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Research Foundation. ALL RIGHTS RESERVED. No part of this
presentation may be copied, reproduced, or otherwise utilized
without permission.
Evaluation and Optimization of Cyanotoxin Analytical Methods
July 13, 2017
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Cyanotoxin Webcast Series
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Cyanobacterial Blooms and Cyanotoxins: Monitoring, Control,
and Communication Strategies
Focus Area Objectives:
1. Source water monitoring strategies
2. Robust analytical methods
3. Cost-effective control options
4. Public outreach and communication strategies and tools
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EPA ActivitiesTimeline Action
CCL 1, 2, 3 and 4
June 2015 Drinking Water Health Advisories
June 2015 Recommendations for Public Water Systems to Manage
Cyanotoxins
Nov. 2015 Strategic Plan for Assessing and Managing Risks
Associated with Cyanotoxins in Drinking Water
Jan. 2017 UCMR4-10 Cyanotoxins/Groups included
Jan. 2017 Recreational water AWQC
< 6 year old > 6 year old and adults
Microcystins (total) 0.3 g/l 1.6 g/l
Cylindrospermopsin 0.7 g/ 3.0 g/l
Microcystins (total): 4.0 g/l Cylindrospermopsin 8.0 g/l
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ELISA versus LC/MS/MS
ELISA LC/MS/MS
Characteristics Measure groups of variants
Measure individual variants
Quantitation Semi-quantitative QuantitativeSample volume
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Evaluation and Optimization of Cyanotoxin Analytical Methods
(Project # 4647)
ObjectiveInvestigate and determine the ability of commonly used
methods to quantify microcystins (MC) at part-per-million levels or
lower
Expected Completion Date December 2017
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Research TeamPrincipal Investigator
Mark Citriglia, Manager of Analytical Services, Northeast Ohio
Regional Sewer District (NEORSD)
Co-Principal InvestigatorJudy Westrick Ph.D., Director of
Lumigen Instrumentation Center, Wayne State University
Team MembersJohnna Birbeck Ph.D. WSU-LICDebmalya Bhattacharyya
Ph.D. NEORSDRosemarie Read Ph.D. NEORSDSheela Agrawal Ph.D.
NEORSDDeborah Schordock NEORSD
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Study Goals Investigate and determine the ability of
commonly used methods to quantify microcystins (MC) at
part-per-million levels or lower Ohio EPA Method 701.0: Total
(Extracellular and Intracellular)
Microcystins ADDA by ELISA Analytical Methodology
US EPA Method 546: Determination of Total Microcystins and
Nodularins in Drinking Water and Ambient Water by Enzyme-Linked
Immunosorbent Assay
US EPA Method 544: Determination of Microcystins and Nodularin
in Drinking Water by SPE and LC-MS/MS Detection
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Study Outcomes Create a final report to assist WRF subscribers
and
utilities with method selection and data evaluation
Findings can be used to design an inter-laboratory validation
study for the recently promulgated EPA methods and in-house
LC/MS/MS methods currently being used within the industry
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Presentation Outline Quantification of MC congeners Survey
summary (ELISA and LC/MS/MS) ELISA Method Review
Method variability and cross-reactivity
LC/MS/MS Method ReviewComparison of standards and samples
MC Biodegradation and Oxidation by-products
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Quantification of MC Congeners
Judy Westrick Ph.D.Director of Lumigen Instrumentation
Center
Wayne State University
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What are the most prevalent Microcystins in USA?
Three types of publications LC/MS/MS with all available
microcystins Foss and Abel (Ohio, US)
High resolution mass spectrometry Two studies Green Lake,
Seattle, WA and Homer Lake, IL
HPLC-PDA California Studies investigating
des-methyl MC-LR
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Summary More research needs to be performed to determine
which MCs predominate in the United States. Data suggest that
some of the disagreement
between ELISA and LC/MS/MS is because of the limited number of
MCs in Method 544 and the limited MC standards available.
