EPA/600/R-12/581 | August 2012 | www.epa.gov/ord
United States
Environmental Protection
Agency
Technical Report for Surface
Analysis of Nitrogen Mustard
Degradation Products by
Liquid Chromatography/
Tandem Mass Spectrometry
(LC/MS/MS)
EDEA &DEA
n.46 MDEA
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-12/581
August 2012
Technical Report for Surface Analysis of Nitrogen Mustard Degradation
Products by Liquid Chromatography/Tandem Mass Spectrometry
(LC/MS/MS)
Revision 1
United States Environmental Protection Agency
National Homeland Security Research Center
Cincinnati, OH 45268
and
Centers for Disease Control and Prevention
National Institute for Occupational Safety and Health
Cincinnati, OH 45213
Last Revised: 9/11
Effective Date:
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Disclaimer
The information in this technical document is comprised of multiple experiments with intent to develop a
single-laboratory-developed sampling and analytical procedure (SAP) for the surface analysis, using
wipes, of nitrogen mustard degradation products using LC-MS/MS (EPA 600/R-l 1/143). The research
has been funded wholly or in part by the U.S. Environmental Protection Agency (EPA), Office of
Research and Development (ORD), National Homeland Security Research Center (NHSRC) and in
collaboration with 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 and
EP10C000016. This document has been subjected to the Agency's review and has been approved for
publication. Note that approval does not signify the content necessarily reflects 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 and Method Development
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 organizations for their contributions towards
the development and/or review of this method. Erin Silvestri and Lukas Oudejans from the United States
Environmental Protection Agency (EPA), Office of Research and Development, National Homeland
Security Research Center; April Dupre from the Office of Water, Office of Ground Water and Drinking
Water, Standards and Risk Management Division; Larry Zintek from EPA region 5; Terry Smith from the
Office of Emergency Management; and Jack Pretty and Robert Striecher from the Centers for Disease
Control and Prevention, National Institute for Occupational Safety and Health.
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EXECUTIVE SUMMARY
The U.S. Environmental Protection Agency (EPA) is charged with developing tools and methodologies
that enable the rapid characterization of indoor and outdoor areas and water systems following terrorist
attacks or natural or manmade disasters. Chemical warfare agents (CWAs) and their degradation
products remain a high-priority concern due to the presence of primary warfare agents in the U.S.
chemical warfare agent inventory and some CWA degradation products can be as toxic as the parent
compounds. Nitrogen mustard agents are vesicant CWAs which can break down into environmentally
persistent degradation products. Sample stability studies suggest nitrogen mustard degradation products
can persist in the environment for several weeks, and probably much longer depending on the associated
environmental conditions. If an incident were to occur within an indoor setting, versatile sampling
procedures are needed to detect CWA degradation products from various CWAs, including nitrogen
mustard, from multiple types of contaminated surfaces (e.g., walls, floors and furniture).
Several different wipes were tested, but only the filter paper wipe was considered viable straight out of
the box. Filter paper wipes were selected over other wipes (including cotton gauze and non-woven
polyester fibers) because they did not contain peaks that interfered with the target analytes, resulted in the
highest percent recoveries and the lowest background levels during sample analysis. For nitrogen
mustard and its degradates, cotton gauze would be an inappropriate choice unless the gauze is pre-cleaned
and treated prior to use, a time-consuming and potentially costly approach, due to the contamination of
TEA and DEA within the wipe. Sampling kits provided to samplers in the field, equipped with pre-
packaged cotton gauze, would need to be tested to ensure that targeted analytes are not present, whereas
no pretreatment is needed for filter paper.
Selective analysis methods must be employed to detect the appropriate degradation products from the
environmental sample. The described sampling and analysis procedure employs the use of LC/MS
because of its versatility, which will aid laboratories with the enhanced capability and capacity to analyze
certain environmental matrices for polar CWA degradation products. Gas chromatography-mass
spectrometry analysis requires an extra derivatization step, which is often problematic. Although LC-MS
analytical methods currently exist for nitrogen mustard degradation products in water, there are no known
wipe sampling collection and analysis protocol for the detection of nitrogen mustard degradation products
from contaminated surfaces.
This report describes experimental details for the research method development and application, by a
single laboratory, to assess the recoveries of nitrogen mustard degradation products from porous (vinyl
tile, painted drywall, wood) and nonporous (laminate, galvanized steel, glass) surfaces. Performance data
(method detection limit and precision and accuracy) are available to demonstrate the fitness-for-purpose
towards developing a protocol for nitrogen mustard degradation products in that single laboratory.
Analysis of blank samples revealed the presence of TEA and DEA on all tested surfaces, most notably in
metal, glass, painted drywall and wood. This was expected given the common commercialization of TEA
and DEA in industrial applications (e.g., metal working fluids, soaps, foaming agents, cleaning agents,
etc.). Samples are collected from spiked surfaces with wipes and carried through methanol extraction by
sonication, filtration, and concentration 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 from the application of wipes to a laminate surface,
following 40 CFR Part 136, Appendix B, as part of EPA's guidelines for determining a method detection
limit. Percent recoveries for the laminate surface were 66-109% for all targeted nitrogen mustard
degradation products. The resulting method detection limits obtained from the wipes were 0.12 ng/cm2
for triethanolamine (TEA), 0.06 ng/cm2 for 7V-ethyldiethanolamine (EDEA), 0.07 ng/cm2 for 7V-
methyldiethanolamine (MDEA), and 0.04 ng/cm2 for diethanolamine (DEA).
iv
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Table of Contents
Disclaimer ii
Foreword iii
Acknowledgments iii
Executive Summary iv
List of Figures vi
List of Tables vii
List of Acronyms and Abbreviations viii
1.0 Introduction 1
2.0 Determination of Analytes by LC/MS/MS 2
2.1 Mobile Phase Composition and Gradient Conditions 3
2.2 Mass Spectrometric Parameters 3
2.3 Establishing an Instrument Detection Limit (IDL) 5
3.0 Selection of Wipe Materials 6
4.0 Determination of the Recovery of Analytes from Surface Materials 8
4.1 Sample Collection/Extraction/Concentration Procedures 9
4.1.1 Sample Collection 9
4.1.2 Extraction 10
4.1.3 Concentration 11
4.2 Identification and Quantitation 11
4.3 Waste Handling and Prevention 11
5.0 Results and Discussion 11
5.1 Detection Limit Determination 11
5.1.1 EPA DL 12
5.1.2NIOSHMDL 13
5.2 Precision and Accuracy Determination 16
5.3 Hoi ding Time Study 20
5.4 Sonication Study 21
6.0 Conclusion 22
7.0 References 23
Appendix A 25
Appendix B: Statistical Data and Calculations for Holding Time Studies 28
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List of Figures
Figure 1. Chromatogram depicting the IDLs for nitrogen mustard degradation compounds (DBA,
surrogate DEA-d8, MDEA, EDEA, and TEA, respectively, from top to bottom) in methanol. (Analyte
names and MRM transitions are listed on the top right of each chromatogram.) 6
Figure 2. Illustration depicting the spiking pattern on a 100 cm2 surface coupon 10
Figure 3. Illustration of wiping pattern on 100 cm2 surface 10
VI
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List of Tables
Table 1. Calibration levels for nitrogen mustard degradation analytes 2
Table 2. Liquid chromatography gradient conditions and parameters 3
Table 3. MRM ion transitions and variable mass spectrometer parameters for each analyte 4
Table 4. ESI+ MS/MS operating conditions 4
Table 5. Retention times (RTs), instrument detection limits (IDLs) and signal:noise (S/N) ratios of
nitrogen mustard degradation analytes 5
Table 7. Materials tested for the wipe analysis of nitrogen mustard degradation products 9
Table 8. List of consumable materials used during sampling 9
Table 9. Student's t-statistic value as it relates to the number (n) of replicate samples 12
Table 10. EPA calculation for DL and LOQ in ng/cm2 and ng/mL for nitrogen mustard degradation
analytes on surfaces 13
Table 11. NIOSH calculation for DL and LOQ determination for nitrogen mustard degradation analytes
on surfaces 14
Table 12. Precision andAccuracy (P&A) data for wipe analysis of nitrogen mustard degradation analytes
on surfaces 16
Table 13. Holding time sample stability of wipes spiked with nitrogen mustard degradation analytes.... 21
Table 14. Analyte recovery from analyte spiked wipes at various sonication intervals 22
Table A-l. Reagents and CAS numbers 25
Table A-2. Detection limit (DL) results for (n=7) samples for wiping the surface of coupons with a 100
cm2 area 26
Table B-l. F-test analysis for nitrogen mustard degradation analytes 28
Table B-2. T-test analysis for nitrogen mustard degradation analytes 29
VII
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List of Acronyms and Abbreviations
ACN Acetonitrile
CAS Chemical Abstract Service
CWA Chemical Warfare Agent
DHHS U.S. Department of Health and Human Services
DBA Diethanolamine
DL Detection Limit
EDEA TV-Ethyldiethanolamine
EPA U.S. Environmental Protection Agency
ESI Electrospray lonization
HILIC Hydrophobic Interaction Liquid Chromatography
HPLC High performance liquid chromatography
IDL Instrument Detection Limit
LC Liquid Chromatography
LC-MS/MS Liquid Chromatography Coupled with Tandem Mass Spectrometry
LOD Limit of Detection
LOQ Limit of Quantitation
MDEA TV-Methyldiethanolamine
MDL Method Detection Limit
MRM Multiple Reaction Monitoring
MS Mass Spectrometry
MSDS Material Safety Data Sheets
MS/MS Tandem Mass Spectrometry
NFL^OAc Ammonium Acetate
NHSRC National Homeland Security Research Center
NIOSH National Institute for Occupational Safety and Health
NMAM NIOSH Manual of Analytical Methods
ORD Office of Research and Development
PPB Parts per billion
PPE Personal Protection Equipment
PPM Parts per million
P&A Precision and Accuracy
PVDF Polyvinylidene Fluoride
PTFE Polytetrafluoroethylene
QAPP Quality Assurance Project Plan
QA Quality Assurance
QC Quality Control
R2 Correlation Coefficient
RSD Relative Standard Deviation
RT Retention Time
RTS Retention Time Shift
SAM Selected Analytical Methods for Environmental Remediation and Recovery
SAP Sampling and Analytical Procedure
SD Standard Deviation
S/N Signal to Noise
SOP Standard Operating Procedure
TEA Triethanolamine
TQD Triple Quadrupole Detector
UPLC Ultra-Performance Liquid Chromatography
VIM
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IX
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1.0 Introduction
After the terrorist attacks of September 11, 2001, and the anthrax attacks in the fall of 2001, reports prepared by the
Environmental Protection Agency (EPA) identified several research gaps that needed to be addressed to improve the
country's preparedness in the event of a terrorist attack. A critical area identified was the need for a list of standardized
analytical methods to be used by all laboratories when analyzing samples from a homeland security incident. 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 Follow ing Homeland Security Events [1], which 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 that are analyzing a
large number of samples associated with chemical, biological, biotoxin or radiological contamination in environmental
matrices. 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, while some
methods are standardized for selected chemicals in specific matrices, not all of the analytical methods listed in the SAM
document address all possible matrices (e.g., water, soil, air, surfaces) encountered in sample collection following an
incident. Some of the analytical methods in SAM have been verified in a single laboratory, but most still need to undergo
verification with respect to a specific contaminant in association with a specific matrix.
