www.epa.gov/or
United States
Environmental Protection
Agency
Surface Analysis of Nerve Agent
Degradation Products by Liquid
Chromatography/Tandem Mass
Spectrometry (LC/MS/MS)
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-13/224
September 2013
SURFACE ANALYSIS OF NERVE AGENT DEGRADATION PRODUCTS BY LIQUID
CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY (LC/MS/MS)
Sampling and Analytical Method for Wipe Analysis of Surfaces
Revision 1
United States Environmental Protection Agency
National Homeland Security Research Center
26 W. Martin Luther King Jr. Drive
Cincinnati, OH 45268
and
Centers for Disease Control and Prevention
National Institute for Occupational Safety and Health
5555 Ridge Ave
Cincinnati, OH 45213
Last Revised: 12/12
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DISCLAIMER
The United States Environmental Protection Agency through its Office of Research and Development
(ORD), National Homeland Security Research Center (NHSRC), funded and managed the research
described here (IA #DW-75-922440001-0) in collaboration with the National Institute of Occupational
Safety and Health (NIOSH), Centers for Disease Control and Prevention (CDC), a division of the U.S.
Department of Health and Human Services (DHHS). It has been subjected to the Agency's administrative
review and approved for publication. The views expressed in this paper do not necessarily reflect the
views or policies of the Agency. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
Questions concerning this document or its application should be addressed to:
Stuart Willison, Ph.D.
Project Officer
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
Robert Streicher, Ph.D.
Project Officer
National Institute for Occupational Safety and Health Laboratories
Alice Hamilton Laboratory
5555 Ridge Avenue
Cincinnati, OH 45213
513-841-4296
Rps3@cdc.gov
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ACKNOWLEDGMENTS
We would like to acknowledge the following individuals and organization for their contributions towards
the development and/or review of this method.
United States Environmental Protection Agency (EPA)
Office of Research and Development, National Homeland Security Research Center
Stuart Willison, Project Officer and Method Development
Matthew Magnuson, Technical Reviewer
United States Environmental Protection Agency (EPA)
Office of Emergency Management
Terry Smith, Technical Reviewer
United States Environmental Protection Agency (EPA)
Office of Ground Water and Drinking Water
April Dupre, Technical Reviewer
Centers for Disease Control and Prevention
National Institute for Occupational Safety and Health
Jack Pretty, Laboratory Advisor
Robert Streicher, Project Officer
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EXECUTIVE SUMMARY
The sampling and analytical method described herein was developed and tested within the same
laboratory to assess the recoveries of nerve agent degradation products from various porous (vinyl tile,
painted drywall, wood) and mostly nonporous (laminate, galvanized steel, glass) surfaces. Performance
data (method detection limit and precision and accuracy data) are available to demonstrate the fitness-for-
purpose regarding the development of a method for nerve agent degradation products in that single
laboratory. Samples are collected from surfaces using wipes, the wipes are spiked with a surrogate
compound and carried through extraction with distilled water by sonication and filtration steps followed
by analysis using liquid chromatography electrospray ionization/tandem mass spectrometry (LC/ESI-
MS/MS) by direct injection without derivatization. Detection limit data were generated using wipes on a
laminate surface following the procedures of 40 CFR Part 136, Appendix B, as part of EPA's guidelines
for determining a method detection limit.
Gauze wipes were selected over other tested wipes (i.e., filter paper, glass fiber filters, nonwoven
polyester fiber) because gauze wipes were physically robust during the wiping procedure, contained low
background levels, produced no peaks that interfered with the target analytes, and produced the highest
percent recoveries of all wipes tested during sample analysis. Percent recoveries were highest for the
laminate surface and ranged from 65-87 % for all of the nerve agent degradation products analyzed in ESI
negative mode. The resulting equivalent method detection limits obtained from wiping the laminate
surface were 0.04 |o,g/cm2 for isopropyl methylphosphonic acid (IMPA), 0.07 |o,g/cm2 for
methylphosphonic acid (MPA), 0.05 |o,g/cm2 for ethyl methylphosphonic acid (EMPA), 0.07 |o,g/cm2 for
ethyl hydrogen dimethylamidophosphate, sodium salt (EHDMAP) and 0.02 |o,g/cm2 for pinacolyl
methylphosphonic acid (PMPA). Diisopropyl methylphosphonate (DIMP) was not recovered unless the
surfaces were wiped immediately after spiking due to the volatile nature of this compound. Other
complications are presented in the method in section 14.4. Precision and accuracy data were generated
from each tested surface fortified with these analytes.
IV
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SURFACE ANALYSIS OF NERVE AGENT DEGRADATION PRODUCTS BY LIQUID
CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY (LC-MS/MS)
TABLE OF CONTENTS
SECTION PGNO.
DISCLAIMER ii
ACKNOWLEDGMENTS iii
EXECUTIVE SUMMARY iv
LIST OF TABLES vii
LIST OF ACRONYMS AND ABBREVIATIONS viii
1. INTRODUCTION 1
2. SCOPE AND APPLICATION 1
3. SUMMARY OF METHOD 3
4. DEFINITIONS 4
5. INTERFERENCES 5
6. HEALTH AND SAFETY 6
7. EQUIPMENT AND SUPPLIES 6
8. REAGENTS AND STANDARDS 7
9. SAMPLE COLLECTION, PRESERVATION AND STORAGE 9
10. QUALITY CONTROL 10
11. INSTRUMENT CALIBRATION AND STANDARDIZATION 15
12. ANALYTICAL PROCEDURE 16
13. DATA ANALYSIS AND CALCULATIONS 18
14. METHOD PERFORMANCE 18
15. POLLUTION PREVENTION 19
16. WASTE MANAGEMENT 19
17. REFERENCES 21
18. TABLES AND VALIDATION DATA 22
19. ATTACHMENTS 28
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LIST OF TABLES
Table 1. Materials Tested for the Wipe Analysis of Nerve Agent Degradation Products 2
Table 2. Holding Time Sample Stability of Nerve Agent Degradation Analytes of Wipe Samples in ESI Negative
Mode 2
Table 3. Method Parameters for Nerve Agent Degradation Products 3
Table 4a. ESI (+) MRM Ion Transitions, Retention Time (RT) and Variable Mass Spectrometer Parameters 4
Table 4b. ESI (-) MRM Ion Transitions, Retention Time (RT) and Variable Mass Spectrometer
Parameters 25
Table 5. ESI (+) and (-) MS/MS Conditions 5
Table 6. Liquid Chromatography Gradient Conditions 5
Table 7. Target Concentrations of Calibration Standards Used During the Development of this Method (ng/mL) 6
VI
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LIST OF ACRONYMS AND ABBREVIATIONS
AS Analyte Stock Standard (Solution)
CAL Calibration Standard
CAS Chemical Abstracts Service
CCC Continuing Calibration Check
CDC Centers for Disease Control and Prevention
CID Collisionally Induced Dissociation
CWA Chemical Warfare Agent
DL Detection Limit
DHHS U.S. Department of Health and Human Services
DIMP Diisopropyl Methylphosphonate
DQO Data Quality Objective
EHDMAP Ethyl Hydrogen Dimethylamidophosphate, sodium salt
EMPA Ethyl Methylphosphonic Acid
EPA U.S. Environmental Protection Agency
ERLN Environmental Response Laboratory Network
ESI (+) Electrospray lonization in Positive Mode
ESI (-) Electrospray lonization in Negative Mode
FD Field Duplicate
IDC Initial Demonstration of Capability
IDL Instrument Detection Limit
IMPA Isopropyl Methylphosphonic Acid
LC Liquid Chromatography
LC/MS/MS Liquid Chromatography Coupled with Tandem Mass Spectrometry
LFB Laboratory Fortified Blank
LFSM Laboratory Fortified Sample Matrix
LFSMD Laboratory Fortified Sample Matrix Duplicate
LMB Laboratory Method Blank
MDL Method Detection Limit
MPA Methylphosphonic Acid
MRL Minimum Reporting Limit
MRM Multiple Reaction Monitoring
MS Mass Spectrometer(try)
MSDS Material Safety Data Sheet
MS/MS Tandem Mass Spectrometry
NHSRC National Homeland Security Research Center
NIOSH National Institute for Occupational Safety and Health
NIST National Institute of Standards and Technology
ORD U.S. EPA's Office of Research and Development
OSHA Occupational Safety and Health Administration
PIR Prediction Interval of Result
PMPA Pinacolyl Methylphosphonic Acid
ppb Parts Per Billion
ppm Parts Per Million
P&A Precision and Accuracy
PVDF Polyvinylidene Fluoride
QC Quality Control
r2 Coefficient of determination
VII
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REC Percent Recovery
RL Reporting Limit
RPD Relative Percent Difference
RSD Relative Standard Deviation
RT Retention Time
SAM Selected Analytical Methods for Environmental Restoration Following Homeland
Security Events
SD Standard Deviation
S/N Signal to Noise
SS Surrogate Standard
SSS Stock Standard Solution
VOA Volatile Organic Analysis
X Average Percent Recovery
a Standard Deviation
VIM
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1. INTRODUCTION
1.1. The U.S. Environmental Protection Agency (EPA) is responsible for developing tools and
methodologies which will enable the rapid characterization of indoor and outdoor areas and
water systems following a deliberate/accidental release or a natural disaster. EPA's National
Homeland Security Research Center (NHRSC), published Selected Analytical Methods for
Environmental Remediation and Recovery (SAM), formerly referred to as the Standardized
Analytical Methods for Environmental Restoration Following Homeland Security Events (1),
which is a compendium of methods that informs sample collection and analysis during the
response to an all-hazards incident. Chemical warfare agents (CWAs) and their degradation
products remain a high-priority concern due to the potential for the intentional or unintentional
release of these agents. Nerve agents are very dangerous CWAs, which can break down into
degradation products sufficiently persistent and toxic to be of interest during site remediation
after a release. Accordingly, if an incident were to occur, versatile sampling procedures are
needed to detect CWA degradation products from various CWAs and help determine the spread
and concentration of these agents and degradation products in contaminated areas. Multiple
types of contaminated surfaces from an indoor setting (e.g., walls, posts, windows, floors and
furniture) will need to be extensively tested within the contaminated areas. Direct extraction
may be a possibility; however, the laboratory procedures can be tedious, complex, and require
the destruction of the material being analyzed. Wipe sampling is preferred because it can be
performed quickly and easily in a manner less destructive to the tested surface when direct
extraction is not feasible.
1.2. After sample collection, selective analysis methods must be implemented to detect and quantify
the appropriate agent and/or degradation products in the environmental sample. The appropriate
procedure should account for possible contaminants already present within the sample as well as
other matrix complications that may arise during analysis to ensure sample integrity and to
ensure that the analysis method is applicable to the matrix of interest. Liquid chromatography-
tandem mass spectrometry (LC-MS/MS) is often the most appropriate and powerful analysis
technique for polar nonvolatile compounds. LC-MS/MS affords laboratories an enhanced
capability to analyze specific environmental matrices for CWA degradation products while
avoiding complications that may arise from derivatization, a step more commonly needed for gas
chromatography/mass spectrometry analysis. Although LC-MS analysis methods do exist for
nerve agent degradation products from water, currently no known wipe sample collection and
analysis protocol for the detection of nerve agent degradation products from contaminated
surfaces is documented in the scientific literature.
2. SCOPE AND APPLICATION
2.1. This sampling and analytical procedure was developed and tested in the same laboratory
to investigate nerve agent degradation products, which may persist at a contaminated site,
via surface wiping followed by analytical characterization. The performance data
presented demonstrate the fitness-for-purpose regarding surface analysis in that single
laboratory. Surfaces (laminate, glass, galvanized steel, vinyl tile, painted drywall and
treated wood) were wiped with cotton gauze wipes, sonicated, extracted with distilled
water, and filtered. Samples were analyzed with direct injection electrospray ionization
liquid chromatography tandem mass spectrometry (ESI-LC-MS/MS) without
derivatization. Detection limit data were generated for all analytes of interest on a
laminate surface. Accuracy and precision data were generated from each surface fortified
with these analytes. The following analytes have been determined using this procedure:
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Analyte CAS Registry Number
Diisopropylmethylphosphonate (DIMP) 1445-75-6
Ethyl Hydrogen Dimethylamidophosphate, sodium salt (EHDMAP) 2632-86-2
Ethyl Methylphosphonic acid (EMPA) 1832-53-7
Isopropyl Methylphosphonic acid (IMPA) 1832-54-8
Methylphosphonic acid (MPA) 993-13-5
Pinacolyl Methylphosphonic Acid (PMPA) 616-52-4
2.2. Wipe sampling can be performed quickly and easily when direct extraction is not feasible
(e.g., walls, posts, windows, floors and furniture) as wipe sampling can be performed
without the destruction of the tested surface. Porous surfaces may have lower recoveries
and less precision because the contaminants may sorb into the material. Wipe sampling
will recover analyte only from the surface of the analyzed material. It is, therefore,
important to understand wipe efficiencies and the materials being wiped. This procedure
assesses the recoveries from several porous and nonporous surfaces using wipes.
