EPA/600/R-16/120 | June 2016
www.epa.gov/homeland~security-research
United States
Environmental Protection
Agency
oEPA
Experiments with the LECO Pegasus®
Gas Chromatograph/Time-of-Flight
Mass Spectrometer Phase 1: Fast GC
Separations and Comparison of the
GC/TOF-MS with Conventional
Quadrupole GC/MS and Fast
Quadrupole GC/MS
Office of Research and Development
Homeland Security Research Program
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EPA/600/R-16/120
June 2016
Experiments with the
LECO Pegasus® Gas Chromatograph/
Time-of-Flight Mass Spectrometer Phase 1:
Fast GC Separations and Comparison of the
GC/TOF-MS
with Conventional Quadrupole GC/MS and Fast
Quadrupole GC/MS
UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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Disclaimer
Neither the United States government, nor Lawrence Livermore National Security, LLC, nor
any of their employees, makes any warranty, expressed or implied, or assumes any legal
liability or responsibility for the accuracy, completeness, or usefulness of any information,
apparatus, product, or process disclosed, or represents that its use would not infringe privately
owned rights. Reference herein to any specific commercial product, process, or service by trade
name, trademark, manufacturer, or otherwise does not constitute or imply its endorsement,
recommendation, or favoring by the United States government or Lawrence Livermore
National Security, LLC. The U.S. Environmental Protection Agency does not endorse the
purchase or sale of any commercial products or services. The views and opinions of authors
expressed herein do not necessarily state or reflect those of the United States government or
Lawrence Livermore National Security, LLC, and shall not be used for advertising or product
endorsement purposes.
Questions concerning this document or its application should be addressed to:
Romy Campisano
U.S. Environmental Protection Agency
Office of Research and Development
National Homeland Security Research Center (NG16)
26 West Martin Luther King Drive
Cincinnati, OH 45268
(513)569-7016
campisano.romy@epa.gov
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Acknowledgments
The research team wishes to acknowledge the support of all those who helped plan and prepare
this report. The U.S. Environmental Protection Agency, Office of Research and Development,
National Homeland Security Research Center (NHSRC) funded this work and Oba Vincent, Rob
Rothman, and Romy Campisano of this organization provided helpful conversations and
contributions. We thank Eric Graybill, EPA, for providing helpful review comments.
We thank Farai Rukunda, Spectrometer Training Specialist, of LECO Corporation, for his
support in resolving hardware and software issues and for providing many helpful conversations
that allowed us to complete this study. We also thank Chris Retarides of Virginia Division of
Consolidated Laboratory Services (Richmond, VA) for helpful discussions.
This document was prepared as an account of work sponsored by an agency of the United States
government. This work performed under the auspices of the U.S. Department of Energy by
Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344; the research
team was comprised of Heather Mulcahy, Carolyn Koester, and Roald Leif.
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Abbreviations/Acronyms
1-D - One dimensional, specifically in reference to gas chromatography
2-D - Two dimensional, specifically in reference to gas chromatography
AMDIS - Automated Mass Spectral Deconvolution and Identification System
CCV - Continuing Calibration Verification
CWA - Chemical Warfare Agent, in the context of this report, the CWAs of interest are HD,
GB, GD, GF, and VX
DC - Direct current
DFTPP - Decafluorotriphenylphosphine
ECBC - Edgewood Chemical Biological Center (ECBC), Aberdeen Proving Grounds, MD
EPA - United States Environmental Protection Agency
ERLN - Environmental Response Laboratory Network
FBP - 2-Fluorobiphenyl
GB - Sarin
GC - Gas Chromatography
GC/MS - Gas Chromatography/Mass Spectrometry
GC/TOF-MS - Gas Chromatography coupled with Time-of-Flight Mass Spectrometry
GD - Soman
GF - Cyclosarin
HD - Distilled sulfur mustard
IAG - Interagency Agreement
i.d. - Internal diameter (of a gas chromatography column)
IDL - Instrument Detection Limit
LLNL - Lawrence Livermore National Laboratory
MDL - Method Detection Limit
MS - Mass spectrometry
m/z - Mass to charge ratio, with reference to an ion
NB-ds - Deuterated (ds) nitrobenzene
ND - Not Detected
NHSRC - EPA's National Homeland Security Research Center, Cincinnati, OH
NIST - National Institute of Standards and Technology
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NMR - Nuclear Magnetic Resonance Spectroscopy
PCP-ds - Deuterated (ds) phencyclidine
PFTBA - Perfluorotributylamine
ppb - Part(s) per billion
ppm - Part(s) per million
QAPP - Quality Assurance Project Plan
RF - Radio-frequency
SIM - Selected Ion Monitoring (operating mode of a mass spectrometer)
S:N - Signal-to-Noise ratio
TEA - Triethylamine
TIC - Total Ion Chromatogram (produced by GC/MS analysis)
TIC - Toxic Industrial Compounds
Ter-di4 - Deuterated (di4) terphenyl
TOC - Total organic carbon
TOF - Time of flight
TPP - Triphenyl phosphate
UD-CWA - Ultra-dilute (10 ppm) Chemical Warfare Agent standards
VX - 6>-ethyUY-[2-(diisopropylamino)ethyl] methylphosphonothioate
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Executive Summary
Conventional analysis by gas chromatography/quadrupole mass spectrometry (GC/MS)
can be time-consuming (30-60 minutes) and prone to interferences. The use of fast gas
chromatography coupled with time-of-flight mass spectrometry (GC/TOF-MS) offers the
advantages of faster (13 min) analysis times, improved GC resolution afforded by the use of
narrower (0.1- 0.18 mm i.d.) columns, and improved mass resolution and data acquisition speed
provided by the TOF-MS. In addition, GC/TOF-MS offers the promise of better detection limits
than quadrupole GC/MS, while still providing the full mass spectral data which offers an
additional level of confidence in analyte identification.
In this study, the LECO Pegasus® 4D GC/TOF-MS was used to detect chemical warfare
agents (CWAs) and to compare this instrument's performance to the speed of analysis and
detection limits of conventional quadrupole-based GC/MS. Analytes studied were sulfur
mustard (HD), sarin (GB), soman (GD), cyclosarin (GF), and (9-ethyl-S-[2-
(diisopropylamino)ethyl] methylphosphonothioate (VX). Measured concentrations of analytes
were determined by GC/MS and GC/TOF-MS and compared for reagent water, surface water,
sand, three types of soils, and wipes. Limited comparisons between analyte concentrations
measured with GC/TOF-MS and fast GC/MS were also performed.
Instrument detection limits (DDLs) for GC/TOF-MS were lower than those observed for
GC/MS; DDLs were analyte-dependent and ranged from 0.0025 to 0.025 ng. The
reproducibilities of retention times for replicate (n=7) injections of 0.5 ng each CWA were
within 0.4% and reproducibilities of peak areas were less than 3%. In general, matches between
TOF-MS spectra and those contained in the NIST database were good (i.e., greater than 700 out
of 1000), even at the lowest levels detected in standards.
Analyte concentrations determined by GC/TOF-MS were reasonably comparable to those
measured by quadrupole GC/MS for standards, water, soils, and wipe extracts. On average,
analyte concentrations determined for control and water samples by GC/TOF-MS and
conventional GC/MS were comparable (agreement within <20%). For sand and soils,
concentrations measured by GC/MS were often higher than those measured by GC/TOF-MS and
were not always in good agreement. For most (but not all) analytes in wipes, concentrations
measured by GC/TOF-MS and GC/MS agreed within 30%. The reasons for the differences are
unclear, but the fact that some recoveries of analytes from wipes were >150%) when measured by
GC/MS suggests that the GC/MS might be prone to matrix interferences that are not being
satisfactorily separated by the conditions used for GC/MS.
GC/TOF-MS appears to be a good technique to measure concentrations of CWAs in
environmental matrices. Such data can be produced with faster analysis times (by a factor of
three) than with conventional GC/MS. And GC-TOF-MS provides low detection limits while
retaining full mass spectral data. The collection of a complete mass spectrum provides greater
confidence in correct analyte identification. Currently, the only disadvantage of GC/TOF-MS is
that many analysts do not have sufficient experience with the technique; however, such expertise
can be developed by knowledgeable GC/MS operators. In addition, standard mass spectrometric
tune criteria (i.e., based on decafluoro-triphenylphosphine (DFTPP) ions of specified relative
abundances) used to determine that quadrupole GC/MS systems are operating correctly must be
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adapted to allow the use of GC/TOF-MS. Once tune criteria are met, the GC/TOF-MS has been
observed to operate well for several months, with no need to "retune" the system. Data suggest
that GC/TOF-MS can be used routinely for the analysis of sample extracts containing CWAs.