Contamination by Chemical Warfare Agents (CWAs) and their degradation products remain an environmental concern
due to their persistence, toxicity and presence of primary warfare agents in the U.S. CW agent inventory [2] and the
possible threat of the use of these agents in a homeland security-type incident. While compiling methods for SAM, CWAs
and their degradation products were selected for inclusion based on environmental persistence and toxicity. Many of the
CWA degradation products are in need of more appropriate methods that will enhance sampling and analysis capability to
improve lab capacity by using better analysis techniques, such as liquid chromatography-mass spectrometry (LC-MS), an
appropriate and powerful technique for polar CWA degradation product analysis. Such degradation products are not
analyzed well using GC-MS without a derivatization step, a tedious and time-consuming process when throughput of
numerous samples is critical. For instance, derivatization typically does not result in complete transformation of the target
analytes with respect to their analysis products, especially when water is present, resulting in detection limit
complications. Establishing a collaborative effort with additional technical expertise and research capabilities in
analytical methods to that of the U.S. EPA seemed appropriate for CWA degradation products involving specific matrices.
The National Institute for Occupational Safety and Health (NIOSH) was chosen because of their capabilities in analytical
methods for several chemicals using High Performance Liquid Chromatography (HPLC), which can be found in the
NIOSH Manual of Analytical Methods (NMAM) [3]. NIOSH has used LC/MS to measure chemotherapeutic drugs,
isocyanates, and components of metal-working fluids, all polar analytes. Furthermore, Ultra-Performance Liquid
Chromatography (UPLC) is also becoming a powerful tool in the field of analytical chemistry for its enhanced ability for
rapid throughput of samples. Because of the power and versatility of LC-MS, application to CWA degradation products
may provide laboratories with improved capability to analyze certain environmental matrices after an incident.
The information within this technical document is comprised of multiple experiments resulting in the successful efforts, as
well as complications, that may arise when working with nitrogen mustard degradation products on surfaces associated
with the single-laboratory-developed sampling and analysis procedure (SAP) using LC-MS/MS (EPA 600/R-l 1/143).
Experimental details described wthin will help fill data gaps related to the need for wipe sampling and analysis during or
after an incident involving CWAs and their degradation products. Wipe sampling is the preferred collection method
because there is less destruction of the tested surface, and wipe sampling can be performed quickly and easily when direct
extraction is not always possible. The purpose of the SAP was to develop a method for the detection and recovery of
CWA degradation products, specifically ethanolamine-based nitrogen mustard degradation products, from various porous
(vinyl tile, painted drywall, wood) and mostly nonporous (laminate, galvanized steel, glass) surfaces using wipes with
proper quality assurance objectives set forth in the Quality Assurance Project Plan (QAPP). Filter paper wipes were
selected over other wipes (including cotton gauze, glass fiber filter and non-woven polyester fiber wipes) because the
filter paper wipes did not produce chromatographic peaks that interfered with the target analytes, resulting in the highest
percent recoveries and lowest background levels for the filter paper wipes during sample analysis. Research investigating
the various wipes was described in report EPA 600/R-l 1/143 [5]. The method detection limits obtained from the filter
paper wipes with a laminate surface were 0.12 ng/cm2 for triethanolamine (TEA), 0.06 ng/cm2 for 7V-ethyldiethanolamine
1
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(EDEA), 0.07 ng/cm2 for 7V-methyldiethanolamine (MDEA), and 0.04 ng/cm2 for diethanolamine (DEA). Precision and
accuracy data were generated from each tested surface fortified with these analytes. Various parameters, including the
selection of surface materials, instrumental factors, limits of detection and quantitation and recoveries of the analytes
from actual surfaces, were investigated and used to demonstrate the fitness-for-purpose of the method for nitrogen
mustard degradation products in a single laboratory.
2.0 Determination of Analytes by LC/MS/MS
Four compounds known to be degradation products of nitrogen mustard CWAs are triethanolamine (TEA), 7V-
ethyldiethanolamine (EDEA), 7V-methyldiethanolamine (MDEA) and diethanolamine (DEA). Neat standards of each
compound (TEA, DEA, EDEA, MDEA and DEA-d8) were used to prepare methanol solutions containing all analytes.
Approximate 1000 parts per million (ppm) stock standard solutions containing each individual compound were prepared
(e.g., 44.4 nL TEA, 45.87 jiL DEA, 49.31 jiL EDEA, 48.08 jiL MDEA and 45.87 jiL DEA- d8 in 50 mL of methanol),
diluted to make 10 ppm solutions and each 10 ppm stock solution was utilized to develop a calibration solution containing
all of the target analytes at an approximate concentration of 500 ng/mL (Level 7) (e.g., 1.25 mL of each 10 ppm stock
solution added to a 25 mL flask and diluted to the mark with methanol). The remaining concentration levels 1 through 6
were prepared from the level 7 solution, with all approximate concentrations in parts per billion (ppb) exhibited in Table
1. All spiking and calibration solutions were stored in amber volumetric flasks at 4 °C (± 2 °C). Holding time study data
on the stability of the solutions can be found in Section 5.3. A calibration curve was generated from analyte concentration
levels 1-7 and was qualitatively and quantitatively determined by LC-MS/MS in a low to high order to ensure that no
carryover would occur.
Table 1. Suggested target calibration levels for nitrogen mustard degradation analytes (ng/mL)
Analyte/Surrogate
Triethanolamine
/V-Ethyldiethanolamine
/V-Methyldiethanolamine
Diethanolamine
Diethanolamine-ds
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
After the calibration curve has passed all quality control verifications described in the SAP and Section 4.2, sample
collection and processing procedures were followed as described in Section 4.1. For example, solutions of appropriate
concentration were used to spike a surface. The sample was allowed to dry, collected using a filter wipe, extracted,
filtered, concentrated and analyzed to determine the presence of nitrogen mustard degradation products. Direct injection
without derivatization, using liquid chromatography/tandem mass spectrometry (LC-MS/MS) provided complete analysis
of each sample. Diethanolamine-ds (DEA-d8) served as the surrogate standard for this project because its properties are
similar to all of the analytes of interest and the deuterated compound is unlikely to be present in environmental samples.
Although certain LC-MS effects such as ESI suppression/enhancement are not taken into account, the deuterated
compound still satisfies the quality control provisions, i.e., when added at a known concentration to the sample prior to
processing, the deuterated compound provides a measure of the overall efficiency of the recovery. The analyzed data
suggest that the surrogate is an appropriate choice at this time. The instrument's software served as a valuable resource
tool for providing accurate recovery results. At the time, it was more advantageous to use the deuterated compound as a
surrogate for the sampling process rather than the internal standard. All qualitative and quantitative control parameters
were monitored to ensure all that quality assurance protocols were met.
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2.1 Mobile Phase Composition and Gradient Conditions
Alternative mobile phase compositions for possible use with the target analytes were tested, including ammonium acetate
and ammonium formate buffers. Although ammonium formate did produce a lower background signal than the acetate
constituent, the ammonium formate solution produced chromatographic peaks for the four compounds of interest that
overlapped chromatographically. As a result, the ammonium acetate was chosen over the formate. Different
concentrations of ammonium acetate (10 mM, 15 mM and 25 mM) were also investigated. Sensitivity of each analyte
peak increased as ammonium acetate concentrations were increased; however, higher molar concentrations of the buffer
may result in retention time shifts and/or result in blockage of the column or guard column. The highest concentration of
25 mM ammonium acetate was chosen for sensitivity reasons, but if any of the problems mentioned above occur, lower
concentrations should be used.
In addition to the use of ammonium acetate mobile phase for the analysis of DBA, TEA, MDEA, EDEA and DEA-d8,
acetonitrile was also used to produce a binary mixture of the two solutions under gradient conditions. For this experiment,
the instrument was equipped with a binary solvent system; however, in some cases, instruments can be outfitted with
ternary solvent systems for performing gradient elutions and the ternary system may be used, if applicable. Due to the
manufacturer's suggestion for operation of the Hydrophobic Interaction Liquid Chromatography (HILIC) column, the
ammonium acetate solution was buffered to below pH 5 with acetic acid. Deterioration of the column, working outside
this range, would otherwise occur quicker than anticipated. Other HILIC columns may have a wider pH range tolerance
capable of using the solvents described herein without the need for buffering, which may be worth using, when applicable.
The binary solvent system for the proposed gradient consisted of two different solutions (A and B). Solution A consisted
of 25 mM ammonium acetate at pH 4.2 (buffered with glacial acetic acid) and 5% acetonitrile added to prevent microbial
growth (A: 95% 25 mM ammonium acetate at pH 4.2 and 5% acetonitrile). Solution B consisted of 95% acetonitrile and
5% 25 mM ammonium acetate to achieve the overall approximate composition of 25 mM for ammonium acetate.
