Method 545: Determination of Cylindrospermopsin and 

Anatoxin-a in Drinking Water by Liquid Chromatography 

Electrospray Ionization Tandem Mass Spectrometry 

(LC/ESI-MS/MS)
Questions concerning this document should be addressed to:

Steven C. Wendelken, PhD

U.S. EPA, Office of Ground Water and Drinking Water, Standards and Risk Management DivisionTechnical Support Center, 26 W. Martin Luther King Dr. Cincinnati, OH 45268

Phone: (513) 569-7491

wendelken.steve@epa.gov

Office of Water (MS-140)

EPA 815-R-15-009

EPA contract EP-C-12-013

April 2015

i
Acknowledgements

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

ii
Table of Contents

Acknowledgements....................................................................................................................................... ii

1 Scope and Application...........................................................................................................................1

1.1 Method .........................................................................................................................................1

1.2 Analyte List....................................................................................................................................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 Ionization Tandem Mass Spectrometry System (LC/ESIMS/MS) .....................................................................................................................................................5

6.8.1 LC System..............................................................................................................................5

6.8.2 Analytical Column .................................................................................................................5

6.8.3 Electrospray Ionization 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

iii
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-Ascorbic Acid, (C6H8O6, CASRN 50-81-7)..........................................................................6

7.1.6 Reagent Water......................................................................................................................6

7.1.7 Sodium Bisulfate, (NaHSO4, 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
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

v
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 Water a (n=7) ...................................22

Table 8. Precision and Accuracy in Fortified Moderate TOC Chlorinated Surface Water a (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
1 Scope and Application

1.1 Method

Method 545 is a liquid chromatography, electrospray ionization, tandem mass spectrometry (LC/ESIMS/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 Chemical Abstracts Services Registry Number 

(CASRN)

Anatoxin-a 64285-06-9

Cylindrospermopsin 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 (μg/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.1 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).

1
2 Summary of Method

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.

3 Definitions

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.

2
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.

3
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 o

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.

6 Equipment 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.

4
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 (μL).

6.4 Analytical Balance

Capable of weighing to the nearest 0.0001 gram (g).

6.5 Disposable Pasteur Pipettes

5 3⁄4-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-μm pore size PVDF filters (Fisher Cat No. 09-910-13, or equivalent).

6.8 Liquid Chromatography Electrospray Ionization 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 HSS T3 2.1 x 150 mm, 3.5-μm 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 Ionization 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 

5
regression or quadratic regression calibration curve and calculate analyte concentrations using the 

internal standard technique.

7 Reagents and Standards

7.1 Gases, Reagents and 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, (CH3OH, CASRN 67-56-1)

LC/MS Grade (Fisher Optima, Fisher Cat. No. A456, or equivalent).

7.1.5 L-Ascorbic Acid, (C6H8O6, 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, (NaHSO4, 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. 

6
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 CASRNa Catalog No.

Uracil-d4, neat material 24897-55-0 C/D/N Isotopes D-5135

L-phenylalanine-d5, neat 

material

56253-90-8 Cambridge Isotopes Labs DLM1258-1 a CASRN = Chemical Abstract Registry Number.

7.2.1.1 Internal Standard Stock Standards (1000 μg/mL)

Uracil-d4 and L-phenylalanine-d5 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-d4) 

and methanol/reagent water (1:1) (L-phenylalanine-d5).

7.2.1.2 Internal Standard Primary Dilution Standard (IS PDS) (0.250–1.00 μg/mL)

The IS PDS is prepared in methanol/reagent water (1:1) and is stored at a temperature <-15 o

C. Use 20 

μL of the IS PDS to fortify the final 1-mL samples. This will yield a final concentration of 5.00 ng/mL Lphenylalanine-d5 and 20.0 ng/mL Uracil-d4 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 Conc. of IS Stock (μg/mL) Final Conc. of IS PDS (μg/mL)

Uracil-d4 1000 1.00

L-phenylalanine-d5 1000 0.250

7.2.2 Method Analyte Standard Solutions

7.2.2.1 Analyte Stock Standard Solution (1000 μg/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

Measured mass

MW 

MW Corrected mass

anatoxin a fumarate

anatoxin a

= ×

7
7.2.2.2 Analyte Primary Dilution Standard (Analyte PDS) (1.00 μg/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 Stock Concentration 

(μg/mL)

Analyte PDS 

Concentration 

(μg/mL)a

Calibration Range 

(ng/mL)

b

Cylindrospermopsin 100 1.00 0.050–10.0

Anatoxin-a 1000 (as fumarate) 1.00 (as fumarate) 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).

8 Sample Collection, Preservation and Storage

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 Amount Purpose

Sodium bisulfate 1.0 g/L Acidic microbial inhibitor

Ascorbic acid 0.10 g/L 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.

