United States
Environmental Protection
Agency
Method 545: Determination of Cylindrospermopsin and
Anatoxin-a in Drinking Water by Liquid Chromatography
Electrospray lonization Tandem Mass Spectrometry
(LC/ESI-MS/MS)
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Questions concerning this document should be addressed to:
Z.JAta
U.S. EPA, Office of Ground Water and Drinking Water, Standards and Risk Management Division-
Technical Support Center, 26 W. Martin Luther King Dr. Cincinnati, OH 45268
Phone:(513)569-7491
wendelken.steve@epa.gov
Off ice of Water (MS-140)
EPA815-R-15-009
EPA contract EP-C-12-013
April 2015
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Acknov- ;- ,;;, --;-••• •
The following people are acknowledged for their support in development of this method:
Ralph Hindle, Vogon Laboratory Services
Rebecca Trenholm, Southern Nevada Water Authority
Joshua Whitaker, Eurofins Eaton Analytical (UL)
Andrew Eaton, PhD, Eurofins Eaton Analytical
Ali Haghani, Eurofins Eaton Analytical
Brett Vanderford, Southern Nevada Water Authority
Yongtao (Bruce) Li, PhD, Eurofins Eaton Analytical (UL)
Joe Weitzel, Agilent
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Acknowledgements ii
1 Scope and Application 1
1.1 Method 1
1.2 AnalyteList 1
1.3 Supporting Data 1
1.3.1 Precision and Accuracy 1
1.3.2 Single Laboratory Lowest Concentration Minimum Reporting Levels 1
1.4 Method Flexibility 1
2 Summary of Method 2
3 Definitions 2
4 Interferences 4
4.1 Clean Glassware 4
4.2 Reagent and Equipment Interferences 4
4.3 Sample Matrix Interferences 4
4.4 IS Purity 4
5 Safety 4
6 Equipment and Supplies 4
6.1 Sample Containers 5
6.2 Autosampler Vials 5
6.3 Micro Syringes 5
6.4 Analytical Balance 5
6.5 Disposable Pasteur Pipettes 5
6.6 Disposable Syringes 5
6.7 Syringe Filters 5
6.8 Liquid Chromatography Electrospray lonization Tandem Mass Spectrometry System (LC/ESI-
MS/MS) 5
6.8.1 LC System 5
6.8.2 Analytical Column 5
6.8.3 Electrospray lonization Tandem Mass Spectrometer (ESI-MS/MS) 5
6.8.4 MS/MS Data System 5
7 Reagents and Standards 6
7.1 Gases, Reagents and Solvents 6
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7.1.1 Acetic Acid, (CH3COOH, CASRN 64-19-7) 6
7.1.2 Collision Gas 6
7.1.3 Desolvation Gas 6
7.1.4 Methanol, (CH3OH, CASRN 67-56-1) 6
7.1.5 L-AscorbicAcid, (C6H8O6, CASRN 50-81-7) 6
7.1.6 Reagent Water 6
7.1.7 Sodium Bisulfate, (NaHSCu, CASRN 7681-38-1) 6
7.2 Standard Solutions 6
7.2.1 Internal Standards 7
7.2.2 Method Analyte Standard Solutions 7
7.2.3 Procedural Calibration (CAL) Standards 8
8 Sample Collection, Preservation and Storage 8
8.1 Sample Bottle Preparation 8
8.1.1 Sample Containers 8
8.1.2 Addition of Preservatives 8
8.2 Sample Collection 8
8.3 Sample Shipment and Storage 9
8.4 Sample Holding Times 9
9 Quality Control 9
9.1 Initial Demonstration of Capability (IDC) 9
9.1.1 Demonstration of Low System Background 9
9.1.2 Demonstration of Precision 9
9.1.3 Demonstration of Accuracy 10
9.1.4 Minimum Reporting Level (MRL) Confirmation 10
9.1.5 Quality Control Sample (QCS) 10
9.2 Ongoing QC Requirements 11
9.2.1 Laboratory Reagent Blank (LRB) 11
9.2.2 Continuing Calibration Check (CCC) 11
9.2.3 Laboratory Fortified Blank (LFB) 11
9.2.4 Internal Standards (IS) 11
9.2.5 Laboratory Fortified Sample Matrix (LFSM) 12
9.2.6 Field Duplicate or Laboratory Fortified Sample Matrix Duplicate (FD or LFSMD) 12
9.2.7 Quality Control Sample 13
IV
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9.3 Method Modification QC Requirements 13
9.3.1 Repeat the Procedures of the IDC and Verify all QC 13
9.3.2 Document Method Performance 13
9.3.3 Document and Assess before Analyzing Field Samples 14
10 Calibration and Standardization 14
10.1 LC/ESI-MS/MS Calibration and Optimization 14
10.1.1 Mass Calibration 14
10.1.2 Optimizing MS Parameters 14
10.1.3 Liquid Chromatography Instrument Conditions 15
10.1.4 Establish LC/ESI-MS/MS Retention Times and MRM Segments 15
10.2 Initial Calibration 15
10.2.1 Procedural Calibration Standards 15
10.2.2 Calibration 15
10.2.3 Calibration Acceptance Criteria 15
10.3 Continuing Calibration Checks (CCCs) 16
10.3.1 Aliquot Injection and Analysis 16
10.3.2 Verify Quantitation Ions 16
10.3.3 Calculate Concentration 16
10.4 Corrective Action 16
11 Procedure 16
11.1 Sample Preparation 16
11.1.1 Triple Freeze and Thaw Process 16
11.1.2 Dechlorinating and Preservation Agents 17
11.1.3 Fortify with PDS 17
11.1.4 Measure, Mix, Filter, etc 17
11.2 Sample Analysis 17
11.2.1 Establish LC/ESI-MS/MS Operating Conditions 17
11.2.2 Establish Initial Calibration 17
11.2.3 Analyze Field and QC Samples 17
11.3 The Analysis Batch 17
11.3.1 Analyze Initial CCC 17
11.3.2 Analyze Final CCC 18
12 Data Analysis and Calculations 18
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12.1 Establish a Retention Time Window 18
12.2 Identify Peaks of Interest 18
12.3 Calculate Analyte Concentrations 18
12.4 Round Concentrations 18
12.5 Review 18
12.6 Exceeding the Calibration Range 18
13 Method Performance 19
13.1 Precision, Accuracy and LCMRL 19
13.2 Analyte Stability Study 19
14 Pollution Prevention 19
15 Waste Management 19
16 References 19
17 Tables, Diagrams, Flowcharts and Validation Data 20
Table 1. HPLC Conditions 20
Table 2. Positive Mode ESI-MS/MS Method Conditions 20
Table 3. Analyte Retention Times, Ions, Cone Voltage, Collision Energy and IS Assignments 21
Table 4. IS Retention Times, Ions, Cone Voltage and Collision Energy 21
Table 5. Lowest Concentration Minimum Reporting Levels (LCMRLs) 21
Table 6. Precision and Accuracy in Fortified Reagent Water (n=7) 21
Table 7. Precision and Accuracy in Fortified Chlorinated Ground Watera (n=7) 22
Table 8. Precision and Accuracy in Fortified Moderate TOC Chlorinated Surface Watera (n=7) 22
Table 9. Aqueous Sample Holding Time Data 23
Table 10. Comparison of Fortified Reagent Water Samples 24
Table 11. Initial Demonstration of Capability (IDC) Quality Control Requirements 24
Table 12. Ongoing Quality Control Requirements 25
Figure 1. Example Chromatogram of ESI (+) Transitions for Method 545 Analytes 27
VI
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ai
1.1 Method
Method 545 is a liquid chromatography, electrospray ionization, tandem mass spectrometry (LC/ESI-
MS/MS) method for the determination of the algal toxins, cylindrospermopsin and anatoxin-a, in
finished drinking water. Method 545 requires the use of MS/MS in Multiple Reaction Monitoring (MRM)
mode to enhance selectivity. This method is intended for use by analysts skilled in the operation of
LC/ESI-MS/MS instrumentation and the interpretation of the associated data.
