. epa.gov/resear
United States
Environmental Protection
Agency
Adaptation of the Conditions of U.S
EPA Method 538 for the Analysis
of a Toxic Degradation Product of
Nerve Agent VX (EA2192) in Water
by Direct Aqueous Injection- Liquid
Chromatography/Tandem Mass
Spectrometry
FINAL REPORT
CH(CH3)2
HO-P-SCH2CH2-N
CH,
\
CH(CH3)2
S-(2-Diisopropylaminoethyl)
methyl phosphonothioate
(EA2192)
Office of Research and Development
National Homeland Security Research Center
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DISCLAIMER
The U.S. Environmental Protection Agency through its Office of Research and Development
funded and managed the research described here under Contract No. EP-C-11-03, Task Order
Number 0007 to Tetra Tech, Inc., Cincinnati, Ohio. 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. Mention of trade names, products, or services does
not convey official EPA approval, endorsement, or recommendation.
Questions concerning this document or its application should be addressed to:
Stuart Willison, Ph.D.
U.S. Environmental Protection Agency
National Homeland Security Research Center
26 W. Martin Luther King Drive, MS NG16 Cincinnati, OH 45268
513-569-7253
Willison.Stuart@epa.gov
Matthew Magnuson, Ph.D.
U.S. Environmental Protection Agency
National Homeland Security Research Center
26 W. Martin Luther King Drive, MS NG16 Cincinnati, OH 45268
513-569-7321
Magnuson.matthew@epa.gov
11
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Ill
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EXECUTIVE SUMMARY
The objective of this study was to evaluate U.S. EPA's Method 538 for the assessment of
drinking water exposure to the nerve agent degradation product, EA2192, the most toxic
degradation product of nerve agent VX. As a result of the similarities in sample preparation and
analysis that Method 538 uses for nonvolatile chemicals, this method is applicable to the
nonvolatile chemical warfare agent (CWA) degradation product, EA2192, in drinking water. The
method might be applicable to other nonvolatile CWAs and their respective degradation products
as well, but the method will need extensive testing to verify compatibility. Gaps associated with
the need for analysis methods applicable to such analytes were addressed by adapting the EPA
538 method for this CWA degradation product. Many laboratories have the experience and
capability to run the already rigorous method for nonvolatile compounds in drinking water.
Increasing the number of laboratories capable of carrying out these methods serves to
significantly increase the surge laboratory capacity to address sample throughput during a large
exposure event. The approach desired for this study was to start with a proven high performance
liquid chromatography tandem mass spectrometry (HPLC/MS/MS) method for nonvolatile
chemicals in drinking water and assess the inclusion of a similar nonvolatile chemical, EA2192.
Two analytes that are currently in Method 538, methamidophos and acephate, were used as
reference standards to determine method acceptability. Methamidophos-d6 was used as an
internal standard.
An HPLC/MS/MS assay for the quantitation of EA2192 in deionized (DI) water was evaluated in
a series of studies reported here. DI water samples fortified with EA2192 were analyzed
following Method 538 procedures. The samples were analyzed on an Applied Biosystems API-
4000 Mass Spectrometer, coupled with a Shimadzu Liquid Chromatography system. The
objectives and procedures used for sample preparation and analysis are described in EPA
Method 538. The only modification to Method 538 was the inclusion of a flow diversion valve to
reduce source contamination.
The method accuracy, precision, reproducibility, linearity, detection limit and quantitation limit
for EA2192 in DI water were evaluated and found to be within the acceptance criteria of
Method 538. Additionally, EA2192 was stable following 28 days at refrigerated temperatures (5
°C ± 3 °C) in all tested water types except chlorinated water.
The method was evaluated to determine if filtering water samples prior to analysis affected
EA2192 concentrations. No loss of EA2192 was observed after filtering the spiked samples.
Preliminary method development was performed to determine if the current HPLC/MS/MS
method could be transferred to ultra-high performance chromatography tandem mass
spectrometry (UPLC/MS/MS). Modifying this method to incorporate UPLC analysis would
drastically shorten the analytical run time from the current 30 minute method to 5 minutes or
less. A method was developed for two of the analytes currently monitored in Method 538,
methamidophos and acephate, along with EA2192. Methamidophos-d6 was used as an internal
standard. Further method development efforts are required to determine the feasibility of
transferring all Method 538 analytes to UPLC/MS/MS, followed by an Independent
Demonstration of Capability to transfer the method.
IV
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TABLE OF CONTENTS
DISCLAIMER ii
ACKNOWLEDGMENTS iii
EXECUTIVE SUMMARY iv
LIST OF TABLES vii
LIST OF FIGURES viii
LIST OF ACRONYMS AND ABBREVIATIONS ix
INTRODUCTION 1
SCOPE AND APPLICATION 1
SUMMARY OF METHOD 2
DEFINITIONS 2
INTERFERENCES 3
HEALTH AND SAFETY 4
EQUIPMENT AND SUPPLIES 4
REAGENTS AND STANDARDS 5
SAMPLE COLLECTION, PRESERVATION, AND STORAGE 9
QUALITY CONTROL 11
INSTRUMENT CALIBRATION AND STANDARDIZATION 13
ANALYTICAL PROCEDURE 14
DATA ANALYSIS AND CALCULATION 14
METHOD PERFORMANCE 16
POLLUTION PREVENTION 19
WASTE MANAGEMENT 19
REFERENCES 19
TABLES AND VALIDATION DATA 20
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ATTACHMENTS 42
19.1: UPLC Method Development and Results from Adapting the Conditions of U.S. EPA
Method 538 for Ultra High Performance Liquid Chromatography/Tandem Mass Spectrometry
(UPLC/MS/MS) Analysis of EA2192 in Water A-l - A-4
19.2: Certificates of Analysis - EA2192, Methamidophos, and Acephate B-l - B-5
VI
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LIST OF TABLES
Table 1. Initial Demonstration of Capability Testing Summary 20
Table 2. Calibration Standards 20
Table 3. Continuing Calibration Check Standards 21
Table 4. Laboratory Fortified Sample Matrix Preparation 21
TableS. Water Conditions 21
Table 6. Water Sample Parameters upon Collection 21
Table 7. Measured Water Parameters at Time of Sample Preparation 22
Table 8. Water Sample Preparation 22
Table 9. HPLC Method Parameters 22
Table 10. HPLC Gradient 23
Table 11. MS/MS Method Parameters 23
Table 12. MRM Transitions 23
Table 13. IDC Calibration Curve Standards—EA2192 24
Table 14. IDC Calibration Curve Standards—Methamidophos 25
Table 15. IDC Calibration Curve Standards—Acephate 26
Table 16. EA2192 Detect!on Limit Determination 27
Table 17. EA2192 Method Reporting Limit Determination 27
Table 18. EA2192 Initial Determination of Precision and Accuracy 28
Table 19. EA2192 Holding Time Study—DI Water 28
Table 20. EA2192 Stability Study 29
Table 21. Filtered Water Comparison Study (HPLC) 31
Table 22. Filtered Water Comparison Study (UPLC) A-5
vn
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LIST OF FIGURES
Figure 1. Representative EA2192 Calibration Curve 32
Figure 2. Representative Methamidophos Calibration Curve 33
Figure 3. Representative Acephate Calibration Curve 34
Figure 4. Representative Chromatogram of a Matrix Blank without IS (DOLSB) 35
Figure 5. Representative Chromatogram of a Matrix Blank with Internal Standard 36
Figure 6. Representative Chromatogram of a Calibration Standard at the MRL 37
Figure 7. Representative EA2192 Chromatograms of Source No. 1 Water 38
Figure 8. Representative EA2192 Chromatograms of Source No. 2 Water 39
Figure 9. Representative EA2192 Chromatograms of Source No. 3 Water 40
Figure 10. Representative EA2192 Chromatograms of Source No. 4 Water 41
Figure 11. CAL1 Standard, UPLC/MS/MS Analysis A-3
Figure 12. CAL7 Standard, UPLC/MS/MS Analysis A-4
Vlll
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LIST OF ACRONYMS AND ABBREVIATIONS
ACC
Acephate
°C
CAL
CCC
CWA
DI
DAI-LC/MS/MS
DL
EA2192
EPA
ESI
HPLC/MS/MS
HSS
IDA
IDC
IDP
IS
ISPDS
LC
LFB
LFSM
LFSMD
LRB
MEOH PDS
Methamidophos
MRL
MRM
ms
NA
PDS
PIR
PPE
psi
QC
QCS
r
RE
RSD
RT
SD
SDS
% Accuracy
N-(methoxy-methylsulfanylphosphonyl) acetamide
degree(s) Celsius
Calibration Standard
Continuing Calibration Check
Chemical Warfare Agent
Deionized (Water)
Direct Aqueous Injection - Liquid Chromatography/Tandem Mass
Spectrometry
Detection Limit
S,2-diisopropylaminoethyl methylphosphonothioic acid
Environmental Protection Agency
Electrospray lonization
High-Performance Liquid Chromatography/Tandem Mass Spectrometry
Half Range for the Prediction Interval of Results
High Strength Silica
Initial Demonstration of Accuracy
Initial Demonstration of Capability
Initial Demonstration of Precision
Internal Standard
Internal Standard Primary Dilution Standard
Liquid Chromatography
Laboratory Fortified Blank
Laboratory Fortified Sample Matrix
Laboratory Fortified Sample Matrix Duplicate
Laboratory Reagent Blank
Methanolic Analyte Primary Dilution Standard
O, S-dimethyl phosphoramidothioate
Minimum Reporting Level
Multiple Reaction Monitoring
millisecond(s)
Not Applicable
Primary Dilution Standard
Prediction Interval of Result
Personal Protective Equipment
Pounds per Square Inch
Quality Control
Quality Control Sample
Correlation Coefficient
Relative Error
Relative Standard Deviation
Retention Time
Standard Deviation
Safety Data Sheet
IX
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SOP Standard Operating Procedure
SSS Stock Standard Solution
TOC Total Organic Carbon
UPLC/MS/MS Ultra High Performance Chromatography Tandem Mass Spectrometry
V Voltage
VX O-ethyl S-[2-ethyl] methylphosphonothioate
WATER PDS Aqueous Analyte Primary Dilution Standard
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1. INTRODUCTION
1.1 The U.S. Environmental Protection Agency's (EPA's) Method 538 is a direct aqueous
injection-liquid chromatography/tandem mass spectrometry (DAI-LC/MS/MS) method
for the determination of selected nonvolatile chemical contaminants in drinking water.
The purpose of this study was to evaluate EPA Method 538 for its applicability to the
assessment of nerve agent degradation exposure by analyzing S,2-
diisopropylaminoethyl methylphosphonothioic acid (EA2192), a degradation product of
O-ethyl S-[2-ethyl] methylphosphonothioate (VX). EA2192 was evaluated following
the criteria outlined in U.S. EPA Method 538 across a concentration range of 0.05-20
Hg/L (See the Attachment 20.1 for U.S. EPA Method 538). The sample preparation,
analysis, and quantitation were performed according to Method 538. The method
accuracy, precision, reproducibility, linearity, and quantitation limits in deionized (DI)
water were evaluated. Holding time studies in a variety of water types were evaluated
for 28 days. Two chemicals that are currently included in Method 538, methamidophos
and acephate, were included in the analysis as reference standards to verify method
functionality. Methamidophos-d6 was used as the internal standard. EPA's Method 538
conditions can be used to analyze for EA2192.