Gradient conditions are displayed in Table 2. Preliminary studies using Ultra-performance liquid chromatography
(UPLC) technology can shorten run times to less than 10 minutes when throughput becomes an issue using a specialty
HILIC UPLC column and LC unit capable of handling higher pressures. Since most laboratories are not equipped with an
LC unit capable of handling such pressures, the data are not included in this study report.
Table 2. Liquid chromatography gradient conditions and parameters
Time
(min)
0
1
2
12
16
17
18
21
Flow
(|j,L/min)
300
300
300
300
300
300
300
300
%
Solution A*
10
10
13
13
15
30
10
10
%
Solution Bft
90
90
87
87
85
70
90
90
fA: 95/5 - 25mM Ni^OAC (pH 4.22)/Acetonitrile (ACN)
ftB: 95/5 - ACN/25mM NtLjOAC
* Column Temperature: 30 °C
*Autosampler Temperature: 15 °C
* Equilibration time: 3 minutes
*Column: Atlantis™HILIC silica, 100 mm x 2.1 mm, 3 jam particle size
2.2 Mass Spectrometer Parameters
Optimal transitions for cone voltages and collision energies were determined by two different techniques. The first
technique involved manually tuning the instrument for each of the five analytes, resulting in proper cone voltages with the
strongest signal intensity for each analyte. The strongest signal intensity was defined as having the greatest signal
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intensity with uniform width over a span of 1 Dalton at half the peak height. Daughter ions were chosen based on the
collision energy that provided the greatest signal intensity of the intended daughter peak relative to the parent ion peak.
The second technique used the MassLynx™ software specific to the Waters TQD instrument (AutoTune Wizard™) and
required only the input of the original masses for each of the analytes of interest. The instrument software was capable of
accurately determining the proper cone voltages resulting in the strongest signal intensities for each analyte as well as the
appropriate collision energies for each daughter ion. Automatic tuning performed by the instrument's software was the
preferred tuning method because it was quick and efficient and found the same MRM mass transitions as the manual
tuning technique. If an incorrect or undesired daughter ion was chosen by the program during the automatic tuning
process, the program was re-run until the correct daughter ion was found. Manual tuning can also be performed, as
described above. Mass transitions and variable mass spectrometer parameters (Table 3 and 4) were selected by the
automatic tuning software for the analysis of the nitrogen mustard degradation analytes. The process by which all the
analytes can be tuned simultaneously is quick, requiring only a couple of minutes, making the simultaneous tuning
technique the preferred method. The use of the instrument capabilities to tune properly to the target analytes will save
time, an efficiency that can be crucial when large quantities of samples need to be analyzed and quick throughput is
necessary.
Table 3. MRM ion transitions and variable mass spectrometer parameters for each analyte
Analyte
Triethanolamine
/V-Ethyldiethanolamine
/V-Methyldiethanolamine
Diethanolamine
Diethanolamine-ds (Surrogate)
Cone
voltage
30
30
30
30
30
MRM mass transition
(parent — > daughter)
150.09^132.10
134.02 -» 116.10
120.03^102.00
106.00^88.10
114.20^96.22
Collision
energy (eV)
12
14
12
12
12
Table 4. ESI+ MS/MS operating conditions
MS Parameter (ESI+)
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
Setting
1.0 kV
See Table 3
2 Volts
0.2 Volts
150°C
300 °C
800 L/hr
50 L/hr
14.5
14.5
0.5 eV
1
See Table 3
1
15.0
15.0
0.5 eV
-560
3.0x10'J Torr
0.005 seconds
0.005 seconds
1
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Span
Dwell
0.1 Daltons
0.3 Seconds
2.3 Establishing an Instrument Detection Limit
The determination of the capability of an instrument to detect target analytes at very low levels is important and can be
accomplished by establishing the instrument's detection limit (IDL). Successive decreases in solution concentration
starting with a known concentration containing all five compounds followed by analysis at each concentration were used
to observe signal:noise (S/N) ratios at each concentration. A signal:noise ratio of at least 3:1 was achieved with low level
concentrations to ascertain the IDL with a Waters Acquity™ and tandem quadrupole detector (TQD), for liquid
chromatography and mass spectrometric analysis, respectively. Table 5 and Figure 1 depict the IDLs obtained for the five
nitrogen mustard degradation compounds using a Waters Acquity and TQD system.
Table 5. Retention times (RTs), approximate instrument detection limit (IDL) concentrations and signahnoise (S:N) ratios of
nitrogen mustard degradation analytes
Analyte of Interest
Triethanolamine (TEA)
/V-Ethyldiethanolamine (EDEA)
/V-Methyldiethanolamine (MDEA)
Diethanolamine (DEA)
Diethanolamine-d8 (DEA-d8)
RT*
(minutes)
9.7
11.1
13.0
12.1
12.2
IDL
(ng/mL)
1.0
1.0
1.0
1.0
1.0
Signal: Noise
Ratio (S:N)
8.36
5.68
5.77
4.07
5.49
Retention times should fall within 5% of the given value; otherwise re-analysis may be necessary.
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Etham+IS-STD-1 ppb-8-10-09-3
S'N PtP=4 07
1 MRM of 5 Channels ES+
105 96 > 88 3 (DEA)
268e3
,s 820913994.Ji32J1.03
iOO 1000
Etham+IS-STD-1 ppb-8-10-09-3
1200 1400
S/N PtP=5 49
•800 2000
1 MRM 0(5 Channels ES+
1142 > 96 22 (DEA-dS)
1 13e3
X277 1400 1434 16334654 " 35 13 66 19 21 20 10 2082
800 1000
Etham*IS-STD-1 ppb-8-10-09-3
1200
1400
Etham
*
Etham
• 8ft
4
800 1000
•HS-STD-1ppb-B-10-09-3
S'M PtP=5 77
1332
IS 00 1800 2000
1: MRM of 5 Channels ES*
120.03 > 102.2 (N-Me-DEA)
1 9663
1200
1400
S/N PtP=5 S8
11,1 55 12 35
1600 1800 2000
1 MRM of 5 Channels ES-*-
13402> 1163(N-Et-DEA)
5 02e3
800 1000
•IS-STD-1 ppb-8-10-09-3
S/N PtP=8 36
689 740 3 12 334 TT 1045
1200
11 72
1400
1315 1455
2Q32
1600 1800 2000
1 MRM of 5 Channels ES+
15003 > 132 3 (TEA:
245e3
1599 1697 184° 193\1930 2082
Time
800 1000 1200 1400 H600 1800 2000
Figure 1. Chromatogram depicting the IDLs for nitrogen mustard degradation compounds (DEA, surrogate DEA-d8, MDEA,
EDEA, and TEA, respectively from top to bottom) in methanol. (Analyte names and MRM transitions are listed on the top
right of each chromatogram)
3.0 Selection of Wipe Materials
In general, wipes are comprised of different materials from various manufacturers. Furthermore, wipe sampling kits can
be assembled and are intended to contain pre-cleaned materials. Although wipe sampling kits were not tested in this
experiment, the sterile wipes that were tested in this study were found to contain contaminants that produced
chromatographic interferences with the analytes of interest. Therefore investigation of pre-cleaned kits should be
explored to ensure that no interferences or contamination is present. In an effort to understand wipe sampling methods,
analytes were tested on several wipe materials to determine wipe efficacy assessing two different factors, recovery and
possible interferences with target analytes. The analysis of different types of wipes evaluated which wipe would have the
least amount of interference with the compounds of interest and provide the lowest background signal for a blank extract
from a wipe in methanol solution. Wipes tested in this experiment were both common wipes, typically used in wipe
methods, and not-so-common wipes. Common wipes included sterile cotton gauze pads (Certi-Gauze Pad™), Ghost™
wipes (used for metals analysis), and an Alphawipe® (a continuous synthetic polyester, low particle, high absorptivity
wipe). Alphawipes are laundered and packaged in a clean room. Less common wipes used for experimentation were
Millipore™ nitrocellulose fiber filters, Whatman® glass microfiber filter paper, Reeve Angel glass microfiber filter paper,
Whatman® filter paper, and a nonwoven polyester fiber cloth from National Nonwovens Textile (thick absorbent cloth).
The nitrocellulose filter deteriorated in methanol resulting in a cloudy solution and clogged filters and was not used for the
remainder of the experiment. Ghost™ wipes were also dropped due to complications from the extraction procedure and
low recoveries of the target analytes.
Performance results from each wipe material spiked with nitrogen mustard degradation products are shown in Table 6 and
Figure 2. Cotton gauze and non-woven polyester fiber cloth wipes exhibited high levels of TEA and DEA contamination
present in blank and known spiked low concentrations of the target analytes. Recoveries of the target analytes from
-------
Alphawipes were low and exhibited a peak possible interfering with TEA (Figure 2). Both glass fiber and filter paper
wipes did not contain any interference peaks associated with the target analytes and appeared to be free of any
contaminants that would affect the background of a blank sample. Glass fiber filter wipes were not as robust as filter
paper wipes, disintegrating upon wiping any surface. Filter paper was the preferred wipe to use on all surfaces for this
study because of the lack of necessity for a pre-cleaning step because of interferences and contaminants and good
recoveries of the targeted analytes.
