanol, if available. These stock standards were stable for at
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least six months when stored at -15 °C or less in amber glass screw cap
vials.
7.2.2.2 ANALYTE PRIMARY DILUTION STANDARD (PDS) SOLUTION
(0.94-5.0 ng/|oL) - 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 methanol at
concentrations of 0.94-5.0 ng/|aL The Analyte PDS is prepared by dilution
of the combined Analyte Stock Standard Solutions (Sect.7.2.2.1) and is used
to prepare CAL standards, and fortify LFBs, LFSMs, LFSMDs and FDs
with the method analytes. The Analyte PDS has been shown to be stable for
one month when stored at -15 °C or less in amber glass screw cap vials.
Microcystin-LR
Mi crocy stin-RR
Microcystin-YR
Microcystin-LY
Microcystin-LF
Nodularin-R
Microcystin-LA
Cone, of Analyte
Stock (ug/mL)
500
10.3
100
100
100
10.3
100
Vol. of Analyte
Stock (uL)
40.0
910
200
200
200
950
500
Final Vol. of
Analyte PDS
(mL)
lOmL
Final Cone, of PDS
(ng/jiL)
2.0
0.94
2.0
2.0
2.0
0.98
5.0
7.2.3 CALIBRATION STANDARDS (CAL) - Prepare a series of at least five
concentrations of calibration solutions in methanol containing 10% water, from
dilutions of the Analyte PDS (Sect 7.2.2.2). The suggested concentrations in this
paragraph are a description of the concentrations used during method
development, and may be modified to conform with instrument sensitivity.
Concentration ranges used during method development were 10- 400 |ig/L, except
for MC-RR (4.7-187.5 |ig/L), nodularin-R (4.9-195.7 |ig/L) and MC-LA (25-1000
|ig/L). Larger concentration ranges will require more calibration points. The SUR
is added to CAL standards at a constant concentration. During method
development, the concentration of the SUR was 129.8 |ig/L in the standard
(259.6 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 shown to be stable for two weeks when stored at -4 °C or less.
Longer storage times are acceptable provided appropriate QC measures are
documented demonstrating the CAL stability.
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8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 SAMPLE BOTTLE PREPARATION
8.1.1
8.1.2
Collect 500-mL samples in amber glass bottles (Sect. 6.1) fitted with teflon-lined
screw caps. Do not use sample bottles larger than 500-mL (rinse steps were not
optimized for larger bottle sizes). Smaller sample sizes may be used, but no less
than 100-mL, provided the MRL can be met. The entire sample volume in the
bottle must be used (e.g., a 100-mL aliquot must not be drawn off a 500-mL
sample because the sample bottle must be rinsed).
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
Trizma
2-Chloroacetamide
Ascorbic acid
Ethylenediaminetetraacetic acid
trisodium salt
Amount
7.75 g/L
2g/L
lOOmg/L
0.35 g/L
Purpose
buffering reagent
antimicrobial
dechlorinating agent
inhibit binding of the targets to
metals
8.2 SAMPLE COLLECTION
8.2.1 Open the cold water tap and allow the system to flush until the water temperature
has stabilized (approximately 3 to 5 min). Collect samples from the flowing
system.
8.2.2 Fill sample bottles, taking care not to flush out the sample preservation reagents.
Samples do not need to be collected headspace free.
8.2.3 After collecting the sample, cap the 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.
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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 9 and 10.
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
that must be met in order to meet EPA quality objectives. QC criteria discussed in the
following sections are summarized in Tables 11 and 12. 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, or 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 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.
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9.2.4 MINIMUM REPORTING LEVEL (MRL) CONFIRMATION - Establish a target
concentration for the MRL based on the intended use of the method. 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 the MRL following
the procedure outlined below.
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
where
s = 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 +_ HRpm) meet the upper and lower recovery limits as shown
below
The Upper PIR Limit must be < 150% recovery.
M*™+™** xlOO%<150%
Fortified Concentrat ion
The Lower PIR Limit must be > 50% recovery.
Mean - HR „,„
xlOO%>50%
Fortified Concentrat ion
9.2.4.3 The MRL is validated if both the Upper and Lower PIR Limits meet the
criteria described above (Sect. 9.2.4.2). 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 9.3.7 to confirm the accuracy of the standards/calibration curve.
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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 ifDL 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
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.
