544

- 1 EPA Document #: EPA/600/R-14/474

METHOD 544. DETERMINATION OF MICROCYSTINS AND NODULARIN IN

DRINKING WATER BY SOLID PHASE EXTRACTION AND LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY (LC/MS/MS)

Version 1.0 February 2015

J.A. Shoemaker US EPA, Offi ce of Research and Development, National Exposure Research Laboratory

D.R. Tettenhorst US EPA, Office of Rese arch and Development, National Exposure Research Laboratory

A. de la Cruz US EPA, Office of Re search and Development, National Exposure Research Laboratory

NATIONAL EXPOSURE RESEARCH LABORATORY

OFFICE OF RESEARCH AND DEVELOPMENT

U. S. ENVIRONMENTAL PROTECTION AGENCY CINCINNATI, OHIO 45268544

- 2 METHOD 544

DETERMINATION OF MICROCYSTINS AND NODULARIN IN DRINKING WATER

BY SOLID PHASE EXTRACTION AND LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY (LC/MS/MS)

1. SCOPE AND APPLICATION

1.1 This is a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for determination of microcystins and nodularin (combined intra cellular and extracellular) in drinking water. Accuracy and precision da ta have been generated in reagent water, and finished ground and surface waters for compounds listed in the table below.

Analyte Chemical Abstract Services Registry Number (CASRN)

microcystin-LA (MC-LA) 96180-79-9

microcystin-LF (MC-LF) 154037-70-4

microcystin-LR (MC-LR) 101043-37-2

microcystin-LY (MC-LY) 123304-10-9

microcystin-RR (MC-RR) 111755-37-4

microcystin-YR (MC-YR) 101064-48-6

nodularin-R (NOD) 118399-22-7

1.2. The Minimum Reporting Level (MRL) is the lowest analyte concentration that meets Data Quality Objectives (DQOs) that are deve loped based on the intended use of this method. The single laboratory lowest concentr ation 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 list ed in Table 5. The procedure used to

determine the LCMRL is described elsewhere. 1

1.3. Laboratories using this method will not be re quired to determine the LCMRL for this method, but will need to demonstrate that their laboratory MR L meets the requirements described in Section 9.2.4.

1.4. Determining the Detection Limit (DL) for an alytes in this method is optional (Sect. 9.2.6). Detection limit is defined as the sta tistically calculated minimum concentration

that can be measured with 99% confidence th at the reported value is greater than zero. 2
The DL is compound dependent and is depe ndent on extraction efficiency, sample matrix, fortification concentr ation, and instrument performance. DLs for analytes in this method range from 1.2-4.6 ng/L, and are listed in Table 5.544

- 3 1.5. This method is intended for use by analys ts 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 colum n, 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 preser vation (Sect. 8), sample extraction steps (Sect. 11), or to quality cont rol requirements (Sect. 9). Method modifications should be considered only to improve method perfor mance. Modifications that are introduced

in the interest of re ducing cost or sample processing time, but result in poorer method performance, should not be used. Anal ytes must be adequately resolved chromatographically in order to permit th e mass spectrometer to dwell on a minimum number of compounds eluting within a rete ntion 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 wher e method modifications are proposed, the analyst must perform the procedures outlined in the initial demons tration of capability (IDC, Sect. 9.2), verify that all Quality C ontrol (QC) acceptance cr iteria (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 pr ovide detailed information of specific portions of the method that may be modifi ed. If there is any perceived conflict between the general method flexibility st atement in Section 1.6 and specific information in Sections 4-12, S ections 4-12 supe rsede Section 1.6.

2. SUMMARY OF METHOD

A 500-mL water sample (fortified with a surrogat e) is filtered and bot h the filtrate and the filter are collected. The filter is placed in a solution of methanol containing 20% reagent

water and held for at least one hour at -20 oC to release the intr acellular 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 intracellula r toxin solution) is passed through a solid phase extraction (SPE) car tridge to extract the method analytes and surrogate. Analytes are eluted from the so lid phase with a small amount of methanol containing 10% reagent water. The extract is concentrated to dryness by evaporation with nitrogen in a heated water bath, and then adjusted to a 1-mL volume with methanol containing 10% reagent water. A 10-μL injectio n is made into an LC equipped with a C 8 column that is interfaced to an MS/MS. Anal ytes are separated and identified by comparing the acquired mass spectra and retention times to reference spectra and retention times for calibration standards acquired under identical LC/MS/MS conditions. The concentration of each analyte is determined by external standard calibration.544

- 4 3. DEFINITIONS

3.1. ANALYSIS BATCH – A set of samples that is analyzed on the same instrument during a 24-hour period, including no more than 20 field samples, that begins and ends with the analysis of the a ppropriate Continuing Calibrati on Check (CCC) standards. Additional CCCs may be required depending on th e 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 stoc k standard solution, and the surrogate. The CAL solutions are used to calibrate the instrument respons e with respect to an alyte concentration.

3.3. COLLISIONALLY ACTIVATED DISSOCI ATION (CAD) – The process of converting the translational energy of th e precursor ion into internal energy by collisions with neutral gas molecules to bring about dissociati on into product ions.

3.4. CONTINUING CALIBRATION CHECK (CCC) – A calibration standard containing the method analytes, and surrogate(s). The CCC is analyzed periodi cally to verify the accuracy of the existing calibration for those analytes.

3.5. DETECTION LIMIT (DL) – The minimum con centration of an analyte that can be identified, measured, and repor ted 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 fiel d samples (not including QC samples) extracted together by the same person duri ng a work day using the same lot of SPE devices, solvents, surrogate, and fortifyi ng solutions. Required QC samples include Laboratory Reagent Blank, Laboratory Fortif ied Blank, Laboratory Fortified Sample Matrix, and either a Field Duplicate or Labor atory Fortified Sample Matrix Duplicate.

3.7. FIELD DUPLICATES (FD1 and FD2) – Two se parate 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 collecti on, 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 th e method analytes and all the preservation compounds are added in the la boratory. The LFB is analy zed exactly like a sample, and its purpose is to determine whether th e methodology is in cont rol, and whether the laboratory is capable of making accu rate and precise measurements.

3.9. LABORATORY FORTIFIED SAMPLE MATR IX (LFSM) – A preserved field sample to which known quantities of the me thod analytes are added in the laboratory. The LFSM is processed and analyzed exact ly like a sample, and its purpose is to544

- 5 determine whether the sample matrix contri butes 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 pr epare the LFSM. The LFSMD is fortified, extracted, and analyzed identically to the LFSM. The LFSMD is used instead of the Field Duplicate to assess me thod precision when the occurre nce 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 pr eservatives, and surrogates that are used in the analysis batch. The LRB is used to determine if method analytes or other interferences are present in th e 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, heal th 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 valu e for a method analyte in a sample following analysis.

This defined concentration can be no lowe r 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 ve rifying a laboratory’s MRL is provided in

Section 9.2.4.

3.15. PRECURSOR ION – For the purpose of th is 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 fragm ented by CAD to produce distinctive product

ions of smaller m/z ratio.

