EPA 600/R-11/143 | November 2011 | www.epa.gov/ord
United States
Environmental Protection
Agency
] National Institute for
f Occupational Safety and Health
Surface Analysis Using
Wipes for the Determination
of Nitrogen Mustard
Degradation Products by Liquid
Chromatography/Tandem Mass
Spectrometry (LC/MS/MS)
SAMPLING AND ANALYTICAL
PROCEDURE FOR ANALYSIS OF
SURFACES USING WIPES
REVISION 2
Office of Research and Development
National Homeland Security Research Center
Centers for Disease Control and Prevention
National Institute for Occupational Safety and Health
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EPA/600/R-11/143
SURFACE ANALYSIS USING WIPES FOR THE DETERMINATION OF NITROGEN
MUSTARD DEGRADATION PRODUCTS BY LIQUID
CHROMATOGRAPHY/TANDEM MASS SPECTROMETRY (LC/MS/MS)
Sampling and Analytical Procedure for Analysis of Surfaces Using Wipes
Revision 2
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: 3/11
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DISCLAIMER
The information in this document is a single-laboratory-developed sampling and analytical procedure
(SAP) that has been funded wholly or in part by the U.S. Environmental Protection Agency (EPA) and in
collaboration with the National Homeland Security Research Center, part of EPA's Office of Research
and Development, and the National Institute of Occupational Safety and Health (NIOSH), a division of
the U.S. Department of Health and Human Services (DHHS), under IA #DW-75-922440001-0. The
method development and document preparation were supported under contract number EP08C000010.
This document has been subjected to the Agency's review and has been approved for publication. Note
that approval does not signify that the contents necessarily reflect the views of the Agency. NIOSH and
EPA do not endorse the purchase or sale of any commercial products or services.
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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FOREWORD
The National Homeland Security Research Center (NHSRC), part of U.S. EPA's Office of Research and
Development (ORD), is focused on developing and delivering scientifically sound, reliable, and
responsive products. These products are designed to address homeland security information gaps and
research needs that support the Agency's mission of protecting public health and the environment. A
portion of NHSRC's research is directed at decontamination of indoor surfaces, outdoor areas, and water
infrastructure. This research is conducted as part of EPA's response to chemical, biological, and
radiological (CBR) contamination incidents. NHSRC has been charged with delivering tools and
methodologies (e.g. sampling and analytical methods, sample collection protocols) that enable the rapid
characterization of indoor and outdoor areas, and water systems following terrorist attacks, and more
broadly, natural and manmade disasters.
The Selected Analytical Methods for Environmental Remediation and Recovery (SAM) document is a
compendium of methods that informs sample collection and analysis during the response to an incident.
SAM can be used by public and private laboratories which are analyzing a large number of samples
associated with chemical, biological, or radiological contamination. Even though some of the analytes in
SAM already have existing analytical methods, others are in need of improvements that enhance
analytical capability and meet more rigorous performance criteria. Furthermore, not all of the analytical
methods listed in the SAM document address all possible matrices (e.g., water, soil, air, glass)
encountered in sample collection following an incident. The analytical methods in SAM have been
verified in a single laboratory, but most still need to undergo multi-laboratory validation with respect to a
specific contaminant in association with a specific matrix.
The single-laboratory-developed Sampling and Analytical Procedure, described herein, demonstrates the
procedure for analysis of nitrogen mustard degradation products on surfaces using wipes by liquid
chromatography/tandem mass spectrometry (LC/MS/MS). A companion study report (Companion
Document for Surface Analysis Using Wipes for the Determination of Nitrogen Mustard Degradation
Products by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS)), describes the
development efforts, a synopsis of the supporting data collected, and scientific justifications for the
decisions made. NHSRC welcomes your comments as we move one step closer to achieving our
homeland security mission, and our overall mission of protecting human health and the environment.
Jon Herrmann,
Director National Homeland Security Research Center
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ACKNOWLEDGMENTS
Contributions of the following individuals and organizations to the development and review of this
method are acknowledged.
United States Environmental Protection Agency (EPA)
Office of Research and Development, National Homeland Security Research Center
Stuart Willison, Project Officer and Method Development
Erin Silvestri, Project Officer
EPA Regions
Lawrence Zintek (Region 5), Technical Reviewer
Lukas Oudejans (RTP), Technical Reviewer
Lawrence Kaelin (OSWER), Technical Reviewer
Centers for Disease Control and Prevention
National Institute for Occupational Safety and Health
Jack Pretty
Robert Streicher
IV
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EXECUTIVE SUMMARY
Wipe sampling can be performed quickly and easily when direct extraction is not always possible (e.g.,
walls, floors and furniture). As a result, wipe sampling is preferred for analysis without destruction of the
tested surface. However, wipe sampling can remove analytes only from the surface of a material, which
could result in lower recoveries and produce less reliable quantitative data from porous surfaces. It is
therefore important to understand wipe efficiencies and the materials being wiped. This procedure
assesses the recoveries from various porous and nonporous surfaces to determine the presence of nitrogen
mustard degradation products. Wipes were analyzed using 100% methanol extraction by sonication,
filtration, and concentration followed by analysis by liquid chromatography electrospray
ionization/tandem mass spectrometry (LC/ESI-MS/MS) by direct injection without derivatization. Data
generated from a formica surface resulted in detection limits of 0.12 |ig/cm2 for TEA, 0.06 |ig/cm2 for
EDEA, 0.07 |ig/cm2 for MDEA, and 0.04 |ig/cm2 for DEA. Accuracy and precision data were generated
from each tested surface fortified with these analytes, then qualitatively and quantitatively determined.
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SURFACE ANALYSIS USING WIPES FOR THE DETERMINATION OF NITROGEN MUSTARD
DEGRADATION PRODUCTS BY LIQUID CHROMATOGRAPHY/TANDEM MASS
SPECTROMETRY (LC/MS/MS)
TABLE OF CONTENTS
SECTION PGNO.