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=sxt(n-l,l-a=0.99)
where
s = standard deviation of replicate analyses
t («-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.
9.3 ONGOING QC REQUIREMENTS - This section summarizes ongoing QC criteria
that must be followed when processing and analyzing field samples.
9.3.1 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 inter-
fere with the measurement of method analytes must be below 1/3 of the MRL. If
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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.
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. 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 SURROGATE RECOVERY - The SUR standard is fortified into all samples,
CCCs, LRBs, LFBs, LFSMs, LFSMDs, and FD 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 the recovery
(%R) for the SUR using the following equation
( A\
%/?= — xlOO
UJ
where
A = measured SUR concentration for the QC or Field Sample
B = fortified concentration of the SUR.
9.3.4.1 SUR recovery in extracts must be in the range of 60-130%. SUR recovery in
CCCs must be 70-130%. When SUR recovery does not meet these criteria,
check 1) calculations to locate possible errors, 2) standard solutions for
degradation, 3) contamination, and 4) instrument performance. Correct the
problem and reanalyze the extract.
9.3.4.2 If the extract reanalysis meets the SUR recovery criterion, report only data
for the reanalyzed extract.
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9.3.4.3 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, extraction of the
sample should be repeated provided the sample is still within the holding
time. If the re-extracted sample also fails the recovery criterion, 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, collect a new sample
and re-analyze.
9.3.5 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 Field Duplicate (FD) (Sect. 9.3.6);
however, infrequent occurrence of method analytes would hinder this assessment.
If the occurrence of method analytes in samples is infrequent, or if historical
trends are unavailable, a second LFSM, or LFSMD, must be prepared, extracted,
and analyzed from a duplicate of the Field Sample. Extraction batches that
contain LFSMDs will not require extraction of a FD. If a variety of different
sample matrices are analyzed regularly, for example, drinking water from ground
water and surface water sources, method performance should be established for
each. Over time, LFSM data should be documented by the laboratory for all
routine sample sources.
9.3.5. 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. 7.2.2.2). 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.
9.3.5.2 Calculate percent recovery (%R) for each analyte using the equation
C
where
A = measured concentration in the fortified sample
B = measured concentration in the unfortified sample
C = fortification concentration.
9.3.5.3 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,
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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 the results are suspect due to matrix effects.
9.3.6 FIELD DUPLICATE OR LABORATORY FORTIFIED SAMPLE MATRIX
DUPLICATE (FD or LFSMD) - Within each extraction batch (not to exceed 20
field samples, Sect. 3.6), a minimum of one FD or LFSMD must be analyzed.
Duplicates check the precision associated with sample collection, preservation,
storage, and laboratory procedures. If method analytes are not routinely observed
in field samples, an LFSMD should be analyzed rather than an FD.
9.3.6. 1 Calculate relative percent difference (RPD) for duplicate measurements
(FD1 and FD2) using the equation
(FD\ + FD2)/2
9.3.6.2 RPDs for FDs should be < 30%. Greater variability may be observed when
the matrix is fortified at analyte concentrations at or near the MRL (within a
factor of two times the MRL concentration). At these concentrations, FDs
should have RPDs that are < 50%. 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 the results are suspect due to matrix effects.
9.3.6.3 If an LFSMD is analyzed instead of a FD, calculate the relative percent
difference (RPD) for duplicate LFSMs (LFSM and LFSMD) using the
equation
\LFSM -LFSMD\
RPD =-^ - J— xlOO
(LFSM + LFSMD)/ 2
9.3.6.4 RPDs for duplicate LFSMs should be < 30% for samples fortified at or
above their native concentration. Greater variability may be observed when
the matrix is fortified at analyte concentrations at or near the MRL (within a
factor of two times the MRL concentration). LFSMs fortified at these
concentrations should have RPDs that are < 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 the results are suspect due to matrix effects.
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9.3.7 QUALITY CONTROL SAMPLES (QCS) - As part of the IDC (Sect. 9.2), each
time a new Analyte PDS (Sect. 7.2.2.2) 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.
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. 3.15; [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. 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.
CAUTION: During multi-laboratory verification studies, 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 the 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).
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10.2.1.3 Optimize the product ion (Sect. 3.17) for each 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. 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.
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. 3.15) 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, although these will be instrument dependent.
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 7.2.3. 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 external 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.
NOTE: External calibration is used due to the lack of appropriate internal
standards. More frequent calibration may be necessary with external
standard calibration.