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Table of Contents
Disclaimer i
Acknowledgments ii
Abbreviations/Acronyms iii
Executive Summary v
List of Tables viii
List of Figures ix
1.0 Introduction and Background 1
2.0 Study Objectives 2
3.0 Experimental Conditions 2
3.1 Standards 2
3.2 Sample preparation 3
3.3 GC/TOF-MS conditions 4
3.4 GC/MS conditions (conventional quadrupole) 5
3.5 GC/MS conditions (fast separations, quadrupole MS) 6
3.6 Quality Assurance (QA) 7
4.0 Results 8
4.1 Chromatographic separation of CWAs 8
4.2 Instrument detection limits 14
4.3 Calibration 16
4.4 Analyses of sample extracts 18
4.4.1 Analyses of water sample extracts 20
4.4.2 Analyses of soil extracts 27
4.4.3 Analyses of wipes 31
5.0 Conclusions and Recommendations 32
6.0 References 33
Appendix A: Autosampler Method for LECO GC/TOF-MS 35
Appendix B: GC Method for LECO GC/TOF-MS, setup for 2-D instrument 36
Appendix C: GC Method for LECO GC/TOF-MS, setup for 1-D instrument 39
Appendix D: MS Method for LECO GC/TOF-MS 43
Appendix E: Data Analysis Method for LECO GC/TOF-MS 46
Appendix F: Analysis Method for LECO GC/TOF-MS, Modified ECBC Conditions 49
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List of Tables
Table 1. Average Retention Times (± Standard Deviations) and Average Analyte Responses (±
Standard Deviations) for Seven Replicate Injections of 0.5 ng of each CWA into the GC/TOF-
MS 10
Table 2. DFTPP Key Ions and Ion Abundance Criteria Used by Different Methods to Verify
GC/MS Tune 11
Table 3. IDLs, Quantification (Quant) and Qualifying (Qual) Ions, and S:N Determined by
GC/TOF-MS 14
Table 4. Comparative Data for IDLs in Nanograms 15
Table 5. Average (N=3) Match Factors (Forward Fit), Relative to the NIST Database,
Determined for Various Analytes at the High and Low Points of the Calibration Curve 17
Table 6. Calibration Data for CWAs: Average Relative Response Factors (RRFs), Percent
Relative Standard Deviations (RSDs) for RRFs, and R2 Values (Linear Regression); Five
Calibration Levels 18
Table 7. Average (n=7) Concentrations (Ave Cone) with Standard Deviations (Std Dev)
and Percent Recoveries (Rec) of CWAs (14 |ig/L) and Surrogates (56 |ig/L) Using GC/MS
Configurations 23
Table 8. Average Percent Differences in Analyte Concentrations in Reagent Water Using
GC/MS Configurations 24
Table 9. Average (n=3) Matrix-based Concentrations of CWAs and Surrogates from Spiked
Surface Water Using GC/MS Configurations 27
Table 10. Average (n=7) Water Concentrations (Ave Cone) with Standard Deviations (Std
Dev) and Percent Recoveries (Rec) of CWAs (500 ng) and Surrogates (1000 ng) From Spiked
E&Hmalg GC/MS Configurations in SIM Mode 28
Table 11. Average (n=3) Soil Concentration (Cone) With Standard Deviation (Std Dev)
and percent recoveries (rec) for CWA (250 ng) and Surrogate (500 ng) From Spiked Soils
GfiflJglS Configurations in SIM mode 29
Table 12. Average (n=7) Mass per Wipe, With Standard Deviation (Std Dev), and Percent
Recoveries (Rec) For CWAs (250 ng) And Surrogates (500 ng) From Spiked Wipes Using
GC/TOF-MS and GC/MS (SIM Mode) 31
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List of Figures
Figure 1. GC/TOF-MS TIC of 0.5 ng each CWA and 1 ng each surrogate standard 9
Figure 2. Example of successful DFTPP tune check (printed by LECO's software) 12
Figure 3. Comparison of CWA and surrogate concentrations in three control samples measured
by GC/MS-TOF and GC/MS 19
Figure 4. Percent differences between the GC/TOF-MS and GC/MS concentrations in control
samples as a function of average concentration 20
Figure 5. CWA and surrogate concentrations, in seven reagent water extracts, measured by
GC/MS-TOF and GC/MS 22
Figure 6. Percent differences between the GC/TOF-MS and GC/MS concentrations measured in
reagent water extracts as a function of average concentration 22
Figure 7. Comparison of CWA and surrogate concentrations, in three surface water extracts,
measured by GC/MS-TOF and GC/MS 25
Figure 8. Percent differences between the GC/TOF-MS and conventional GC/MS (SIM mode)
concentrations measured in reagent water extracts as a function of average concentration 25
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1.0 Introduction and Background
Gas chromatography/mass spectrometry (GC/MS) is the method of choice for the
analysis of volatile and semivolatile organic compounds in environmental samples. Most
laboratories performing this technique use quadrupole mass spectrometers. The quadrupole
GC/MS performs mass filtering of ions based on changing direct current (DC) and radio-
frequency (RF) fields and has low detection limits. It is capable of detecting low- to sub-
nanogram quantities of chemicals when operated in the full scan mode and low picogram
amounts of materials when operated in the selected ion monitoring (SIM) mode. However, in
order to achieve the low detection limits of SIM, the collection of full mass spectral data is
sacrificed. Quadrupole GC/MS is commonly used in laboratories because advances in software
made by instrument manufacturers have simplified the use of such instruments — it is easy to
collect data with quadrupole mass spectrometers and the systems are rugged, reliable, and
relatively inexpensive (-$85,000, for a basic Agilent GC/MS). In addition, mass spectral
libraries have been developed for use with quadrupole GC/MS to assist in the identification of
unknown compounds and software packages assist in the processing and presentation of
quantitative data. The main disadvantages of quadrupole GC/MS are that a typical analysis takes
30-60 minutes and that some older systems do not possess the electronic components necessary
to provide the fast scan speeds required for operation with fast GC separations.
Gas chromatography coupled with time-of-flight mass spectrometry (GC/TOF-MS)
offers the promise of improved analytical speed. Because its principle of operation differs from
that of a quadrupole GC/MS (i.e., in GC/TOF-MS, ions of different masses travel through the
flight tube at different speeds, thus reaching the detector at different times), TOF-MS does not
have to "scan" a mass spectrum (by changing DC and RF fields) as does the quadrupole MS.
Due to this operational difference, the cycle of ion production, acceleration, and detection is
faster (on the order of 100 |isec) for TOF-MS than for quadrupole MS. The speed of the cycle
makes the TOF-MS an ideal instrument to couple with fast GC separations (i.e., separations that
provide improved GC resolution afforded by the use of narrow-bore [0.1- 0.18 mm i.d.] capillary
columns). Complete analyses using a fast GC method can be performed in less than half the time
required for separations using conventional 30 m x 0.25 mm i.d. columns. Thus, the GC/TOF-
MS is expected to be a valuable tool in situations where a large number of sample analyses are
required in a short amount of time. In addition, GC/TOF-MS provides low picogram detection
limits, retains complete mass spectral data for each compound it detects, and is comparable in
price to the quadrupole GC/MS ($101,000 for a basic LECO TruTOF® HT, LECO Corporation,
St. Joseph, MI). Retention of complete mass spectral data offers an additional level of
confidence in analyte identification (i.e., the more ions upon which to base analyte identification,
the greater the confidence in that identification)
In this study, the LECO Pegasus® 4D GC/TOF-MS was used to detect chemical warfare
agents (CWAs) and to compare this instrument's performance, with regards to speed of analysis
and detection limits, to conventional quadrupole GC/MS. Analytes studied were sulfur mustard
(HD), sarin (GB), soman (GD), cyclosarin (GF), and (9-ethykY-[2-(diisopropylamino)ethyl]-
methylphosphonothioate (VX). The use of the GC/TOF-MS was also compared to quadrupole
GC/MS for analysis of CWAs spiked in various matrices, including waters, sand, soils, and
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wipes. Limited data were also collected with fast GC separations coupled with quadrupole
GC/MS.
2.0 Study Objectives
The focus of this work was to determine how best to utilize the GC/TOF-MS (with
electron ionization) for the analysis of CWAs. Specifically, the goals were:
1) To establish appropriate separation conditions for the analysis of HD, GB, GD, GF, and
VX by GC/TOF-MS, while minimizing the analysis time.
2) To determine instrument detection limits (DDLs) by GC/TOF-MS (electron ionization
mode) for HD, GB, GD, GF, and VX.
3) To establish calibration curves and response factors using EPA Method 8270 internal
standards.
4) To compare analytical concentrations determined for analytes in sample extracts
measured by GC/MS (quadrupole system) and GC/TOF-MS. Sample extracts were
derived from various spiked matrices, including water, sand, soils, and wipes.
3.0 Experimental Conditions
The experimental strategy used in these studies was to first optimize separation and
analysis conditions for HD, GB, GD, GF, and VX and then to analyze the same standard
solutions and sample extracts by GC/TOF-MS and by quadrupole GC/MS.
3.1 Standards
CWA standards used for this study were synthesized by LLNL and were characterized
for purity by nuclear magnetic resonance spectroscopy (NMR) and GC/MS analyses. Dilute
standards were prepared gravimetrically from neat materials (stock solutions of 100 ppm
concentrations of each individual CWA were made new every 6 months; dilute solutions used for
calibrations were made from the 100 ppm stock solutions as needed, but typically used within
two weeks). As determined by proton NMR, the purities for GB, GD, GF, HD, and VX were
97.2%, 92.9%, 94.4%), 94.0%>, and 94.0%, respectively.
Surrogates and internal standards used were those of EPA Method 8270D (2) and those
suggested by a previous Battelle study (3). The surrogate standard mix included nitrobenzene-ds
(NB-ds), 2-fluorobiphenyl (FBP), phencyclidine-ds (PCP-ds), terphenyl-di4(Ter-di4), and
triphenyl phosphate (TPP). Specific solutions purchased for this work included: Base/Neutrals
Surrogate Standard, 1000 |ig/mL, in dichloromethane (Catalog number ERB-076, Cerilliant,
Round Rock, TX), Triphenylphosphate, 5000 |ig/mL, in methyl tert-butyl ether (Catalog number
ERT-108S, Cerilliant), andPCP-ds (phencyclidine-ds), 1000 |ig/mL, in methanol (Catalog
number P-006, Cerilliant).
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Internal standards used included l,4-dichlorobenzene-d4, naphthalene-dx,
acenaphthene-dio, phenanthrene-dio, chrysene-di2, and perylene-di2. These standards were
purchased as a Semivolatile Internal Standard Mix, 2000 |ig/mL, in dichloromethane (Catalog
number 861238, Supelco, Bellefonte, PA). Internal standards were spiked into all sample
extracts such that their concentrations were 1 ng/|iL for all analyses.
Decafluorotriphenylphosphine (DFTPP) was used to verify that the GC/MS systems were
functioning optimally. DFTPP was purchased as a solution with a concentration of 1000 |ag/m L
in acetone (Catalog number 47941, Supelco, Bellefonte, PA).
All standards and samples were stored at 4-8 °C.
3.2 Sample preparation
The sample preparation procedures, described below, were consistent with a CWA
analytical protocol study now under development at EPA. The extraction materials and
protocols, described below, have been previously employed by the laboratory (4).
Soil samples
Briefly, 10-g aliquots of sand and soils were spiked with 500 ng of surrogates (see
Section 3.1 above) and extracted for one-hour by waterbath sonication with 25.00 mL of
25/50/25 (v/v/v) acetone/dichloromethane/ethyl acetate. The resulting extract was separated
from the sand or soil by centrifugation and the supernatant removed. The sand and soils were
then extracted for a second time, as described above, with 5% triethylamine (TEA) in ethyl
acetate. Extracts from the two extraction procedures were kept separate, reduced in volume to
1.00 mL [using nitrogen and a Rapid Vap unit, customized to accommodate 40-mL vials
(LabConco, Kansas City, MO) and a Pierce Reacti-Therm III, #188 evaporation module
(ThermoScientific, Hudson, NH)] and spiked with internal standards (also per Section 3.1) prior
to analysis.
Soil samples used included Nebraska Aglands Ap soil (5.1% sand, 57.5% silt, 31.7%
clay, and 1.9% TOC), Georgia Bt2 soil (46% sand, 22% silt, 32% clay, and 0.2% TOC), and
Virginia soil (64.5% sand, 28% silt, 7.5% clay, and 2.6% TOC). All soils were obtained from
National Exposure Research Laboratory, US EPA, Las Vegas, NV.
Water samples
Water samples (35-mL) were spiked with 2 |ig each surrogate (see Section 3.1 above)
and extracted by vortexing for 2 minutes with 2.00 mL dichloromethane. Measured amounts of
the resulting extract were spiked with internal standards (also per Section 3.1) and analyzed. The
pH of the water samples was measured; pH of the reagent water (HPLC-grade, Aldrich, P/N
27,073-3) used was 9.85; pH of Milli-Q™ water was 8.14; and surface water was pH 7.80. The
surface water used in this study was collected at the Zone 7 Water Agency Water Quality
Laboratory, Livermore, California, and came from the South Bay Aqueduct, which collects water
from the Sacramento River Delta and includes snowmelt water from the northern Sierra Nevada
Mountains.