1.2 Analyte List
Method 545 is applicable for the measurement of the following analytes:
Analyte
Anatoxin-a
Cylindrospermopsin
Chemical Abstracts Services Registry Number
(CASRN)
64285-06-9
143545-90-8
1.3 Supporting Data
1,3,1 Precision and Accuracy
Precision and accuracy data were generated in reagent water and finished drinking water for both
ground water and surface water sources (Sect. 17, Tables 6 to 8).
1,3,2 Single Laboratory Lowest Concentration Minimum Reporting Levels
Single laboratory lowest concentration minimum reporting levels (LCMRLs) in this method were 0.018
and 0.063 micrograms per liter (u.g/L) for anatoxin-a and cylindrospermopsin, respectively (Sect. 17,
Table 5). The LCMRL is the lowest spiking concentration such that the probability of spike recovery in the
50% to 150% range is at least 99%. The procedure used to determine the LCMRL is described
elsewhere.- Laboratories using this method are not required to determine LCMRLs, but they must
demonstrate that the Minimum Reporting Level (MRL) for each analyte meets the requirements
described in Section 9.1.4.
1.4 Method Flexibility
The laboratory is allowed to select LC columns, LC conditions, and MS conditions different from those
used to develop the method. The two internal standards listed in this method must be used. However, if
isotopically labeled target analytes become available (they were not available at the time of method
development), they may be used to replace the internal standards listed in the method. Changes may
not be made to sample collection and preservation (Sect. 8) or the quality control (QC) requirements
(Sect. 9). Single quadrupole instruments are not permitted. Method modifications that improve method
performance are allowed. Modifications that are considered in the interest of reducing cost or sample
processing time, but result in poorer method performance, may not be used. Analytes should have
sufficient chromatographic resolution to allow the mass spectrometer to acquire a minimum of 10 scans
across a chromatographic peak. When method modifications are made, the analyst must perform the
procedures outlined in the Initial Demonstration of Capability (IDC, Sect. 9.1) and verify that all QC
acceptance criteria in the method (Sect. 17, Tables 11 and 12) are met. Additionally, the analyst must
verify method performance in a representative sample matrix (Sect. 9.3.2).
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2 , ' • >d
In the field, samples are added to bottles or vials containing ascorbic acid (dechlorinating agent) and
sodium bisulfate (microbial inhibitor). In the laboratory, aliquots (1 ml) of sample are taken for analysis,
and internal standards are added. An aliquot of the sample is injected into an LC equipped with an
analytical column that is interfaced to an MS/MS. The analytes are separated and identified by
comparing retention times and signals produced by unique mass transitions to retention times and
reference signals for procedural calibration standards acquired under identical LC-MS/MS conditions.
The concentration of each analyte is determined using the integrated peak area and the internal
standard technique.
Analysis Batch - A set of samples that are analyzed on the same instrument during a 24 hour period 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 the number of field
samples.
Calibration Standard - A solution of the method analytes and internal standards that is prepared from
the Primary Dilution Standards. The calibration standards are used to calibrate the instrument response
with respect to analyte concentration.
Continuing Calibration Check (CCC) - A calibration standard that is analyzed periodically to verify the
accuracy of the existing calibration.
Field Duplicates (FD) - Separate samples collected at the same time, shipped, and stored under identical
conditions. Method precision, including the contribution from sample collection procedures, is
estimated from the analysis of FDs. For the purposes of this method, Field Duplicates are necessary to
conduct repeat analyses if the original field sample is lost, or to conduct repeat analyses in the case of
QC failures associated with the analysis of the original field sample. Field Duplicates are used to prepare
Laboratory Fortified Sample Matrix and Laboratory Fortified Sample Matrix Duplicate QC samples.
Internal Standard (IS) - A pure compound that is added to all standard solutions and samples in a
known amount and used to measure the relative response of other method analytes that are
components of the same solution. The internal standard must respond similarly to the method analytes,
have no potential to be present in water samples, and not be a method analyte.
Ion Suppression/Enhancement-An observable decrease or increase in analyte response in complex
(field) samples as compared to the response obtained in standard solutions.
Laboratory Fortified Blank (LFB) - A volume of reagent water, containing method preservatives, to
which known quantities of the method analytes are added. The LFB is used during the IDC (Sect. 9.1) to
verify method performance for precision and accuracy.
Laboratory Fortified Sample Matrix (LFSM) - A field sample containing preservatives to which known
quantities of the method analytes are added in the laboratory. The LFSM is processed and analyzed as a
sample, and its purpose is to determine whether the sample matrix contributes bias to the analytical
results.
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Laboratory Fortified Sample Matrix Duplicate (LFSMD) - A Field Duplicate of the sample used to
prepare the LFSM which is fortified and analyzed identically to the LFSM. The LFSMD is used instead of
the Field Duplicate to assess method precision when the method analytes are rarely found at
concentrations greater than the MRL.
Laboratory Reagent Blank (LRB) - A volume of reagent water that is processed exactly as a sample
including exposure to all glassware, equipment, solvents and reagents, sample preservatives, and
internal standards. The LRB is used to determine if the method analytes or other interferences are
present in the laboratory environment, the reagents, or the apparatus.
Lowest Concentration Minimum Reporting Level (LCMRL) - The lowest true concentration for which
the future recovery is predicted to fall between 50% to 150% with 99% confidence.1
Material Safety Data Sheets (MSDS) - Written information provided by vendors concerning a chemical's
toxicity, health hazards, physical properties, fire and reactivity data, storage instructions, spill response
procedures, and handling precautions.
Minimum Reporting Level (MRL) - The minimum concentration that can be reported by a laboratory as
a quantified value for the method analyte in a sample following analysis. This concentration must meet
the criteria defined in Section 9.1.4 and must be no lower than the concentration of the lowest
calibration standard for each method analyte.
Multiple Reaction Monitoring (MRM) - A mass spectrometric technique in which a precursor ion is first
isolated, then subsequently fragmented into a product ion(s). Quantitation is accomplished by
monitoring a specific product ion. As described in Section 10.1.2, MS parameters must be optimized for
each precursor ion and product ion.
Precursor Ion - The gas-phase species corresponding to the method analyte that is produced in the ESI
interface. In MS/MS, the precursor ion is mass selected and fragmented by collision-activated
dissociation to produce distinctive product ions of smaller mass/charge (m/z) ratio.
Primary Dilution Standard (PDS) - A solution that contains the method analytes (or internal standards)
prepared from Stock Standard Solutions. A PDS solution is diluted to prepare calibration standards and
sample fortification solutions.
Procedural Calibration Standard - A calibration technique in which calibration standards are processed
through the entire method, including sample preparation and addition of preservatives. For this
method, reagent water is used as the aqueous medium.
Product Ion - One of the fragment ions that is produced in MS/MS by collision-activated dissociation of
the precursor ion.
