EPA/600/R-18/056 | May 2021
www.epa.gov/emergency-response-research
United States
Environmental Protection
Agency
oEPA
Direct Aqueous Injection of
the Fluoroacetate Anion in
Potable Water for Analysis by
Liquid Chromatography/
Tandem Mass Spectrometry
Office of Research and Development
Homeland Security Research Program

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EPA Report Number EPA/600/R-18/056
Date: May 2021
Direct Aqueous Injection of the
Fluoroacetate Anion in Potable Water for
Analysis by Liquid
Chromatography/Tandem Mass
Spectrometry
Stuart Willi son and Emily Parry
U.S. Environmental Protection Agency
Office of Research and Development
Homeland Security Research Program
Cincinnati, OH 45268

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Disclaimer
The U.S. Environmental Protection Agency (EPA) through its Office of Research
and Development, National Homeland Security Research Center (NHSRC)
funded and managed the research described herein under IA #DW-75-958671 in
collaboration with the National Institute of Occupational Safety and Health
(NIOSH), Centers for Disease Control and Prevention (CDC), a division of the
U.S. Department of Health and Human Services (DHHS). It has been subjected to
the Agency's review and has been approved for publication. Note that approval
does not signify that the contents necessarily reflect the views of the Agency. Any
mention of trade names, products, or services does not imply an endorsement by
the U.S. Government or EPA. The EPA does not endorse any commercial
products, services, or enterprises.

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Table of Contents
Disclaimer	 ii
List of Tables	iv
List of Figures	v
Abbreviations and Acronyms	vi
Acknowledgments	viii
Executive Summary 	ix
I.0	Introduction	 1
2.0 Scope and Application	 1
3.0 Summary of Method	 2
4.0 Definitions	 2
5.0 Interferences	 4
6.0 Health and Safety	 4
7.0 Equipment and Supplies	 4
8.0 Reagents and Standards	 6
9.0 Sample Collection, Preservation, and Storage	 7
10.0 Quality Control	 8
II.0	Instrument Calibration and Standardization	 10
12.0 Analytical Procedure	 11
13.0 Data Analysis and Calculations	 12
14.0 Method Performance	 13
15.0 Pollution Prevention	 14
16.0 Waste Management	 14
17.0 References	 14
18.0 Tables and Validation Data	 16
Appendix A: Methyl Fluoroacetate Analysis	 24
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List of Tables
Table 1: Summary of IDC with RW	16
Table 2: Parameters tested during method development	16
Table 3: Calibration Standards	17
Table 4: Water Quality Parameters for the four water samples used in this study	17
Table 5: LC Parameters	17
Table 6: LC Gradient	18
Table 7: ESI and MS Parameters	18
Table 8: Compound specific MRM parameters	18
Table 9: DL Calculation	20
Table 10: Half-Range Prediction Interval Calculation	20
Table 11: Initial Demonstration of Accuracy and Precision	21
Table 12: % FAA remaining compared to Day 0	22
Table 13: Sample Filtration	23
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List of Figures
Figure 1 Formation of fluoroacetate anion (FAA)	2
Figure 2: Representative FAA Calibration Curve. Response is the area of the QT (57.26 m/z) divided by the
internal standard quantification ion (36.13 m/z)	19
Figure 3: Blank and 1 |ig/L sample chromatogram. Extracted Ion Chromatogram (EIC) of 57.26 m/z	19
Figure 4:Low Standard Chromatograms for FAA	21
Figure 5: Hydrolysis of MFA to FAA in four different water treatments	25
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Abbreviations and Acronyms
ANOVA
Analysis of Variance
CAL
Calibration Standard
CCC
Continuing Calibration Check
CF
Qualification Ion
CI
Confidence Interval
DAI
Direct Aqueous Injection
DL
Detection Limit
EIC
Extracted Ion Chromatogram
EPA
U.S. Environmental Protection Agency
ESI
Electrospray Ionization
ESI-
Electrospray Ionization, negative mode
FAA
Fluoroacetate anion
FAA L
Labeled fluoroacetate ion (13C2, 99%; 2-2D2, 98%)
FAU
Formazin Attenuation Units
LC
Liquid Chromatography
HRpir
Half Range for the Prediction Interval of Results
IDA
Initial Demonstration of Accuracy
IDC
Initial Demonstration of Capability
IDP
Initial Demonstration of Precision
IRIS
Integrated Risk Information System
IS
Internal Standard
LC
Liquid Chromatograph(y)
LRB
Laboratory Reagent Blank
LFSM
Laboratory Fortified Sample Matrix
LFSMD
Laboratory Fortified Sample Matrix Duplicate
MeCN
Acetonitrile
MeOH
Methanol
MFA
Methyl Fluoroacetate
MRL
Method Reporting Limit
MRM
Multiple Reaction Monitoring
MS/MS
Tandem Mass Spectrometry
m/z
Mass to Charge Ratio
NTU
Nephelometric Turbidity Unit
PIR
Prediction Interval of Results
PPE
Personal Protective Equipment
PTFE
Polytetrafluoroethylene
PVDF
Polyvinylidene fluoride
QC
Quality Control
QT
Quantification Ion
% R
% Recovery
R2
Coefficient of Determination
RE
Relative Error
RPD
Relative Percent Difference
RSD
Relative Standard Deviation
RT
Retention Time
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RW
Reagent Water
s
Standard Deviation
SAM
Selected Analytical Methods for Environmental Remediation and Recovery
SB
Sequence Blank
SDS
Safety Data Sheet
sss
Stock Standard Solution
TOC
Total Organic Carbon
VOA
Volatile Organic Analysis
WS
Water Source
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Acknowledgments
We would like to acknowledge the following individuals and organizations for their contributions
towards the development and/or review of this method.
United States Environmental Protection Agency (EPA)
Office of Research and Development, National Homeland Security Research Center
Stuart Willison, Project Officer
Emily Parry, Method Development
Matthew Magnuson and Jim Starr, Reviewers
Centers for Disease Control and Prevention
National Institute for Occupational Safety and Health
Jack Pretty, Laboratory Advisor
Robert Streicher, Project Officer
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Executive Summary
Sodium fluoroacetate, also known as Compound 1080, is a pesticide registered for use in livestock protection
collars, and it is acutely toxic to humans. In water, it dissociates into the fluoroacetate anion (FAA) and could
greatly impact human health if it was released into a water system, either deliberately or accidently. The U.S.
Environmental Protection Agency (EPA) is responsible for supporting water utilities in their response to water
system contamination. In support of this objective, EPA's National Homeland Security Research Center publishes
Selected Analytical Methods for Environmental Remediation and Recovery (SAM), which provides suggested
methods for numerous hazardous chemicals in a variety of matrices including drinking water. Sodium
fluoroacetate, fluoroacetic acid, and methyl fluoroacetate are all recommended in SAM to be analyzed as FAA.
However, the current revision of SAM does not include a verified method for analysis in drinking water. The
purpose of this report is to describe a quick, robust method to quantitate FAA in drinking water.
In this method, samples are preserved with ascorbic acid (for dechlorination) and sodium omadine (an anti-
microbial) and filtered prior to analysis. They are analyzed by liquid chromatography tandem mass spectrometry
in negative ion mode (electrospray ionization, ESI-). Total analysis time, including column re-equilibration, is 32
minutes. An initial demonstration of capability (IDC) in reagent water (RW) showed linearity over two orders of
magnitude (1 |ig/L to 100 jx/L), a detection limit (DL) of 0.4 |ig/L. and a minimum reporting level (MRL) of 2
|ig/L. Accuracy and precision were 2.5% (relative error, RE) and 3.4% (relative standard deviation, RSD),
respectively. FAA stability was investigated in RW, as well as in four different drinking water samples with
varying characteristics (e.g., organic content, hardness, etc.). FAA appeared stable in RW and the tested drinking
water sources. Also included, is application of the method for the analysis of methyl fluoroacetate after its
hydrolysis to FAA. Further testing would be required before implementation.
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1.0 Introduction
The U.S. Environmental Protection Agency (EPA) is responsible for supporting the response to a
deliberate/accidental chemical release or natural disaster damaging water and related infrastructure. In support of
this objective, EPA's National Homeland Security Research Center publishes Selected Analytical Methods for
Environmental Remediation and Recovery (SAM) [1] which provides suggested methods for numerous hazardous
chemicals. SAM recommends that fluoroacetic acid, fluoroacetate salts, and methyl fluoroacetate all be analyzed
as the fluoroacetate anion (FAA). However, SAM does not contain a verified method for performing this analysis
in drinking water. The purpose of this report is to describe a quick, robust method to quantitate FAA in drinking
water.
One source of FAA is Compound 1080, registered for use in the United States (RN# 56228-22) for livestock
protection collars. Predators attacking the livestock bite the collars, leading to ingestion of Compound 1080 and
subsequent morbidity or mortality of the predator. Compound 1080 is the sodium fluoroacetate salt that
dissociates in water to fluoroacetate anion (FAA). FAA is also present after pH-dependent deprotonation of
fluoroacetic acid, as well as through the hydrolysis of methyl fluoroacetate, a precursor used in pharmaceutical
synthesis. Human toxicity occurs at low levels 0.7-2.1 mg/kg [2], EPA's Integrated Risk Information System
(IRIS) lists a reference dose of 0.00002 mg/kg/day [3], and 0.4 |ig/L in tap water is listed in the regional screening
level tables [4], However, remediation goals would be site-specific.
Reports of FAA analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) are available;
however, the reported analyses require multiple sample preparation steps [5-6], and a high-throughput method
would be advantageous. This report describes a direct aqueous injection (DAI) method that minimizes sample
preparation time. Drinking water samples are separated on a reverse phase octylsilane (C8) column. MS/MS
fragmentation provides identification and quantitation. An isotopically labeled version of FAA, 13C2, 99%; 2-2D2,
98%, (FAA L) is used as an internal standard (IS) for quantitation and quality control purposes. The method has
been tested for accuracy, precision, linearity, and detection limit in reagent grade water (RW). Four additional
drinking water sources were evaluated to assess their effects on method performance and FAA stability.
2.0 Scope and Application
Figure 1 shows several ways the fluoroacetate anion can form in water. The dissociation of sodium fluoroacetate,
the deprotonation of fluoroacetic acid, and the hydrolysis of methyl fluoroacetate can all occur, depending on the
environmental conditions. The scope of this study was determination of FAA by DAI-LC-MS/MS. The method
was developed and evaluated in the same laboratory. Initial Demonstration of Capability (IDC) results are
summarized in Table 1. Additional water samples were tested, representing a variety of drinking water types
(chlorinated, chloraminated, etc.), to determine the effect on the method or stability of FAA. Quantitation and
quality control includes the use of isotopically labeled FAA as an internal standard. The following analyte was
tested:
Chemical Name:	fluoroacetate
Abbreviation:	FAA
Empirical Formula:	C2H2FO2
Molecular mass:	77.035 g/mol
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o
'OH
Fluoroacetic acid
CAS: 144-49-0
o
o Na
Sodium fluoroacetate
CAS: 62-74-8
o