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
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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.
10.3 CONTINUING CALIBRATION CHECK (CCC) - Minimum daily calibration
verification is as follows. 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, FDs and LFSMDs 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 this 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 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 should be taken
(Sect. 10.3.3) 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.
10.3.3 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, etc., requires recalibration (Sect 10.2) and verification of sensitivity by
analyzing a CCC at or below the MRL (Sect 10.3).
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11. PROCEDURE
11.1 This procedure may be performed manually or in an automated mode using a robotic
or automatic sample preparation device. Data presented in Tables 5-10 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 the
manufacturer's operating instructions, but all extraction and elution steps must be the
same as in the manual procedure. Extraction and/or elution steps may not be changed
or omitted to accommodate the 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
meetLRB 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 refurbished for reuse in
subsequent analyses.
11.2 SAMPLE PREPARATION
11.2.1 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 extraction, 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. Before extraction, mark the level of
the sample on the outside of the sample bottle for later sample volume
determination (Sect. 11.6). If using weight to determine volume, weigh the bottle
with collected sample before extraction.
NOTE: The solvent volumes in Sections 11.3 and 11.4 were optimized for 500-
mL sample bottles. The use of larger sample bottles for the QC Samples
is not recommended as this may adversely affect analyte recoveries.
NOTE: Section 8 allows smaller sample sizes to be used provided the MRL can
be met. The same sample size must be used for the LFB, LFB, FD,
LFSM and LFSMD as for the Field Sample and all QC in Section 9 must
be met for the smaller sample size.
11.2.2 Add an aliquot of the SUR PDS (Sect. 7.2.1.1) to each sample to be extracted, cap
and invert to mix. During method development, a 20-|oL aliquot of the 6.49 ng/|jL
SUR PDS (Sect. 7.2.1.1) was added to 500 mL for a final concentration of
259.6 ng/L in the aqueous sample.
11.2.3 In addition to SUR and preservatives, if the sample is an LFB, FD, LFSM, or
LFSMD, add the necessary amount of Analyte PDS (Sect. 7.2.2.2). Cap and invert
each sample to mix.
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11.3 INTRACELLULAR TOXIN RELEASE PROCEDURE
11.3.1 Filter the 500-mL water sample using a Nuclepore filter (Sect. 6.3) with the shiny
side up; collect the filtrate into a 500 mL amber glass bottle (Sect. 6.2.1) for
extraction in Sect. 11.4.
11.3.2 Rinse sample bottle with 5 mL of 10% reagent water in methanol. Pour bottle
rinsate into filter apparatus and combine the rinsate with the filtered water sample
in Sect. 11.3.1.
11.3.3 Rinse the sides of the funnel with another 2.5 mL of 10% reagent water in
methanol and combine with the filtered water sample in Sect. 11.3.1.
11.3.4 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 small enough to fit into a glass test tube (Sect.
6.4). Push the filter to the bottom of the glass test tube using a glass pipet.
11.3.5 Add 2 mL of 20% reagent water in methanol to the test tube containing the filter
(ensure that the filter is covered with liquid) and manually swirl the tube gently a
few times.
11.3.6 Place the test tube containing the 2 mL filter solution and the filter in a freezer
at -20 °C for 1 to 16 hours. Do not exceed 16 hours in the freezer. If the filter is
kept frozen for more than 2 hours, the 500-mL aqueous filtrate from Section
11.3.1 must be kept refrigerated at <6 °C until completion of the toxin release
procedure.
11.3.7 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
500 mL water sample collected in Section 11.3.1.
11.3.8 Rinse the filter and test tube by adding another 2 mL of 20% reagent water in
methanol 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 500 mL water sample
collected in Section 11.3.1.
11.3.9 Rinse the filter a second time by adding another 1 mL of 20% reagent water in
methanol 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 500 mL water sample
collected in Section 11.3.1. Swirl the 500 mL sample several times to homogenize
the sample.
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11.4 CARTRIDGE SPE PROCEDURE
11.4.1 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
sample transfer tubes (Sect. 6.9.3), turn on the vacuum, and begin adding filtered
sample (containing the released intracellular toxins) to the cartridge.
11.4.2 SAMPLE EXTRACTON - 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.
11.4.3 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 and 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).