Quality Control Sample (QCS) - A solution containing the method analytes at a known concentration
that is obtained from a source external to the laboratory and different from the source of calibration
standards. The purpose of the QCS is to verify the accuracy of the primary calibration standards.
Reagent Water - Purified water that does not contain any measurable quantity of the method analytes
or interfering compounds at or above 1/3 the MRL.
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Stock Standard Solution - A concentrated standard solution that is prepared in the laboratory using
assayed reference materials or that is purchased from a commercial source with a certificate of analysis.
4 Interferences
4.1 Clean Glassware
All glassware must be meticulously cleaned. Wash glassware with detergent and tap water, rinse with
tap water, followed by reagent water. Non-volumetric glassware may be heated in a muffle furnace at
400 °C for two hours or solvent rinsed. Volumetric glassware should be solvent rinsed and allowed to air
dry.
4.2 Reagent and Equipment Interferences
Method interferences may be caused by contaminants in solvents, reagents (including reagent water),
sample bottles and caps, and other sample processing hardware. These interferences may lead to
discrete artifacts or elevated baselines in the chromatograms or both. All laboratory reagents and
equipment must be routinely demonstrated to be free from interferences (less than 1/3 the MRL for the
target analytes) under the conditions of the analysis. This may be accomplished by analyzing LRBs as
described in Section 9.2.1.
4,3 Sample Matrix Interferences
Matrix interferences may be caused by contaminants that are present in the sample. The extent of
matrix interferences will vary considerably from source to source depending upon the nature of the
water. Matrix components may directly interfere by producing a signal at or near the retention time of
an analyte peak. Humic or fulvic material from environmental samples or both can cause enhancement
or suppression in the electrospray ionization source or both. Total organic carbon (TOC) is an indicator
of the humic content of a sample. Analysis of LFSMs (Sect. 9.2.5) provides evidence for the presence (or
absence) of matrix effects.
4,4 IS Purity
Depending on the source and purity, labeled analogs used as internal standards may contain a small
percentage of the corresponding native analyte. Such a contribution may be significant when attempting
to determine MRLs. The labeled internal standards must meet the purity requirements stated in the IDC
(Sect. 9.1.1).
5 Safety
Each chemical should be treated as a potential health hazard and exposure to these chemicals should be
minimized. Each laboratory is responsible for maintaining an awareness of OSHA regulations regarding
safe handling of chemicals used in this method. A reference file of MSDSs should be made available to
all personnel involved in the chemical analysis. Guidance for the handling of chemicals in the workplace
is available on the OSHA website.
G FquipmcMit and Supplies
References to specific brands or catalog numbers are included as examples only and do not imply
endorsement of the product. Such reference does not preclude the use of other vendors or suppliers.
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6.1 Sample Containers
Amber glass vials/bottles fitted with polytetrafluoroethylene (PTFE)-lined screw caps.
6.2 Autosampler Vials
Amber glass vials with PTFE/silicone septa.
6.3 Micro Syringes
Suggested sizes include 5, 10, 25, 50, 100, 250, 500, and 1000 microliters (ul).
6.4 Analytical Balance
Capable of weighing to the nearest 0.0001 gram (g).
6.5 Disposable Pasteur Pipettes
5 %-inch or 9-inch borosilicate glass, used to transfer samples to autosampler vials and for sample
preparation (Fisher Cat. No. 13-678-20B, 13-678-20C, or equivalent).
6.6 Disposable Syringes
3-mL, polypropylene, Luer Lock syringes for use in filtering standards and samples (Fisher Cat No. 03-
377-27, or equivalent).
6.7 Syringe Filters
13 mm, 0.2-u.m pore size PVDF filters (Fisher Cat No. 09-910-13, or equivalent).
6.8 Liquid Chromatography Electrospray lonization Tandem Mass Spectrometry System
(LC/ESI-MS/MS)
6.8.1 LC System
The LC system must provide consistent sample injection volumes and be capable of performing binary
linear gradients at a constant flow rate.
6.8.2 Analytical Column
The method was developed using a Waters XSelect HSST3 2.1 x 150 mm, 3.5-u.m column (Waters Part
No. 186006466). Any column that provides adequate resolution, peak shape, capacity, accuracy and
precision (Sect. 9), and does not exacerbate suppression or enhancement of analyte responses may be
used.
6.8.3 Electrospray lonization Tandem Mass Spectrometer (ESI-MS/MS)
The mass spectrometer must be capable of electrospray ionization. The system must be capable of
performing MS/MS to produce unique product ions for the method analytes within specified retention
time segments. A minimum of 10 scans across the chromatographic peak is needed to ensure adequate
precision.
6.8.4 MS/MS Data System
An interfaced data system is required to acquire, store and output MS data. The computer software
must have the capability of processing stored data by recognizing a chromatographic peak within a given
retention time window. The software must allow integration of the abundance of any specific ion
between specified time or scan number limits. The software must be able to construct a linear
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regression or quadratic regression calibration curve and calculate analyte concentrations using the
internal standard technique.
7 Reagents and Standards
7.1 Gases, Reagents Solvents
Reagent grade or better chemicals must be used. Unless otherwise indicated, all reagents will conform
to the specifications of the Committee on Analytical Reagents of the American Chemical Society (ACS),
where such specifications are available. Other grades may be used if the reagent is demonstrated to be
free of analytes and interferences and all requirements of the IDC (Sect. 9.1) are met when using these
reagents.
7.1.1 Acetic Acid; (CH3COOH, CASRN 64-19-7)
Glacial, HPLC grade (Fisher Cat. No. A35-500, or equivalent). Added to eluent as a mobile phase
modifier.
7,1,2 Collision Gas
High purity compressed gas (e.g., nitrogen or argon) used in the collision cell of the mass spectrometer.
The specific type of gas, purity and pressure requirements will depend on the instrument
manufacturer's specifications.
7.1.3 Desolvation Gas
High purity compressed gas (e.g., nitrogen or zero-air) used for desolvation in the mass spectrometer.
The specific type of gas, purity, and pressure requirements will depend on the instrument
manufacturer's specifications.
7.1.4 Methanol, (CH30H, CASRN 67-56-1)
LC/MS Grade (Fisher Optima, Fisher Cat. No. A456, or equivalent).
7,1,5 L-Ascorbic Acid; (C6H806; CASRN 50-81-7)
>99% (Sigma-Aldrich Cat. No. 255564, or equivalent). Added to reduce residual chlorine in finished
waters.
7,1,6 Reagent Water
Purified water that does not contain measurable quantities of any method analytes or interfering
compounds greater than 1/3 the MRL for each method analyte of interest.
7,1,7 Sodium Bisulfate, (NaHS04; CASRN 7681-38-1)
~95% (Sigma Cat. No. 71656, or equivalent). Used to inhibit microbial growth in dechlorinated water
samples.
7.2 Standard Solutions
The solution concentrations listed in this section were used to develop this method and are included
only as examples. The concentrations chosen for standards are at the discretion of the analyst to further
optimize the method (Sect. 1.4) as long as the IDC and ongoing QC requirements are met. PDS and
calibration standards were found to be stable for, at least, one month during method development.
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Laboratories should use standard QC practices to determine when standards need to be replaced. The
target analyte manufacturer's guidelines may be helpful when making the determination.
7.2,1 Internal Standards
Two isotopically enriched internal standards must be used in the method. The following table lists the
internal standards used in method development. If isotopically labeled target analytes are available,
they may be used in place of the standards below.