DISCLAIMER i
FOREWORD ii
ACKNOWLEDGMENTS iii
EXECUTIVE SUMMARY v
LIST OF TABLES vii
LIST OF ACRONYMS AND ABBREVIATIONS viii
1. SCOPE AND APPLICATION 1
2. SUMMARY OF METHOD 2
3. DEFINITIONS 3
4. INTERFERENCES 4
5. HEALTH AND SAFETY 5
6. EQUIPMENT AND SUPPLIES 5
7. REAGENTS AND STANDARDS 7
8. SAMPLE COLLECTION, PRESERVATION AND STORAGE 8
9. QUALITY CONTROL 9
10. INSTRUMENT CALIBRATION AND STANDARDIZATION 13
11. ANALYTICAL PROCEDURE 14
12. DATA ANALYSIS AND CALCULATIONS 16
13. METHOD PERFORMANCE 17
14. POLLUTION PREVENTION 18
15. WASTE MANAGEMENT 18
16. REFERENCES 18
17. TABLES AND VALIDATION DATA 20
18. ATTACHMENTS 25
VI
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LIST OF TABLES
Table 1. Method Parameters 21
Table 2. Holding Time Sample Stability of Nitrogen Mustard Degradation Analytes 21
Table 3. Concentrations of Calibration Standards (ng/mL) 22
Table 4. MRM Ion Transitions, Retention Time and Variable Mass Spectrometer Parameters 22
Table 5. Gradient Conditions for Liquid Chromatography 23
Table 6. ESI+-MS/MS Conditions 23
Table 7. Materials Tested for the Wipe Analysis of Nitrogen Mustard Degradation Products 24
Table 8. List of Consumable Materials Used During Sampling 24
VII
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LIST OF ACRONYMS AND ABBREVIATIONS
ACN Acetonitrile
AS Analyte Standard
CAL Calibration Standard
CAS Chemical Abstracts Service
CBR Chemical, Biological and/or Radiological
CCC Continuing Calibration Check
CID Collisionally Induced Dissociation
CV Calibration Verification
CWA Chemical Warfare Agent
DBA Diethanolamine
DHHS U.S. Department of Health and Human Services
DL Detection limit
EDEA TV-Ethyldiethanolamine
EPA U.S. Environmental Protection Agency
ERLN Environmental Response Laboratory Network
ESI Electrospray lonization
IDC Initial Demonstration of Capability
IDL Instrument Detection Limit
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
LRB Laboratory Reagent Blank
MDEA TV-Methyldiethanolamine
MDL Method Detection Limit
MRL Minimum Reporting Limit
MRM Multiple Reaction Monitoring
MS Mass Spectrometry
MSDS Material Safety Data Sheet
MS/MS Tandem Mass Spectrometry
MSP Method Specific Parameter
NFL^OAc Ammonium Acetate
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
PPB Parts per Billion
PPM Parts per Million
P&A Precision and Accuracy
PVDF Polyvinylidene Fluoride
QA Quality Assurance
QC Quality Control
QL Quantitation Limit
QMP Quality Management Plan
REC Percent Recovery
RL Reporting Limit
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VIM
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RSD Relative Standard Deviation
RT Retention Time
RTS Retention Time Shift
SAM Standardized Analytical Methods for Environmental Restoration Following Homeland
Security Events
SAP Sampling and Analytical Procedure
SD Standard Deviation
S/N Signal to Noise
SS Surrogate Standard
SSS Stock Standard Solution
TC Target Compound
TEA Triethanolamine
UPLC Ultra-Performance Liquid Chromatography
X Average Percent Recovery
IX
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1. SCOPE AND APPLICATION
1.1. This procedure covers the determination of nitrogen mustard degradation products on
surfaces using wipes. Surfaces were wiped and wipes were analyzed using 100%
methanol extraction by sonication, filtration, and concentration followed by analysis by
liquid chromatography electrospray ionization/tandem mass spectrometry (LC/ESI-
MS/MS) by direct injection without derivatization. Detection limit data were generated
for all analytes of interest on surfaces. Accuracy and precision data were generated from
each surface fortified with these analytes, then qualitatively and quantitatively
determined. The following analytes were determined using this procedure:
Analyte CAS Registry Number
Triethanolamine (TEA) 102-71-6
7V-Ethyldiethanolamine (EDEA) 139-87-7
7V-Methyldiethanolamine (MDEA) 105-59-9
Diethanolamine (DEA) 111 -42-2
1.2. Wipe sampling can be performed quickly and easily when direct extraction is not always
possible (e.g., walls, floors and furniture). As a result, wipe sampling is preferred for
analysis without destruction of the tested surface. However, wipe sampling can remove
analyte only from the surface of a material, which could result in lower recoveries and
produce less reliable quantitative data from porous surfaces. It is therefore important to
understand wipe efficiencies and the materials being wiped. This procedure assesses the
recoveries from various porous and nonporous surfaces using wipes.
1.3. Detection limit (DL) metrics were presented using EPA conventions1"3. The detection
limit was defined as the statistically calculated minimum concentration that can be
measured with 99% confidence that the reported value is greater than zero. The
statistical procedure, utilizing the Laboratory Fortified Sample Matrix samples (LFSM)
and their duplicates, will be used to calculate uncertainty. Precision and accuracy (P&A)
studies should be performed as an initial demonstration of capability (IDC) and after any
modifications to the procedure, including changes in instrumentation and operating
conditions. These studies will evaluate whether the reporting limits and calibration
standard concentrations are appropriate.
1.4. The Minimum Reporting Level (MRL) is the lowest analyte concentration that meets data
quality objectives that are developed based on the intended use of this sampling and
analytical procedure (SAP). The MRL is the lowest true concentration for which the
future recovery is predicted (between 50 and 150% recovery) and is listed as the lowest
calibration level (Level 1).
1.5. This method was intended for use by analysts skilled in the operation of LC/MS/MS
instruments 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.
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1.6. 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
conditions, and wipe/collection materials can be used for analysis as long as similar
performance is demonstrated and the quality control measures can be observed.
2. SUMMARY OF METHOD
2.1. Samples are collected from surfaces with wipes and stored at 4 °C (± 2 °C) for samples
not immediately analyzed within a 24-hour time period. When the samples are ready to
be analyzed, samples are spiked with a surrogate compound, the appropriate solvent is
added, the sample solution is sonicated, the solution is extracted with a syringe filter unit,
then the extract is concentrated and analyzed directly by LC/MS/MS operated in the
positive electrospray ionization (ESI+) mode.
2.2. Each target compound was separated and identified by retention time and by comparing
the sample primary multiple reaction monitoring (MRM) transition to the known standard
MRM transition from reference spectra under identical LC/MS/MS conditions. The
retention time for the analytes of interest must fall within the retention time of the
standard (within ± 5%). The concentration of each analyte is determined by the
instrumentation software using external calibration.
2.3. The detection limit (DL) and quantitation limit (QL) for these compounds were
calculated using an EPA approach and are listed in Table 1 and Attachment 18.1. The
precision and accuracy (P&A) quality control acceptance criteria are shown in
Attachment 18.2. Stability studies suggest samples can be stored up to 28 days (Table 2)
at 4 °C (± 2 °C). The concentrations of the calibration standards are listed in Table 3 and
the retention times, mass transitions, and mass spectrometer parameters are listed in
Table 4. The gradient conditions and ESI-MS/MS conditions are listed in Tables 5 and 6,
respectively. This SAP was tested on several wipes in previous studies to establish that
filter paper provided the highest recoveries with the least interference for any targeted
analytes. Analytes spiked onto surfaces were wiped and the recoveries from both porous
and nonporous surfaces were reported.
The overall performance of the filter paper, in terms of analyte recovery and fewest
interferences, suggested that the filter paper was an appropriate wipe for recovering the
analytes from a surface while analyzing the described nitrogen mustard degradation
products. Other wipes such as cotton gauze would require a pre-cleaning step due to the
presence of interferences to the targeted analytes.
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3. DEFINITIONS
3.1 ANALYSIS BATCH - A set of samples analyzed on the same instrument, not
exceeding 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 in
the analysis batch and/or the number of field samples.
3.2 CALIBRATION STANDARD (CAL) - A solution prepared from the analyte stock
standard solution and the internal standard. The CAL solutions are used to calibrate
the instrument response with respect to analyte concentration.
3.3 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.
3.4 CONTINUING CALIBRATION CHECK (CCC) - A calibration standard containing
the method analytes and surrogate standard. The CCC is analyzed periodically to
verify the accuracy of the existing calibration for those analytes at or near the mid-
level concentrations.
3.5 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.
3.6 SURROGATE STANDARD (SS) - A pure chemical 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. This method uses a deuterated
analyte.
3.8 LABORATORY FORTIFIED BLANK (LFB) - A volume of solvent or other 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.
3.9 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) - A preserved field
sample to which known quantities of the method analytes are added in the laboratory.
The LFSM is processed and analyzed exactly like a sample, and its purpose is to
determine whether the sample matrix contributes bias to the analytical results. The
background concentrations of the analytes in the sample matrix must be determined
in a separate sample and the measured values in the LFSM must be corrected for
background concentrations.
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3.10 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 occurrence of method analytes is low.
3.11 LABORATORY REAGENT BLANK (LRB) - An aliquot of solvent or other blank
matrix that is treated exactly as a sample including exposure to all glassware,
equipment, solvents and reagents and surrogate standards that are used in the analysis
batch. The LRB is used to determine if method analytes or other interferences are
present in the laboratory environment, the reagents, or the apparatus.