11.4.4 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 methanol
containing 10% reagent water and elute the analytes from the cartridges by
pulling the 5 mL of methanol (used to rinse the bottles) through the sample
transfer tubes and the cartridges. 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 methanol containing 10% reagent water.
11.5 EXTRACT CONCENTRATION - Concentrate the extract to dryness under a gentle
stream of nitrogen in a heated water bath (60 °C). Add 1 mL of methanol containing
10% reagent water to the collection vial and vortex. Transfer an aliquot to an
autosampler vial.
11.6 SAMPLE VOLUME DETERMINATION - If the level of the sample was marked on
the sample bottle, use a graduated cylinder to measure the volume of water required to
fill the original sample bottle to the mark made prior to extraction. Determine to the
nearest 10 mL. If using weight to determine volume, weigh the empty bottle to the
nearest 10 g and determine the sample weight by subtraction of the empty bottle
weight from the original sample weight (Sect. 11.2.1). Assume a sample density of
1.0 g/mL. In either case, the sample volume will be used in the final calculations of the
analyte concentration (Sect. 12.2).
544-25
-------
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.
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 methanol containing 10% water. Re-inject the diluted extract.
Incorporate the dilution factor into the final concentration calculations.
Acceptable SUR performance (Sect. 9.3.4) should be determined from the
undiluted sample extract. The resulting data should be documented as a dilution
and MRLs should be adjusted accordingly.
544-26
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12 DATA ANALYSIS AND CALCULATION
12.1 Complete chromatographic resolution is not necessary for accurate and precise
measurements of analyte concentrations using MS/MS. In validating this method,
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 11.6.
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. 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 three water matrices:
reagent water (Table 6); chlorinated (finished) ground water (Table 7); chlorinated
(finished) surface water (Table 8).
13.2 SAMPLE STORAGE STABILITY STUDIES - An analyte storage stability study was
conducted by fortifying the analytes into chlorinated surface 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 9.
13.3 EXTRACT STORAGE STABILITY STUDIES - Extract storage stability studies
were conducted on extracts obtained from a chlorinated surface 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 10.
13.4 SECOND LABORATORY DEMONSTRATION - Performance of this method was
demonstrated by multiple laboratories, with results similar to those reported in
Section 17. The authors wish to acknowledge the assistance of the analysts and
laboratories for their participation in the multi-laboratory verification studies: 1) Dr.
William A. Adams of CB&I Federal Services under EPA contract EP-C-12-013 and 2)
Dr. Andrew Eaton and Mr. Ali Haghani of Eurofms Eaton Analytical, Inc.
544-27
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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 that may be applicable to laboratory
operations, consult "Less is Better: Guide to Minimizing Waste in Laboratories"
available from the American Chemical Society's Department of Government Relations
and Science Policy, 1155 16th Street N.W., Washington, D.C., 20036 or on-line at
http://portal.acs.org/portal/fileFetch/CAVPCP 012290/pdf/WPCP 012290.pdf
(accessed February 2015).
15. WASTE MANAGEMENT
15.1 Analytical procedures described in this method generate relatively small amounts of
waste since only small amounts of reagents and solvents are used. The matrices of
concern are finished drinking water or source water. 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 lonization/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 lonization Mass
Spectrometry." J. Chromatogr. A, 2004, 1041, 171-180.
5. Draper, W.M., Xu, D., Perera, S.K. "Electrolyte-Induced lonization Suppression and
Microcystin Toxins: Ammonium Formate Suppresses Sodium Replacement Ions and
544-28
-------
Enhances Protiated and Ammoniated Ions for Improved Specificity in Quantitative LC-MS-
MS."Anal. Chem. 2009, 81, 4153-4160.
6. "Biosafety in Microbiological and Biomedical Laboratories", 5th edition, Appendix I—
Guidelines for Work with Toxins of Biological Origin. U.S. Department of Health and
Human Services, Public Health Service Centers for Disease Control and Prevention, National
Institutes of Health. Available at http://www.cdc.gov/biosafety/publications/bmbl5/
BMBL5_appendixI.pdf (accessed February 2015).
7. "Prudent Practices in the Laboratory: Handling and Disposal of Chemicals," National
Academies Press (1995), available at http://www.nap.edu (accessed May 2014).
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," American Chemical Society Publication,
Committee on Chemical Safety, 7th Edition. Available online at
http ://portal. acs.org/portal/PublicWeb Site/about/governance/committees/chemicalsafety/publ
ications/WPCP 012294 (accessed February 2015).