Internal Standard
Uracil-c/4, neat material
L-phenylalanine-c/s, neat
material
CASRNa
24897-55-0
56253-90-8
Catalog No.
C/D/N Isotopes D-5135
Cambridge Isotopes Labs DLM-
1258-1
° CASRN = Chemical Abstract Registry Number.
7,2,1,1 internal Standard Stock Standards (1000 [ig/mL)
Uracil-c/4 and L-phenylalanine-c/s stock standard solutions are prepared by weighing 10 mg of the solid
material into a tared 10-mL volumetric flask and diluting to volume with hot reagent water (uracil-c/4)
and methanol/reagent water (1:1) (L-phenylalanine-c/s).
7,2.1.2 Internal Standard Primary Dilution Standard (IS PDS) (0.250-1.00 |ig/mL)
The IS PDS is prepared in methanol/reagent water (1:1) and is stored at a temperature <-15 °C. Use 20
ulof the IS PDS to fortify the final 1-mL samples. This will yield a final concentration of 5.00 ng/mL L-
phenylalanine-c/s and 20.0 ng/mL Uracil-c/4 in the samples. Analysts are permitted to use other IS PDS
concentrations and volumes provided all samples and calibration standards contain the same final
concentration of the internal standards and adequate signal is obtained.
Internal Standard
Uracil-d4
L-phenylalanine-d5
Cone, of IS Stock (ng/mL)
1000
1000
Final Cone, of IS PDS (ng/mL)
1.00
0.250
7.2,2 Method Analyte Standard Solutions
7.2.2.1 Analyte Stock Standard Solution (1000 |ig/mL)
Obtain the analytes listed in the table in Section 1.2 as ampoulized solutions or as neat materials.
Prepare stock standards individually by weighing 10 mg of the solid standards into tared 10-mL
volumetric flasks and diluting to volume with methanol/reagent water (1:1). If only limited quantities of
neat material are available, stock standards may be prepared by adding a known volume of
methanol/reagent water (1:1) directly to manufacturers' vials and calculating the concentration based
on the mass provided by the manufacturer.
Note: Anatoxin-a may not be available as a solution or neat material. If another form of anatoxin-a is
used to prepare stock solutions (for example anatoxin-a fumarate), the analyst should correct for the
mass difference. For example
_ . MW anatoxma -_ T .
Corrected mass = x Measured mass
JVLW anatoxin a fumarate
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7.2.2.2 Analyte Primary Dilution Standard (Analyte PDS) (1.00 u.g/mL)
Prepare the Analyte PDS by diluting the Analyte Stock Standard solutions into methanol/reagent water
(1:1). An example preparation of the Analyte PDS that was used to collect data presented in Section 17
is provided in the table below. Analyte PDS is used to prepare calibration standards, and to fortify LFBs,
LFSMs, and LFSMDs with the method analytes.
Analyte Stock
Cylindrospermopsin
Anatoxin-a
Stock Concentration
(ug/mL)
100
1000 (asfumarate)
Analyte PDS
Concentration
(ug/mL)a
1.00
1.00 (asfumarate)
Calibration Range
(ng/mL)b
0.050-10.0
0.029-5.87C
a Multiple dilutions of the Analyte PDS were used in the preparation of calibration standards.
b Calibration curve concentration ranges used in method development.
c Corrected for anatoxin-a.
7,2.3 Procedural Calibration (CAL) Standards
The preparation of calibration standards requires the use of reagent water containing all sample
preservatives. Prepare at least five calibration standards over the concentration range of interest by
adding aliquots of Analyte PDS with the reagent water matrix and diluting to 1 ml. The lowest
calibration standard must be at or below the MRL Add a constant amount of the IS PDS to each 1-mL
calibration standard. The CAL standards may also be used as CCCs (Sect. 9.2.2).
ge
le Collects
8.1 Sample Bottle Preparation
8.1.1 Sample Containers
At a minimum, ten-milliliter amber glass bottles or vials with PTFE-lined screw caps. Collect additional
samples to fulfill the QC requirements for the frequency of field duplicates, LFSM, and LFSMD QC
samples (Sect. 9.2.5 and 9.2.6).
8.1.2 Addition of Preservatives
Preservation reagents, listed in the table below, are added to each sample container prior to shipment
to the field (or prior to sample collection).
Compound
Sodium bisulfate
Ascorbic acid
Amount
1.0 g/L
O.lOg/L
Purpose
Acidic microbial inhibitor
Reducing agent for chlorine
8.2 Sample Collection
When sampling from a cold water tap, remove the aerator, open the tap, and allow the system to flush
until the water temperature has stabilized (approximately three to five minutes). Invert the bottles or
vials several times to mix the sample with the preservation reagents. Fill sample bottles or vials taking
care not to flush out the preservatives. It is acceptable to leave head-space in the container.
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8,3 Sample Shipment arid Storage
Samples must be chilled during shipment and must not exceed 10 °C during the first 48 hours after
collection. Samples must be at or below 10 °C when they are received at the laboratory. In the
laboratory, samples must be stored at or below 6 °C and protected from light until analysis. Samples
must not be frozen.
8,4 Sample Holding Times
Results of the sample storage stability study (Sect. 17, Table 9) indicated that all compounds listed in the
method have adequate stability for 28 days when collected, preserved, shipped and stored as described
in Sections 8.1-8.3. Therefore, samples should be analyzed as soon as possible, but must be analyzed
within 28 days.
9 Quality Control
QC requirements include the IDC and ongoing QC requirements. This section describes each QC
parameter, its required frequency, and the performance criteria that must be met in order to satisfy EPA
quality objectives. The QC criteria discussed in the following sections are summarized in Section 17,
Tables 11 and 12. These QC requirements are considered the minimum acceptable QC.
9,1 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 generate an acceptable initial calibration following the procedure outlined in
Section 10.2.
9,1,1 Demonstration of Low System Background
Analyze a LRB. Confirm that the blank is free of contamination as defined in Section 9.2.1.
9,1,1,1 IS Purity
Depending on the source and purity, labeled internal standards may contain a small percentage of the
corresponding native analyte. Therefore, the analyst must demonstrate that the internal standards do
not contain the unlabeled analytes at a concentration >l/3 of the MRL when added at the selected
concentration to samples.
9.1.1.2 Carry-over
The system should also be checked for carry-over by analyzing a LRB immediately following the highest
CAL standard. If this sample does not meet the criteria outlined in Section 9.2.1, then carry-over is
present and should be eliminated.
9,1,2 Demonstration of Precision
Prepare and analyze four to seven replicate LFBs. Fortify these samples near the midrange of the initial
calibration curve. The method preservatives must be added to the LFBs as described in Section 8.1.2.
The percent relative standard deviation (%RSD) of the results of the replicate analyses must be <20%.
n, _ „_ Standard Deviation of Measured Concentrations ,
%RSD = xlOO
Average Concentration
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9,1,3 Demonstration of Accuracy
Using the same set of replicate data generated for Section 9.1.2, calculate the average percent recovery
(%R). The average percent recovery for each analyte must be within ±30% of the true value.
„._. Average Measured Concentration ,nn
%R = xlOO
Fortified Concentration
9,1,4 Minimum Reporting Level (MRL) Confirmation
Establish a target concentration for the MRL based on the intended use of the method. Analyze an
initial calibration following the procedures in Section 10. The lowest calibration standard used to
establish the initial calibration (as well as the low-level CCC) 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.