2.3. Method detection limit (MDL) metrics are presented using EPA conventions (2-3). The
detection limit is defined as the statistically calculated minimum concentration that can
be measured with 99% confidence that the reported value is greater than zero (4). The
MDL is compound-dependent and reliant on sample preparation, sample matrix,
concentration and instrument performance. The statistical procedure, utilizing the
Laboratory Fortified Sample Matrix samples (LFSM) and LFSM duplicates (LFSMDs),
is used to calculate recovery. Precision and accuracy (P&A) studies are performed as an
initial demonstration of capability (IDC) and ongoing demonstration of capability to
perform the procedure, including changes in instrumentation and operating conditions.
These studies evaluate whether the reporting limits (RLs) and calibration standard
concentrations are appropriate.
2.4. This procedure is intended for use by analysts skilled in the operation of LC-MS/MS
instrumentation and the interpretation of the associated data. Due to the inherent
complexities of LC-MS/MS analysis, including the need to relate sample characteristics
to analytical performance, laboratories should update their initial estimates of
performance and should strive to tighten their quality control limits as more experience is
gained with this particular procedure.
2.5. METHOD FLEXIBILITY
Many variants of liquid chromatography (LC) and Tandem Mass Spectrometry (MS/MS)
technology are currently in operation. In addition, variability exists in the sources of
wipe materials, wipe composition, and compatibility of various wipe materials with some
surfaces. This procedure was developed using a triple quadrupole LC-MS/MS, with
optimized LC conditions and wipe materials. The procedure has been verified using only
the specified equipment and conditions. Other types of LC-MS/MS instrumentation, LC
and/or ESI-MS/MS conditions, sample collection and processing steps, and
wipe/collection materials can be used for analysis as long as similar performance is
demonstrated and the quality control measures outlined in section 10 of this report are
implemented.
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3. SUMMARY OF METHOD
3.1. Samples are collected from surfaces with wipes and stored at 4 °C (± 2 °C) if samples are
not to be analyzed within a 24-hour time period. When the samples are analyzed,
samples are spiked with the appropriate surrogate compounds, the appropriate solvent
volume is added, the sample solution is sonicated, extracted with a syringe filter unit,
then the extract is analyzed directly by LC-MS/MS operated simultaneously in positive
and negative electrospray ionization modes, (ESI+) and (ESI-) respectively. Data
described in this procedure refer to ESI (-) mode because some complications can occur
in ESI (+) mode.
3.2. Each target compound is separated chromatographically and identified by retention time.
Comparison of the sample primary multiple reaction monitoring (MRM) transition to the
known standard MRM transition from reference spectra under identical LC-MS/MS
conditions is used to identify analytes. The retention time for the analytes of interest must
fall within the retention time window of the standard (within ±5%). The concentration
of each analyte is determined by the instrumentation software using external calibration.
Surrogate analytes are added to samples to monitor extraction efficiency of the method
analytes from the wipe and extraction process.
3.3. This procedure utilizes cotton gauze wipes, which were determined to provide the highest
recoveries with the least interference for any targeted analyte. Other wipes such as filter
paper or glass fiber filters did have comparable recoveries and might be an appropriate
alternative but would not be as robust during the wiping procedure for the targeted
analytes.
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4. DEFINITIONS
4.1. ANALYSIS BATCH - A set of samples analyzed on the same instrument within a 24-hour
period and including no more than 20 field samples, beginning and ending with the analysis of
the appropriate continuing calibration check (CCC) standards. Additional CCCs may be required
depending on the number of samples (excluding QC samples) in the analysis batch and/or the
number of field samples.
4.2. CALIBRATION STANDARD (CAL) - A solution prepared from the analyte stock standard
solution and the surrogate/internal standard(s). The CAL solutions are used to calibrate the
instrument response with respect to analyte concentration.
4.3. COLLISIONALLY INDUCED DISSOCIATION (CID) - The process of converting the
precursor ion's translational energy into internal energy by collisions with neutral gas molecules
to bring about dissociation into product ions.
4.4. CONTINUING CALIBRATION CHECK (CCC) - A calibration standard containing the method
analytes and surrogate standard(s). The CCC is analyzed periodically to verify the accuracy of
the existing calibration for those analytes at or near the mid-level concentrations. Low
calibration concentrations can be added, in addition to mid-level concentrations, for further
accuracy, but are not required.
4.5. 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.
4.6. EXTRACTION BATCH - A set of up to twenty field samples (excluding quality control [QC]
samples) extracted together using the same solvents, surrogate(s), fortifying solutions, and
sampling devices.
4.7. FIELD DUPLICATE (FD) - Separate samples collected at the same time and place, under
identical circumstances and treated exactly the same as other field samples throughout field
and/or laboratory procedures. Analyses of FDs will give a measure of the precision associated
with sample collection, preservation, and storage, as well as laboratory procedures.
4.8. LABORATORY FORTIFIED BLANK (LFB) - A blank matrix to which known quantities of
the method analytes are added in the laboratory. The LFB is analyzed exactly like a sample, and
its purpose is to demonstrate that the methodology is in control and that the laboratory is capable
of making accurate and precise measurements.
4.9. LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - A field sample to which known
quantities of the method analytes are added in the laboratory. The LFSM is processed and
analyzed exactly like a sample, and its purpose is to determine whether the sample matrix
contributes bias to the analytical results. The background concentrations of the analytes in the
sample matrix must be determined in a separate sample.
4.10. 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. The LFSMD is used to assess method precision when the observed concentrations of
method analytes are low.
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4.11. LABORATORY METHOD BLANK (LMB) - A blank matrix that is treated exactly the same as
a sample including exposure to all glassware, equipment, solvents and reagents and surrogate
standards that are used in the analysis batch. The LMB is used to determine if method analytes or
other interferences are present in the laboratory environment, the reagents, or the apparatus.
4.12. MATERIAL SAFETY DATA SHEET (MSDS) - 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. 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.
4.14. PRECURSOR ION - For the purpose of this method, the precursor ion is the protonated
molecule ([M+H]+) or adduct ion of the method analyte. In MS/MS, the precursor ion is mass-
selected and fragmented by collisionally induced dissociation (CID) to produce distinctive
product ions of lower mass.
4.15. PRODUCT ION - For the purpose of this method, a product ion is one of the fragment ions
produced in MS/MS by CID of the precursor ion.
4.16. SURROGATE STANDARD (SS) - A pure chemical(s) added to a standard solution in a known
amount(s) and used to measure the relative response of other method analytes that are
components of the same solution. The surrogate standard must be a chemical that is structurally
similar to the method analytes, has no potential to be present in samples, and is not a method
analyte.
4.17. 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
Procedural interferences can be caused by contaminants in solvents, reagents, glassware and other
apparatus that lead to discrete artifacts or elevated baselines in the selected ion current profiles. All
of these materials must routinely be demonstrated to be free from interferences by analyzing
Laboratory Method Blanks (LMBs) (Section 10.4.1) under the same conditions as the samples (5).
Subtraction of blank values from sample results is not performed.
5.1. All reagents and solvents should be of pesticide grade purity or higher to minimize interference
problems. All glassware should be cleaned and demonstrated to be free from interferences.
5.2. Matrix interferences may be caused by contaminants from the sample matrix, sampling devices
or storage containers. The extent of matrix interferences will vary considerably from sample
source to sample source, depending upon variations in the sample matrix. Wipe matrix
interferences and contaminants are likely to be present and may have an effect on the recoveries
for the analytical procedure. These interferences lead to elevated baselines and artifacts that may
be interpreted as positives. Wipes were not pre-cleaned but were analyzed to ensure that there
were no interferences present. Any wipe materials containing interferences with the analytes of
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interest were not used.
5.3. Matrix effects are known phenomena of ESI-MS techniques, especially for coeluting
compounds. Managing the unpredictable suppression and enhancement caused by these effects
is recognized as an integral part of the performance and verification of an ESI-MS procedure.
The data presented in this procedure were designed to demonstrate that the procedure is capable
of functioning with realistic samples. Each analyst is encouraged to observe appropriate
precautions and follow the described QC procedures to help minimize the influence of ESI-MS
matrix effects on the data reported. Matrix effects include ion suppression/enhancement, high
background and improper ion ratios.
6. HEALTH AND SAFETY
The toxicity and carcinogenicity of each reagent used in this method have not been defined precisely.
However, each chemical compound was treated as a health hazard. Exposure to these chemicals
should be reduced to the lowest possible level and proper protective equipment should be worn for
skin, eyes, etc. Each laboratory is responsible for maintaining an awareness of Occupational Safety
and Health Administration (OSHA) regulations regarding the safe handling of chemicals used in this
method. A reference file of MSDSs that address the safe handling of the chemicals should be made
available to all personnel involved in the chemical analyses or subject to potential exposure.
Additional references are available (6-9).
7. EQUIPMENT AND SUPPLIES
References to specific brands of equipment and catalog numbers are provided solely as examples and
do not constitute an endorsement of the use of such products or suppliers. Materials tested for the
wipe analysis of nerve agent degradation products are described in Table 1.
7.1 LC-MS/MS APPARATUS
7.1.1 LIQUID CHROMATOGRAPHY (LC) SYSTEM - An analytical system complete
with a temperature programmable liquid chromatograph with a solvent mixer
(Waters, Milford, MA - Acquity™ or equivalent able to perform the analyses as
described) and all required accessories including syringes, solvent degasser, and
autosampler.
7.1.2 ANALYTICAL COLUMN - Atlantis® - dC18, 100 mm x 2.1 mm, 3 |^m particle size
(Waters, Milford, MA, Catalog # 186001299), or equivalent.
7.1.3 TANDEM MASS SPECTROMETER (MS/MS) SYSTEM - An MS/MS instrument
(Waters TQD™ or similar instrument) can be used for analysis of the target analytes.
A mass spectrometer capable of MRM analysis with the capability to obtain at least
10 scans over a peak with adequate sensitivity is required.
7.1.4 DATA SYSTEM - Waters' MassLynx™ software (or similar software) interfaced to
the LC/MS that allows the continuous acquisition and storage on machine-readable
media of all mass spectra obtained throughout the duration of the chromatographic
program. Waters' QuanLynx™ (or similar software) is used for all quantitative
analysis for data generated from the LC-MS unit.
7.2 EXTRACTION DEVICE
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7.2.1 SONICATOR (Fisher Scientific Catalog # 15-335-112) or equivalent.
7.3 GLASSWARE AND MISCELLANEOUS SUPPLIES
7.3.1 AUTOSAMPLER VIALS - Amber 2-mL autosampler vials with pre-slit Teflon®-
lined screw tops (Waters Corp., Milford, MA), or equivalent.
7.3.2 DISPOSABLE STERILE SYRINGES - 10.0 mL ± 1% accuracy BD Safery-Lok™
syringes (Catalog No. 14-829-32, Fisher Scientific, Pittsburgh, PA), or equivalent.
7.3.3 AUTO PIPETTES - 10.0 mL, 1000 uL, 100 uL and 10 uL ± 1% accuracy.
7.3.4 DESOLVATION GAS - Nitrogen gas generator or equivalent nitrogen gas supply.
Aids in the generation of an aerosol of the ESI liquid spray and should meet or
exceed instrument manufacturer's specifications.
7.3.5 COLLISION GAS - Argon gas used in the collision cell in MS/MS instruments and
should meet or exceed instrument manufacturer's specifications.
7.3.6 ANALYTICAL BALANCE - accurate to 0.1 mg; reference weights traceable to
Class S or S-l weights.
7.3.7 National Institute of Standards and Technology (NIST)-traceable thermometer.
7.3.8 STANDARD SOLUTION FLASKS - Class A volumetric glassware
7.3.9 SYRINGE FILTER - Millex® GV Syringe-driven polyvinylidene fluoride (PVDF) 13
mm filter unit , 0.22 jam (Millipore Corporation, Billerica, MA, Catalog #
SLGV013NL).