o
F^o-
Fluoroacetate anion
CAS: 513-62-2
'O
Methyl fluoroacetate
CAS: 453-18-9
Figure 1 Formation of fluoroacetate anion (FAA)
3.0 Summary of Method
A 40-mL drinking water sample was collected in a bottle and preserved with a chlorine neutralizer (ascorbic acid)
and an anti-microbial agent (sodium omadine). An aliquot of 990 |iL of sample was filtered with a 0.22-|im filter,
placed into an autosampler vial, and the internal standard was added. A 20-|iL injection was made into an LC
equipped with a C8 column interfaced to an MS/MS operated in negative electrospray ionization (ESI-) mode.
FAA was identified by comparing the mass spectra, fragment ratios, and retention times to that of FAA
calibration standards analyzed under identical conditions. FAA quantification was achieved by isotope dilution.
4.0 Definitions
4.1.	ANALYSIS BATCH
An Analysis Batch consists of a set of samples analyzed on the same instrument within a 24-hour period
and included no more than 20 field samples, beginning and ending with the appropriate continuing
calibration check (CCC) standards. Additional CCCs may be required depending on the number of
samples (excluding quality control (QC) samples) in the analysis batch and/or the number of field
samples.
4.2.	CALIBRATION STANDARD
The Calibration Standard (CAL) is a solution prepared from the analyte stock standard solution, the
internal standard and diluted with water. The CAL solutions are used to calibrate the instrument response
with respect to analyte concentration.
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4.3. CONTINUING CALIBRATION CHECK
The Continuing Calibration Check (CCC) is a calibration standard containing the method analyte and
surrogate standard. The CCC is analyzed periodically to verify the accuracy of the existing calibration.
4.4.	DETECTION LIMIT
The Detection Limit (DL) is the minimum concentration of an analyte that can be identified, measured,
and reported with 99% confidence that the analyte concentration is greater than zero.
4.5.	INTERNAL STANDARD
The Internal Standard (IS) is a pure chemical added to a standard solution in a known amount and is used
to measure the relative response of other method analytes that are components of the same solution. The
internal standard (IS) is a chemical that is structurally similar to the method analyte, has no potential to be
present in the samples, and is not a method analyte.
4.6.	LABORATORY FORTIFIED SAMPLE MATRIX
The Laboratory Fortified Sample Matrix (LFSM) is a field sample to which known quantities of the
method analytes are added in the laboratory. The LFSM is processed and analyzed exactly like a sample,
and its purpose is to determine whether the sample matrix contributes bias to the analytical results. The
background concentrations of the analytes in the sample matrix must be determined in a separate sample.
4.7.	LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE
The Laboratory Fortified Sample Matrix Duplicate (LFSMD) is fortified and analyzed identically to the
LFSM. The LFSMD is used to assess method precision when the observed concentrations of the method
analytes are low.
4.8.	LABORATORY REAGENT BLANK
The Laboratory Reagent Blank (LRB) is a blank matrix treated exactly the same as a sample including
exposure to all glassware, equipment, solvents, reagents, preservatives, and surrogate standards used in
the analysis batch. The LRB is used to determine if method analytes or other interferences are present in
the laboratory environment, the reagents, or the apparatus.
4.9.	MINIMUM REPORTING LEVEL
The minimum reporting level (MRL) is the minimum concentration that can be reported as a quantitated
value for a method analyte in a sample following analysis. This defined concentration can be no lower
than the concentration of the lowest calibration standard for that analyte and can be used only if
acceptable QC criteria for this standard are met.
4.10.	PRECURSOR ION
For the purpose of this report, the precursor ion is the deprotonated molecule ([M-H]") or adduct ion of
the method analyte. In tandem mass spectrometry (MS/MS), the precursor ion is mass-selected and
fragmented by collision induced dissociation to produce distinctive product ions of lower mass.
4.11.	PRODUCT ION
For the purpose of this report, a product ion is one of the fragment ions produced in MS/MS by collision
inducted dissociation of the precursor ion.
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4.12. SAFETY DATA SHEET
The Safety Data Sheet (SDS) contains written information provided by vendors concerning a chemical's
toxicity, health hazards, physical properties, fire, and reactivity data including storage, spill and handling
precautions.
4.13.	SEQUENCE BLANKS
Sequence blanks (SBs) are prepared with the same dilutant and IS as the CAL standards. They are
included in all injection sequences to ensure that no contamination or interferences have occurred in the
preparation of the CAL standards. They also provide verification that there is no carryover between
sample injections.
4.14.	STOCK STANDARD SOLUTION
The Stock Standard Solution (SSS) is a concentrated solution containing the method analyte prepared in
the laboratory using assayed reference materials or purchased from a reputable commercial source.
Interferences
Method Interferences can be caused by contaminants in solvents, reagents (including reagent water
(RW)), sample bottles and caps, and other laboratory supplies or hardware that lead to discrete artifacts
and/or elevated baselines in the chromatograms. All items contacting the sample were routinely
demonstrated to be free from interferences (less than 1/3 the DL) under the conditions of the analysis by
analysis of a LRB. Subtracting blank values from sample results was not permitted.
Relatively large quantities of the preservatives were added to added to sample bottles. Trace-level organic
contaminants could be present in these reagents. Interferences were monitored by analysis of LRBs.
Health and Safety
FAA toxicity is documented, and other chemicals used in the method have known carcinogenicity. All
were treated as a potential health hazards and exposure was minimized through the proper use of personal
protective equipment (PPE) and engineering controls. PPE consisted of close-toed shoes, eye protection,
lab coat, and nitrile gloves A reference file of SDSs was made available to all personnel involved in
chemical analyses.
Equipment and Supplies
References to specific equipment brands and catalog numbers are provided as examples and do not
constitute an endorsement of the use of such products or suppliers. Glassware, reagents, supplies,
equipment, and settings other than those listed in this report may be employed, provided that method
performance is demonstrated and documented. Analyses may be performed with any system capable of
performing LC-MS/MS, as long as performance criteria are met.
7.1. LC-MS/MS INSTRUMENT
7.1.1. Liquid Chromatography (LC) System
An Accela 1250 (Thermo, San Jose, CA) LC pump and autosampler with a CTO-IOAVP (Shimadzu,
Columbia, MD) column oven were used for method development any equivalent is acceptable.
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7.1.2.	Analytical Column
Sunfire™ -dC8, 150 mm x 4.6 mm, 5 |im particle size (Waters, Milford, MA, Catalog #186002737), or
equivalent. Additional columns tested are listed in Table 2.
7.1.3.	Tandem Mass Spectrometer (MS/MS) System
A Thermo Quantum TSQ (San Jose, CA) was used for method development. Any equivalent system may
be used.
7.1.4.	Data System
An interfaced data system is required to acquire, store, reduce, and output mass spectral data. Xcalibur 2.2
(Thermo, San Jose CA) software was used for method development any equivalent compatible with the
LC-MS/MS instrument may be used.
7.2. GLASSWARE AND MISCELLANEOUS SUPPLIES
7.2.1.	Autosampler Vials
Amber 2-mL autosampler vials with pre-slit Teflon® - lined screw tops (Waters Corp., Milford, MA), or
equivalent.
7.2.2.	Disposable Sterile Syringes
10.0 mL ± 1% accuracy BD Safety-Lok™ syringes (Catalog No. 14-829-32, Fisher Scientific, Pittsburgh,
PA), or equivalent.
7.2.3.	Auto Pipettes
1000 (j,L, 100 (iL and 10 (iL ± 1% accuracy.
7.2.4.	Desolvation Gas
Nitrogen gas generator, or equivalent nitrogen gas supply that aids in the generation of an aerosol for the
ESI liquid spray and should meet or exceed instrument manufacturer's specifications.
7.2.5.	Collision Gas
Argon gas used in the collision cell in the MS/MS instruments and should meet or exceed instrument
manufacturer's specifications.
7.2.6.	Analytical Balance
Accurate to 0.1 mg; reference weights traceable to Class S or S-l weights.
7.2.7.	Standard Solution Flasks
Class A volumetric glassware.
7.2.8.	Syringe Filter
Millex® GV Syringe-driven polyvinylidene fluoride (PVDF) 13-mm filter unit, 0.22 |im (Millipore
Corporation, Billerica, MA, Catalog # SLGV013NL), or equivalent.
7.2.9.	Sample Collection Containers
Twenty- or 40-mL volatile organic analysis (VOA) vials with polytetrafluoroethylene (PTFE) screw caps
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(Environmental Sampling Supply, product no. 0040-0310-PC, San Leandro, CA), or equivalent.
7.2.10.	pHMeter
Accumet ® pH Meter 50 with electrode (Fisher Scientific, Pittsburg, PA), or equivalent.
7.2.11.	Vortex mixer
Maxi Mix II (Thermo Scientific, San Jose, CA), or equivalent.
8.0 Reagents and Standards
8.1. STANDARDS, SOLVENTS AND REAGENTS
All reagents used during the course of this study were analytical grade or equivalent.
8.1.1.	Standards
FAA and FAA_L were purchased from Cambridge Wasotopes (Tewskbury, MA).
8.1.2.	Solvents and Chemicals
Solvents utilized for this study were acetonitrile (MeCN), methanol (MeOH), and reagent grade water
(RW) (Fwasher, Optima®, LC-MS grade) (Waltham, MA) and were demonstrated to be free of analytes
and interferences. Chemicals included acetic acid (Fwasher, Optima™, >99.7%), sodium omadine (Sigma
Aldrich, >96%) and L (+)-ascorbic acid (Acros Organics™, 99%).
8.1.3.	Mobile Phase A
Prepared by adding 50 |iL of acetic acid to a mobile phase bottle containing 1 L of RW. Mixed and stored
at room temperature. The solution expired in five days.
8.1.4.	Mobile Phase B
Prepared by adding 50 |iL of acetic acid to a mobile phase bottle containing 1 L of LC/MS grade MeCN.
Mixed and stored at room temperature. The solution expired in 1 month.
8.1.5.	Sodium Omadine Solution
Prepared by transferring 0.8 g (±0.1g) of sodium omadine, accurately weighed, into a 25 mL, Class A
volumetric flask. Diluted with RW and mixed by vortex. Resulting nominal concentration was 32 g/L.
Prepared a lOx dilution of the stock solution (3.2 g/L). Stored solutions at 4°C (± 3°C)
8.1.6.	Ascorbic Acid Solution
Prepared by transferring 0.0625 g (±0.001 g) ascorbic acid, accurately weighed, into a 25 mL, Class A
volumetric flask. Dissolved and diluted with RW. Mixed by vortex. Resulting nominal concentration was
2.5 g/L. Stored solutions at 4 °C (±3 °C) and prepared fresh daily.
8.1.7.	Needle Wash 1
Combined 190 mL of RW, 10 mL of MeCN and 10 (iL of acetic acid. Mixed well and stored at room
temperature for up to 30 days.
8.1.8.	Needle Wash 2
Combined 100 mL of RW and 100 mL of MeOH. Mixed and stored at room temperature for up to 30
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days.
8.2. STANDARD SOLUTIONS
8.2.1.	FAA Standard Solutions
Prepared 10 mg/L FAA stock solution by diluting 100 |iL of the purchased 1000 mg/L FAA solution (>
98% purity) into 10 mL of RW. Transferred the remaining purchased stock to a 4-mL vial. Prepared 1-
mg/L by diluting 1 mL of the 10-mg/L FAA solution into 10 mL of RW. Prepared 50-(ig/L by diluting of
500 |iL of 10 mg/L solution into 10 mL of RW. Peak areas and calibration slopes were monitored for
consistency to ensure integrity of the solutions. Stored all solutions at 4 °C (± 3°C) for a maximum of six
months.
8.2.2.	IS Solutions
Prepared 10 mg/L FAA_L stock solution by diluting 100 |iL of the purchased 1000-mg/L FAA_L
solution into 10 mL of RW. Transferred the remaining purchased stock to a 4-mL vial. Prepared 2.5-mg/L
standard by diluting 2.5 mL of the 10 mg/L FAA solution into 10 mL of RW. Stored all solution at 4 °C
(± 3 °C) for a maximum of six months.
8.2.3.	Calibration Standards
Prepared the calibration (CAL) standards by transferring a set amount of FAA dilution stocks into 1-mL
vials. Added the 3.2-g/L sodium omadine solution (20 jxL) and 2.5-/L ascorbic acid solution (100 |iL) to
each CAL standard. Added the 2.5-mg/L FAA L IS solution (10 jxL) to each vial, and diluted to volume
with RW and mixed by vortex. Table 3, Calibration Standards, details the dilution series. These samples
were also used as the CCC.
9.0 Sample Collection, Preservation, and Storage
9.1.	SAMPLE COLLECTION FROM TREATMENT UTILITIES
Treated water samples were obtained from four drinking water utilities across the U.S. Utilities were
selected to provide samples that had varying water quality parameters (Total Organic Carbon (TOC),
conductivity, etc.) and residual disinfectant (chlorine or chloramine) to determine whether these
parameters had any effect on FAA stability or the method. Tested water quality parameters for the four
water sources (WS) are listed in Table 4.
9.2.	SAMPLE PRESERVATION
Drinking water samples were preserved to prevent any compound degradation from residual chlorine and
microbial degradation. Dechlorination was achieved with ascorbic acid (250 mg/L, final concentration)
and sodium omadine (64 mg/L, final concentration) served as an anti-microbial. Additional additives that
were tested, but did not achieve optimal performance are listed in Table 2.
9.3.	WATER FILTRATION STUDY
High and low FAA concentrations in RW were prepared to assess loss during filtration. An aliquot of
each sample was analyzed, filtered and unfiltered. Samples were prepared by adding sodium omadine (80
|iL) and ascorbic acid (10 mg) to two separate 40-mL VOA vials filled with RW. Either 16 or 160 jxL of
10 mg/L FAA stock was added to create a low (4 |ig/L) or high (40 |ig/L) concentration sample. Four
aliquots were removed, filtered, and placed in LC vials. Four additional replicates were removed from the
VOA vial (unfiltered). All replicates were fortified with FAA_L, mixed by vortex, and analyzed.
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9.4. HOLDING TIME STUDY IN REAGENT WATER
A holding time study was performed to determine the stability of RW samples spiked with FAA for up to
28 days. The following preparation scheme was used:
1.	Added 40 mL of RW to a VOA vial. Preserved by adding 80 jxL of 32 g/L of sodium
omadine and 10 mg of ascorbic acid with final concentrations of 64 mg/L and 250 mg/L,
respectively. Added 40 mL of RW and mixed by vortex.
2.	Added either 16 (iL or 160 (iL of 10 mg/L FAA solution to obtain concentrations of
either 4 |ig/L or 40 |ig/L. respectively. Mixed by vortex.
3.	Withdrew ~ 4 mL of sample solution with a syringe, fixed a 0.22-|im filter onto the
syringe, and dispensed the solution into a clean container. Aliquoted 990 jxL of filtered
solution into each LC vial (n=3).
4.	Added 10 jxL of 2.5 mg/L FAA L solution to each LC vial. Capped and mixed by vortex.
5.	Placed the remaining sample solution into 4 °C (± 3 °C) storage conditions.
6.	Analyzed samples and appropriate QCs.
7.	Repeated steps 3-6 for each subsequent time point (1, 3, 7, 14, 21, 28 days).
9.5. WATER STABILITY STUDY IN TAP WATERS
Water from four different sources representing a variety of water types (chlorinated, chloraminated, hard
water, organic content) was prepared and tested with the procedure described in Section 9.4.
10.0 Quality Control
Quality control (QC) requirements included the Initial Demonstration of Capability (IDC) and ongoing
QC requirements to be met when preparing and analyzing samples. This section describes the QC
parameters, their required frequency, and the performance criteria required to meet quality control
objectives.
10.1. INITIAL DEMONSTRATION OF CAPABILITY
The Initial Demonstration of Capability (IDC) was successfully performed prior to analyzing any field
samples. Prior to conducting the IDC, an acceptable initial calibration following the procedure outlined in
Section 11.3 was generated. The following subsections describe how the IDC was generated for this
report.
10.1.1.	Initial demonstration of low system background
A Laboratory Reagent Blank (LRB) was prepared by analyzing blank RW prepared with the same
additives as a standard (i.e., ascorbic acid and sodium omadine) and internal standards. To be acceptable,
method analytes were not detected in the LRB at concentration > 1/3 of the DL.
10.1.2.	Minimum Reporting Level Confirmation
Seven replicates of the minimum reporting level were prepared at the CAL 2 level (see Table 3) and
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analyzed. The mean measured concentration and standard deviation for the method analytes in the seven
replicates were calculated, and the Half Range for the Prediction Interval of Results (HRPir) was
determined using the following formula:
HRpir	=3.963s
where: s	= standard deviation
1.963	= a constant value for seven replicates
The upper and lower limits for the Prediction Interval of Result (PIR) were required to meet the following
upper and lower recovery limits based on the following formulas:
The upper PIR limit requirement was < 150% recovery
Mean + HRpir
Fortified Concentration
The lower PIR limit requirement was > 50% recovery
Mean — HR
x 100% < 150%
PIR x 100% > 50%
Fortified Concentration
10.1.3. Detection Limit Determination
The Detection Limit (DL) was verified by the preparation and analysis of seven (7) replicates of a
standard concentration 1 Ox the noise limit, and 7 LRB replicates were prepared in tandem over the course
of three (3) days. The DL was calculated using the following formula:
DL	— S * t(n-l, l-a=0.99)
where: s	= standard deviation of replicate analyses
t(n-l, l-a=0.99)	=3.143
n	= number of replicates
10.1.4.	Initial Demonstration of Precision
Seven (7) replicates of CAL 5 (Table 3) were prepared for the Initial Demonstration of Precision (IDP)
study and analyzed. To pass acceptability criteria, the calculated relative standard deviation from the
replicate analyses was required to be < 20%.
10.1.5.	Initial Demonstration of Accuracy
The same seven (7) replicates of CAL 5 that were generated for the IDP study were used for the Initial
Demonstration of Accuracy (IDA) study. To pass acceptability criteria, the calculated mean recovery
from the replicate analyses was required to be ± 30%.
10.2 ONGOING QC REQUIREMENTS
10.2.1. Laboratory Reagent Blanks
An LRB was required with each analysis batch (Sect. 4.8) to confirm that background contaminants were
not interfering with the identification or quantitation of FAA. If the LRB showed a peak within the RT
window of FAA, the source of the contamination was determined and eliminated before processing
samples. Background from FAA or other contaminants that interfere with the measurement must be
below 1/3 of the MRL. Blank contamination was estimated by extrapolation, if the concentration was
below the lowest CAL standard. This extrapolation procedure was not allowed for sample results as it
may not meet data quality objectives. If FAA was detected in the LRB concentrations equal to or greater
9