9.1.4.1 Fortify and Analyze Seven Replicate LFBs at or Below the Proposed MRL Concentration.
The LFBs must contain the method preservatives as specified in Section 8.1.2. Calculate the mean
(Mean) and standard deviation for these replicates. Determine the Half Range for the Prediction Interval
of Results (HRp/j?) using the equation:
HRpm = 3.963s
Where s is the standard deviation and 3.963 is a constant value for seven replicates.1
9.1.4.2 Confirm Upper and Lower Limits for the PIR
Confirm that the Upper and Lower limits for the Prediction Interval of Results (PIR = Mean +HRP/j?) meet
the upper and lower recovery limits as shown below.
The Upper PIR Limit must be <150 percent recovery.
Mean + HR
pm
—
Fortified Concentration
The Lower PIR Limit must be >50 percent recovery.
Mean - HRPIR
x 100 < 150%
Fortified Concentration
x 100> 50%
9.1.4.3 MRL Criteria
The MRL is validated if both the Upper and Lower PIR Limits meet the criteria described above. If these
criteria are not met, the MRL has been set too low and must be confirmed again at a higher
concentration.
Note: These equations are only valid for seven replicate samples.
9,1,5 Quality Control Sample (QCS)
Analyze a mid-level Quality Control Sample (Sect. 9.2.7) to confirm the accuracy of the primary
calibration standards.
10
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9,2 Ongoing QC Requirements
This section describes the ongoing QC elements that must be included when processing and analyzing
field samples.
9,2,1 Laboratory Reagent Blank (LRB)
Analyze a LRB with each Analysis Batch. The LRB must contain the method preservatives at the same
concentration as field samples. Background from method analytes or contaminants that interfere with
the measurement of method analytes must be
-------
9.2,5 Laboratory Fortified Sample Matrix (LFSM)
Within each Analysis Batch, analyze a minimum of one LFSM for every 20 samples. The background
concentrations of the analytes in the sample matrix must be determined in a separate aliquot and
subtracted from the measured values in the LFSM. If various sample matrixes are analyzed regularly, for
example, drinking water processed from ground water and surface water sources, performance data
should be collected for each source.
9.2.5,1 Prepare LFSM
Prepare the LFSM by fortifying a Field Duplicate with an appropriate amount of the Analyte PDS.
Generally, select a spiking concentration that is greater than or equal to the native concentration for
most analytes. Selecting a duplicate aliquot of a sample that has already been analyzed aids in the
selection of an appropriate spiking level. If this is not possible, use historical data when selecting a
fortifying concentration.
9.2,5,2 Calculate the %R
Calculate the %R using the equation:
c
Where:
A = measured concentration in the fortified sample,
B = measured concentration in the unfortified sample, and
C = fortification concentration.
9,2,5,3 Recoveries
Recoveries for samples fortified at concentrations near or at the MRL (within a factor of two times the
MRL concentration) must be within ±50% of the true value. Recoveries for samples fortified at all other
concentrations must be within ±30% of the true value. If the accuracy for any analyte falls outside the
designated range, and the laboratory performance for that analyte is shown to be in control in the CCCs
and in the LFB, the recovery is judged matrix biased. Report the result for the corresponding analyte in
the unfortified sample as "suspect/matrix."
Note: In order to obtain meaningful percent recovery results, correct the measured values in the LFSM
and LFSMDforthe native levels in the unfortified samples, even if the native values are less than the
MRL. This situation and the LRB background estimation are the only permitted uses of analyte results
below the MRL.
9.2,6 Field Duplicate or Laboratory Fortified Sample Matrix Duplicate {FD or LFSMD)
Within each Analysis Batch, analyze a minimum of one Field Duplicate or one Laboratory Fortified
Sample Matrix Duplicate. If the method analytes are not routinely observed in field samples, analyze an
LFSMD rather than an FD.
9.2.6,1 Relative Percent Difference Calculation
Calculate the relative percent difference (RPD) for duplicate measurements (FDi and FD2) using the
equation:
12
-------
FD, -FD7
RPD
7 - - -
(FDj+FDj/2
9.2.6.2 RPDs for Field Duplicates
RPDs for Field Duplicates should be <30% for each analyte. Greater variability may be observed when
Field Duplicates have analyte concentrations that are near or at the MRL (within a factor of two times
the MRL concentration). At these concentrations, Field Duplicates must have RPDs that are <50%. If the
RPD of an analyte falls outside the designated range, and the laboratory performance for the analyte is
shown to be in control in the CCC and in the LFB, the precision is judged matrix influenced. Report the
result for the corresponding analyte in the unfortified sample as "suspect/matrix."
9.2.6.3 RPDs for LFSMD
If an LFSMD is analyzed instead of a Field Duplicate, calculate the RPD for the LFSM and LFSMD using the
equation:
LFSM -LFSMD
(LFSM + LFSMD)/2
9.2.6.4 RPDs for Duplicate LFSMs
RPDs for duplicate LFSMs should be <30% for each analyte. Greater variability may be observed when
the matrix is fortified at analyte concentrations near or at the MRL (within a factor of two times the MRL
concentration). LFSMs at these concentrations must have RPDs that are <50%. If the RPD of an analyte
falls outside the designated range, and the laboratory performance for the analyte is shown to be in
control in the CCC and in the LFB, the precision is judged matrix influenced. Report the result for the
corresponding analyte in the unfortified sample as "suspect/matrix."
9.2.7 Quality Control Sample
As part of the IDC (Sect. 9.1), 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. Fortify the QCS near
the midpoint of the calibration range. The acceptance criteria for the QCS are the same as the mid- and
high-level CCCs (Sect. 10.3). If the accuracy for any analyte fails the recovery criterion, prepare fresh
standard dilutions and repeat the QCS evaluation.
9,3 Method Modification QC Requirements
The analyst is permitted to modify the separation technique, LC column, mobile phase composition, LC
conditions, and MS/MS conditions.
9.3.1 Repeat the Procedures of the IDC and Verify all QC
Each time method modifications are made, the analyst must repeat the procedures of the IDC (Sect. 9.1)
and verify that all QC criteria can be met in ongoing QC samples (Sect. 9.2).
9.3.2 Document Method Performance
The analyst is also required to evaluate and document method performance for the proposed
modifications in real matrixes that span the range of waters that the laboratory analyzes. This additional
step is required because modifications that perform acceptably in the IDC, which is conducted in reagent
13
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water, could fail ongoing method QC requirements in real matrixes. This is particularly important for
methods subject to matrix effects, such as LC/MS-based methods. For example, a laboratory may
routinely analyze drinking water from municipal treatment plants that process ground water, surface
water, or a blend of surface and ground water. In this case, this modification requirement could be
accomplished by assessing precision and accuracy (Sects. 9.1.2 and 9.1.3) in a surface water with
moderate to high total organic carbon (for example, 2 mg/L or greater) and a hard ground water (for
example, 250 mg/L as calcium carbonate (CaCO3) equivalent, or greater).
9,3,3 Document and Assess before Analyzing Field Samples
The results of Sections 9.3.1 and 9.3.2 must be appropriately documented by the analyst and
independently assessed by the laboratory's QA officer prior to analyzing field samples. When
implementing method modifications, it is the responsibility of the laboratory to closely review the
results of ongoing QC, and in particular, the results associated with the LFSM (Sect. 9.2.5), FD (Sect.