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Wipe samples
Wipes (3" x 3", Kendall-Curity, 12-ply, P/N 1903, available from Tyco Healthcare Group
LP, Mansfield, MA) were spiked with 500 ng surrogates (see Section 3.1 above) extracted by
waterbath sonication for 30 minutes (twice) with 15.00 mL 25/50/25 (v/v/v)
acetone/dichloromethane/ethyl acetate. The resulting extracts were combined, evaporated to
1.00 mL, spiked with internal standards (also per Section 3.1), and analyzed.
All sample extracts were stored at 4-8 °C until the time of analysis and each batch of
sample extracts was analyzed with corresponding method blanks.
3.3 GC/TOF-MS conditions
GC/TOF-MS experiments were performed with an Agilent 6890 gas chromatograph
(Agilent Technologies, Inc., Santa Clara, CA) coupled with a LECO Pegasus® 4D mass
spectrometer (LECO Corp., St. Joseph, MI). Prior to use, the GC/TOF-MS was tuned with the
vendor's standard protocols and perfluorotributylamine (PFTBA) as a calibrant. An injection of
15 ng decafluorotriphenylphosphine (DFTPP) was used to check the performance of the
instrument prior to analyzing samples. The amount of DFTPP used for instrument checks was
lower than the 50 ng amount used to check the performance of a quadrupole GC/MS system.
The reduction in DFTPP was necessary so as not to overload the GC column and TOF-MS
detector. Experimental data were collected using the same instrument conditions, including
electron multiplier voltages, as those used to produce the DFTPP check samples. During
analysis sequences, a continuing calibration verification (CCV) standard near the midpoint of the
calibration range was analyzed every 10 samples. The CWA concentrations calculated for the
CCV, using the most recent calibration curve, were required to be within 20% of the expected
value in order for the data collected between CCV checks to be considered valid.
Standard operating parameters for the GC/TOF-MS were as described below:
Injection size:
Inlet type:
Injection mode:
Pulse pressure:
Purge time:
Carrier gas:
GC injection port:
GC columns:
1 |iL
Split/splitless
Pulsed-splitless
40 psi for 0.5 min
35 sec at 30 mL/min
He with constant flow of 1.2 mL/min
250 °C
15 m x 0.18 mm i.d. x 0.18 |im film thickness, HP5-MS UI
(Agilent Technologies, Inc., Santa Clara, CA)
1 m x 0.1 mm i.d. x 0.1 |im film thickness, Rxi-17 (Restek,
Bellefonte, PA)
GC oven (primary): 55 °C held for 0.5 min, 20 °C/min to 100 °C, 40 °C/min to 280 °C,
held for 2.75 min
GC oven (secondary): 70 °C held for 0.5 min, 20 °C/min to 115 °C, 40 °C/min to 295 °C,
held for 1.64 min
GC transfer line: 295 °C
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The following MS conditions were used for detection.
MS filament delay: 1.5 min
MS scan range: 35-500, at a data acquisition rate of 15 spectra/sec
MS source: 250 °C
Electron energy: 70 eV
Although the GC/TOF-MS used for this study was capable of performing two-
dimensional (2-D) GC separations, it was operated to perform only one-dimensional (1-D)
separations, using the HP5-MS UI column. In 2-D chromatography, a second GC column with a
different chemical phase than that of the first GC column is used to provide additional separation
of analytes as they elute from the first column. Because the chemistry of the second GC column
is different from the first, compounds that co-elute from the first column may be easily resolved
after a separation on the second GC column (i.e., the peak capacity of the system is increased and
the specificity of analyte detection is improved). In order to maintain a configuration that
allowed for easy transition between 1-D and 2-D modes of operation (i.e., did not require the
venting and down-time of the TOF-MS associated with column changes and installations), the
Rxi-17 column was left in place when 1-D experiments were performed. The modulator and
secondary oven conditions were optimized so that the second GC column acted as a transfer line
into the TOF-MS. No separations occurred on the secondary GC column. By keeping the
secondary GC oven at a temperature 15 °C higher than the primary oven (to ensure that analytes
did not condense in the secondary column) and by ensuring that the modulator was not used to
cryofocus effluent from the primary GC column, the Rxi-17 column segment performed the
function of a transfer line into the mass spectrometer.
To collect GC/TOF-MS data, separate autosampler, GC, MS, and data processing
methods were created and linked. The method parameters routinely used to collect and process
data are recorded in Appendices A, B, D, and E of this report. Because the values of the
parameters that were entered into the LECO software were of great importance, screen capture
images of the method setup pages have been provided so that the conditions of the analyses can
be replicated. Appendix A contains the autosampler method used for all experiments and
Appendix B contains the GC conditions used by LLNL. Because the GC/TOF-MS used in this
study was capable of operating with 2-D GC separations, there were additional parameters that
would not be used with a GC/TOF-MS that is capable of only 1-D GC separations. For this
reason, also included in Appendix C are parameters that would be used to replicate the methods
on a GC/TOF-MS that is capable of performing a chromatographic separation using a single GC
column. Appendix D contains the relevant MS method and Appendix E contains the data
analysis method used in this study.
3.4 GC/MS conditions (conventional quadrupole)
GC/MS was performed with an Agilent 6890 GC coupled with an Agilent 5973 MS (Agilent
Technologies, Inc., Santa Clara, CA). Prior to use, the GC/MS was tuned with the vendor's
standard protocols and PFTBA as a calibrant. An injection of 50 ng DFTPP was used to check
the performance of the instrument prior to sample analysis. CCVs were also performed every 10
samples, as prescribed by EPA protocols, during the course of run sequences. Acceptance
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criteria used for the CCVs required that their measured concentrations were 80-120% of the
expected values.
The standard GC parameters were:
were:
Carrier gas: Helium, at a constant flow of 32 cm/s
Injection mode: Splitless for 0.75 min
Injector temperature: 250 °C
Sample injection volume: 1 |iL
GC Column: Agilent HP-5MS, (5%-phenyl)-methylpolysiloxane
Column dimensions: 30 m x 0.25 mm x 0.25 |im (length x i.d. x film thickness)
GC temperature program: 40 °C held for 3 min, 10 °C/min to 150 °C, 25 °C/min to
280 °C, held for 10.8 min
The standard MS conditions for full scan analyses performed in electron ionization mode
MS transfer line temperature: 280 °C
MS source temperature: 230 °C
MS quadrupole temperature: 150 °C
Solvent delay time: 3 min
Scan range: 35-500 m/z
Electron energy: 70 eV
Scan time: 3.15 scans/sec
Ionization polarity: Positive
The standard MS conditions for selected ion monitoring analyses performed in electron
ionization mode were:
MS transfer line temperature: 280 °C
MS source temperature: 230 °C
MS quadrupole temperature: 150 °C
Electron energy: 70 eV
Ion dwell time: 100 msec per ion (each analyte was assigned its own SIM
group; depending on the number of ions monitored, cycle
times ranged from 1.44 - 2.86 cycles/sec)
Ionization polarity: Positive
3.5 GC/MS conditions (fast separations, quadrupole MS)
GC/MS analyses were performed with an Agilent 5973 system, which was tuned with
DFTPP and checked with 50 ng DFTPP. During analyses, CCVs (continuing calibration
verification) were analyzed at a frequency of every 10 samples. Acceptance criteria used for the
CCVs required that their measured concentrations were 80-120% of the expected values.
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The GC conditions were modified to allow faster chromatographic separations.
Parameters that were changed from the conditions of Section 3.4 are shown below:
Carrier gas:
Injection mode:
Injector temperature:
Sample injection volume:
GC Column:
Column dimensions:
GC temperature program:
Helium, at a constant pressure of 17.8 psi
Splitless for 0.75 min
250 °C
1 |iL
Agilent HP-5MS-UI, (5%-phenyl)-methylpolysiloxane
20 m x 0.18 mm x 0.18 |im (length x i.d. x film thickness)
40 °C for 1.25 min, 24 °C/min to 150 °C, 45 °C/min to 280
°C, held for 7.3 min
The standard MS conditions for full scan analyses performed in electron ionization mode
were:
MS transfer line temperature: 280 °C
MS source temperature: 230 °C
MS quadrupole temperature: 150 °C
Solvent delay time: 3 min
Scan range: 35-500 m/z
Electron energy: 70 eV
Scan time: 5.92 scans/sec
Ionization polarity: Positive
The standard MS conditions for selected ion monitoring analyses performed in electron
ionization mode were:
MS transfer line temperature: 280 °C
MS source temperature: 230 °C
MS quadrupole temperature: 150 °C
Electron energy: 70 eV
Ion dwell time: 40 - 300 msec per ion (each analyte was assigned its own
SIM group; depending on the number of ions monitored,
cycle times ranged from 1.05 - 5.88 cycles/sec)
Ionization polarity: Positive
3.6 Quality Assurance (QA)
Data limitations. The study was performed per the approved quality assurance project
plan (QAPP) (1); Tasks 1 through 5 of that study plan were completed and discussed in this
report. The LECO Pegasus® 4D GC/TOF-MS was used to detect CWAs and to compare this
instrument's performance to the speed of analysis and detection limits of the quadrupole-based,
Agilent 5973 GC/MS. While data presented in this report are only valid for the specific
instrument systems cited, the applicability of quadrupole and time-of-flight mass spectrometers
of other vendors to the analysis of CWA can be inferred. However, instrument and method
detection limits will vary and will be instrument-specific.
7
-------
Deviations from study plan. Analytes studied were HD, GB, GD, GF, and VX.
According to the original QAPP, tabun (GA) was also to be studied. When the QAPP was
written, it was thought that there was sufficient stock of existing GA of acceptable purity to
include in this study. As GA was not available at the time the study was initiated, it was decided
to study only the five CWAs being provided to the Environmental Response Laboratory Network
(ERLN) laboratories.
The original QAPP called for the use of 2 ng DFTPP to document the good performance
of both the GC/TOF-MS and the GC/MS. During this study, 15 ng DFTPP were used to access
the performance of the GC/TOF-MS and 50 ng DFTPP were used to access the performance of
the GC/MS. This change was made to be consistent with the quality checks used with these
instruments for other EPA-sponsored work that was ongoing at the time of this study. The
change in concentration did not affect the ability to demonstrate that the mass spectrometers
were in working condition prior to commencing analyses.
Measured concentrations of analytes were determined by GC/MS and GC/TOF-MS and
compared for reagent water, surface water, sand, three types of soils, and wipes, per the original
QAPP. Method detection limit studies were not performed.
4.0 Results
4.1 Chromatographic separation of CWAs
Using the GC/TOF-MS, with conditions described above, chromatographic analysis of
CWAs was performed in less than 13 minutes; see Figure 1. This speed of chromatographic
analysis represents a two- to three-fold reduction in time from the 30-minute analysis that was
required using GC/MS with a 30 m x 0.25 mm i.d. GC column. This analysis time was longer
than the six-minute separation reported by ECBC, using a 10 m x 0.25 mm i.d. GC column (5).
Originally the ECBC method was implementedwas found to not be practical with the LECO
Pegasus® IV for several reasons. First, because the LECO Pegasus® IV was configured for 2-D
separations, the instrument could not physically accommodate the oven insert that was needed to
provide consistent GC oven heating with higher temperature ramp rates. The secondary oven
and modulator occupied space in the GC oven which prohibited the installation of the oven
insert. Because the instrument used by ECBC was configured for 1-D separations, ECBC was
able to use an oven insert to reduce GC oven volume, thereby obtaining consistent temperature
ramp rates near the vendor's recommended maximum values for an Agilent 240V fast, 6890 GC.
