EPA-OGWDW/TSC: 545: Determination of Cylindrospermopsin and Anatoxin-a in Drinking Water by LC/ESI-MS/MS

Summary
Analytes
Revision
Data and Sites

Official Method Name

Method 545: Determination of Cylindrospermopsin and Anatoxin-a in Drinking Water by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC/ESI-MS/MS)

Current Revision

2015

Media

WATER

Instrumentation

Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry

Method Subcategory

Biotoxin

Method Source

EPA-OGWDW/TSC

Citation

Method 545: Determination of Cylindrospermopsin and Anatoxin-a in Drinking Water by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC/ESI-MS/MS)

Brief Method Summary

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.

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.

Scope and Application

This 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.

Applicable Concentration Range

Cylindrospermopsin: 0.050 - 10.0 ng/mL; Anatoxin-a: 0.029 - 5.87 ng/mL

Interferences

(A) Glassware contamination: All glassware must be meticulously cleaned with detergent and rinsed with tap water and then by reagent water. Non-volumetric glassware may be heated in a muffle furnace. (B) Reagent and Equipment Interferences: Contaminants can be found in solvents, reagents (including reagent water), sample bottles and caps, and other sample processing hardware and must be routinely demonstrated to be free from interferences. (C) Matrix interferences may be caused by contaminants that are present in the sample and include humic or fulvic material. Total organic carbon (TOC) is an indicator of the humic content of a sample. Analysis of LFSMs provides evidence for the presence (or absence) of matrix effects. (D) Internal standards Purity: The labeled internal standards must meet the purity requirements stated in the method.

Quality Control Requirements

Calibration Standard, Continuing Calibration Check (CCC), Field Duplicates (FD), Internal Standard (IS), Internal Standard Stock Standards, Internal Standard Primary Dilution Standard (IS PDS), Laboratory Fortified Blank (LFB), Laboratory Fortified Sample Matrix (LFSM), Laboratory Fortified Sample Matrix Duplicate (LFSMD), Laboratory Reagent Blank (LRB), Procedural Calibration Standard, Quality Control Sample (QCS), Analyte Stock Standard Solution, Analyte Primary Dilution Standard (Analyte PDS), Procedural Calibration (CAL) Standards,

Sample Handling

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. Preservatives are added to each sample container prior to shipment to the field (or prior to sample collection) and include Sodium bisulfate 1.0 g/L and Ascorbic acid 0.10 g/L if chlorine is present. 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. 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. All compounds listed in the method have adequate stability for 28 days when collected, preserved, shipped and stored as described in the method. Therefore, samples should be analyzed as soon as possible, but must be analyzed within 28 days.

Maximum Holding Time

28 days

Relative Cost

Sample Preparation Methods

Triple freeze and thaw process (see method)

This method has 2 analytes associated with it.

Analyte	Detection Level	Bias	Precision	Pct False Positive	Pct False Negative	Spiking Level
Anatoxin a(64285-06-9) Anatoxin a	0.063 ug/L	109% Rec (SL)	11.00 % RSD (SL)
Cylindrospermopsin(143545-90-8) Cylindrospermopsin	0.018 ug/L	111% Rec (SL)	7.40 % RSD (SL)

Precision Descriptor Notes:	Single laboratory method performance data were collected using a Waters Acquity liquid chromatograph coupled to a Micromass Quattro Premier XE triple quadrupole mass spectrometer. Precision and accuracy are presented for three water matrixes, each with 7 replicates: reagent water; chlorinated (finished) groundwater; and moderate TOC chlorinated (finished) surface water.
Detection Level Note:	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 this procedure, 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., 2) Calculate the mean (Mean) and standard deviation for these replicates, 3) Determine the Half Range for the Prediction Interval of Results (HRPIR) using the equation listed in the method, 4) 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 in the method. 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.

Precision Descriptor Notes:

Single laboratory method performance data were collected using a Waters Acquity liquid chromatograph coupled to a Micromass Quattro Premier XE triple quadrupole mass spectrometer. Precision and accuracy are presented for three water matrixes, each with 7 replicates: reagent water; chlorinated (finished) groundwater; and moderate TOC chlorinated (finished) surface water.

Detection Level Note:

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 this procedure, 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., 2) Calculate the mean (Mean) and standard deviation for these replicates, 3) Determine the Half Range for the Prediction Interval of Results (HRPIR) using the equation listed in the method, 4) 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 in the method. 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.

Revision	PDF/Link
2015

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EPA-OGWDW/TSC: 545: Determination of Cylindrospermopsin and Anatoxin-a in Drinking Water by LC/ESI-MS/MS

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