10. Winslow, S. D. , Pepich, S. D. , Bassett, M. V., Wendelken, S. C., Munch, D. I, Sinclair, J.
L. "Microbial Inhibitors for U.S. EPA Drinking Water Methods for the Determination of
Organic Compounds." Environ. Sci. Techno!., 2001, 35, 4103-4110.
11. Hyenstrand, P., Metcalf, J. S., Beattie, K. A., Codd, G. A. "Effects of Adsorption to Plastics
and Solvent Conditions in the Analysis of the Cyanobacterial Toxin Microcystin-LR by High
Perormance Liquid Chromatography." Wat. Res., 2001, 35, 3508-3511.
544-29
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17. TABLES, DIAGRAMS, FLOWCHARTS AND VALIDATION DATA
TABLE 1. LC METHOD CONDITIONS
Time (min)
Initial
2.0
16
16.1
22.0
22.1
26.0
% 20 mM
Ammonium Formate
90
90
20
10
10
90
90
% Methanol
10
10
80
90
90
10
10
Phenomenex Kinetex Cs column, 2.6 jam, 2.1 x 100 mm
Flow rate of 0.3 mL/min
10 |aL partial loop injection into a 20 jiL loop
TABLE 2. ESI-MS/MS METHOD CONDITIONS
ESI Conditions
Polarity
Capillary needle voltage
Cone gas flow
Nitrogen desolvation gas
Desolvation gas temp.
Positive ion
4kV
25 L/hr
lOOOL/hr
350 °C
544-30
-------
TABLE 3. METHOD ANALYTE SOURCE AND RETENTION TIMES (RTs)
Analyte
MC-YR
Nodularin-R
MC-RR
MC-LR
MC-LA
MC-LY
MC-LF
C2D5-MC-LR (SUR)
Method Analyte Source8
GreenWater Laboratories
National Research Council Canada
National Research Council Canada
GreenWater Laboratories
GreenWater Laboratories
Enzo Life Sciences
Enzo Life Sciences
Synthesized under contractb
RT
(min)
11.07
11.08
11.33
11.49
12.41
12.51
14.05
14.3
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.
b Synthesized by Dr. Judy Westrick, Wayne State University, EPA Contract
#EP13C000079.
544-31
-------
TABLE 4. MS/MS METHOD CONDITIONS3
Segment11
1
1
1
1
2
2
3
3
Analyte
MC-YR
Nodularin-R
MC-RR
MC-LR
MC-LA
MC-LY
MC-LF
C2D5-MC-LR (SUR)
Precursor Ion c
(m/z)
523.4 TM+2H12+
825.4 TM+H1+
519.9 TM+2H12+
995.5 TM+H1+
910.5 TM+H1+
1002.5 TM+H1+
986.5 TM+H1+
1028.6 TM+H1+
Product
Ionc'd (m/z)
134.9
134.9
134.9
134.9
776.4
134.9
134.9
134.9
Cone
Voltage (v)
20
45
35
60
40
40
40
55
Collision
Energy6 (v)
15
55
30
65
20
60
60
60
a An LC/MS/MS chromatogram of the analytes is shown in Figure 2.
Segments are time durations in which single or multiple scan events occur.
c 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.
d Ions used for quantitation purposes.
e Argon used as collision gas at a flow rate of 0.3 mL/min.
TABLE 5. DLs AND LCMRLs IN REAGENT WATER
Analyte
MC-YR
Nodularin-R
MC-RR
MC-LR
MC-LA
MC-LY
MC-LF
Fortified
Cone. (ng/L)a
8.0
3.9
3.8
8.0
20
8.0
8.0
DLb(ng/L)
4.6
1.8
1.2
4.3
4.0
2.2
3.4
LCMRLC
(ng/L)
22
7.3
5.6
6.6
2.9
4.6
3.5
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.