MCs that need to be included in Method 544:
Commercially available - [Asp3]MC-RR, [Dha7]MC-RR, [Asp3]MC-LR,
[Dha7]MC-LR, MC-HilR, MC-WR, MC-HtyR
Not available but needed based on literature search MC-MhtyR and
MC-FR
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Survey Summary ELISA & LC/MS/MS
Mark CitrigliaManager of Analytical Services, NEORSD
Judy Westrick Ph.D.Director of Lumigen Instrumentation Center
Wayne
State University
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Survey Goals Determine how laboratories are performing MC
analysis Consistency between procedures
Types of methods and instrumentation
Quality control practices (types and frequency)
Sample processing procedures
Sample collection and preservation
Method modification or custom methods
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ELISA Survey 24 laboratories participated
64% Public Utilities, 36% Regulatory, private or
universities
Conducted in November 2016 Experience
57% 2 years or less (30% < 1 year) 43% > 2 years (33% >
5 years)
ELISA methods 45% Ohio ADDA-ELISA Method 701.0 21% Manufacturers
instructions 33% Combination of manufacturers
instructions and Method 701.0 Sample Collection
57% amber glass containers, 39% PETG (mostly Ohio labs)
ELISA Format 82% use manual microtiter plate, 14% use
an automated system
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ELISA Survey Findings Positive Findings
Minimum QC is being performed (LRB, LCRC,QCS, and sample
duplicate)
Majority of labs are using 4-parameter curve fit Sample
collection, preservation, holding-time and lysing
procedures are consistent among labs Manual microtiter plate
most widely used
Opportunities for Improvement Control charting Tracking
calibration and lot information MDL verification varied from 40 CFR
Part 136 Independent Calibration Verification (ICV) (same vendor)
Color development time varied 10 30 minutes
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LC/MS/MS Survey 12 laboratories participated (16
Respondents)
CA (2), RI, KY, IL, AR, OH (3), FL (2)
Six (6) of those laboratories have implemented EPA 544, Section
1.6, Method Flexibility 2 laboratories have automated the SPE step
using a
Thermo Fisher Scientific Dionex AutoTrace 280 Solid-Phase
Extraction system
1 laboratory uses a Turbo Vap to reduce the SPE volume 1
laboratory had to increase the injection volume and
decreased the MeOH:water ratio from 90:10 to 50:50 Several
different solvent systems were mentioned. 2 types of columns: C18
and C8
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Participants Responses to a Multi-choice Evaluation of Method
544
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LC/MS/MS In-house Methods Most laboratories are using C18
columns 8 laboratories using HPLC and 4 using UHPLC Certified
Reference Materials are needed for all MC
Microcystin Calibrator (Standard) QA/QC Method Number of
Laboratories
Published extinction coefficient 1
Validate all new standards purchased against current calibration
standards 9
Check for impurities by scanning with PDA or HR MS/MS 2
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LC/MS/MS Survey Findings
Positive FindingsLaboratories have created in-house methods
to
QA/QC purchased microcystinsSeveral of the laboratories have
already
implemented EPA Method 544 Opportunities for Improvement
Standard QA/QC for calibrators, until reference materials are
available.
Automation of the sample preparationShortening the
chromatography run time
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ELISA Method Review
Mark CitrigliaManager of Analytical Services, NEORSD
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ELISA Method Review Sample Preservation
Demonstration of Capabilities
Method Sample container Quenching Total Cl2 pH (SU) Hold Time
(days) Temp (C)
546 Amber glass, PTFE1-lined
cap; single-useSodium
Thiosulfate DPD
< MDL n/a3 14 Transport: < 10
Storage: frozen, -20
701Glass or PETG;
may reuse if wash with validation
Sodium Thiosulfate
DPD, ortest strips < 0.1 mg/L
5 - 11 S.U. 5 0 to 4
544Amber glass w/PTFE
lined cap
Trizma2-Chloroacetamide
Ascorbic Acid EDTA
NA 7.0 0.5
28-days to ExtractAnalysis
28-days after Extraction
Transport: < 10 Storage: < 6 Extract: 4
Method holding-times 701.0 vs 546 sample containers,
preservation and hold times
IDC Requirement Method 701.0 Method 546Minimum Reporting Level
(MRL) No YesMethod Detection Limit (MDL) Yes NoPrecision and
Accuracy Study No YesAcceptable System Background No Yes
Method 701.0 vs 546 IDC requirements
Source: Ohio EPA 2015, Zaffiro et al. 2016
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ELISA Method Review Method Comparison
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ELISA Method Review Summary