2. SCOPE AND APPLICATION
2.1 The scope of this study was to determine if EA2192, a degradation product of VX,
could be analyzed under similar conditions as reported in Method 538. Method 538 was
evaluated for accuracy, precision, reproducibility, linearity, and quantitation limits for
EA2192 in water. (See Table 1 for a summary of results.) The detection limit for
EA2192 is 0.0130 |J,g/L. Holding time studies in DI water and drinking water were
evaluated for a period of 28 days. Additionally, water samples were tested, representing
a variety of water types (chlorinated, chloraminated, hard water, etc.), to determine the
stability of EA2192. Methamidophos and acephate, compounds currently included in
Method 538, were included in the testing as reference standards. Methamidophos-d6
was used as the internal standard. The following analyte was tested:
Chemical Name:
Code Name:
Empirical Formula:
Lot Number:
Purity:
Storage Conditions:
Structure:
S-[2-(diisopropylamino) ethyl] methylphosphonic acid
EA2192
C9H22NO2PS
NA
94.2 % by NMR (Appendix C)
2-8 °C
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3. SUMMARY OF METHOD
3.1 A 40-mL water sample was collected in a bottle containing sodium omadine
(antimicrobial agent) and ammonium acetate (to bind free chlorine in sample). An
aliquot of the sample was placed in an autosampler vial with the internal standard
added. A 50-jiL injection was made into an LC equipped with a CIS column
interfaced to an MS/MS operated in the electrospray ionization (ESI) mode. The
analytes were separated and identified by comparing the acquired mass spectra and
retention times to reference spectra and retention times for calibration standards
acquired under identical LC/MS/MS conditions. The concentration of each analyte
was determined by internal standard calibration using procedural standards.
4. DEFINITIONS
4.1 CALIBRATION STANDARD (CAL) - A solution prepared from the primary
dilution standard solution and/or stock standard solution and the internal standard.
The CAL solutions are used to calibrate the instrument response with respect to
analyte concentration.
4.2 CONTINUING CALIBRATION CHECK (CCC) - A calibration standard
containing the method analytes and internal standard. The CCC is analyzed
periodically to verify the accuracy of the existing calibration for those analytes.
4.3 DETECTION LIMIT (DL) - The minimum concentration of an analyte that can be
identified, measured, and reported with 99 % confidence that the analyte
concentration is greater than zero. The DL is a statistical determination of precision
and accurate quantitation is not expected at this level.
4.4 INTERNAL STANDARD (IS) - A pure chemical dissolved in a standard solution in
a known amount and used to measure the relative response of other method analytes
that are components of the same solution. The internal standard should be a chemical
that is structurally similar to the method analytes, has no potential to be present in
water samples, and is not a method analyte.
4.5 LABORATORY FORTIFIED BLANK (LFB) - A volume of reagent water or other
blank matrix to which known quantities of the method analytes and all the
preservation reagents are added in the laboratory. The LFB is analyzed exactly like a
sample, and its purpose is to determine whether the methodology is in control, and
whether the laboratory is capable of making accurate and precise measurements.
4.6 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - A preserved 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
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determined in a separate sample and the measured values in the LFSM corrected for
background concentrations.
4.7 LABORATORY FORTIFIED SAMPLE MATRIX DUPLICATE (LFSMD) - A
duplicate of the Field Sample used to prepare the LFSM. The LFSMD is fortified,
and analyzed identically to the LFSM.
4.8 LABORATORY REAGENT BLANK (LRB) - An aliquot of reagent water or other
blank matrix that is treated exactly as a sample including exposure to all glassware,
equipment, solvents and reagents, sample preservatives, and internal standards that
are 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 (MRL) - 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 PRIMARY DILUTION STANDARD SOLUTION - A solution containing the
analytes prepared in the laboratory from stock standard solutions and diluted as
needed to prepare calibration solutions and other needed analyte solutions.
4.11 QUALITY CONTROL SAMPLE (QCS) - A solution of method analytes of known
concentrations that is obtained from a source external to the laboratory and different
from the source of calibration standards. The QCS is used to check calibration
standard integrity.
4.12 SAFETY DATA SHEET (SDS) - 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 STOCK STANDARD SOLUTION (SSS) - A concentrated solution containing one
or more method analytes prepared in the laboratory using assayed reference
materials or purchased from a reputable commercial source.
5. INTERFERENCES
5.1 Method interferences could be caused by contaminants in solvents, reagents
(including reagent water), sample bottles and caps, and other laboratory supplies or
hardware that lead to discrete artifacts and/or elevated baselines in the
chromatograms. All items such as these were routinely demonstrated to be free from
interferences (less than Vs the DL) under the conditions of the analysis by analysis of
an LRB. Subtracting blank values from sample results is not permitted.
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5.2 Relatively large quantities of the preservatives were added to sample bottles. The
potential existed for trace-level organic contaminants in these reagents. Interferences
from these sources were monitored by analysis of LRBs.
6. HEALTH AND SAFETY
6.1 The toxicity or carcinogenicity of each reagent used in this method had not been
defined precisely. Each chemical was treated as a potential health hazard and
exposure to these chemicals was minimized through the proper use of personal
protective equipment (PPE). A reference file of SDSs was made available to all
personnel involved in the chemical analyses.
7. EQUIPMENT AND SUPPLIES
7.1 GLASSWARE AND SUPPLIES - all equipment used was calibrated and validated
(if applicable) according to standard operating procedures (SOPs).
7.1.1 ANALYTICAL BALANCE - Balances used included:
Mettler Toledo AX26DR (Mettler-Toledo Inc., Columbus, OH)
Mettler Toledo XS205DU (Mettler-Toledo Inc., Columbus, OH)
Mettler Toledo UMX2 microbalance (Mettler-Toledo Inc., Columbus, OH)
7.1.2 AUTOPIPETTES - 10 |iL, 100 |iL, 1,000 |iL ± 1 % accuracy
7.1.3 CLASS A VOLUMETRIC GLASSWARE - various sizes
7.1.4 SAMPLE COLLECTION CONTAINERS - Clean 100 mL Nalgene® (Thermo
Fisher Scientific Inc., Waltham, MA) polypropylene containers
7.1.5 AUTOSAMPLER VIALS - 2-mL autosampler vials with pre-slit screw tops
7.1.6 COLORIMETER - Hach Pocket Colorimeter II, Chlorine, MR and HR, with
Hach Voluette® Analytical Standards, chlorine concentration: 64.8 ± 0.2 mg/L
(Hach Company, Loveland, CO)
7.1.7 pH PAPER - Fisher Brand™ (Pittsburgh, PA) pH paper rolls (catalog no. 13-
640-507)
7.1.8 FILTERS - Acrodisc® filters (Pall Corporation, Port Washington, NY), GHP,
25 mm, 0.45 jim
7.1.9 REFRIGERATOR - 5 °C ± 3° C
7.1.10 FREEZER --20 °C± 10 °C
7.2 LC/MS/MS APPARATUS
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7.2.1 LIQUID CHROMATOGRAPHY (LC) SYSTEM - The LC system had
programmable solvent mixers capable of delivering a flow rate of 0.3 mL/min.
The LC system had all requisite accessories including injection syringe,
degasser, and temperature-controlled autosampler. The LC system used in this
analysis was a Shimadzu Solvent Delivery Module (LC-10 ADvp) (Shimadzu
Inc., Columbia, Maryland) with a SIL-5000 autosampler.
7.2.2 ANALYTICAL COLUMN - Waters (Milford, MA) Atlantis T3, 150 x 2.1
mm, 5 |im particle size.
7.2.3 TANDEM MASS SPECTROMETER (MS/MS) SYSTEM - The mass
spectrometer for the analyses (Applied Biosystems AP1-4000) (Waltham, MA)
utilized positive ion ESI ionization and was capable of performing MS/MS
analyses, producing unique product ions with a minimum of 10 scans across
each chromatographic peak.
7.2.4 DATA SYSTEM - Analyst Version 1.5.1 software was used to acquire, store,
reduce and output mass spectral data. The computer software had the capability
of processing stored LC/MS/MS data by recognizing an LC peak within any
given retention time window. The software allowed integration of the ion
abundance of any specific ion within specified time or scan number limits. The
software was able to construct linear regression calibration curves and
calculate analyte concentrations. The LC was controlled using Waters Acquity
(Milford, MA) Version 1.40.
7.2.5 ULTRA-HIGH PERFORMANCE LIQUID CHROMATOGRAPH - The
UPLC used for the method development was a Waters Acquity UPLC System
(Milford, MA), which included the temperature-controlled autosampler,
injection syringe, and degasser. The UPLC was capable of delivering a flow
rate of 0.6 mL/min.
8. 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 - EA2192 was supplied by in-house supply. See the Attachment
20.2 for the EA2192 Certificate of Analysis. Methamidophos (CAS No. 10265-
92-6, Lot No. SZBD011XV) and acephate (CAS No. 30560-19-1, Lot No.
SZBA083XV) were supplied by Sigma-Aldrich (St. Louis, MO).
Methamidophos-d6 (Lot No. 20515 AC) was procured from EQ Laboratories
(Atlanta, GA).
8.1.2 SOLVENTS AND CHEMICALS - Solvents utilized for this study were
acetonitrile (Fisher, HPLC Grade) (Waltham, MA), methanol (Burdick and
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Jackson, HPLC Grade) (Morristown, NJ), and DI water (in-house supply), and
were demonstrated to be free of analytes and interferences. Chemicals included
ammonium acetate (Sigma-Aldrich, > 97 %), sodium omadine (Sigma-Aldrich,
> 96 %) and ammonium formate (Sigma-Aldrich, > 99.995 %).
8.1.3 MOBILE PHASE A - Prepared by adding 1.26 g of ammonium formate,
accurately weighed (±0.1 g) to a mobile phase bottle and dissolving in 1 L of
high-purity water. The mobile phase was mixed well and stored at room
temperature. The solution expired in 48 hours.
8.1.4 MOBILE PHASE B - 100 % methanol.
8.1.5 SODIUM OMADINE SOLUTION - Prepared by transferring ~ 0.8 g (±0.1 g)
of sodium omadine, accurately weighed, into a 25 mL, Class A, volumetric
flask. The compound was dissolved and diluted with DI water and mixed by
inversion. The nominal concentration of the resulting solution was 32 g/L. The
solution was stored at 5 °C (±3 °C). The solution was prepared fresh daily.
8.1.6 AMMONIUM ACETATE SOLUTION - Prepared by transferring ~ 15.4 g of
ammonium acetate into a 100 mL, Class A, volumetric flask. The mixture was
diluted to volume with DI water and mixed by inversion. This 2 mM solution
was stored at 5 °C (±3 °C). The solution was prepared fresh daily.