3.12 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.
3.13 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 quality control
(QC) criteria for this standard are met. A procedure for verifying a laboratory's MRL
is provided in Section 9.2.4.
3.14 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.
3.15 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.
3.16 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.
4. INTERFERENCES
Procedural interferences may 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 LRBs
(Section 9.4.1) under the same conditions as the samples.4 Subtraction of blank values from sample
results is not performed.
4.1 All reagents and solvents should be of pesticide grade purity or higher to minimize
interference problems. All glassware should be cleaned and demonstrate to be free from
interferences.
4.2 Matrix interferences may be caused by contaminants from the sample, sampling devices
or storage containers. The extent of matrix interferences will vary considerably from
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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.
4.3 Matrix effects are well known phenomena of ESI-MS techniques, especially for co-
eluting 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.
5. 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 safe handling of chemicals used in this
method. A reference file of material safety data sheets (MSDSs) that address the safe handling of the
chemicals should be made available to all personnel involved in the chemical analyses. Additional
references are available.5"7
6. EQUIPMENT AND SUPPLIES
References to specific brands of equipment and catalog numbers were provided solely as examples
and do not constitute an endorsement of the use of such products or suppliers.
6.1 LC/MS/MS APPARATUS
6.1.1 LIQUID CHROMATOGRAPH (LC) SYSTEM - An analytical system complete
with a temperature programmable liquid chromatograph with a solvent mixer
(Waters - Acquity™ or equivalent able to perform the analyses as described) and all
required accessories including syringes, solvent degasser, and autosampler.
6.1.2 ANALYTICAL COLUMN - Waters - Atlantis™ HILIC Silica, 100 mm x 2.1 mm, 3
|im particle size, or equivalent.
6.1.3 TANDEM MASS SPECTROMETER (MS/MS) SYSTEM - A 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.
6.1.4 DATA SYSTEM - 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. QuanLynx™ (or similar software) is used for all quantitation for data
generated from the LC/MS unit.
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6.2 NITROGEN EVAPORATOR
6.2.1 A nitrogen evaporation device, such as the N-Evap 24 - port device (Organomation
Associates, Inc.) equipped with a water bath that can be maintained at 50 °C for final
analyte concentration (< 10 mL volume). Evaporation times are expected to increase
without this feature. Other nitrogen devices, which do not have a temperature control
feature, may be used as long as QC criteria can still be reached.
6.3 EXTRACTION DEVICE
6.3.1 SONICATOR (Fisher Scientific Catalog #: 15-335-112) or equivalent.
6.4 GLASSWARE AND MISCELLANEOUS SUPPLIES
6.4.1 AUTOSAMPLER VIALS - Amber 2-mL autosampler vials with TeflonD-lined
screw tops (Waters Corp., Milford, MA), or equivalent.
6.4.2 DISPOSABLE STERILE SYRINGES - 10.0 mL ± 1% accuracy (Fisher Scientific,
Pittsburgh, PA), or equivalent.
6.4.3 AUTO PIPETTES - 10.0 mL, 1000 uL, 100 uL and 10 uL ± 1% accuracy.
6.4.4 DESOLVATION GAS - Ultra Pure 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.
6.4.5 COLLISION GAS - Ultra Pure Argon gas used in the collision cell in MS/MS
instruments and and should meet or exceed instrument manufacturer's specifications.
6.4.6 ANALYTICAL BALANCE - accurate to 0.1 mg; reference weights traceable to
Class S or S-l weights.
6.4.7 National Institute of Standards and Technology (NIST)-traceable thermometer.
6.4.8 STANDARD SOLUTION FLASKS - Class A volumetric glassware
6.4.9 SYRINGE FILTER - Millex® GV Syringe-driven filter unit (PVDF) 0.22 |im
(Millipore Corporation, Catalog # SLGV013NL).
6.4.10 WIPES - Whatman 42 ashless, 55 mm filter paper (Fisher Scientific, Pittsburgh, PA,
Catalog # 09-845A).
6.4.11 SAMPLE COLLECTION CONTAINERS - Clean Nalgene containers with screw
cap (Fisher Scientific, Pittsburgh, PA, Catalog # 11-815-10C), or equivalent.
6.4.12 SAMPLE CONCENTRATION CONTAINERS - Sterile 15 mL conical tubes (Fisher
Scientific, Pittsburgh, PA, Catalog # 05-538-59A), or equivalent.
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7. REAGENTS AND STANDARDS
7.1 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 of
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.
7.1.1 SOLVENTS - Acetonitrile (CAS # 75-05-8), Methanol (CAS # 67-56-1), and Water
(CAS # 7732-18-5), HPLC mass spectrometry pesticide grade or equivalent,
demonstrated to be free of analytes and interferences.
7.1.2 AMMONIUM ACETATE (CAS # 631-61-8, ACS Reagent Grade or equivalent
demonstrated to be free of analytes and interferences.)
7.1.3 ACETIC ACID (CAS # 64-19-7, Concentrated, ACS Reagent Grade or equivalent
demonstrated to be free of analytes and interferences.)
7.1.4 MOBILE PHASE A - Solution A consisted of 95% of 25 mM ammonium acetate at
pH 4.2, and 5% of acetonitrile to prevent microbial growth. To prepare 1 L, add 1.93
g of ammonium acetate to water, adjust to pH 4.2 with acetic acid and dilute to 1 L
mark. Add 950 mL of the 25 mM ammonium acetate at pH 4.2 solution to a 1L
container. Add 50 mL of acetonitrile. This solvent system is still prone to some
microbial growth and should be replaced once a week.
7.1.5 MOBILE PHASE B- Solution B was comprised of 95% acetonitrile and 5% 25 mM
ammonium acetate. To prepare 1 L, add 1.93 g of ammonium acetate to 1 L of water.
Add 950 mL of acetonitrile to a 1L container. Add 50 mL of the 25 mM ammonium
acetate solution.
7.1.6 TARGET ANALYTES - Triethanolamine (CAS # 102-71- 6), 7V-ethyldiethanolamine
(CAS # 139-87-7), 7V-methyldiethanolamine (CAS # 105-59-9) and diethanolamine
(CAS# 111-42-2).
7.1.7 SURROGATE ANALYTE- Bis(2-hydroxyethyl)-d8-amine (Diethanolamine-d8)
(CAS# 103691-51-6)
7.2 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 vials) and at 4 °C (± 2 °C). Standards were estimated to be stable for
at least a month. Although stability times are suggested, laboratories should utilize QC
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practices to determine when standards should be replaced.
7.2.1 SURROGATE STOCK STANDARD SOLUTION (SSS) (10-1000 |ig/mL)
A standard solution may be prepared from a certified commercially available neat
compound. Isotopically-labeled surrogate, diethanolamine-d8 (CAS # 103691-51-6),
was obtained from CDN Isotopes. The surrogate was added to a 50 mL volumetric
flask in order to achieve a concentration of 1000 |ig/mL in solution (i.e., 50 mg or
45.8 |iL of diethanolamine-d8 was added to a 50 mL volumetric flask and diluted to
mark with methanol). Further dilutions of the 1000 |ig/mL concentration were used
to obtain 100 and 10 |ig/mL solutions in methanol. Surrogate stock standard solutions
were stable for at least a month when stored at 4 °C.
(NOTE: Although diethanolamine-d8 was used as a surrogate in this SAP,
diethanolamine-d8 could be used as an internal standard for diethanolamine for
quantitation purposes. However, further evaluation would be necessary to ensure that
diethanolamine-dg is a viable internal standard and meets QC requirements.)