Manufacturers instruction good starting point
Ohio EPA Method 701.0 added the minimum QC requirements for data
quality without being burdensome
U.S EPA 546 added the essential QA/QC requirements needed for
data validation, essential batch QC, and demonstration of
capabilities
Not enough focus placed on method variability with regards to
calibration parameters, and Equivalent/Effective Concentrations
(EC) and their effect on sample quantification
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ELISA Method Variability
Debmalya Bhattacharyya Ph.D. Biologist, NEORSD
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ELISA Method Variability The accuracy of ELISA analysis is
highly
dependent upon:Calibration curve fit and Equivalent
Concentrations (EC)Storage conditions of the test kitReagent,
standard, and ELISA kit lotsTime and temperature sensitive assay
(color
development critical step)Analyst technique
Precision and accuracy of pipetting (reagent volumes)
Use of alternate vendor standards (non-kit)
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Calibration Equation y= B/B0 normalized absorbance; x =
concentration, A1 = absorbance at bottom asymptote; A2 = absorbance
at top asymptote; x0= concentration at the inflection point (EC50);
P = slope at inflection point
Equivalent Concentrations (EC) Concentration on the x-axis
related to
20,40,60,80% of the maximum absorbance
EC20 Upper limit of useful measurement
EC40 Upper limit of most reliable measurement
EC50 Concentration at the inflection point
EC60 - lower limit of most reliable measurement
EC80 - Upper limit of useful measurement
4-parameter logistic fit of the curves
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Calibration Curve Variability Calibration 6/8/2016
High EC50 = 1.20 g/L Slope = -0.98
Calibration 9/8/2015 EC50 = 0.76 g/L Low Slope = -0.62
Calibration 8/11/2016 EC50 = 0.44 g/L Slope = -1.03
Average EC50 = 0.51 g/L Slope = -0.99
Average EC50 0.51g/L
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Calibration Curve Variability
0.001 0.01 0.1 1 10
0.4
0.8
1.2
1.6
2.0 Actual (1) EC20-80 (1) Actual (2) EC20-80 (2)
Abso
rban
ce
Concentration (g/L)
EC80
EC80
EC80
EC80
Std-1 0.15 g/L
Std-5 5.0 g/L
EC20
EC20
MRL
0.1
8 g
/L
1 2 CC
EC20 1.53 12.23 2.24EC40 0.63 3.11 0.78EC50 0.44 1.77 0.51EC60
0.30 1.00 0.34EC80 0.12 0.26 0.12Slope -1.11 -0.72 -0.99
EC50
EC50
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Kit Variability Over Time
0.1 10.2
0.4
0.6
0.8
1.0
1.2 Day 0 Day 14 Day 30 Day 60
Abso
rban
ce
Concentration (g/L)
Multiple calibrations and QC samples were analyzed on the same
kit at Day 0, 14, 30 and 60
Observed a decrease in overall absorbance on the 60th day
Calibration curve and QA/QC standards met method criteria
EC20 EC80 were within laboratory control limits
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Color Development Time
0.1 1
0.2
0.4
0.6
0.8
1.0
1.2
1.4 10 min 15 min 20 min 25 min 30 min 35 min
Abso
rbanc
e
Concentration (g/L)
Mmanufacturer states 20 30 minutes
Survey ranged from 10 30 minutes
Absorbance increased over time
R2 and QC within method limits EC50 maximized between 15 -
20 minutes
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ELISA Variability Across Wells (CAAS)
Values lower than 1 g/L Higher deviation from 1 g/L
Plate Location: 1-26 27-52 53-78Mean 0.998 0.945 0.974
SD 0.030 0.046 0.067%CV 3.034 4.845 6.916
p value (1-sample T-test) 0.802 0.000 0.064
1 g/L MC-LR standard was analyzed across 78 wells to show
variability across the microtiter plate
33% of the plate had values that were statistically
significantly different from the mean
Variability could be a combination of both plate variability and
analysis time
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Variability among ELISA kit lots
The equivalent concentrations EC20 EC80) were calculated for
each calibration curve and lot of ELISA Kits
High variability appears to exist among the kit lots
Cannot differentiate between lot variability and analyst
variability
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ELISA Variability between Analysts 2015 Manual
5 MDL studies Calibration standard 0.40 g/L
2016 Manual / CAAS 9 MDL studies Vendor prepared MDL
Standard 0.40 g/L
2017 CAAS / Manual 7 MDL studies Performed across multiple
batches and days Full method (freeze-thaw) Lab Prepared 0.40
g/L
(Abraxis)
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Automated ELISA (CAAS) Reduces analyst
variability EC20 EC80 Calibration parameters
Increased laboratory efficiency
Manual ELISA CAAS
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ELISA Method Variability Summary
The ELISA assay is time and temperature sensitive Analyst