8.1.7 10 % METHANOL IN WATER SOLUTION - Prepared by combining 10 mL
of methanol with 90 mL of DI water. The solution was mixed well and stored at
room temperature for up to 30 days.
8.1.8 NEEDLE WASH A - Prepared by transferring 500 mL of methanol into a
mobile phase bottle and mixing with 500 mL of water. The wash solution was
mixed well and stored at room temperature for up to 30 days.
8.1.9 NEEDLE WASH B - 100 % methanol.
8.2 STANDARD SOLUTIONS
8.2.1 STOCK STANDARD SOLUTIONS (SSS) - The methamidophos stock
solution was prepared by transferring ~ 10.0 mg (± 0.5 mg) of methamidophos,
accurately weighed into a weighing pan on a microbalance in an argon-purged
glove box and transferred to a 10 mL, Class A, volumetric flask. The compound
was dissolved and diluted with methanol and mixed by inversion. The nominal
concentration of the resulting solution was 1 g/L. The stock solution was stored
in amber 4-dram vials at -20 °C (±10 °C) for up to six months.
The acephate stock solution was prepared by transferring ~ 10.0 mg (± 0.5 mg)
of acephate, accurately weighed into a weighing pan on a microbalance in an
argon-purged glove box and transferred to a 10 mL, Class A, volumetric flask.
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The compound was dissolved and diluted with methanol and mixed by
inversion. The nominal concentration of the resulting solution was 1 g/L. The
stock solution was stored in 4-dram amber vials at -20 °C (±10 °C) for up to six
months.
The EA2192 stock solution was prepared by transferring ~ 10.0 mg (±0.5 mg)
of EA2192, accurately weighed, into a 10 mL, Class A, volumetric flask. The
compound was dissolved and diluted with acetonitrile and mixed by inversion.
The nominal concentration of the resulting solution was 1 g/L. The stock
solution was stored in 4-dram amber vials at 5 °C (±3 °C). Assessment of the
stock solution stability of EA2192 was not part of the EPA Scope of Work for
this project; however, EA2192 is known to be very stable. The EA2192 stock
solution was 308 days old for the final stability testing batch.
To verify the stock solution preparation, a second EA2192 stock solution was
prepared by transferring -7.5 mg (±0.5 mg) of EA2192, accurately weighed,
into a 10 mL, Class A, volumetric flask. The compound was dissolved and
diluted with acetonitrile and mixed by inversion. The nominal concentration of
the resulting solution was 0.75 g/L. The stock solution was stored at 5 °C (±3
The two independent stock solutions were diluted to concentrations within the
calibration curve, internal standard was added, and the solutions were then
analyzed by LC/MS/MS. The analysis showed a < 5 % difference between the
concentrations of the stock solutions. Once verified, one stock solution was used
for preparation of the standards.
8.2.2 METHANOLIC ANALYTE PRIMARY DILUTION STANDARD - The
Methanolic Analyte Primary Dilution Standard (MEOH PDS) Solution was
prepared by transferring 40 jiL of the methamidophos stock solution, 40 jiL of
the acephate stock solution, and 40 jiL of the EA2192 stock solution into a
1 mL, Class A, volumetric flask. The mixture was diluted to volume with
methanol and mixed by inversion. The nominal concentration for each
compound was 40 mg/L. The MEOH PDS solution was stored at -20 °C (±10
°C). Expiration was set at one month although stability was not tested for
EA2192 in solution.
8.2.3 AQUEOUS ANALYTE PRIMARY DILUTION STANDARD - The Aqueous
Analyte Primary Dilution Standard (WATER PDS) Solution was prepared by
transferring 62 jiL of the MEOH PDS into a 10 mL, Class A, volumetric flask.
The mixture was diluted to volume with 10 % methanol in water and mixed by
inversion. The nominal concentration of the resulting solution was 250 |ig/L for
each compound. The MEOH PDS solution was stored at -20 °C (±10 °C).
Expiration was set at one month although stability was not tested for EA2192 in
solution.
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8.2.4 IS PRIMARY DILUTION STANDARD - The Internal Standard Primary
Dilution Standard (IS PDS) was prepared by transferring 40 jiL of
methamidophos-d6 stock solution into a 10 mL, Class A, volumetric flask. The
solution was diluted to volume with acetonitrile and mixed by inversion. The
nominal concentration of the resulting solution was 400 |ig/L. This solution was
stored at 5 °C (±3 °C). Method 538 indicates that this solution is stable for up to
six months.
8.2.5 CALIBRATION STANDARDS - The calibration (CAL) standards were
prepared by transferring a set amount of the WATER PDS solution into 10 mL,
Class A, volumetric flasks. The 2M ammonium acetate solution (100 jiL) and
20 jiL of the 32 g/L sodium omadine solution were added to each CAL
standard. The CAL standard was diluted to volume with DI water and mixed by
inversion. Table 2, Calibration Standards, details the dilution series.
8.2.6 CONTINUING CALIBRATION CHECK STANDARDS - The Continuing
Calibration Check (CCC) Standards were prepared by transferring a set amount
of the WATER PDS solution into 10 mL, Class A, volumetric flasks. The 2M
ammonium acetate solution (100 jiL) and 20 jiL of the 32 g/L sodium omadine
solution were added to each CCC standard. The CCC standard was diluted to
volume with DI water and mixed by inversion. Table 3, Continuing Calibration
Check Standards, details the dilution series. These dilution schemes were also
used for the Detection Limit (DL), Minimum Reporting Limit (MRL), Initial
Demonstration of Precision (IDP), and Initial Demonstration of Accuracy (IDA)
study sample preparations.
8.2.7 MATRIX BLANKS - Matrix blanks from each water source were prepared
without preservatives and analyzed. Blanks were prepared fresh for each
analysis.
8.2.8 MATRIX SPIKES - A Laboratory Fortified Sample Matrix (LFSM) and a
Laboratory Fortified Sample Matrix Duplicate (LFSMD) were prepared from
each water source according to Table 4, then mixed well by inversion. Matrix
spikes were prepared fresh for each analysis.
8.2.9 STABILITY SAMPLES - Stability studies were performed in accordance with
Method 538 to determine if water samples from different sources (representing
a variety of water conditions) spiked with EA2192 were stable for 28 days.
Water samples were received from four different sources (determined by the
EPA); see Table 5 for the representative water conditions. Water samples were
received at the laboratory on blue ice (5 °C ± 3 °C) and stored under
refrigerated conditions (5 °C ± 3 °C) prior to sample preparation. See Table 6
for the water source parameters (pH, turbidity, conductivity, alkalinity,
hardness, free chlorine, chloramine, and total organic carbon) and their
measurements for the four source waters. The free chlorine concentration was
measured for each bulk water sample using a Hach colorimeter immediately
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prior to Time 0, the time of the sample preparation (See Table 7); pH was also
measured using pH strips.
9. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
9.1 HOLDING TIME STUDY IN DEIONIZED WATER - A holding time study was
performed using Method 538 conditions to determine if DI water samples that were
spiked with EA2192 were stable for up to 28 days. The following preparation
scheme was used for sample preparation:
1. Mix 1 mL of 2M ammonium acetate and 200 jiL of sodium omadine (32 g/L) into
lOOmLofDIwater.
2. Confirm pH of this solution using pH paper.
3. Aliquot 10 mL of the solution into amber vials (n = 6).
4. Spike 20 jiL of the WATER PDS solution into each vial and mix by inversion.
5. Prepare one vial immediately as Time 0 sample in accordance with the sample
preparation procedure, n = 7 replicates from Time 0 sample.
6. Place remaining vials into 5 °C (±3 °C) conditions for stability testing.
Stability time points were taken on Day 7, Day 14, and Day 28. After the allotted
storage, the vial was removed from the storage condition and allowed to reach room
temperature. Seven (7) aliquots of each sample were then prepared and analyzed in
accordance with the method.
9.2 WATER STABILITY STUDY IN TAP WATER- Water from four different sources
(100 mL), representing a variety of water types (chlorinated, chloraminated, hard
water, etc.) was transferred into two wide mouth iChem™ jars with caps (Thermo
Scientific), one to represent the low concentration sample (at the CAL2 level, 0.125
|ig/L) and one to represent the high concentration sample (at the CAL 6 level, 2.50
|ig/L). One mL of 2M ammonium acetate and 200 jiL of 32 g/L sodium omadine
were added to each iChem™ jar and mixed well by inversion.
Each of the four low concentration samples was spiked with 50 jiL of WATER PDS
and mixed well. Each low concentration sample was then split into 10 mL aliquots
(six per sample source). Five aliquots were stored at 5 °C (± 3 °C). The remaining
aliquot (the Time 0 sample) was left at room temperature for immediate sample
preparation.
Each of the four high concentration samples was spiked with 1,000 jiL of WATER
PDS and mixed well. Each high concentration sample was then split into 10 mL
aliquots (six per sample source). Five aliquots were stored at 5 °C (± 3 °C). The
-------
remaining aliquot (the Time 0 sample) was left at room temperature for immediate
sample preparation. See Table 8, Water Sample Preparation, for the dilution series.
Stability analyses were performed at Time 0, Day 7, Day 14, and Day 28. After the
allotted storage, the vial was removed from the storage conditions and allowed to
reach room temperature. Seven (7) aliquots of each sample were then prepared and
analyzed in accordance with the method.
9.3 EFFECTS OF RESIDUAL CHLORINE ON EA2192 - (NOTE: This is an
additional investigation (not presented in Method 538) to investigate residual
chlorine effects on EA2192.) The purpose of this study was to determine the stability
of EA2192 in water samples with a free chlorine level of- 1 mg/L, representative of
a common concentration of free chlorine in a distribution system. A water sample
was received and stored at 5 °C (± 3 °C) until sample preparation. Immediately prior
to sample preparation, the free chlorine level was adjusted to 1 mg/L using the Hach
colorimeter chlorine standard kit using the following procedure:
1. Verify the calibration of the Hach colorimeter using the supplied Hach
standards.
2. Measure 1 L of water and transfer to an iChem™ jar.
3. Add 10 mL of chlorine standard to the water sample. Mix well for -30 seconds.
4. Transfer 10 mL of the chlorinated water to a Hach vessel and confirm the
reading is 1 (± 0.2) mg/L.
The chlorinated water was transferred into two iChem™ jars (100 mL each), one to
represent the low concentration sample and one to represent the high concentration
sample.
The low concentration sample was spiked with 50 jiL of WATER PDS and mixed
well. The low concentration sample was then split into 10 mL aliquots (n=7). Five
aliquots were stored at 5 °C (± 3 °C) for future time points. One aliquot was left at
room temperature for a three-hour time point. The remaining aliquot (the Time 0
sample) was prepared immediately.
The high concentration sample was spiked with 1,000 jiL of WATER PDS and
mixed well. The high concentration sample was then split into 10 mL aliquots
(n = 7). Five aliquots were stored at 5 °C (± 3 °C) for future time points. One aliquot
was left at room temperature for the three-hour time point. The remaining aliquot
(the Time 0 sample) was prepared immediately.