7.2.2 ANALYTE STOCK STANDARD SOLUTION (AS)
Standard solutions may be prepared from certified, commercially available neat
compounds. All neat compounds are viscous liquids at room temperature. Neat
materials of triethanolamine and diethanolamine were obtained from Chem Service
(West Chester, PA). 7V-ethyldiethanolamine and 7V-methyldiethanolamine were
obtained from Aldrich as neat materials. A standard solution concentration of 1000
|lg/mL for each compound was obtained in 50 mL volumetric flasks (e.g., 44.4 |iL of
TEA, 45.87 |iL of DEA, 49.31 |iL of EDEA and 48.08 |iL of MDEA were each
added to separate 50 mL volumetric flasks and diluted to the mark with methanol) .
Further dilutions of the 1000 |ig/mL concentration can be used to obtain 100 and 10
|lg/mL solutions in methanol. The calibration standards were made from appropriate
dilution concentration of these stock standards.
(NOTE: All spiking solutions should be within ten times the DL).
7.2.3 CALIBRATION STANDARD SOLUTION (CAL)
A calibration stock standard solution (Level 7) was prepared from the Analyte
Standard (AS) solution concentrations, containing, triethanolamine, 7V-
ethyldiethanolamine, 7V-methyldiethanolamine, diethanolamine and the surrogate
diethanolamine-d8 in methanol (i.e., 250 |iL of TEA, DEA, EDEA, MDEA and DEA-
ds of a 100 |ig/mL solution was added to a 50 mL volumetric flask and diluted to
mark with methanol). From Level 7, further dilutions were performed to prepare
Levels 6 through 1 as shown in Table 3. All concentrated stock standard solutions
were made in methanol.
8. SAMPLE COLLECTION. PRESERVATION AND STORAGE
8.1 SAMPLE COLLECTION
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8.1.1 The exact choice of sampling vessel and procedure is not critical for the analysis and
can be adjusted to meet the needs of the situation as long as the different materials
have been tested and show no presence of the target analytes. As an example for
field samples, the field samplers would collect samples with the appropriate wetted
wipe (methanol) and place the wipes in a jar 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.
8.1.2 Wipe samples were collected using Whatman 42 ashless 55 mm circle filter paper.
The required analyte spike solution containing the four analytes of interest was added
to the surface, allowed to dry, and wiped with each wipe separately. Two wipes were
separately wetted with approximately 300 |iL of methanol. The first wipe is used to
wipe the surface in a Z-like pattern horizontally across a defined surface (100 cm2)
(Figure 18.3). The second wipe is used to wipe the same surface in a Z-like pattern
vertically across a defined surface (100 cm2). Then both wipes are placed into a 125
mL Nalgene polypropylene straight-sided jar with a polypropylene screw cap.
Surrogate (DEA-d8) and methanol solvent (10 mL) are added to the jar. Because the
wipe can lie flat on the bottom of the jar, the solvent fully immerses the wipes. Field
and/or matrix blanks are needed, according to conventional sampling practices.
8.2 SAMPLE STORAGE AND HOLDING TIMES
8.2.1 Samples should be analyzed as soon as possible. All samples were refrigerated at 4
°C (± 2 °C) from the time of collection until analysis unless the samples were
analyzed within a 24-hour time period. At the laboratory, samples were stored in the
refrigerator at 4 °C (± 2 °C) until requested for analysis. Samples should be analyzed
within 48 hours of collection or as soon as possible. Samples from a particular site
should be carefully characterized to ensure that there is no interaction with the wipe
or specific surface to cause interferences or degradation of the analytes after 48
hours. After injection in the LC/MS, the vial septa were replaced and the vials were
stored in a refrigerator in case further analysis was needed. Samples can be stored up
to 28 days (Table 2) in the refrigerator at 4 °C (± 2 °C).
9. QUALITY CONTROL
9.1 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 18.2 Attachment) as well as a detection limit study (DL, as shown in
Table 1 and Section 18.1 Attachments) must be performed to demonstrate laboratory
capability or whenever a major modification is made to this SAP. Laboratories are
encouraged to institute additional QC practices to meet their specific needs.
9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC)
The IDC must be successfully performed prior to the analysis of field samples. Prior to
conducting an IDC, an acceptable Initial Calibration must be generated as outlined in
Section 10.2.
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9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND
Any time a new lot of solvents, reagents and autosampler vials is used, the
laboratory reagent blank (LRB) must be demonstrated to be reasonably free of
contamination and that criteria are met in Section 9.4.1. The LRB was used to ensure
that analytes of interest or other interferences were not present in the laboratory
environment, the solvent, or the apparatus.
NOTE: Good laboratory practices indicate the use of a blank during calibration of
instrumentation to ensure that no carryover occurs between samples. If the required
criteria were 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.
9.2.2 INITIAL DEMONSTRATION OF PRECISION AND ACCURACY (P&A)
NOTE: Because porosity 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 Formica®
(Formica, Cincinnati, OH) surface because the Formica surface has been shown to
be mostly free of contamination and is a relatively nonporous surface.
For a precision and accuracy study (P&A), prepare a check standard containing
triethanolamine, jV-ethyldiethanolamine, jV-methyldiethanolamine, diethanolamine
and diethanolamine-d8, 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 were chosen with seven samples each.
The check samples were analyzed according to Section 11.
9.2.3 The average percent recovery (X), standard deviations (a) and the percent relative
standard deviation (%RSD) of the recoveries were calculated for each analyte. The
% RSD value of < 25% should be applied to all analytes.
9.2.4 MINIMUM REPORTING LEVEL (MRL)
Establish a target concentration for the MRL based on the intended use of the
method. Establish an Initial Calibration (Section 10.2). 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.
The MRL is reported in this study as the lowest calibration level.
9.2.5 Calibration verification (CCV)
A mid-level sample 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.
9.3 DETECTION AND QUANTITATION LIMITS (DL and QL)
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The procedure for the determination of the laboratory detection and quantitation limits for
the EPA approach follows 40 CFR Part 136 Appendix B as described in the
Environmental Response Laboratory Network (ERLN). Detection limits (DLs) 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). Quantitation limits (QLs) represent the smallest detectable
concentration of analyte greater than the detection limit, where the precision and bias
achieve program objectives. The DL and QL were determined for each target analyte.
9.3.1 Determination of laboratory instrument detection limits (IDLs)
Laboratory instrument detection limits (IDLs) were determined for each instrument
used for analyses. Although the determination of the laboratory IDL is not an EPA
requirement, the laboratory IDL can be used to establish an estimate of the initial
spiking concentration used for determination of the DL. The laboratory IDL was
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 were injected until the
S/N ratio was in the range of 3:1 - 5:1. Replicates were 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: S/N ratios must be demonstrated; linearity of S/N ratio with increasing or
decreasing concentration cannot be assumed.)
9.3.2 Determination of laboratory MDL
DLs represent the optimal detection achieved by a laboratory in a matrix of interest.
Formica coupons were used for the determination of the MDL for surface samples.
The 40 CFR Part 136, Appendix B procedure was followed, particularly with regard
to spike levels used. Replicate reference matrix samples were spiked at a level
between 1-5 times the estimated detection level (e.g., the IDL, 3 times the standard
deviation of replicate instrument measurements of the analyte in desired solvent, or
the region where there is a break in the slope at the low end of the standard curve).
The resulting DL must be within 10 times the spike level used, or the DL
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 were 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):
MDL = t („-!, l-a = 0.99) X SD
where
MDL = method detection limit
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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 DLs are shown in Table 1 and Attachment 18.1.
9.4 ONGOING QC REQUIREMENTS
9.4.1 LABORATORY REAGENT BLANK (LRB)
A reagent blank was prepared and analyzed with each analysis batch, using methanol,
for confirmation that there were no background contaminants interfering with the
identification or quantitation of the target analytes. If there was 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.