-------
than 1/3 the MRL, then all data must be considered invalid for all samples in the analysis batch.
10.2.2.	Sequence Blanks
Sequence Blanks (SBs) were run in tandem with the CCCs, bracketing every five samples, to periodically
verify a background free of interferences and FAA contamination/carryover.
10.2.3.	FAA verification
FAA identification was confirmed by elution of the sample peak within ± 1.5 % of the average RT
observed in the current calibration. Additional confirmation was provided by the presence of the CF/QT
ratio comparable (± 30 %) to the CF/QT ratio in the most recent calibration.
10.2.4.	Continuing Calibration Check
The calibration was confirmed by analysis of a CCC at the beginning and end of a sample analysis batch.
CCCs were then injected after every five samples alternating between a high level (CAL 8) and a low
level (CAL 2) to ensure instrument sensitivity and stability.
The following requirements were required to be met for a batch to meet acceptability criteria:
1.	The absolute area counts of the IS had to be within 50-150% of the average area
measured in the most recent calibration.
2.	The calculated amount for FAA in the high level CCCs had to be within ± 30% of the
true value.
3.	The calculated amount for FAA in the low level CCCs had to be within ± 50% of the true
value.
10.2.5.	Internal Standard
The analyst monitored internal standard (IS) areas in all injections during the analysis. The IS peak areas
in any chromatographic run were within 50-150% of the average IS area in the most recent calibration
curve. If the IS areas did meet this criterion, inject a second aliquot from the same autosampler vial was
injected.
10.2.6.	Laboratory Fortified Sample Matrix
Analysis of a Laboratory Fortified Sample Matrix (LFSM) was required in each analysis batch and was
used to determine that the sample matrix did not adversely affect method accuracy. Assessment of method
precision was accomplished by analysis of a Laboratory Fortified Sample Matrix Duplicate (Sect. 4.7).
10.5.	STABILITY STUDIES
All stability batches must meet CCC requirements to be acceptable. The concentrations of the stored
(stability) samples were compared to the concentrations analyzed at time zero and % R calculated. To
generate a % R for the reference value, Day 0 replicates were compared to their mean value and a % R
generated. One-way analysis of variance (ANOVA) across RW and drinking water samples relating % R
and time as a factor must show no significance (a =0.05) for FAA to be considered stable.
10.6.	WATER FILTRATION STUDY
The average concentration of the filtered samples was compared to the average concentration of the non-
filtered samples at both concentrations. To be reported as comparable, the sample sets should show no
significance when compared via a one-sided t-test with a =0.05.
11.0 Instrument Calibration and
10

