544-1 

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
 

 544-2 

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.

Analyte 

Chemical Abstract Services 

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 requirements 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. 
 

 544-3 

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. 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 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 oC 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-μL 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 external standard calibration. 
 

 544-4 

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 
 

 544-5 

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 
 

 544-6 

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. 
 

 544-7 

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 and Biomedical 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).
 

 544-8 

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 μm, (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-μL 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 Nvinylpyrrolidone 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). 
 

 544-9 

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-μL 

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 μm C8 solid phase particles (Phenomenex Kinetex #00D-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. 
 

 544-10 

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(hydroxymethyl)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. 
 

 544-11 

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. 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, d5 (C2D5-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/μL) – The SUR PDS was prepared by diluting 64.9 μg 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 μL of this 6.49 ng/μL 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 μg/mL) – Neat 

cyanotoxins are typically purchased in quantities of 10-500 μg. 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 μg) for a final concentration of 10-

500 μg/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 
 

 544-12 

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/μL) – 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/μL 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. 

Conc. of Analyte 

Stock (μg/mL) 

Vol. of Analyte 

Stock (μL) 

Final Vol. of 

Analyte PDS 

(mL) 

Final Conc. of PDS 

(ng/μL) 

Microcystin-LR 500 40.0 

10 mL 

2.0 

Microcystin-RR 10.3 910 0.94 

Microcystin-YR 100 200 2.0 

Microcystin-LY 100 200 2.0 

Microcystin-LF 100 200 2.0 

Nodularin-R 10.3 950 0.98 

Microcystin-LA 100 500 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 μg/L, except 

for MC-RR (4.7-187.5 μg/L), nodularin-R (4.9-195.7 μg/L) and MC-LA (25-1000 

μg/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 μg/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.

 
 

 544-13 

8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE 

8.1 SAMPLE BOTTLE PREPARATION 

8.1.1 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). 

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

2-Chloroacetamide 2 g/L antimicrobial 

Ascorbic acid 100 mg/L dechlorinating agent 

Ethylenediaminetetraacetic acid 

trisodium salt 0.35 g/L 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. 
 

 544-14 

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. 
 

 544-15 

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 + HRPIR) meet the upper and lower recovery limits as shown 

below 

 The Upper PIR Limit must be ≤ 150% recovery. 

100% 150% 

Fortified Concentrat ion

Mean HRPIR

 The Lower PIR Limit must be  50% recovery. 

100%  50% 

Fortified Concentrat ion

Mean HRPIR

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. 

HRPIR = 3.963s 
 

 544-16 

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 

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 

 

 

 where 

 s = standard deviation of replicate analyses 

 t (n-1, 1-α=0.99) = 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 interfere with the measurement of method analytes must be below 1/3 of the MRL. If 

( 1,1 0.99)  n 

DL s t
 

 544-17 

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 

 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. 

% 100 

 



  B

A R
 

 544-18 

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 

 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, 

  % 100   C

A B R
 

 544-19 

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 

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 

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. 

  100

1 2 / 2

1 2





  FD FD

FD FD

RPD

  100

/ 2





  LFSM LFSMDLFSM LFSMD

RPD
 

 544-20 

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/μL 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). 
 

 544-21 

10.2.1.3 Optimize the product ion (Sect. 3.17) for each analyte by infusing 

approximately 1-5 ng/μL 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 
 

 544-22 

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, nondetects 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). 
 

 544-23 

 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 

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 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-μL aliquot of the 6.49 ng/L 

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. 
 

 544-24 

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. 

 

 
 

 544-25 

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). 
 

 544-26 

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 μL 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. 

 
 

 544-27 

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 Eurofins Eaton Analytical, Inc. 

 
 

 544-28 

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/C/WPCP_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 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 
 

 544-29 

Enhances Protiated and Ammoniated Ions for Improved Specificity in Quantitative LC-MSMS.” 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/PublicWebSite/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. 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. 

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. 
 