0 LCMRLs were calculated according to the procedure in reference 1.
544-32
-------
ABLE 6. PRECISION AND ACCURACY DAT^
FORTIFIED IN REAGENT WATER (
Analyte
MC-YR
Nodularin-R
MC-RR
MC-LR
MC-LA
MC-LY
MC-LF
C2D5-MC-LR (SUR)
Fortified
Cone. (ng/L)
400.0
195.7
187.5
400.0
1000
400.0
400.0
259.6
Mean %
Recovery
87.4
89.5
95.8
85.5
81.8
83.8
101
83.8
V FOR METHOD ANALYTES
n=4)
% RSD
5.3
1.8
3.1
4.7
2.7
3.5
3.4
4.9
Fortified
Cone. (ng/L)
40.0
19.6
18.8
40.0
100.0
40.0
40.0
259.6
Mean %
Recovery
87.5
91.9
97.3
87.5
94.7
92.8
85.0
87.2
% RSD
7.1
3.1
9.5
5.6
6.0
6.4
4.1
2.9
TABLE 7. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
FORTIFIED IN FINISHED DRINKING WATER FROM A GROUND WATER
SOURCE8 (n=4)
Analyte
MC-YR
Nodularin-R
MC-RR
MC-LR
MC-LA
MC-LY
MC-LF
C2D5-MC-LR (SUR)
Fortified
Cone. (ng/L)
400.0
195.7
187.5
400.0
1000
400.0
400.0
259.6
Mean %
Recovery
93.2
93.5
92.1
90.1
87.0
83.9
87.9
90.4
% RSD
8.0
7.7
9.8
7.4
3.8
6.8
6.1
6.0
Fortified
Cone. (ng/L)
40.0
19.6
18.8
40.0
100.0
40.0
40.0
259.6
Mean %
Recovery
114
99.5
116
99.8
99.5
97.7
92.9
94.0
% RSD
14
3.4
6.2
6.1
4.1
2.3
3.4
1.6
TOC = 0.48 mg/L and hardness = 360 mg/L as calcium carbonate.
544-33
-------
TABLE 8. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES
FORTIFIED IN FINISHED DRINKING WATER FROM A SURFACE WATER
SOURCE8 (n=4)
Analyte
MC-YR
Nodularin-R
MC-RR
MC-LR
MC-LA
MC-LY
MC-LF
C2D5-MC-LR (SUR)
Fortified
Cone. (ng/L)
400.0
195.7
187.5
400.0
1000
400.0
400.0
259.6
Mean %
Recovery
88.7
97.3
100
105
92.1
94.6
92.7
92.2
% RSD
9.0
1.3
0.9
1.7
0.9
2.5
2.1
2.4
Fortified
Cone. (ng/L)
40.0
19.6
18.8
40.0
100.0
40.0
40.0
259.6
Mean %
Recovery
82.4
103
106
117
112
106
104
94.0
% RSD
11
1.9
2.9
5.0
3.2
5.9
8.7
2.9
a TOC = 2.49 mg/L and hardness =137 mg/L as calcium carbonate.
544-34
-------
TABLE 9. AQUEOUS SAMPLE HOLDING TIME DATA FOR SAMPLES OF FINISHED DRINKING WATER FROM A
SURFACE WATER SOURCE3, FORTIFIED WITH METHOD ANALYTES AND PRESERVED AND STORED
ACCORDING TO SECTION 8 (n=4)
Analyte
MC-YR
Nodularin-R
MC-RR
MC-LR
MC-LA
MC-LY
MC-LF
C2D5-MC-LR (SUR)b
Fortified
Cone.
(ng/L)
400.0
195.7
187.5
400.0
1000
400.0
400.0
259.6
DayO
Mean
%Rec
101
91.7
91.7
89.4
91.2
88.4
89.1
86.9
%
RSD
8.7
3.9
2.1
2.3
0.9
1.8
2.3
5.4
Day 7
Mean
%Rec
92.5
92.0
94.6
87.2
90.6
87.9
86.4
92.8
%
RSD
2.2
3.2
3.4
2.5
2.3
2.0
1.6
0.7
Day 14
Mean
%Rec
89.0
94.4
94.9
89.4
87.2
89.3
86.6
89.7
%
RSD
6.7
1.6
1.7
2.3
1.8
1.1
1.2
3.3
Day 21
Mean
%Rec
96.4
96.5
94.9
90.2
90.2
89.9
86.6
92.8
%
RSD
1.5
2.3
1.7
2.1
1.7
1.1
2.2
3.4
Day 28
Mean
%Rec
95.1
91.6
90.3
86.4
88.1
88.1
85.4
92.0
%
RSD
6.1
3.3
0.6
1.3
1.5
1.9
2.1
4.1
a TOC = 0.9 mg/L and hardness = 120 mg/L as calcium carbonate.
b Surrogate was not added to samples until the day of extraction.