In addition, because the insert effectively reduced the volume of the GC oven, the insert allowed
ECBC to reduce GC cycle times and, therefore, increase sample throughput.
8
-------
•i; •*—'
Y Jl -c
E £S
to-£ 01
=3 -C ">
F Q 5<
f I
Figure 1. GC/TOF-MS TIC of 0.5 ng each CWA and 1 ng each surrogate standard.
Second, analyte separations using a 10 m GC column (HP-5MS UI10 m x 0.18mm i.d. x
0.18 |im film thickness) and fast temperature ramps were not reproducible between equivalent
GC columns. While the separation conditions used by ECBC (5) provided complete resolution
of all analytes in the calibration solution on one GC column (see additional data provided in
Appendix F), complete separation of HD, naphthalene-d8, and GF was not achieved when two
other GC columns of the same stationary phase and column dimensions were used. Subsequent
discussion with two other CWA labs suggested that they, too, observed similar separation
problems. Even when complete separation of analytes was observed, because the separation
between HD and naphthalene-d8 was only approximately 1 second, there were concerns that the
compression of the chromatography would not be sufficient to allow separation of the analytes
from the many components expected to be present in environmental matrices. Thus, the less
aggressive GC temperature ramp rates reported in Section 3.3 were used, which resulted in a 13-
minute separation (in contrast to the 6-minute separation shown in Appendix F).
Third, there was a concern that a GC temperature ramp rate that was too fast would not
provide adequate chromatographic separation of analytes from the many interfering compounds
expected to be present in complex sample matrices. While LECO's software includes algorithms
that aid in the deconvolution of spectral data, this software deconvolution is no substitute for
good chromatographic separation. In addition to good chromatographic separation, a secondary
concern is to make sure that there are a sufficient number of data points to adequately define
each chromatographic peak present. When developing any GC-based method for analyte
detection and quantification, care must be taken to collect an adequate number of data points
across a GC peak to accurately define its shape. One common rule of thumb is that at least 10
data points are needed to define a chromatographic peak. LECO (GC/TOF-MS vendor)
recommends that 15-20 mass spectra be collected across each chromatographic peak. Without
an adequate number of data points to accurately define a chromatographic peak, peak shape,
reproducibility (which affects quantitation), the ability to use LECO's deconvolution software,
and the potential to later perform 2-D separations (which can provide important information
when confirmatory analyses are needed) are compromised. Thus, the strategy used to develop a
GC/TOF-MS method was to first optimize the chromatography and then to make sure that the
data acquisition rate of the TOF-MS was set so that 15-20 mass spectra would be collected
across the narrowest GC peak observed. The collection of 15-20 mass spectra allows LECO's
deconvolution software to assign the proper spectra and signal intensities to any coeluting peaks.
9
-------
Using the chromatographic conditions previously described, peak widths on the order of 0.8-1
second were typically observed and data acquisition rates on the order of 20-25 mass spectra per
minute were found to be adequate. While the GC/TOF-MS is capable of acquisition speeds of
500 spectra/sec, there are trade-offs between the number of spectra collected and the ease of data
processing and the number of spectra collected and the size of the data file that is collected
during the course of an analysis.
Using the separation conditions previously discussed (i.e., Section 3.3), reproducibility of
retention times and analyte responses for seven replicate injections of a standard containing 0.5
|ig/mL of CWAs were documented and are shown in Table 1. As was evident from the data, for
seven replicate injections, retention times were stable and varied by less than 0.4% (relative
standard deviation) for all compounds. This stability is reasonably consistent with reports that a
sample of 32 Agilent 6890 Plus GCs consistently demonstrated relative standard deviation of
less than 0.1 percent, with some lower than 0.01 percent (6). All analyte responses, measured as
peak areas, were also reproducible and varied by less than 3%.
Table 1. Average Retention Times (± Standard Deviations) and Average Analyte Responses
(± Standard Deviations) for Seven Replicate Injections of 0.5 ng of each CWA into the
GC/TOF-MS
Analyte
Average
Retention
Time (sec)
Average Analyte Response
(peak area in arbitrary
units)
GB
152.49 ±0.67
1267684 ±23832
GD 1
272.68 ±0.83
318556 ±6419
GD 2
275.54 ±0.83
284388 ±4875
GF
361.51 ±0.65
874301 ±22988
HD
352.45 ±0.68
803696±18309
VX
494.59 ±0.22
66214± 1580
Before data could be collected using established separation conditions, acceptable
performance of the GC/MS-TOF needed to be documented. As for other GC/MS-based methods
used by EPA, DFTPP was used to check acceptability of the system tune. DFTPP mass spectral
attributes required to document acceptable performance of the GC/TOF-MS were evaluated.
Several sets of DFTPP criteria are in use for various EPA methods; see Table 2. DFTPP criteria
include those which were derived for contract laboratory program work, those which were used
by EPA Methods 8270D (2) and 527 (7), and those suggested by LECO (8, 9). While
performing instrument detection limit and calibration studies, the DFTPP tune check was
observed to predictably fail against the criteria (abundance of m/z 442 was outside the allowed
range and the abundances of m/z 365 and m/z 441 were almost outside their allowed ranges).
The DFTPP check also failed the requirements of Method 8270D (abundances of m/z 441 and
m/z 442 failed and m/z 365 was near failure). The DFTPP check suggested by LECO always met
acceptance criteria if the instrument was operating properly. LECO also found this to be the case
and, in 2005, petitioned to have their DFTPP criteria accepted by the US EPA (10).
10
-------
Subsequently, the DFTPP criteria of EPA Method 527 were modified so that both quadrupole
GC/MS and GC/TOF-MS would be able to meet them. Figure 2 shows an example of a tune
report used to determine if the GC/TOF-MS was working properly. Samples were analyzed only
if these DFTPP tune criteria were met.
Table 2. DFTPP Key Ions and Ion Abundance Criteria Used by Different Methods to
Verify GC/MS Tune
Ion Abundance Criteria
Mass
Purpose
EPA Method
8270D
Draft EPA
protocol
9/2008
LECO
EPA Method
527
51
Low-mass
10-80% of base
10-80% of
10-85% of
10-85% of base
sensitivity
peak
m/z 198
m/z 198
peak
68
Low-mass resolution
< 2% m/z 69
< 2% m/z 69
< 2% m/z 69
< 2% m/z 69
69
Not used
Present
Not used
Not used
70
Low-mass resolution
< 2% m/z 69
< 2% m/z 69
< 2% m/z 69
< 2% m/z 69
127
Low-mid-mass
10-80% of base
10-80% of
10-80% of
10-80% of base
resolution
peak
m/z 198
m/z 198
peak
197
Mid-mass resolution
<2% m/z 198
<2% m/z 198
<2% m/z 198
<2% m/z 198
198
Mid-mass resolution
& sensitivity
Base peak or
> 50% m/z 442
Base peak
Base peak
Base peak or
> 50% m/z 442
199
Mid-mass resolution
& isotope ratio
5-9% m/z 198
5-9% m/z 198
5-9% m/z 198
5-9% m/z 198
275
Mid-high-mass
10-60% of base
10-60% of m/z
10-60% of
10-60% of base
sensitivity
peak
198
m/z 198
peak
365
Baseline threshold
> 1% m/z 198
> 1% m/z 198
> 0.5% m/z
198
> 0.5% m/z 198
441
High-mass
Present, < 24%
Present, < m/z
< 150%
< 150%
resolution
of m/z 442
443
m/z 443
m/z 443
442
High-mass
Base peak or
> 50 - 100%
> 30% of m/z
Base peak or
resolution &
> 50% m/z 198
m/z 198
198
> 30% m/z 198
sensitivity
443
High-mass
resolution & isotope
ratio
15-24% m/z 442
15-24%
m/z 442
15-24%
m/z 442
15-24% m/z 442
11
-------
Ilk-
DFTPP Tune Check
Lawrence uvermore National Laboratory Operator: Heather Mulcany
Sample Name: 15 ng/uL DFTPP (EPA-STD&M2-1) Data File Name: DFTPP:99
Date: 6/8/2009 Time: 7:57:02 AM
Leco Pegasus IV GC-TOF/MS Model: 614-200-700 SN: 3271
Result: Passed
Mass
Criteria
Reference Mass
Min Rel. Abundance
Max Rel, Abundance
Relative Abundance
Pass/Fail
51
>10.00% and <85.00
% of Base Inn
Base
10. DO
85.00
4B.00
Passed
S3
<2_DO% of mass 59
69
2. DO
1.18
Passed
70
<2_DO% of mass 99
69
2.DO
0.32
Passed
127
>10.00% and <30.00
% of Base Ion
Base
10. DO
80.00
3-8.63
Passed
187
<2_D0%of mass 198
198
2.DO
0.21
Passed
168
Base Ion
442
50. DO
100.DO
Passed
196
>5_DO% and 10.00% and <80.00
% of Base Ion
Base
10.DO
eo.oo
13.63
Passed
365
>0.50% of mass 198
198
0.5D
0.76
Passed
441
<150.00% of mass 443
443
150.00
10Z9S
Passed
442
>30.00% of mass 188
198
30. DO
45.94
Passed
443
>15.00% and <24.00
% of mass 442
442
15.DO
24.00
20.30
Passed
800000
700000
600000
500000-
400000 -
300000 -
200000-
100000-
Qc Result - DFTPP:99
lOOOn 198
800-
600-
400-
200-
I I I I [ T
Time (s) 430 435 440 445 450 455
198 I
110
255
d
iLA I f*p-|VtVrYn [1
100 200 300 400
442
iVrr
500
Figure 2. Example of successful DFTPP tune check (printed by LECO's software).
12
-------
In summary, when choosing GC/TOF-MS separation conditions, several factors were
considered including:
1) The method must have the ability to be performed on a basic GC/TOF-MS,
without additional investment in equipment such as a GC oven insert.
2) The selected temperature ramp rates must be well within the limits of the GC
oven.
3) Selected conditions should provide reasonable chromatographic separation of
analytes for sample extracts that contain a large number of matrix interferences.
4) Analyte retention times and peak areas must be reproducible.
5) A minimum of 15-20 mass spectra must be collected across each
chromatographic peak.
The final method that was proposed met all of these criteria and provided separation of
analytes in just less than 13 minutes (see Section 3.3). Using this LLNL-developed GC/TOF-MS
method (1-D separation, while two GC columns are installed in GC/TOF-MS), an estimated
throughput of 82 analyses per 24 hours could be achieved using a single instrument. During
this same time period, only 35 samples could be analyzed using conventional GC/MS
(quadrupole), which required an analysis time of approximately 30 minutes. However, should a
higher sample throughput be desired, there are several options that could be explored, including:
1) Use a GC oven insert to reduce the oven volume so that the GC column heats
more quickly, yielding faster analyte separations. The reduced oven volume also
allows faster cooling of the GC, thus reducing the overall cycle time and
increasing throughput. This option is currently available only for GC/TOF-MS
systems that perform exclusively 1-D separations.