7.3.10 WIPES - Dukal™, 2" x 2" - 12-ply sterile cotton gauze pads, individually packaged
(Fisher Scientific, Pittsburgh, PA, Catalog # 17986468).
7.3.11 SAMPLE COLLECTION CONTAINERS - Clean 125 ml Nalgene polypropylene
straight-side jars with screw caps (Fisher Scientific, Pittsburgh, PA, Catalog #11-
815-1OC), or equivalent.
7.3.12 SAMPLE CONCENTRATION CONTAINERS - Sterile 15 mL conical graduated
plastic centrifuge tubes (Fisher Scientific, Pittsburgh, PA, Catalog # 05-5 3 8-5 9A), or
equivalent.
8 REAGENTS AND STANDARDS
8.1 REAGENTS AND STANDARDS
When compound purity is assayed to be 98% or greater, the weight may be used without
correction to calculate the concentration of the stock standard. Expiration times for
7
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prepared solutions are suggested below, but laboratories should follow standard QC
procedures to determine when the standards should be replaced. Label all standards and
verify the correct grade of solvents. Traceability of standards is established by the
manufacturer's specifications provided at time of purchase.
8.1.1 SOLVENTS, REAGENTS and GASES - Acetonitrile (CAS # 75-05-8), Methanol
(CAS # 67-56-1), and LC-MS grade Water (CAS # 7732-18-5), HPLC mass
spectrometry pesticide grade or equivalent, demonstrated to be free of analytes and
interferences. Formic Acid (Chemical Abstracts Service (CAS) # 64-18-6). Nitrogen
is used for the generation of aerosol of the ESI liquid spray, and purity should meet
instrument manufacturer's specifications. Argon is used as the collision gas in
MS/MS applications, and purity should meet instrument manufacturer's
specifications.
8.1.2 MOBILE PHASE A - Solution A consisted of LC-MS grade water and 0.2% of
formic acid to prevent microbial growth. To prepare 0.5 L, add 1 mL of formic acid
and dilute to 0.5 L mark with water. This solvent system is prone to some microbial
growth and should be replaced at least once a week.
8.1.3 MOBILE PHASE B- Solution B was comprised of acetonitrile and 0.2% of formic
acid. To prepare 0.5 L, add 1 mL of formic acid and dilute to 0.5 L mark with
acetonitrile.
8. 1 .4 TARGET ANALYTES - MPA (Catalog #: 289868) and EMPA (Catalog #: 1 12062)
were purchased from Sigma-Aldrich (St. Louis , MO). IMPA (Catalog #: ERI-015),
DIMP (Catalog #: ERD-083), PMPA (Catalog #: ERP-083), and EHDMAP (Catalog
#: ULM-6091-1.2) were purchased from Cerilliant (Round Rock, TX).
8.1.5 SURROGATE ANALYTES - MPA-d3 (Catalog #: DLM-6196-1.2), PMPA-13C6
(Catalog #: CLM-6620-1.2)and DIMP-d14 (Catalog #: ERD-086) were purchased
from Cerilliant.
8.2 STANDARD SOLUTIONS
When compound purity is assayed to be at least 98% or greater, the weight can be used
without correction to calculate the concentration of the stock standard. Stock standards
and all subsequent solutions should be replaced when analyzed solution concentrations
deviate more than ± 20% from the prepared concentration. Standards are stored protected
from light (amber flasks) and at 4 °C (± 2 °C). Standards are estimated to be stable for at
least a month as long as water is not present. Although stability times are suggested,
laboratories should utilize QC practices to determine when standards should be replaced.
8.2.1 SURROGATE STOCK STANDARD SOLUTION (Surrogate SSS) (10-1000
A standard solution may be prepared from certified commercially available methanol
•6
solutions or neat compounds. Isotopically-labeled surrogates (MPA-d3, PMPA- C
and DIMP-di4) were purchased as methanol solutions. The surrogate is added to a 10
mL volumetric flask to achieve a concentration of approximately ten times the
highest calibration concentration (ten times calibration 7) in solution (i.e., 900 |oL of
8
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MPA-d3 and PMPA-13C6 and 18 nL of DIMP-d14 were added to a 10 mL volumetric
flask and diluted to the mark with methanol). Surrogate stock standard solutions are
stable for at least a month when stored at 4 °C.
(NOTE: Although the listed analytes were used as surrogates in this method, they
could also be used as internal standards for quantitation purposes. However, further
evaluation would be necessary to ensure that they are viable internal standards and
meet QC requirements.)
8.2.2 ANALYTE STOCK STANDARD SOLUTION (AS)
Standard solutions may be prepared from certified commercially available neat
compounds. MPA and BMP A were purchased as a neat solid and liquid,
respectively. Separate methanol solutions (1000 |o,g/mL) containing MPA and
EMPA were used to make the analyte stock standard solution. DIMP, EHDMAP,
IMPA, and PMPA were purchased as methanol solutions. A standard methanol
solution with a concentration of 3 |o,g/mL (ppm) was made in a 25 mL volumetric
flask containing DIMP, EMPA, MPA, PMPA, and IMPA (i.e., 18 jiL of DIMP, 30
HL of EMPA, 75 jiL of MPA, 18 jiL of PMPA and 90 jiL of IMPA are each added to
a 25 mL volumetric flask and diluted to the mark with methanol). EHDMAP is not
added to the initial stock standard solution because it is not stable over the suggested
OO
stability period when added to the methanol solution. EHDMAP and the surrogate
analytes are added to calibration standard solutions only when the solutions are ready
for use. The calibration standards and spike solutions are made from the appropriate
dilution of this analyte stock standard. The analyte stock standard solution is stable
for at least a month when stored at 4 °C.
8.2.3 CALIBRATION STANDARD SOLUTION (CAL)
Dilution of the 3 jog/mL methanol solution can be used to obtain a 750 ng/mL (ppb)
solution in water. A calibration stock standard solution (Level 7) is prepared from
the Analyte Stock Standard Solution (AS) and SSS by adding, 2.5 mL of AS, 9 joL of
EHDMAP, and 1 mL of the SSS (i.e., 2.5 mL of the AS containing DIMP, EMPA,
MPA, PMPA, and IMPA, 9 nL of EHDMAP, and 1 mL of the SSS are added to a 10
mL volumetric flask and diluted to the mark with LC-MS grade water). From Level
7, further dilutions are performed with LC-MS grade water to prepare Levels 6
through 1, as shown in Table 2.
SAMPLE COLLECTION. PRESERVATION AND STORAGE
9.1 SAMPLE COLLECTION
9.1.1 Volatile organic analysis (VOA) vials and Nalgene containers were both used for
sample collection and both were deemed adequate for use, but Nalgene containers
were specifically used in this method. Other vessels may be used as long as they are
tested and verified to ensure they do not contain any interfering compounds. As an
example for field samples, the field samplers would collect samples with the
appropriate water-wetted wipe and place the wipes in ajar with a cap (e.g., 125 mL
Nalgene polypropylene straight-sided jar with a polypropylene screw cap) and ship
the jar containing the sample to the laboratory. The Nalgene containers did not
present contamination problems nor did the results suggest that the analytes of
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interest adhere to the jars, so Nalgene containers can be used instead of glass VOA
vials used in standard practice.
9.1.2 The wipe is wetted with 1 mL of LC-MS grade water, sufficient to wet the wipe. The
surface is wiped in a Z-like pattern horizontally across a defined surface (100 cm2)
(Attachment 19.3), folded, then used to wipe the same surface in a Z-like pattern
vertically across a defined surface (100 cm2). The wipe is placed into a 125 mL
Nalgene polypropylene straight-sided jar with a polypropylene screw cap. Surrogates
(66.6 joL of the SSS) and LC-MS grade water (5 mL) are added to the jar. Field
and/or matrix blanks are needed, according to conventional sampling practices;
therefore, one blank sample coupon was analyzed in every sample extraction batch.
9.2 SAMPLE STORAGE AND HOLDING TIMES
9.2.1 Wipe samples should be extracted as soon as possible after collection but must be
extracted within 30 days of collection. Samples not immediately analyzed from a
particular site should be carefully characterized to ensure there is no interaction with
the wipe or a specific surface to cause interferences or degradation of the analytes.
An LFSM can be generated for the appropriate time period to verify such an
occurrence. Samples can be stored up to 30 days (Table 2) at 4 °C (± 2 °C).
10 QUALITY CONTROL
10.1 QC requirements include the performance of an initial demonstration of capability (IDC)
and ongoing QC requirements that must be met to generate data of acceptable quality when
preparing and analyzing samples. This section describes the QC parameters, their required
frequencies and performance criteria. A precision and accuracy study (P&A, as shown in
section 19.2) as well as a Detection Limit (DL) study (Table 3 and section 19.1) must be
performed to demonstrate laboratory capability. Laboratories are encouraged to institute
additional QC practices to meet their specific needs.
10.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC)
The IDC must be performed successfully prior to the initiation of analysis of field
samples. Prior to conducting an IDC, an acceptable Initial Calibration must be generated
as outlined in Section 11.2.
10.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND
Any time a new lot of solvents, reagents, filters and autosampler vials is used, the
LMB must be demonstrated to be reasonably free of contamination (i.e., that the
criteria are met as stipulated in Section 10.4.1). The LMB is used to ensure that
analytes of interest or other interferences are not present in the laboratory
environment, the solvent, or the apparatus.
NOTE: Good laboratory practices indicate the use of a blank before and after
analyzing a calibration curve for an instrument to ensure that no carryover will occur.
If the required criteria are not met and samples were not free of contamination, then
the source of the contamination should be identified and eliminated before the
performance of any analysis.
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10.2.2 INITIAL DEMONSTRATION OF PRECISION AND ACCURACY (P&A)
NOTE: Because porosity of the wiped surface will inevitably have an effect on analyte recovery from the
surface, accuracy results between calculated values and true values may differ from surface to surface.
The precision and accuracy results are based on the wipe used on the laminate (Formica®, Formica
Corp., Cincinnati, OH) surface because (1) the laminate surface has been shown to be free of
contamination, (2) this surface results in minimal surface interaction between the chemical and the
surface, and (3) the laminate is a relatively nonporous surface.
For a P&A, prepare a check standard containing DIMP, EMPA, MPA, PMPA,
EHDMAP, and IMPA near or below the midpoint concentration of the calibration
range. This check standard should be analyzed with a minimum of four replicates.
For this study, four different concentrations are chosen with seven samples each. The
check samples are analyzed according to Section 12.
10.2.3 The average percent recovery (X), standard deviations (a) and the percent relative
standard deviation (%RSD) of the recoveries are calculated for each analyte. The %
RPD limit of < 30% should be applied to all replicate analyses.
10.2.4 MINIMUM REPORTING LEVEL (MRL)
Establish a target concentration for the MRL based on the intended use of the
method. Establish an Initial Calibration (Section 11.2). The lowest CAL standard
used to establish the initial calibration must be at or below the MRL concentration. If
the MRL concentration is too low, ongoing QC requirements may fail repeatedly,
and the MRL must be determined again at a higher concentration. The MRL
reported in this study is the lowest calibration level. The MRL is validated following
the procedure below.
10.2.4.1 Fortify, extract, and analyze seven replicate LFBs at the proposed MRL
concentration. Calculate the mean measured concentration (Mean) and
standard deviation for these replicates. Determine the Half Range for the
prediction interval of results (HRPIR) using the equation below
HRPIR = 3.963s
where
s = the standard deviation
3.963 = a constant value for seven replicates (10).
10.2.4.2 Confirm that the upper and lower limits for the Prediction Interval of Result
(PIR = Mean j_ HRPIR) meet the upper and lower recovery limits as shown
below
The Upper PIR Limit must be <150% recovery.
Mean + HRP1R
FortifiedConcentration
The Lower PIR Limit must be > 50% recovery.
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Mean-HRP1R
FortifiedConcentration
10.2.5 CALIBRATION VERIFICATION
Mid-level and low-level samples from the calibration curve should be analyzed to
confirm the accuracy of the fit of the calibration curve/standards after the end of
sample batches.
10.3 METHOD DETECTION LIMITS (MDL)
The procedure for the determination of the laboratory detection and quantitation limits for
the EPA approach follows 40 CFR Part 136, Appendix B. MDLs represent the minimum
concentration at which there is a high degree of statistical confidence that, when the
method reports that an analyte is present, that analyte is actually present (i.e., a low risk
of false positives).