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Standardization
11.1.	LC INSTRUMENT AND PARAMETERS
The LC method parameters are in Table 5 and the gradient is described in Table 6.
11.2.	ESI-MS/MS
The [M-H]" signal was optimized for FAA and FAAL by infusing approximately 1 |ig/m L of each
analyte directly into the MS. The MS parameters were optimized for the precursor m/z, the product m/z
value and the collision energies were determined. See Table 7 for the optimized ESI-MS/MS conditions
and Table 8 for the Multiple Reaction Monitoring (MRM) Transitions.
11.3.	INITIAL CALIBRATION
Nine CAL standards were used in this report. The lowest CAL was required to be at or below the MRL.
The curve was created by normalizing FAA peak area by the IS response. The LC-MS/MS data system
software was used to generate a linear regression calibration curve with 1/x weighting.
11.4.	CALIBRATION CURVE
Linearity was studied over two orders of magnitude. Calibration curves consisted of nine nonzero samples
(each at different concentrations) covering the nominal concentration range of 1 |ig/L - 100 |ig/L. An SB
was analyzed at the beginning and end of the series of CAL injections. Plots of the peak area divided by
internal standard area versus nominal standard concentration were constructed using a best-fit line
determined by regression analysis. A curve-weighting factor of 1/x with linear regression was utilized.
Linearity was acceptable at a coefficient of determination (R2) > 0.99
11.5.	CALIBRATION ACCEPTANCE CRITERIA
Each calibration point (except the lowest point) should calculate to be within 70-130% of its true value.
The lowest CAL point should calculate to be within 50-150% of its true value.
11.6.	CONTINUING CALIBRATION CHECK
The initial calibration must be verified at the beginning and end of each group of analyses, and after every
fifth sample. Continuing Calibration Checks (CCCs) alternated between a low and medium concentration
CAL standard. The absolute areas of the quantitation ions of the IS must be within 50% - 150% of the
average areas measured in the most recent calibration. Additionally, the calculated amount for each
analyte for medium level CCCs must be within ± 30% of the true value and ± 50% at the lowest
calibration level.
12.0 Analytical Procedure
The following procedure was used for preparation of samples for analysis (i.e., Stability Samples, LFSM
and LFSMD, etc.)
1.	Filter > 1 mL of sample water into a clean container
2.	Transfer 990 |iL of filtered sample into an autosampler vial.
3.	Add 10 fxL of FAA L to each samples.
11