After the required storage time, 100 jiL of 2 M ammonium acetate and 20 jiL of
32 g/L sodium omadine were added to each sample and mixed well by inversion,
followed by the preparation scheme.
10
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9.4 WATER FILTRATION STUDY - A study was performed to determine if there was
a loss of analyte upon filtration of water samples spiked with EA2192. DI water was
transferred into four wide mouth iChem™ jars with caps (100 mL each), two to
represent the low concentration samples (one filtered and one non-filtered) and two
to represent the high concentration samples (one filtered and one non-filtered). One
mL of 2 M ammonium acetate and 200 jiL of 32 g/L sodium omadine were added to
each iChem™ jar and mixed well by inversion.
The low concentration samples were spiked with 50 jiL of WATER PDS and mixed
well. Each of the four high concentration samples was spiked with 1,000 jiL of
WATER PDS and mixed well. The samples were then split into individual 10 mL
aliquots (seven per sample source). For the filtered samples, the 10-mL aliquots were
filtered individually and transferred to a syringe attached with a GUP Acrodisc prior
to sample preparation (GHP Acrodisc, 25 mm, 0.45 jim).
10. QUALITY CONTROL
10.1 Quality control (QC) requirements include the Initial Demonstration of Capability
(IDC) and ongoing QC requirements that were met when preparing and analyzing
samples. This section describes the QC parameters, their required frequencies, and
the performance criteria that were met to meet EPA quality objectives.
10.2 CALIBRATION CURVE - Calibration curves consisted of at least five nonzero
samples (each at a different concentration) covering the nominal concentration range
of 0.05-20 |ig/L. A blank DI water sample (collected at the same time as the DI
water sample used for standard preparation) was also analyzed. Plots of the peak
area response versus gravimetric standard concentration were constructed using a
best-fit line determined by a regression analysis. A curve-weighting factor of 1/x
with linear regression was utilized.
10.3 CONTINUING CALIBRATION CHECK - The calibration was confirmed by
analysis of a CCC at the beginning and end of a sample analysis batch. The
beginning CCC was required to be at or below the MRL (typically at CCC2 level)
(refer to Table 3) to verify instrument sensitivity. CCCs were then injected after
every ten samples and after the last sample, alternating between a mid-level (CCC4)
and a high-level (CCC7).
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 areas measured in the most recent calibration.
2. The calculated amount for each analyte for medium and high level CCCs
had to be within ±30 % of the true value.
3. The calculated amount for each analyte for low level CCCs had to be
within ±50 % of the true value.
11
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10.4 INITIAL DEMONSTRATION OF CAPABILITY
10.4. 1 DETECTION LIMIT DETERMINATION - The Detection Limit (DL) was
verified with the preparation and analysis of seven (7) replicates of a standard
at the CCC1 concentration (see Table 3) over the course of three (3) days. This
concentration was estimated by selecting a concentration that was
approximately two to five times the noise level. The DL was calculated using
the following formula:
DL = S X t („-!, l-a=0.99)
where: s = standard deviation of replicate analyses
t (H-I, i-a=o.99) = Student' s t value for the 99 % confidence lev el
with n-l degrees of freedom
n = number of replicates
10.4.2 MINIMUM REPORTING LEVEL CONFIRMATION - Seven replicates of
the Minimum Reporting Level (MRL) were prepared at the CCC2 level (see
Table 3) and 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 (HRp/s) was determined
using the following formula (per Method 538):
where: s = standard deviation
3.963 = 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
Fortified Concentration
The lower PIR limit requirement was > 50 % recovery
12
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x lOQo/o > 500/0
Fortified Concentration
10.4.3 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND - Any
time a new lot of solvents, reagents, or autosampler vials was used, a
Laboratory Reagent Blank (LRB) was prepared to demonstrate that the new lot
was reasonably free of contamination. To demonstrate the freedom from
contamination, an LRB was prepared by analyzing blank DI water prepared
with the same additives as a standard (i.e., ammonium acetate and sodium
omadine) and internal standards. To be acceptable, method analytes could not
be detected in the LRB at concentrations > l/3 the DL.
10.4.4 INITIAL DEMONSTRATION OF PRECISION - Seven (7) replicates of
CCC4 were prepared for the Initial Demonstration of Precision (TDP) study as
described in Table 3 and analyzed. To pass acceptability criteria, the calculated
relative standard deviation from the replicate analyses was required to be < 20
10.4.5 INITIAL DEMONSTRATION OF ACCURACY - The same seven (7)
replicates of CCC4 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 %.
10.5 STABILITY STUDIES - The concentrations of the stored (stability) samples were
compared to the concentrations of the samples analyzed at Time 0. To be reported as
stable, the concentration of the stored samples could not deviate from the
concentration of the samples analyzed at time 0 by more than ±30 %. In addition,
replicate stability samples at a given stability condition must have a % RSD value of
< 15 % to be acceptable. For the stability batches to be acceptable, the batch must
meet CCC requirements.
10.6 WATER FILTRATION STUDY - The concentrations of the filtered samples were
compared to the concentrations of the non-filtered samples. To be reported as
comparable, the concentration of the filtered samples could not deviate from the
concentration of the non-filtered samples by more than ± 30 %. In addition, replicate
samples at a given condition required a % RSD value of < 15 % to be acceptable.
For the batch to be acceptable, the batch had to meet the CCC standard requirements.
11. INSTRUMENT CALIBRATION AND STANDARDIZATION
11.1 HPLC INSTRUMENT AND PARAMETERS - The HPLC method parameters are
listed in Table 9. The HPLC gradient is listed in Table 10.
1 1 .2 ESI-MS/MS TUNING - The [M±H]+ signal was optimized for each method analyte
by infusing approximately 1 |ig/mL of each analyte directly into the MS. The MS
13
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parameters were varied until optimal analyte responses were determined. Once the
MS parameters were optimized, the product ions and MS/MS parameters were
determined. See Table 11 for the optimized ESI-MS/MS conditions and Table 12 for
the Multiple Reaction Monitoring (MRM) transitions.
11.3 INITIAL CALIBRATION - The initial calibration curve consisted of seven CAL
standards. The lowest CAL was required to be at or below the MRL. The curve was
calibrated using the IS technique. The LC/MS/MS data system software was used to
generate a linear regression calibration curve with 1/x weighting.
11.4 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.
11.5 CONTINUING CALIBRATION CHECK (CCC) - The initial calibration was
verified at the beginning and end of each group of analyses, and after every tenth
sample. The beginning CCC of each analysis batch was required to be at or below
the MRL to verify instrument sensitivity prior to any analyses. Subsequent CCCs
alternated between a medium and high concentration CAL standard. The absolute
areas of the quantitation ions of the IS had to be within 50 %-150 % of the average
areas measured in the most recent calibration. Additionally, the calculated amount
for each analyte for medium and high level CCCs had to be within ±30 % of the true
value and ±50 % at the lowest calibration level.
12. ANALYTICAL PROCEDURE
The following procedure was used for the preparation of samples for analysis (i.e., CALs,
CCC standards, IDC samples, Stability Samples, Water Matrix Blanks, LFSM, etc.).
Volumes were delivered with calibrated adjustable pipettes:
12.1 Transfer 990 jiL of sample into an autosampler vial (except for blanks—add 1,000
jiL of sample).
12.2 Add 10 |iL of IS PDS to each sample (do not add IS PDS to blanks).
12.3 Mix by inversion and cap for analysis.
13. DATA ANALYSIS AND CALCULATION
13.1 DESCRIPTIVE STATISTICS - Descriptive statistics [mean, standard deviation
(SD), relative standard deviation (% RSD), percent accuracy (% ACC), relative error
(% RE), and percent difference], were calculated for this method.."
The following formulas were used during the course of this study:
13.1.1 Results were expressed as a concentration based on the calibration curve. The
concentration was calculated as follows:
, _ , . T. I (response-y mi) }
Sample Concentration (ng/mL) = -— -
^ Slope J
14
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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 was expressed as percent relative error (% RE) which was
calculated based on the gravimetric concentration as follows:
% Relative Error = ^ ' ^ x 100
E
where: D = determined concentration
E = expected (gravimetric) concentration
13.1.3 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
UJ
where: o = standard deviation
X = mean
13.1.4 To evaluate stability, the mean concentration after the storage time was
compared to the mean concentration at Time 0 as follows:
%of Time 0 = — x 100
Y
where: X = mean concentration after storage time
Y = mean determined concentration at Time 0
13.1.5 To evaluate percent difference between LFSM and LSFSMD samples, the
determined concentration of individually prepared LFSM and LFSMD
solutions were compared to each other:
% Difference =
(X-Y)
(X + Y)/2
xlOO
where: X = determined concentration of LFSM
Y = determined concentration of LFSMD
15
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14. METHOD PERFORMANCE
14.1 LINEARITY - Coefficients of correlation (r) were at least 0.9996 for EA2192. The
percent accuracy (% RE) for EA2192 met Initial Demonstration of Capability (IDC)
criteria ranging from 96.4 % to 105 % for CAL1 and from 92.4 % to 107 % at all
other concentrations (see Table 13). A representative EA2192 calibration curve is
shown in Figure 1, where linearity is demonstrated over the tested calibration range.
Coefficients of determination (r) were 0.9999 for methamidophos. The percent
accuracy (% RE) for methamidophos ranged from 91.6 % to 107 % for CAL1 and
from 91.8 % to 107 % at all other concentrations (Table 14). A representative
methamidophos calibration curve is shown in Figure 2, where linearity is
demonstrated over the tested calibration range.
Coefficients of determination (r) were at least 0.9988 for acephate. The percent
accuracy (% RE) for acephate ranged from 94.1 % to 106 % for CAL1 and from
89.5 % to 109 % at all other concentrations (Table 15). A representative acephate
calibration curve is shown in Figure 3, where linearity is demonstrated over the
tested calibration range.
14.2 CONTINUING CALIBRATION CHECKS - Continuing calibration checks were
analyzed during each batch to verify that the current calibration was still meeting
acceptability criteria. A CCC2 sample (at the MRL) was initially analyzed to verify
sensitivity, followed by a CCC4 and CCC7 to verify the accuracy of the sequence in
comparison to the current calibration. CCCs were reanalyzed after every ten samples
and/or at the end of the sequence to verify there was no loss in sensitivity.
For EA2192, CCCs in all batches passed the acceptability criteria. Methamidophos
and acephate CCC results were calculated to verify instrument performance and to
determine if the sensitivity and chromatography were acceptable. In two batches, the
final grouping of CCCs for acephate failed the acceptability criteria of ±30 % of its
true value. In both cases, the accuracy was within ± 40 % and in both cases, the
value was a response that was higher than expected. The methamidophos CCCs
passed acceptability criteria for all batches. Because EA2192 passed acceptability
criteria for these batches, and because the purpose of this study was to incorporate
EA2192 into the method, the batches were accepted. The acephate CCC failures
could indicate that the instrument requires cleaning or that a new calibration curve is
required. The failures were identified and corrective actions were taken to remedy
the issue in subsequent batches by cleaning the instrument, analyzing a new
calibration curve, and replacing the analytical column. Further steps included the
addition of a diversion valve to remedy source contamination during longer analysis
run sequences (section 14.9) and reduce the risk of further sample failures. After the
IDC and initial holding time studies were completed, it was decided to eliminate
acephate from the method for the subsequent studies.