3.16. PRIMARY DILUTION STANDARD (PDS) SO LUTION – A solution containing the analytes prepared in the labor atory from stock standard solu tions and diluted as needed to prepare calibration solutions a nd other needed analyte solutions.

3.17. PRODUCT ION – For the purpose of this met hod, a product ion is one of the fragment ions produced in MS/MS by C AD of the precursor ion.

3.18. QUALITY CONTROL SAMPLE (QCS) – A solu tion of method analytes of known

concentrations that is obtained from a sour ce external to the laboratory and different544

- 6 from the source of calibration standards. Th e second source stock standard solution is used to fortify the QCS at a known con centration. The QCS is used to check calibration standard integrity.

3.19. STOCK STANDARD SOLUTION (SSS) – A con centrated solution containing one or more method analytes prepared in the labor atory using assayed reference materials or purchased from a reputable commercial source.

3.20. SURROGATE ANALYTE (SUR) – A pure chem ical 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 ot her method analytes. The purpose of the SUR is to monitor method performance with each sample.

4. INTERFERENCES

4.1. All glassware must be meticulously cleane d. 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 fu rnace 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 contam inants in solvents, reagents (including reagent water), sample bottles and caps, a nd 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 de scribed in Section 9.3.1. Subtracting blank values from sample results is not permitted.

4.3. Matrix interferences may be caused by contam inants 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 electr ospray 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 developmen t, suppression of analyte signals due to electrolyte-induced ioni zation 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 reduci ng the occurrence of this phenomenon.

4.5. Relatively large quantities of the preservatives (Sect. 8.1.2) are added to sample bottles. The potential exists for trace-level organic contaminants in these reagents.544

- 7 Interferences from these sources should be monitored by analysis of laboratory reagent blanks (Sect. 9.3.1), particularly when new lots of reagents are acquired.

4.6. SPE cartridges can be a sour ce of interferences. Analysis of field and laboratory reagent blanks can provide important inform ation regarding the pr esence or absence of

such interferences. Brands and lots of SPE devices should be tested to ensure that contamination does not preclude analyt e identification and quantitation.

5. SAFETY

5.1. Each chemical should be treated as a poten tial health hazard, and exposure to these

chemicals should be minimized. Each laborat ory is responsible for maintaining an awareness of OSHA regulations regarding sa fe handling of chemicals used in this

method. A reference file of MSDSs should be made available to al l personnel involved in the chemical analysis. Toxin decontamin ation/inactivation guidelines may be found

in Biosafety in Microbiological and Biomedical Laboratories , 5 th edition. 6

Additional references to labor atory 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 s hould be taken not to breathe the vapors or in gest the materials.

6. EQUIPMENT AND SUPPLIES (Brand names and/or cat alog numbers are included for illustration only, and do not imply endorsement of the product.)

6.1 SAMPLE CONTAINERS – 500-mL ambe r glass bottles fitted with polytetrafluoroethylene (PTFE)-lined screw caps.

6.2 SAMPLE FILTER APPARATUS (See Figure 1)

6.2.1 CONTAINERS FOR COLLEC TING FILTRATE – 500-mL amber glass bottles (Fisher #02-542-4C or equivalent) a nd 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 tubi ng adapter for filtration apparatus (Kimble Chase #736400-1413 or equivalent).544

- 8 6.2.6 METAL CLAMP – 47 mm aluminum clamp (Kimble Chase #953753-0000 or equivalent).

6.2.7 FUNNEL – 47 mm, 300 mL glass f unnel (Kimble Chase #953751-0000 or equivalent).

6.3 MEMBRANE FILTER – 47 mm Nuclepore poly carbonate filter membranes, pore size

0.4 μ m, (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 cent rifuge tubes (Corning #8082-15) or other glassware suitable for colle ction 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 equi valent) with caps containing PTFE-faced septa (National Scientific #C4000-53 or equivalent).

6.7 MICRO SYRINGES – Suggested sizes include 5, 10, 25, 50, 100, 250, 500 and 1000-μL syringes.

6.8 ANALYTICAL BALANCE – Capable of weighing to the nearest 0.0001 g.

6.9 SOLID PHASE EXTRACTION (SPE) A PPARATUS FOR USING CARTRIDGES

6.9.1 SPE CARTRIDGES – Waters Oasis HL B, 150 mg, 6cc divinylbenzene Nvinylpyrrolidone copolymer (Waters # 186003365).

6.9.2 VACUUM EXTRACTION MANIFOLD

6.9.2.1 Manual Extraction – A manual vacuum manifold with Visiprep  large volume sampler (Supelco #57030 and #57275 or equivalent) for cartridge

extractions.

6.9.2.2 SAMPLE DELIVERY SYSTEM – Use of a transfer tube system (Supelco “Visiprep,” #57275 or equivalent), which transfers the sample directly from the sample container to the SPE cartridge is recommended.

6.10 EXTRACT CONCENTRATION SYSTEM – Extracts are concentrated by evaporation with nitrogen usi ng a water bath set no higher than 60 °C (Meyer N-Evap, Model 111, Organomation Associat es, Inc. or equivalent).544

- 9 6.11 LABORATORY OR ASPIRA TOR 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- μ L aliquots, and performing binary linear grad ients 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 i onization (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 co llected using a triple quadrupole mass

spectrometer.

6.12.3 DATA SYSTEM – An interfaced data syst em 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 tim e 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 C 8 column (2.1 x 100 mm) packed

with 2.6 μ m C 8 solid phase particles (Phe nomenex Kinetex #00D-4497-AN) was used. Any equivalent column that provides adequate resolution, peak shape, capacity, accuracy, and precis ion (Sect. 1.6 and 9) may be used.

7. REAGENTS AND STANDARDS

7.1 GASES, REAGENTS, AND SOLVENTS – Reagen t grade or better chemicals should be used. Unless otherwise indicated, it is in tended that all reagen ts shall conform to the specifications of the Committee on Analy tical Reagents of the American Chemical Society, where such specifications are ava ilable. Other grades may be used, provided it is first determined that the reagent is of sufficiently high purity to permit its use without lessening the quality of the determination.544

- 10 7.1.1 REAGENT WATER – Purified water whic h does not contain any measurable quantities of any method analytes or in terfering compounds greater than 1/3 the MRL for each method analyte of interest.

7.1.2 METHANOL (CH 3 OH, CAS#: 67-56-1) – High purity, demonstrated to be free of analytes and interferen ces (Fisher Optima LC/MS grade or equivalent).

7.1.3 AMMONIUM FORMATE (CH 5 O 2 N, 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 leas t every 48 hours.

7.1.5 SAMPLE PRESERVATION REAGENTS – Th e 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 pr emixed blend of Tris [Tris(hydroxymethyl)aminomethane] and Tris HC L [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 reag ent water. Trizma functions as a buffer (Sect. 8.1.2).