TABLE 10. EXTRACT HOLDING TIME DATA FOR SAMPLES OF FINISHED DRINKING WATER FROM A
SURFACE WATER SOURCE, FORTIFIED WITH METHOD ANALYTES AND PRESERVED AND STORED
ACCORDING TO SECTION 8 (n=4)
Analyte
MC-YR
Nodularin-R
MC-RR
MC-LR
MC-LA
MC-LY
MC-LF
C2D5-MC-LR (SUR)
Fortified
Cone.
(ng/L)
400.0
195.7
187.5
400.0
1000
400.0
400.0
259.6
DayO
Mean
%Rec
101
91.7
91.7
89.4
91.2
88.4
89.1
86.9
%
RSD
8.7
3.9
2.1
2.3
0.9
1.8
2.3
5.4
Day 7
Mean
%Rec
98.4
93.9
98.6
91.1
93.1
92.4
92.6
90.9
%
RSD
2.4
2.4
1.7
2.4
2.5
2.5
0.9
5.2
Day 14
Mean
%Rec
90.7
94.9
97.0
94.4
90.0
94.4
89.0
89.1
%
RSD
5.3
1.8
2.2
3.8
0.5
2.2
1.8
1.4
Day 21
Mean
%Rec
91.3
95.8
92.6
90.4
91.7
91.6
90.8
90.5
%
RSD
2.8
2.4
2.9
2.7
0.7
1.8
1.9
3.2
Day 28
Mean
%Rec
95.0
95.1
92.5
90.8
92.5
93.4
91.0
93.2
%
RSD
4.9
1.3
2.1
1.1
1.9
1.6
2.5
3.6
544-35
-------
TABLE 11. INITIAL DEMONSTRATION OF CAPABILITY QUALITY CONTROL REQUIREMENTS
Method
Reference
Sect. 9.2.1
and 9.3.1
Sect. 9.2.2
Sect. 9.2.3
Sect. 9.2.4
Sect. 9.2.5
and 9.3.7
Requirement
Initial Demonstration of
Low System Background
Initial Demonstration of
Precision (IDP)
Initial Demonstration of
Accuracy (IDA)
Minimum Reporting Limit
(MRL) Confirmation
Quality Control Sample
(QCS)
Specification and Frequency
Analyze LRB prior to any other IDC steps.
Analyze four to seven replicate LFBs fortified near
the midrange calibration concentration.
Calculate average recovery for replicates used in
IDP.
Fortify, extract and analyze seven replicate LFBs
at the proposed MRL concentration. Calculate the
Mean and the Half Range (HR). Confirm that the
upper and lower limits for the Prediction Interval
of Result (Upper PIR, and Lower PIR, Sect.
9.2.4.2) meet the recovery criteria.
Analyze a standard from a second source, as
part of IDC.
Acceptance Criteria
Demonstrate that all method analytes are below 1/3 the MRL
and that possible interferences from extraction media do not
prevent the identification and quantification of method
analytes.
%RSDmustbe <30%
Mean recovery + 30% of true value
Upper PIR < 150%
Lower PIR > 50%
Results should be within 70-130% of true value.
NOTE: Table 11 is intended as an abbreviated summary of QC requirements provided as a convenience to the method user. Because the information has been
abbreviated to fit the table format, there may be issues that need additional clarification, or areas where important additional information from the method text
is needed. In all cases, the full text of the QC in Section 9 supersedes any missing or conflicting information in this table.
544-36
-------
TABLE 12. ONGOING QUALITY CONTROL REQUIREMENTS (SUMMARY)
Method
Reference
Sect. 8.4
Sect. 8.4
Sect. 9.3.1
Sect. 9.3.3
Sect. 9.3.4
Sect. 9.3.5
Sect. 9.3.6
Sect. 9.3.7
Requirement
Sample Holding Time
Extract Holding Time
Laboratory Reagent Blank
(LRB)
Laboratory Fortified Blank
(LFB)
Surrogate Standards
(SUR)
Laboratory Fortified
Sample Matrix (LFSM)
Field Duplicates (FD) or
Laboratory Fortified
Sample Matrix Duplicate
(LFSMD)
Quality Control Sample
(QCS)
Specification and Frequency
28 days with appropriate preservation and storage as
described in Sections 8.1-8.4.