16
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14.3 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND - Method
analytes were not detected in an LRB spiked with preservatives and internal
standards at concentrations that were > lh of the DL. A representative chromatogram
of a blank sample without internal standard is shown in Figure 4, and a
representative chromatogram of a blank with internal standard is shown in Figure 5.
14.4 DETECTION LIMIT DETERMINATION - The EA2192 DL was determined from
seven replicates of samples at the CCC1 level, batches prepared over three days. The
DL was calculated to be 0.0130 |ig/L, using a t value of 3.143. The DL calculation is
presented in Table 16.
14.5 MINIMUM REPORTING LEVEL CONFIRMATION - MRL was determined from
seven replicates of samples at the CCC2 level. The HRp/R was determined to be
0.0353 ng/L. Based on this result, the Lower PIR was calculated to be 86.3 % and
the Upper PIR was calculated to be 145 %. 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 17. A representative
chromatogram of the CAL2 standard at the MRL is shown in Figure 6.
14.6 INITIAL DEMONSTRATION OF PRECISION - The IDP was determined from
seven replicates at the CCC4 concentration level, calculated versus a calibration
curve. The precision (% RSD) was 9.61 %. This value was within the % RSD
acceptability criteria of < 20 %. The IDP results are summarized in Table 18.
14.7 INITIAL DEMONSTRATION OF ACCURACY - The IDA was determined from
the same seven CCC4 replicates that were used for the IDP study, calculated vs. a
calibration curve. The IDA (% RE) was 21.8 %. This value was within the % RE
acceptability criteria of ± 30 %. The IDA results are summarized in Table 18.
14.8 HOLDING TIME STUDY IN DEIONIZED WATER - The average concentration
of EA2192 after storage under refrigerated conditions (5 °C ± 3 °C) when compared
to Time 0 was 81.6 % (8, % RSD) after seven days, 97.4 % (3, % RSD) after 14
days, and 86.7 % (4, % RSD) after 28 days. The holding time study results are
summarized in Table 19.
The Day 14 batch failed upon initial analysis. The response of the internal standard
was less than 50 % of the average internal standard area counts of the initial
calibration. Remedial action was taken by cleaning the instrument, replacing the
analytical column, and preparing and analyzing a fresh calibration curve. The
Day 14 samples were reanalyzed compared to the new calibration curve to obtain the
reported value, but these samples were analyzed more than 24 hours after sample
preparation. A stability study of the samples on the autosampler has not been
performed on EA2192. Such an investigation would be necessary prior to including
EA2192 in Method 538 due to Day 14 batch reanalysis past 24 hours; however,
holding time study data in source water samples are sufficient for Method 538
holding time parameters.
17
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14.9 WATER SAMPLE STABILITY STUDY IN TAP WATER - During the Time 0
analysis, CCC standards for EA2192 started to fail acceptance criteria after 48
injections. The IS area counts were decreasing throughout the analysis which caused
the CCC standards to calculate higher than the acceptance criteria allowed. A
diversion valve was added to divert waste for the first three minutes of the analysis
(prior to analyte elution) as well as after 20 minutes into the analysis (after elution of
the last analyte in Method 538). The source was thoroughly cleaned and the
diversion was used to help keep the source clean through the longer analyses. After
instituting these processes, the remaining analyses passed all acceptance criteria for
both EA2192 and methamidophos (tested for up to 105 injections per batch).
Water was received from four individual utility companies. The associated water
parameters are listed in Table 6. The water was spiked with EA2192, and the
concentration of the compound was determined after storage under refrigerated
conditions (5 °C ± 3 °C) for 28 days. The pH and free chlorine levels were measured
immediately prior to sample preparation at Day 0 and are listed in Table 7. When
compared to Time 0, the concentration was 81.7%-117% after seven days, 98.5 %-
118 % after 14 days, and 79.7 %-l 19 % after 28 days. The highest Relative Standard
Deviation (%RSD) of the triplicate samples was 11.9 %. The water sample stability
study results are summarized in Table 20. Representative figures for each of the four
water sample types are included in Figures 7-10, which include a matrix blank, low
concentration sample and high concentration sample and water sample parameters
are provided in Tables 5, 6, and 7.
The IS area counts for two of the four method blanks (from Water Sources No. 1 and
No. 4) were not ± 50 % of the average IS response from the initial calibration (both
instances failed low). This failure was observed in two separate preparations and
analyses (Stability Day 0 and Day 7, data not shown). Method blanks were then
prepared with ammonium acetate and sodium omadine for all four water types. The
IS response passed acceptance criteria with the inclusion of additives.
14.10 EFFECTS OF RESIDUAL CHLORINE ON EA2192 - Water was received from
Water Source No. 1 and the free chlorine concentration was adjusted to 0.93 mg/L,
measured using a Hach Colorimeter, immediately prior to sample preparation.
EA2192 was not detected in the Time 0 samples or the three-hour samples.
Therefore, the remaining stability time points were not prepared. Because bleach is
used for the decontamination of CWAs, it is assumed that the higher level of
chlorine present in this water sample led to the rapid degradation of EA2192.
14.11 WATER FILTRATION STUDY - The average concentration of the filtered Low
samples was 100 % of the non-filtered samples. The average concentration of the
filtered High samples was 118 % of the non-filtered samples. The highest %RSD of
the triplicate samples was 15.6 %. The water filtration study results are summarized
in Table 21. Filtering the samples at either high or low concentrations did not affect
the recovery of the target analyte.
18
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15. POLLUTION PREVENTION
15.1 This method utilized ESI-LC/MS/MS for the analysis of method analytes in water.
The method required the use of very small volumes of organic solvent and very
small quantities of pure analytes, thereby minimizing the potential hazards to both
the analyst and the environment.
16. WASTE MANAGEMENT
16.1 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 and/or source water. Laboratory
waste management practices were conducted consistent with all applicable rules and
regulations, and 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. REFERENCES
17.1 All data obtained from the study were evaluated in accordance with the following
EPA methods or published SOPs:
17.1.1 U.S. EPAMethod 538, "Determination of Selected Organic Contaminants in
Drinking Water by Direct Aqueous Injection-Liquid Chromatography/Tandem
Mass Spectrometry (DAI-LC/MS/MS)," Version 1.0, November 2009, EPA
Document No. EPA/600/R-09-149
19
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18. TABLES AND VALIDATION DATA
Table 1. Initial Demonstration of Capability Testing Summary
Test Article
Matrix
Quantitation
Regression Type
Linear Range
IDC Tests (EA2192)
Coefficient of Determination (r)
Minimum Reporting Level
Detection Limit Determination
Initial Demonstration of Low System
Background
Initial Demonstration of Precision
Initial Demonstration of Accuracy
EA2192 Holding Time Study
Day 7
Day 14
Day 28
EA2192
Dl Water
LC/MS/MS
Linear (1/x)
0.05 ug/L to 20 ug/L
Acceptance criteria
NA
< 150% Upper PIR Limit
> 50 % Lower PIR Limit
NA
Background < 1/3 of
minimum reporting level
< 20 % RSD
±30 % mean recovery (RE)
Acceptance criteria
70-130%
70-130%
70-130%
Results
>0.9996
Upper Limit = 145 %
Lower Limit = 86.3 %
0.0130ug/L
Non-detect
9.61 %
21.8%
% Recovery from Day 0
81.6%
97.4 %
86.7 %
NA = Not applicable; RE = Relative Error; RSD = Relative Standard Deviation; PIR = Prediction Interval of Result.
Table 2. Calibration Standards
Calibration
Solution Name
CAL1
CAL2
CAL3
CAL4
CAL5
CAL6
CAL7
CAL8
CAL9
Source Solution
WATER PDS
WATER PDS
WATER PDS
WATER PDS
WATER PDS
WATER PDS
WATER PDS
WATER PDS
WATER PDS
Source
Solution
Volume
(ML)
2
5
10
20
40
100
200
400
800
(2M)
Ammonium
Acetate
Volume (uL)
100
100
100
100
100
100
100
100
100
(32 g/L)
Sodium
Omadine
Volume
(ML)
20
20
20
20
20
20
20
20
20
Final
Volume of
Solution
(mL)
10
10
10
10
10
10
10
10
10
Nominal
Solution
Cone.
(M9/L)
0.050
0.125
0.250
0.500
1.00
2.50
5.00
10.0
20.0
CAL = Calibration; PDS = Primary dilution standard.
20
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Table 3. Continuing Calibration Check Standards
Solution Name
CCC 1
(DL Study)
CCC 2
(MRL Study)
CCC 4
(IDP and IDA
Studies)
CCC 7
Source Solution
Name
WATER PDS
WATER PDS
WATER PDS
WATER PDS
Source
Solution
Volume
(ML)
2
5
20
200
(2M)
Ammonium
Acetate
Volume (uL)
100
100
100
100
(32 g/L)
Sodium
Omadine
Volume
(ML)
20
20
20
20
Final
Volume
of
Solution
(mL)
10
10
10
10
Nominal
Solution
Cone.
(Mg/L)
0.050
0.125
0.500
5.00
CCC = Continuing calibration check; PDS = Primary dilution standard.
Table 4. Laboratory Fortified Sample Matrix Preparation
Solution Name
[Source Number]
LFSM1
Source
Solution
WATER PDS
Source
Solution
Volume
(ML)
5
Ammonium
Acetate
Volume
(ML)
100
Sodium
Omadine
Volume
(ML)
20
Final
Volume
(mL)
10
Nominal
Solution
Cone.
(M9/L)
0.125
1 Each water source was prepared as listed in this table.
LFSM = Laboratory fortified sample matrix; PDS = Primary dilution standard.
Table 5. Water Conditions
Source Number
1
2
3
4
Representative Water Condition
LowTOC, chlorinated surface water
High TOC, chloraminated surface water
LowTOC, chloraminated surface water
High hardness, chlorinated ground water
TOC = Total organic carbon.
Table 6. Water Sample Parameters upon Collection
Source
Number
1
2
3
4
PH
8.7
9.18
7.4
7.24
Turbidity
(NTU)
0.02
0.10
0.07
0.15
Conductivity
525 uS/cm
414 uS/cm
400 uS/cm
998 uS/cm
Alkalinity
(mg/L)
88
—
118
333
Hardness
(mg/L)
157
—
165
461
Free
Chlorine
(mg/L)
1.21
3.10
0
0.38
Chloramine
(mg/L)
—
3.40
3.5
-
Total
Organic
Carbon
(mg/L)
0.80
7.98
1.3
__ 1
1 Reported to have total organic carbon below the detection limit as expected for groundwater not under the
influence of surface water.
Parameters were measured upon water collection.
"—" indicates that a value was not reported.