8
8.3 Sample Shipment and Storage

Samples must be chilled during shipment and must not exceed 10 °C during the first 48 hours after 

collection. 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 >1/3 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%.

100

AverageConcentration

Standard Deviation of Measured Concentrations % RSD = ×

9
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.

100

Fortified Concentration

Average Measured Concentration %R = ×

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 (HRPIR) using the equation:

HRPIR = 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 +HRPIR) meet 

the upper and lower recovery limits as shown below.

The Upper PIR Limit must be ≤150 percent recovery.

Mean HR

FortifiedConcentration

PIR +

× 100 ≤ 150%

The Lower PIR Limit must be ≥50 percent recovery.

Mean HR

FortifiedConcentration

PIR − × 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
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 ≤1/3 of the MRL. If method analytes are detected in the 

LRB at concentrations greater than this level, then all data for the problem analyte(s) must be 

considered invalid for all samples in the Analysis Batch. Subtracting blank values from sample results is 

not permitted.

Note: Although quantitative data below the MRL may not be accurate enough for data reporting, such 

data are useful in determining the magnitude of background interference. Therefore, blank 

contamination levels may be estimated by extrapolation when the concentration is below the MRL.

9.2.2 Continuing Calibration Check (CCC)

Analyze CCC standards at the beginning of each Analysis Batch, after every ten field samples, and at the 

end of the Analysis Batch. See Section 10.3 for concentration requirements and acceptance criteria for 

CCCs.

9.2.3 Laboratory Fortified Blank (LFB)

Since this method utilizes procedural calibration standards, which are fortified reagent waters, there is 

no difference between the LFB and the continuing calibration check standard. Consequently, the 

analysis of a separate LFB is not required as part of the ongoing QC; however, the acronym LFB is used 

for clarity in the IDC. 

9.2.4 Internal Standards (IS)

The analyst must monitor the peak areas of the internal standards in all injections of the Analysis Batch. 

The internal standard responses (as indicated by peak areas) for any chromatographic run must not 

deviate by more than ±50% from the average areas measured during the initial calibration for the 

internal standards. If the IS areas in a chromatographic run do not meet these criteria, check the 

corresponding IS of the most recent CCC and proceed as follows:

9.2.4.1 IS Failure in Sample but not CCC

If the IS criterion is met in the CCC but not in the sample, reanalyze the sample in the same or 

subsequent Analysis Batch. If the reanalyzed sample produces an acceptable IS response, report results 

for that injection. If the IS area count fails to meet the acceptance criterion in the repeated analysis but 

still passes the most recent CCC, report the sample results as “suspect/matrix.” Alternatively, analyze a 

FD or collect a new sample and reanalyze.

9.2.4.2 IS Failure in Sample and CCC

If both the original sample and the CCC fail the IS criteria, take corrective action (e.g., Sect. 10.4). It 

might be helpful to check the integrity of the IS solution and the fortification technique before 

reanalyzing the sample in a subsequent Analysis Batch. After corrective action, re-inject the sample in a 

subsequent Analysis Batch. If the IS area fails to meet the acceptance criterion in the repeat analysis, 

but passes in the most recent CCC, report the sample results as “suspect/matrix.”

11
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:

( ) 100

C

A - B %R = ×

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 LFSMD for the 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 (FD1 and FD2) using the 

equation:

12
( ) 100

FD FD / 2

FD FD

RPD

1 2

1 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:

( ) 100

LFSM LFSMD /2

LFSM LFSMD

RPD ×

+

− =

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
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 μg/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
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 μg/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 MRM 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 ≤MRL, the result for each analyte should be within 

±50% of the true value. All other calibration points should calculate to be within ±30% of their true 

value. If these criteria cannot be met, the analyst will have difficulty meeting ongoing QC criteria. In this 

case, reanalyze the calibration standards or restrict the range of calibration. If the cause for failure to 

15
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 ≤MRL 

must calculate to be within ±50% of the true value. The calculated concentration of the method analytes 

in CCCs fortified at all other levels must be within +30%. If these limits are exceeded, then all data for 

the failed analytes must be considered invalid. Any field samples analyzed since the last acceptable CCC 

that are still within holding time must be reanalyzed after an acceptable calibration has been restored.

10.4 Corrective Action

Failure to meet the CCC QC performance criteria requires corrective action. Acceptable method 

performance may be restored simply by flushing the column with 100% methanol. Following this and 

other minor remedial action, check the calibration with a mid-level CCC and a CCC at the MRL, or 

recalibrate according to Section 10.2. If internal standard and calibration failures persist, maintenance 

may be required, such as servicing the ESI-MS/MS system or replacing the LC column. These latter 

measures constitute major maintenance, and the analyst must return to the initial calibration step (Sect. 

10.2).