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4. Mix by vortex and cap for analysis.
13.0 Data Analysis and Calculations
13.1. DESCRIPTIVE STATISTICS
Descriptive statistics [mean, standard deviation (s), relative standard deviation (% RSD), relative error
RE) and percent difference] were calculated for this method.
13.1.1.	Sample concentration
Results were expressed as a concentration based on the calibration curve. The concentration was
calculated as follows:
,	¦ / rr \ ((response-v int)\
Sample concentration (|ig/L) = ^	)
where:	response = Peak area of the analyte versus IS in the sample
y int =	y-intercept obtained from the calibration curve
slope =	slope obtained from the calibration curve
13.1.2.	Method Accuracy
Method accuracy was expressed as percent relative error (% RE) which was calculated based on the
nominal concentration as follows:
% Relative error = N^ x 100
N
where:	D=	determined concentration
N=	nominal concentration
13.1.3.	Method Precision
Method precision was expressed as percent relative standard deviation (% RSD) when the number of
samples (n) > 3 and was calculated as follows:
% Relative Standard Deviation = x 100
where:	s=	standard deviation
X=	mean
13.1.4.	Confidence Interval
To provide estimation of probability of sample mean, the 95% confidence interval was calculated as
follows:
95% Confidence Interval = X ± 1.96 -7=
\]n
where:	X=	mean
s=	standard deviation
n=	number of samples
13.1.5.	Stability
To evaluate stability, the mean concentration after the storage time was compared to the mean
concentration at Time 0 as follows:
% of Time 0=	-xl00
Y
where:
X=	mean concentration after storage time
Y=	mean determined concentration at Time 0.
12