21
-------
Table 7. Measured Water Parameters at Time of Sample Preparation
Source Number
1
2
3
4
PH
6 to 6.5
6 to 6. 5
6 to 6.5
6 to 6. 5
Free Chlorine (mg/L)
0.74
0.03
0.08
0.19
Parameters were measured immediately prior to sample preparation.
pH was measured using pH strips.
Free chlorine was measured using Hach Colorimeter.
Table 8. Water Sample Preparation
Solution Name
[Source Number]
Low Concentration 1
[Source Number]
High Concentration 1
Source
Solution
WATER
PDS
WATER
PDS
Source
Solution
Volume
(ML)
50
1,000
Ammonium
Acetate
Volume
(ML)
1,000
1,000
Sodium
Omadine
Volume
(ML)
200
200
Water
Source
Volume
(mL)
100
100
Nominal
Solution
Cone.
(M9/L)
0.123
2.45
Each water source was prepared as listed in this table, then split into six aliquots.
Table 9. HPLC Method Parameters
Setting Name
Liquid Chromatograph
Autosampler
Column
Mobile Phase A
Mobile Phase B
Flow Rate
Injection Volume
Run Time
Sample Temperature
Needle Wash A
Needle Wash B
Column Temperature
Value
Shimadzu Solvent Delivery Module LC-10
ADvp
Shimadzu SIL-5000
Waters Atlantis T3 5 u, 150 x 2.1 mm
20mM Ammonium Formate
100%Methanol
0.3 mL/min
50 uL
30.0 min
5°C
50/50 (v/v) Methanol/Water
100%Methanol
Ambient
22
-------
Table 10. HPLC Gradient
Time
(min)
0
3.0
5.0
8.0
20.0
20.1
25.0
25.1
%A
90
90
70
70
30
10
10
90
%B
10
10
30
30
70
90
90
10
HPLC Flow
To Waste
To Instrument
(3 to 20 min)
To Waste
Table 11. ESI-MS/MS Method Parameters
Setting Name
Mass Spectrometer
Software
lonization Mode
Scan Mode
Curtain Gas
Collision (CAD) Gas
Ion Spray Voltage (IS)
Temperature
Ion Source (GS1)
Ion Source (GS2)
Interface heater (ihe)
Entrance Potential
Collision Cell Exit Potential
Value
Applied Biosystems API-4000
Analyst V. 1.5.1
Turbo ionspray, positive
Multiple Reaction Monitoring (MRM)
20 psi
4 psi
5000V
450 °C
30 psi
30 psi
on
10V
12V
Table 12. MRM Transitions
Compound Name
EA2192
Ace p hate
Methamidophos
Methamidophos-d6
Monitored
Transition
240.4 > 128.1
184.2 > 143.0
142.0 > 94.0
148.0 > 97.0
Dwell Time
(ms)
100
100
100
100
Declustering
Potential
(V)
45
35
30
30
Collision
Energy
(V)
25
12
20
20
Retention
Time
(min)
5.6
5.7
3.6
3.7
23
-------
Table 13. IDC Calibration Curve Standards—EA2192
Standard Name
CAL1
CAL2
CAL3
CAL4
CAL5
CAL6
CAL7
CAL8
CAL9
Standard
Concentration
Level
(ug/L)
0.0481
0.120
0.241
0.481
0.962
2.41
4.81
9.62
19.2
Determined
Concentration
(ug/L)
0.0464
0.0467
0.0479
0.123
0.124
0.120
0.242
0.468
0.961
2.42
5.02
9.52
19.1
% Accuracy
96.4
97.1
99.6
102
104
100
100
97.3
99.9
100
104
99.0
99.4
correlation coefficient (r) value: 0.9998
CAL = Calibration
Standard Name
CAL1
CAL2
CAL3
CAL4
CAL5*
CAL6
CAL7
CAL8
CAL9
Standard
Concentration
Level
(ug/L)
0.0481
0.120
0.241
0.481
0.962
2.41
4.81
9.62
19.2
Determined
Concentration
(M9/L)
0.0505
0.111
0.236
0.483
1.35
2.40
5.15
9.46
19.0
% Accuracy
105
92.4
98.0
100
140
99.8
107
98.3
99.2
rvalue: 0.9996
CAL = Calibration; r = Correlation coefficient.
* Point excluded from calibration curve.
24
-------
Table 14. IDC Calibration Curve Standards—Methamidophos
Standard Name
CAL1
CAL2
CAL3
CAL4
CAL5
CAL6
CAL7
CAL8
CAL9
Standard
Concentration
Level
(ug/L)
0.0472
0.118
0.236
0.472
0.944
2.36
4.72
9.44
18.9
Determined
Concentration
(ug/L)
0.0505
0.0455
0.0432
0.127
0.127
0.119
0.236
0.440
0.901
2.35
4.74
9.40
19.0
% Accuracy
107
96.3
91.6
107
107
101
100
93.3
95.4
99.7
101
99.6
100
rvalue: 0.9999
CAL = Calibration
r = Correlation coefficient.
Standard Name
CAL1
CAL2
CAL3
CAL4
CAL5*
CAL6
CAL7
CAL8
CAL9
Standard
Concentration
Level
(ug/L)
0.0472
0.118
0.236
0.472
0.944
2.36
4.72
9.44
18.9
Determined
Concentration
(ug/L)
0.0505
0.108
0.227
0.476
1.21
2.39
4.89
9.43
18.7
% Accuracy
107
91.8
96.4
101
128
101
104
99.9
99.0
rvalue: 0.9999
CAL = Calibration; ; r = Correlation coefficient.
* Point excluded from calibration curve.
25
-------
Table 15. IDC Calibration Curve Standards—Acephate
Standard Name
CAL1
CAL2
CAL3
CAL4
CAL5
CAL6
CAL7
CAL8
CAL9
Standard
Concentration
Level
(ug/L)
0.0496
0.124
0.248
0.496
0.992
2.48
4.96
9.92
19.8
Determined
Concentration
(ug/L)
0.0478
0.0497
0.0528
0.125
0.136
0.129
0.240
0.475
0.925
2.42
4.90
9.93
20.0
% Accuracy
96.3
100
106
101
109
104
96.7
95.7
93.2
97.4
98.8
100
101
rvalue: 0.9999
CAL = Calibration
r = correlation coefficient.
Standard Name
CAL1
CAL2
CAL3
CAL4
CAL5*
CAL6
CAL7
CAL8
CAL9
Standard
Concentration
Level
(ug/L)
0.0496
0.124
0.248
0.496
0.992
2.48
4.96
9.92
19.8
Determined
Concentration
(ug/L)
0.0467
0.111
0.250
0.522
1.52
2.54
5.42
10.2
19.0
% Accuracy
94.1
89.5
101
105
153
102
109
103
95.9
rvalue: 0.9988
CAL = Calibration; r= Correlation coefficient.
* Point excluded from calibration curve.
26
-------
Table 16. EA2192 Detection Limit Determination
Sample Name
DL CAL 1 Day 1
DL CAL 1 Day 1
DL CAL 1 Day 1
DL CAL 1 Day 2
DL CAL 1 Day 2
DL CAL 1 Day 3
DL CAL 1 Day 3
Gravimetric
Concentration
Level
(M9/L)
0.0481
Average
Std. Dev.
n
degrees of freedom
t value
Detection Limit
Determined
Concentration
(M9/L)
0.0553
0.0561
0.0605
0.0558
0.0591
0.0622
0.0667
0.0594
0.00415
7
6
3.143
0.0130
DL CAL = Detection limit Calibration; n= number of samples; Std.
value
Dev. = standard deviation; t value = Student's t-
Table 17. EA2192 Method Reporting Limit Confirmation
Sample Name
CAL 2 (MRL)
CAL 2 (MRL)
CAL 2 (MRL)
CAL 2 (MRL)
CAL 2 (MRL)
CAL 2 (MRL)
CAL 2 (MRL)
Gravimetric
Concentration
Level
(M9/L)
0.120
Average
Std. Dev.
HRPIR
Lower PIR Limit
Upper PIR Limit
Determined
Concentration
(M9/L)
0.130
0.137
0.145
0.137
0.154
0.141
0.128
0.139
0.00890
0.0353
86.3 %
145%
27
-------
Table 18. EA2192 Initial Demonstration of Precision and Accuracy
Sample Name
IDP/IDACal4
IDP/IDACal4
IDP/IDACal4
IDP/IDACal4
IDP/IDACal4
IDP/IDACal4
Gravimetric
Concentration
Level
(M9/L)
0.481
Average
Std. Dev.
Precision (%RSD)
Accuracy (%RE)
Determined
Concentration
(M9/L)
0.584
0.588
0.634
0.557
0.497
0.655
0.586
0.0563
9.61
21.8
IDP/IDA Cal = Initial demonstration of precision/initial demonstration of accuracy calibration
Table 19. EA2192 Holding Time Study—DI Water
Time, Days
0
7
14
28
Average*
(M9/L)
Standard
Deviation
% RSD
0.513
0.0181
3.52
0.419
0.0317
7.57
0.500
0.0147
2.95
0.455
0.0166
3.73
% of Day 0
-
81.6
97.4
86.7
NOTE: Gravimetric Concentration: 0.481 ug/L.
*NOTE: Seven replicates were prepared and
analyzed for each concentration at each time point.
28
-------
Table 20. EA2192 Stability Study in Tap Water
Source No. 1
Low Concentration
(ug/L)
Time,
days
0
7
14
28
Average*
Std. Dev.
% RSD
0.120
0.004
3.7
0.104
0.005
4.7
0.131
0.006
4.3
0.099
0.004
4.4
%of
DayO
-
86.9
110
82.9
High Concentration
(ug/L)
Time,
days
0
7
14
28
Average*
Std. Dev.
% RSD
2.43
0.170
7.0
2.44
0.220
9.2
2.39
0.060
2.5
2.42
0.070
2.9
%of
DayO
-
101
98.5
99.7
Source No. 2
Low Concentration
(ug/L)
Time,
days
0
7
14
28
Average*
Std. Dev.
% RSD
0.125
0.011
9.0
0.102
0.003
2.6
0.132
0.005
3.8
0.100
0.003
2.7
%of
DayO
-
81.7
106
79.7
High Concentration
(ug/L)
Time,
days
0
7
14
28
Average*
Std. Dev.
% RSD
2.28
0.030
1.4
2.62
0.160
6.0
2.27
0.150
6.5
2.44
0.040
1.8
%of
DayO
-
115
99.5
107
*NOTE: Seven replicates were prepared and analyzed for each
concentration at each time point.
29
-------
Table 20. EA2192 Stability Study in Tap Water (cont.)
Source No. 3
Low Concentration
(ug/L)
Time,
days
0
7
14
28
Average*
Std. Dev.
% RSD
0.121
0.014
11.9
0.104
0.002
2.2
0.126
0.004
3.3
0.105
0.005
4.3
%of
DayO
-
85.6
104
87.0
High Concentration
(ug/L)
Time,
days
0
7
14
28
Average*
Std. Dev.