9.4.2 CONTINUING CALIBRATION CHECK (CCC)
CCC standards (near the midpoint of the calibration range) are analyzed at the
beginning of each analysis batch, after every twenty field samples, and at the end of
the analysis batch.
9.4.3 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM)
A LFSM is analyzed to determine that spike accuracy for a sample matrix is not
adversely affected. If a variety of sample matrices is analyzed, performance should
be established for each surface.
9.4.3.1 Within each analysis batch, a 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 analyte AS (Section 7.2.2).
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 is used. If the accuracy does not fall within this range, check
with a CCC or prepare a fresh AS solution for analysis.
9.4.4 SURROGATE STANDARD
All samples were spiked with surrogate standard spiking solution as described in
Section 7.2.1. An average percent recovery of the surrogate compound and the
standard deviation of the percent recovery were calculated and updated regularly.
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9.4.5 MATRIX SPIKE (MS) OR LABORATORY FORTIFIED SAMPLE MATRIX
DUPLICATE (LFSMD)
Within each analysis batch, a minimum of one MS or LFSMD should be analyzed.
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. MS/LFSMD samples must be incorporated into the field sampling plan.
If the laboratory did not receive MS samples for determination of site-specific
precision and accuracy (P&A), the laboratory will evaluate the site data quality based
on the Laboratory Fortified Sample Matrix (LFSM) data, if there is sufficient sample
in the site samples to conduct an analysis. MS/LFSMD recovery results will be used
for site-specific precision and accuracy (P&A) data. LFSM data were used as
MS/LFSMD sample data for this study. RSD values should be < 30% for samples.
9.4.6 METHOD MODIFICATION QC REQUIREMENTS
The analyst may modify the separation technique, LC column, mobile phase
composition, LC conditions and MS conditions so as long as all QC and ongoing QC
criteria are met. It is the laboratory's responsibility to review the results when
method modifications are implemented. If repeated failure occurs, the modification
must be abandoned.
10. 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.8 Verification for 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 should be performed at the beginning and end of each analysis batch.
10.1 CALIBRATION OF MAS S SPECTROMETER
Mass calibration of the mass spectrometer (Waters Acquity™ or equivalent) is performed
monthly or when mass shifts of more than 0.5 daltons are noticed by the analyst. The
mass calibration file is saved in the mass spectrometer software file folder (MassLynx™
or similar software). The mass calibration solution used is a mixture of NaCsI provided
by the manufacturer. Other calibration solutions can also be used per instrument
manufacturer's specifications. The detailed procedure for mass calibration of the mass
spectrometer can be found in the software instruction manual provided by the
manufacturer.
10.2 INITIAL CALIBRATION FOR ANALYTES
10.1.1 Optimize the [M+H]+ ion for each analyte by infusing a 500 ng/mL methanol
solution directly into the MS at a flow rate of 0.3 mL/min. The MS parameters
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(voltages, temperatures, gas flows, etc.) are varied until optimal analyte responses are
achieved. Optimize the product ion by following the same procedures 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 are presented in Tables 4 and 6.
10.1.2 Establish LC operating conditions that will optimize peak resolution and shape.9
Suggested LC conditions (listed in Table 5) may not be optimal for all LC systems.
10.1.3 The initial calibration contains a seven-point curve using the analyte concentrations
prepared in section 7.2.3 and are shown in Table 3. The lowest calibration curve
standard must be at the MRL. 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. The coefficient of
determination (r2) of the linear fit should be greater than or equal to 0.98. The
coefficient of determination (r2) of the quadratic curve should be greater than or equal
to 0.99. A calibration curve and an instrument blank will be analyzed at the
beginning of each batch or daily to ensure instrument stability.10 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 saved
and used to quantify all samples.
10.3 QUANTITATION OF ANALYTES
The quantitation of the target analytes is accomplished with quantitation software as it
relates to each specific instrument (QuanLynx™ or similar software).11 An external
calibration is used along with monitoring diethanolamine-d8 surrogate recovery. Refer to
Table 4 for the MRM transitions and retention times.
11. ANALYTICAL PROCEDURE
11.1 SAMPLE PREPARATION
11.1.1 Samples were collected and stored as described in Section 8. Surrogate (DEA-d8)
and methanol solvent (10 mL) were added to the jar. Sonicate each jar containing the
methanol solution for approximately 15 minutes in a water bath at room temperature
with no heat required.
11.1.2 After sonication, decant the extraction solvent into a 10 cc lock-tip sterile fitted
syringe with a Millex® GV syringe driven filter unit, polyvinylidene fluoride (PVDF)
filter (0.22 |om), transferring the filtered sample to a sterile 15-mL polypropylene
tube (or equivalent).
11.1.3 Place the 15-mL polypropylene tube on the nitrogen evaporator and set the
temperature of the water bath to 50 °C (± 5 °C).
11.1.4 Concentrate sample in the 15 mL polypropylene tube to < 2 mL using the N-Evap
(Thomson Instrument Co., Clear Brook, VA) concentrator.
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11.1.5 Dilute the sample to 2 mL (± 5% accuracy) final volume using methanol. (NOTE:
More suitable glassware with better accuracy may be used to ensure an exact 2 mL
final volume.)
11.1.6 Transfer (via pipette) to a standard 2 mL sample vial.
NOTE: Calibration standards are not filtered through the syringe-driven filter units
since no particulates are present. The filters 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.
11.2 SAMPLE ANALYSIS/ANALYTICAL SEQUENCE
11.2.1 Establish Liquid Chromatography/Mass Spectrometry conditions as per guidance
described in Section 10 and summarized in Tables 4, 5 and 6.
11.2.2 Prepare a sequence that includes all QC samples and surface samples. The first
sample to be analyzed is a 5 uL injection on column of a blank (methanol).
11.2.3 The calibration standards, Levels 1 through 7, are analyzed next. 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 as necessary. Print
quantitation reports for the calibration standards.
11.2.4 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 and regenerate
a calibration report. Alternatively, re-analyze "nonconforming" calibration level(s)
and repeat the above procedures.
11.2.5 The first sample analyzed after the calibration curve is a blank to ensure there is no
carryover.9 If the initial calibration data are acceptable, begin analyzing samples,
including QC and blank samples, at their appropriate frequency injecting the same
size aliquots (5 |iL) 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.
11.2.6 EXCEEDING THE CALIBRATION RANGE: If the absolute amount of a target
compound exceeds the working range of the LC/MS system (see Level 7 in Table 3),
the prepared sample is diluted with methanol and re-analyzed. 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 afterwards to demonstrate no carryover will occur.
11.2.7 At the conclusion of the data acquisition, use the same software that was used in the
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calibration procedure to identify peaks of interest from the retention time windows.
Use the data software to examine the ion abundances of the peaks in the
chromatogram and compare retention times with the retention time of the
corresponding peak in an analyte standard.
11.2.8 All qualitative and quantitative measurements are performed as described in Sections
9.2 and 9.3. When not being analyzed, samples are stored in the refrigerator at 4 °C
(± 2 °C) and protected from light in screw cap top vials equipped with Teflon-lined
septa.
12. DATA ANALYSIS AND CALCULATIONS
12.1 QUALITATIVE AND QUANTITATIVE ANALYSIS
12.1.1 An external calibration is used when monitoring the MRM transitions of each
analyte. Quantitation software (such as QuanLynx™) is utilized to conduct the
quantitation of the target analytes and surrogate standard. 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.11
12.1.2 Computer programs used for analysis of data include instrumentation and
quantitation software (e.g., MassLynx™ with QuanLynx™). The manufacturer's
quantitation software manual should be consulted to ensure the proper use of the
software. The quantitation method is set as an external calibration using the peak
areas in ng/mL as long as the analyst is consistent. 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 match the integration
of other peaks and/or known calibration peaks. Any manual integration should be
carried out by a qualified analyst and checked against quality control procedures
(sections 9 and 10.3).
