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

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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

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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

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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

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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

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  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

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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

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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

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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

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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

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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

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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

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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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