Table 6. Average recoveries from three different approximate spike concentrations tested on different wipe materials
COTTON GAUZE
Spike
Concentration
(ng/mL)
50
25
10
TEA
Recovered
(ng/mL)
74
64
68
%
Recovery
148
254
682
EDEA
Recovered
(ng/mL)
14
7
4
%
Recovery
28
27
35
MDEA
Recovered
(ng/mL)
15
9
5
%
Recovery
30
37
49
DEA
Recovered
(ng/mL)
217
173
168
%
Recovery
433
691
1680
NON-WOVEN POLYESTER FIBER CLOTH
Spike
Concentration
(ng/mL)
50
25
10
TEA
Recovered
(ng/mL)
484
548
508
%
Recovery
968
2190
5080
EDEA
Recovered
(ng/mL)
41
27
13
%
Recovery
82
107
126
MDEA
Recovered
(ng/mL)
38
25
12
%
Recovery
75
99
120
DEA
Recovered
(ng/mL)
97
42
37
%
Recovery
193
168
373
ALPHAWIPE™
Spike
Concentration
(ng/mL)
50
25
10
TEA
Recovered
(ng/mL)
11
5
0.9
%
Recovery
23
19
9
EDEA
Recovered
(ng/mL)
11
5
2
%
Recovery
22
21
18
MDEA
Recovered
(ng/mL)
11
5
2
%
Recovery
21
20
17
DEA
Recovered
(ng/mL)
9
4
1
%
Recovery
18
16
10
WHATMAN GLASS FIBER
Spike
Concentration
(ng/mL)
50
25
10
TEA
Recovered
(ng/mL)
8
3
0.1
%
Recovery
16
14
1
EDEA
Recovered
(ng/mL)
53
24
9
%
Recovery
106
97
86
MDEA
Recovered
(ng/mL)
28
11
3
%
Recovery
56
45
34
DEA
Recovered
(ng/mL)
18
6
2
%
Recovery
36
24
19
WHATMAN FILTER
Spike
Concentration
(ng/mL)
50
25
10
TE
Recovered
(ng/mL)
41
20
7
A
%
Recovery
82
78
74
ED
Recovered
(ng/mL)
50
26
9
EA
%
Recovery
101
103
87
MC
Recovered
(ng/mL)
42
22
8
IEA
%
Recovery
83
89
76
DE
Recovered
(ng/mL)
38
20
6
A
%
Recovery
77
80
62
7
-------
DBA
Ethanolamines500+IS-50ppb-Blank-5-8-09
Elham+IS-STD-500ppb-5-8-09-2
1
9.64
l\
08-May-200915:12:45
1: MRM of 5 Channels ES+
TIC
5.78e5
20.00
1: MRM of 5 Channels ES+
TIC
2.25eS
20.00
1: MRM of 5 Channels ES+
TIC
3.99*3
20.00
1: MRM of 5 Channels ES+
TIC
7.14e4
20.00
1: MRM of 5 Channels ES+
TIC
1.79e3
20.00
1: MRM of 5 Channels ES-f
TIC
1.81e3
20.00
1: MRM of 5 Channels ES+
TIC
1.91e3
Time
Figure 2. MRM chromatograms exhibiting contaminants within wipe materials with the addition of only methanol to the
wipe. Sample a) is strictly for comparison, a) Nitrogen mustard degradation products stock standard at 500 ng/mL, b) Cotton
gauze blank in methanol, c) Alphawipe® blank in methanol, d) Nonwoven polyester fiber wipe blank in methanol, e) Reeve®
glass fiber wipe blank in methanol, f) Whatman® glass fiber wipe blank in methanol, g) Whatman® filter paper blank in
methanol.
4.0 Determination of the Recovery of Analytes from Surface Materials
A variety of surface materials, both porous and nonporous, were tested to emulate what would possibly be encountered at
common urban settings. Porous surfaces (vinyl tile, wood, painted drywall) and nonporous surfaces (glass, steel,
laminate) and are listed in Table 7. All materials could be obtained from commercial suppliers and/or manufacturers.
Other materials such as carpet and concrete were part of the tested materials but were subsequently dropped from the
experiment because they did not produce recoveries above the analytical protocol detection limit. Further investigation
with the described porous materials is needed to determine if recovery from such a surface is possible. A pre-determined
area of 100 cm2 (10 cm x 10 cm) was selected for surface wiping. Analytes were spiked onto each surface to be tested
and then wiped with the chosen filter paper wipe to assess wipe efficacy and recovery. This approach was selected to
mimic an actual field sampling event. Sample collection, extraction and processing procedures using the filter paper are
discussed in the subsequent sections.
8
-------
Table 7. Surface materials tested for the wipe analysis of nitrogen mustard degradation products
Surface Material
Glass
Vinyl Tile
Formica
Pretreated Pine Wood (2" x 4" pine)
Galvanized steel
Painted Drywall (paint & primer in
one, single coat, acrylic)
Manufacturer/Vendor
Carolina Glass Co./Lowe's
Armstrong/Home Depot
Wilsonart® Laminate/Home Depot
Home Depot
McMaster-Carr
BEHR/Home Depot
4.1 Sample Collection/Extraction/Concentration Procedure
All wipe sampling and collection materials used during the sampling and processing of samples were tested to ensure that
none of the target analyte species were native components of any of the sampling materials used in the method and that no
significant loss (>10%) of analyte species occurred. Materials used in sampling are listed in Table 8. Polypropylene
sampling containers were used over conventional glassware because preliminary studies suggest target analytes may have
a propensity to adhere to the glassware affecting recoveries. If different sampling materials than those described within
this report are used, it will be necessary to test the alternate materials to ensure they do not contain any of the targeted
analytes of concern or result in significant losses.
Table 8. List of consumable materials used during sampling
Sampling and Collection 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 urn PVDF
Waters 1 .8 ml amber glass vials with pre-slit
silicone polytetrafluoroethylene (PTFE) screw cap
Manufacturer
GE Healthcare Life
Sciences (Piscataway, NJ)
Nalge Nunc International
(Rochester, NY)
Becton, Dickinson and
Company (Franklin Lakes,
NJ)
Corning Incorporated
(Corning, NY)
EMD Millipore (Billerica,
MA)
Waters Corp. (Milford, MA)
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)
4.1.1 Sample Collection
Sample coupon sizes of the various surface materials (i.e., glass, stainless steel/metal, formica, vinyl, and wood) were cut
to provide a 10 cm x 10 cm (100 cm2) template. Each coupon was spiked with the appropriate concentration of a solution
containing DBA, TEA, MDEA and EDEA in a pattern comprised of five equivalent spots (Figure 3). The solution was
allowed to dry on the surface for approximately 5-10 minutes to ensure complete solvent evaporation. Two separate wipes
were used to wipe the surface, with each wipe wetted with 300 u,L of methanol (sufficient to wet the entire wipe).
Coupons were wiped in a Z-wipe pattern (Figure 4), with the first wipe used in a horizontal Z-wipe pattern and placed in a
125 mL Nalgene® polypropylene straight-sided jar with a polypropylene screw cap. The second wipe was used in a
9
-------
vertical Z-wipe pattern, placed in the same jar and capped.
O O
o
O O
Figure 3. Illustration depicting the spiking pattern on a 100 cm surface coupon.
ra
Q.
CM
ro
Q.
P
co
1
03
Q_
jr
Figure 4. Illustration of wiping pattern on 100 cm surface.
4.1.2 Extract!on
When the samples were ready to be analyzed, the deuterated surrogate, DEA-d8, was added to the jar and, after 5 minutes,
approximately 10 mL of methanol was added to the jar. The jar was subsequently capped. The solvent volume fully
immerses the wipes as they lie flat on the bottom of the jar. The jar was sonicated for 10-15 minutes in a water sonication
bath at room temperature. Sonication studies (section 5.4) suggest optimal extraction time periods were approximately
10-15 minutes. Following sonication, the resulting solution was drawn into a 10 mL disposable Luer-lok syringe with a
lock tip, fitted with a 0.22 (im polyvinylidene fluoride (PVDF) filter and filtered into a sterile 15 mL tube. Solvent
recovery was approximately 80-90% of the original 10 mL solution.
10
-------
4.1.3 Concentration
The solution was concentrated using a N2-evaporation apparatus with a temperature-controlled water bath, maintained at
45-50 °C for final analyte concentration (< 10 mL volume). Solutions were concentrated to below 2 mL, then diluted with
methanol to the 2 mL mark. The accuracy of the volume added to the tubes was comparable to that of an calibrated
pipette. Other collection vials/tubes with more accurate markings may be desired if lower detection is necessary. The
solution was lightly shaken, added to a 2 mL amber sample vial and analyzed by LC-MS/MS. Any remaining solution
was stored in a refrigerator at or below 4 °C (±2 °C).
4.2 Identification and Quantitation
Nitrogen mustard degradation compounds were analyzed at different concentrations, within the range of the calibration
curve (10-500 ng/mL). Some sample concentrations were different from the actual values of the curve to demonstrate
accuracy over various points of the calibration curve. (NOTE: All chosen concentrations were within the calibration
range to ensure analyte response linearity.) Even with a linear response function (where the R2 value is above 0.99), the
concentrations that were not identical to the calibration levels fell within the linear range with little to no bias illustrating
how well the wipe analysis concentrations fit along the curve. Qualitatively, a positive identification of an analyte
molecule required the retention time window of the MRM transition to be within 5% of the retention time of the analyte
standard. Analysis of seven replicate samples at each concentration demonstrates the precision of the method at various
concentrations.
Quantitative analysis of samples was accomplished by the performance of a linear regression of the peak areas for each
nitrogen mustard degradation product in the seven calibration curve standards. The instrument software, QuanLynx™,
was used to generate a polynomial calibration curve, which provided a mathematical relationship between peak area and
analyte concentration, along with an accompanying correlation coefficient, R2. If the polynomial type is linear or
quadratic and excludes the point of origin, a fit weighting of 1/X was used to more heavily weight lower concentrations
values, associated with low detection limits, over the higher concentration values. The SAP quality assurance/quality
control (QA/QC) guidelines stipulate a minimum R2 value of 0.98 for linear fits and 0.99 for quadratic fits. All of the
calibration curves for the nitrogen mustard degradation SAP in this study had a correlation coefficient (R2) value of
greater than 0.99 for a linear fit to ensure quality performance. Although no points needed to be excluded, if a calibration
standard, other than the high or low standard, causes the curve's correlation coefficient to fall below the stipulated
minimum of 0.98 or 0.99, the standard should be re-injected and a new calibration curve must be generated and analyzed.
An external calibration was used to monitor the MRM transitions of each analyte. Quanlynx™ software was utilized for
the quantitation of the target analytes and external standard. The MRM transitions of each analyte were used for
quantitation and confirmation by isolating the parent ion, fragmenting the parent ion to the daughter ion and relating the
transition to the retention time in the calibration standard.
4.3 Waste Handling and Prevention
A waste container for all compatible chemicals used in these studies was dated and labeled prior to the addition of any
waste. The waste container's label should be an accurate log of the container's chemical contents as well as the
approximate amount of each species added to the container and was maintained throughout its use.