 544-30 

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 20 80 

16.1 10 90 

22.0 10 90 

22.1 90 10 

26.0 90 10 

Phenomenex Kinetex C8 column, 2.6 μm, 2.1 x 100 mm 

Flow rate of 0.3 mL/min 

10 μL partial loop injection into a 20 μL loop 

TABLE 2. ESI-MS/MS METHOD CONDITIONS

ESI Conditions 

Polarity Positive ion 

Capillary needle voltage 4 kV 

Cone gas flow 25 L/hr 

Nitrogen desolvation gas 1000 L/hr 

Desolvation gas temp. 350 C
 

 544-31 

TABLE 3. METHOD ANALYTE SOURCE AND RETENTION TIMES (RTs)

Analyte Method Analyte Sourcea RT (min) 

MC-YR GreenWater Laboratories 11.07 

Nodularin-R National Research Council Canada 11.08 

MC-RR National Research Council Canada 11.33 

MC-LR GreenWater Laboratories 11.49 

MC-LA GreenWater Laboratories 12.41 

MC-LY Enzo Life Sciences 12.51 

MC-LF Enzo Life Sciences 14.05 

C2D5-MC-LR (SUR) Synthesized under contractb 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. 
 

 544-32 

TABLE 4. MS/MS METHOD CONDITIONSa

Segmentb Analyte 

Precursor Ion c

(m/z)

Product 

Ionc,d (m/z)

Cone 

Voltage (v) 

Collision 

Energye (v)

1 MC-YR 523.4 [M+2H]

2+ 134.9 20 15 

1 Nodularin-R 825.4 [M+H]

+ 134.9 45 55 

1 MC-RR 519.9 [M+2H]

2+ 134.9 35 30 

1 MC-LR 995.5 [M+H]

+ 134.9 60 65 

2 MC-LA 910.5 [M+H]

+ 776.4 40 20 

2 MC-LY 1002.5 [M+H]

+ 134.9 40 60 

3 MC-LF 986.5 [M+H]

+ 134.9 40 60 

3 C2D5-MC-LR (SUR) 1028.6 [M+H]

+ 134.9 55 60 a

 An LC/MS/MS chromatogram of the analytes is shown in Figure 2. 

b 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 

Fortified 

Conc. (ng/L)a DLb (ng/L)

LCMRLc

(ng/L) 

MC-YR 8.0 4.6 22 

Nodularin-R 3.9 1.8 7.3 

MC-RR 3.8 1.2 5.6 

MC-LR 8.0 4.3 6.6 

MC-LA 20 4.0 2.9 

MC-LY 8.0 2.2 4.6 

MC-LF 8.0 3.4 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. 

c

 LCMRLs were calculated according to the procedure in reference 1. 
 

 544-33 

TABLE 6. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES 

FORTIFIED IN REAGENT WATER (n=4)

Analyte 

Fortified 

Conc. (ng/L)

Mean % 

Recovery % RSD 

Fortified 

Conc. (ng/L) 

Mean % 

Recovery % RSD 

MC-YR 400.0 87.4 5.3 40.0 87.5 7.1 

Nodularin-R 195.7 89.5 1.8 19.6 91.9 3.1 

MC-RR 187.5 95.8 3.1 18.8 97.3 9.5 

MC-LR 400.0 85.5 4.7 40.0 87.5 5.6 

MC-LA 1000 81.8 2.7 100.0 94.7 6.0 

MC-LY 400.0 83.8 3.5 40.0 92.8 6.4 

MC-LF 400.0 101 3.4 40.0 85.0 4.1 

C2D5-MC-LR (SUR) 259.6 83.8 4.9 259.6 87.2 2.9 

TABLE 7. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES 

FORTIFIED IN FINISHED DRINKING WATER FROM A GROUND WATER 

SOURCEa (n=4)

Analyte 

Fortified 

Conc. (ng/L)

Mean % 

Recovery % RSD 

Fortified 

Conc. (ng/L) 

Mean % 

Recovery % RSD 

MC-YR 400.0 93.2 8.0 40.0 114 14 

Nodularin-R 195.7 93.5 7.7 19.6 99.5 3.4 

MC-RR 187.5 92.1 9.8 18.8 116 6.2 

MC-LR 400.0 90.1 7.4 40.0 99.8 6.1 

MC-LA 1000 87.0 3.8 100.0 99.5 4.1 

MC-LY 400.0 83.9 6.8 40.0 97.7 2.3 

MC-LF 400.0 87.9 6.1 40.0 92.9 3.4 

C2D5-MC-LR (SUR) 259.6 90.4 6.0 259.6 94.0 1.6 a

 TOC = 0.48 mg/L and hardness = 360 mg/L as calcium carbonate. 

