EPA-NERL: 544: Microcystins and Nodularin in drinking water by SPE and LC/MS/MS

Summary
Analytes
Revision
Data and Sites

Official Method Name

DETERMINATION OF MICROCYSTINS AND NODULARIN IN DRINKING WATER BY SOLID PHASE EXTRACTION AND LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY (LC/MS/MS)

Current Revision

2015

Media

WATER

Instrumentation

Liquid Chromatography with Tandem Mass Spectrometry

Method Subcategory

Biotoxin

Method Source

EPA-NERL

Citation

DETERMINATION OF MICROCYSTINS AND NODULARIN IN DRINKING WATER BY SOLID PHASE EXTRACTION AND LIQUID CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY (LC/MS/MS)

Brief Method Summary

This is a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for determination of microcystins and nodularin (combined intracellular and extracellular) in drinking water. Accuracy and precision data have been generated in reagent water, and finished ground and surface waters for compounds listed on the analytes tab and in the linked method documentation.

METHOD FLEXIBILITY – In recognition of technological advances in analytical systems and techniques, the laboratory is permitted to modify the evaporation technique, separation technique, LC column, mobile phase composition, LC conditions and MS and MS/MS conditions (Sect. 6.12, 9.1.1, 10.2, and 12.1). Changes may not be made to sample collection and preservation (Sect. 8), sample extraction steps (Sect. 11), or to quality control requirements (Sect. 9). Method modifications should be considered only to improve method performance. Modifications that are introduced in the interest of reducing cost or sample processing time, but result in poorer method performance, should not be used. See linked full method for more information.

Scope and Application

This is a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for determination of microcystins and nodularin (combined intracellular and extracellular) in drinking water.

Applicable Concentration Range

Concentration ranges used during method development were 10- 400 µg/L, except for MC-RR (4.7-187.5 µg/L), nodularin-R (4.9-195.7 µg/L) and MC-LA (25-1000 µg/L).

Interferences

(A) Glassware contamination: All glassware must be meticulously cleaned. Non-volumetric glassware can be heated in a muffle furnace for a minimum of 90 min at 400°C. Volumetric glassware should be solvent rinsed and heated in an oven no hotter than 120 °C. (B) Contaminants in solvents, reagents (including reagent water), sample bottles and caps, SPE cartridges, and other sample processing hardware: All items must be routinely demonstrated to be free from interferences (less than 1/3 the MRL for each method analyte), (C) Matrix interferences: Humic and/or fulvic material can be co-extracted during SPE and high levels can cause signal enhancement and/or suppression in the electrospray ionization source and low recoveries on the SPE sorbent. Total organic carbon (TOC) is a good indicator of humic content of the sample, (D) Dissolved salts in the mobile phase: Although not observed during method development, suppression of analyte signals due to electrolyte-induced ionization caused by dissolved salts in the mobile phase has been reported in the literature. The addition of ammonium formate to the mobile phase in this method aids in reducing the occurrence of this phenomenon, (E) Contamination in the preservative: Interferences from these sources should be monitored by analysis of laboratory reagent blanks.

Quality Control Requirements

CALIBRATION STANDARD (CAL), CONTINUING CALIBRATION CHECK (CCC), FIELD DUPLICATES (FD1 and FD2), LABORATORY FORTIFIED BLANK (LFB), LABORATORY FORTIFIED SAMPLE MATRIX (LFSM), LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD), LABORATORY REAGENT BLANK (LRB), PRIMARY DILUTION STANDARD (PDS) SOLUTION, QUALITY CONTROL SAMPLE (QCS), SURROGATE ANALYTE (SUR), ANALYTE PRIMARY DILUTION STANDARD (PDS) SOLUTION, CALIBRATION STANDARDS (CAL),

Sample Handling

A 500-mL water sample (fortified with a surrogate) is filtered and both the filtrate and the filter are collected. The filter is placed in a solution of methanol containing 20% reagent water and held for at least one hour at -20 ºC to release the intracellular toxins from cyanobacteria cells captured on the filter. The liquid is drawn off the filter and added back to the 500-mL aqueous filtrate. The 500-mL sample (plus the intracellular toxin solution) is passed through a solid phase extraction (SPE) cartridge to extract the method analytes and surrogate. Analytes are eluted from the solid phase with a small amount of methanol containing 10% reagent water. The extract is concentrated to dryness by evaporation with nitrogen in a heated water bath, and then adjusted to a 1-mL volume with methanol containing 10% reagent water.

Maximum Holding Time

Sample (28 days with appropriate preservation and storage), Extract (28 days when stored at ≤ -4 degrees C).

Relative Cost

Sample Preparation Methods

This method has 2 analytes associated with it.

Analyte	Detection Level	Bias	Precision	Pct False Positive	Pct False Negative	Spiking Level
Microcystin LA(96180-79-9) Cyanoginosin-LA Microcystin LA Microcystin-leucine-alanine Toxin BE 4 (Microcystis aeruginosa)	4.000 ng/L	95% Rec	6.00 % RSD			100.00 ng/L
Nodularin(118399-22-7) Nodularin	1.800 ng/L	92% Rec	3.10 % RSD			19.60 ng/L

Precision Descriptor Notes:	Precision and accuracy were determined for three water matrices: reagent water; chlorinated (finished) ground water; and chlorinated (finished) surface water. Four replicate LFBs fortified near the midrange of the initial calibration curve with preservatives were prepared in each matrix and reported separately.
Detection Level Note:	Establish a target concentration for the MRL based on the intended use of the method. Establish an Initial Calibration following the procedure outlined in the method. The lowest CAL standard used to establish 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 as follows: 1) fortify, extract, and analyze seven replicate LFBs at the proposed MRL concentration. These LFBs must contain all method preservatives, 2) calculate the mean measured concentration (Mean) and standard deviation for these replicates. 3) determine the Half Range for the prediction interval of results (HRPIR) using the equation specified in the method, and 4) confirm that the upper and lower limits for the Prediction Interval of Result (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 in the method. If these criteria are not met, the MRL has been set too low and must be determined again at a higher concentration.

Precision Descriptor Notes:

Precision and accuracy were determined for three water matrices: reagent water; chlorinated (finished) ground water; and chlorinated (finished) surface water. Four replicate LFBs fortified near the midrange of the initial calibration curve with preservatives were prepared in each matrix and reported separately.

Detection Level Note:

Establish a target concentration for the MRL based on the intended use of the method. Establish an Initial Calibration following the procedure outlined in the method. The lowest CAL standard used to establish 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 as follows: 1) fortify, extract, and analyze seven replicate LFBs at the proposed MRL concentration. These LFBs must contain all method preservatives, 2) calculate the mean measured concentration (Mean) and standard deviation for these replicates. 3) determine the Half Range for the prediction interval of results (HRPIR) using the equation specified in the method, and 4) confirm that the upper and lower limits for the Prediction Interval of Result (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 in the method. If these criteria are not met, the MRL has been set too low and must be determined again at a higher concentration.

Revision	PDF/Link
2015

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EPA-NERL: 544: Microcystins and Nodularin in drinking water by SPE and LC/MS/MS

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