% RSD
2.22
0.080
3.6
2.57
0.110
4.2
2.32
0.030
1.5
2.49
0.030
1.3
%of
DayO
-
116
105
112
Source No. 4
Low Concentration
(ug/L)
Time,
days
0
7
14
28
Average*
Std. Dev.
% RSD
0.106
0.002
2.1
0.124
0.014
10.9
0.125
0.008
6.0
0.112
0.003
2.6
%of
DayO
-
117
118
106
High Concentration
(ug/L)
Time,
days
0
7
14
28
Average*
Std. Dev.
% RSD
2.27
0.090
4.0
2.53
0.070
2.8
2.35
0.040
1.9
2.69
0.130
4.7
%of
DayO
-
112
104
119
*NOTE: Seven replicates were prepared and analyzed for each
concentration at each time point.
30
-------
Table 21. Filtered Water Comparison Study (HPLC)
Low Concentration
(M9/L)
Condition
Non-Filtered
Filtered
Average*
Std. Dev.
% RSD
0.123
0.0114
9.3
0.123
0.0113
9.2
%of
Non-
Filtered
-
100%
High Concentration
(M9/L)
Condition
Non-Filtered
Filtered
Average*
Std. Dev.
% RSD
3.27
0.512
15.6
3.85
0.202
5.2
%of
Non-
Filtered
-
118%
*NOTE: Seven replicates were prepared and analyzed for each concentration at each time point.
-------
EA2192: "Linear" Regression ("1 /x" weighting): y = 2.16x + 0.0212
(r= 0.9998)
7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0
0.0 1.0 2.0 3.0 4.0 5.0
Figure 1. Representative EA2192 calibration curve: x-axis, analyte concentration/internal
standard concentration.; y-axis, analyte area/internal standard area.
32
-------
Methamidophos: "Linear" Regression ("1 /x" weighting): y = 0.151 x + 0.00178
(r = 0.9999)
00 10 £0 3.0 +JO Sti &0 TO S.O
13J5 W.O 1S.O 1S.O 1TJO 1»JO 1SJO
Figure 2. Representative Methamidophos calibration curve: x-axis, analyte
concentration/internal standard concentration; y axis, analyte area/internal standard area.
33
-------
Acephate: "Linear" Regression ("1 / x" weighting): y = 1.55 x + 0.00609
(r= 0.9998)
00 10 2JO 3JO 4O SJO SO 7.0 iO 90 100 110 110 1iJO tt-0 1S.O 16.0 170 1S.O 1S 0
Figure 3. Representative Acephate calibration curve: x-axis, analyte
concentration/internal standard concentration; y axis, analyte area/internal standard area.
34
-------
P .T-«^l =:I4i > =.-00 TK .
MemamMophos
1077
.13.12
19Si,19S3
f . lli. J M Ulll-j t .1 .. Jl J ,.l
—.54
J,
M^ = T-
Figure 4. Representative Chromatogram of a Matrix Blank without Internal Standard
Chromatogram order:
EA2192 (top)
Methamidophos
Acephate
Methamidophos-d6 (bottom)
35
-------
JSkfct
,:• 5.TQ
EA2192
92 \
f}^JW <>*JaHlf^**^1^^»s}lW Kj#^+
10
T "e "in
i::
1CC
,
wf • .
F/einani dooiws
1*^* 16 s
10
r -e "in
xco1-t.i=9.i.:*jars
1 .; 5 i at - i-ii- 1 5 tf ioi»5i3TOSw-iTj» ara,;.
Acepnale
DOO 3a D =^O "\ 54-T5 ft 1 •. =r 5 fi
2.054
1.054
mfe ma I Standard
10 C
T "c "in
Figure 5. Representative Chromatogram of a Matrix Blank with Internal Standard: x-axis,
time (minutes); y axis, intensity (counts)
Chromatogram order:
EA2192 (top)
Methamidophos
Acephate
Methamidophos-d6 (bottom)
36
-------
XCo'-f.' =».'•:* Mr* 14C4CCU=-10C^= D E-
4:::
-ICC
lice
;:. a" lC1iC6i6TCi=,wriTJ» =;:"/• sVno:
EA2VJ2
Mat 61*5 C =3
1C
T "e "ir
XCa1-*IRM.:*
n:
iCC
ICC-
3 1C1 iCiiiTOi w" .J
Metiam Hop nos
= :H
11=0 uos 1+JS
. J ,^ B ..... Jm^^jL 4
10
TTntmri
XC a- -MRM .;+ par* 1 itaiO'ttiDOC 3a D ac»-iBK'ra~i =a-as 1 .;CA- i - i-S- 1 i; a- id XiliTOii wr .;TJ» 3orai'> =noa
A M 5 ' =S
1C
~ -t ~r
^•I: o* -t.1 =J.l •;* Mrs 1 +*txc T ccc 3a D 5To "i Ba"^ i 1 •:a^_l - =rli-1 i> o* ^
Merarn Hop Hos-d6
I rue ma I Standard
Figure 6. Representative Chromatogram of a Calibration Standard at the Minimum
Reporting Level: x-axis, time (minutes); y axis, intensity (counts)
Chromatogram order:
EA2192 (top)
Methamidophos
Acephate
Methamidophos-d6 (bottom)
37
-------
XO a1 -t.1 =».1 .> Mr* HCWQ'tlilOODa I) EM19iTo-lEa-l3C 1 OjIVAW Hjl DC- I; a1 iC1i11iiTC;7 wf il
1 i
1 G
XC O^-M^M -[I Mrs IK- KC.'t* ICC 3fl ^ EM 151 "TO"'! 5a"1D~e 1 'i'j^'AVs' j^A' X - 1} tf i?1 i11iTTCT7 W - -T
7715
P.I* 771 i Q :
-ICC
IICI
CA-2
10
r "c "in
Figure 7. Representative EA2192 Chromatograms of Source No. 1 Water : x-axis, time
(minutes); y axis, intensity (counts)
Chromatogram order:
High Concentration Time 0 (top)
Low Concentration Time 0 (middle)
Matrix Blank (bottom)
38
-------
KOa1 -M=».1 .;* Mrs i4C4CC.ti1CC Ha D EM19£"ro-iEa-i3E 1 .;=WJHIT30-1!a1 201J1122TC64
CA-6
CA-2
DflsdPnpc 1 '.frt J Ms.i
Figure 8. Representative EA2192 Chromatograms of Source No. 2 Water: x-axis, time
(minutes); y axis, intensity (counts)
Chromatogram order:
High Concentration Time 0 (top)
Low Concentration Time 0 (middle)
Matrix Blank (bottom)
39
-------
Mat 1 4c=- IDS
CAJ-:
—3:
e:::
i:cc
i:::
10
T *t -*ir
11 10
Mat HOOcta
11: 11 c 13 c uc uc isc 17 c lie 19 c 100
Figure 9. Representative EA2192 Chromatograms of Source No. 3 Water: x-axis, time
(minutes); y axis, intensity (counts)
Chromatogram order:
High Concentration Time 0 (top)
Low Concentration Time 0 (middle)
Matrix Blank (bottom)
40
-------
D: EM19C1IW15jmt 1 vMWJ m*l DO- 1;
CA-6
10 1i U 1E 1i 20
T-e -in
10
T "c "in
11 U
M* 1 4K: C :
Figure 10. Representative EA2192 Chromatograms of Source No. 4 Water: x-axis, time
(minutes); y axis, intensity (counts)
Chromatogram order:
High Concentration Time 0 (top)
Low Concentration Time 0 (middle)
Matrix Blank (bottom)
41
-------
19. ATTACHMENTS
19.1 Ultra-High Performance Chromatography (UPLC) Method Development and Results
by Adapting of the Conditions of U.S. EPA Method 538 for Ultra-High Performance
Liquid Chromatography/Tandem Mass Spectrometry (UPLC/MS/MS) Analysis of
EA2192 in Water
19.2 CERTIFICATES OF ANALYSIS - EA2192, Methamidophos, and Acephate
42
-------
19.1 ULTRA-HIGH PERFORMANCE CHROMATOGRAHPY (UPLC) METHOD
DEVELOPMENT AND RESULTS
19.1.1 UPLC/MS/MS METHOD DEVELOPMENT - A preliminary method was
developed to transfer the adapted conditions from U.S. EPA Method 538 using
High-performance liquid chromatography/tandem mass spectrometry
(HPLC/MS/MS) to UPLC/MS/MS for the analysis of EA2192. Modifying this
method to incorporate UPLC analysis would drastically shorten the analytical run
time from the current 30 minute method to 5 minutes or less. In case of a time
sensitive environmental incident, the shorter analysis time could be vital for
increasing laboratory efficiency. The flow diversion valve was included in the
method due to a decrease in sensitivity over the course of longer analyses. By
switching to UPLC/MS/MS, the analysis of 100 injections could be accomplished in
under ten hours, whereas by HPLC/MS/MS, this same analysis would take over 50
hours.
Standards of EA2192, methamidophos, acephate, and methamidophos-d6 were
injected as a sub-set of the Method 538 analytes to determine the feasibility of
transferring the method to UPLC. Once a method was developed, the samples
prepared for the Water Filtration Study (Table 22) were injected using the developed
UPLC/MS/MS method to compare the two analytical methods.
19.1.2 UPLC SYSTEM AND PARAMETERS - The following UPLC system and
parameters were used in the UPLC/MS/MS method. The UPLC gradient program is
detailed below. The gradient used was derived from the U.S. EPA Method 538
gradient and optimized for UPLC analysis.
UPLC: Waters Acquity
Column: Waters Acquity HSS (high strength silica) T3, 1.8 \i,
100 x 2.1 mm
Mobile Phase A: 20mM Ammonium formate in water
Mobile Phase B: 100 % Methanol
Flow Rate: 0.6mL/min
Injection Volume: 30 jiL
RunTime: S.Omin
Sample Temperature: 5 °C
Column Temperature: 45 °C
Needle Wash A: 50/50 (v/v) Methanol/Water
Needle Wash B: 100 % Methanol
A-l
-------
UPLC/MS/MS Gradient
Time
0
1.3
3.5
3.51
4.2
4.21
5
%A
90
70
70
10
10
90
90
%B
10
30
30
90
90
10
10
19.1.3 MASS SPECTROMETER SYSTEM AND PARAMETERS - The mass
spectrometer parameters stayed consistent with the parameters used for
HPLC/MS/MS analyses (Table 11 and Table 12).
19.1.4 UPLC/MS/MS METHOD DEVELOPMENT RESULTS - The retention times (RTs)
of the four analytes are listed below. See Figure 11 and Figure 12 for representative
chromatograms of UPLC analyses.
Analyte
Methamidophos
Acephate
EA2192
Methamidophos-d6
HPLC RT
(min)
3.3
5.2
5.4
3.7
UPLC RT
(min)
1.0
1.2
1.2
1.0
RT= retention time
The average concentration of the filtered low concentration samples was 105 % of
the non-filtered samples. The average concentration of the filtered high
concentration samples was 107 % of the non-filtered samples. The UPLC water
filtration study results are summarized in Table 22. The results obtained from the
UPLC and HPLC analyses (see Table 21) were very similar.