28 days when stored at < -4 °C.
One LRB with each extraction batch of up to 20 field
samples.
One LFB is required for each extraction batch of up
to 20 field samples. Rotate the fortified
concentrations between low, medium, and high
amounts.
The surrogate is added to all CAL standards and
samples, including QC samples. Calculate SUR
recoveries.
Analyze one LFSM per extraction batch (20 samples
or less) fortified with method analytes at a
concentration greater than or equal to the native
concentration, if known. Calculate LFSM recoveries.
Extract and analyze at least one FD or LFSMD with
each extraction batch (20 samples or less). A LFSMD
may be substituted for a FD when the frequency of
detects are low. Calculate RPDs.
Analyze at least quarterly or when preparing new
standards, as well as during the IDC.
Acceptance Criteria
Sample results are valid only if samples are extracted within the
sample holding time.
Extract results are valid only if extracts are analyzed within the
extract holding time.
Demonstrate that all method analytes are below 1/3 the MRL, and
confirm that possible interferences do not prevent quantification of
method analytes. If targets exceed 1/3 the MRL or if interferences
are present, results for these subject analytes in the extraction batch
are invalid.
Results of LFB analyses must be 70-130% of the true value for each
method analyte for all fortified concentrations except the lowest
CAL point. Results of the LFBs corresponding to the lowest CAL
point for each method analyte must be 50-150% of the true value.
SUR recovery in extracts must be 60-130% of the true value. SUR
recovery in CCCs must be 70-130% of the true value. If a SUR fails
these criteria, report all results for sample as suspect/SUR recovery.
Recoveries at mid and high levels should be within 60-140% and
within 50-150% at the low-level fortified amount (near the MRL). If
these criteria are not met, results are labeled suspect due to matrix
effects.
See Sect. 9.3.5 and 9.3.6 for instructions on the interpretation of
LFSM and FD results.
Results should be within 70-130% of true value.
544-37
-------
TABLE 12. (Continued)
Method
Reference
Requirement
Specification and Frequency
Acceptance Criteria
Sect. 10.2
Initial Calibration
Use external calibration technique to generate a
linear or quadratic calibration curve for each
analyte. Use at least five standard concentrations.
Check the calibration curve as described in
Sect. 10.2.7.
When each CAL standard is calculated as an unknown using
the calibration curve, the analyte results must be 70-130% of
the true value for all except CAL standards < MRL, which
must be 50-150% of the true value. If this criterion is not met
reanalyze the CAL standards, restrict the range of
calibration, or select an alternate method of calibration.
Sect. 9.3.2
and Sect.
10.3
Continuing Calibration
Check (CCC)
Verify initial calibration by analyzing a low level
(at the MRL or below) CCC prior to analyzing
samples. CCCs are then injected after every 10
field samples and after the last sample, rotating
concentrations to cover the calibrated range of the
instrument.
Recovery for each SUR must be within 70-130% of the true
value in all CCCs. Each analyte fortified at a level < MRL
must calculate to be within ± 50% of the true value. The
calculated concentration of the method analytes in CCCs
fortified at all other levels must be within ± 30%.
NOTE: Table 12 is intended as an abbreviated summary of QC requirements provided as a convenience to the method user. Because the information has been
abbreviated to fit the table format, there may be issues that need additional clarification, or areas where important additional information from the method text
is needed. In all cases, the full text of the QC in Section 8-10 supersedes any missing or conflicting information in this table.
544-38
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FIGURE 1. DIAGRAM OF FILTER APPARATUS WITH PART NUMBERS (SECT 6.2)
953751-0000
953753-0000 •.:.-:;-?>
953752-5047
736400-141
410170-4534
410171-4226
02-542-4C (Fisher)
544-39
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FIGURE 2. EXAMPLE CHROMATOGRAM (OVERLAID MS/MS SEGMENTS) OF A CALIBRATION STANDARD WITH
METHOD 544 ANALYTES AT CONCENTRATION LEVELS OF 187.5-1000 ng/L.
MC-RR
NOD
MC-YR
MC-LA
MC-LR
MC-LF
MC-LY
SUR
10.50 10.75 11.00 11.25 11.50 11.75 12.00 12.25 12.50 12.75 13.00 13.25 13.50 13.75 1400 14.25 14.50 14.75 15.00
Retention Time, min
544-40
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