10.3.1 DETERMINATION OF LABORATORY INSTRUMENT DETECTION LIMITS
(IDLs)
The laboratory IDL can be used to establish an estimate of the initial spiking
concentration used for determination of the MDL, although other approaches for
determining the initial spiking concentration may be used. The laboratory IDL is
determined for each analyte as a concentration that produced an average signal-to-
noise (S/N) ratio in the range of 3:1 - 5:1 for at least three replicate injections. For
example, successively lower concentrations of the analytes are injected until the S/N
ratio is in the range of 3:1 - 5:1. Replicates are then injected at that target
concentration to ensure that the average S/N of the replicates was within the 3:1 - 5:1
range. Note that since linearity of S/N ratio with increasing or decreasing
concentration cannot be assumed, the concentrations determined via this procedure
are necessarily approximate.
10.3.2 DETERMINATION OF LABORATORY METHOD DETECTION LIMIT (MDL)
Method Detection Limits (MDLs) represent the optimal detection achieved by a
laboratory in a matrix of interest. The analyte spiking solution, containing all six
analytes, was added to the surface (section 19.3). The solution on the surface was
allowed to completely dry and wiped using a wetted-cotton gauze wipe. Wipe
extracts from the laminate coupons are used for the determination of the MDL for
surface samples. The 40 CFR Part 136, Appendix B procedure is followed,
particularly with regard to spike levels used. Replicate reference matrix samples are
spiked at a level between 1-5 times the estimated detection level (e.g., suggested by
the IDL procedure in 9.3.1). The resulting MDL must be within 10 times the spike
level used, or the MDL determination would be repeated using a more appropriate
spike level. Full method sample preparation procedures to prepare and analyze at
least seven replicates of the spiked clean matrix of interest are used. Apply the
following equation to the analytical results (Student's t-factor is dependent on the
number of replicates used; the value 3.14 assumes seven replicates):
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MDL =!(„_!,!_„ = 0.99) XSD
where
MDL = method detection limit
t = Student's t value for the 99% confidence level with n-1 degrees of
(n-l,l-a = 0.99)
freedom (for seven replicate determinations, the Student's t value is 3.143 at a 99%
confidence level),
n = number of replicates, and
SD = standard deviation of replicate analyses.
a = standard deviation of the percent recovery
Data for MDLs are shown in Table 3 and Section 19.1.
10.4 ONGOING QUALITY CONTROL (QC) REQUIREMENTS
10.4.1 LABORATORY FORTIFIED BLANK (LFB)
An LFB is required with each extraction batch to confirm that potential background
contaminants are not interfering with identification or quantitation of the target
analytes. If there is a contaminant within the retention time window preventing the
determination of the target analyte, the source of the contamination should be
determined and eliminated before processing samples. LFBs include cotton gauze
wipes wetted with water.
10.4.2 LABORATORY METHOD BLANK (LMB)
An LMB is prepared and analyzed with each extraction batch, using LC-MS grade
reagent water, for confirmation that there are no background contaminants interfering
with the identification or quantitation of the target analytes. If there is a contaminant
within the retention time window preventing the determination of the target analyte,
the source of the contamination should be determined and eliminated before
processing samples. LMBs include the extracted wipe used to wipe the surface
coupon.
10.4.3 CONTINUING CALIBRATION CHECK (CCC)
CCC standards are analyzed at the beginning and end of each analysis batch. The
CCC is analyzed periodically to verify the accuracy of the existing calibration for
analytes near the midpoint of the calibration range and/or near the MRL. CCC
values should be specified by the sample submitter's Data Quality Objectives
(DQOs) or fulfill other QC requirements, such as LFSM acceptance).
10.4.4 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM)
A LFSM is analyzed to determine that spike accuracy for a particular sample matrix
is not adversely affected by chemical interactions between target analytes and
experimental matrices (i.e., coupon/wipe materials). If a variety of sample matrices
are analyzed, performance should be established for each surface.
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10.4.4.1Within each analysis batch, an LFSM is prepared and analyzed at a frequency of one
sample matrix for every twenty samples. The LFSM is prepared by spiking a sample
with the appropriate amount of AS (Section 8.2.2). Select a spiking concentration that
is greater than or equal to the matrix background concentration, if known. Records
are maintained of the surface target compound spike analyses, and the average percent
recovery (X) and the standard deviation of the percent recovery (a) are calculated.
Analyte recoveries may exhibit bias for certain matrices. Acceptable recoveries are
50-150% if a low-level concentration near or at the MRL (within a factor of 3) is used.
If the recovery does not fall within this range, check with a CCC or prepare a fresh AS
solution for analysis. If the recovery of any analyte still falls outside the designated
range and the laboratory performance for that analyte is shown to be in control in the
CCCs, the recovery is judged to be matrix biased. The result for that analyte in the
unfortified sample is labeled suspect/matrix to inform the data user that the results are
suspect due to matrix effects.
10.4.5 SURROGATE STANDARD
All samples (CCCs, LFBs, LMBs, LFSMs, LFSMDs, FDs, and CAL standards) are
spiked with surrogate standard spiking solution as described in Section 8.2.1. An
average percent recovery of the surrogate compound and the standard deviation of
the percent recovery (REC) are calculated and updated regularly.
10.4.6 FIELD DUPLICATE (FD) OR LABORATORY FORTIFIED SAMPLE
MATRIX DUPLICATE (LFSMD)
Within each analysis batch, a minimum of one FD or LFSMD should be analyzed for
every twenty samples. Target compound spike accuracy in the sample matrix is
monitored and updated regularly. Duplicates check the precision associated with
sample collection, storage and laboratory procedures. Records are maintained of
spiked matrix analyses and the average percent recovery (X) and corresponding
standard deviation (a) are calculated. FD/LFSMD samples must be incorporated into
the field sampling plan. If the laboratory did not receive FD samples for
determination of site-specific P&A, the laboratory will evaluate the site data quality
based on the LFSM data, if there is sufficient sample in the site samples to conduct
an analysis. FD/LFSMD recovery results will be used for site-specific P&A data.
LFSM data are used as FD/LFSMD sample data for this study. RPD values should
be < 30% for FD/LFSMD samples.
10.4.6.1 Calculate the relative percent difference (RPD) for duplicate measurements
(FDj and FD2) using the equation:
FD, -FD9
RPD = 7
+FD2)/2
RPDs for Field Duplicates should be < 30% for each analyte. Greater variability
may be observed when Field Duplicates have analyte concentrations at or near
the MRL (within a factor of two times the MRL concentration). At these
concentrations, FDs must have RPDs that are < 50%. If the RPD of an analyte
falls outside the designated range and the laboratory performance for the analyte
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is shown to be in control in the CCC and in the LFB, the precision is judged
matrix influenced. Report the result for the corresponding analyte in the
unfortified sample as "suspect/matrix."
10.4.6.2 If an LFSMD is analyzed instead of an FD, calculate the RPD for the LFSM
and
LFSM-LFSMDl
using the RPD =
equation: (LFSM + LFSMD)/2
RPDs for duplicate LFSMs should be < 30% for each analyte. Greater
variability may be observed when the matrix is fortified at analyte
concentrations at or near the MRL (within a factor of two times the MRL
concentration). LFSMs at these concentrations must have RPDs that are < 50%.
If the RPD of an analyte falls outside the designated range and the laboratory
performance for the analyte is shown to be in control in the CCC and in the
LFB, the precision is judged matrix influenced. Report the result for the
corresponding analyte in the unfortified sample as "suspect/matrix."
11 INSTRUMENT CALIBRATION AND STANDARDIZATION
All laboratory equipment should be calibrated according to manufacturer's protocols. Demonstration
and documentation of acceptable mass spectrometer (MS) tuning and initial calibration is necessary
prior to sample analysis. Verification of the tuning of the MS must be repeated each time instrument
modification/maintenance is performed and prior to analyte calibration. After initial calibration is
successful, a CCC ( at the appropriate concentration described in section 10.4.2) should be performed
at the beginning and end of each analysis batch.
11.1 CALIBRATION OF MASS SPECTROMETER
Calibrate the mass scale of the mass spectrometer as prescribed by the manufacturer.
The mass calibration file is saved in the mass spectrometer software file folder
(MassLynx™ or similar software). The mass calibration solution used in this method is a
mixture of NaCsI provided by the manufacturer. Other calibration solutions can also be
used per instrument manufacturer's specifications.
11.2 INITIAL CALIBRATION FOR ANALYTES
11.2.1 ESI negative mode is the preferred choice for this method due to the optimal
conditions and advantages (e.g., greater peak intensity, few interferences, and
lower background) in ESI negative mode over ESI positive mode. However,
ESI positive mode may be used if matrix interferences become problematic. The
data are presented in both modes in some tables, but for clarification, only ESI
negative mode will be discussed in the method.
11.2.2 Optimize the [M-H]~ ion in ESI negative mode for each analyte by infusing an
appropriate calibration solution at a flow rate similar to that the flow rate used for
15
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the LC separation. Adjust MS parameters (voltages, temperatures, gas flows,
etc.) until optimal analyte responses are achieved. Optimize the product ion by
following the same procedures as for the [M-H]~ ion. Ensure that there are at
least 10 scans across the peak for optimal precision. ESI-MS and MS/MS
parameters utilized during development of this method are presented in Tables 4a
and 4b and 5.
11.2.3 Establish LC operating conditions that will optimize peak resolution and shape.
Suggested LC conditions (listed in Table 6) may not be optimal for all LC
systems.
11.2.4 The initial calibration contains a seven-point curve using the analyte
concentrations prepared in section 8.2.3 and shown in Table 7. The lowest
calibration curve standard must be at the MRL. The calibration curve and all
samples should be analyzed in a low to high concentration regimen so carryover
is less of a concern in case the LC cleaning cycle does not clean the system
adequately between injections. Verify that all analytes have been properly
identified and quantified using software programs. Integrate manually, if
necessary, in accordance with laboratory quality assurance plans Depending on
the instrument, sensitivity and calibration curve responses may vary. At a
minimum, a five-point linear or a six-point quadratic calibration curve will be
utilized for all analytes. If the polynomial type excludes the point of origin, use a
fit weighting of 1/X to give more weighting to the lower concentrations. The
coefficient of determination (r2) of the linear fit should be greater than or equal to
0.98. If one of the calibration standards other than the high or low standard
causes the r2 to be <0.98, this point must be re-injected or a new calibration
curve must be analyzed. If the low and/or high point is excluded, a six-point
curve is acceptable but the calibration range and reporting limits must be
modified to reflect this change. The r2 of the quadratic curve should be greater
than or equal to 0.99. If one of the calibration standards other than the high or
low standards causes the r2 to be <0.99, follow the same procedure given above
for a linear fit. A calibration curve and an instrument blank will be analyzed at
the beginning of each batch or daily to ensure instrument stability (9). When
quantitated, each calibration point for each analyte should calculate to be within
70-130% of its true value. The lowest CAL standard should calculate to be within
50-150% of its true value. A new curve will be generated daily. The calibration
method is used to quantify all samples.
11.3 QUANTITATION OF ANALYTES
The quantitation of the target analytes is accomplished with quantitation software as it
relates to each specific instrument (9). An external calibration is used along with
monitoring MPA-d3, PMPA-13C6 and DIMP-di4 surrogate recoveries. Refer to Tables 4a
and 4b for the MRM transitions and retention times utilized during the development of
this method.
12 ANALYTICAL PROCEDURE
12.1 SAMPLE PREPARATION
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12.1.1 Samples were collected and stored as described in Section 9. Surrogates (MPA-
d3, PMPA-13C6 and DIMP-d14) are added first, then LC-MS grade water (5 mL) is
added to the jar. Sonicate each jar containing the solution for approximately 15
minutes in a water bath at room temperature with no heat required.
12.1.2 After sonication, decant the extraction solvent into a 10 cc lock-tip sterile fitted
®
syringe with a Millex GV syringe driven filter unit, PVDF filter (0.22 jam),
transferring the filtered sample to a sterile 15-mL polypropylene tube (or
equivalent).
12.1.3 Transfer (via pipette) to a standard 2 mL sample vial.
NOTE: Calibration standards are not filtered through the syringe-driven filter units
because no particulates are present. The filters and syringes used in this study were
not shown to affect analyte concentrations. If alternate filtering is incorporated, the
filters should be subjected to QC requirements to ensure they do not introduce
interferences or retain the target analytes.
12.2 SAMPLE ANALYSIS/ANALYTICAL SEQUENCE
12.2.1 Use the same Liquid Chromatography/Mass Spectrometry conditions established
per guidance described in Section 11 and summarized in Tables 4a, 4b, 5 and 6.