9.2.6), CCCs (Sect. 10.3), and the internal standard area counts (Sect. 9.2.4). If repeated failures are
noted, the modification must be abandoned.
10 Calibration and Standardization
Demonstration and documentation of acceptable MS calibration and initial analyte calibration are
required before performing the IDC (Sect. 9.1) and prior to analyzing field samples. The initial calibration
should be repeated each time a major instrument modification or maintenance is performed.
10.1 LC/ESI^MS/MS Calibration and Optimization
10,1,1 Mass Calibration
Calibrate the mass spectrometer with the calibration compounds and procedures specified by the
manufacturer.
10,1,2 Optimizing MS Parameters
Each LC/ESI-MS/MS system will have different optimal conditions, which are influenced by the source
geometry and system design. Due to the differences in design, follow the recommendations of the
instrument manufacturer when tuning the instrument. During the development of this method,
instrumental parameters were optimized for the precursor and product ions listed in Section 17, Table 3.
Product ions other than those listed may be selected; however, the analyst is cautioned to avoid using
ions with lower mass or common ions or both, that may not provide sufficient discrimination between
the analytes of interest and co-eluting interferences.
10.1.2.1 Optimize the Response of the Precursor Ion
Optimize the response of the precursor ion for each analyte by split infusion at the analytical flow rate
using approximately 1 u.g/mL of each analyte. Vary the MS parameters (source voltages, source and
desolvation temperatures, gas flows, etc.) until optimal analyte responses are obtained. The target
analytes may have different optimal instrument parameters, thus requiring some compromise on the
final operating conditions. See Section 17, Table 2 for the ESI-MS/MS conditions used in method
development.
14
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10.1.2.2 Optimize the Response of the Product Ion
Optimize the response of the product ion for each analyte by split infusion at the analytical flow rate
using approximately 1 u.g/mL of each analyte. Vary the MS/MS parameters (collision gas pressure,
collision energy, etc.) until optimal product ion responses are determined.
10,1,3 Liquid Chromatography Instrument Conditions
Establish LC operating parameters that optimize resolution. Suggested LC operating conditions are
described in Section 17, Table 1. Conditions different from those listed (for example, LC columns and
mobile phase compositions) may be used if the QC criteria in Sections 9.1, 9.2, and 9.3 are met and
chromatographic separation of the method analytes is achieved.
Note: Chromatographic separation as defined does not include isotopically enriched internal standards
(if used), which are mass separated. Co-elution of the internal standards with their analogous method
analytes helps mitigate matrix suppression or enhancement effects or both.
10,1,4 Establish LC/ESI-MS/MS Retention Times and Segments
Inject a mid- to high-level calibration standard under optimized LC/ESI-MS/MS conditions to obtain the
retention times of each method analyte. Divide the chromatogram into segments that contain one or
more chromatographic peaks. For maximum sensitivity in subsequent MS/MS analyses, minimize the
number of MRM transitions that are simultaneously monitored within each segment.
10,2 Initial Calibration
During method development, daily calibrations were performed; however, it is permissible to verify the
calibration with daily CCCs. Calibration must be performed using peak areas and the internal standard
technique. Calibration using peak heights or external standard calibration is not permitted.
10,2,1 Procedural Calibration Standards
Prepare a set of at least five calibration standards as described in Section 7.2.3. The analyte
concentrations in the lowest calibration standard must be at or below the MRL. Field samples must be
quantified using a calibration curve that spans the same concentration range used to collect the IDC
data (Sect. 9.1), that is, analysts are not permitted to use a restricted calibration range to meet the IDC
criteria and then use a larger dynamic range during analysis of field samples.
10,2,2 Calibration
Calibrate the LC/ESI-MS/MS and fit the calibration points with either a linear regression or quadratic
regression (response vs. concentration). Weighting may be used. Forcing the calibration curve through
the origin is not recommended. The MS/MS instruments used during method development were
calibrated using weighted (1/x) quadratic curves. Internal standard assignments appropriate for each
method analyte are presented in Section 17, Table 3. The MRM transitions for the internal standards are
provided in Section 17, Table 4.
10,2,3 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
-------
meet the criteria is due to contamination or standard degradation, prepare fresh CAL standards and
repeat the initial calibration.
10.3 Continuing Calibration Checks (CCCs)
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. The beginning CCC for each Analysis Batch must be
at or below the MRL This CCC verifies instrument sensitivity prior to the analysis of samples.
Subsequent CCCs should alternate between a medium and high concentration CAL standard.
10,3,1 Aliquot Injection and Analysis
Inject an aliquot of the appropriate concentration CAL standard and analyze with the same conditions
used during the initial calibration.
10,3,2 Verify Quantitation Ions
Verify that the absolute areas of the quantitation ions of each of the internal standards have not
changed by more than ±50% from the average areas measured during the initial calibration. If this limit
is exceeded, corrective action is necessary (Sect. 10.4).
10,3,3 Calculate Concentration
Calculate the concentration of each method analyte in the CCC. Each analyte fortified at a level
-------
(polyethylene barrel/polypropylene plunger) to allow for an efficient transfer and filtration of the
sample. The results of the freeze and thaw process and a comparison of plastic syringes and glass vials
can be seen in Section 17, Table 10.
11.1.2 Dechlorinating and Preservation Agents
Samples are dechlorinated, preserved, collected and stored as described in Section 8. All field and QC
samples must contain the dechlorinating and preservation agents listed in Section 8.1.2, including the
LRB.
11.1.3 Fortify with PDS
Fortify LFBs, LFSMs, or LFSMDs, with an appropriate volume of Analyte PDS (Sect. 7.2.2.2). Cap and
invert each sample several times to mix.
11,1,4 Measure, Mix, Filter, etc,
Measure 1 ml of each field or QC sample. Add the IS PDS (Sect. 7.2.1.2) and mix well. Filter each 1-mL
solution using 0.2 u.m PVDF filters and disposable syringes. Place the filtered solution in an autosampler
vial and cap. The filters used for calibration standards and samples must be of the same lot. If a new lot
of filters is used for subsequent Analysis Batches, the analyst must ensure all QC requirements are still
met.
11.2 Sample Analysis
11,2,1 Establish LC/ESI-MS/MS Operating Conditions
Establish LC/ESI-MS/MS operating conditions equivalent to those summarized in Tables 1-4 of Section
17 as per the guidance in Section 10.1. Column choice and instrument parameters should be optimized
prior to initiation of the IDC (Sect. 9.1).
11,2,2 Establish Initial Calibration
Establish a valid initial calibration following the procedures in Section 10.2 or confirm that the
calibration is still valid by analyzing a CCC (Sect. 10.3). If establishing an initial calibration for the first
time, complete the IDC as described in Section 9.1 prior to analyzing field samples.
11,2,3 Analyze Field and QC Samples
Analyze field and QC samples at appropriate frequencies in a properly sequenced Analysis Batch as
described in Section 11.3.
113 The Analysis
An Analysis Batch is a sequence of samples, analyzed within a 24-hour period, of no more than 20 field
samples and includes all required QC samples (LRB, CCCs, the LFSM and LFSMD (or FD)). The required QC
samples are not included in counting the maximum field sample total of 20. LC-MS/MS conditions for
the Analysis Batch must be the same as those used during calibration.