7.1.5.2 L-ASCORBIC ACID (CAS# 50-81-7) – Re duces free chlorine at the time of sample collection (Sigma-Ald rich #255564 or equivalent). 10

7.1.5.3 2-CHLOROACETAMIDE (CAS# 79-07-2) – Inhibits microbial growth and analyte degradation (Sigma-A ldrich #C0267 or equivalent). 10

7.1.5.4 ETHYLENEDIAMINETETRAACETI C ACID, TRISODIUM SALT (Trisodium EDTA, CAS# 10378-22-0) – I nhibits metal-catalyzed hydrolysis of analytes. The trisodium salt is used instead of the disodium salt because the trisodium salt solution pH is clos er 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 speci fications. Nitrogen gas may be used as collision gas provided sufficient sensitivity (product ion formation) is achieved.544

- 11 7.2 STANDARD SOLUTIONS – When the purity of a compound is assayed to be 95% or greater, the weight can be used without co rrection 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 fortificat ion 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 s ections, laboratories should use standard QC practices to determine when thei r standards need to be replaced.

NOTE: Pipets using polypropylene tips must not be used for dispensing solutions containing method analytes as adsorp tion 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, d 5 (C 2 D 5 -MC-LR; see Table 3). This isotopically labeled SUR standard was carefully c hosen during method development because it contains similar functional groups as the method analytes. Although alternate SUR standards may be used provided th ey are isotopically labeled compounds with similar functional groups as met hod analytes, the analyst must have documented reasons for using alternate SU R standards. In addition, alternate SUR standards must meet the QC requirements in Section 9.3.4.

7.2.1.1 SURROGATE PRIMARY DILUTION STANDARD (SUR PDS; 6.49 ng/μL) – The SUR PDS was prepared by diluting 64.9 μg of neat material

with 10 mL of methanol. Th is solution is used to fortify all QC and field samples. The PDS has been shown to be stable for at least one month when

stored at -15  C or less. Use 20 μL of this 6.49 ng/μL SUR PDS to fortify the 500 mL aqueous QC and field samp les 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 concentratio n may be adjusted to accommodate instrument sensitivity.

7.2.2 ANALYTE STANDARD SOLUTIONS – Analyt e standards may be purchased commercially as ampulized solutions or prep ared from neat materials (see Table 3 for analyte sources used during method development).

7.2.2.1 ANALYTE STOCK STANDARD SOLUTI ON (10-500 μg/mL) – Neat cyanotoxins are typically purchased in quantities of 10-500 μg. Due to the small quantity and toxicity of these analytes, weighing the cyanotoxins is not feasible. If preparing from neat ma terial, simply add 1 mL of methanol to the purchased neat ma terial (10-500 μg) for a final concentration of 10- 500 μg/mL. Repeat for each method analyte prepared from neat material. Alternatively, purchase commercially available stock standards of the analytes, preferably in methanol, if available. These stock standards were stable for at544

- 12 least six months when stored at -15  C or less in amber glass screw cap

vials.

7.2.2.2 ANALYTE PRIMARY DILUTION STANDARD (PDS) SOLUTION (0.94-5.0 ng/ μ L) – 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 devel opment, 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/ μ L The Analyte PDS is prepared by dilution of the combined Analyte Stock Standa rd 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 PD S has been shown to be stable for

one month when stored at -15  C or less in amber glass screw cap vials.

Conc. of Analyte

Stock (μg/mL) Vol. of Analyte Stock (μL) Final Vol. of Analyte PDS (mL) Final Conc. of PDS (ng/μL)

Microcystin-LR 500 40.0

10 mL 2.0 Microcystin-RR 10.3 910 0.94 Microcystin-YR 100 200 2.0 Microcystin-LY 100 200 2.0 Microcystin-LF 100 200 2.0

Nodularin-R 10.3 950 0.98 Microcystin-LA 100 500 5.0

7.2.3 CALIBRATION STANDARDS (CAL) – Prep are a series of at least five

concentrations of calibration solutions in methanol containing 10% water, from dilutions of the Analyte PDS (Sect 7.2.2.2). The suggested concentrations in this paragraph are a description of the concentrations used during method development, and may be modified to conform with instrument sensitivity. Concentration ranges used during method development were 10- 400 μg/L, except for MC-RR (4.7-187.5 μg/L), nodularin-R (4.9-195.7 μg/L) and MC-LA (25-1000 μg/L). Larger concentration ranges will require more calibration points. The SUR

is added to CAL standards at a c onstant concentration. During method development, the concentration of th e SUR was 129.8 μg/L in the standard (259.6 ng/L in the aqueous sample). The lo west concentration CAL standard must be at or below the MRL, which may de pend on system sensitivity. CAL standards may also be used as CCCs (Sect. 9.3.2). During method development, CAL

standards were shown to be stable for two weeks when stored at -4  C or less. Longer storage times are acceptable provided appropriate QC measures are documented demonstrating the CAL stability.544

- 13 8. SAMPLE COLLECTION, PRES ERVATION, AND STORAGE

8.1 SAMPLE BOTTLE PREPARATION

8.1.1 Collect 500-mL samples in amber glass bot tles (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). Smalle r sample sizes may be used, but no less than 100-mL, provided the MRL can be met. The entire sample volume in the bottle must be used (e.g., a 100-mL aliquot must not be drawn off a 500-mL sample because the sample bottle must be rinsed).

8.1.2 Preservation reagents, listed in the table below, are added to each sample bottle as a solid prior to shipment to the field (or prior to sample collection).

Compound Amount Purpose

Trizma 7.75 g/L buffering reagent

2-Chloroacetamide 2 g/L antimicrobial

Ascorbic acid 100 mg/L dechlorinating agent

Ethylenediaminetetraacetic acid

trisodium salt 0.35 g/L inhibit binding of the targets to

metals

8.2 SAMPLE COLLECTION

8.2.1 Open the cold water tap and allow the syst em to flush until the water temperature has stabilized (approximately 3 to 5 mi n). 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 fi rst 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 la b 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 sh ipped with sufficient ice to meet the above requirements.544

- 14 8.4 SAMPLE AND EXTRACT HOLDING TIMES – Water samples should be extracted as soon as possible after collection but must be extracted within 28 days of collection.

Extracts must be stored at ≤ -4  C and analyzed within 28 days after extraction. Sample and extract holding time data are presented in Tables 9 and 10.

9. QUALITY CONTROL

9.1 QC requirements include the Initial Demons tration of Capability (IDC) and ongoing QC requirements that must be met when pr eparing and analyzing field samples. This section describes QC parameters, their re quired 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 Tabl es 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 – Th e 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 CAPABI LITY (IDC) – The IDC must be successfully performed prior to analyzing any field sample s. 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, solven ts, 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 PRECISIO N (IDP) – Prepare, extract, and analyze four to seven replicate LFBs for tified 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 devi ation (RSD) of the results of re plicate 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 r ecovery. The average recovery of replicate values must be within ± 30% of the true value.544

- 15 9.2.4 MINIMUM REPORTING LEVEL (MRL) CONF IRMATION – Establish a target

concentration for the MRL based on th e intended use of the method. The MRL may be established by a laboratory for th eir specific purpose or may be set by a regulatory agency. Establish an Initia l 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. Establishi ng 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 re plicate LFBs at the proposed MRL

concentration. These LFBs must contai n all method preservatives described

in Section 8.1.2. Calculate the m ean measured concentration ( Mean ) and standard deviation for these replicates. Determine the Half Range for the prediction interval of results ( HR PIR ) 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 In terval of Result ( PIR = Mean + HR PIR ) meet the upper and lower recovery limits as shown

below

The Upper PIR Limit must be ≤ 150% recovery.

