Evaluation and Optimization of Cyanotoxin Analytical … · Evaluation and Optimization of...

2017 Water Research Foundation. ALL RIGHTS RESERVED. 2017 Water 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

2017 Water Research Foundation. ALL RIGHTS RESERVED.

Cyanotoxin Webcast Series


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


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


ELISA versus LC/MS/MS

ELISA LC/MS/MS

Characteristics Measure groups of variants

Measure individual variants

Quantitation Semi-quantitative QuantitativeSample volume


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


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


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


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


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

2017 Water Research Foundation. ALL RIGHTS RESERVED. 2017 Water Research Foundation. ALL RIGHTS RESERVED.

Quantification of MC Congeners

Judy Westrick Ph.D.Director of Lumigen Instrumentation Center

Wayne State University


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


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


Survey Summary ELISA & LC/MS/MS

Mark CitrigliaManager of Analytical Services, NEORSD

Judy Westrick Ph.D.Director of Lumigen Instrumentation Center Wayne

State University


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


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


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


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


Participants Responses to a Multi-choice Evaluation of Method 544


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


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


ELISA Method Review

Mark CitrigliaManager of Analytical Services, NEORSD


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


ELISA Method Review Method Comparison


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


ELISA Method Variability

Debmalya Bhattacharyya Ph.D. Biologist, NEORSD


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)


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


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


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


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


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


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


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


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)


Automated ELISA (CAAS) Reduces analyst

variability EC20 EC80 Calibration parameters

Increased laboratory efficiency

Manual ELISA CAAS


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


ELISA Cross-Reactivity

Debmalya Bhattacharyya Ph.D.Biologist, NEORSD


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


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


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


LC/MS/MS Standard Accuracy and Purity

Judy Westrick Ph.D., DirectorJohnna Birbeck Ph.D., Manager MS Laboratory

Lumigen Instrumentation CenterWayne State University


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


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


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


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


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


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


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


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


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


MC Degradation and OxidationBy-products

Judy Westrick Ph.D., DirectorLumigen Instrumentation Center

Wayne State University


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


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


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.


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)


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


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


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


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


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


Q&A


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

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