2) Eliminate the late-eluting acenaphthene-dio, chrysene-di2 and perylene-di2 as
internal standards that are currently proposed by the draft CWA protocol under
development so that analytical run time would be reduced.
3) Use cryo-cooling to reduce GC cycle time. Experiments in the laboratory (data
not shown) have suggested that the use of cryo-cooling can reduce GC cycle
times by more than 50%. Note that this strategy is only feasible when a single
GC column is being used.
13
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4.2 Instrument detection limits
Instalment detection limits (DDLs) were determined by making successive injections of
individual standards of decreasing analyte concentrations until a signal-to-noise ratio (S:N) of
approximately 3:1-5:1, as determined manually, was obtained for the analyte peak of the second
confirmation ion present in the chromatogram (detection of an analyte required the presence of
the quantitation ion and two qualifying ions). The analyte mass at which a S :N of 3:1-5:1 was
obtained for the second qualifying ion of three successive injections was reported as the DDL.
Blank samples (i.e., clean solvent) were analyzed before the determination of the final DDLs to
ensure that carryover of higher concentrations of analytes did not influence DDL determinations.
As shown in Table 3, GC/TOF-MS DDLs ranged from 0.0025-0.025 ng. These DDLs are
reasonably comparable to GC/TOF-MS DDLs observed by Virginia Division of Consolidated
Laboratory Services (Richmond, VA) (data unpublished); see Table 4. Some differences in DDLs
were noted and might be attributed to differences in the methods used to determine S:N.
Table 3. IDLs, Quantification (Quant) and Qualifying (Qual) Ions, and S:N Determined by
GC/TOF-MS
Analyte
LLNLIDL
TOF
(ng)
Quant.
Ion
1st Qual.
Ion
2nd Qual.
Ion
Avg. S/N
(n=3)
2nd Qual. Ion
GB
0.025
99
125
81
4.3
GD1+GD2
0.005
99
126
82
7.7
GF
0.0025
99
67
81
5.3
HD
0.0025
109
111
63
9.8
VX
0.0025
114
72
127
5.4
Note: S:N values for the second qualifying ion were determined by manual integration
14
-------
Table 4. Comparative Data for IDLs in Nanograms
Analvlc
1 .ahoraloi'Y
\ A
I.I.M.
I.I.M.
I.I.M.
I'.qui
niicnl Tested
GC/TOF-
MS
GC/TOF-
MS
Quadupole
GC/MS, FS*
Quadupole
GC/MS, SIM*
GB
0.005
0.025
0.2
0.01
GD1+GD2
0.0025
0.005
0.05
0.01
GF
0.005
0.0025
0.2
0.02
HD
0.002
0.0025
0.05
0.01
VX
0.0025
0.0025
0.2
0.05
*Data from Ref 4
Acronyms: VA, Virginia Division of Consolidated Laboratory Services; LLNL,
Lawrence Livermore National Laboratory; SIM, selected ion monitoring mode; FS, full
scan mode
Note: GC/TOF-MS experiments performed with 15 m, 0.18 mm i.d., GC columns and
GC/MS experiments performed with 30 m, 0.25 mm i.d., GC columns.
During the course of this work, obvious differences in manual and automated S:N
determinations were observed. It is important to understand the differences in the methods used
to calculate S:N to ensure that DDLs are determined based on comparable numbers. Manual S:N
determinations were calculated for an ion of a preselected mass and were determined based on
peak height and baseline noise in the region of the ion chromatogram that immediately preceded
the peak of interest. Manual S:N determinations were usually (but not always) lower than those
determined using LECO's software. LECO's software provided two automated methods of S:N
determinations; both of these methods calculated noise based on the background signal of the
entire chromatogram. LECO's "Quant S:N" mode calculated S:N for a selected peak based on
baseline, peak height, and the standard deviation of the noise of the baseline at a specified mass-
to-charge ratio (m/z). LECO's "S:N" mode calculated S:N based on the signal for an unique
mass and baseline selected by the software's deconvolution software and was used for peak find
and peak purity calculations. "S:N" baseline was not necessarily the same baseline as that used
for "Quant S:N" calculations, so the two methods of S:N determinations did not always provide
equivalent results. To calculate "S:N", LECO's software allowed the selection of an unique ion.
In contrast, while using "Quant S:N", the software determined an ion that was both of strong
abundance and was not subject to interferences from neighboring peaks.
To provide an illustration of the results produced by different methods of S:N
determination, consider the S:N values calculated when 0.025 ng of VX were introduced into the
GC/TOF-MS. Manual S:N calculation for m/z 127 (2nd qualifying ion) yielded S:N = 136. S:N
values of 346 and 353 were determined using the instrument's software when the "S:N" and
"Quant S:N" modes, respectively, were used. In this situation, using the software's S:N
determination algorithms, and basing DDL on S:N, would produce an DDL that was
approximately a factor of two lower than DDL based on the manual S:N determination. For this
reason, manual S:N calculations were used to determine DDLs that are reported in Table 3 (i.e., at
15
-------
DDL, a manually determined S :N of approximately 3:1-5:1 was obtained for the peak of the
analyte's second confirmation ion). Manual integration was also chosen so that the method of
DDL determination was comparable to that used for quadrupole GC/MS studies (4).
As shown in Table 4, GC/TOF-MS DDLs were lower than those obtained by GC/MS
(quadrupole) operated both in full-scan and selected ion monitoring modes. The lower detection
limits were attributed to both the different MS detector and the fact that GC/TOF-MS
experiments used 0.18 mm i.d. GC columns. When equivalent amounts of analyte were
introduced onto the GC column, the 0.18 mm i.d. column of the GC/TOF-MS produced narrower
and taller chromatographic peaks than did the 0.25 mm i.d. column of the quadrupole GC/MS.
The GC/TOF-MS produced lower DDLs than quadrupole GC/MS and, unlike GC/MS operated in
the selected ion monitoring mode (which provides optimum detection limits for GC/MS by
detecting only pre-selected ions), provided full mass spectral data, which, as noted in the
introduction, offers an additional level of confidence in analyte identification (i.e., the more ions
upon which to base analyte identification, the greater the confidence in that identification).
4.3 Calibration
Once DDLs were determined, calibration curves were established. At the low and high
points of the calibration curve (calibration ranges were chosen to reflect expected concentrations
in environmental samples), the mass spectra of the analytes of interest were determined and
compared with those of the NIST library. Match factors, describing how well generated mass
spectra fit to those reported in the NIST library, were reported; see Table 5. At higher
concentrations, match factors were good—typically better than 840 out of a possible 1000. At
the lowest detectable concentrations, the library matches for GB and GF, respectively, at 905 and
913, were good; however, the goodness-of-fit in spectral data for the other CWAs ranged from
590-650. Thus, at the lower concentrations, confidence in correct analyte assignment based on
spectral data alone was less certain. For the CWAs, at 2-3 times the DDL, the match factors were
better than 700 and S:N values were greater than or equal to 10 (by manual determination).
Although these data represent a best case scenario (i.e., match factors for similar concentrations
of CWAs in complex matrices are not expected to be as good), they are useful because they
provide some perspective for data evaluation.
16
-------
Table 5. Average (N=3) Match Factors (Forward Fit), Relative to the NIST Database,
Determined for Various Analytes at the High and Low Points of the Calibration Curve
Analyte
Retention Time
(sec.)
Curve
Point
Forward
Fit
GB
107.7
Low - 0.01 ng
905
High - 0.5 ng
931
GD 1
194.3
Low - 0.0025 ng
663
High-0.125 ng
921
GD 2
196.0
Low - 0.0025 ng
655
High-0.125 ng
916
GF
248.4
Low - 0.01 ng
913
High - 0.5 ng
929
HD
242.5
Low - 0.005 ng
650
High - 0.25 ng
922
VX
353.5
Low - 0.01 ng
592
High - 0.5 ng
842
Note: calibration ranges were chosen to reflect expected
concentrations in environmental samples
Using the calibration data, average relative response factors, percent relative standard
deviations, and R2 values (linear regression) for five calibration levels were determined; see
Table 6. Calibration ranges were chosen to reflect the largest possible range available using the
ultradilute chemical agent standards. Using practices that were consistent with EPA Method
8000C (11) and the draft CWA protocol, a procedure for quantifying GB, GD, GF, HD, and VX
was implemented using the internal standards of Method 8270D, which are 1,4-dichlorobenzene-
d4, naphthalene-d8, acenaphthene-dio, phenanthrene-dio, chrysene-di2, and perylene-di2.
Because the percent relative standard deviations for the relative response factor values were not
always less than or equal to 20% (based on the guidance required by U.S. EPA's 8000-series
methods), all quantitation for the CWAs was based on linear regression. At 2-3 times the DDL,
the differences between the expected and measured concentrations for standards (based on
calibration curve values) were < 15%.
17
-------
Table 6. Calibration Data for CWAs: Average Relative Response Factors (RRFs), Percent
Relative Standard Deviations (RSDs) for RRFs, and R2 Values (Linear Regression); Five
Calibration Levels
Analyte
Internal Standard
Calibration
Range
(ng/jiL)
Mean
RRF
% RSD of
RRF
R2
GB
1,4-Dichlorobenzene, d4
0.025 - 10
0.976
20.6
0.9994
GD
1,4-Dichlorobenzene, d4
0.025 - 5
0.428
18.3
0.9999
GF
Napthalene, ds
0.025 - 10
0.312
24.6
0.9999
HD
Napthalene, ds
0.025 - 5
0.176
30.4
0.9994
VX
Phenanthrene, dio
0.05 - 10
0.316
34.6
0.9996
Note: Calibration ranges were chosen to reflect the largest possible range available using the ultradilute
chemical agent standards.
4.4 Analyses of sample extracts
Selected matrices were spiked with CWAs and surrogates, extracted, and analyzed using
the GC/TOF-MS. The same sample extracts were also analyzed by quadrupole GC/MS (30 m x
0.25 mm i.d. x 0.25 |im film thickness GC column). In order to provide somewhat similar S:N
ratios for selected peaks analyzed by both GC/MS and GC/TOF-MS, all GC/MS analyses of
sample extracts were performed using the SIM mode. Extracts of laboratory reagent water,
surface water, clean sand, three soil types, and wipes were extracted and analyzed by both
GC/TOF-MS and GC/MS to produce comparative data.
To provide a simple comparison of analyte concentrations measured by GC/TOF-MS and
GC/MS, a plot of analyte concentrations (CWAs and surrogates) measured by GC/TOF-MS
versus analyte concentrations measured by GC/MS (SIM mode) was generated for the data
collected from three control samples analyzed during the course of the study. Each of these
control samples contained five CWAs and five surrogate compounds. In plotting these
concentration data, the population variances of measurements by GC/TOF-MS and by GC/MS
were assumed to be equal. We also assumed, to a first approximation, that all analytes behaved
similarly in their ability to be detected by GC/MS and GC/TOF-MS. Thus, all analytes were
plotted on the same graph. Control samples consisted of dichloromethane that was spiked
directly with CWAs and surrogates, and represented the cleanest possible samples (i.e., samples
with no interfering compounds introduced from the sample matrix). The specific CWAs and
surrogates in the samples have previously been described in Sections 2.0 and 3.1 of this report.