technique is critical Storage and use of kits is critical
Automation reduces analyst variability at a cost
Recommendations The used of Equivalent Concentrations (EC20
EC80) for calibration
and data evaluation should be encouraged Control charting should
include calibration parameters like the
slope and the absorbance at the top and bottom plateau to help
identify variability
Samples with results greater than the upper limit of useful
measurement (EC20) should be diluted
The lower limit of useful measurement (EC80) should be reported
with for each calibration with respect to the MDL and MRL
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ELISA Cross-Reactivity
Debmalya Bhattacharyya Ph.D.Biologist, NEORSD
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ADDA-ELISA Cross-Reactivity The Abraxis Total Microcystin and
Nodularin ADDA-ELISA assay
is an indirect, competitive ELISA assay which uses a polyclonal
antibody to target the ADDA group
The ADDA group is present in most MC congeners; the assay is
designed to have limited cross-reactivity among the congeners
MC-LR is used as a primary standard and all sample results are
reported as MC-LR equivalents
Cross-reactivity data has been published for a limited number of
MC congeners
Experiments were performed to determine the cross-reactivity of
13 MC congeners
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Interpretation of Binding Curves
MC-LA and MC-LR would be overestimated: ~ 0.6 g/L of MC-LA and
0.5 g/L of [D-Asp3] MC-LR interpreted as 1 g/L MC-LR equivalent
MC-RR congener would be underestimated: ~1.5 g/L MC-RR
interpreted as 1.0 g/L The amount of over or under estimation is
dependent upon the Equivalent Concentration
EC20 EC40 EC50 EC60 EC80MC-LR 100% 100% 100% 100% 100%MC-LA 163%
124% 111% 99% 75%MC-LY 194% 139% 122% 106% 76%MC-YR 140% 107% 95%
85% 65%MC-RR 66% 64% 63% 62% 59%MC-WR 128% 99% 90% 81% 62%MC-LF 83%
73% 69% 66% 58%
Nodularin 90% 86% 85% 83% 79%MC-LW 180% 123% 106% 90% 62%
dmMC-LR 175% 136% 123% 111% 86%[D-Asp3]MC-LR 190% 156% 143% 132%
108%[D-Asp3]MC-RR 182% 130% 114% 99% 71%
MC-HTyr 177% 144% 132% 122% 99%MC-HiLR 65% 74% 78% 82% 93%
Congeners% Cross reactivity
EC20
EC80
EC50
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ADDA-ELISA Cross-Reactivity Summary
Differential cross-reactivity exist between the 13 MC congeners
studied
ELISA assay will over (False +) or under (False-) estimate the
amount of MC-LR equivalents in the sample depending on the
diversity of MC congeners
False positives and overestimates can be financially burdensome
for utilities
The disagreement in LC/MS/MS and ELISA data can be due to
cross-reactivity predominant congeners
The variation in total MC values with dilution effect can be due
to cross-reactivity of the congeners present
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LC/MS/MS Standard Accuracy and Purity
Judy Westrick Ph.D., DirectorJohnna Birbeck Ph.D., Manager MS
Laboratory
Lumigen Instrumentation CenterWayne State University
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MC Concentration by Beers LawAbsorbance = C l
ISO Method 170706 recommends using the Extinction Coefficient to
calculate the concentration of the stock MC standard
L mol-1 cm-1
MCReferenced
Extinction CoefficientReference
Extinction CoefficientUsed
MC-LR 39800/36500
Harada et al., 1990 /
Honkanen et al., 1990 *39800
MC-YR 38100/41100 Blom et al., 2001 *38100
MC-RR 39800 Harada et al., 1990 *39800
MC-LA 36500
unpublished data by
Carmichael 36500
D-Asp3-LR 31600 Harada et al., 1990 31600
D-Asp3, E-Dhb7 MC-RR 50400 Blom et al., 2001 50400
[Dha7] - LR 46800 Harada et al., 1990 46800
39800MC-LW, MC-WR, MC-LF,MC-LY, MC-HtyR, D-Asp3-RR, C2D5
MC-LR
* Used by NEORSD
Sheet1
MCReferenced Extinction CoefficientReferenceExtinction
CoefficientUsed
MC-LR39800/36500Harada et al., 1990 / Honkanen et al.,
1990*39800
MC-YR38100/41100Blom et al., 2001*38100
MC-RR39800Harada et al., 1990*39800
MC-LA36500unpublished data by Carmichael36500
D-Asp3-LR31600Harada et al., 199031600
D-Asp3, E-Dhb7 MC-RR50400Blom et al., 200150400
[Dha7] - LR46800Harada et al., 199046800
MC-LW, MC-WR, MC-LF,MC-LY, MC-HtyR, D-Asp3-RR, C2D5 MC-LR
39800
* Used by NEORSD
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Comparison of LC/MS/MS EPA 544 Methods
Standard Curves Made with extinction
coefficient correction for LR, RR, YR (every experiment)
EPA method 544 HPLC C8 column Water, ammonium
formate/Methanol
Standard Curves Extinction coefficients
correction made monthly for the bottle for all MC, using
published or MCLR
EPA method 544 using Method Flexibility HPLC C18 column
Water/Acetonitrile with
trace formic acid
All samples were extracted and prepared at NEORSD.