12.2.2 Prepare an analytical batch that includes all QC samples and surface samples.
The first sample to be analyzed is a 10 uL injection of a blank (LC-MS grade
reagent water) on column followed by the calibration curve.
12.2.3 Update the calibration file and print a calibration report. Review the report for
calibration outliers and make area corrections by manual integration, if necessary
and appropriate. If corrections have been made, update the calibration file,
noting the changes, and regenerate a calibration report. Alternatively, re-analyze
"nonconforming" calibration level(s) and repeat the above procedures.
12.2.4 The first sample analyzed after the calibration curve is an additional blank (LC-
MS grade reagent water) to ensure there is no carryover (11). If the initial
calibration data are acceptable, begin analyzing samples, including QC and blank
samples, at their appropriate frequency injecting the same size aliquots (10 joL)
under the same conditions used to analyze CAL standards. The ending CCC
must have each analyte concentration within 30% of the calculated true
concentration or the affected analytes from that run must be qualified as
estimates or the samples must be re-analyzed with passing criteria to remove the
qualification.
12.2.5 If the absolute amount of a target compound exceeds the working range of the
LC-MS system (see Level 7 in Table 7), the prepared sample is diluted with
water and re-analyzed along with additional samples that may have run after the
sample known to exceed the calibration range, because of the possibility of
carryover. Care must be taken to ensure that there is no carryover of the analyte
that has exceeded the calibration range. If the amount of analyte exceeds the
calibration range, a blank sample should be analyzed afterward to demonstrate no
carryover will occur.
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12.2.6 At the conclusion of the data acquisition, use the same software that is used in the
calibration procedure to identify peaks of interest from the predetermined
retention time windows. Use the data software to examine the ion abundances of
the peaks in the chromatogram to identify and compare retention times in the
sample chromatogram with the retention time of the corresponding analyte peak
in an analyte standard.
13 DATA ANALYSIS AND CALCULATIONS
13.1 QUALITATIVE AND QUANTITATIVE ANALYSIS
13.1.1 Complete chromatographic resolution is not needed for accurate and precise
measurements of analyte concentrations when using MS/MS. An external
calibration is used when monitoring the MRM transitions of each analyte.
Quantitation software is utilized to conduct the quantitation of the target analytes
and surrogate standards. The MRM transitions of each analyte are used for
quantitation and confirmation. The MRM transition serves as a confirmation by
isolating the precursor ion, fragmenting the precursor ion to the product ion, and
relating the transition to the retention time in the calibration standard (9).
13.1.2 Computer programs used for analysis of data include instrumentation and
quantitation software. Manual integration may be necessary for some peak areas
if the peak area is not integrated properly (i.e., the integration for the peak is not
fully performed by the instrument's software, which will be noticeable by visual
inspection of each peak). Inspect all integrated peaks for visible integration
errors and manually integrate as necessary to ensure consistent integration of
other peaks and/or known calibration peaks. Any manual integration should be
carried out by a qualified analyst, noted, and checked against quality control
procedures (sections 10 and 11.3).
13.2 Prior to reporting data, the chromatogram should be reviewed for any incorrect peak
identifications. The retention time window of the MRM transitions must be within 5% of
the retention time of the analyte standard. If this is not true, the calibration curve needs to
be re-analyzed to see if there was a shift in retention times during the analysis and the
sample needs to be re-injected. If the retention time is still incorrect in the sample, the
analyte is referred to as an unknown. If peaks need to be manually adjusted due to incorrect
integration by the program, clarification of where professional judgment was used to alter
the peaks should be documented during the data reduction and verification process.
14 METHOD PERFORMANCE
14.1 PRECISION, ACCURACY AND DETECTION LIMITS
14.1.1 Tables for precision, accuracy and detection limit results for a single laboratory
study are presented in Sections 19.1 and 19.2 and Table 3.
14.2 RECOVERIES AND PRECISION FOR OTHER SURFACE TYPES
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14.2.1 Section 19.2 lists recoveries and precision of target analytes for a variety of other
surfaces.
14.3 WIPE STORAGE STABILITY STUDY
14.3.1 Extract storage was conducted on the laminate surface fortified with the targeted
method analytes. Precision and accuracy (n = 4) of the extracts were analyzed on
days 0, 2, 3, 7, 14, 21, and 30 days and are reported in Table 2.
14.4 PROBLEM ANALYTES AND SURFACES
14.4.1 TARGET ANALYTES ON UNCLEANED SURFACES
DIMP and some EHDMAP recoveries may be problematic due to the volatility or
rapid decomposition of these specific compounds (12). Analysts should be aware
that these two specific compounds may not be present within the tested sample
matrix and plan accordingly. EHDMAP detection limits in this method are based on
samples extracted within the same day. Due to the degradation of EHDMAP,
samples analyzed after 24 hours may reflect different results. Furthermore, ESI (+)
analysis results are problematic for certain compounds (e.g., IMPA and MPA) due to
possible electrospray enhancement/suppression effects, whereas ESI (-) results tend
to be more reliable. Both ESI (+) and (-) results are presented. However, detection
limits are based on ESI (-) data. Wood surfaces resulted in poor recoveries outside
the range of this procedure. As a result, the method should not be used to identify
these analytes on a wood surface. Although porosity of the surface is most likely the
culprit for low recoveries, further analysis should be performed to determine
definitive reasons for poor recoveries from the surface. Direct extraction of the
analytes from wood could be used to elucidate whether or not chemical interactions
are occurring between target analytes and compounds found in a wood matrix.
15 POLLUTION PREVENTION
15.1 This method utilizes small volumes of organic solvent and small quantities of pure analytes,
thereby minimizing the potential hazards to both analyst and environment.
15.2 For information about pollution prevention that may be applicable to laboratory operations,
consult "Less is Better: Laboratory Chemical Management for Waste Reduction" available
from the American Chemical Society's Department of Government Relations and Science
Policy, 1155 16th Street N.W., Washington, D.C., 20036 or on-line at
http://www.acs.org/content/dam/acsorg/about/governance/committees/chemicalsafety/public
ations/less-is-better.pdf (accessed August 15, 2013).
16 WASTE MANAGEMENT
16.1 The analytical procedures described in this procedure generate relatively small amounts of
waste since only small amounts of reagents and solvents are used. Laboratory waste
management practices must be conducted consistent with all applicable rules and
regulations, and laboratories should protect the air, water, and land by minimizing and
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controlling all releases from fume hoods and bench operations. Also, compliance with any
sewage discharge permits and regulations is required, particularly the hazardous waste
identification rules and land disposal restrictions.
16.2 Each laboratory should determine with federal and local officials how to safely dispose of
field and QC samples. Waste containers should be properly labeled to identify the
contents. Remember to attach the appropriate chemical waste label, date the beginning of
collection before using the container and follow all appropriate federal and local waste
disposal requirements.
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17 REFERENCES
1. U.S. Environmental Protection Agency (EPA), 2012. Selected Analytical Methods for
Environmental Restoration Following Homeland Security Events (SAM). EPA/600/R-12/555
July 2012. Cincinnati, Ohio: United States Environmental Protection Agency, Office of
Research and Development, National Homeland Security Research Center.
2. Code of Federal Regulations, 40 CFR Part 136, Appendix B. Definition and Procedure for the
Determination of the Method Detection Limit - Revision 1.11.
3. Federal Advisory Committee on Detection and Quantitation Approaches and Uses in Clean
Water Act Programs. Final Report. (Submitted to U.S. Environmental Protection Agency.)
December 28, 2007.
4. Glaser, J.A., D.L. Foerst, G.D. McKee, S.A. Quave, W.L. Budde, "Trace Analyses for
Wastewaters." Environ. Sci. Technol. 1981, 15, 1426-1435.
5. Standard Practices for Preparation of Sample Containers and for Preservation of Organic
Constituents, ASTM Annual Book of Standards, Part 31, D3694-78. Philadelphia: American
Society for Testing and Materials.
6. OSHA Safety and Health Standards, General Industry (29CRF1910). Occupational Safety and
Health Administration, OSHA 2206 (Revised, July 1, 2001).
7. Carcinogens-Working with Carcinogens, Publication No. 77-206. Atlanta, Georgia:
Department of Health, Education, and Welfare, Public Health Service, Center for Disease
Control, National Institute of Occupational Safety and Health, August 1977.
8. Safety in Academic Chemistry Laboratories, American Chemical Society Publication,
Committee on Chemical Safety, 7th Edition.
9. "Prudent Practices in the Laboratory: Handling and Disposal of Chemicals," National
Academies Press (1995), available at http://www.nap.edu (accessed August 15, 2013).
10. Winslow, S.D., Pepich, B.V., Martin, J.J., Hallberg, G.R, Munch, D.J., Frebis, C.P., Hedrick,
E.J., Krop, R.A. "Statistical Procedures for Determination and Verification of Minimum
Reporting Levels for Drinking Water Methods." Environ. Sci. Technol. 2006, 40, 281-288.
11. Peters, F.T.; Drummer, O.H.; Musshoff, F. "Validation of New Methods", Forensic Science
International 2007, 165 (2): 216-224
12. ASTM Standard D7597 - 09el, 2009 "Standard Test Method for Determination of Diisopropyl
Methylphosphonate, Ethyl Hydrogen Dimethylamidophosphate, Ethyl Methylphosphonic
Acid, Isopropyl Methylphosphonic Acid, Methylphosphonic Acid and Pinacolyl
Methylphosphonic Acid in Water by Liquid, Chromatography/Tandem Mass Spectrometry"
West Conshohocken, PA: ASTM International. 2009, DOI: 10.1520/D7597-09E01 ,
www.astm.org.
21
-------
18 TABLES AND VALIDATION DATA
Item Title
Table 1. Materials Tested for the Wipe Analysis of Nerve Agent Degradation Products 2
Table 2. Holding Time Sample Stability of Nerve Agent Degradation Analytes of Wipe Samples in ESI Negative Mode 2
Table 3. Method Parameters for Nerve Agent Degradation Products 3
Table 4a. ESI (+) MRM Ion Transitions, Retention Time (RT) and Variable Mass Spectrometer Parameters 4
Table 4b. ESI (-) MRM Ion Transitions, Retention Time (RT) and Variable Mass Spectrometer Parameters 4
Table 5. ESI (+) and (-) MS/MS Conditions 5
Table 6. Liquid Chromatography Gradient Conditions 5
Table 7. Target Concentrations of Calibration Standards Used During the Development of this Method (ng/mL) 6
-------
Table 1. Materials Tested for the Wipe Analysis of Nerve Agent Degradation Products
Material
Glass
Vinyl Tile
Laminate
Wood (southern pine, pre-treated)
Galvanized steel
Painted Drywall (BEHR® latex paint)
Manufacturer/Vendor
Carolina Glass Co./Lowe's
Armstrong/Home Depot
Wilsonart Laminate/Home Depot
Home Depot
McMaster-Carr
BEHR/Home Depot
Table 2. Holding Time Sample Stability of Nerve Agent Degradation Analytes of Wipe Samples in ESI Negative Mode
ESI (-) Mode
Concentration
ng/mL
Holding Time
(days)
0
2
3
7
14
21
30
IMPA
175
Average
%
Recovery
87.8
72.0
77.3
85.7
75.3
78.7
75.3
%
RSD
5
3
2
9
4
2
7
MPA
175
Average
%
Recovery
86.1
70.9
77.2
77.8
72.1
75.7
74.4
%
RSD
2
2
2
4
3
3
7
MPA-d3
175
Average
%
Recovery
78.9
72.7
73.8
75.6
71.6
72.3
73.5
%
RSD
3
1
4
3
3
4
7
EMPA
70
Average
%
Recovery
74.7
65.7
77.4
75.1
68.5
74.9
78.7
%
RSD
2
4
4
2
3
1
7
EHDMAP
175
Average
%
Recovery
106
24.4
20.8
28.2
29.5
33.4
48.8
%
RSD
3
2
3
15
3
3
54
PMPA
35
Average
%
Recovery
79.9
74.6
77.7
74.4
74.6
79.8
73.5
%
RSD
2
1
1
4
3
3
4
PMPA-13C6
175
Average
%
Recovery
79.5
71.3
75.4
74.4
74.8
70.6
72.6
%
RSD
2
1
1
1
2
2
4
RSD, relative standard deviation
-------
Table 3. Method Parameters for Nerve Agent Degradation Products
LAMINATE
Analyte
IMPA
EMPA
EHDMAP
MPA
PMPA
DIMP
MDL*
ng/cm2t
0.042
0.050
0.067
0.065
0.017
-
ng/mL
4.2
5.0
6.7
6.5
1.7
-
MRL
ng/mL
25
10
25
25
5
-
*Final DL Study-8/12. ESI" ionization mode provided the method detection limit (MDL) and minimum reporting level (MRL) values.