150% % 100   

ion Concentrat Fortified HR Mean PIR

The Lower PIR Limit must be  50% recovery.

50% % 100   

ion Concentrat Fortified HR Mean PIR

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 crit eria 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. HR P IR = 3.963s544

- 16 9.2.6 DETECTION LIMIT DETERMINATION (optional)

– While DL determination is not a specific requirement of this method, it may be required by various regulatory bodies associated with complian ce monitoring. It is the responsibility of the laboratory to determine if DL determination is required based upon the intended use of the data.

Replicate analyses for this procedure shoul d 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 se ven replicate LFBs at a concentration

estimated to be near the DL. This con centration 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 va rious concentrations as in dicated in the table. Appropriate fortification concentrations will be dependent upon the sensitivity of the LC/MS/MS system used. All preserva tion 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 m eets these requirements, a DL may be

calculated from the MRL confirmation data, and no additional analyses

are necessary.

Calculate the DL using the following equation

where

s = standard deviation of replicate analyses

t ( n -1, 1- α =0.99) = Student's t value for the 99% confidence level with n-1 degrees of freedom

n = number of replicates.

NOTE: Do not subtract blank values when performing DL calculations.

9.3 ONGOING QC REQUIREMENTS – This se ction summarizes ongoing QC criteria that must be followed when proces sing 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 backgro und contaminants are not interfering with identific ation or quantitation of met hod analytes. If more than 20 field samples are included in a batch, an alyze an LRB for every 20 samples. If the LRB produces a peak within the rete ntion time window of any analyte that would prevent determination of that analyte, determine the source of

contamination and eliminate the inte rference before processing samples. Background contamination must be reduced to an acceptable level before proceeding. Background from method analytes or other contaminants that interfere with the measurement of method analyt es must be below 1/3 of the MRL. If 

### ) 99

. 0 1

, 1 ( 

     n t s DL544

- 17 method analytes are detected in the LRB at concentrations equal to or greater than this level, then all data for the problem analyte(s) must be considered invalid for all samples in the extraction batch. Blank contamination is estimated by extrapolation, if the concentration is below the lowest CAL standard. This extrapolation procedure is not allowed for sample results as it may not meet data quality objectives.

9.3.2 CONTINUING CALIBRATION CHECK ( CCC) – CCC standards are analyzed at the beginning of each analysis batch (Sect. 3.1), after every 10 field samples, and at the end of the analysis batc h. 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 fortif ied 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 con centration LFB should be near the high end of the calibration range established during the initia l calibration (Sect. 10.2). Results of low-level LFB analyses must be 50-150% of the true value. Results of medium and high-level LFB analyses must be 70-130% of the true value. If LFB

results do not meet these criteria for method analytes, then all data for the problem analyte(s) must be considered invalid for all samples in the extraction

batch.

9.3.4 SURROGATE RECOVERY – The SUR standard is fortified into all samples, CCCs, LRBs, LFBs, LFSMs, LFSMDs, and FD prior to extraction. It is also

added to CAL standards. The SUR is a means of assessing method performance from extraction to final chromatographic measurement. Calculate the recovery (%R) for the SUR using the following equation

where

A = measured SUR concentration for the QC or Field Sample

B = fortified concentration of the SUR.

9.3.4.1 SUR recovery in extracts must be in the range of 60-130%. SUR recovery in

CCCs must be 70-130%. When SUR recovery does not meet these criteria, check 1) calculations to locate possibl e 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 SU R recovery criterion, report only data for the reanalyzed extract. 100 %       

 B A R544

- 18 9.3.4.3 If the extract reanalysis fails the 60- 130% recovery criterion, the analyst

should check the calibration by injecting the last CA L standard that passed.

If the CAL standard fails the criteri a of Section 10.3, re calibration is in order per Section 10.2. If the CAL standard is acceptable, extraction of the sample should be repeated provided th e 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 rec overy to inform the data user that the results are suspect due to SUR recover y. Alternatively, collect a new sample and re-analyze.

9.3.5 LABORATORY FORTIFIED SAMPLE MATR IX (LFSM) – Analysis of an LFSM is required in each extraction batc h and is used to determine that the sample matrix does not adversely aff ect method accuracy. Assessment of method precision is accomplished by analysis of a Field Duplicate (FD) (Sect. 9.3.6); however, infrequent occurrence of method an alytes would hinder this assessment. If the occurrence of method analytes in sa mples 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 concen tration that is greater than or equal to the matrix background concentr ation, if known. Use historical data and rotate through low, mid and high concentrations when selecting a fortifying concentration.

9.3.5.2 Calculate percent recovery (% R ) for each analyte using the equation

where

A = measured concentration in the fortified sample

B = measured concentration in the unfortified sample

C = fortification concentration.

9.3.5.3 Analyte recoveries may exhibit matrix bias. For samples fortified at or above their native concentration, rec overies 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 analyt e falls outside the designated range, 

##   100 %  

 C B A R544

- 19 and laboratory performance for that anal yte is shown to be in control in CCCs, the recovery is judged to be matr ix 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 LABORATO RY 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 associat ed with sample collection, preservation, storage, and laboratory procedures. If me thod analytes are not routinely observed in field samples, an LFSMD should be analyzed rather than an FD.

9.3.6.1 Calculate relative percent difference ( RPD ) for duplicate measurements ( FD1 and FD2 ) using the equation

9.3.6.2 RPDs for FDs should be ≤ 30%. Greater variability may be observed when the matrix is fortified at analyte concen trations at or near the MRL (within a

factor of two times the MRL concentr ation). At these concentrations, FDs

should have RPDs that are ≤ 50%. If the RPD of any analyte falls outside the designated range, and laboratory perf ormance for that analyte is shown to be in control in the CCC, the recove ry is judged to be matrix biased. The result for that analyte in the unfortified sample is labeled suspect/matrix to inform the data user that the results are suspect due to matrix effects.

9.3.6.3 If an LFSMD is analyzed instead of a FD, calculate the relative percent difference (RPD) for duplicate LFSMs (LFSM and LFSMD) using the equation

9.3.6.4 RPDs for duplicate LFSMs should be ≤ 30% for samples fortified at or

above their native concentration. Greate r variability may be observed when the matrix is fortified at analyte concen trations at or near the MRL (within a

factor of two times the MRL concen tration). 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 performan ce 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 ar e suspect due to matrix effects. 

##   100 2 / 2 1 2

1






 FD FD FD

FD RPD



##   100 2 / 

 

 LFSMD LFSM LFSMD

LFSM RPD544

- 20 9.3.7 QUALITY CONTROL SAMPLES (QCS) – As part of the IDC (Sect. 9.2), each time a new Analyte PDS (Sect. 7.2.2.2) is pr epared, 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 availabl e, 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 acceptabl e initial calibration is required before any samples are analyzed. The MS tune check and initial calibration must be repeated each time a major instrument modification is made, or maintenance is performed.