11,3,1 Analyze Initial CCC
After a valid calibration is established, begin every Analysis Batch by analyzing an initial low-level CCC at
or below the MRL This initial CCC must be within ±50% of the true value for each method analyte and
must pass the IS area criterion (Sect. 10.3.2). The initial CCC confirms that the calibration is still valid.
Failure to meet the QC criteria may indicate that recalibration is required prior to analyzing samples.
After the initial CCC, continue the Analysis Batch by analyzing an LRB, followed by field and QC samples
17
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at appropriate frequencies (Sect. 9.2). Analyze a mid- or high-level CCC after every ten field samples and
at the end each Analysis Batch. Do not count QC samples (LRBs, FDs, LFSMs, LFSMDs) when calculating
the required frequency of CCCs.
113,2 Analyze Final CCC
A final CCC completes the Analysis Batch. The acquisition start time of the final CCC must be within 24
hours of the acquisition start time of the initial low-level CCC at the beginning of the Analysis Batch.
More than one Analysis Batch within a 24-hour period is permitted.
12 Data Analysis and Calculations
12.1 Establish a Retention Time Window
Establish an appropriate retention time window for each analyte to identify them in the resulting
chromatograms. Base this assignment on measurements of actual retention time variation for each
compound in standard solutions over the course of time. The suggested variation is plus or minus three
times the standard deviation of the retention time for each compound for a series of injections. The
injections from the initial calibration and from the IDC (Sect. 9.1) may be used to calculate the retention
time window. However, the experience of the analyst should weigh heavily on the determination of an
appropriate range.
12,2 Identify Peaks of Interest
At the conclusion of data acquisition, use the same software settings established during the calibration
procedure to identify peaks of interest in the predetermined retention time windows. Confirm the
identity of each analyte by comparison of its retention time with that of the corresponding analyte peak
in an initial calibration standard or CCC.
12.3 Calculate Analyte Concentrations
Calculate analyte concentrations using the multipoint calibration established in Section 10.2. Report only
those values that fall between the MRL and the highest calibration standard.
12,4 Round Concentrations
Calculations must use 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.
12.5 Review
Prior to reporting the data, the chromatograms must be reviewed for any incorrect peak identifications
or improper integration. The laboratory is responsible for ensuring that QC requirements have been met
and that any appropriate qualifier is assigned.
12.6 Exceeding the Calibration
The analyst must not extrapolate beyond the established calibration range. If an analyte result exceeds
the range of the initial calibration curve, the sample may be diluted using reagent water containing all
preservatives and the appropriate amount of internal standard added to match the original level. Re-
inject the diluted sample. Incorporate the dilution factor into final concentration calculations. The
resulting data must be annotated as a dilution, and the reported MRLs must reflect the dilution factor.
18
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13 Method Performance
Single laboratory method performance data were collected using a Waters Acquity liquid
chromatograph coupled to a Micromass Quattro Premier XE triple quadrupole mass spectrometer.
13.1 Precision, Accuracy and LCMRL
Tables for single laboratory data are presented in Section 17. LCMRLs for each method analyte are
presented in Section 17, Table 5. Precision and accuracy are presented for three water matrixes: reagent
water (Sect. 17, Table 6); chlorinated (finished) groundwater (Sect. 17, Table 7); and moderate TOC
chlorinated (finished) surface water (Sect. 17, Table 8).
13.2 Analyte Stability Study
Chlorinated (finished) surface water samples, inoculated with diluted local microbial rich ambient water
and fortified with method analytes at 1.47-2.50 u.g/L (Anatoxin-a/Cylindrospermopsin), were preserved
as required in Section 8 and stored over a 28-day period. The percent change from the initial analyzed
concentration, observed after 7, 14, 21 and 28 days storage, is presented in Section 17, Table 9.
14 Po Prevention
For information about pollution prevention applicable to laboratory operations described in this
method, consult: Less is Better, Guide to Minimizing Waste in Laboratories, a web-based resource
available from the American Chemical Society website.
15 V\a-t> Manri^Hiiem
The analytical procedures described in this method generate relatively small amounts of waste since
only small amounts of reagents and solvents are used. The matrix of concern is finished drinking water.
However, the Agency requires that laboratory waste management practices 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. In addition, compliance
is required with any sewage discharge permits and regulations, particularly the hazardous waste
identification rules and land disposal restrictions.
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." Environmental Science & Technology 2006, 40, 281.
19
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17 Tables, Diagrams, Flowd
Table 1. HPLC Conditions
HPLCa
Column: Waters XSelect® HSS T3, 2.1 x 150 mm, 3.5 urn
Column temperature: 30 °C
Column flow rate: 0.20 mL/min
Autosampler temperature: 10 °C
Injection volume: 50 ul
Elution: Step Gradient
Time(min)
0.00
0.50
8.50
13.50
%100 mM acetic acid in reagent
waterb
100
90
100
100
%MeOH
0
10
0
0
a Waters Acquity LC system
b Preparation of 100 mM acetic acid in reagent water: Combine 5.8 mL concentrated acetic acid and dilute to
volume with reagent water in a 1 L volumetric flask.
Table 2, Positive Mode ES1-MS/MS Method Conditions
MS Parameter
Polarity
Capillary Voltage, kV
Source Temperature, °C
N2 Desolvation Temperature, °C
N2 Desolvation Gas Flow, L/hr
Cone Gas Flow, L/hr
Extractor Lens, V
RF Lens, V
MS/MSa
Positive ion
electrospray
4.00
120
350
800
100
2.00
0.1
Micromass Quattro Premier XE triple quadrupole mass spectrometer
20
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Table 3. Analyte Retention Times, Ions, Cone Voltage, Collision Energy and IS Assignments
Analyte
Cylindrospermopsin
Anatoxin-a
Ret. Time
(min)
4.57
5.74
ESI Mode
ESI+
ESI+
Precursor
Ion
416.2
165.8
Product Ion
194.0
148.8
Cone
Voltage (V)
35
25
Collision
Energy (eV)
35
15
Dwell Time
(s)
0.250
0.250
Internal Standard
Uracil-c/4
L-phenylalanine-c/s
Table 4. IS Retention Times, Ions, Cone Voltage Collision Energy
Internal Standard
Uracil-c/4
L-phenylalanine-c/s
Ret. Time
(min)
3.85
8.00
ESI Mode
ESI+
ESI+
Precursor Ion
114.8
170.8
Product Ion
97.8
124.8
Cone Voltage
(V)
28
18
Collision
Energy (eV)
15
10
Dwell Time (s)
0.250
0.250
Table 5. Lowest Concentration Minimum Reporting Levels (LCMRLs)
Analyte
Cylindrospermopsin
Anatoxin-a
LCMRL (u,g/L)a
0.063
0.018
a LCMRLs were calculated according to the procedure in reference 1 with the following modification: Instead of evaluating seven replicates at four
concentration levels, LCMRLs are now obtained by analyzing four replicates at seven concentration levels.
Table 6. Precision Accuracy in Fortified Reagent Water (n=7)
Low Concentration
Analyte
Cylindrospermopsin
Anatoxin-a
Fortified Concentration (ug/L)
0.100
0.059
Avg. %Recovery
109
111
%RSD
11
7.4
High Concentration
Analyte
Cylindrospermopsin
Anatoxin-a
Fortified Concentration (ug/L)
2.50
1.47
Avg. %Recovery
109
109
%RSD
3.6
2.9
21
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Table ?, Precision and Accuracy in Fortified Chlorinated Ground Watera (n=7)
Low Concentration
Analyte
Cylindrospermopsin
Anatoxin-a
Fortified Concentration (ng/L)
0.100
0.059
Avg. %Recovery
126
95.4
%RSD
5.2
5.3
High Concentration
Analyte
Cylindrospermopsin
Anatoxin-a
Fortified Concentration (ng/L)
2.50
1.47
Avg. %Recovery
98.0
95.0
%RSD
2.2
1.8
a Ground water physical parameters: total hardness = 325 milligrams/liter (mg/L) (as CaCOs); free chlorine = 0.69 mg/L; total chlorine = 0.97 mg/L; conductivity
= 798 u.S.