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14.0 Method Performance
14.1.	LINEARITY
The coefficient of determination (R2) for the calibration standards of a particular batch was required to be
0.99 or greater for the batch to be acceptable. Calculated concentrations for each point should be within
70-130 % of the nominal value and 50-150% for the lowest CAL point. An example calibration curve is
shown in Figure 2. In the displayed instance, R2 = 0.9983, and the greatest deviation from the nominal
concentration was 116% (16% RE) at the lowest calibration level, 1 (.ig/L.
14.2.	CONTINUING CALIBRATION CHECKS
CCC primarily performed within parameters. If a CCC failed, the samples bracketed by the failing check
were re-run.
14.3.	INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND
FAA was not detected in a sample spiked with preservatives and internal standards at concentrations that
were > 1/3 of the DL. A representative chromatogram of a blank sample is shown in Figure 3.
14.4.	MINIMUM REPORTING LEVEL CONFIRMATION
The MRL was determined from seven replicates of samples at the CAL 2 level. The HRpir was
determined to be 0.65 |ig/L. Based on this result, the lower PIR was calculated to be 57.0% and the upper
PIR to be 122%. These values meet both the upper and lower PIR limit requirements of < 150% for the
upper PIR and > 50% for the lower PIR. The MRL confirmation is shown in Table 10. Representative
chromatograms of both 1 |ig/L and 2 |ig/L. the MRL, are shown in Figure 4.
14.5.	DETECTION LIMIT DETERMINATION
FAA detection limit was determined from seven replicates of samples at the CAL 3 level, with batches
prepared over three days. The DL was calculated to be 0.4 |ig/L. using at value of 3.143 for n=7. The DL
calculation is presented in Table 9.
14.6.	INITIAL DEMONSTRATION OF PRECISION
The IDP was determined from seven replicates at the CAL 5 concentration level, calculated versus a
calibration curve. The precision (% RSD) was 3.4%. This value was within the % RSD acceptability
criteria of < 20 %. The IDP results are summarized in Table 11.
14.7.	INITIAL DEMONSTRATION OF ACCURACY
The IDA was determined from the same seven CAL 5 replicates that were used for the IDP study,
calculated vs. a calibration curve. The IDA (% RE) was 2.46%. This value was within the % RE
acceptability criteria of ± 30%. The nominal value was within the confidence interval. The IDA results
are summarized in Table 11.
14.8.	HOLDING TIME STUDY IN REAGENT WATER
Both concentration levels (40 and 4 |ig/L) of FAA in RW were compared across all time points. One-way
ANOVA analysis showed no significant difference (p=0.34) in the recovery during the 28-day test period.
The data set is available in Table 12.
13

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14.9. HOLDING TIME STUDY IN DRINKING WATER
Stability was tested at two concentrations in four different drinking waters. The data set is available in
Table 11. FAA met the stability criteria (Section 10.5) for all drinking water samples (WS 1-4) across the
28-day time frame with a one-way ANOVA showing no statistical difference (p=0.08).
14.10. WATER FILTRATION
One-sided t-tests (a=0.05) showed no difference in the means of the unfiltered and filtered water samples
for both concentration levels. The water filtration study results are summarized in Table 13. Filtering the
samples at either high or low concentrations did not affect the recovery of the target analyte.
15.0 Pollution Prevention
This method utilized ESI-LC/MS/MS for the analysis of FAA in water. The method required the use of small
volumes of organic solvent and small quantities of pure analytes, thereby minimizing the potential hazards to both
the analyst and the environment. All waste was collected and disposed of in accordance with regulations.
16.0 Waste Management
The analytical procedures described in this method generated relatively small amounts of waste since only small
amounts of reagents and solvents were used. The matrices of concern were finished drinking water. Laboratory
waste management practices were in adherence with all applicable rules and regulations. The laboratory protected
the air, water and land by minimizing and controlling all releases from fume hood and bench operations.
Compliance with any sewage discharge permits and regulations, particularly the hazardous waste identification
rules and land disposal restrictions were followed.
17.0 References
1.	U.S. EPA. Selected Analytical Methods for Environmental Remediation and Recovery (SAM) 2012.
U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-12/555, 2012
2.	Connolly, G. Technical Bulletin for the Sodium Fluoroacetate (Compound 1080) Livestock
Protection Collar EPA Registration Number: 56228-22; U.S. Department of Agriculture, Animal and
Plant Health Inspection Service, Wildlife Services: Riverdale, MD, February 2017.
3.	U.S. EPA. 1991. Integrated Risk Information System. Retrieved from US EPA website:
http://www.epa.gov/iris/subst/0469.htm [11/01/2017],
4.	U.S. EPA. Regional Screening Levels (RSLs) - Generic Tables (June 2017). Retrieved from US EPA
website: https://www.epa.gov/risk/regional-screening-levels-rsls-generic-tables-iune-2017
5.	Bessaire, T., Tarres, A., Goyon, A., Mottier, P., Dubois, M., Tan, W.P., Delatour, T. Quanitative
determination of sodium monofluoroacetate "1080" in infant formulas and dairy products by isotope
dilution LC-MS/MS. Food Additives & Contaminants: Part A, 2015, 32 (11) 1885-1892.
6.	Hamelin, E.I., Mawhinney, D.B., Parry, R., Kobeleski, R.J. Quantification of monofluoroacetate and
monochloroacetate in human urine by isotope dilution liquid chromatography tandem mass
spectrometry. Journal of Chromatography B, 2010, 878, 1045-1050.
14

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7. CSID:5046, http://www.chemspider.com/Chemical-Structure.5046.html (accessed 15:06, Nov 30,
2017)
15

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18.0 Tables and Validation Data
Table 1: Summary of IDC with RW
Test Parameter
Fluoroacetate Anion
Matrix
RW

Quantitation
LC-MS/MS

Regression Type
Linear (1/x)

Linear Range
1 |jg/L to 100 |jg/L
IDC Tests
Acceptance Criteria
Results
Coefficient of Determination
NA
>0.99
(R2)


Minimum Reporting Level
<150% Upper PIR Limit
> 50% Lower PIR Limit
Upper limit = 122 %
Lower Limit = 56.9%
Detection Limit Determination
NA
0.41 (jg/L
Initial Demonstration of Low
Background < 1/3 of minimum
Non-detect
System Background
reporting Level

Initial Demonstration of
< 20 % RSD
3.4 %
Precision


Initial Demonstration of
± 30 % Mean Recovery (RE)
2.5 %
Accuracy


FAA Holding Time Study3
Test
p-valueb
Reagent Water (RW)
One-way ANOVA
0.34
Drinking Water (WS 1-4)
One-way ANOVA
0.09
statistical results for both RW and drinking water samples across all time points
bResults considered significant if p < 0.05
Table 2: Parameters tested during method development
Test Parameters	Test System	Performance
Ammonium acetate (2	Added as a dechlorinating Caused FAA peak
.. M) - Final	agent with sodium	broadening
Preservatives
concentration in	omadine as an anti-
sample 1.5 g/L	microbial
Columns
Waters BEH Amide
column
SIELC Primesep B2
Water + 0.01 % formic
acid + 5mM ammonium
formate, gradient elution
Direct aqueous injection is
incompatible with hydrophilic
interaction chromatography
60:40 MeCN: Water +
0.05% Acetic acid
FAA RT not stable among
waters with different pH
values.
16