10.2 INITIAL CALIBRATION

10.2.1 ESI-MS/MS TUNE

10.2.1.1 Calibrate the mass scale of the MS with the calibration compounds and procedures prescribed by the manufacturer.

10.2.1.2 Optimize the precursor ion (Sect. 3.15; [M+H] + or [M+2H] 2+ ) for each method analyte by infusing approximately 1-5 ng/μL of each analyte (prepared in the initial m obile phase conditions) direc tly 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 vari ed 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 v erification studies, desolvation temperature was identified as a parameter that can affect the degree of analyte suppressi on observed in matrices. Desolvation temperature is applied in different ways to different instruments; a heated gas or a heated stainless steel capillary is used in ESI source designs. Thus, it is highly recommended that the desolvation temperature be minimized and that temperatures of ≤ 400 °C be used for the heated gas source designs and ≤ 275 °C for heated capillary source designs (these recommended temperatures

are based on LC conditions employed during development of this method).544

- 21 10.2.1.3 Optimize the product ion (Sect. 3.17) for each analyte by infusing approximately 1-5 ng/μL of each analyte (prepared in the initial mobile phase conditions) directly into the MS at the chosen LC mobile phase flow rate (approximately 0.3 mL/min). This tune can be done on a mix of method analytes. MS/MS parameters (collision gas pressure, collision energy, etc.) are varied until optimal analyte responses are determined. See Table 4 for MS/MS conditions used in method development.

10.2.2 Establish LC operating parameters that optimize resolution and peak shape. Suggested LC conditions can be found in Table 1. LC conditions listed in Table 1 may not be optimum for all LC systems and may need to be optimized by the analyst.

10.2.3 Inject a mid-level CAL standard under LC/MS conditions to obtain retention times of each method analyte. Divide the chromatogram into retention time windows (segments) each of which contains one or more chromatographic peaks. During MS/MS analysis, fragment a small number of selected precursor ions ([M+H] + or [M+2H] 2+ ; Sect. 3.15) for the analytes in each window and choose the most abundant product ion. Product ions (a lso quantitation ions) chosen during method development are in Table 4, alt hough 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 op timized 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 p eak for optimum precision.

10.2.5 Prepare a set of at least five CAL st andards 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 reco mmended that at least four of the CAL standards are at a concentration gr eater than or equal to the MRL.

10.2.6 The LC/MS/MS system is calibrated usi ng 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 appr opriate internal standards. More freque nt calibration may be n ecessary with external

standard calibration.

10.2.7 CALIBRATION ACCEPTANCE CRITERIA – Validate the initial calibration by calculating the concentration of each anal yte as an unknown against its regression equation. For calibrati on 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 will544

- 22 have difficulty meeting ongoing QC criteria . It is recommende d that corrective action is taken to reanalyze the CAL standa rds, 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 anal ysis 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 wind ow 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 ever y 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 analys is batch must be at or below the MRL in order to verify instrument sensitivity pr ior 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 th e 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 con centration CAL standard and analyze with the same conditions used during the initial calibration.

10.3.2 Calculate the concentration of each anal yte 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 an alytes in CCCs fortified at all other

levels must be within ± 30%. If these condi tions do not exist, then all data for the problem analyte must be considered inva lid, and remedial action should be taken (Sect. 10.3.3) which may require recalibra tion. Any Field or QC Samples that have been analyzed since the last accep table calibration verification should be reanalyzed after adequate calibration ha s been restored, with the following exception. If the CCC fails because the calcul ated concentration is greater than 130% (150% for the low-level CCC) for a particular method analyte, and Field Sample extracts show no de tection for that method analyte, nondetects may be reported without re-analysis.

10.3.3 REMEDIAL ACTION – Failure to meet CCC QC performance criteria may require remedial action. Major maintenan ce, such as cleaning the electrospray probe, atmospheric pressure ionization s ource, mass analyzer, replacing the LC column, etc., requires recalibration (Sect 10.2) and verification of sensitivity by analyzing a CCC at or below the MRL (Sect 10.3).544

- 23 11. PROCEDURE

11.1 This procedure may be performed manually or in an automated mode using a robotic or automatic sample preparation device. Da ta presented in Tables 5-10 demonstrate data collected by manual extraction. An auto matic/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 al l extraction and elution steps must be the same as in the manual procedure. Extracti on and/or elution steps may not be changed

or omitted to accommodate the use of an automa ted system. If an automated system is used, LRBs should be rotated among the ports to ensure that all valves and tubing meet LRB requirements (Sect. 9.3.1).

NOTE: SPE cartridges described in this section are designe d 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 stor ed 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, veri fy that the sample pH is 7 ± 0.5. If the sample pH does not meet this require ment, discard the sample. If the sample pH is acceptable, proceed with the analys is. Before extraction, mark the level of the sample on the outside of the sa mple 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 Secti ons 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 a dversely affect analyte recoveries.

NOTE: Section 8 allows smaller sample si zes 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- μ L aliquot of the 6.49 ng/  L SUR PDS (Sect. 7.2.1.1) was added to 500 mL for a final concentration of 259.6 ng/L in the aqueous sample.

11.2.3 In addition to SUR and preservatives, if the sample is an LFB, FD, LFSM, or LFSMD, add the necessary amount of An alyte PDS (Sect. 7.2.2.2). Cap and invert each sample to mix.544

- 24 11.3 INTRACELLULAR TOXIN RELEASE PROCEDURE

11.3.1 Filter the 500-mL water sample using a Nu clepore filter (Sect. 6.3) with the shiny side up; collect the filt rate 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 an other 2.5 mL of 10% reagent water in

methanol and combine with the fi ltered 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 gla ss test tube (Sect. 6.4). Push the filter to the bottom of the gla ss 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 li quid) 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, sw irl gently a few times, then draw off the 2 mL of liquid using a glass pipet. Transf er 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 gen tly. Draw off the 2 mL of liquid using a glass pipet and transfer the 2 mL of li quid to the filtered 500 mL water sample

collected in Section 11.3.1.

11.3.9 Rinse the filter a second time by adding a nother 1 mL of 20% reagent water in methanol to the test tube and swirl gen tly. Draw off the 1 mL of liquid using a glass pipet and transfer the 1 mL of li quid to the filtered 500 mL water sample

collected in Section 11.3.1. Swirl the 500 mL sample several times to homogenize the sample.544

- 25 11.4 CARTRIDGE SPE PROCEDURE

11.4.1 CARTRIDGE CLEAN-UP AND CONDITI ONING – 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 allo wing the water to drop below the top edge of the packing. If the cartridge goes dry duri ng the conditioning phase, the conditioning

must be started over. Add 4-5 mL of r eagent 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 ELUTI ON – 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 samp le 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 lo w vacuum such that the solvent exits the cartridge in a dropwise fash ion. Repeat sample bottle ri nse and cartridge elution with a second 5-mL aliquot of meth anol containing 10% reagent water.

