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.
                                 544-12

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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.
                                        544-13

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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.
                                        544-14

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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.

                                  544-15

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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

                                     544-16

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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.
                                  544-17

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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,

                                   544-18

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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.
                                  544-19

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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).
                                        544-20

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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

                                  544-21

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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).
                                     544-22

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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.
                                        544-23

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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.
                                    544-24

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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

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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

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   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

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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

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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

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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

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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

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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

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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

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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

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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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