12.1.3 If the polynomial type is linear and excludes the point of origin, use a fit weighting of
1/X in order to give more weighting to the lower concentrations. The retention time
window of the MRM transitions must be within 5% of the retention time of the
analyte in a mid-range calibration 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. The coefficient of
determination, r2, should be > 0.98 for each analyte. If one of the calibration
standards other than the high or low standard causes the curve 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.
12.1.4 If the polynomial type is quadratic, the point of origin is excluded and a fit weighting
of 1/X is used in order to give more weighting to the lower concentrations. The
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retention time window of the MRM transitions must be within 5% of the retention
time of the analyte in a mid-range calibration 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. The coefficient
of determination, r2, should be > 0.99 for each analyte. If one of the calibration
standards other than the high or low standard causes the curve to be <0.99, this point
must be re-injected or a new calibration curve should be analyzed. If the low or high
point is excluded, a six-point curve is acceptable using a quadratic fit. An initial
seven-point curve over the calibration range is suggested in the event the low and/or
high point must be excluded to obtain a coefficient of determination > 0.99. In this
event, the calibration range and detection limits must be modified to reflect this
change.
12.2 Prior to reporting data, the chromatogram should be reviewed for any incorrect peak
identification. 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.
12.3 All data packages will be verified by a qualified analyst to ensure incorrect peak
identifications or poor integrations were properly identified. The qualified analyst will
sign off on the narrative and checklist.
13. METHOD PERFORMANCE
13.1 PRECISION, ACCURACY AND DETECTION LIMITS
13.1.1 Tables for precision, accuracy and detection limit results for a single laboratory study
are presented in Sections 18.1 and 18.2 and Table 1.
13.2 RECOVERIES AND PRECISION FOR OTHER SURFACE TYPES
13.2.1 Section 18.2 lists recoveries and precision of target analytes for a variety of other
surfaces.
13.3 PROBLEM ANALYTES AND SURFACES
13.3.1 TARGET ANALYTES ON PRE-CLEANED AND UNCLEANED SURFACES
Target analytes were spiked on surfaces and the wipe samples were tested for
differences between pre-cleaned surfaces, using methanol, and wipe samples from
uncleaned surfaces (used as received). When surfaces are cleaned prior to analysis,
noticeable differences in TEA and DEA recoveries may occur due to a pre-existing
presence/contamination of surfaces with these specific two compounds (matrix
blanks will confirm the presence of TEA and DEA). Potential matrix effects are also
indicated, suggesting laboratories should seek to understand matrix effects occurring
in specific samples through thoughtful choice of MS materials. Although pre-
cleaning surfaces would provide a more accurate analysis of the true recovery of
TEA and DEA, it is not practical in a real scenario. Analysts should be aware that
these two specific compounds may already be present within the tested sample matrix
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and plan accordingly. Wood and painted drywall surfaces resulted in poor recoveries
outside the range of this procedure. As a result, the SAP should not be used to
identify these analytes in relation to these specific surfaces. Although porosity of the
surface is most likely the culprit for low recoveries, further analysis should be
performed to determine definitive reasoning of poor recoveries from the surface.
14. POLLUTION PREVENTION
14.1 This method utilizes the use of small volumes of organic solvent and small quantities of
pure analytes, thereby minimizing the potential hazards to both analyst and environment.
14.2 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://membership.acs.org/c/ccs/pub_9.htm (accessed November 2009).
15. WASTE MANAGEMENT
15.1 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 that laboratories protect the air, water, and land by minimizing and
controlling all releases from fume hoods and bench operations. Also, compliance is
required with any sewage discharge permits and regulations, particularly the hazardous
waste identification rules and land disposal restrictions.
15.2 Each laboratory should determine with 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 and date the beginning of
collection before using the container.
16. REFERENCES
1. Code of Federal Regulations, 40 CFR Part 136, Appendix B. Definition and Procedure for the
Determination of the Method Detection Limit - Revision 1.11.
2. Federal Advisory Committee on Detection and Quantitation Approaches and Uses in Clean
Water Act Programs. Final Report. December 28, 2007.
3. Lloyd Currie. "Quantitative Determination: Application to Radiochemistry." 1968.
Anal.Chem 40(3):586-593L.
4. Standard Practices for Preparation of Sample Containers and for Preservation of Organic
Constituents, American Society for Testing and Materials, Philadelphia. ASTM Annual Book
of Standards, Part 31, D3694-78.
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5. OSHA Safety and Health Standards, General Industry (29CRF1910). Occupational Safety and
Health Administration, OSHA 2206 (Revised, July 1, 2001).
6. Carcinogens-Working with Carcinogens, Publication No. 77-206, Department of Health,
Education, and Welfare, Public Health Service, Center for Disease Control, National Institute
of Occupational Safety and Health, Atlanta, Georgia, August 1977.
7. Safety in Academic Chemistry Laboratories, American Chemical Society Publication,
Committee on Chemical Safety, 7th Edition. Information on obtaining a copy is available at
http://membership.acs.org/C/CCS/pub_3.htm (accessed November, 2009). Also available by
request at OSS@acs.org.
8. Eichelberger, J.W.; Harris, L.E.; Budde, W.L. "Reference Compound to Calibrate Ion
Abundance Measurement in Gas Chromatography-Mass Spectrometry Systems" Analytical
Chemistry 1975, 47(7): 995-1000. http://pubs.acs.org/doi/abs/10.1021/ac60357a018.
9. McNair, N.M.; Bonelli, E.J. Basic Chromatography; Consolidated Printing: Berkeley, CA,
1969; p. 52.
10. Burke, J.A. "Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects", J.
Assoc. Off. Anal. Chem. 1965,48:1037-1058.
11. Peters, F.T.; Drummer, O.H.; Musshoff, F. "Validation of New Methods", Forensic Science
International 2007, 165(2):216-224.
19
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17. TABLES AND VALIDATION DATA
NHSRC/NIOSH SAP
Revision Date: 3/30/11
Page 20 of 3 8
Item
Table 1
Table 2
Table 3
Table 4
Title
Method Parameters
Holding Time Study for Nitrogen Mustard
Degradation Analytes
Concentration of Calibration Standards
MRM Retention Times and MRM Ions and
Number
of Pages
1
1
1
1
Revision
Number
1
1
1
1
Date
Revised
3/2011
3/2011
3/2011
3/2011
Variable Mass Spectrometer Parameters
Table 5 Gradient Conditions for Liquid
Chromatography
Table 6 ESf-MS/MS Conditions
Table 7 Materials Tested for the Wipe Analysis of
Nitrogen Mustard Degradation Products
Table 8 List of Consumable Materials Used During
Sampling
3/2011
3/2011
3/2011
3/2011
20
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NHSRC/NIOSH SAP
Revision Date: 3/30/11
Page 21 of 38
Table 1. Method Parameters
FORMICA
Analyte
TEA
EDEA
MDEA
DEA
DL*
ng/fcm2t
0.12
0.06
0.07
0.04
ng/mL
12.32
6.25
6.85
4.37
LOQ
ng/fcm2t
1.23
0.63
0.69
0.44
ng/mL
123.2
62.6
68.5
43.7
MRL
ng/mL
10
10
10
10
*Last DL Study- March 2011.
fng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area
of the coupon (100 cm2).