11 Procedure

This section describes the procedures for sample preparation and analysis. Important aspects of this 

analytical procedure include proper sample collection and storage (Sect. 8), ensuring that the 

instrument is properly calibrated (Sect. 10), and that all required QC elements are included (Sect. 9).

11.1 Sample Preparation

11.1.1 Triple Freeze and Thaw Process

If necessary, a triple freeze and thaw process may be used on samples prior to filtration and analysis in 

order to address the potential presence of intact algal cells in finished water samples. During method 

development, the process was evaluated by freezing samples for 1 h at -30°C, then thawing samples in a 

40°C water bath for 5 min. The freeze and thaw process was repeated two more times prior to the 

addition of an IS and analysis. Water samples were frozen and thawed inside plastic syringes 

16
(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 μm 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.

11.3 The Analysis Batch

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
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.

11.3.2 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 Range

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. Reinject 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
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 μg/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 Pollution 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 Waste Management

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.

16 References

1. Winslow, S. D.; Pepich, B. V.; Martin, J. J.; Hallberg, G. R.; Munch, D. J.; Frebis, C. P.; Hedrick, E. J.; 

Krop, R. A. "Statistical Procedures for Determination and Verification of Minimum Reporting 

Levels for Drinking Water Methods." Environmental Science & Technology 2006, 40, 281.

19
17 Tables, Diagrams, Flowcharts and Validation Data

Table 1. HPLC Conditions

HPLCa

Column: Waters XSelect® HSS T3, 2.1 x 150 mm, 3.5 μm

Column temperature: 30 o

C

Column flow rate: 0.20 mL/min

Autosampler temperature: 10 o

C

Injection volume: 50 μL

Elution: Step Gradient

Time (min) %100 mM acetic acid in reagent 

waterb

%MeOH

0.00 100 0

0.50 90 10

8.50 100 0

13.50 100 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 ESI-MS/MS Method Conditions

MS Parameter MS/MSa

Polarity Positive ion electrospray

Capillary Voltage, kV 4.00

Source Temperature, oC 120

N2 Desolvation Temperature, oC 350

N2 Desolvation Gas Flow, L/hr 800

Cone Gas Flow, L/hr 100

Extractor Lens, V 2.00

RF Lens, V 0.1 a Micromass Quattro Premier XE triple quadrupole mass spectrometer

20
Table 3. Analyte Retention Times, Ions, Cone Voltage, Collision Energy and IS Assignments

Analyte Ret. Time 

(min)

ESI Mode Precursor 

Ion

Product Ion Cone 

Voltage (V)

Collision 

Energy (eV)

Dwell Time 

(s)

Internal Standard

Cylindrospermopsin 4.57 ESI+ 416.2 194.0 35 35 0.250 Uracil-d4

Anatoxin-a 5.74 ESI+ 165.8 148.8 25 15 0.250 L-phenylalanine-d5

Table 4. IS Retention Times, Ions, Cone Voltage and Collision Energy

Internal Standard Ret. Time 

(min)

ESI Mode Precursor Ion Product Ion Cone Voltage 

(V)

Collision 

Energy (eV)

Dwell Time (s)

Uracil-d4 3.85 ESI+ 114.8 97.8 28 15 0.250

L-phenylalanine-d5 8.00 ESI+ 170.8 124.8 18 10 0.250

Table 5. Lowest Concentration Minimum Reporting Levels (LCMRLs)

Analyte LCMRL (μg/L)a

Cylindrospermopsin 0.063

Anatoxin-a 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 and Accuracy in Fortified Reagent Water (n=7)

Low Concentration

Analyte Fortified Concentration (μg/L) Avg. %Recovery %RSD

Cylindrospermopsin 0.100 109 11

Anatoxin-a 0.059 111 7.4

High Concentration

Analyte Fortified Concentration (μg/L) Avg. %Recovery %RSD

Cylindrospermopsin 2.50 109 3.6

Anatoxin-a 1.47 109 2.9

21
Table 7. Precision and Accuracy in Fortified Chlorinated Ground Water a (n=7)

Low Concentration

Analyte Fortified Concentration (μg/L) Avg. %Recovery %RSD

Cylindrospermopsin 0.100 126 5.2

Anatoxin-a 0.059 95.4 5.3

High Concentration

Analyte Fortified Concentration (μg/L) Avg. %Recovery %RSD

Cylindrospermopsin 2.50 98.0 2.2

Anatoxin-a 1.47 95.0 1.8 a Ground water physical parameters: total hardness = 325 milligrams/liter (mg/L) (as CaCO3); free chlorine = 0.69 mg/L; total chlorine = 0.97 mg/L; conductivity 

= 798 μS.