The resulting plot is shown Figure 3.
Figure 3 shows a comparison of CWA and surrogate concentrations in the three control
samples measured by GC/MS-TOF and GC/MS (30 m x 0.25 mm i.d. x 0.25 |im GC column).
Each point represents a measured concentration of CWA or surrogate in the sample extract. In
this sample set, CWAs were spiked at 0.5 ng/|iL (GD isomers were measured individually at
approximately 0.25 ng/|iL) and surrogates were spiked at 1 ng/|iL. All GC/MS data were
collected in SIM mode.
18
-------
A regression line was calculated for the data displayed in Figure 3; its R2 value was 0.82,
its slope was 0.82 (with a standard error of 0.068), and its intercept was 0.068 (with a standard
error of 0.048). The data show a statistically significant correlation of concentration measured
by GC/MS and GC/TOF-MS (p-value « 0.001). On average, concentrations measured by
GC/MS and GC/TOF-MS agreed within 16%. However, there were two apparent outliers. Two
of the concentrations measured for PCP-ds by GC/MS were noticeably lower than the expected 1
ng/|iL, The reason for observation of these lower concentrations is unclear.
Comparison of Control Sample Concentrations
GC/MS, 30 m column (ng/nL)
Figure 3. Comparison of CWA and surrogate concentrations in three control samples
measured by GC/MS-TOF and GC/MS.
19
-------
1.4
Figure 4. Percent differences between the GC/TOF-MS and GC/MS concentrations in
control samples as a function of average concentration.
Because the slope of the regression line of Figure 3 suggested that slightly lower analyte
concentrations were measured by GC/TOF-MS (a slope of exactly 1 would be observed if
concentrations measured by the different instruments were equivalent), the percent differences in
the GC/TOF-MS and GC/MS concentrations were plotted as a function of average concentration
to study the bias; see Figure 4. Figure 4 shows the percent differences between average
concentrations measured by GC/TOF-MS and GC/MS (percent difference = 100*(GC/MS cone
- GC TOF-MS cone) / [(GC/MS cone + GC-TOF-MS conc)/2]). Visual inspection of the plot
shown in Figure 4 suggests that no clear bias in the measured concentrations; however, there is
considerable scatter in the data.
4.4.1 Analyses of water sample extracts
Reagent water was spiked with CWA and surrogates, extracted, and analyzed by
GC/TOF-MS and GC/MS (SIM mode). Figure 5 provides a plot of analyte concentrations in
sample extracts measured by GC/MS (30 m x 0.25 mm i.d. x 0.25 |im film thickness GC
column) versus extract concentrations measured by GC/TOF-MS, generated from the data
collected from seven replicate samples that were spiked, extracted, and analyzed. Each point
represents a measured concentration of CWA or surrogate in the sample extract. In this sample
set, CWAs, assuming 100% recovery, would be present at approximately 0.25 ng/|iL (GD
isomers were measured individually at approximately 0.14 ng/|iL and VX concentrations were
high at -0.5 ng/|iL) and surrogates would be present at approximately 1 ng/|iL. All GC/MS data
were collected in SIM mode.
Using the data in Figure 5, a regression line was calculated and its R2 value was 0.92, its
slope was 0.86 (with a standard error of 0.030), and its intercept was 0.059 (with a standard error
of 0.027). The data show a statistically significant correlation of concentration measured by
GC/MS and GC/TOF-MS (p-value « 0.001). On average, concentrations measured by GC/MS
and GC/TOF-MS agreed within 16%.
50
C
-------
As in the analysis of the previous data, the percent differences in the GC/TOF-MS and
GC/MS concentrations were plotted as a function of average concentration to study the bias
(percent difference = 100*(GC/MS cone - GC/TOF-MS cone) / [(GC/MS cone + GC/TOF-MS
conc)/2]). Visual inspection of the plot shown in Figure 6 suggests that there was bias in the
measured concentrations. Concentrations measured by GC/MS were slightly higher than those
measured by GC/TOF-MS, as was evident from the greater number of data points that reside
above the zero line of the y-axis. When plotting these data, several outliers became apparent.
Two measured GB concentrations were 50% lower when measured by GC/TOF-MS and two
concentration measurements of TPP were higher when measured by GC/MS.
Table 7 shows the analyte concentrations in reagent water determined with the
GC/TOF-MS and also comparative data generated by analyzing the same sample extracts by
quadrupole GC/MS (SIM mode), operated with both a 30 m x 0.25 mm i.d. x 0.25 |im film
thickness GC column (GC/MS, conventional) and a 15 m x 0.18 mm i.d. x 0.18 |im film
thickness GC column (GC/MS, fast). Average (n=7) concentrations of CWAs and surrogates
(i.e., NB-d5, FBP, PCP-d5, Ter-dl4, and TPP ) were measured in spiked (14 |ig/L of each CWA
and 56 |ig/L each of surrogates) reagent water. Final sample extracts (total volume 2 mL) were
spiked with 0.5 |ag/m L of each internal standard. Examining these data another way, average
percent differences in analyte concentrations were calculated; see Table 8. (Percent difference
calculated as follows: 100*(GC/MS cone - GC/TOF-MS cone) / [(GC/MS cone) / [(GC/MS cone
+ GC/TOF-MS conc)/2]. Paired t-tests were performed to determine (p<0.01) if differences
between concentrations were statistically significant.) On average, matrix-based concentrations
measured by GC/TOF-MS differed by 21% from conventional GC/MS and by 9% from fast
GC/MS. Paired t-tests were performed to test if significant differences were noted between the
average concentrations measured by each detection system. We performed paired t-tests on the
log-transformed, matrix-based concentrations. Statistical analyses were performed on the
logarithms of the measured concentrations because variability of concentration units increases
with concentration, whereas variability in terms of percent change tends to be more stable as a
function of concentration. As shown in Table 8, some of the differences were statistically
significant, even when the average measured concentrations were less than ±20%. It is generally
accepted in the environmental community that, for low measured concentrations, numbers within
±20%) are considered to be in reasonably good agreement.
21
-------
1.8
—1
1 fi
1—s
tXQ
C
1.4
c
o
1.2
nj
1
+¦>
c
0.8
-------
Table 7. Average (n=7) Concentrations (Ave Cone) with Standard Deviations (Std Dev) and
Percent Recoveries (Rec) of CWAs (14 jug/L) and Surrogates (56 jug/L) Using GC/MS
Configurations
GC/TOF-MS
GC/MS, conventional
GC/MS, fast
Analyte
Ave
Cone
(ng/uL)
Std Dev
Cone
(ng/uL)
Rec
(%)
Ave
Cone
(ng/uL)
Std Dev
Cone
(ng/uL)
Rec
(%)
Ave
Cone
(ng/uL)
Std Dev
Cone
(ng/uL)
Rec
(%)
GB
9.0
0.6
64
6.3
2.1
45
6.2
0.4
44
GDI
9.8
0.9
140
5.8
1.7
83
10
1.5
147
GD2
8.4
0.5
120
4.8
1.0
68
8.6
1.2
122
HD
15
3.5
106
15
3.7
109
14
4.2
101
GF
12
1.1
86
12
5.3
82
11
1.4
78
VX
37
5.2
262
33
1.7
238
35
5.6
247
NB-d5
63
2.4
113
66
2.3
118
68
4.0
122
FBP
63
1.2
113
54
3.0
97
60
2.4
107
PCP-d5
82
4.4
147
58
2.7
104
75
6.8
134
Ter-dl4
73
5.6
130
72
11
128
69
4.8
124
TPP
65
5.4
116
72
16
127
72
7.2
129
Redone 2/15/2011
23
-------
Table 8. Average Percent Differences in Analyte Concentrations in Reagent Water Using
GC/MS Configurations
Percent Difference
Between
Matrix-Based
Concentrations
Determined by
Conventional GC/MS
and GC/TOF-MS
Differences
Between
Conventional
GC/MS and
GC/TOF-MS
statistically
significant
(p<0.01)?
Percent Difference*
Between
Matrix-Based
Concentrations
Determined by Fast
GC/MS and
GC/TOF-MS
Differences
Between Fast
GC/MS and
GC/TOF-MS
statistically
significant
(p<0.01)?
GB
-35
Yes
-36
Yes
GDI
-51
Yes
2
Yes
GD2
-54
Yes
2
No
HD
3
No
-6
No
GF
-5
No
-10
No
VX
-11
Yes
-6
No
N B-tls
5
Yes
8
Yes
FBP
-15
Yes
-5
No
PCP-ds
34
Yes
-9
No
Tcr-di4
-1.3
Yes
-5
No
TPP
10
Yes
10
Yes
Average
Absolute Value
of Difference
21
9
* Percent difference calculated as follows: 100*(GC/MS cone - GC/TOF-MS cone) / [(GC/MS cone) / [(GC/MS
cone + GC/TOF-MS conc)/2]. Paired t-tests were performed to determine (p<0.01) if differences between
concentrations were statistically significant.
We also extracted and compared concentrations of CWAs and surrogates (28 |ig/L of
each CWA and 56 |ig/L of each surrogate) measured in surface water. Figure 7 provides a plot
of analyte concentrations in sample extracts measured by GC/TOF-MS versus concentrations
measured by GC/MS (30 m x 0.25 mm i.d. x 0.25 |im film thickness GC column), generated
from the data collected from three replicate samples that were spiked, extracted, and analyzed.
Each point represents a measured concentration of CWA or surrogate in the sample extract. In
this sample set, CWAs were spiked so that concentrations, at 100% recovery, would be 0.5
ng/|iL for CWAs (except for each GD isomer, which was expected to be 0.25 ng/|iL) and 1
ng/|iL for surrogates. All GC/MS data were collected in SIM mode. A regression line was
calculated and its R2 value was 0.94, its slope was 0.94 (with a standard error of 0.042), and its
intercept was 0.031 (with a standard error of 0.036). The data show a statistically significant
correlation of concentration measured by GC/MS and GC/TOF-MS (p-value « 0.001). On
average, concentrations measured by GC/MS and GC/TOF-MS agreed within 9%.
24
-------
Measured Extract Concentrations
Spiked Zone 7 Water
1.5
2.
bfl
c
to
u
O
l
0.5
y = 0.9447x + 0.031
R2 = 0.9415
0.2 0.4 0.6 0.8 1 1.2
GC/MS, 30 m column (ng/i*L)
1.4
Figure 7. Comparison of CWA and surrogate concentrations, in three surface water
extracts, measured by GC/MS-TOF and GC/MS.
We also plotted the percent differences (as previously defined) in the GC/TOF-MS and
conventional GC/MS concentrations as a function of average concentration to study the bias.