Table 8, Precision and Accuracy in Fortified Moderate TOC Chlorinated Surface Watera (n=7)
Low Concentration
Analyte
Cylindrospermopsin
Anatoxin-a
Fortified Concentration (ng/L)
0.100
0.059
Avg. %Recovery
117
96.9
%RSD
10
6.2
High Concentration
Analyte
Cylindrospermopsin
Anatoxin-a
Fortified Concentration (ng/L)
2.50
1.47
Avg. %Recovery
108
100
%RSD
1.6
2.8
a Surface water physical parameters: total hardness = 142 milligrams/liter (mg/L) (as CaCOs); free chlorine = 0.64 mg/L; total chlorine = 1.14 mg/L; conductivity
= 344 u.S; TOC = 3.04 ppm.
22
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Table 9. Aqueous Sample Time
For samples from chlorinated surface watera, fortified with method analytes and preserved and stored according to method section 8 (n=3).
Analyte
Cylindrospermopsin
Anatoxin-a
Cylindrospermopsin
Anatoxin-a
Cylindrospermopsin
Anatoxin-a
Cylindrospermopsin
Anatoxin-a
Cylindrospermopsin
Anatoxin-a
Fortified
Concentration (ng/L)
2.50
1.47
2.50
1.47
2.50
1.47
2.50
1.47
2.50
1.47
Day
0
0
7
7
14
14
21
21
28
28
Average Measured Concentration
(Hg/L)
2.57
1.49
Not applicable.
Not applicable.
Not applicable.
Not applicable.
Not applicable.
Not applicable.
Not applicable.
Not applicable.
% Change from Day Ob
Not applicable.
Not applicable.
-5.4
0.67
-6.2
-7.4
-5.1
-0.67
-1.6
-1.3
%RSD
3.3
2.6
2.4
4.4
1.5
2.4
1.2
0.91
3.5
2.1
a Surface water physical parameters: total hardness = 142 milligrams/liter (mg/L) (as CaCOs); free chlorine = 0.64 mg/L; total chlorine = 1.14 mg/L; conductivity
= 344 u.S; TOC = 3.04 ppm.
b % Change from Day 0 calculation: (Day x mean concentration - Day 0 mean concentration) / Day 0 mean concentration * 100%, where x is the analysis day.
23
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Table 10. Comparison of Fortified Reagent Water Samples
Comparison of fortified reagent water samples in contact with plastic and glass during a triple freeze and thaw process a (n=4).
Plastic Contact
Analyte
Cylindrospermopsin
Anatoxin-a
Fortified Concentration (ng/L)
2.50
1.47
Avg. %Recovery
110
94.1
%RSD
4.3
1.0
Glass Contact
Analyte
Cylindrospermopsin
Anatoxin-a
Fortified Concentration (ng/L)
2.50
1.47
Avg. %Recovery
111
98.4
%RSD
4.1
2.4
a Samples were frozen at -30°C for 1 h, then thawed in a 40°C water bath for 5 min. The process was repeated two more times prior to the addition of an IS
and analysis.
Table 11. Initial Demonstration of Capability (IDC) Quality Control
'~ments
Method Reference
Section 9. 1.1
Section 9. 1.2
Section 9. 1.3
Requirement
Demonstration of low system
background
Demonstration of precision
Demonstration of accuracy
Specification and Frequency
Analyze a Laboratory Reagent
Blank (LRB) prior to any other IDC
steps and after the highest CAL
standard to check for carry-over.
Prepare and analyze 4-7
replicate Laboratory Fortified
Blanks (LFBs) fortified near the
midrange concentration.
Calculate average percent
recovery for replicates used in
Section 9. 1.2.
Acceptance Criteria
Demonstrate that all method
analytes are
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Method Reference
Section 9. 1.4
Section 9. 1.5
Requirement
MRL confirmation
Quality Control Sample (QCS)
Specification and Frequency
Fortify and analyze 7 replicate
LFBs at the proposed MRL
concentration. Confirm that the
Upper Prediction Interval of
Results (PIR) and Lower PIR (Sect.
9.1.4.1 and Sect. 9.1.4.2) meet
the recovery criteria.
Analyze mid-level QCS.
Acceptance Criteria
Upper PIR <150%; Lower PIR
>50%
Results must be within ±30% of
the true value.
Table 12. Ongoing Quality Control Requirements
Method Reference
Requirement
Specification and Frequency
Acceptance Criteria
Section 10.2
Initial calibration
Use the internal standard
calibration technique to generate
a linear or quadratic calibration
curve. Use at least five calibration
concentrations. Validate the
calibration curve as described in
Section 10.2.3.
When each calibration standard
is calculated as an unknown using
the regression equation, the
lowest level standard must be
within ±50% of the true value. All
other points must be within
±30% of the true value.
Section 9.2.1
Laboratory Reagent Blank (LRB)
Analyze one LRB with each
Analysis Batch.
Demonstrate that all method
analytes are
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Method Reference
Section 10.3
Section 9.2.4
Section 9.2.5
Section 9.2.6
Section 9.2.7
Requirement
Continuing Calibration Check
(CCC)
Internal standard (IS)
Laboratory Fortified Sample
Matrix (LFSM)
Laboratory Fortified Sample
Matrix Duplicate (LFSMD) or Field
Duplicate (FD)
Quality Control Sample (QCS)
Specification and Frequency
Verify initial calibration by
analyzing a low-level CCC at the
beginning of each Analysis Batch.
Subsequent CCCs are required
after every 10 field samples, and
at the end of the Analysis Batch.
Internal standards are added to
all standards and samples.
Analyze one LFSM per Analysis
Batch. Fortify the LFSM with
method analytes at a con-
centration greater than the
native concentrations.
Analyze at least one LFSMD or FD
with each Analysis Batch.
Analyze mid-level QCS at least
quarterly.
Acceptance Criteria
The lowest level CCC must be
within ±50% of the true value. All
other points must be within
±30% of the true value. Internal
standards must be ±50% of the
average peak areas in the initial
calibration. Results for field
samples that are not bracketed
by acceptable CCCs are invalid.
Peak area counts for each IS must
be within ±50% of the average
peak areas in the initial
calibration.
For LFSMs fortified at
concentrations <2 x MRL, the
calculated recovery must be
within +50% of the true value. At
concentrations greater than the 2
x MRL, the recovery must be
±30% of the true value.
ForLFSMDsorFDs, the
calculated relative percent
difference must be <30%. (<50%
if concentration <2 x MRL.)
Results must be ±30% of the true
value.
26
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Figure 1. Example Chromatogram of ESI (+) Transitions for Method 545 Analytes
100n
UraciW4
(IS)
L-phenylalanine-^5
(IS)
ANA
CYN
000
ri r ' • i '
2.00 4.00
t:00 800 10,00
i ' ' ' ' i
' i ' ' ' ' i ' ' Time
12.00
27
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