 
 

 544-34 

TABLE 8. PRECISION AND ACCURACY DATA FOR METHOD ANALYTES 

FORTIFIED IN FINISHED DRINKING WATER FROM A SURFACE WATER 

SOURCEa

 (n=4)

Analyte 

Fortified 

Conc. (ng/L)

Mean % 

Recovery % RSD 

Fortified 

Conc. (ng/L) 

Mean % 

Recovery % RSD 

MC-YR 400.0 88.7 9.0 40.0 82.4 11 

Nodularin-R 195.7 97.3 1.3 19.6 103 1.9 

MC-RR 187.5 100 0.9 18.8 106 2.9 

MC-LR 400.0 105 1.7 40.0 117 5.0 

MC-LA 1000 92.1 0.9 100.0 112 3.2 

MC-LY 400.0 94.6 2.5 40.0 106 5.9 

MC-LF 400.0 92.7 2.1 40.0 104 8.7 

C2D5-MC-LR (SUR) 259.6 92.2 2.4 259.6 94.0 2.9 a

 TOC = 2.49 mg/L and hardness =137 mg/L as calcium carbonate. 
 544-35 

TABLE 9. AQUEOUS SAMPLE HOLDING TIME DATA FOR SAMPLES OF FINISHED DRINKIN

SURFACE WATER SOURCEa, FORTIFIED WITH METHOD ANALYTES AND PRESEACCORDING TO SECTION 8 (n=4)

Fortified 

Conc. 

(ng/L) 

Day 0 Day 7 Day 14 Day 21 

Analyte Mean 

%Rec 

% 

RSD 

Mean 

%Rec 

% 

RSD 

Mean 

%Rec 

% 

RSD 

Mean 

%Rec 

% 

RSD

MC-YR 400.0 101 8.7 92.5 2.2 89.0 6.7 96.4 1.5 Nodularin-R 195.7 91.7 3.9 92.0 3.2 94.4 1.6 96.5 2.3 MC-RR 187.5 91.7 2.1 94.6 3.4 94.9 1.7 94.9 1.7 MC-LR 400.0 89.4 2.3 87.2 2.5 89.4 2.3 90.2 2.1 MC-LA 1000 91.2 0.9 90.6 2.3 87.2 1.8 90.2 1.7 MC-LY 400.0 88.4 1.8 87.9 2.0 89.3 1.1 89.9 1.1 MC-LF 400.0 89.1 2.3 86.4 1.6 86.6 1.2 86.6 2.2 C2D5-MC-LR (SUR)b 259.6 86.9 5.4 92.8 0.7 89.7 3.3 92.8 3.4 

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 WATE

SURFACE WATER SOURCE, FORTIFIED WITH METHOD ANALYTES AND PRESEACCORDING TO SECTION 8 (n=4)

Fortified 

Conc. 

(ng/L) 

Day 0 Day 7 Day 14 Day 21 

Analyte Mean 

%Rec 

 % 

RSD 

Mean 

%Rec 

 % 

RSD 

Mean 

%Rec 

% 

RSD

Mean 

%Rec

% 

RSD 

MC-YR 400.0 101 8.7 98.4 2.4 90.7 5.3 91.3 2.8 Nodularin-R 195.7 91.7 3.9 93.9 2.4 94.9 1.8 95.8 2.4 MC-RR 187.5 91.7 2.1 98.6 1.7 97.0 2.2 92.6 2.9 MC-LR 400.0 89.4 2.3 91.1 2.4 94.4 3.8 90.4 2.7 MC-LA 1000 91.2 0.9 93.1 2.5 90.0 0.5 91.7 0.7 MC-LY 400.0 88.4 1.8 92.4 2.5 94.4 2.2 91.6 1.8 MC-LF 400.0 89.1 2.3 92.6 0.9 89.0 1.8 90.8 1.9 C2D5-MC-LR (SUR) 259.6 86.9 5.4 90.9 5.2 89.1 1.4 90.5 3.2 
 544-36 

TABLE 11. INITIAL DEMONSTRATION OF CAPABILITY QUALITY CONTROL REQUIREMENTS 

Method 

Reference Requirement Specification and Frequency Acceptance C

Sect. 9.2.1 

and 9.3.1 

Initial Demonstration of 

Low System Background Analyze LRB prior to any other IDC steps. 