11.5 EXTRACT CONCENTRATION – Concentrate th e 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 extrac tion. Determine to the nearest 10 mL. If using weight to determ ine volume, weigh the empty bottle to the nearest 10 g and determine the sample we ight 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 w ill be used in the final calculations of the analyte concentration (Sect. 12.2).544

- 26 11.7 EXTRACT ANALYSIS

11.7.1 Establish operating conditions equivalent to those summ arized in Tables 1-4 of

Section 17. Instrument conditions and co lumns 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 chro matogram. Thus, diverting the early port ion 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 retenti on time variation for each method analyte

in CAL standard solutions an alyzed on the LC over the c ourse of time. A value of plus or minus three times the standard de viation of the retent ion time obtained for each method analyte while establishing th e initial calibration and completing the IDC can be used to calculate a sugges ted 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 follo wing the procedures outlined in Sect. 10.2

or confirm that the calibration is still va lid by running a CCC as described in Sect. 10.3. If establishing an initi al calibration, complete th e IDC as described in

Section 9.2.

11.7.4 Begin analyzing field samples, includi ng QC samples, at their appropriate frequency by injecting the same size aliquots (10 μ L was used in method development), under the same conditions us ed 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 calib ration range. If an analyte peak area exceeds the range of th e initial calibration curve, the extract may be diluted with methanol containing 10% water. Re-inject the diluted extract. Incorporate the dilution f actor into the final concentration calculations. Acceptable SUR performance (Sect. 9.3.4) should be determined from the undiluted sample extract. The resulting da ta should be documented as a dilution and MRLs should be adjusted accordingly.544

- 27 12. DATA ANALYSIS AND CALCULATION

12.1 Complete chromatographic resolution is not necessary for accurate and precise measurements of analyte concentrations using MS/MS. In validating this method, concentrations were calculated by measuri ng 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 us ing the multipoint cali bration established in Section 10.2. Do not use daily calibration ve rification data to qua ntitate analytes in samples. Adjust final analyte concentra tions 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 pr ecision, but final reported concentrations should be rounded to an appr opriate number of significant figures (one digit of uncertainty), typica lly two, and not more than three significant figures.

13. METHOD PERFORMANCE

13.1 PRECISION, ACCURACY, AND MINIMU M REPORTING LEVELS – Tables for these data are presented in Section 17. LCMRLs and DLs for each method analyte are presented in Table 5. Precis ion and accuracy are presente d for three water matrices: reagent water (Table 6); chlorinated (fini shed) 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 desc ribed 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 wa ter fortified with method analytes. Precision and mean recove ry (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 acknowle dge the assistance of the analysts and laboratories for their participation in the mu lti-laboratory verification studies: 1) Dr.

William A. Adams of CB&I Federal Services under EPA contract EP-C-12-013 and 2) Dr. Andrew Eaton and Mr. Ali Haghani of Eurofins Eaton Analytical, Inc.544

- 28 14. POLLUTION PREVENTION

14.1 This method utilizes SPE to extract analytes from water. It requires the use of very small volumes of organic solv ent and very small quantitie s of pure analytes, thereby minimizing potential hazards to both the analyst and the environment as compared to the use of large volumes of organic solven ts in conventional li quid-liquid extractions.

14.2 For information about pollution prevention that may be applicable to laboratory operations, consult “Less is Better: Guid e to Minimizing Waste in Laboratories”

available from the American Chemical Soci ety’s Department of Government Relations and Science Policy, 1155 16 th Street N.W., Washington, D.C., 20036 or on-line at http://portal.acs.org/ portal/fileFetch/C/W PCP_012290/pdf/WPCP_012290.pdf (accessed February 2015).

15. WASTE MANAGEMENT

15.1 Analytical procedures desc ribed 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 c onsistent with all a pplicable rules and regulations, and that laborato ries protect the air, wate r, 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 Proce dures 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., Fu rlong, E.T., Ferrer, I. “Molecular Resolution and Fragmentation of Fulvic Acid by El ectrospray Ionization/Mu ltistage 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 Chro matography Electrospray Ionization Mass Spectrometry.” J. Chromatogr. A

, 2004, 1041 , 171-180.

5. Draper, W.M., Xu, D., Perera, S.K. “El ectrolyte-Induced Ioni zation Suppression and Microcystin Toxins: Ammonium Formate Suppresses Sodium Replacement Ions and544

- 29 Enhances Protiated and Ammoniated Ions fo r Improved Specificity in Quantitative LC-MSMS.” Anal. Chem . 2009, 81 , 4153-4160.

6. “Biosafety in Microbiological and Biomedical Laboratories”, 5 th edition, Appendix I— Guidelines for Work with Toxins of Biological Origin

. U.S. Department of Health and

Human Services

, Public Health Service Centers for Di sease 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 La boratory: Handling and Disposal of Chemicals,” National Academies Press (1995), available at http://www.nap.edu (accessed May 2014).

8. “OSHA Safety and Health Standards, Genera l Industry,” (29CFR1910), Occupational Safety and Health Administration, OS HA 2206, (Revised, July 2001).

9. “Safety in Academic Chemistry Laboratories, ” American Chemical Society Publication, Committee on Chemical Safety, 7t h Edition. Available online at http://portal.acs.org/portal/PublicWebSite/about /governance/committees/chemicalsafety/publ ications/WPCP_012294 (accessed February 2015).

10. Winslow, S. D. , Pepich, S. D. , Bassett, M. V., Wendelken, S. C., Munch, D. J., Sinclair, J.

L. “Microbial Inhibitors for U.S. EPA Dri nking Water Methods for the Determination of Organic Compounds.” Environ. Sci. Technol. , 2001, 35 , 4103-4110.

11. Hyenstrand, P., Metcalf, J. S., Beattie, K. A., Codd, G. A. “Effects of Adsorption to Plastics and Solvent Conditions in the Analysis of the Cyanobacterial Toxin Microcystin-LR by High Perormance Liquid Chromatography.” Wat. Res. , 2001, 35 , 3508-3511.544

- 30 17. TABLES, DIAGRAMS, FLOW CHARTS AND VALIDATION DATA

TABLE 1. LC METHOD CONDITIONS

Time (min) % 20 mM

Ammonium Formate % Methanol

Initial 90 10

2.0 90 10

16 20 80

16.1 10 90

22.0 10 90

22.1 90 10

26.0 90 10

Phenomenex Kinetex C 8 column, 2.6 μ m, 2.1 x 100 mm

Flow rate of 0.3 mL/min

10 μ L partial loop injection into a 20 μL loop

TABLE 2. ESI-MS/MS METHOD CONDITIONS

ESI Conditions

Polarity Positive ion

Capillary needle voltage 4 kV

Cone gas flow 25 L/hr

Nitrogen desolvation gas 1000 L/hr

Desolvation gas temp. 350  C544

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

Analyte Method Analyte Source a RT (min)

MC-YR GreenWater Laboratories 11.07

Nodularin-R National Research Council Canada 11.08

MC-RR National ResearchCouncil Canada 11.33

MC-LR GreenWater Laboratories 11.49

MC-LA GreenWater Laboratories 12.41

MC-LY Enzo Life Sciences 12.51

MC-LF Enzo Life Sciences 14.05

C 2 D 5 -MC-LR (SUR) Synthesized under contract b 14.3

a Data presented in this method were obt ained 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, Wa yne State University, EPA Contract