5.0 Results and Discussion
5.1 Detection Limit Determination
11
-------
MDLs and LOQs were determined using EPA and NIOSH conventions. For simplicity and to avoid confusion, only EPA
detection and quantitation limits were displayed in the SAP, but both are available in this report. The EPA-preferred
protocol utilized guidelines in 40 CFR Part 136, Appendix B - Definition and Procedure for the Determination of the
Method Detection Limit - Revision 1.11 [5]. NIOSH guidelines require the use of Standard Operating Procedure (SOP)
018 and 504 for chemical analysis [6, 7].
5.1.1EPAMDL
Calculation of the method detection limit (MDL) for the nitrogen mustard degradation compounds was performed
according to guidelines in 40 CFR Part 136, Appendix B - Definition and Procedure for the Determination of the Method
Detection Limit (Table 9). Protocol required the resulting MDL to be within ten times the spike level. Subsequent lower
concentration levels for a mixture containing all analytes of interest were spiked onto each coupon surface, carried
through the extraction/concentration process (Section 4.1) and analyzed until a S/N ratio of at least 3:1 was obtained.
MDL levels were selected by evaluating three separate low concentration level samples in order to determine the lowest
concentration level to be reproducibly recovered (< 20% RSD). Once the S/N ratio of approximately 3:1 was achieved,
samples were subjected to full method preparation, extraction and analytical procedures for at least seven replicates for
the matrix of interest. The MDL was determined by using the standard deviation (a) of the seven replicates multiplied by
the Student's t-factor for seven replicate samples (Student's t-factor is dependent on the number of replicates used; 3.143
assumes seven replicates) as shown in Table 9. The MDL was calculated using the formula below and the resulting data
are displayed in Table 10 and Appendix A. Subsequent LOQ determination was calculated by multiplying the standard
deviation of the MDL results by a factor often.
Table 9. Student's t-statistic value as it relates to the number of replicate samples
Number of Replicates (n)
7
Student's t-statistic
3.143
MDL = 3.143xSD [5]
n = number of replicates = 7
3.143 = Student's t-value for (n-1) = 6 degrees of freedom at 99% confidence
SD = standard deviation of replicate analyses.
* NOTE: The value used for the Student's t-statistic will change if the number of samples analyzed or the
confidence level is altered.
Unspiked coupons were wiped and analyzed for the presence of nitrogen mustard degradation analytes that may be native
to the surface materials as well as interferences. In each case, coupons were spiked with only the surrogate and not with
the targeted analytes, and were taken through the same sampling and analysis procedure. Data from the analysis of
unspiked (blank) coupons are shown in Table 12 as part of the precision and accuracy (P&A) data. Large relative
standard deviations suggest the native analyte contamination/interference content of the coupon surfaces was not uniform.
Furthermore, background subtraction for blank analyte levels was not performed. Analysis of blank samples revealed the
presence of TEA and DEA (and/or interferences) on all tested surfaces, most notably in metal, glass, painted drywall and
wood. This observation was not too surprising given the commonality and commercialization of TEA and DEA in
industrial applications (e.g., metal working fluids, soaps, foaming agents, cleaning agents, etc.). Although the data were
affected by the contamination at lower concentrations and detection limit levels, higher concentrations (still below levels
of concern) will be less likely to contribute to error. For this reason, the standard deviation is believed to contribute to
higher MDLs and LOQs than what the SAP was capable of and can be found if the tested surfaces were cleaned prior to
examination. Pre-cleaning surfaces does not, however, mimic a real-world scenario. Therefore all surfaces were used as
received and the data represent an actual collection of analytes from an uncleaned surface with the knowledge that very
low levels will show the presence of TEA and DEA and should be noted. Recovery levels of EDEA and MDEA were not
affected by the contamination levels of TEA and DEA, suggesting that the SAP can be used for EDEA and MDEA as
written. Further investigation of TEA and DEA is still needed to verify that the method can be used for its fitness-for-
purpose.
12
-------
Table 10. EPA calculation for MDL and LOQ in ng/cm and ng/mL for nitrogen mustard degradation analytes on a laminate
surface. Concentration levels (ng/mL) were divided by the surface area (100 cm2) to achieve (ng/cm2) results
Analyte
TEA
EDEA
MDEA
DEA
LAMINATE
MDL
ng/cm2
0.12
0.06
0.07
0.04
ng/mL
12.3
6.3
6.9
4.4
LOQ
ng/cm2
0.39
0.20
0.22
0.14
ng/mL
39.2
19.9
21.8
13.9
5.1.2NIOSHMDL
NIOSH defined its detection limit as the mass of an analyte which gives a mean signal three times (3ab) above the mean
blank signal, where ab is the standard deviation of the blank signal. The LOQ was defined as the mass corresponding to
the mean blank signal + 10ab (i.e., ± 30% uncertainty, which would be 3.33 x MDL) or the mass above which recovery is
> 75%.
Four or more low-level concentrations were spiked onto the sampling media to cover a range from the expected MDL to
no greater than ten times the anticipated MDL. Of the concentrations being used for a low-level experiment, 1-2 should
be at or below the expected Limit of Detection (LOD), 1-2 at or near the mid-range, and 2-3 should be at or near the mid-
range up to 10 x MDL. These samples were extracted and analyzed under the same conditions as would be encountered
for field samples. A graph was compiled of the responses of the concentrations vs. the mass (or concentration) used. A
linear regression equation was obtained and the standard error of the regression (sy) was calculated (as explained in SOP
018). The MDL was determined as 3sy/slope. The LOQ for this study was reported as 3.33 x MDL due to the fact that
some recoveries were below 75%, whereas other recoveries for the compounds were all above 75% even at the lowest
concentrations. Contamination of the surfaces from the pre-existing presence of the analytes made it difficult to
determine an appropriate MDL and LOQ using either EPA or NIOSH technique. As with the EPA determination, blank
subtraction of the analytes was not performed in this experiment. The data are displayed in Table 11.
Painted Drywall and Wood MDL values were not calculated using the NIOSH determination because only the highest
concentration level was used within the calibration range, and recoveries from that spike level were still low for all
targeted analytes (1-20%).
13
-------
Table 11. NIOSH calculation for MDL and LOQ determination for nitrogen mustard degradation analytes on surfaces.
LAMINATE
TEA
Spike
(ng/mL)
50
75
100
150
Sy
Slope
Slope RSD
LOD
LOQ
LOD
LOQ
Recovered
(ng/mL)
75
71
81
124
12.25
0.52
0.31
ng/mL
71
235
ng/cm2
0.71
2.35
EDEA
Spike
(ng/mL)
50
75
100
150
Sy
Slope
Slope RSD
LOD
LOQ
LOD
LOQ
Recovered
(ng/mL)
37
31
65
91
11.61
0.61
0.26
ng/mL
57
190
ng/cm2
0.57
1.90
MDEA
Spike
(ng/mL)
50
75
100
150
Sy
Slope
Slope RSD
LOD
LOQ
LOD
LOQ
Recovered
(ng/mL)
40
47
67
95
9.15
0.25
0.49
ng/mL
110
366
ng/cm2
1.10
3.66
DEA
Spike
(ng/mL)
50
75
100
150
Sy
Slope
Slope RSD
LOD
LOQ
LOD
LOQ
Recovered
(ng/mL)
57
49
69
99
10.48
0.47
0.29
ng/mL
67
223
ng/cm2
0.67
2.23
METAL
TEA
Spike
(ng/mL)
50
75
100
150
Sy
Slope
Slope RSD
LOD
LOQ
Recovered
(ng/mL)
147
145
172
175
9.79
0.32
0.41
ng/mL
92
306
EDEA
Spike
(ng/mL)
50
75
100
150
Sy
Slope
Slope RSD
LOD
LOQ
Recovered
(ng/mL)
42
78
72
91
13.01
0.41
0.42
ng/mL
94
314
MDEA
Spike
(ng/mL)
50
75
100
150
Sy
Slope
Slope RSD
LOD
LOQ
Recovered
(ng/mL)
43
59
70
72
7.97
0.27
0.39
ng/mL
89
295
DEA
Spike
(ng/mL)
50
75
100
150
Sy
Slope
Slope RSD
LOD
LOQ
Recovered
(ng/mL)
64
81
86
98
4.52
0.31
0.20
ng/mL
44
146
14
-------
GLASS
TEA
Spike
(ng/mL)
50
75
100
150
Sy
Slope
Slope RSD
LOD
LOG
Recovered
(ng/mL)
219
256
256
261
15.54
0.34
0.62
ng/mL
137
457
EDEA
Spike
(ng/mL)
50
75
100
150
Sy
Slope
Slope RSD
LOD
LOG
Recovered
(ng/mL)
26
50
53
78
6.19
0.49
0.17
ng/mL
38
126
MDEA
Spike
(ng/mL)
50
75
100
150
Sy
Slope
Slope RSD
LOD
LOG
Recovered
(ng/mL)
30
57
55
84
7.97
0.49
0.22
ng/mL
49
163
DEA
Spike
(ng/mL)
50
75
100
150
Sy
Slope
Slope RSD
LOD
LOG
Recovered
(ng/mL)
58
92
77
119
14.63
0.54
0.37
ng/mL
81
271
VINYL TILE
TEA
Spike
(ng/mL)
100
150
200
300
Sy
Slope
Slope RSD
LOD
LOG
Recovered
(ng/mL)
30
137
143
224
32.6
0.88
0.25
ng/mL
111
370
EDEA
Spike
(ng/mL)
100
150
200
300
Sy
Slope
Slope RSD
LOD
LOG
Recovered
(ng/mL)
29
14
52
87
16.74
0.34
0.34
ng/mL
148
492
MDEA
Spike
(ng/mL)
100
150
200
300
Sy
Slope
Slope RSD
LOD
LOG
Recovered
(ng/mL)
29
16
64
75
18.07
0.28
0.44
ng/mL
194
645
DEA
Spike
(ng/mL)
100
150
200
300
Sy
Slope
Slope RSD
LOD
LOG
Recovered
(ng/mL)
36.