Table 2. Holding Time Sample Stability of Nitrogen Mustard Degradation Analytes
Concentration 50 ng/mL (n =5)
Holding Time
(days)
0
7
14
21
28
TEA
Average
%
Recovery
92.98
93.57
74.93
75.30
78.35
% RSD
6
6
10
7
5
EDEA
Average
%
Recovery
97.56
85.15
87.06
83.84
87.77
% RSD
7
9
6
10
5
MDEA
Average
%
Recovery
94.52
82.36
82.38
82.97
86.60
% RSD
6
8
8
10
5
DEA
Average
%
Recovery
93.47
85.06
82.11
77.84
74.16
% RSD
5
7
6
10
12
DEA-d,
o
Average
%
Recovery
96.32
89.75
85.77
83.51
90.08
% RSD
7
9
11
11
9
21
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NHSRC/NIOSH SAP
TableS. Concentrations of Calibration Standards (ng/tnL)
Revision Date: 3/30/11
Page 22 of 3 8
Analyte /Surrogate
Triethanolamine
/VEthyldiethanolamine
/VMethyldiethanolamine
Diethanolamine
Diethanolamine-d8
Level
1
10
10
10
10
10
Level
2
25
25
25
25
25
Level
3
50
50
50
50
50
Level
4
100
100
100
100
100
Level
5
250
250
250
250
250
Level
6
350
350
350
350
350
Level
7
500
500
500
500
500
Table 4. MRM Ion Transitions, Retention Time and Variable Mass Spectrometer Parameters
Analyte
Triethanolamine
/V-Ethyldiethanolamine
/V-Methyldiethanolamine
Diethanolamine
Diethanolamine-d8 (Surrogate)
Cone
voltage
30
30
30
30
30
MRM mass transition
(parent -product)
150.09 -132.10
134.02 -116.10
120.03 -402.00
106.00 -88.10
114.20 -96.22
Collision
energy (eV)
12
14
12
12
12
Rr
(minutes)
9.7
11.1
13.0
12.1
12.2
Retention times should fall within 5% of the given value; otherwise re-analysis may be necessary.
22
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NHSRC/NIOSH SAP
Revision Date: 3/30/11
Page 23 of 3 8
Table
5. Gradient Conditions for Liquid Chromatography
Time
(min)
0
1
2
12
16
17
18
21
Flow
(pl/min)
300
300
300
300
300
300
300
300
%
Solution Af
90
90
87
87
85
70
90
90
%
Solution Bft
10
10
13
13
15
30
10
10
fA: 95% - ACN / 5% - 25mM M^OAC
f fB: 95% - 25mM NH4OAC (pH 4.22) / 5% - ACN
Injection volume - 5|iL (recommended)
* Column Temperature: 30 °C
*Autosampler Temperature: 15 °C
* Equilibration time: 3 minutes
*Column: Atlantis™HILIC silica, 100mm x 2.1mm, 3|im particle size
Table 6. ESI+-MS/MS Conditions
MS Parameter (ESO
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
1.0 kV
See Table 4
2 Volts
0.2 Volts
150 °C
300 °C
800 L/hr
50 L/hr
14.5
14.5
0.5
1
See Table 4
1
15.0
15.0
0.5
-560
3.0 x 10'3 Torr
0.005 seconds
0.005 seconds
1
0.1 Daltons
0.3 Seconds
23
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NHSRC/NIOSH SAP
Revision Date: 3/30/11
Page 24 of 3 8
Table 7. Materials Tested for the Wipe Analysis of Nitrogen Mustard Degradation Products
Material
Glass
Vinyl Tile
Formica
Wood (southern pine, pre-treated)
Galvanized steel
Painted Drywall (BEHR latex paint)
Manufacturer/Vendor
Carolina Glass Co. /Lowe's
Armstrong/Home Depot
Wilsonarf Laminate/Home Depot
Home Depot
McMaster-Carr
BEHR/Home Depot
Table 8. List of Consumable Materials Used During Sampling
Material
Whatman 42 ashless circle filters, 55 mm
125 ml Nalgene polypropylene straight-side jars with
screw caps
10 ml BD safety-lok syringes
Corning 15 ml graduated plastic centrifuge tubes
Millipore 13 mm Millex filter, 0.22 pm PVDF
Waters 1 .8 ml amber glass vials with pre-slit silicone
PTFE screw cap
Vendor
Fisher Scientific (Pittsburgh, PA)
Fisher Scientific (Pittsburgh, PA)
Fisher Scientific (Pittsburgh, PA)
Fisher Scientific (Pittsburgh, PA)
Fisher Scientific (Pittsburgh, PA)
Waters Corp. (Milford, MA)
24
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NHSRC/NIOSH SAP
Revision Date: 3/30/11
Page 25 of 3 8
18. ATTACHMENTS
18.1 Detection and Quantitation Limits
18.2 Preci sion and Accuracy
18.3 Illustration depicting the wiping pattern on a 100 cm2 surface
25
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NHSRC/NIOSH SAP
Revision Date: 3/30/11
Page 26 of 3 8
18.1 DL AND QL CALCULATIONS
PL Calculations for Seven Replicates for Nitrogen Mustard Degradation Analytes
FORMICA
Average Spike
Concentration
(ngyrnL) (n=7)
50.00
Formica Blank
Average Spike
Concentration
(ngyfcm2) (n=7)
0.50
Formica Blank
TEA
Average
Recovery
ng/inL
54.45
26.30
Average
Recovery
(ngyfcm2)
0.54
0.26
%
Recovery
108.9
%
Recovery
108.9
%
RSD
7
%
RSD
7
EDEA
Average
Recovery
ngymL
32.13
0
Average
Recovery
(ngyfcm2)
0.32
0
%
Recovery
64.27
%
Recovery
64.27
%
RSD
6
%
RSD
6
MDEA
Average
Recovery
ngymL
36.92
0
Average
Recovery
(ngyfcm2)
0.37
0
%
Recovery
73.84
%
Recovery
73.84
%
RSD
6
%
RSD
6
fng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area
of the coupon (100 cm2).
FORMICA
Average Spike
Concentration
(ngyhiL) (n=7)
50.00
Formica Blank
Average Spike
Concentration
(ngyfcm2) (n=7)
0.50
Formica Blank
DEA
Average
Recovery
(ng/hiL)
41.05
5.42
Average
Recovery
(ngyfcm2)
0.41
0.05
%
Recovery
82.09
%
Recovery
82.09
%
RSD
3
%
RSD
3
DEA
-------
NHSRC/NIOSH SAP
Revision Date: 3/30/11
Page 27 of 3 8
18.2 PRECISION AND ACCURACY
Precision and Accuracy (P&A) data for wipe analysis of nitrogen mustard degradation
analytes on surfaces, (n = 7 samples at each concentration)
FORMICA
Average Spike
Concentration
(ngAnL) (n=7)
50
75
100
150
Average
Formica Blank
Average Spike
Concentration
(ng/tm2)t
(n=7)
0.50
0.75
1.00
1.50
Average
Formica Blank
TEA
Average
Recovery
(ngAnL)
75.32
71.12
81.11
123.96
23.41
Average
Recovery
(ngytm2)
0.75
0.71
0.81
1.24
0.23
%
Recovery
150.63*
94.83
81.11
82.64
-
%
Recovery
150.63*
94.83
81.11
82.64
-
%
RSD
8
8
6
10
-
%
RSD
8
8
6
10
-
EDEA
Average
Recovery
(ngAnL)
37.11
31.27
65.46
91.24
0
Average
Recovery
(ng/cm2)
0.37
0.32
0.66
0.91
0
%
Recovery
74.23
41.69
65.46
60.82
-
%
Recovery
74.23
41.69
65.46
60.82
-
%
RSD
17
8
4
14
-
%
RSD
17
8
4
14
-
MDEA
Average
Recovery
(ngAnL)
39.49
46.76
66.82
95.14
0
Average
Recovery
(ngytm2)
0.40
0.47
0.67
0.95
0
%
Recovery
78.98
62.34
66.82
63.43
-
%
Recovery
78.98
62.34
66.82
63.43
-
%
RSD
7
11
5
9
-
%
RSD
7
11
5
9
-
*TEA recoveries >150% are consistent with TEA being a native species to this material, as evidenced by
blank coupon samples. TEA and DEA are not present in sovent blank or wipe blank samples, but are
detected at low levels on the material blank sample, suggesting that TEA and DEA exist for this
material.fng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the
test area of the coupon (100 cm2).