#EP13C000079.544

- 32 TABLE 4. MS/MS METHOD CONDITIONS a

Segment b Analyte Precursor Ion c (m/z) Product

Ion c,d ( m/z ) Cone Voltage (v) Collision Energy e (v)

1 MC-YR 523.4 [ M+2H ] 2+ 134.9 20 15

1 Nodularin-R 825.4 [ M+H ] + 134.9 45 55

1 MC-RR 519.9 [ M+2H ] 2+ 134.9 35 30

1 MC-LR 995.5 [ M+H ] + 134.9 60 65

2 MC-LA 910.5 [ M+H ] + 776.4 40 20

2 MC-LY 1002.5 [ M+H ] + 134.9 40 60

3 MC-LF 986.5 [ M+H ] + 134.9 40 60

3 C 2 D 5 -MC-LR (SUR) 1028.6 [ M+H ] + 134.9 55 60

a An LC/MS/MS chromatogram of th e analytes is shown in Figure 2.

b Segments are time durations in which single or multiple scan events occur.

c During MS and MS/MS optimization, the anal yst 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 produc t ion masses (with one decimal place) should be used in the MS/MS method for all analyses.

d Ions used for quantitation purposes.

e Argon used as collision gas at a flow rate of 0.3 mL/min.

TABLE 5. DLs AND LCMRLs IN REAGENT WATER

Analyte Fortified Conc. (ng/L) a DL b (ng/L) LCMRL c (ng/L)

MC-YR 8.0 4.6 22

Nodularin-R 3.9 1.8 7.3

MC-RR 3.8 1.2 5.6

MC-LR 8.0 4.3 6.6

MC-LA 20 4.0 2.9

MC-LY 8.0 2.2 4.6

MC-LF 8.0 3.4 3.5

a Spiking concentration used to determine DL.

b Detection limits were determined by analyzing seven rep licates over three

days according to Section 9.2.6.

c LCMRLs were calculated according to the procedure in reference 1.544

- 33 TABLE 6. PRECISION AND ACCURAC Y DATA FOR METHOD ANALYTES FORTIFIED IN REAGENT WATER (n=4)

Analyte Fortified Conc. (ng/L) Mean % Recovery % RSD Fortified Conc. (ng/L) Mean % Recovery % RSD

MC-YR 400.0 87.4 5.3 40.0 87.5 7.1

Nodularin-R 195.7 89.5 1.8 19.6 91.9 3.1

MC-RR 187.5 95.8 3.1 18.8 97.3 9.5

MC-LR 400.0 85.5 4.7 40.0 87.5 5.6

MC-LA 1000 81.8 2.7 100.0 94.7 6.0

MC-LY 400.0 83.8 3.5 40.0 92.8 6.4

MC-LF 400.0 101 3.4 40.0 85.0 4.1

C 2 D 5 -MC-LR (SUR) 259.6 83.8 4.9 259.6 87.2 2.9

TABLE 7. PRECISION AND ACCURAC Y DATA FOR METHOD ANALYTES

FORTIFIED IN FINISHED DRINKING WATER FROM A GROUND WATER

SOURCE a (n=4)

Analyte Fortified Conc. (ng/L) Mean % Recovery % RSD Fortified Conc. (ng/L) Mean % Recovery % RSD

MC-YR 400.0 93.2 8.0 40.0 114 14

Nodularin-R 195.7 93.5 7.7 19.6 99.5 3.4

MC-RR 187.5 92.1 9.8 18.8 116 6.2

MC-LR 400.0 90.1 7.4 40.0 99.8 6.1

MC-LA 1000 87.0 3.8 100.0 99.5 4.1

MC-LY 400.0 83.9 6.8 40.0 97.7 2.3

MC-LF 400.0 87.9 6.1 40.0 92.9 3.4

C 2 D 5 -MC-LR (SUR) 259.6 90.4 6.0 259.6 94.0 1.6

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

.544

- 34 TABLE 8. PRECISION AND ACCURAC Y DATA FOR METHOD ANALYTES
FORTIFIED IN FINISHED DRINKING WATER FROM A SURFACE WATER

SOURCE a (n=4)

Analyte Fortified Conc. (ng/L) Mean % Recovery % RSD Fortified Conc. (ng/L) Mean % Recovery % RSD