32
71
79
13.56
0.25
0.37
ng/mL
163
542
15
-------
5.2 Precision and Accuracy Determination
Initial demonstration of a laboratory's capability to generate data of acceptable quality will be possible through the
performance of a precision and accuracy (P&A) study. P&A sample data sets were collected at four concentration levels
using a standard containing triethanolamine, jV-ethyldiethanolamine, TV-methyldiethanolamine and diethanolamine.
Solutions were spiked onto the coupons at various concentrations, ranging from at or below the midpoint concentration in
the calibration curve (with the exception of porous surfaces such as wood and painted drywall), generally different from
those chosen for calibration standards. All chosen concentration levels must fall within levels 1-7 of the calibration
standards listed in Table 1 to ensure analyte response linearity. One blank sample was added to each of the four sample
sets to determine the presence of native species within the selected materials. As discussed in section 4.1, unspiked
coupons were wiped and analyzed for the presence of nitrogen mustard degradation analytes that may be native to the
substrate materials. Data from the analysis of unspiked (blank) coupons are shown in Table 12. As in section 5.1, TEA
and DBA were present in every substrate material. Large relative standard deviations indicated that the native analyte
content of the coupon surfaces was not uniform. Although background subtraction for blank analyte levels was not
performed, such an analysis could be carried out for TEA and/or DBA recoveries. As referenced in Section 5.1.1,
recovery levels of EDEA and MDEA were not affected by the contamination levels of TEA and DEA, suggesting that the
SAP can be used for EDEA and MDEA as written. Seven replicates were used for this study for added statistical value.
The average recoveries and standard deviations were calculated as described in Section 5.1 and are displayed in Table 12.
Nonporous surfaces should yield higher recoveries of the analytes. Porous materials are expected to result in lower
recoveries due to analyte permeation into the material, requiring direct extraction of the entire test coupon as the better
alternative for increased analyte recoveries. Alternative solvents used to extract target analytes from porous surfaces may
result in improved recoveries; however, this experiment was not tested at this time. Due to the low recoveries on porous
materials (painted drywall and wood) a concentration (500 ng/mL) higher than the midpoint of the calibration curve was
used for P&A studies. Formica, metal and glass all provide acceptable recoveries at the 50 ng/mL level. Vinyl tile
recoveries were not as high due to the porosity of the surface. As expected, painted drywall and wood produced the
lowest recoveries and further testing would be needed to obtain better recoveries for these substrates, most likely direct
extraction or a better sampling technique, including alternative solvents or wipes, for porous surfaces is needed.
Table 12. Precision &Accuracy (P&A) data for wipe analysis of nitrogen mustard degradation analytes on surfaces.
LAMINATE
Average Spike
Concentration
(ng/mL) (n=7)
50
75
100
150
Average
Formica Blank
TEA
Average
Recovery
(ng/mL)
75
71
81
124
23
%
Recovery
151
95
81
83
-
%
RSD
8
8
6
10
-
EDEA
Average
Recovery
(ng/mL)
37
31
66
91
0
%
Recovery
74
72
66
61
-
%
RSD
17
8
4
14
-
MDEA
Average
Recovery
(ng/mL)
40
47
67
95
0
%
Recovery
79
62
67
63
-
%
RSD
6
11
5
9
-
16
-------
LAMINATE
Average Spike
Concentration
(ng/mL) (n=7)
50
75
100
150
Average
Formica Blank
DEA
Average
Recovery
(ng/mL)
57
49
69
99
2
%
Recovery
114
66
69
66
-
%
RSD
25
6
7
10
-
DEA-ds
Average
Recovery
(ng/mL)
38
41
62
93
27
%
Recovery
75
55
62
62
54
%
RSD
6
3
5
12
-
METAL
Average Spike
Concentration
(ng/mL) (n=7)
50
75
100
150
Average Metal
Blank
TEA
Average
Recovery
(ng/mL)
147
146
172
175
118
%
Recovery
294
194
172
117
-
%
RSD
10
18
13
9
-
EDEA
Average
Recovery
(ng/mL)
42
58
72
91
0
%
Recovery
85
78
72
61
-
%
RSD
7
7
9
14
-
MDEA
Average
Recovery
(ng/mL)
43
59
70
72
0
%
Recovery
86
79
70
48
-
%
RSD
6
6
6
14
-
METAL
Average Spike
Concentration
(ng/mL) (n=7)
50
75
100
150
Average Metal
Blank
DEA
Average
Recovery
(ng/mL)
64
81
86
98
16
%
Recovery
129
108
86
65
-
%
RSD
5
5
5
4
-
DEA-ds
Average
Recovery
(ng/mL)
45
69
73
96
50
%
Recovery
91
92
73
64
100
%
RSD
10
6
4
3
-
17
-------
GLASS
Average Spike
Concentration
(ng/mL) (n=7)
50
75
100
150
Average Glass
Blank
TEA
Average
Recovery
(ng/mL)
219
256
256
261
203
%
Recovery
439
342
256
174
-
%
RSD
6
10
12
8
-
EDEA
Average
Recovery
(ng/mL)
26
50
53
78
0
%
Recovery
52
66
53
52
-
%
RSD
7
5
14
12
-
MDEA
Average
Recovery
(ng/mL)
30
57
55
84
0
%
Recovery
61
76
55
56
-
%
RSD
6
4
14
10
-
GLASS
Average Spike
Concentration
(ng/mL) (n=7)
50
75
100
150
Average Glass
Blank
DEA
Average
Recovery
(ng/mL)
58
92
77
119
19
%
Recovery
115
122
77
80
-
%
RSD
14
4
19
8
-
DEA-ds
Average
Recovery
(ng/mL)
30
61
55
92
53
%
Recovery
59
81
55
61
105
%
RSD
11
5
21
11
-
VINYL TILE
Average Spike
Concentration
(ng/mL) (n=7)
100
150
200
300
Average Vinyl
Blank
TEA
Average
Recovery
(ng/mL)
30
137
143
225
18
%
Recovery
30
92
71
75
-
%
RSD
25
7
7
10
-
EDEA
Average
Recovery
(ng/mL)
29
14
52
87
0
%
Recovery
29
9
26
29
-
%
RSD
11
11
8
15
-
MDEA
Average
Recovery
(ng/mL)
29
16
64
75
0
%
Recovery
29
11
32
28
-
%
RSD
9
7
7
16
-
18
-------
VINYL TILE
Average Spike
Concentration
(ng/mL) (n=7)
100
150
200
300
Average Vinyl
Blank
DEA
Average
Recovery
(ng/mL)
36
32
71
79
7
%
Recovery
36
21
35
26
-
%
RSD
13
19
12
10
-
DEA-ds
Average
Recovery
(ng/mL)
28
18
46
60
54
%
Recovery
28
12
23
20
107
%
RSD
19
4
6
14
-
WOOD
Average Spike
Concentration
(ng/mL) (n=7)
500
Average Wood
Blank
TEA
Average
Recovery
(ng/mL)
100
73
%
Recovery
20
-
%
RSD
16
-
EDEA
Average
Recovery
(ng/mL)
10
0
%
Recovery
2
-
%
RSD
28
-
MDEA
Average
Recovery
(ng/mL)
16
0
%
Recovery
3
-
%
RSD
30
-
WOOD
Average Spike
Concentration
(ng/mL) (n=7)
500
Average Wood
Blank
DEA
Average
Recovery
(ng/mL)
37
19
%
Recovery
7
-
%
RSD
22
-
DEA-ds
Average
Recovery
(ng/mL)
10
57
%
Recovery
2
115
%
RSD
32
-
19
-------
PAINTED DRYWALL
Average Spike
Concentration
(ng/mL) (n=7)
500
Average Drywall
Blank
TEA
Average
Recovery
(ng/mL)
110
73
%
Recovery
22
-
%
RSD
10
-
EDEA
Average
Recovery
(ng/mL)
75
0
%
Recovery
15
-
%
RSD
17
-
MDEA
Average
Recovery
(ng/mL)
85
0
%
Recovery
17
-
%
RSD
18
-
PAINTED DRYWALL
Average Spike
Concentration
(ng/mL) (n=7)
500
Average
Drywall Blank
DEA
Average
Recovery
(ng/mL)
87
23
%
Recovery
17
-
%
RSD
18
-
DEA-ds
Average
Recovery
(ng/mL)
57
40
%
Recovery
11
80
%
RSD
20
-
5.3 Holding Time Study
An evaluation of a holding time study provided a measurement of the stability of the wipe medium with the nitrogen
mustard degradation analytes. Holding time studies consisted of five sets of eight samples: three blank wipe samples
(spiked with neither analyte nor surrogate) and five analyte-spiked wipe samples. In blank samples, each wipe was wetted
with 400 (iL of methanol and placed into the sample jars. Samples were spiked with analyte to achieve a final analyte
concentration of 50 ng/mL, using a 500 ng/mL spiking standard, with equal volume of the spiking standard added to each
wipe of the sample. Each sample vial contained two filter papers. Following wetting or spiking, each sample set was
analyzed after its designated holding time period of 0, 7, 14, 21, or 28 days. Each set of samples was stored at 4 °C (±2
°C) until processed and analyzed and the data are displayed in Table 13.
Determination of the variance between data sets being statistically different from the variance at time 0 data set was
accomplished by performing the f-test. F-test results suggested no significant differences in the variances for each analyte
at the different time frames. A paired t-test compared the mean recovery of each analyte at each holding time to its
corresponding mean recovery at time 0 with 95% confidence. T-test results indicated no significant differences between
the mean recovery values of any analyte to its counterpart at time 0. Analysis of results from these two tests suggests that
both sample and sampling media were stable for time periods up to and including 28 days. Corresponding statistical data
are presented in Appendix B.