Demonstrate that all method analy

and that possible interferences fro

prevent the identification and q

analyte

Sect. 9.2.2 Initial Demonstration of Precision (IDP) Analyze four to seven replicate LFBs fortified near the midrange calibration concentration. %RSD must bSect. 9.2.3 Initial Demonstration of Accuracy (IDA) Calculate average recovery for replicates used in IDP. Mean recovery  30% Sect. 9.2.4 Minimum Reporting Limit (MRL) Confirmation 

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. 

Upper PIR  

Lower PIR  

Sect. 9.2.5 

and 9.3.7 

Quality Control Sample 

(QCS) 

Analyze a standard from a second source, as 

part of IDC. Results should be within 70

NOTE: Table 11 is intended as an abbreviated summary of QC requirements provided as a convenience to the method user. Because the i

abbreviated to fit the table format, there may be issues that need additional clarification, or areas where important additional infor

is needed. In all cases, the full text of the QC in Section 9 supersedes any missing or conflicting information in this table.
 544-37 

TABLE 12. ONGOING QUALITY CONTROL REQUIREMENTS (SUMMARY) 

Method 

Reference Requirement Specification and Frequency Acceptance 

Sect. 8.4 Sample Holding Time 28 days with appropriate preservation and storage as described in Sections 8.1-8.4. Sample results are valid only if s

sample hold

Sect. 8.4 Extract Holding Time 28 days when stored at ≤ -4 C. Extract results are valid only if e

extract hold

Sect. 9.3.1 Laboratory Reagent Blank (LRB) One LRB with each extraction batch of up to 20 field samples. 

Demonstrate that all method analy

confirm that possible interferences 

method analytes. If targets exceed 

are present, results for these subjec

are inv

Sect. 9.3.3 Laboratory Fortified Blank (LFB) 

One LFB is required for each extraction batch of up 

to 20 field samples. Rotate the fortified 

concentrations between low, medium, and high 

amounts. 

Results of LFB analyses must be 70

method analyte for all fortified co

CAL point. Results of the LFBs c

point for each method analyte mu

Sect. 9.3.4 Surrogate Standards (SUR) The surrogate is added to all CAL standards and samples, including QC samples. Calculate SUR recoveries. SUR recovery in extracts must be 

recovery in CCCs must be 70-130% 

these criteria, report all results for 

Sect. 9.3.5 Laboratory Fortified Sample Matrix (LFSM) 

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. 

Recoveries at mid and high levels 

within 50-150% at the low-level fo

these criteria are not met, results a

effec

Sect. 9.3.6 

Field Duplicates (FD) or 

Laboratory Fortified 

Sample Matrix Duplicate 

(LFSMD) 

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. 

See Sect. 9.3.5 and 9.3.6 for instr

LFSM and F

Sect. 9.3.7 Quality Control Sample (QCS) Analyze at least quarterly or when preparing new standards, as well as during the IDC. Results should be within 
 544-38 

TABLE 12. (Continued) 

Method Reference Requirement Specification and Frequency Acceptance C

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 calcu

the calibration curve, the analyte r

the true value for all except CAL 

must be 50-150% of the true value

reanalyze the CAL standard

calibration, or select an alterna

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 w

value in all CCCs. Each analyte 

must calculate to be within ± 50

calculated concentration of the m

fortified at all other levels m

NOTE: Table 12 is intended as an abbreviated summary of QC requirements provided as a convenience to the method user. Because the i

abbreviated to fit the table format, there may be issues that need additional clarification, or areas where important additional infor

is needed. In all cases, the full text of the QC in Section 8-10 supersedes any missing or conflicting information in this table. 
 544-39 

FIGURE 1. DIAGRAM OF FILTER APPARATUS WITH PART NUMBERS (SECT 6.2) 

410171-4226 

02-542-4C (Fisher)

953751-0000 

953753-0000 

953752-5047 

410170-4534 

736400-1413
 544-40 

FIGURE 2. EXAMPLE CHROMATOGRAM (OVERLAID MS/MS SEGMENTS) OF A CALIBRATION ST

METHOD 544 ANALYTES AT CONCENTRATION LEVELS OF 187.5-1000 ng/L. 

Retention Time, min

MC-YR

NOD

MC-RR

MC-LR

MC-LA

MC-LY

MC-LF

SUR