Visual inspection of the plot shown in Figure 8 suggests that there may be some bias in the data,
as concentrations measured by GC/MS appear, by visual inspection, slightly higher than those
measured by GC/TOF-MS. This behavior is consistent with the behavior observed for reagent
water (see Figure 6).
v
u
c
v
<4-
+->
c
V
u
v
Q-
25
20
15
10
5
0
-5
-10
-15
-20
-25
GD-2
HD
0"
I ~~
~
PCP-cL
0.2 0.4 0.6 0.8 1 1.2
Average concentration (ng/nL)
1.4
Figure 8. Percent differences between the GC/TOF-MS and conventional GC/MS (SIM
mode) concentrations measured in reagent water extracts as a function of average
concentration.
25
-------
Detailed comparative data for these water samples are shown in Table 9. The CWA were
measured in Zone 7 surface water spiked with with 28 |ig/L of each CWA and 56 |ig/L each of
surrogate. The final sample extracts (total volume 2 mL) were spiked with 0.5 ng/|iL of each
internal standard. Data represent the average concentration (Cone), standard deviation in concentration
(Std Dev Cone), and percent recovery (Rec) for three replicate samples that were spiked, extracted, and
analyzed by GC/TOF-MS or GC/MS. Percent differences (Diff) between GC/TOF-MS and conventional
GC/MS, SIM mode, and GC/TOF-MS and fast GC/MS, SIM mode, are provided. Concentrations of
CWAs and surrogates were in good agreement. On average, concentrations in sample extracts
measured by GC/MS and GC/TOF-MS agreed within 4%. Paired t-tests on the log-transformed,
matrix-based concentrations were performed as previously described. For all analytes, with the
exceptions of VX and TPP, concentrations determined by GC/TOF-MS and conventional
GC/MS were statistically equivalent. Concentrations determined by GC/TOF-MS and fast
GC/MS for all analytes were statistically equivalent.
26
-------
Table 9. Average (n=3) Matrix-based Concentrations of CWAs and Surrogates from Spiked
Surface Water Using GC/MS Configurations
GC-TOF-MS
GC/MS, Conventional
GC/MS, Fast
Cone
(ms/l)
Std
Dev
Cone
(ms/l)
Rec
(%)
% Diff from
GC/MS,
conventionala
% Diff
from
GC/MS,
fastb
Cone
(ms/L)
Std
Dev
Cone
(ms/l)
Rec
(%)
Cone
(ms/l)
Std
Dev
Cone
(ms/l)
Rec
(%)
GB
29
1.2
105
-4
-4
27
0.3
95
27
0.3
97
GDI
16
0.3
117
6
3
18
0.3
125
17
0.6
118
GD2
15
0.6
110
6
0
17
0.3
124
15
0.6
110
HD
13
3.0
47
0
4
13
1.7
48
14
1.7
49
GF
33
0.3
117
0
3
33
0.6
118
35
0.3
125
VX
32
1.2
116
-3a
3
30
3.1
106
34
0.9
121
NB-ds
64
2.8
112
6
2
72
1.2
126
66
1.5
115
FBP
62
4.4
108
0
-1
62
3.1
109
61
3.0
107
PCP-ds
74
3.6
129
5
3
62
0.3
108
76
3.8
134
Ter-di4
63
1.0
111
4
8
69
2.5
121
67
1.0
118
TPP
61
5.8
116
-4a
-4
66
5.3
116
71
2.9
124
Average
Absolute
Value of
Difference
4
3
Acronyms: Cone, average concentration; Diff, Percent differences Rec, percent recovery; Std Dev Cone, standard
deviation in concentration
Notes: a Paired t-tests indicated that only concentrations for VX and TPP determined by the two techniques were
statistically different (P<0.01).
b Paired t-tests indicated that the differences in concentrations, for all analytes, determined by the two
techniques were not statistically significant (p<0.01).
4.4.2 Analyses of soil extracts
Sand and soils were spiked with CWA and surrogates, extracted, and analyzed by
GC/TOF-MS and GC/MS. Table 10 provides data for analyte concentrations measured in sand
by fast GC/MS (15 m x 0.18 mm i.d. x 0.18 |im film thickness GC column) and GC/TOF-MS.
No conventional GC/MS data are presented in Table 10 because subsequent review of the data
showed that the CCVs (continuing calibration verifications) were outside the acceptable range
and, for this reason, all previously-collected data were rejected. Because fast GC/MS data were
acceptable, these data were compared to those generated by GC/TOF-MS. Table 10 shows that
some concentration values measured for sand with GC/TOF-MS agreed well with those
measured by fast GC/MS and some did not. Paired t-tests on the log-transformed matrix-based
concentrations were performed, as previously described. For all analytes, with the exceptions of
27
-------
FBP and PCP-ds, concentrations determined by GC/TOF-MS and conventional GC/MS were
statistically different (p«0.01). The measurement of VX was especially problematic. Data
collected by analyses of different soil types, presented in Table 11, also showed this trend. Table
11 presents comparative data generated for 250 ng of each agent and 500 ng of each surrogate
spiked on and extracted from 10 g of various soils (1 |ag/m L internal standard in sample extract).
Soil samples used included Virginia (VA) soil, Nebraska Aglands Ap (NeAp) soil, and Georgia
Bt2 (GaBt) soil. All GC/MS analyses were performed in SIM mode. Our experience suggests
that VX quantification is complicated by both co-eluting interferences and matrix enhancement
effects; this problem could possibly be studies in greater detail in the future.
Table 10. Average (n=7) Water Concentrations (Ave Cone) with Standard Deviations (Std
Dev) and Percent Recoveries (Rec) of CWAs (500 ng) and Surrogates (1000 ng) From
Spiked Sand Using GC/MS Configurations in SIM Mode
GC/TOF-MS
GC/MS, fast
% Diff h
Analyte
Cone, on
sand
(ji«/k«)
Std Dcv
Cone,
fag/kg)
Rec
(%)
Cone, on
sand
frig/kg)
Std Dev
Cone,
frig/kg)
Rec (%)
GB
35
5
69
0
0
0
N/A
GDI
9
1
36
19
2
78
71
GD2
9
1
36
20
3
78
75
HD
o o
J J
o
3
65
42
5
84
24
GF
36
4
71
41
5
83
13
VXa
14
1
29
31
4
62
76
NB-ds
35
4
35
71
9
71
70
FBP
81
10
81
79
9
79
->
-J
PCP-ds a
28
5
28
34
3
34
19
Ter-di4
78
7
78
88
11
88
12
TPP
77
7
77
94
11
94
-20
a Note that concentrations for VX and PCP-ds represent the sum of these analytes in the first and second solvent
extracts of sand.
b Percent difference is calculated as follows:
100*( GC/MS cone - GC/TOF-MS cone ) / [(GC/TOF-MS cone + GC/MS conc)/2].
For all analytes, with the exceptions of FBP and PCP-ds, concentrations determined by GC-TOF-MS and fast
GC/MS were statistically different (P«0.01).
28
-------
Table 11. Average (n=3) Soil Concentration (Cone) With Standard Deviation (Std Dev) and
percent recoveries (rec) for CWA (250 ng) and Surrogate (500 ng) From Spiked Soils Using
GC/MS Configurations in SIM mode.
VA soil, GC/TOF-MS
VA soil, conventional GC/MS
Soil
Cone
(fig/kg)
Soil
Cone
Std Dev
(jig/kg)
Rec (%)
Soil
Cone
(fig/kg)
Soil
Cone
Std Dev
(jig/kg)
Rec (%)
% Diff
from
GC/MSb
GB
3
0.6
11
18
0.58
73
143c
GDI
8
0.0
67
9
1
75
12
GD2
9
1.2
78
13
1.15
111
36
HD
19
0.6
78
13
0.58
53
-38 c
GF
28
1.2
115
27
1
108
-4
VXa
25
2.6
99
41
5
172
48c
NB-ds
23
1.0
48
39
3.06
77
52 c
FBP
28
1.2
58
30
1.53
59
7
PCP-d5a
108
4.4
217
56
4.2
111
-63c
Ter-di4
91
9.3
190
39
1.53
79
-80c
TPP
164
9.1
342
51
1.53
103
-105c
NeAp, GC-TOF-MS
NeAp, GC/MS
Soil
Cone
(fig/kg)
Soil
Cone
Std Dev
(jig/kg)
Rec (%)
Soil
Cone
(fig/kg)
Soil
Cone
Std Dev
(jig/kg)
Rec (%)
% Diff
from
GC/MSb
GB
3
1.5
14
15
0.58
61
133
GDI
8
0.0
67
10
0.58
81
22
GD2
8
0.6
69
10
0.58
81
22
HD
17
1.0
71
11
0.58
43
-43c
GF
26
1.0
108
23
0.58
91
-12
VXa
29
5.0
119
0
0
0d
N/A
NB-ds
25
0.6
53
40
2.65
80
46c
FBP
31
1.2
65
33
1
66
6
PCP-d5a
12
4.2
26
12
3.6
24
0
Ter-di4
99
4.0
206
43
1.53
85
-79c
TPP
159
5.2
331
57
4.93
115
-94c
29
-------
Table 11, continued.
GaBt, GC-TOF-MS
GaBt, conventional GC/MS
Soil
Cone
(fig/kg)
Soil
Cone
Std Dev
(jig/kg)
Rec (%)
Soil
Cone
(fig/kg)
Soil
Cone
Std Dev
(jig/kg)
Rec (%)
% Diff
from
GC/MSb
GB
2
0.0
8
0
0
0
N/A
GDI
2
0.0
17
0
0
0
N/A
GD2
2
0.0
17
0
0
0
N/A
HD
13
5.8
56
8
0.05
31
-48
GF
5
0.6
19
0
0
0
N/A
VXa
7
0.6
31
26
2
103
115 c
NB-ds
19
1.0
40
25
0.01
50
27
FBP
24
1.7
50
25
0.02
50
4 c
PCP-d5a
49
0.6
101
46
4
93
-6 c
Ter-di4
77
4.6
160
36
0.01
72
-73c
TPP
106
5.0
221
43
0.03
87
-85
a Note that concentrations for VX and PCP-ds represent the sum of these analytes in the first and second
solvent extracts of sand.
b Percent difference is calculated as follows:
100*(GC/MS cone - GC/TOF-MS cone) / [(GC/MS cone + GC/TOF-MS conc)/2],
0 Paired t-tests on the log-transformed, matrix-based concentrations were performed as previously described,
and concentrations determined by GC/TOF-MS and conventional GC/MS were determined to be
statistically different (p<0.01).
d GC/MS peaks for VX yielded S:N values less than 3:1, and therefore VX recovery was reported as "0".
30
-------
4.4.3 Analyses of wipes
Wipes were also spiked directly, extracted, and analyzed by GC/TOF-MS and by GC/MS
with a conventional GC column; see Table 12. In many cases, agreement was within 30%.
However, some anomalies were noted. By GC/MS, interferences or matrix effects appeared to
be more pronounced for GF and many of the surrogates. TPP, with a recovery of 323% by
conventional GC/MS, was most problematic. Such high recoveries were not observed with the
GC/TOF-MS.