20
-------
Table 13. Holding time sample stability of wipes spiked with nitrogen mustard degradation analytes
Concentration 50 ng/mL (n = 5)
Holding Time
(days)
0
7
14
21
28
TEA
Average
%
Recovery
93
94
75
75
78
% RSD
6
6
10
7
5
EDEA
Average
%
Recovery
98
85
87
84
88
% RSD
7
9
6
10
5
MDEA
Average
%
Recovery
95
82
82
83
87
% RSD
6
8
8
10
5
DEA
Average
%
Recovery
93
85
82
78
74
% RSD
5
7
6
10
12
DEA-ds
Average
%
Recovery
96
90
86
84
90
% RSD
7
9
11
11
9
5.4 Sonication Study
To decrease sample processing time in the event of a national security incident, variable sonication times involving spiked
wipes were investigated to determine the optimal recovery of nitrogen mustard degradation products. The initial proposal
of a 25 minute sonication time for the extraction of ethanolamine-based analytes from wipes (either direct-wipe spiking or
after wiping spiked surfaces) was proposed. Data were collected at 25 minutes to serve as a control as the existing
protocol calls for 25 minutes of sonication time. Data sets consisting of three samples, containing two Whatman 42
filters per sample, were spiked to achieve a final analyte concentration of 50 ng/mL, with equal volumes of the spiking
standard added to each wipe of the sample. Sonication times of 5, 10, 15, and 25 minutes were analyzed to determine if
comparable analyte recovery could be achieved with shorter sonication times.
Data presented in Table 14 indicated that a ten minute sonication time was sufficient for quantitative extraction of the
desired analytes from the wipes. The recovery of the analytes at all sonication times was nearly quantitative, and five
minute sonication time periods may be sufficient for analysis. Additional replicates for added statistical power would be
needed to assess the effect of sonication time on analyte recovery from spiked wipes more effectively. A data set
evaluating samples not subjected to sonication could also provide valuable input. Unless larger (or multiple) sonication
baths are accessible or smaller sample vials are used, the number of samples available to simultaneously process was a
limiting factor in sample turnaround time. As a result, shorter sonication times, larger shaker tables, alternative sampling
vials, or all of the above may be suitable replacements for the materials used in this study.
21
-------
Table 14. Analyte recovery from analyte spiked wipes at various sonication intervals.
Concentration 50 ng/mL (n = 3)
Sonication time
(min)
5
10
15
25
TEA
Average
%
Recovery
89
100
85
94
%
RSD
7
5
5
6
EDEA
Average
%
Recovery
92
108
94
107
%
RSD
10
3
3
8
MDEA
Average
%
Recovery
91
104
98
103
%
RSD
9
8
2
7
DEA
Average
%
Recovery
94
111
96
111
%
RSD
13
5
2
9
DEA-ds
Average
%
Recovery
97
120
105
116
%
RSD
9
3
3
8
6.0 Conclusion
A procedure for recovering nitrogen mustard degradation analytes from surfaces through wiping was developed and
characterized on laminate, metal/stainless steel, glass, vinyl, painted drywall and wood. Several different wipes were
tested, but only one wipe was considered viable straight out of the box when analysis of these analytes is being performed.
Cotton gauze wipes were individually packaged and considered sterile, meaning they had been cleaned during the
manufacturing process. Not only did cotton gauze wipes tested in this specific experiment contain very high levels of
TEA and DEA, other cotton gauze wipes would also contain similarly high levels. The most preferred wipe used during
sample analysis is typically cotton gauze. However, in this case it would not be appropriate to use a cotton gauze wipe
unless further pre-cleaning and treatment has occurred. Furthermore, sampling kits provided to samplers in the field are
equipped with pre-packaged wipes. Even if the wipes are pre-cleaned, testing on wipe materials is needed to ensure that
targeted analytes were not present. For nitrogen mustard and its degradates, cotton gauze would need to be properly
treated prior to use to remove all contaminations and interferences, a time-consuming and potentially costly approach,
whereas no pretreatment is needed for filter paper. As a result, the preferred wipes used for the SAP were the Whatman®
filter papers. Although glass fiber wipes are an alternative to the preferred wipe, they may not fare well when the rigors
of wiping a surface are encountered. Whatman® filter paper wipes not only produce little to no interferences and low
background blank levels, the overall recoveries using Whatman® filter paper wipes were reasonably high as well.
In addition to testing different wipes, alternative solvent systems were also explored. Both ammonium formate and
ammonium acetate were found to be suitable for the analysis of the ethanolamine-based nitrogen mustard degradation
compounds. If ammonium formate were to be used, full scale workup and procedures are needed in order to determine if
this solvent system produces similar results. Ammonium acetate was chosen for this particular experiment because of the
chromatographic separation of the targeted analytes. Such separation would allow for preliminary and possibly future
analyses of the compounds on different instrumentation, such as an ion trap mass spectrometer. Various concentrations
were also explored to observe the effect of eluent concentration. Although 25 mM ammonium acetate concentration was
chosen for this study, such a high concentration of buffer might result in retention time shifts and clogging of the
analytical column. The highest concentration of 25 mM ammonium acetate was chosen for sensitivity reasons, but if any
of the problems mentioned above occur, caution should be noted and levels can be adjusted accordingly if such issues
occur.
Recovery of the tested analytes from commonly encountered surfaces through wipe sampling was possible at low levels.
Holding time studies indicated that the analytes and sampling media are stable for at least 28 days, but the target analytes
are expected to persist much longer. Wipe sampling was a viable means of screening all tested surfaces (except painted
drywall and wood) for the presence of degradation analytes. However, the capacity of wipe sampling for quantitation from
surfaces was unclear due to incomplete recovery and sometimes poor reproducibility because of the presence of native
TEA and DEA on surfaces. Qualitatively, the data could be used to determine the presence of EDEA and MDEA on all
surfaces where recoveries were above the detection limit. Higher concentrations and/or a larger calibration curve range or
22
-------
direct extraction may be necessary for analysis on more porous surfaces. For more porous surfaces, a better sampling
procedure needs to be developed to convey the presence of such targeted analytes adequately.
All surfaces exhibited some type of TEA and DBA contamination at different levels. Surfaces with the highest TEA and
DBA contamination were glass and metal, not surprising since most metal working fluids and cleaners are known to
contain TEA and/or DBA. For this reason, the standard deviation is believed to contribute to higher MDLs and LOQs
than what the SAP truly indicates. Lower MDLs and LOQs could be found if the tested surfaces were cleaned prior to
examination. However, such a process would not be indicative of a real-world scenario. All surfaces were therefore used
as received and the data represent an actual collection of analytes from an uncleaned surface with the knowledge that very
low levels will show the presence of TEA and DBA and should be noted. Analytical investigation of blank samples
detected no presence of EDEA and MDEA on any of the surfaces, suggesting the SAP will work well for those specific
compounds at low levels. Only for analyses at low levels would the presence of TEA and DEA be an issue, possibly
during site characterization for samples collected following decontamination, degradation rates, etc. However, since these
specific compounds are common in real world applications and are not considered to be as toxic, higher MDL and LOQ
levels are suggested (10-100-fold current MDLs used within the corresponding SAP) to prevent problems with recovery
and standard deviation.
While the performance demonstration may be compatible for EDEA and MDEA, it is possible that further
experimentation and alteration of experimental parameters may lead to better recoveries and lower detection limits, if
needed, especially for TEA and DEA. Similarly, selection of alternate columns with wider pH ranges may lead to a
simplified elution gradient. As instrumentation continues to improve, so can the analytical detection limits for any
targeted compound. UPLC-MS/MS is an example of faster run times and potentially better results. If any of the
experimental conditions are changed, the laboratory should rigorously establish the performance of the analytical
approach operated under the modified conditions. Further studies will also be needed to test other nonporous surfaces to
determine how well this particular wipe can perform on any surface that may need to be tested during a remediation event.
The data serve as a proof-of-concept for the use of Whatman® filter paper in sampling procedures for the analysis of
nitrogen mustard degradation products on most surfaces. Filter paper wipes appear to be an easier alternative for the
sampling and analysis of nitrogen mustard degradation products than the commonly used cotton gauze wipe.
7.0 References
1. U.S. Environmental Protection Agency (EPA), 2010. Standardized Analytical Methods for Environmental
Restoration Following Homeland Security Events (SAM). EPA/600/R-10/122 September 2010. Cincinnati, Ohio:
United States Environmental Protection Agency, Office of Research and Development, National Homeland
Security Research Center.
2. Munro, N.B., S.S. Talmage, G.D. Griffin, L.C. Waters, A.P. Watson, J.F. King, and V. Hauschild. (1999). The
Sources, Fate, and Toxicity of Chemical Warfare Agent Degradation Products. Environmental Health
Perspectives. 102(12): 933-974.
3. NIOSH Manual of Analytical Methods (NMAM®), 4th ed., DHHS (NIOSH) Publication 94-113 (August, 1994),
3rd Supplement 2003-154.
23
-------
4. U.S. Environmental Protection Agency (EPA), 2011. Surface Analysis Using Wipes for the Determination of
Nitrogen Mustard Degradation Products by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS)
EPA 600/R-l 1/143, November 2011. Cincinnati, Ohio: United States Environmental Protection Agency, Office of
Research and Development, National Homeland Security Research Center.
5. Code of Federal Regulations, 40 CFR Part 136, Appendix B. Definition and Procedure for the Determination of
the Method Detection Limit - Revision 1.11
6. SOP 018: Standard Operating Procedures for Industrial Hygiene Sampling and Chemical Analysis,
NIOSH/DPSE Quality Assurance Manual, July, 1994.