Table 8. Precision and Accuracy in Fortified Moderate TOC Chlorinated Surface Water a (n=7)

Low Concentration

Analyte Fortified Concentration (μg/L) Avg. %Recovery %RSD

Cylindrospermopsin 0.100 117 10

Anatoxin-a 0.059 96.9 6.2

High Concentration

Analyte Fortified Concentration (μg/L) Avg. %Recovery %RSD

Cylindrospermopsin 2.50 108 1.6

Anatoxin-a 1.47 100 2.8 a Surface water physical parameters: total hardness = 142 milligrams/liter (mg/L) (as CaCO3); free chlorine = 0.64 mg/L; total chlorine = 1.14 mg/L; conductivity 

= 344 μS; TOC = 3.04 ppm.

22
Table 9. Aqueous Sample Holding Time Data

For samples from chlorinated surface water a, fortified with method analytes and preserved and stored according to method section 8 (n=3).

Analyte Fortified 

Concentration (μg/L)

Day Average Measured Concentration 

(μg/L)

% Change from Day 0b % RSD

Cylindrospermopsin 2.50 0 2.57 Not applicable. 3.3

Anatoxin-a 1.47 0 1.49 Not applicable. 2.6

Cylindrospermopsin 2.50 7 Not applicable. -5.4 2.4

Anatoxin-a 1.47 7 Not applicable. 0.67 4.4

Cylindrospermopsin 2.50 14 Not applicable. -6.2 1.5

Anatoxin-a 1.47 14 Not applicable. -7.4 2.4

Cylindrospermopsin 2.50 21 Not applicable. -5.1 1.2

Anatoxin-a 1.47 21 Not applicable. -0.67 0.91

Cylindrospermopsin 2.50 28 Not applicable. -1.6 3.5

Anatoxin-a 1.47 28 Not applicable. -1.3 2.1 a Surface water physical parameters: total hardness = 142 milligrams/liter (mg/L) (as CaCO3); free chlorine = 0.64 mg/L; total chlorine = 1.14 mg/L; conductivity 

= 344 μS; 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
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 Fortified Concentration (μg/L) Avg. %Recovery %RSD

Cylindrospermopsin 2.50 110 4.3

Anatoxin-a 1.47 94.1 1.0

Glass Contact

Analyte Fortified Concentration (μg/L) Avg. %Recovery %RSD

Cylindrospermopsin 2.50 111 4.1

Anatoxin-a 1.47 98.4 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 Requirements

Method Reference Requirement Specification and Frequency Acceptance Criteria

Section 9.1.1 Demonstration of low system 

background

Analyze a Laboratory Reagent 

Blank (LRB) prior to any other IDC 

steps and after the highest CAL 

standard to check for carry-over.

Demonstrate that all method 

analytes are ≤1/3 of the 

Minimum Reporting Level (MRL) 

and that possible interferences 

from reagents and glassware do 

not prevent the identification 

and quantitation of method 

analytes.

Section 9.1.2 Demonstration of precision Prepare and analyze 4–7 

replicate Laboratory Fortified 

Blanks (LFBs) fortified near the 

midrange concentration.

Percent relative standard 

deviation must be ≤20%.

Section 9.1.3 Demonstration of accuracy Calculate average percent 

recovery for replicates used in 

Section 9.1.2.

Average percent recovery within 

±30% of the true value.

24
Method Reference Requirement Specification and Frequency Acceptance Criteria

Section 9.1.4 MRL confirmation 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.

Upper PIR ≤150%; Lower PIR 

≥50%

Section 9.1.5 Quality Control Sample (QCS) Analyze mid-level QCS. 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 ≤1/3 the Minimum 

Reporting Level (MRL), and that 

possible interferences from 

reagents and glassware do not 

prevent identification and 

quantitation of method analytes.

25
Method Reference Requirement Specification and Frequency Acceptance Criteria

Section 10.3 Continuing Calibration Check 

(CCC)

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.

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.

Section 9.2.4 Internal standard (IS) Internal standards are added to 

all standards and samples.

Peak area counts for each IS must 

be within ±50% of the average 

peak areas in the initial 

calibration.

Section 9.2.5 Laboratory Fortified Sample 

Matrix (LFSM)

Analyze one LFSM per Analysis 

Batch. Fortify the LFSM with 

method analytes at a concentration greater than the 

native concentrations.

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.

Section 9.2.6 Laboratory Fortified Sample 

Matrix Duplicate (LFSMD) or Field 

Duplicate (FD)

Analyze at least one LFSMD or FD 

with each Analysis Batch.

For LFSMDs or FDs, the 

calculated relative percent 

difference must be ≤30%. (≤50% 

if concentration ≤2 x MRL.)

Section 9.2.7 Quality Control Sample (QCS) Analyze mid-level QCS at least 

quarterly.

Results must be ±30% of the true 

value.

26
Figure 1. Example Chromatogram of ESI (+) Transitions for Method 545 Analytes

27