MC-YR 400.0 88.7 9.0 40.0 82.4 11

Nodularin-R 195.7 97.3 1.3 19.6 103 1.9

MC-RR 187.5 100 0.9 18.8 106 2.9

MC-LR 400.0 105 1.7 40.0 117 5.0

MC-LA 1000 92.1 0.9 100.0 112 3.2

MC-LY 400.0 94.6 2.5 40.0 106 5.9

MC-LF 400.0 92.7 2.1 40.0 104 8.7

C 2 D 5 -MC-LR (SUR) 259.6 92.2 2.4 259.6 94.0 2.9

a TOC = 2.49 mg/L and hardness =137 mg/L as calcium carbonate

.544-35TABLE 9. AQUEOUS SAMPLE HOLDINGTIME DATA FOR SAMPLES OF FINISHED DRINKING WATER FROM ASURFACE WATER SOURCEa, FORTIFIED WITH METHOD ANALYTES AND PRESERVED AND STOREDACCORDING TO SECTION 8 (n=4)FortifiedConc.(ng/L)Day 0Day 7Day 14Day 21Day 28AnalyteMean%Rec%RSDMean%Rec%RSDMean%Rec%RSDMean%Rec%RSDMean%Rec%RSDMC-YR 400.0 101 8.7 92.5 2.2 89.0 6.7 96.4 1.5 95.1 6.1Nodularin-R 195.7 91.7 3.9 92.0 3.2 94.4 1.6 96.5 2.3 91.6 3.3MC-RR 187.5 91.7 2.1 94.6 3.4 94.9 1.7 94.9 1.7 90.3 0.6MC-LR 400.0 89.4 2.3 87.2 2.5 89.4 2.3 90.2 2.1 86.4 1.3MC-LA 1000 91.2 0.9 90.6 2.3 87.2 1.8 90.2 1.7 88.1 1.5MC-LY 400.0 88.4 1.8 87.9 2.0 89.3 1.1 89.9 1.1 88.1 1.9MC-LF 400.0 89.1 2.3 86.4 1.6 86.6 1.2 86.6 2.2 85.4 2.1C2D5-MC-LR (SUR)b259.6 86.9 5.4 92.8 0.7 89.7 3.3 92.8 3.4 92.0 4.1aTOC = 0.9 mg/L and hardness = 120 mg/Las calcium carbonate.bSurrogate was not added to samples until the day of extraction.TABLE 10. EXTRACT HOLDING TIME DATA FOR SAMPLES OF FINISHED DRINKING WATER FROM ASURFACE WATER SOURCE, FORTIFIED WITH METHOD ANALYTES AND PRESERVED AND STOREDACCORDING TO SECTION 8 (n=4)FortifiedConc.(ng/L)Day 0Day 7Day 14Day 21Day 28AnalyteMean%Rec%RSDMean%Rec%RSDMean%Rec%RSDMean%Rec%RSDMean%Rec%RSDMC-YR 400.0 101 8.7 98.4 2.4 90.7 5.3 91.3 2.8 95.0 4.9Nodularin-R 195.7 91.7 3.9 93.9 2.4 94.9 1.8 95.8 2.4 95.1 1.3MC-RR 187.5 91.7 2.1 98.6 1.7 97.0 2.2 92.6 2.9 92.5 2.1MC-LR 400.0 89.4 2.3 91.1 2.4 94.4 3.8 90.4 2.7 90.8 1.1MC-LA 1000 91.2 0.9 93.1 2.5 90.0 0.5 91.7 0.7 92.5 1.9MC-LY 400.0 88.4 1.8 92.4 2.5 94.4 2.2 91.6 1.8 93.4 1.6MC-LF 400.0 89.1 2.3 92.6 0.9 89.0 1.8 90.8 1.9 91.0 2.5C2D5-MC-LR (SUR) 259.6 86.9 5.4 90.9 5.2 89.1 1.4 90.5 3.2 93.2 3.6544-36TABLE 11. INITIAL DEMONSTRATION OF CAPABILITY QUALITY CONTROL REQUIREMENTSMethodReferenceRequirementSpecification and FrequencyAcceptance CriteriaSect. 9.2.1and 9.3.1Initial Demonstration ofLow System BackgroundAnalyze LRB prior to any other IDC steps.Demonstrate that all method analytes are below 1/3 the MRLand that possible interferencesfrom extraction media do notprevent the identification and quantification of methodanalytes.Sect. 9.2.2Initial Demonstration ofPrecision (IDP)Analyze four to seven replicate LFBs fortified nearthe midrange calibration concentration.%RSD must be <30%Sect. 9.2.3Initial Demonstration ofAccuracy (IDA)Calculate average recoveryfor replicates used inIDP.Mean recovery30% of true valueSect. 9.2.4Minimum Reporting Limit(MRL) ConfirmationFortify, extract and analyze seven replicate LFBsat the proposed MRL concentration. Calculate theMean and the Half Range (HR). Confirm that theupper and lower limits for the Prediction Intervalof Result (Upper PIR, and Lower PIR, Sect.9.2.4.2) meet the recovery criteria.Upper PIR150%Lower PIR50%Sect. 9.2.5and 9.3.7Quality Control Sample(QCS)Analyze a standard from a second source, aspart of IDC.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 beenabbreviated to fit the table format, there may be issues thatneed additional clarification, orareas where important additionalinformation from the method textis needed. In all cases, the full text of the QC in Section 9supersedes any missing or conflicting information in this table.544-37TABLE 12.ONGOING QUALITY CONTROL REQUIREMENTS (SUMMARY)MethodReferenceRequirementSpecification and FrequencyAcceptance CriteriaSect. 8.4 Sample Holding Time28 days with appropriate preservation and storage asdescribed in Sections 8.1-8.4.Sample results are valid only if samples are extracted within thesample holding time.Sect. 8.4 Extract Holding Time28 days when stored at≤-4C.Extract results are valid only ifextracts are analyzed within theextract holding time.Sect. 9.3.1Laboratory Reagent Blank(LRB)One LRB with each extractionbatch of up to 20 fieldsamples.Demonstrate that all method analytes are below 1/3the MRL, andconfirm that possible interferences do not prevent quantification ofmethod analytes. If targets exceed 1/3 the MRL or if interferencesare present, results for these subjectanalytes in the extraction batchare invalid.Sect. 9.3.3Laboratory Fortified Blank(LFB)One LFB is required for each extraction batch of upto 20 field samples. Rotate the fortifiedconcentrations between low, medium, and highamounts.Results of LFB analyses must be70-130% of the true value for eachmethod analyte for all fortified concentrations except the lowestCAL point. Results of the LFBs corresponding to the lowest CALpoint for each method analyte mustbe 50-150% of the true value.Sect. 9.3.4Surrogate Standards(SUR)The surrogate is added to all CAL standards andsamples, including QC samples. Calculate SURrecoveries.SUR recovery in extracts must be60-130% of the true value. SURrecovery in CCCs must be 70-130% of the true value. If a SUR failsthese criteria, report all results for sample as suspect/SUR recovery.Sect. 9.3.5Laboratory FortifiedSample Matrix (LFSM)Analyze one LFSM per extraction batch (20 samplesor less) fortified with method analytes at aconcentration greater than or equal to the nativeconcentration, if known. Calculate LFSM recoveries.Recoveries at mid and high levels should be within 60-140% andwithin 50-150% at the low-level fortified amount (near the MRL). Ifthese criteria are not met, resultsare labeled suspect due to matrixeffects.Sect. 9.3.6Field Duplicates (FD) orLaboratory FortifiedSample Matrix Duplicate(LFSMD)Extract and analyze at least one FD or LFSMD witheach extraction batch (20 samples or less). A LFSMDmay be substituted for a FD when the frequency ofdetects are low. Calculate RPDs.See Sect. 9.3.5 and 9.3.6 for instructions on the interpretation ofLFSM and FD results.Sect. 9.3.7Quality Control Sample(QCS)Analyze at least quarterly or when preparing newstandards, as well as during the IDC.Results should be within 70-130% of true value.544-38TABLE 12.(Continued)MethodReferenceRequirementSpecification and FrequencyAcceptance CriteriaSect. 10.2 Initial CalibrationUse external calibration technique to generate alinear or quadratic calibration curve for eachanalyte. Use at least five standard concentrations.Check the calibration curve as described inSect. 10.2.7.When each CAL standard is calculated as an unknown usingthe calibration curve, the analyte results must be 70-130% ofthe true value for all except CAL standards≤MRL, whichmust be 50-150% of the true value. If this criterion is not metreanalyze the CAL standards, restrict the range ofcalibration, or select an alternate method of calibration.Sect. 9.3.2and Sect.10.3Continuing CalibrationCheck (CCC)Verify initial calibration by analyzing a low level(at the MRL or below) CCC prior to analyzingsamples. CCCs are then injected after every 10field samples and after the last sample, rotatingconcentrations to cover the calibrated range of theinstrument.Recovery for each SUR must be within 70-130% of the truevalue in all CCCs. Each analyte fortified at a level≤MRLmust calculate to be within ± 50% of the true value. Thecalculated concentration of the method analytes in CCCsfortified 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 beenabbreviated to fit the table format, there may be issues thatneed additional clarification, orareas where important additional information from the method textis needed. In all cases, the full text of the QC in Section 8-10 supersedes any missing or conflicting information in this table.544-39FIGURE 1. DIAGRAM OF FILTER APPARATUS WITH PART NUMBERS (SECT 6.2)410171-422602-542-4C(Fisher)953751-0000953753-0000953752-5047410170-4534736400-1413544-40FIGURE 2. EXAMPLE CHROMATOGRAM (OVERLAID MS/MS SEGMENTS) OF A CALIBRATION STANDARD WITHMETHOD 544 ANALYTES AT CONCENTRATION LEVELS OF 187.5-1000 ng/L.Retention Time, minMC-YRNODMC-RRMC-LRMC-LAMC-LYMC-LFSUR