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Table 3: Calibration Standards
Calibration
Source
Source
2.5 g/L
3.2 g/L
2.5 mg/L
Final
Nominal
Solution
Solution
Solution
Ascorbic
Sodium
FAA L
Volume
Solution
Name

Volume
Acid
Omadine
Standard
of
Con-


(ML)
Solution
Solution
Solution
Solution
centration



Volume
(ML)
(ML)
(mL)
(mq/l)



(ML)




CAL 1
0.05 mg/L FAA
20
100
20
10
1
1
CAL 2
0.05 mg/L FAA
40
100
20
10
1
2
CAL 3
0.05 mg/L FAA
80
100
20
10
1
4
CAL 4
0.05 mg/L FAA
160
100
20
10
1
8
CAL 5
1 mg/L FAA
12
100
20
10
1
12
CAL 6
1 mg/L FAA
20
100
20
10
1
20
CAL 7
1 mg/L FAA
40
100
20
10
1
40
CAL 8
1 mg/L FAA
60
100
20
10
1
60
CAL 9
1 mg/L FAA
100
100
20
10
1
100
Table 4: Water Quality Parameters for the four water samples used in this study

WS 1
WS 2
WS 3
WS 4
Date Collected
2/22/2017
2/23/2017
8/01/2017
8/10/2017
PH
9.35
8.8
7.14
7.69
Turbidity3
0.27 NTU
0.05 NTU
0.28 NTU
0 FAU
Conductivity (MS/cm)
348
287
1124
388
Alkalinity (mg/L)
76
63
346
65
Hardness (mg/L)
76
114
436
127
Free chlorine (mg/L
0.03
1.3
0.4
0.07
Chloramine (mg/L)
NA
NA
NA
3.11 (mono)
Total Organic Carbon (mg/L)
7.85
1.13
NA
NR
aNTU=Nephelometric Turbidity Unit, FAU=Formazin Attenuation Units
Table 5: LC Parameters
Parameter
Setting
Mobile Phase A
0.005% Acetic Acid in water
Mobile Phase B
0.005% Acetic Acid in MeCN
Column
Sunfire™ -dC8,

150 mm x 4.6 mm, 5 |jm

particle size or equivalent
Column
37 °C
Temperature

Injection Volume
20 (jL
17

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Table 6: LC Gradient
TIME %A
%B
FLOW LC FLOW
(MIN)

RATE


(mL/min)
0 95
5
0.5 Waste
1 95
5
0.5
11 60
40
0.5 Instrument (10 to 21
14 0
100
0.5 min)
19 0
100
0.5
20 95
5
0.5
21 80
20
1 Waste
24 0
100
1
30 95
5
1
30.5 95
5
0.5
32 95
5
0.5
Die 7: ESI and MS Parameters


Parameter

Value
Mass Spectrometer

Thermo Quantum TSQ
Software

Thermo Xcalibur 2.2 SP1.48
Ionization Mode

Heated Electrospray, negative
Scan Mode

Multiple reaction monitoring (MRM)
Spray Voltage

3300 V
Vaporizer Temperature

250 °C
Sheath Gas Pressure

50
Ion Sweep Gas

0
Auxiliary Gas Pressure

0
Capillary Temperature

275 °C
Capillary Offset

-85
Tube Lens Offset

-90
Collision Gas Pressure

0.7
Table 8: Compound specific MRM parameters
COMPOUND NAME
FLUOROACETATE
13C2,2-2D2
FLUOROACETATE
aQT=Quantification transition, CF=Confirmation transition
MONITORED
DWELL
COLLISION
RETENTION
TYPE
TRANSITION
TIME (MS)
ENERGY (EV)
TIME (MIN)

77.1 > 57.26
100
14
19.4
QT
77.1 > 33.04
100
14
19.4
CF
80.95 >36.13
100
14
19.4
QT
80.95 > 59.9
100
14
19.4
CF
18

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y=0.01736-0.0154x
R2=0.9983
w:1/x
40 60 80
Concentration (jjg/L)
100
120
Figure 2: Representative FAA Calibration Curve. Response is the area of the QT (57.26 m/z)
divided by the internal standard quantification ion (36.13 m/z).
4000
1 ppb FAA
blank
£>• £>' -T 4?'	^
Time
Figure 3: Blank and 1 pg/L sample chromatogram. Extracted Ion Chromatogram (EIC) of 57.26 m/z.
19

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Table 9: DL Calculation

Nominal
Determined
Sample
Concentration
Concentration

(Mg/L)
(Mg/L)
DL CAL 3 Day 1
4
4.2
DL CAL 3 Day 2
4.1
DL CAL 3 Day 2
3.8
DL CAL 3 Day 2
4.2
DL CAL 3 Day 3
4.0
DL CAL 3 Day 3
4.1
DL CAL 3 Day 3
4.0

Average
4.0

s
0.13
Calculated DL	0.4
Table 10: Half-Range Prediction Interval Calculation
Sample Name
Nominal
Determined


Concentration
Concentration


Level (Mg/L)
(Mg/L)
DL CAL 2
1

1.7
DL CAL 2
2

2.0
DL CAL 2
3

2.0
DL CAL 2
4
2
1.7
DL CAL 2
5

1.6
DL CAL 2
6

1.9
DL CAL 2
7

1.6


Average
1.8


s
0.2


HRpir
0.65
20

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Table 11: Initial Demonstration of Accuracy and Precision
Sample
Nominal
Concentration
(mq/l)
Determined
Concentration
1 DP/I DA Cal 5_1

12.1
I DP/I DA Cal 5_2

11.5
I DP/I DA Cal 5_3

11.3
I DP/I DA Cal 5_4
12
11.2
I DP/I DA Cal 5_5

12.2
I DP/I DA Cal 5_6

11.7
I DP/I DA Cal 5_7

11.9

Average
11.7

s
0.397

Precision (% RSD)
3.4%

Accuracy (% RE)
2.5%
95% Confidence	11.7 ±0 .29
interval
3000
2500
2000
g 1500
1000
500
1 |jg/L CF
1 |jg/L QT
0

16.0 16.8 17.7 18.6 19.5 20.4
Time (min)
17.7 18.6
Time (min)
20.4
Figure 4:Low Standard Chromatograms for FAA
21

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Table 12: % FAA remaining compared to Day 0.
Day 0 Day 1 Day 3 Day 7 Day 14 Day 21 Day28/31a
RW Low3 Average (Mg/L)
3.3
3.7
3.9
3.7
3.7
3.5
3.5
% RSD
5
9
6
8
11
5
12
% of day 0
ref
112
120
114
111
107
108

RW High Average (Mg/L)
44.9
39.0
43.4
39.0
40.0
41.9
38.2
% RSD
8
3
6
13
8
4
13
% of day 0
ref
87
97
87
89
93
85

