Office of Research and DevelopmentNational Exposure Research Laboratory
Jody A. Shoemaker, Daniel R. Tettenhorst, Armah de la Cruz
EPA Drinking Water Method 544
Disclaimers: Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
The views expressed in this presentation are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency.
OAWWA-2016
Office of Research and DevelopmentNational Exposure Research Laboratory
Outline
• Method Development Background–Compliance methods–Methods for Unregulated Contaminant Monitoring Regulations
(UCMR)
• General Method Development Process
• EPA Method 544–SPE-LC/MS/MS method for 6 MCs and nodularin in drinking water
Office of Research and DevelopmentNational Exposure Research Laboratory
CCL & UCMR Background The 1996 amendments to the SDWA required EPA to establish a
Drinking Water Contaminant Candidate List (CCL)
The first CCL was published in 1998 - updated every 5 years
CCL 3 was published in October 2009 and contains 104 chemicals or chemical groups; Draft CCL 4 proposed in January 2015
Nationwide occurrence data needed on CCL chemicals and/or contaminants of emerging concern
EPA’s OGWDW collects occurrence data under the Unregulated Contaminant Monitoring Regulation (UCMR)
Rugged, standardized EPA methods for drinking water contaminants needed for UCMR
If contaminants are regulated, methods usually suitable for compliance monitoring
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Toxin Release (extract intracellular toxin from bacterial cells)
Optimize Instrumentation (chromatography, mass calibrate, tune, evaluate instrument stability, calibration including IS and SUR)
Method Development Process
YESWrite Method
Potential Use inFuture UCMR
Multi-Lab Verification
Determine Aqueous & Extract Holding Times
(ideally ≥14 days)
Revise Technical Approach (re-evaluate sensitivity,
precision, accuracy, interferences and challenging
matrices)
Does Method Meet DQOs? (70-130% REC <30%
RSD)
NO
Consider Analyte Properties and Prior Work(chemical: volatile/nonvolatile, polar/nonpolar, etc.physical: solubility, boiling pointliterature?)
Determine Preservatives(select antimicrobial, dechlor, buffer)
Determine Interferences (determine tolerance limits of method against predetermined QC criteria in challenging matrices)
Internal and External Peer Review
Develop SPE(optimize extraction, accuracy and precision in reagent water)
Determine LCMRL (ideally less than HRL)
HRL = health reference level
LCMRL = Lowest concentration minimum reporting level
Office of Research and DevelopmentNational Exposure Research Laboratory
Development ofCyanotoxin LC/MS/MS Methods
Cyanobacteria & cyanotoxins have become a worldwide concern
Blooms are ecologically disruptive, have economic impacts and produce toxins at relevant health levels
Lack of rugged, multi-lab verified, standardized methods for cyanotoxins
MC-LR, cylindrospermopsin and anatoxin-a on CCL 3
What’s the problem?
Solution Method 544 – SPE-LC/MS/MS method for 6 MCs and nodularin
Method 545 – direct aqueous injection LC/MS/MS method for cylindrospermopsin and anatoxin-a
Office of Research and DevelopmentNational Exposure Research Laboratory
Method 544 Challenges Lack of certified standards – quality control standard (QCS)
difficulties
Release of intracellular toxins with a 500-mL sample MCs are largely intracellular - need to extract toxins from cyanobacteria Conventional cell lysis - 3 freeze/thaw cycles Cannot perform freeze/thaw cycles efficiently on large volume samples
Extract composition – extract composition affects some recoveries
Lack of sufficient isotopically-labeled MCs to use as internal standards and surrogates
Effect of mobile phase on MC recoveries in tap water (matrix effects)
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Method 544 Toxin Release Procedure
Filtrate collected
Filter collected
SPE after toxin release
Freeze 1-16 h at -20 ºC
2 mL 80:20 MeOH:DI
500-mL water sample filtered using a 0.4
micron polycarbonate filter
Filter
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0
20
40
60
80
100
120M
ean
Rec
over
y, %
Glass Fiber (n=4) Polyethersulfone (n=2) Polycarbonate (n=4)
Evaluation of Filter Types
Results: Glass fiber filters disintegrated during F/T cycles Polycarbonate overall highest recoveries
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Effectiveness of Toxin Release ProcedureCells + H2O
3 F/T in H2O
MeOH:H2O
Freeze 1 h
ELISA (n=3) 0.206
MeOH:H2O
Freeze 1 h
0.258
Cells on FilterCells + H2O
MeOH:H2O
Freeze 1 h
0.276
Demonstrates: A single freeze of the
filter in MeOH:H2O is equivalent to 3 freeze/thaw (F/T) cycles of a water sample
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Method 544 SPE Procedure
10 µL injection
Elution with 9:1 MeOH:water
Oasis HLBcartridge
N2 Evaporate to dryness Add 1 mL
9:1 MeOH:water
Surrogate & preservatives
Preservatives2-chloroacetamide
ascorbic acidEDTAtrizma
SurrogateC2D5-MC-LR
Rinse cartridge and bottle withreagent water
LC/MS/MSExternal Calibration
500 mLSample
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Recovery & Precision Acid & Neutral pH Mobile Phases (MP)
preserved and fortified at 0.2-1 µg/L (n=4)
Demonstrates: Importance of evaluating challenging matrices
Neutral MP/LFSMNeutral MP/LFBAcidic MP/LFSMAcidic MP/LFB
0
20
40
60
80
100
120
% R
ecov
ery
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MC-YR
MC-YR
m/z200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050
%
0
100
Acidic vs Neutral pH LC Mobile PhaseTap water extract - Full Scan MS
Normalized to max intensity of 7.7x108
mass spectrumm/z 200-1050
pH ~3
pH ~6
Demonstrates: LC parameters can
affect recoveries in matrices
Modifications, without evaluating challenging matrices, may affect ruggedness
Office of Research and DevelopmentNational Exposure Research Laboratory
40
50
60
70
80
90
100
110
8/29
/13
9/3/
13
9/17
/13
9/18
/13
10/2
2/13
10/2
3/13
11/2
/13
11/1
3/13
11/2
6/13
1/7/
14
1/16
/14
2/18
/14
3/6/
14
3/17
/14
% R
ecov
ery
Fortified DI water extract CCC
Demonstrates: Ruggedness is
not determined in a day or a week!