NEORSD WSU
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Determination of Cyanotoxin Concentration by UV Spectra
MC-LRAverage
MC-RRAverage
MC-YRAverage
MC-LAAverage
MC-LFAverage
MC-LYAverage
C2D5MC-LR
Average
D-Asp3-RR
Average
MC-HilRAverage
MC-WRAverage
MC-HtyRAverage
MC-LWAverage
D-Asp3-LR
AverageEnzo-Min%R 78% 61% 106% 105% 95% 80% 90% 100% 113% 82%
90% 101% 108%
Enzo-Max%R 151% 137% 240% 141% 191% 204% 128% 142% 229% 200%
178% 158% 175%
Enzo-Avg%R 109% 97% 156% 122% 125% 137% 105% 120% 153% 136% 140%
120% 141%
# of Standards 48 48 48 9 9 9 5 5 5 8 5 9 17
0.00%
50.00%
100.00%
150.00%
200.00%
250.00%
Recovery of Enzo MC-Standards based on Extinction
CoefficientEnzo-Min%R Enzo-Max%R
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A Comparison between UV and MS Quantifications
Inter-laboratory
% Re
cove
ry
Suppliers
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
180.00
200.00
APExBIO
Beagle BioProducts
Cayman Chem
icals
NRCC
Beagle BioProducts
Cayman Chem
icals
Cyano BioTech Gm
bH
NRCC
Sigma-Aldrich
Beagle BioProducts
Cyano BioTech Gm
bH
Sigma-Aldrich
% REC-UV %REC-MS
MC-RR MC-YRMC-LR
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Comparison on UV Calibration Method
-80
-60
-40
-20
0
20
40
60
MC-LR
MC-RR
MC-YR
%
Rela
tive
Per
cent
Dif
fere
nce
Suppliers
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Chromatography of the Microcystins
NEORSD WSUName RT (min)
Precursor Ion Quant Ion Qual Ion
Nodularin 11.53 825 135 389YR 11.52 523 135 507
HtyR 11.56 530 135 103RR 11.76 520 135 103
Dasp3RR 11.79 513 135 103LR 12.02 498 135 482
WR 12.33 534 135 103Dha7LR 12.46 491 135 103
HilR 12.53 505 135 103Dasp3LR 12.71 491 135 103
LA 12.95 910 776 135LY 13.01 1002 135 375LW 13.95 1025 135 375LF
14.54 986 135 478
surr 14.83 515 135 499
Name RT(min)Precursor
Ion Quant Ion Qual IonD-Asp3-RR 0.67 513 135 213
MC-RR 0.71 520 135 440Nodularin 0.99 825 135 389
MC-YR 1.2 1046 213 136MC-HtyR 1.27 1059 135 617
MC-LR 1.36 995 135 213D-Asp3-LR 1.4 981 135 675MC-HilR 1.65 1009
135 213MC-WR 1.76 1068 135 626
surr 2.61 1029 135 163MC-LA 2.98 910 375 402MC-LY 3.21 1002 494
868MC-LW 3.93 1026 517 891MC-LF 4.11 986 852 478
[Dha7] - LR n/a n/a n/a n/a
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Chromatography Order
Different solvent systems
Different media
m/z+1 vs m/z+2
MC- RR
MC-YR
Nodularin MC-HtyR
MC-LRD-Asp3LR
MC-LAMC-WR
D-Asp3RR
MC-LFMC-LWMC-LY
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Total MC by LC/MS/MS WSU & NEORSD 24 Samples with
positive
ELISA results were analyzed by LC/MS/MS by NEORSD and WSU using
two different LC/MS/MS method
WSU Modified EPA 544
NEORSD EPA 544
Both methods have more microcystin congeners
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LC/MS/MS Summary The concentration determined by the
manufacturers mass did not agree
with concentration determined by UV Our data suggests that using
the extinction coefficient to determine the
concentration of the standards provides a more precise and
robust method MCs that have the least variability in concentration
based on UV
absorbance are: LA, C2D5-LR, and D-Asp3-RR. MCs that have the
