. 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

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       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

-------
                     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

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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

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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

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                          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

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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

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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

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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

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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

-------