See section 19.1 for complete DL data in both ionization modes.
tng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area of the coupon (100 cm2).
-------
Table 4a. ESI (+) MRM Ion Transitions, Retention Time (RT) and Variable Mass Spectrometer
Parameters
Analyte
DIMP
IMPA
EMPA
EHDMAP
MPA
DIMP-d14
MPA-d3
Cone
voltage
22
22
26
28
45
24
48
MRM mass transition
(parent — > product)
1 81 . 33 -» 139.25
139.29^96.80
125.22^96.82
154.29^125.82
97.25 -> 79.20
195.45^147.20
100.20^82.00
Collision
energy (eV)
7
18
12
16
15
7
16
RT*
(minutes)
7.6
6.6
4.0
3.2
1.8
7.6
1.8
Retention times should fall within 5% of the given value; otherwise re-analysis may be necessary.
Table 4b. ESI (-) MRM Ion Transitions, Retention Time (RT) and Variable Mass Spectrometer
Parameters
Analyte
IMPA
EMPA
EHDMAP
PMPA
MPA
PMPA-13C6
MPA-d3
Cone
voltage
30
26
30
38
45
34
37
MRM mass transition
(parent — > product)
137.18^95.00
123.10^94.95
152.17^78.92
179.20^95.00
95.06^78.95
185.22^94.99
98.00^78.80
Collision
energy (eV)
18
12
12
18
15
18
15
RT*
(minutes)
6.6
4.0
3.2
8.7
1.8
8.7
1.8
Retention times should fall within 5% of the given value; otherwise re-analysis may be necessary.
-------
Table 5. ESI (+) and (-) MS/MS Conditions
MS Parameter
Capillary Voltage
Cone Voltage
Extractor
RF Lens
Source Temperature
Desolvation Temperature
Desolvation Gas Flow
Cone Gas Flow
Low Mass Resolution 1
High Mass Resolution 1
Ion Energy 1
Entrance Energy
Collision Energy
Exit Energy
Low Mass Resolution 2
High Mass resolution 2
Ion Energy 2
Multiplier
Gas Cell Pirani Gauge
Inter-Channel Delay
Inter-Scan Delay
Repeats
Span
Dwell
Setting
4.3 kV
See Table 4a and b
2 Volts
0.2 Volts
150°C
350 °C
600 L/hr
50 L/hr
14.5
14.5
0.5
1
See Table 4a and b
1
15.0
15.0
0.5
-560
3.0x10"J Torr
0.005 seconds
0.005 seconds
1
0.1 Daltons
0.15 Seconds
Table 6. Liquid Chromatography Gradient Conditions*
Time
(min)
0
4
5
9
10
12
13
15
Flow
(jjL/min)
300
300
300
300
300
300
300
300
%
Solution Af
100
100
55
55
40
30
100
100
%
Solution Bft
0
0
45
45
60
70
0
0
fA: Water (0.2% Formic Acid)
f fB: Acetonitrile (0.2% Formic Acid)
^Injection volume - 10 uL( recommended)
*Column Temperature: 30 ° C
*Autosampler Temperature: 15 °C
^Equilibration time: 2 minutes
*Column:, 100 mm x 2.1mm, 3|j,m particle size
-------
Table 7. Target Concentrations of Calibration
Method
Standards Used During the Development of this
(ng/mL)
Analyte/Surrogate
DIMP
IMPA
EMPA
EHDMAP
PMPA
MPA
DIMP-d14
PMPA-13C6
MPA-d3
Level
1
5
25
10
25
5
25
5
25
25
Level
2
10
50
20
50
10
50
10
50
50
Level
3
20
100
40
100
20
100
20
100
100
Level
4
35
175
70
175
35
175
35
175
175
Level
5
50
250
100
250
50
250
50
250
250
Level
6
100
500
200
500
100
500
100
500
500
Level
7
150
750
300
750
150
750
150
750
750
-------
19 ATTACHMENTS
19.1 Method Detection Limit Data and Calculations
19.2 Preci sion and Accuracy
19.3 Illustration depicting the wiping pattern on a 100 cm2 surface
7
-------
19.1
METHOD DETECTION LIMIT (MDL) DATA AND CALCULATIONS
MDL Data for Seven Replicates for Nerve Agent Degradation Analytes
Analyte
IMPA
MPA
EM PA
EHDMAP
DIMP
PMPA
MPA-d3
DIMP-d14
PMPA-13C6
Analyte
IMPA
MPA
EM PA
EHDMAP
DIMP
PMPA
MPA-d3
DIMP-d14
PMPA-13C6
LAMINATE in ESI (+)
mode
Concentration 1*
Average
Recovery
(ng/mL)
54.6
65.3
19.5
39.5
ND
-
88.8
16.8
-
Average
Recovery
(ng/cm2)t
0.546
0.653
0.195
0.395
ND
-
0.888
0.168
-
%
Recovery
91.1
109
81.1
65.9
ND
-
88.8
84.1
-
%
Recovery
91.1
109
81.1
65.9
ND
-
88.8
84.1
-
%
RSD
8
6
8
8
-
-
6
4
-
%
RSD
8
6
8
8
-
-
6
4
-
LAMINATE in ESI (-)
mode
Concentration 1*
Average
Recovery
(ng/mL)
44.7
48.2
20.3
39.3
-
9.9
84.6
-
87.1
Average
Recovery
(ng/cm2)t
0.447
0.482
0.203
0.393
-
0.100
0.846
-
0.871
%
Recovery
74.5
80.3
84.5
65.4
-
82.9
84.6
-
87.1
%
Recovery
74.5
80.3
84.5
65.4
-
82.9
84.6
-
87.1
%
RSD
3
4
8
5
-
5
5
-
1
%
RSD
3
4
8
5
-
5
5
-
1
^Concentration 1 correlates to the following analyte concentrations: 60 ng/mL for IMPA, MPA, and
EHDMAP, 24 ng/mL for EMPA, and 12 ng/mL for DIMP and PMPA. Surrogate recovery concentrations
correspond to the following: 100 ng/mL for MPA-d3and PMPA-13C6, and 20 ng/mL for DIMP-d14.
tng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area
of the coupon (100 cm2).
8
-------
MDL Calculation for Seven Replicates for Nerve Agent Degradation Analytes
LAMINATE in ESI (+) mode
Analyte
IMPA
MPA
EMPA
EHDMAP
DIMP
MDL
ng/cm2f
0.15
0.12
0.047
0.11
ND
ng/mL
15
12
4.7
11
ND
MRL
ng/mL
25
25
10
25
-
MDL, method detection limit; MRL, minimum reporting limit
tng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area
of the coupon (100 cm2).
LAMINATE in ESI (-) mode
Analyte
IMPA
MPA
EMPA
EHDMAP
PMPA
MDL
ng/cm2f
0.042
0.065
0.049
0.067
0.017
ng/mL
4.2
6.5
4.9
6.7
1.7
MRL
ng/mL
25
25
10
25
5
MDL, method detection limit; MRL, minimum reporting limit
tng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area
of the coupon (100 cm2).
-------
19.2 PRECISION AND ACCURACY
Concentration levels correspond to the following final concentrations on the surface: Concentration 1 is
the same as in Attachment 19.1 (60 ng/mL for IMPA, MPA, and EHDMAP, 24 ng/mL for EMPA, and 12
ng/mL for DIMP and PMPA. Surrogate recovery concentrations correspond to the following for
concentrations 1 and 2: 100 ng/mL for MPA-d3 and PMPA-13C6, and 20 ng/mL for DIMP-d14. Surrogate
recovery concentrations correspond to the following for concentrations 3 and 4: 300 ng/mL for MPA-d3
and PMPA-13C6, and 60 ng/mL for DIMP-di4). Concentration 2 is calibration concentration level 3.
Concentrations 3 and 4 are 2.25 and 3 times Concentration 2.
• Table A. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation
Analytes on Laminate Surfaces in ESI (+) Mode
• Table B. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation
Analytes on Laminate Surfaces in ESI (-) Mode
• Table C. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation
Analytes on Metal Surfaces in ESI (+) Mode
• Table D. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation
Analytes on Metal Surfaces in ESI (-) Mode
• Table E. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation
Analytes on Glass Surfaces in ESI (+) Mode
• Table F. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation
Analytes on Glass Surfaces in ESI (-) Mode
• Table G. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation
Analytes on Painted Drywall Surfaces in ESI (+) Mode
• Table H. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation
Analytes on Painted Drywall Surfaces in ESI (-) Mode
• Table I. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation
Analytes on Vinyl Tile Surfaces in ESI (+) Mode
• Table J. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation
Analytes on Vinyl Tile Surfaces in ESI (-) Mode
• Table K. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation
Analytes on Treated Wood Surfaces in ESI (+) and ESI (-) Mode
10
-------
P&A data for wipe analysis of nerve agent degradation analytes on surfaces.
Table A. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation Analytes on Laminate Surfaces in ESI (+)
LAMINATE in ESI (+) mode
Analyte
IMPA
MPA
EMPA
EHDMAP
DIMP
MPA-d3
DIMP-d14
Analyte
IMPA
MPA
EMPA
EHDMAP
DIMP
MPA-d3
DIMP-d14
Concentration 1
Average
Recovery
(ng/mL)*
54.6
65.3
19.5
39.5
ND
88.3
16.8
Average
Recovery
(ng/cm2)t
0.546
0.653
0.195
0.395
ND
0.883
0.168
%
Recovery
91.1
109
81.1
65.9
-
88.3
84.0
%
Recovery
91.1
109
81.1
65.9
-
88.8
84.0
%
RSD
8
6
8
8
-
3
4
%
RSD
8
6
8
8
-
3
4
Concentration 2
Average
Recovery
(ng/mL)*
97.6
115
40.6
72.6
ND
119
18.2
Average
Recovery
(ng/cm2)t
0.976
1.15
0.406
0.726
ND
1.19
0.182
%
Recovery
97.6
115
101
72.6
-
119
91.2
%
Recovery
97.6
115
101
72.6
-
119
91.2
%
RSD
10
8
7
9
-
15
13
%
RSD
10
8
7
9
-
15
13
Concentration 3
Average
Recovery
(ng/mL)*
219
277
90.9
127
ND
321
45.3
Average
Recovery
(ng/cm2)t
2.19
2.77
0.909
1.27
ND
3.21
0.453
%
Recovery
97.4
123
101
56.5
-
107
75.4
%
Recovery
97.4
123
101
56.5
-
107
75.4
%
RSD
6
5
4
6
-
8
8
%
RSD
6
5
4
6
-
8
8
Concentration 4
Average
Recovery
(ng/mL)*
283
263
106
210
ND
286
50.4
Average
Recovery
(ng/cm2)t
2.83
2.63
1.06
2.10
ND
2.86
0.504
%
Recovery
94.2
87.6
88.3
70.1
-
95.4
84.0
%
Recovery
94.2
87.6
88.3
70.1
-
95.4
84.0
%
RSD
18
5
3
9
-
2
8
%
RSD
18
5
3
9
-
2
8
Mode
Concentration 1 is for low concentration of analyte on the surface, See attachments 19.1-19.2 for values; Concentration 2 is calibration
concentration level 3; Concentrations 3 and 4 are 2.25 and 3 times Concentration 2; RSD is relative standard deviation
* (n = 7 samples at each concentration)
t ng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area of the coupon (100 cm2).