MC-LW Ruggedness Issues
CCC= continuing calibration check standard
MC-LW not included in Method 544
Office of Research and DevelopmentNational Exposure Research Laboratory
0
20
40
60
80
100
120
140
MC
-YR
Nod
ular
in-R
MC
-RR
MC
-LR
MC
-LA
MC
-LY
MC
-LF
C2D
5-M
C-L
R (S
UR
)
% R
ecov
ery
LFB LFSM-GW source LFSM-SW source
Method 544 Performance Datafortified at 18.8-100 ng/L
LFB QC CriteriaLFSM QC Criteria
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Aqueous & Extract Holding Time Study (n=4)Chlorinated tap water, fortified at 188-1000 ng/L
0
20
40
60
80
100
120
MC-YR Nodularin MC-RR MC-LR MC-LA MC-LY MC-LF
Mea
n R
ecov
ery,
%
Day Zero Aqueous Day 14 Aqueous Day 28 AqueousDay Zero Extract Day 14 Extract Day 28 Extract
Demonstrates: MCs stable with preservatives for 28 days in aqueous samples MCs stable in extracts for 28 days after extraction
Office of Research and DevelopmentNational Exposure Research Laboratory
Method 544 DLs and LCMRLs (ng/L)Analyte
ng/LDL LCMRL HRL
MC-LR 4.3 6.6 21MC-RR 1.2 5.6MC-LA 4.0 2.9MC-YR 4.6 22 MC-LY 2.2 4.6MC-LF 3.4 3.5NOD 1.8 7.3
DLs based on precision only 7 Laboratory fortified blanks (LFBs) Calculated using student t-test For compliance methods, labs have to
demonstrate detection limits (DLs)
LCMRLs based on precision and accuracy 4 LFBs at 7 concentrations Lowest true concentration for which the future recovery is predicted
to fall, with high confidence (99%), between 50 and 150% recovery Multi-lab LCMRLs used to determine minimum reporting level (MRL) For UCMR, labs do not demonstrate LCMRL - only confirm accuracy
and precision at MRL
LCMRL = Lowest concentration minimum reporting level
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Multi-Laboratory Study
Typically 4-6 labs participate Voluntary participation
Labs provide performance data using draft method LRBs LFBs LFSMs LCMRLs
Evaluate multi-lab data Analytes may be dropped or QC criteria adjusted if
necessary May illuminate where method clarifications needed
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Conclusions
Many parameters evaluated to produce rugged standardized methods that are accurate and precise for use in the UCMR or compliance purposes
Drinking water methods take 2-3 years to develop
Method published on-line at https://www.epa.gov/water-research/epa-drinking-water-research-methods
Office of Research and DevelopmentNational Exposure Research Laboratory
EPA TOUR SNEAK PEAK:Cyanotoxin Ambient Water Methods
EPA Method 544 Modifications for Ambient Water6 Microcystin congeners and Nodularin in DW 13 Microcystin congeners and NodularinSPE-LC/MS/MS Filter pore size increased
Sample volume reduced by 5xInternal Standard added
EPA Method 545 Modifications for Ambient WaterCylindrospermopsin and Anatoxin-a in DW No major modificationsDirect aqueous injection LC/MS/MS (no extraction)
Effects of Copper Sulfate and Oxidant Exposure on Cyanobacterial Cells; Methods Employed Include Toxin Analyses by ELISA, Chlorophyll-a Analyses by Fluorometry, and Cell Analyses by Flow Cytometry
Nick Dugan
ADDA ELISA, Cylindrospermopsin ELISA and MMPB Drinking Water Method Development Steve Wendelken
Cultivation of Cyanobacteria & in vitro Toxicity Assay of Cyanotoxins Armah de la Cruz
Application of the MMPB Method for Measurement of ‘Total’ Microcystin Congeners Toby Sanan
LC/MS/MS Methods for Cyanotoxins in Ambient Water Jody Shoemaker