most variability in concentration based on UV
absorbance are: YR, LF, LY, HilR, and WR Elution order and
retention times of the MCs change with different solvent
systems Both laboratories use quantifier/qualifier ions
Currently investigating the differences in MC concentrations from
inter-
laboratory field samples: Chromatography systems
Quantifier/Qualifier ions Calibration Curve Standardization ELISA
cross-reactivity
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MC Degradation and OxidationBy-products
Judy Westrick Ph.D., DirectorLumigen Instrumentation Center
Wayne State University
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KMnO4 and ADDA-ELISA(Spies and Szlag, Oakland University)
Samples were: Quenched with Sodium
Thiosulfate Microfuged ELISA Protein Phosphatase
Inhibition Assay (PPIA) LC/TOF
PPIA vs TOF and ELISA vs MS
50 g/L MC-LA; 35C; 2 ppm KMnO4
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Predicted Products by MS
NNH
NHNH
NHHN
HN
OHO
O
OH
OO
O
OO
O
O
O
Microcystin LA
OH
O
Mass: 220.146
OxidationProposed Oxidation
Products
NNH
NHNH
NHHN
HN
OHO
O
OH
OO
O
OO
O
O
O
Mass:709.32
and CO2
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Preliminary Data for the Biodegradation of MC-LR(Brandel and
Huntley, University of Toledo)
MC-LR ADDA-ELISA concentrations increase with time.
MC-LR LC-MS concentration starts to decrease after 4 days.
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Following Biodegradation of MC-LR by LC/MS/MS
MC-LR concentration increases over the 22 days; similar to
increased concentration of the 615 m/z and 950 m/z.
0
50000
100000
150000
200000
250000
0
50
100
150
200
250
300
350
5G14 Day 1 5G14 Day 4 5G14 Day 9 5G14 Day 11 5G14 Day 15 5G14
Day 18 5G14 Day 22 LC/M
S/M
S R
espo
nse
MC
LR
g/L
Day
Biodegradation of 5G14
ADDA_ELISA LC/MS/MS_MCLR LC/MS/MS-(615 m/z)
MC_LR_Decarboxyl_(950 m/z)
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Summary: Degradation and Oxidation By-Products
The bacteria system designed by Brandel and Huntley degrades
MC-LR
MC-LR concentration detected ELISA was significantly higher than
the concentration detected by LC/MS/MS.
Two fragment detected by MS; increase at similar rates to the
concentration determined by ELISA
KMnO4 degradation of MC-LA was investigated by Spies and
Szlag
Concentrations of MC-LA by PPIA and MS agreed; however
concentrations of MC-LA by ELISA were elevated
Two dominant fragments determined by MSDominant fragments are
being separated to be analyzed on the ELISA kit and
characterized
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Research TeamPrincipal Investigator
Mark Citriglia, Manager of Analytical Services, Northeast Ohio
Regional Sewer District (NEORSD)
Co-Principal InvestigatorJudy Westrick Ph.D., Director of
Lumigen Instrumentation Center, Wayne State University
Team MembersJohnna Birbeck Ph.D. WSU-LICDebmalya Bhattacharyya
Ph.D. NEORSDRosemarie Read Ph.D. NEORSDSheela Agrawal Ph.D.
NEORSDDeborah Schordock NEORSD
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2017 Water Research Foundation. ALL RIGHTS RESERVED.