11
-------
Table B. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation Analytes on Laminate Surfaces in ESI (-) Mode
LAMINATE in ESI (-) mode
Analyte
IMPA
MPA
EMPA
EHDMAP
PMPA
MPA-d3
PMPA-13C6
Analyte
IMPA
MPA
EMPA
EHDMAP
PMPA
MPA-d3
PMPA-13C6
Concentration 1
Average
Recovery
(ng/mL)*
44.7
48.2
20.3
39.3
9.90
84.6
87.1
Average
Recovery
(ng/cm2)t
0.447
0.482
0.203
0.393
0.0990
0.846
0.871
%
Recovery
74.5
80.3
84.5
65.4
82.9
84.6
87.1
%
Recovery
74.5
80.3
84.5
65.4
82.9
84.6
87.1
%
RSD
3
4
8
5
5
5
1
%
RSD
3
4
8
5
5
5
1
Concentration 2
Average
Recovery
(ng/mL)*
69.2
58.3
38.2
71.4
18.1
68.7
94.6
Average
Recovery
(ng/cm2)t
0.692
0.583
0.382
0.714
0.181
0.687
0.946
%
Recovery
69.2
58.3
95.4
71.4
90.2
68.7
94.6
%
Recovery
69.2
58.3
95.4
71.4
90.2
68.7
94.6
%
RSD
9
6
7
10
3
7
14
%
RSD
9
6
7
10
3
7
14
Concentration 3
Average
Recovery
(ng/mL)*
146
132
86.5
128
41.5
170
245
Average
Recovery
(ng/cm2)t
1.46
1.32
0.865
1.28
0.415
1.70
2.45
%
Recovery
64.8
58.5
96.1
56.9
92.2
56.7
81.5
%
Recovery
64.8
58.5
96.1
56.9
92.2
56.7
81.5
%
RSD
5
5
4
7
5
3
4
%
RSD
5
5
4
7
5
3
4
Concentration 4
Average
Recovery
(ng/mL)*
234
243
108
217
52.3
261
276
Average
Recovery
(ng/cm2)t
2.34
2.43
1.08
2.17
0.523
2.61
2.76
%
Recovery
77.8
80.9
89.9
72.5
87.2
87.0
92.1
%
Recovery
77.8
80.9
89.9
72.5
87.2
87.0
92.1
%
RSD
1
3
2
5
3
2
2
%
RSD
1
3
2
5
3
2
2
Concentration 1 is for low concentration of analyte on the surface, See attachments 19.1-19.2 for values; Concentration 2 is calibration
concentration level 3; Concentrations 3 and 4 are 2.25 and 3 times Concentration 2; RSD is relative standard deviation
* (n = 7 samples at each concentration)
t ng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area of the coupon (100 cm2).
12
-------
Table C. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation Analytes on Metal Surfaces in ESI (+) Mode
METAL in ESI (+) mode
Analyte
IMPA
MPA
EMPA
EHDMAP
DIMP
MPA-d3
DIMP-d14
Analyte
IMPA
MPA
EMPA
EHDMAP
DIMP
MPA-d3
DIMP-d14
Concentration 1
Average
Recovery
(ng/mL)*
65.1
50.0
19.6
16.8
ND
142
18.1
Average
Recovery
(ng/cm2)t
0.651
0.500
0.196
0.168
ND
1.42
0.181
%
Recovery
108
83.3
81.6
28.0
-
142
90.5
%
Recovery
108
83.3
81.6
28.0
-
142
90.5
%
RSD
20
11
12
29
-
4
6
%
RSD
20
11
12
29
-
4
6
Concentration 2
Average
Recovery
(ng/mL)*
162
86.8
32.5
71.5
ND
106
19.1
Average
Recovery
(ng/cm2)t
1.62
0.868
0.325
0.715
ND
1.06
0.191
%
Recovery
162
86.8
81.2
71.5
-
106
95.4
%
Recovery
162
86.8
81.2
71.5
-
106
95.4
%
RSD
17
15
6
14
-
6
5
%
RSD
17
15
6
14
-
6
5
Concentration 3
Average
Recovery
(ng/mL)*
353
182
74.8
138
ND
288
55.1
Average
Recovery
(ng/cm2)t
3.53
1.82
0.748
1.38
ND
2.88
0.551
%
Recovery
157
80.9
83.1
61.4
-
96.1
91.8
%
Recovery
157
80.9
83.1
61.4
-
96.1
91.8
%
RSD
16
17
4
30
-
3
4
%
RSD
16
17
4
30
-
3
4
Concentration 4
Average
Recovery
(ng/mL)*
236
213
85.7
75.9
ND
210
44.2
Average
Recovery
(ng/cm2)t
2.36
2.13
0.857
0.759
ND
2.10
0.442
%
Recovery
78.7
71.0
71.4
25.3
-
69.8
73.6
%
Recovery
78.7
71.0
71.4
25.3
-
69.8
73.6
%
RSD
9
13
7
55
-
5
5
%
RSD
9
13
7
55
-
5
5
Concentration 1 is for low concentration of analyte on the surface, See attachments 19.1-19.2 for values; Concentration 2 is calibration
concentration level 3; Concentrations 3 and 4 are 2.25 and 3 times Concentration 2; RSD is relative standard deviation
* (n = 7 samples at each concentration)
t ng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area of the coupon (100 cm2).
13
-------
Table D. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation Analytes on Metal Surfaces in ESI (-) Mode
METAL in ESI (-) mode
Analyte
IMPA
MPA
EMPA
EHDMAP
PMPA
MPA-d3
PMPA-13C6
Analyte
IMPA
MPA
EMPA
EHDMAP
PMPA
MPA-d3
PMPA-13C6
Concentration 1
Average
Recovery
(ng/mL)*
42.7
28.2
20.0
13.0
8.80
89.0
66.9
Average
Recovery
(ng/cm2)t
0.427
0.282
0.200
0.130
0.0880
0.890
0.669
%
Recovery
71.2
47.0
83.4
21.7
73.0
89.0
66.9
%
Recovery
71.2
47.0
83.4
21.7
73.0
89.0
66.9
%
RSD
8
9
8
83
6
11
3
%
RSD
8
9
8
83
6
11
3
Concentration 2
Average
Recovery
(ng/mL)*
65.3
58.1
35.6
75.6
15.7
67.1
89.8
Average
Recovery
(ng/cm2)t
0.653
0.581
0.356
0.756
0.157
0.671
0.898
%
Recovery
65.3
58.1
89.0
75.6
78.6
67.1
89.8
%
Recovery
65.3
58.1
89.0
75.6
78.6
67.1
89.8
%
RSD
8
12
7
16
4
9
4
%
RSD
8
12
7
16
4
9
4
Concentration 3
Average
Recovery
(ng/mL)*
127
119
76.1
142
35.6
185
266
Average
Recovery
(ng/cm2)t
1.27
1.19
0.761
1.42
0.356
1.85
2.66
%
Recovery
56.2
52.8
84.6
62.9
79.2
61.5
88.6
%
Recovery
56.2
52.8
84.6
62.9
79.2
61.5
88.6
%
RSD
2
10
3
35
4
7
3
%
RSD
2
10
3
35
4
7
3
Concentration 4
Average
Recovery
(ng/mL)*
245
172
96.8
87.8
45.8
185
203
Average
Recovery
(ng/cm2)t
2.45
1.72
0.968
0.878
0.458
1.85
2.03
%
Recovery
81.8
57.3
80.7
29.3
76.3
61.8
67.6
%
Recovery
81.8
57.3
80.7
29.3
76.3
61.8
67.6
%
RSD
4
12
4
48
5
5
2
%
RSD
4
12
4
48
5
5
2
Concentration 1 is for low concentration of analyte on the surface, See attachments 19.1-19.2 for values; Concentration 2 is calibration
concentration level 3; Concentrations 3 and 4 are 2.25 and 3 times Concentration 2; RSD is relative standard deviation
* (n = 7 samples at each concentration)
t ng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area of the coupon (100 cm2).
14
-------
Table E. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation Analytes on Glass Surfaces in ESI (+) Mode
GLASS in ESI (+) mode
Analyte
IMPA
MPA
EMPA
EHDMAP
DIMP
MPA-d3
DIMP-d14
Analyte
IMPA
MPA
EMPA
EHDMAP
DIMP
MPA-d3
DIMP-d14
Concentration 1
Average
Recovery
(ng/mL)*
141
71.4
25.0
44.1
ND
172
17.1
Average
Recovery
(ng/cm2)t
1.41
0.714
0.250
0.441
ND
1.72
0.171
%
Recovery
234
119
104
73.4
-
172
85.7
%
Recovery
234
119
104
73.4
-
172
85.7
%
RSD
13
11
5
6
-
6
10
%
RSD
13
11
5
6
-
6
10
Concentration 2
Average
Recovery
(ng/mL)*
210
105
33.4
121
ND
111
17.4
Average
Recovery
(ng/cm2)t
2.10
1.05
0.334
1.21
ND
1.11
0.174
%
Recovery
210
105
83.5
121
-
111
86.8
%
Recovery
210
105
83.5
121
-
111
86.8
%
RSD
10
7
7
4
-
10
11
%
RSD
10
7
7
4
-
10
11
Concentration 3
Average
Recovery
(ng/mL)*
292
253
84.0
217
ND
286
46.6
Average
Recovery
(ng/cm2)t
2.92
2.53
0.840
2.17
ND
2.86
0.466
%
Recovery
129
112
93.3
96.5
-
95.3
111
%
Recovery
129
112
93.3
96.5
-
95.3
111
%
RSD
31
4
6
5
-
5
8
%
RSD
31
4
6
5
-
5
8
Concentration 4
Average
Recovery
(ng/mL)*
444
347
113
270
ND
275
43.2
Average
Recovery
(ng/cm2)t
4.44
3.47
1.13
2.70
ND
2.75
0.432
%
Recovery
148
116
94.4
90.1
-
91.8
72.0
%
Recovery
148
116
94.4
90.1
-
91.8
72.0
%
RSD
8
4
2
3
-
2
7
%
RSD
8
4
2
3
-
2
7
Concentration 1 is for low concentration of analyte on the surface, See attachments 19.1-19.2 for values; Concentration 2 is calibration
concentration level 3; Concentrations 3 and 4 are 2.25 and 3 times Concentration 2; RSD is relative standard deviation
* (n = 7 samples at each concentration)
t ng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area of the coupon (100 cm2).
15
-------
Table F. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation Analytes on Glass Surfaces in ESI (-) Mode
GLASS in ESI (-) mode
Analyte
IMPA
MPA
EMPA
EHDMAP
PMPA
MPA-d3
PMPA-13C6
Analyte
IMPA
MPA
EMPA
EHDMAP
PMPA
MPA-d3
PMPA-13C6
Concentration 1
Average
Recovery
(ng/mL)*
40.4
33.1
23.1
28.2
10.7
99.2
139
Average
Recovery
(ng/cm2)t
0.404
0.331
0.231
0.282
0.107
0.992
1.39
%
Recovery
67.4
55.2
96.0
47.1
88.8
99.2
139
%
Recovery
67.4
55.2
96.0
47.1
88.8
99.2
139
%
RSD
7
9
9
18
3
2
4
%
RSD
7
9
9
18
3
2
4
Concentration 2
Average
Recovery
(ng/mL)*
51.6
65.5
32.6
113
10.8
62.4
96.3
Average
Recovery
(ng/cm2)t
0.516
0.655
0.326
1.13
0.108
0.624
0.963
%
Recovery
51.6
65.5
81.4
113
54.0
62.4
96.3
%
Recovery
51.6
65.5
81.4
113
54.0
62.4
96.3
%
RSD
2
8
9
12
4
15
7
%
RSD
2
8
9
12
4
15
7
Concentration 3
Average
Recovery
(ng/mL)*
131
145
83.2
225
39.4
169
247
Average
Recovery
(ng/cm2)t
1.31
1.45
0.832
2.25
0.394
1.69
2.47
%
Recovery
58.4
64.3
92.4
100
87.5
56.2
82.3
%
Recovery
58.4
64.3
92.4
100
87.5
56.2
82.3
%
RSD
9
8
2
23
3
8
6
%
RSD
9
8
2
23
3
8
6
Concentration 4
Average
Recovery
(ng/mL)*
163
185
117
307
54.8
149
240
Average
Recovery
(ng/cm2)t
1.63
1.85
1.17
3.07
0.548
1.49
2.40
%
Recovery
54.3
61.5
97.3
102
91.4
49.7
79.9
%
Recovery
54.3
61.5
97.3
102
91.4
49.7
79.9
%
RSD
6
7
5
6
2
9
3
%
RSD
6
7
5
6
2
9
3
Concentration 1 is for low concentration of analyte on the surface, See attachments 19.1-19.2 for values; Concentration 2 is calibration
concentration level 3; Concentrations 3 and 4 are 2.25 and 3 times Concentration 2; RSD is relative standard deviation
* (n = 7 samples at each concentration)
t ng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area of the coupon (100 cm2).