27
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NHSRC/NIOSH SAP
Revision Date: 3/30/11
Page 28 of 38
FORMICA
Average Spike
Concentration
(ng/hiL) (n=7)
50
75
100
150
Average
Formica Blank
Average Spike
Concentration
(ng/cm2)t
(n=7)
0.50
0.75
1.00
1.50
Average
Formica Blank
DEA
Average
Recovery
(ngAnL)
56.97
49.13
68.47
99.18
2.41
Average
Recovery
(ngytm2)
0.57
0.49
0.69
0.99
0.02
%
Recovery
113.93
65.51
68.47
66.12
-
%
Recovery
113.93
65.51
68.47
66.12
-
%
RSD
25
6
7
10
-
%
RSD
25
6
7
10
-
DEA-d,
o
Average
Recovery
(ngAnL)
37.49
41.23
62.29
93.05
27.00
Average
Recovery
(ngyfcm2)
0.37
0.41
0.62
0.93
0.27
%
Recovery
74.97
54.97
62.29
62.03
54.01
%
Recovery
74.97
54.97
62.29
62.03
54.01
%
RSD
6
3
5
12
-
%
RSD
6
3
5
12
-
tng/cm2 calculation was performed by dividing the concentration spiked onto the surface by the test area
of the coupon (100 cm2).
METAL
Average Spike
Concentration
(ng/hiL) (n=7)
50
75
100
150
Average Metal
Blank
TEA
Average
Recovery
(ngAnL)
147.19
145.46
172.36
174.86
117.57
%
Recovery
294.37*
193.95*
172.36*
116.57
-
%
RSD
10
18
13
9
-
EDEA
Average
Recovery
(ngAnL)
42.30
58.43
72.25
91.17
0
%
Recovery
84.60
77.91
72.25
60.78
-
%
RSD
7
8
9
14
-
MDEA
Average
Recovery
(ng/rnL)
42.76
59.01
70.18
71.68
0
%
Recovery
85.52
78.68
70.18
47.79
-
%
RSD
6
6
6
14
-
*TEA recoveries >150% are consistent with TEA being a native species to this material, as evidenced by
blank coupon samples and discussed in section 13.3. TEA and DEA are not present in sovent blank or
wipe blank samples, but are detected at low levels on the material blank sample, suggesting that TEA and
DEA exist for this material.
28
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NHSRC/NIOSH SAP
Revision Date: 3/30/11
Page 29 of 3 8
METAL
Average Spike
Concentration
(ngyhiL) (n=7)
50
75
100
150
Average Metal
Blank
DEA
Average
Recovery
(ng/hiL)
64.41
80.65
85.62
97.95
16.28
%
Recovery
128.81
107.54
85.62
65.30
-
%
RSD
5
5
5
4
-
DEA-d8
Average
Recovery
(ngyhiL)
45.32
69.33
72.87
96.35
50.25
%
Recovery
90.64
92.44
72.87
64.23
100.50
%
RSD
10
6
4
3
-
GLASS
Average Spike
Concentration
(ngyhiL) (n=7)
50
75
100
150
Average Glass
Blank
TEA
Average
Recovery
(ng/hiL)
219.24
256.12
256.18
260.51
202.57
% Recovery
438.48*
341.50*
256.18*
173.67*
-
%
RSD
6
10
12
8
-
EDEA
Average
Recovery
(ngyhiL)
25.93
49.72
52.56
78.22
0
%
Recovery
51.87
66.29
52.56
52.14
-
%
RSD
7
5
14
12
-
MDEA
Average
Recovery
(ng/hiL)
30.28
56.65
55.44
83.81
0
%
Recovery
60.56
75.53
55.44
55.87
-
%
RSD
6
4
14
10
-
*TEA recoveries >150% are consistent with TEA being a native species to this material, as evidenced by
blank coupon samples and discussed in section 13.3. TEA and DEA are not present in sovent blank or
wipe blank samples, but are detected at low levels on the material blank sample, suggesting that TEA and
DEA exist for this material.
29
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NHSRC/NIOSH SAP
Revision Date: 3/30/11
Page 30 of 38
GLASS
Average Spike
Concentration
(ng/hiL) (n=7)
50
75
100
150
Average Glass
Blank
DEA
Average
Recovery
(ngAnL)
57.51
91.64
77.2
119.25
18.83
%
Recovery
115.01
122.19
77.2
79.5
-
%
RSD
14
4
19
8
-
DEA-d,
o
Average
Recovery
(ngAnL)
29.68
60.67
54.96
91.52
52.47
%
Recovery
59.37
80.9
54.96
61.02
104.94
%
RSD
11
5
21
11
-
VINYL TILE
Average Spike
Concentration
(ngAnL) (n=7)
100
150
200
300
Average Vinyl
Blank
TEA
Average
Recovery
(ngAnL)
29.95
137.26
142.59
224.48
17.6
%
Recovery
29.95
91.51
71.3
74.83
-
%
RSD
25
7
7
10
-
EDEA
Average
Recovery
(ngAnL)
29.34
13.77
52.11
86.98
0
%
Recovery
29.34
9.18
26.06
28.99
-
%
RSD
11
11
8
15
-
MDEA
Average
Recovery
(ngAnL)
29.1
15.95
63.96
75.42
0
%
Recovery
29.1
10.63
31.98
27.79
-
%
RSD
9
7
7
16
-
30
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NHSRC/NIOSH SAP
Revision Date: 3/30/11
Page 31 of 38
VINYL TILE
Average Spike
Concentration
(ngAnL) (n=7)
100
150
200
300
Average Vinyl
Blank
DEA
Average
Recovery
(ngAnL)
36.09
31.91
70.5
78.61
7.00
%
Recovery
36.09
21.27
35.25
26.2
-
%
RSD
13
19
12
10
-
DEA
-------
NHSRC/NIOSH SAP
Revision Date: 3/30/11
Page 32 of 38
PAINTEDDRYWALL*
Average Spike
Concentration
(ng/hiL) (n=7)
500
Average Drywall
Blank
TEA
Average
Recovery
(ngAnL)
109.63
72.79
%
Recovery
21.93
-
%
RSD
10
-
EDEA
Average
Recovery
(ngAnL)
74.97
0
%
Recovery
14.99
-
%
RSD
17
-
MDEA
Average
Recovery
(ngAnL)
84.91
0
%
Recovery
16.98
-
%
RSD
18
-
PAINTEDDRYWALL*
Average Spike
Concentration
(ngAnL) (n=7)
500
Average
Drywall Blank
DEA
Average
Recovery
(ngAnL)
87.02
23.11
%
Recovery
17.4
-
%
RSD
18
-
DEA-d8
Average
Recovery
(ngAnL)
56.57
39.76
%
Recovery
11.31
79.52
%
RSD
20
-
*Recoveries of all target analytes from this surface are below the acceptable range provided in this SAP.
As a result, the SAP should not be used to identify these analytes in relation to this specific surface.
Although porosity of the surface is most likely the culprit for low recoveries, further analysis should be
performed to determine definitive reasoning of poor recoveries from the surface.
32
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18.3 Illustration of wiping pattern on 100 cm surface
NHSRC/NIOSH SAP
Revision Date: 3/30/11
Page 33 of 38
in
VI
m
a.
73
OJ
v>
w
ro
Q.
E
co
i
in
VI
03
Q.
*
33
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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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