WS 1 Low3 Average (Mg/L)
3.9
3.9
3.3
4.3
3.7
3.5
3.5
% RSD
13
8
10
5
9
7
8
% of day 0
ref
101
86
110
94
89
91
WS1 High3 Average (Mg/L) 41.1 39.5 36.9 39.0 41.8 38.0	44.2
% RSD	4	5	7	2	3	2	11
% of day 0	ref 96 90 95 102 92	107
WS 2 Low3
4.0
3.8
3.7
3.8
3.2
3.3
3.0
Average (Mg/L)







% RSD
15
7
2
10
15
10
26
% of day 0
ref
94
92
95
80
81
76
WS 2 High3
41.5
39.2
37.6
40.2
38.3
36.8
45.3
Average (Mg/L)







% RSD
2
4
1
4
1
4
8
% of day 0
ref
94
91
97
92
89
109
WS 3 Low Average (Mg/L)
4.0
3.6
2.5
3.1
3.0
3.1
2.4
% RSD
5
13
13
6
33
14
15
% of day 0
ref
89
63
76
74
78
59
WS 3 High Average (Mg/L)	40.3 38.5 36.2 40.0 38.4 44.4	44.2
% RSD	11	3	3	9	8	9	3
% of day 0	ref 96 90 99 95 110	110
WS 4 Low Average (Mg/L)
4.0
4.0
3.9
4.0
4.0
3.5
3.6
% RSD
1
10
21
11
20
9
29
% of day 0
ref
100
99
101
99
88
90

WS 4 High Average (Mg/L)
40.6
37.3
36.6
41.3
37.4
39.8
37.4
% RSD
3
2
5
6
3
2
5
% of day 0
ref
92
90
102
92
98
92
aFinal measurement was conducted after 31 days.
All values are calculated from three replicates. RSD = relative standard deviation. Ref = Day 0 was the
comparison value so recovery is considered 100%.
22

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Table 13: Sample Filtration
Condition

Low
Concentration3
High
Concentration13

Average0
3.4
37.3
Unfiltered
s
0.84
2.22

RSD
25%
6%

Average0
3.0
38.5
Filtered
s
0.61
3.33

RSD
20%
9%
p-valued

0.25
0.29
aLow concentration = 4 |ig/L
bHigh concentration = 40 |ig/L
cn=4
done-tailed t-test, a=0.05
23

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Appendix A: Methyl Fluoroacetate Analysis
The FAA method was evaluated to determine if it could be used to measure Methyl fluoroacetate (MFA), a
hydrolysis product of FAA. MFA spiking experiments verified that the transformed product could be measured
using the FAA method. Preliminary experiments, results and important considerations are discussed below.
A. 1. INTRODUCTION
Methyl fluoroacetate (MFA) is a methylated analog of FAA, which has similar toxicity (3-4 mg/kg,
orally, rat) [1]. MFA is available from U.S. vendors and is used as a pharmaceutical precursor or as a
starting material during consumer product formulation.
MFA is vulnerable to both acid- and base-catalyzed hydrolysis in water, transforming into the free acid,
FAA. Base-catalyzed hydrolysis is purported to be faster. Price and Jackson [2] reported a minimum rate
of hydrolysis between pH of 2-3.5 and a hydrolysis half-life of less than an hour at pH=7. Structure-based
calculations (estimated by EPI Suite and accessed on ChemSpider) estimate a half-life of 9.67 h at pH =8
and a half-life of 4.03 days at pH=7 [3] at 25°C. MFA is also more volatile than FAA with Henry's
constant of 3.49 xlO"4 atm m3/mol [3], making the loss to volatilization from water more likely than with
the free acid.
Preliminary experiments were conducted to verify hydrolysis of MFA in tap water, examine the effect of
water pH, and whether FAA measurements would be a feasible way to characterize contamination levels.
Accordingly, tests were evaluated in RW and WS 2 utilizing the procedures described for FAA analysis.
A.2. MATERIALS AND METHODS
MFA was purchased from TCI America (Portland, OR) and diluted to 10 mg/L in MeCN. Four water
samples were prepared, two with RW and two with WS 2. One of each water type was preserved as
described in the FAA method (Section 9.0). A method blank was also prepared with preservatives. After
mixing, the pH was measured. Samples were fortified to 0.54 (j,M (50 |ig/L) of MFA, and the time was
recorded. Filtered samples were aliquoted into LC vials. Sample handling, analytical and calculation
procedures were as described for FAA. Samples were injected in sets of five (i.e., one injection of each
treatment) interspersed with CCC and blanks. Each set (~ 3.5 h) was marked as one time point and the
total covered approximately 24 h. While awaiting injection, samples were stored in the sample tray at
10°C.
A 3. RESULTS
Water pH ranged from 3.71 to 8.5, and values are listed associated with the treatment in Figure 5. Water
utilities typically try to maintain a basic pH to minimize corrosion, but the pH range can span 6-8.5.
Addition of the ascorbic acid preservative lowered water pH to outside that range, so it is not strictly
representative of tap water other than to show the effect of preservatives/pH. The MFA stock in MeCN
was also analyzed and showed a consistent level of -0.078 (iM (7 |ig/L) FAA, demonstrating the
transformation of MFA without the presence of water.
The method blanks showed a consistent level of FAA. During the FAA method development, no other
method blank samples showed FAA contamination. Given the position of the LRB in sample injection
sequence, two contamination scenarios are plausible. First, the LRB was contaminated during preparation
due to the more volatile nature of MFA. Second, the LRB was run at the end of the five injection series. It
is possible that the untransformed MFA was retained and transformed into FAA on the column and eluted
during subsequent injections. The second explanation seems slightly more likely since the LRB was the
same preparation as the RW plus preservatives (RW + P) and had a relatively low pH.
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Only the MFA in the tap water source (no preservatives) (Tap) displayed complete transformation to FAA
over the course of 24 h. No transformation was observed with MFA in tap water source plus preservatives
(Tap + P) This result is consistent with previous literature on hydrolysis rates and the estimated
physicochemical properties of MFA. In all other water treatments, the pH was too low (<6.5) to facilitate
hydrolysis in the studied time-frame.
Time point
Figure 5: Hydrolysis of MFA to FAA in four different water treatments.
A.4. CONCLUSIONS
1.	The alkaline pH maintained by many water utilities will likely begin MFA conversion to
FAA upon its addition. However, it may take many half-lives (depending on amount
added) to achieve complete transformation.
2.	Due to MFA volatility, sample collection may need to be headspace-free to reduce losses.
3.	If using FAA to estimate MFA contamination, exhaustive hydrolysis of samples may
need to be conducted to ensure complete conversion prior to analysis. Otherwise, toxicity
may be underestimated due to presence of MFA that is not converted. Likely this will
include pH adjustment to pH >8 and vigorous shaking for longer than 24 h.
A.5. REFERENCES
1.	Floss, G.L., The Toxicology and Pharmacology of Methyl Fluoroacetate (MFA) in
Animals with Some Notes on Experimental Therapy. Brit. L. Pharmacol. (1948), 3, 118.
2.	Price, C.C.; Jackson, W.G., Some Properties of Methyl Fluoroacetate and Fluoroethanol,
Journal of the American Chemical Society (1947), 69(5) 1065-1068.
3.	CSID:9565, http://www.chemspider.com/Chemical-Structure.9565.html (accessed 14:57,
Nov 30, 2017)
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