7. SOP 504: Limits of Detection and Quantitation, CEMB Laboratory Services, February, 2004.
24
-------
Appendix A
Table A-l. Reagents and CAS numbers
CHEMICAL NAME
Acetonitrile, ACN, LC/MS grade
Water, H2O, LC/MS grade
Methanol, MeOH, LC/MS grade
Ammonium acetate, NH/iOAc
Glacial acetic acid, HO Ac
Diethanolamine, DEA
Triethanolamine, TEA
7V-Methyldiethanolamine, MDEA
TV-Ethyldiethanolamine, EDEA
bis(2-Hydroxyethyl)-dg-amine
(Diethanolamine-dg), DEA-dg
CAS#
75-05-8
7732-18-5
67-56-1
631-61-8
64-19-7
111-42-2
102-71-6
105-59-9
139-87-7
103691-51-6
Equipment
Waters Acquity TQD LC/MS/MS system, or equivalent
Fisher Scientific FS 140 H ultrasonic cleaner, or equivalent
OA-Sys N-evap 111 nitrogen evaporator, or equivalent
Mettler AE 240 analytical balance, or equivalent
Supplies
Automated Pipettes (100 |iL, 1000 |iL, and 10 mL)
Pipette tips (100 |iL, 1000 |iL, and 10 mL)
Class A volumetric flasks (250 mL, 500 mL, and lOOOmL)
Class A amber glass volumetric flasks (10 mL, 25 mL, and 50mL)
10 cm x 10 cm pre-cut coupons (glass, metal/stainless steel, formica, vinyl, wood)
Whatman 42 ashless circle filters, 55 mm
10 mL BD safety-lok syringes
Millipore 13 mm Millex filter, 0.22 |im PVDF
15 mL graduated polypropylene centrifuge tubes
Waters 1.8 mL amber glass vials with pre-slit silicone PTFE screw cap
Special safety precautions
Experimenters should be familiar with MSDS sheets for all solvents and reagent chemicals used.
Nitrile gloves, safety glasses and other PPE should be worn when working in the laboratory.
25
-------
Procedure
1.
2.
3.
4.
5.
6.
7.
8.
9.
Spike coupons with standard solution, using 5 spots per coupon. Let dry completely.
Wipe coupons with two Whatman 42 55 mm filters:
a. Wet wipe with 300 (iL MeOH.
b. 1st wipe: wipe horizontally in a "Z -pattern".
c. 2nd wipe: wipe vertically in a "Z -pattern".
d. After wiping, place wipes flat in 125 mL Nalgene jar.
e. Add approx. 10 mL of MeOH to each jar to completely cover
wipes.
Sonicate samples in jars for approximately 10 minutes.
Withdraw as much of the solution as possible with a 10 mL disposable Luer-Lok
syringe.
Filter sample through a 13 mm 0.22 (im PVDF filter into a 15 mL graduated
polypropylene centrifuge tube.
Concentrate dilute sample down to < 2 mL in a warm water bath using N2 (being
careful not to evaporate to complete dryness).
Adjust sample volume to 2 mL, if necessary, with MeOH.
Add sample into an amber glass autosampler vial and place into autosampler,
documenting each sample's position in the autosampler tray.
Inject a 5 (iL aliquot of sample into the LC-MS/MS instrument and analyze.
Table A-2. Detection limit (PL) results for (n=7) samples for wiping the surface of coupons with a 100 cm2 area
LAMINATE
Average Spike
Concentration
(ng/mL) (n=7)
50
Formica Blank
Average Spike
Concentration
(ng/cm2) (n=7)
0.50
Formica Blank
TEA
Average
Recovery
ng/mL
54
26
Average
Recovery
(ng/cm2)
0.54
0.26
%
Recovery
109
%
Recovery
109
%
RSD
7
%
RSD
7
EDEA
Average
Recovery
ng/mL
32
0
Average
Recovery
(ng/cm2)
0.32
0
%
Recovery
64
%
Recovery
64
%
RSD
6
%
RSD
6
MDEA
Average
Recovery
ng/mL
37
0
Average
Recovery
(ng/cm2)
0.37
0
%
Recovery
74
%
Recovery
74
%
RSD
6
%
RSD
6
26
-------
LAMINATE
Average Spike
Concentration
(ng/mL) (n=7)
50
Formica Blank
Average Spike
Concentration
(ng/cm2) (n=7)
0.50
Formica Blank
DEA
Average
Recovery
(ng/mL)
41
5
Average
Recovery
(ng/cm2)
0.41
0.05
%
Recovery
82
%
Recovery
82
%
RSD
3
%
RSD
3
DEA-ds
Average
Recovery
(ng/mL)
33
34
Average
Recovery
(ng/cm2)
0.33
0.33
%
Recovery
66
67
%
Recovery
66
67
%
RSD
3
%
RSD
3
27
-------
Appendix B: Statistical Data and Calculations for Holding Time Studies
The f-test was administered to determine if independent populations have significantly different variances. Variations
would have an effect for when pooling the standard deviations and determining the number of degrees of freedom when
conducting at-test. The f-value is calculated using the equation shown below:
Si and s2 represent the standard deviations of the two pools of data being compared and Si is the larger of the two standard
deviations (i.e., F > 1 by definition).
The calculated F-value was compared to the critical F-value at the 95% confidence level where the data sets for si and s2
have (n] - 1) and (n2 - 1) degrees of freedom, respectively. Since 5 replicates were analyzed for all holding times, the
number of degrees of freedom for all data sets was 4. The F-test data is shown in Table B-l. The data indicated no
significant differences in the variances existed for any analyte at time 0 when compared to its counterpart. Consequently,
the pooled standard deviation, spooied, was calculated using the formula below:
H! = number of samples in data set 1
n2 = number of samples in data set 2
Si = standard deviation of data set 1
s2 = standard deviation of data set 2
Spooled, along with the mean recoveries for each analyte at time t (t = 7, 14, 21, or 28 days) and time 0 are used to calculate
a t-value to compare the two means with a paired t-test at 95% confidence using the formula below:
-j with nj-112 - 2 degrees of freedom
xj = mean value of data set 1
X2 = mean value of data set 2
Spooled = pooled standard deviation
H! = number of samples in data set 1
n2 = number of samples in data set 2
degrees of freedom = Tij-nj -2=8
The t-test data is shown in Table B-2. T-test data exhibited no significant difference in the mean for any analyte at any
holding time when compared to its counterpart at its initial holding time at day 0. Since there was no significant
difference in the means, all analytes at every holding time tested showed recoveries that are within 10% of their
counterparts at 0 days holding time. Therefore, all analytes studies were stable for a period of at least 28 days.
Table B-l. F-test analysis for nitrogen mustard degradation analytes
28
-------
TEA F-test
DayO
Day?
DayO
Day 14
DayO
Day 21
DayO
Day 28
Standard Deviation
5.20
5.64
5.20
7.71
5.20
5.20
5.20
3.88
Calculated F-value
1.18
2.20
1.00
1.80
Critical F-value
6.39
6.39
6.39
6.39
Significant?
no
no
no
no
EDEA F-test
DayO
Day?
DayO
Day 14
DayO
Day 21
DayO
Day 28
Standard Deviation
6.48
7.65
6.48
5.16
6.48
8.79
6.48
4.54
Calculated F-value
1.39
1.58
1.84
2.03
Critical F-value
6.39
6.39
6.39
6.39
Significant?
no
no
no
no
MDEA F-test
DayO
Day?
DayO
Day 14
DayO
Day 21
DayO
Day 28
Standard Deviation
5.82
6.34
5.82
6.74
5.82
8.65
5.82
4.64
Calculated F-value
1.19
1.34
2.21
1.57
Critical F-value
6.39
6.39
6.39
6.39
Significant?
no
no
no
no
DBA F-test
DayO
Day?
DayO
Day 14
DayO
Day 21
DayO
Day 28
Standard Deviation
4.31
5.89
4.31
5.16
4.31
7.59
4.31
9.20
Calculated F-value
1.87
1.43
3.11
4.57
Critical F-value
6.39
6.39
6.39
6.39
Significant?
no
no
no
no
DEA-d8 F-test
DayO
Day?
DayO
Day 14
DayO
Day 21
DayO
Day 28
Standard Deviation
6.29
7.72
6.29
9.24
6.29
9.15
6.29
8.27
Calculated F-value
1.51
2.16
2.12
1.73
Critical F-value
6.39
6.39
6.39
6.39
Significant?
no
no
no
no
Table B-2. T-test analysis for nitrogen mustard degradation analytes
TEA t-tests, 95% CI, DoF = 8
29
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DayO
Day?
DayO
Day 14
DayO
Day 21
DayO
Day 28
X
92.98
93.57
92.98
74.93
92.98
75.30
92.98
78.35
s
5.20
5.64
5.20
7.71
5.20
5.20
5.20
3.88
Spooled
5.42
6.58
5.20
4.59
Calculated t-value
0.07
1.74
2.15
2.02
Critical t-value
2.306
2.306
2.306
2.306
Significant?
no
no
no
no
EDEA t-tests, 95% CI, DoF = 8
DayO
Day?
DayO
Day 14
DayO
Day 21
DayO
Day 28
X
97.56
85.15
97.56
87.06
97.56
83.84
97.56
87.77
s
6.48
7.65
6.48
5.16
6.48
8.79
6.48
4.54
^pooled
7.09
5.85
7.72
5.59
Calculated t-value
1.11
1.13
1.12
1.11
Critical t-value
2.306
2.306
2.306
2.306
Significant?
no
no
no
no
MDEA t-tests, 95% CI, DoF = 8
DayO
Day?
DayO
Day 14
DayO
Day 21
DayO
Day 28
X
94.52
82.36
94.52
82.38
94.52
82.97
94.52
86.60
s
5.82
6.34
5.82
6.74
5.82
8.65
5.82
4.64
^pooled
6.09
6.30
7.37
5.26
Calculated t-value
1.26
1.22
0.99
0.95
Critical t-value
2.306
2.306
2.306
2.306
Significant?
no
no
no
no
DBA t-tests, 95% CI, DoF = 8
DayO
Day?
DayO
Day 14
DayO
Day 21
DayO
Day 28
X
93.47
85.06
93.47
82.11
93.47
77.84
93.47
74.16
s
4.31
5.89
4.31
5.16
4.31
7.59
4.31
9.20
^pooled
5.16
4.75
6.17
7.18
Calculated t-value
1.03
1.51
1.60
1.70
Critical t-value
2.306
2.306
2.306
2.306
Significant?
no
no
no
no
DEA-d8 t-tests, 95% CI, DoF = 8
DayO
Day?
DayO
Day 14
DayO
Day 21
DayO
Day 28
X
96.32
89.75
96.32
85.77
96.32
83.51
96.32
90.08
s
6.29
7.72
6.29
9.24
6.29
9.15
6.29
8.27
^pooled
7.04
7.90
7.85
7.35
Calculated t-value
0.59
0.84
1.03
0.54
Critical t-value
2.306
2.306
2.306
2.306
Significant?
no
no
no
no
30
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