16
-------
Table G. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation Analytes on Painted Drywall Surfaces in ESI (+)
Mode
PAINTED DRYWALL in ESI (+) mode
Analyte
IMPA
MPA
EMPA
EHDMAP
DIMP
MPA-d3
DIMP-d14
Analyte
IMPA
MPA
EMPA
EHDMAP
DIMP
MPA-d3
DIMP-d14
Concentration 1
Average
Recovery
(ng/mL)*
40.3
59.0
18.9
33.9
ND
114
14.6
Average
Recovery
(ng/cm2)t
0.403
0.590
0.189
0.339
ND
1.14
0.146
%
Recovery
67.1
98.3
78.7
56.6
-
114
73.1
%
Recovery
67.1
98.3
78.7
56.6
-
114
73.1
%
RSD
6
28
14
10
-
7
13
%
RSD
6
28
14
10
-
7
13
Concentration 2
Average
Recovery
(ng/mL)*
67.0
110
32.5
50.0
ND
123
16.2
Average
Recovery
(ng/cm2)t
0.670
1.10
0.325
0.500
ND
1.23
0.162
%
Recovery
67.0
110.1
81.2
50.0
-
123
81.1
%
Recovery
67.0
110
81.2
50.0
-
123
81.1
%
RSD
3
11
7
6
-
8
9
%
RSD
3
11
7
6
-
8
9
Concentration 3
Average
Recovery
(ng/mL)*
184
223
64.1
163
ND
282
45.7
Average
Recovery
(ng/cm2)t
1.84
2.23
0.641
1.63
ND
2.82
0.457
%
Recovery
81.5
99.1
71.3
72.3
-
94.0
76.1
%
Recovery
81.5
99.1
71.3
72.3
-
94.0
76.1
%
RSD
6
5
3
3
-
7
8
%
RSD
6
5
3
3
-
7
8
Concentration 4
Average
Recovery
(ng/mL)*
236
285
78.6
231
ND
299
47.5
Average
Recovery
(ng/cm2)t
2.36
2.85
0.786
2.31
ND
2.99
0.475
%
Recovery
78.5
95.0
65.5
77.0
-
99.7
79.2
%
Recovery
78.5
95.0
65.5
77.0
-
99.7
79.2
%
RSD
3
8
5
2
-
8
9
%
RSD
3
8
5
2
-
8
9
Concentration 1 is for low concentration of analyte on the surface, See attachments 19.1-19.2 for values; Concentration 2 is calibration
concentration level 3; Concentrations 3 and 4 are 2.25 and 3 times Concentration 2; RSD is relative standard deviation
* (n = 7 samples at each concentration)
t ng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area of the coupon (100 cm2).
17
-------
Table H. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation Analytes on Painted Drywall Surfaces in ESI (-)
Mode
PAINTED DRYWALL in ESI (-) mode
Analyte
IMPA
MPA
EMPA
EHDMAP
PMPA
MPA-d3
PMPA-13C6
Analyte
IMPA
MPA
EMPA
EHDMAP
PMPA
MPA-d3
PMPA-13C6
Concentration 1
Average
Recovery
(ng/mL)*
44.9
22.4
20.4
31.5
13.2
75.6
81.6
Average
Recovery
(ng/cm2)t
0.449
0.224
0.204
0.315
0.132
0.756
0.816
%
Recovery
74.8
37.4
85.2
52.6
110
75.6
81.6
%
Recovery
74.8
37.4
85.2
52.6
110
75.6
81.6
%
RSD
12
13
5
8
6
6
4
%
RSD
12
13
5
8
6
6
4
Concentration 2
Average
Recovery
(ng/mL)*
59.6
33.2
28.6
49.9
13.2
68.0
81.6
Average
Recovery
(ng/cm2)t
0.596
0.332
0.286
0.499
0.132
0.680
0.816
%
Recovery
59.6
33.2
71.5
49.9
65.8
68.0
81.6
%
Recovery
59.6
33.2
71.5
49.9
65.8
68.0
81.6
%
RSD
6
16
9
5
6
9
4
%
RSD
6
16
9
5
6
9
4
Concentration 3
Average
Recovery
(ng/mL)*
155
142
62.3
155
33.7
205
255
Average
Recovery
(ng/cm2)t
1.55
1.42
0.623
1.55
0.337
2.05
2.55
%
Recovery
68.8
63.1
69.3
68.8
74.9
68.3
85.0
%
Recovery
68.8
63.1
69.3
68.8
74.9
68.3
85.0
%
RSD
4
3
3
3
2
4
3
%
RSD
4
3
3
3
2
4
3
Concentration 4
Average
Recovery
(ng/mL)*
216
180
74.1
224
45.4
200
257
Average
Recovery
(ng/cm2)t
2.16
1.80
0.741
2.24
0.454
2.00
2.57
%
Recovery
72.1
30.1
61.7
74.7
75.7
66.6
85.8
%
Recovery
72.1
30.1
61.7
74.7
75.7
66.6
85.8
%
RSD
4
5
2
3
2
7
3
%
RSD
4
5
2
3
2
7
3
Concentration 1 is for low concentration of analyte on the surface, See attachments 19.1-19.2 for values; Concentration 2 is calibration
concentration level 3; Concentrations 3 and 4 are 2.25 and 3 times Concentration 2; RSD is relative standard deviation
* (n = 7 samples at each concentration)
t ng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area of the coupon (100 cm2).
18
-------
Table I. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation Analytes on Vinyl Tile Surfaces in ESI (+) Mode
VINYL TILE in ESI (+) mode
Analyte
IMPA
MPA
EMPA
EHDMAP
DIMP
MPA-d3
DIMP-d14
Analyte
IMPA
MPA
EMPA
EHDMAP
DIMP
MPA-d3
DIMP-d14
Concentration 1
Average
Recovery
(ng/mL)*
57.3
87.5
22.2
42.2
ND
92.0
15.0
Average
Recovery
(ng/cm2)t
0.573
0.875
0.222
0.422
ND
0.920
0.150
%
Recovery
95.5
146
92.6
70.4
-
92.0
75.1
%
Recovery
95.5
146
92.6
70.4
-
92.0
75.1
%
RSD
16
21
6
7
-
9
8
%
RSD
16
21
6
7
-
9
8
Concentration 2
Average
Recovery
(ng/mL)*
95.0
108
32.8
53.6
ND
94.8
15.4
Average
Recovery
(ng/cm2)t
0.950
1.08
0.328
0.536
ND
0.948
0.154
%
Recovery
95.0
108
81.9
53.6
-
94.8
76.8
%
Recovery
95.0
108
81.9
53.6
-
94.8
76.8
%
RSD
26
8
13
7
-
8
7
%
RSD
26
8
13
7
-
8
7
Concentration 3
Average
Recovery
(ng/mL)*
191
136
62.9
164
ND
228
52.4
Average
Recovery
(ng/cm2)t
1.91
1.36
0.629
1.64
ND
2.28
0.524
%
Recovery
84.8
60.6
69.9
73.0
-
76.1
87.4
%
Recovery
84.8
60.6
69.9
73.0
-
76.1
87.4
%
RSD
9
13
3
3
-
2
4
%
RSD
9
13
3
3
-
2
4
Concentration 4
Average
Recovery
(ng/mL)*
290
208
111
231
ND
250
44.7
Average
Recovery
(ng/cm2)t
2.90
2.08
1.11
2.31
ND
2.50
0.447
%
Recovery
96.8
69.4
92.4
76.9
-
83.3
74.5
%
Recovery
96.8
69.4
92.4
76.9
-
83.3
74.5
%
RSD
11
6
5
5
-
2
10
%
RSD
11
6
5
5
-
2
10
Concentration 1 is for low concentration of analyte on the surface, See attachments 19.1-19.2 for values; Concentration 2 is calibration
concentration level 3; Concentrations 3 and 4 are 2.25 and 3 times Concentration 2; RSD is relative standard deviation
* (n = 7 samples at each concentration)
t ng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area of the coupon (100 cm2).
19
-------
Table J. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation Analytes on Vinyl Tile Surfaces in ESI (-) Mode
VINYL TILE in ESI (-) mode
Analyte
IMPA
MPA
EMPA
EHDMAP
PMPA
MPA-d3
PMPA-13C6
Analyte
IMPA
MPA
EMPA
EHDMAP
PMPA
MPA-d3
PMPA-13C6
Concentration 1
Average
Recovery
(ng/mL)*
47.0
41.1
19.0
47.9
8.2
86.9
81.1
Average
Recovery
(ng/cm2)t
0.470
0.411
0.190
0.479
0.0820
0.869
0.811
%
Recovery
78.4
68.5
79.2
79.8
68.5
86.9
81.1
%
Recovery
78.4
68.5
79.2
79.8
68.5
86.9
81.1
%
RSD
12
10
11
10
4
8
4
%
RSD
12
10
11
10
4
8
4
Concentration 2
Average
Recovery
(ng/mL)*
66.9
69.7
27.3
59.1
12.2
80.6
81.8
Average
Recovery
(ng/cm2)t
0.669
0.697
0.273
0.591
0.122
0.806
0.818
%
Recovery
66.9
69.7
68.2
59.1
60.8
80.6
81.8
%
Recovery
66.9
69.7
68.2
59.1
60.8
80.6
81.8
%
RSD
5
7
13
11
6
11
6
%
RSD
5
7
13
11
6
11
6
Concentration 3
Average
Recovery
(ng/mL)*
161
119
65.8
168
33.5
217
250.0
Average
Recovery
(ng/cm2)t
1.61
1.19
0.658
1.68
0.335
2.17
2.50
%
Recovery
71.8
52.9
73.2
74.7
74.4
72.3
83.3
%
Recovery
71.8
52.9
73.2
74.7
74.4
72.3
83.3
%
RSD
2
6
3
3
3
3
3
%
RSD
2
6
3
3
3
3
3
Concentration 4
Average
Recovery
(ng/mL)*
211
165
113
242
43.8
225
266
Average
Recovery
(ng/cm2)t
2.11
1.65
1.13
2.42
0.438
2.25
2.66
%
Recovery
70.4
55.1
94.3
80.6
73.0
74.9
88.7
%
Recovery
70.4
55.1
94.3
80.6
73.0
74.9
88.7
%
RSD
3
4
4
4
3
4
3
%
RSD
3
4
4
4
3
4
3
Concentration 1 is for low concentration of analyte on the surface, See attachments 19.1-19.2 for values; Concentration 2 is calibration
concentration level 3; Concentrations 3 and 4 are 2.25 and 3 times Concentration 2.
RSD is relative standard deviation
* (n = 7 samples at each concentration)
t ng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area of the coupon (100 cm2).
20
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Table K. Precision and Accuracy Data for Wipe Analysis of Nerve Agent Degradation Analytes on Treated Wood Surfaces in ESI (+) and
ESI (-) Mode
Analyte
IMPA
MPA
EM PA
EHDMAP
DIMP
PMPA
MPA-d3
DIMP-d14
PMPA-13C6
Analyte
IMPA
MPA
EM PA
EHDMAP
DIMP
PMPA
MPA-d3
DIMP-d14
PMPA-13C6
TREATED WOOD in ESI (+) mode
Concentration 4
Average Recovery
(ng/ml_)*
20.9
5.70
ND
8.90
ND
-
257
55.5
-
Average Recovery
(ng/cm2)t
0.209
0.570
ND
0.0890
ND
-
2.57
0.555
-
% Recovery
7.00
1.90
ND
3.00
-
-
85.6
92.6
-
% Recovery
7.0
1.9
-
3.0
-
-
85.6
92.6
-
% RSD
17
121
-
14
-
-
5
7
-
% RSD
17
121
-
14
-
-
5
7
-
TREATED WOOD in ESI (-) mode
Concentration 4
Average Recovery
(ng/ml_)*
12.3
10.7
ND
8.1
ND
1.8
238
-
277
Average Recovery
(ng/cm2)t
0.123
0.107
ND
0.0810
ND
0.0180
2.38
-
2.77
% Recovery
4.10
3.60
ND
2.70
-
3.00
79.5
-
92.3
% Recovery
4.1
3.6
-
2.7
-
3.0
79.5
-
92.3
% RSD
9
16
-
21
-
16
8
-
3
% RSD
9
16
-
21
-
16
8
-
3
Concentration 1 is for low concentration of analyte on the surface, See attachments 19.1-19.2 for values; Concentration 2 is calibration
concentration level 3; Concentrations 3 and 4 are 2.25 and 3 times Concentration 2. RSD is relative standard deviation
* (n = 7 samples at each concentration)
t ng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area of the coupon (100 cm2).
21
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19.3 Illustration of wiping pattern on 100 cm surface
The analyte spike solution, containing the six analytes of interest, was added to the surface as shown in
19.3, allowed to completely dry (approximately 60-90 minutes depending on droplet size), and wiped
using wetted-cotton gauze wipes.
22
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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
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