Further work is suggested to complete the transfer of Method 538 conditions for EA
2192 described here to UPLC to include:
• Additional method development for all remaining Method 538 analytes to
confirm retention times and optimize the UPLC gradient.
• Initial Demonstration of Capability (IDC) testing to confirm method
acceptability.
• Addition of other CWA-related chemicals to Method 538.
• Tap waters high in total organic carbon (TOC) and hardness will be evaluated
under the UPLC conditions.
A-2
-------
XICaf+IVRM(4pairs):240.4ayi28.100CBD:EA2192frDmS&tTple1 (test std 1) of 20140503T_005»iff (Tuto Spray)
MEK 1.3e4cps
1.29e4
1.COe4 -
500QOO -
EA2192
4.55
A
0.5
1.0
1.5
2.0
2.5 3.0
Tirre rrin
XICaf+IVRM(4pairs): 142.0CO/94.000DalD rrEthirridophosfromSarrpte 1 (teststd 1)of 201405CBT_C05.wff (TutoSpray)
Mac 710.0cps
600-
400-
200-
0.45
. . 1 Q?1_,
I V1-.?2
1-49,1.55 "
Wtetharridophos
1
T-272. .
3S94.11 4.32446
0.5
1.0
1.5
2.0
2.5
Tima rrin
3.0
3.5
4.0
45
XlCof+IVRM(4pairs): 184.2CO/143.000CBD: acephatsfrcmSarrplel (teststd 1)af20140508T_005™ff (TurboSpray)
MSK. 8030.0 cps
5000 -
fcephste
045 0.67
0.5
1.0
1.5
2.0
2.5
Timg rrin
3.0
3.5
4.0
45
XlCaf+IVRM(4pairs): 14S.OCO/97.000CalD ISfromSarplel (teststd 1) of20140508T_005.wff (TurboSpray)
IV^c 5.7e4cpa
57e4
4.C64 -
2.C64 -
0.0
Wfetharridophos-d6
Irtemal Standard
0.5
1.0
1.5
2.0
2.5
m^ rrin
3.0
3.5
4.0
45
Figure 11. CAL1 Standard, UPLC/MS/MS Analysis
A-3
-------
• XlCrf+IVRM(4pairs):240.4CO/128.100DaD:EA2
7.S65
6.C65 -
4.C65 -
2.C65 -
192 from Sample 1 (test std 7) of 20140503T_007.»iff (Tuto Spray)
EA2192
\^
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Tims rrin
• XlCof+IVRM(4pairs): 142.0CO/94.000CalD tTEthirtidophosfromSartpte 1 (test std 7) of 20140503T_C07.wff (Tubo Spray)
6.C64 i
40s4 ~
Wtetharridophos
2.C64 -
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Tims rrin
• XICof+IVRM(4pairs):184.2CO/143.000CBD:a=e(
5Ee5
4.C65 -
2.C65 -
jhatefrcm Sample 1 (test std 7) of 201 40508T_007.™ff (TurboSpray)
n
Asp hate
\
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Tims rrin
• XlCof+IVRM(4pairs): 1 48.000/97.000 Da ID ISfromSarplel (test std 7) of 20140508T_007.wff (TurboSpray)
6.C64 1 l
4.C64 Wfetharridophos-d6
1 Irtemal Standard
2.C64 - 1
nn^ V
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Tims rrin
IVbc 7.9e5cps,
45
IVbc 6.0e4cps,
45
IV^c 5.5e5cpa
45
IVbc 6.0e4cps,
45
Figure 12. CAL7 Standard, UPLC/MS/MS Analysis
A-4
-------
Table 22. Filtered Water Comparison Study (UPLC)
Low Concentration
(M9/L)
Condition
Non-Filtered
Filtered
Average*
Std. Dev.
% RSD
0.121
0.0126
10.4
0.127
0.0110
8.7
%of
Non-
Filtered
-
105%
High Concentration
(M9/L)
Condition
Non-Filtered
Filtered
Average*
Std. Dev.
% RSD
2.80
0.179
6.4
2.99
0.152
5.1
%of
Non-
Filtered
-
107%
*NOTE: Seven replicates were prepared and analyzed for each concentration at each time point.
A-5
-------
19.2 Certificates of Analysis - EA2192, Methamidophos, and Acephate
Heikutat Sofafwns Mforhbrfcfe Impact
CERTIFICATE OF ANALYSIS
EA2192: [(S-diisopropylaminoOethyI]mett)ylphosphonio acid
MRISIoba! Agent ID: EA2192110301 -DOG-1
Original data is archived under MRIStobal Project No. 61010S.02.002.01
Compound Identification
Product:
Empirical Formula:
Molecular Weight:
ECBCLot*
Primary Standard ID.:
Solvent:
Quality
[(S-dBisopropylaminoOettiyflmethylphosphonic acid
239.32
NA
12632-50-2
AcetonMe-d3
Purfty (%): .
Storage Conditions:
Date of Analysis:
Expiration Date:
Standard Operating Procedure:
94.2%
9, 2013
Requires re-assessment after July S, 2014
MRI-5870, Rev. 5
Experimental Techniques
1) Nuclear Magnetic Resonance (31P-NMR). See data package dated July 24, 2013, for complete
details on the purity assessment.
Approved:
Date:
Smith
Chemical Agent Custodian
CA Group/Test and Evaluation Section/NSSI Division
B-l
-------
'A -AL OffICTH"'
CERTIFICATE OF ANALYSIS
Sigma-ftldrieh Laborchemikalien GtitbE D-3D918 Seelze
felefon: +49 5131 8238-150
Sselze, 21.31,2013/467129/13/01301
Order-No,:
Customer-No,:
Order-Cods;
Quantity:
Production Date: ll.Jan.2013
Expiry Date: ll.Jan.2016
Article/Product: 33395
Methamidophos PESTANAL8
Batch : SZBD011XV
Reference Material (RM)
1, General Information
Formula
CAS-No,
Usage
C2H8N02PS
[10265-92-6]
Acaricide/Insecticide
Molar mass: 141.13 g/Mole
Recomm. storage temp.: -20 "C
The estimated uncertainty ol a single measurement o1 the assay can be expected to be 1 % relative
(confidence level = 95%, n= 6} whereby the assay measurements are calculated by 100% minus found
impurities.
2, Batch Analysis
Identity (NMR)
Assay (HPLC)
Melting range
Water {Karl Fischer)
Date of Analysis
complying
97.7 area
40.0-45.0 °C
0.2 %
18.Jan.2013
3. Advice and Remarks
* fhe expiry date is based on the current knowledge and holds only for proper storage conditions in the
originally closed flasks/ packages,
* Whenever the container is opened for removal of aliqout portions of the substance, the person handling the
substance mast assure, that the integrity of the substance is maintained and proper records of all its
handlings are kept. Special care has to be taken to avoid any contamination or adulteration of the substance,
* We herewith confirm that the delivery is effected according to the technical delivery conditions agreed.
» Particular properties of the products or the suitability for a particular area of application are not assured,
* Na guarantee a proper quality within our General Conditions of Sales,
Sigma-Aldrich Laborchemikalion GmbH
Quality Management SA-LC
This document was produced electronically and is valid without a signature
B-2
-------
HPLC-Method
Article : Methaitidophos
Article-No : 33395
Batch :SZBD01iXV
Column : L=250mm, ID=4,6mm; Supelcosil LC-1S 5pm
Eliient : 40 % Aestonitrile
60 % Water
Flow : Q,8ml/min
Detector :UV-215nm
Injection-Volume : 20(jl
Sample-Preparation : 2mg/ml Acdonitrik
Linearity : checked
Evaluation : Normalisation (uncorrected)
Operator : Schowe
Chromatogiam
Mdhanidophos C:\LabSoMons\Data\Prqjectl \1300763.AIA\SIGNAL01.cdf
"Xooooo-
75000-
50000-
25000-
0-
0.0 2.5
1 DetA Chi /
Chi
PeakS Ret. Time
1 2.270
2 3.765
Total
Area
56
2364
2420
'IDgtACh
125
1.1.0
20.0
mm
PeakTable
Area %
2.298
97.702
100.000
B-3
-------
'A -AL OffICTH"'
CERTIFICATE OF ANALYSIS
Sigma-ftldrieh Laborchemikalien GtitbE D-3D918 Seelze
felefon: +49 5131 8238-150
Sselze, 21.12,2010/153344/10/06407
Order-No,:
Customer-No,:
Order-Cods;
Quantity:
Production Date: 24,Mar.2Q10
Expiry Date: 24.Mar.2tH5
Article/Product: 45315
Acephate PESTANAL®
Batch : SZBA083XV
Reference Material (RM)
1, General Information
Formula
CAS-No,
Usage
C4H10N03PS
[30560-19-1]
Insecticide
Molar mass: 183.17 g/Mole
Recomm. storage temp.: 2-8 "C
The estimated uncertainty ol a single measurement o1 the assay can be expected to be 1 % relative
(confidence level = 95%, n= 6} whereby the assay measurements are calculated by 100% minus found
impurities.
2, Batch Analysis
Identity (NMR)
Assay (GC)
Melting range
Water {Karl Fischer)
Date of Analysis
complying
97.8 area
87.0-90.8 °C
0.07 %
15.Apr.2010
3. Advice and Remarks
* The minimum shelf life is bassd on ths current ]enowl&dge and holds onl^ for proper storage conditions in the
originally closed flasks/ packages,
* Whenever the container is opened for removal of aliqout portions of the substance, the person handling the
substance mast assure, that the integrity of the substance is maintained and proper records of all its
handlings are kept. Special care has to be taken to avoid any contamination or adulteration of tlie substance.
* We herewith confirm that the delivery is effected according to the technical delivery conditions agreed.
» Particular properties of the products or the suitability for a particular area of application are not assured,
* Na guarantee a proper quality within our General Conditions of Sales.
Sigma-Aldrich Laborchemikalion GmbH
Quality Management SA-LC
This document was produced electronically and is valid without a signature
B-4
-------
GLC-Method
Article
Article-No
Batch
Acephato
45315
SZBA083XV
Column
Inj.-temp.
Det.temp.
Oven-temp.
Split
Flow
Inj.v.
Eva1uat ion
Operator
MDN-5, 30m,fs cap.,I.D.=0,32mm,l,0micron df
280°C
330°C
150°(4min)to 3200C(100/min)hold 15min
1:100
1ml He/min
Ipl solution in Dichloromethane
Normalisation(uncorrected)
Schulz
Intensity
50000
40000-
30000-
20000^
10000
\y
10
20
30
Peak Table - Channel 1
Peakf let .Time
1 8,766
2 14,186
3 14,746
Total
Height
53854
1120
1052
56026
Area
263888
3031
2827
269746
Area%
97,8284
1,1236
1,0481
100,0000
B-5
-------
United States
Environmental Protection
Agency
PRESORTED STANDARD
POSTAGE & FEES PAID
EPA
PERMIT NO. G-35
Office of Research and Development (8101R)
Washington, DC 20460
Official Business
Penalty for Private Use
$300
------- |