Acknowledgements Indiana Department of Environmental
Management, Office of Water, Drinking Water Branch
New Jersey American Water - Northern Operating Area
Ohio EPA, Division of Environmental Services Alloway
Environmental Testing Laboratory Marion
Ohio Akron Water Supply Celina Water Treatment Plant Menasha
City Water Plant Capital Regional District Parks and
Environmental
Services
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2017 Water Research Foundation. ALL RIGHTS RESERVED.
RFP # 4716Refinement and Standardization of
Cyanotoxin Analytical Techniques for Drinking Water
Objective: evaluate existing chemical and biological methods for
the analysis of cyanotoxins at low part-per-trillion (ppt)
detection levels in raw and finished drinking water
Proposals due date :11/1/2017
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2017 Water Research Foundation. ALL RIGHTS RESERVED. 2017 Water
Research Foundation. ALL RJGH1'5 RESERVED.
-
2017 Water Research Foundation. ALL RIGHTS RESERVED.
On-Going WRF Projects Performance Evaluation of ELISA Methods
for the Analysis of
Cyanotoxins
CyanoTOX Field Validation and Enhancement Related to Chemical
Kinetics and ELISA Kinetics
Release of Intracellular Cyanotoxins during Oxidation of
Naturally Occurring and Lab Cultured Cyanobacteria
Development of a Risk Communication Toolkit for Cyanotoxins
Benthic Cyanobacterial Risk
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2017 Water Research Foundation. ALL RIGHTS RESERVED. 2017 Water
Research Foundation. ALL RIGHTS RESERVED.
Q&A
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2017 Water Research Foundation. ALL RIGHTS RESERVED. 2017 Water
Research Foundation. ALL RIGHTS RESERVED.
Thank YouComments or questions, please
contact:[email protected]
For more information visit:www.waterrf.org
mailto:[email protected]://www.waterrf.org/
Slide Number 1Cyanotoxin Webcast SeriesCyanobacterial Blooms and
Cyanotoxins: Monitoring, Control, and Communication StrategiesEPA
ActivitiesELISA versus LC/MS/MSEvaluation and Optimization of
Cyanotoxin Analytical Methods (Project # 4647)Research TeamStudy
GoalsStudy OutcomesPresentation OutlineQuantification of MC
CongenersWhat are the most prevalent Microcystins in
USA?SummarySurvey Summary ELISA & LC/MS/MS Survey GoalsELISA
SurveyELISA Survey FindingsLC/MS/MS SurveyParticipants Responses to
a Multi-choice Evaluation of Method 544LC/MS/MS In-house
MethodsLC/MS/MS Survey FindingsELISA Method ReviewELISA Method
ReviewELISA Method ReviewELISA Method ReviewELISA Method
VariabilityELISA Method Variability4-parameter logistic fit of the
curvesCalibration Curve VariabilityCalibration Curve VariabilityKit
Variability Over TimeColor Development TimeELISA Variability Across
Wells (CAAS)Variability among ELISA kit lotsELISA Variability
between AnalystsAutomated ELISA (CAAS)ELISA Method VariabilityELISA
Cross-ReactivityADDA-ELISA Cross-ReactivityInterpretation of
Binding CurvesADDA-ELISA Cross-ReactivityLC/MS/MS Standard Accuracy
and PurityMC Concentration by Beers LawAbsorbance = e C lComparison
of LC/MS/MS EPA 544 MethodsDetermination of Cyanotoxin
Concentration by UV SpectraA Comparison between UV and MS
Quantifications Inter-laboratoryComparison on UV Calibration
MethodChromatography of the Microcystins ChromatographyTotal MC by
LC/MS/MS WSU & NEORSDLC/MS/MS SummaryMC Degradation and
Oxidation By-productsKMnO4 and ADDA-ELISA(Spies and Szlag, Oakland
University)Predicted Products by MSPreliminary Data for the
Biodegradation of MC-LR(Brandel and Huntley, University of
Toledo)Following Biodegradation of MC-LR by LC/MS/MS Summary:
Degradation and Oxidation By-ProductsResearch TeamAcknowledgements
RFP # 4716Refinement and Standardization of Cyanotoxin Analytical
Techniques for Drinking WaterSlide Number 61On-Going WRF
ProjectsQ&AThank You