Table 12. Average (n=7) Mass per Wipe, With Standard Deviation (Std Dev), and Percent
Recoveries (Rec) For CWAs (250 ng) And Surrogates (500 ng) From Spiked Wipes Using
GC/TOF-MS and GC/MS (SIM Mode)
GC/TOF-MS
GC/MS, Conventional
Mass per
Wipe
(ng)
Std Dev
Mass (ng)
Rec
(%)
Mass per
Wipe
(ng)
Std Dev
Mass
(ng)
Rec
(%)
% Diff
in Mass
from
GC/MS3
GB
310
10
122
250
7
100
-21b
GDI
160
20
125
184
13
147
14b
GD2
180
10
145
188
7
151
4
GF
290
30
114
523
36
209
57 b
HD
160
10
63
178
7
71
llb
VX
220
10
89
161
11
64
-31b
NB-ds
410
10
83
463
18
93
12b
FBP
350
20
69
361
16
72
3b
PCP-d5
350
10
71
103
12
20
1
o
vo
cr
Ter-di4
780
90
157
707
24
141
-10
TPP
720
100
145
1617
64
323
77 b
a Percent difference is calculated as follows:
100*(GC/MS cone - GC/TOF-MS cone) / [(GC/MS cone + GC/TOF-MS conc)/2],
b Paired t-tests on the log-transformed, matrix-based concentrations were performed as previously described,
nd concentrations determined by GC-TOF-MS and conventional GC/MS were determined to be statistically
different (p<0.01).
31
-------
5.0 Conclusions and Recommendations
The GC/TOF-MS appears to be a good alternative to quadrupole GC/MS. Both
instruments have comparable costs. The GC/TOF-MS (LECO's Pegasus® 4) provides lower
instrument limits of detection than GC/MS (Agilent 5973), while still retaining full mass spectral
data. The retention of full mass spectral data is expected to be advantageous in assisting in the
process of confirming an analyte's identification. In order for the GC/MS to obtain the relatively
low detection levels used in this study, the GC/MS was operated in the selected ion monitoring
(SIM) mode. Several EPA analysts have expressed concern during teleconferences that they are
not confident using SIM analyses to provide accurate identifications and quantitations and that
they favor the collection of full-scan data. Thus, the GC/TOF-MS would be a desirable detector
for this group. The reproducibility of GC/TOF-MS appears comparable to GC/MS. Fast
separations with the GC/TOF-MS offer increased analytical speeds and the promise of higher
sample throughput; the GC/TOF-MS was observed to be two- to three-times faster than
conventional GC/MS.
Both GC/TOF-MS and GC/MS analyses must be performed by educated operators.
Because GC/MS is most widely used in environmental analyses, many analysts already have the
required knowledge to successfully implement GC/MS methods for the detection of organic
compounds. Currently, there are not as many analysts that are familiar with the proper use of
GC/TOF-MS systems. However, given sufficient training, analysts with the skills needed to
successfully perform GC/MS analyses will be able to perform GC/TOF-MS analyses. One of the
areas most critical to the correct implementation of GC/TOF-MS methods is data analysis.
Because data analysis packages are not as well-developed for the GC/TOF-MS as for the GC/MS
systems, care must be taken with data interpretation. While not a topic of discussion in this
report, it was found that the deconvolution algorithms available for use with the GC/TOF-MS are
useful to assign the proper spectra and correct signal intensity to coeluting peaks and provide
quick, reliable quantification of the analyte in the presence of coeluting species. In addition,
attention needs to be paid to method setup. For example, a GC/TOF-MS operator must ensure
that 20 data points are generated across each chromatographic peak in order to adequately define
the peak and to obtain reproducible data. While the need to have an adequate number of data
points to define a peak is also critical to proper GC/MS analysis, given the maturity of GC/MS
software, such requirements are often forgotten as analysts blindly use default instrument method
values. Once an analyst becomes familiar with GC/TOF-MS operation and data analysis,
GC/TOF-MS could be a useful tool which can be used to perform analyses in support of EPA
missions.
Specific recommendations for using GC/TOF-MS for future work include:
1) Because of the lower detection limits of GC/TOF-MS and the lower capacity
of the narrow-bore GC columns used for fast GC analyses, the amount of
DFTPP required for instrument performance checks could be reduced from 50
ng to 15 ng (or lower).
2) The MS performance criteria for DFTPP listed in the ongoing EPA CWA
study could be changed to those of EPA Method 527 to provide the users of
32
-------
the draft protocol the flexibility to use either a quadrupole GC/MS or
GC/TOF-MS for analyses.
3) Criteria for CCVs requiring that measured concentrations fall within ± 25% of
their expected values can easily be met using GC/TOF-MS.
4) EPA would benefit from further exploration of GC/TOF-MS for routine
analyses.
5) EPA would benefit from additional experiments with fast GC/MS.
6.0 References
1. Quality Assurance Project Plan and Study Plan for Lawrence Livermore National
Laboratory's Experiments with the LECO Pegasus® Gas Chromatograph/Time-of-Flight
Mass Spectrometer for EPA under IAG #DW89922616-01-0, Revision 1.5, LLNL-TR-
408100, Carolyn Koester, January 23, 2009.
2. Method8270D: Semivolatile Organic Compounds by Gas Chromatography/Mass
Spectrometry (GC/MS), Rev. 4, February 2007, U.S. Environmental Protection Agency.
3. Schumacher, B., Characterization and Monitoring Branch, ESD, NERL, US EPA, Las
Vegas, NV, personal communication, December 18, 2006.
4. Year One Report for Lawrence Livermore National Laboratory's Verification of Standard
Analytical Protocol for Extractable Semivolatile Organic Compounds For EPA under IAG
#DW89922616-01-0, Heather Mulcahy, Roald Leif, and Carolyn Koester, LLNL-TR-
412124, Revision 2, July 13, 2009.
5. Rapid Detection and Quantification of Select Chemical Warfare Agents (CWAs) in
Dichloromethane (DCM) Extracts by Gas Chromatography/Time-of-FlightMass
Spectrometry (GC/TOF-MS), J. Oyler, T. Rusek, J. Greene, Document No. AM-181,
Edgewood Chemical/Biological Forensic Analytical Center, May 13, 2008.
6. Agilent Technologies, Inc., Retention Time Reproducibility of the Agilent 6890 Plus GC,
Gas Chromatography Technology Note, August, 1999. Available at
http://www.chem.agilent.com/Library/technicaloverviews/Public/59686420.pdf.
7. Price EK, Prakash B, Domino MM, Pepich BV. Method 527: Determination of Selected
Pesticides and Flame Retardants in Drinking Water By Solid Phase Extraction and
Capillary Column Gas Chromatography/Mass Spectrometry (GC/MS), April 2005, U.S.
Environmental Protection Agency. EPA 815-R-05-005
8. EPA Method8270D—Calibration Curve Development andDFTPP Tuning by GC-TOF-
33
-------
MS, Applications Note, John Heim, LECO Corporation, St. Joseph, MI, March 2008.
9. GC-TOF-MS tune criteria suggested by LECO. See applications note at
http://www.leco.com/resources/application note subs/pdf/separation scienceZ-328.pdf
10. Letter to Jack Cochran of LECO Corporation, William Telliard and Herb Brass, U.S. EPA.
February 9, 2005 available at
http://www.leco.com/resources/pdf/UnitedStatesEnvironmentalProtectionAgencv-
ATPACCEPTANCELETTER020905 pdf.
11. Method8000C: Determinative Chromatographic Separations, Rev. 3, March 2003, U.S.
Environmental Protection Agency.
34
-------
Appendix A: Autosampler Method for LECO GC/TOF-MS
Select; auto sampler; type:;
# Agilent©
O Rail System (CTC, Gerstel, LEAP)
O Shimadzu©
0 7890, 6890N or 6890 with nanoliter adapter enabled
~ Enable overlapped operation when connected to an Agilent 7890 GC
Syringe;Size £ wt;
10
Sample Volume (jjL ):
Number of Sample Pumps ( 0-15
Ei
2
Viscosity Belay ( 0 7 sec }:
0 I
Sample Pre-Wash (0-1 ):
Solvent A Pre-Wati ( 0 15 ;
2
Solvent B Pre-Wash (0-15):
2
Pre-lnjection Delay (0 00-1 00 min ):
0
Post-ii^ectionDetay:(0:00-100 rriiri);:;
Solvent A Post-Wash (0-15 ).
3
Solvent;; BPoftt-Wash; (0-15
3
Slowplunger enable:
Yes • No
Sample skim enable:
Yes • No
35
-------
Appendix B: GC Method for LECO GC/TOF-MS, setup for 2-D instrument
This was the method that was used by LLNL for all 1-D experimental work.
Hardware control
OApJenBTBM
® Agilent® 6890
O Shiinatia® 6C-2D10
O Generic
O Direct Intel
Option:
~ MACHALTM Owen
SlLECaeGCxGC
AgiSent© 7690 Gas Chromalograph
Aglfent© 8S90 Gas Chromaiograph
Shimadzu© GC-201S
Generic Gas Chromatograph
Direct inlet to Calibration Conipouno
CapSary CmlgiiBioBi
Mo praMems detected with column configuration.
IffjrujLj
iSMPIW <1 Ml s f.
Type
Location
Length(m) | tot. Otartisterfj;
Max T»rnf
Film f Mseknewt
Pba&e | Bteei Masses
1"
Ti er
Back
I
;
2
3
4
C a Hillary
GC Oven 115.000
250,0C
350.0
0.25
HP5MS 73149 207 281
Caalllary
Caallar#
si-
ll
0.100
100.0C
326,0
325.0
0.10
R*l-17
Rxi-17
73 14920? 281
73 149207 281
1.000
100.00
0,10
5
Capillar/
Detector o
0.210
100.0C
325.0
0 to
Rxi-17
.¦ ¦ > 207 281
0
Detector
TOF
Add
Delete
Promote
Demote
Copy
~ Enable Flow Pa'ti 2
Mass Selection for Auto Mass Defect Tracking)
® Excluded Masses in Auto Mass Defect Made
O Inducted Masses h Auto Maw Delect Mode
i Set Auto Mass Defect Mode in MS method. Select
masses between 130 to 384 Inclusive. )
f For general unknown analyses. Select column bleed,
matrix, interfering, arw non-target masses. ;
( Generally for target analyses. Seiect significant masses of
target anaiytes, minimum of 2 masses required j
Garner Gas:
36
-------
Appendix B: GC Method for LECO GC/TOF-MS, 2-D instrument, continued.
ttelum
T
Back Inlet Type-.
Spli / Splilest
Bach Inlet Mode:
Pulsed Splitless
Active Intet Location.
O Front ®Baek
; he active inlet must be present in the capiiiary configuration.
0 Coinectee! constant flow via pressure ramps
Use this mode when in GCxGC mode or using
short ( < 5 m t angle column or two columns.
# j Rats (ml/mir.a) | Target Flow (niL/rrtin) j Duration (mil)-
JRats<
IjnitiPl
J 1.20
Entire Bun
Column 2 ' Back ;niet Purse iime { sec i:
The time, after the beginning of the run, when the purge valve wh
open.
%2' 63c* n>et *>urge F'ow ' The flow from the purge vent
-------
Appendix B: GC Method for LECO GC/TOF-MS, 2-D instrument, continued.
Column 2 I Back, inlet Gas Saver
'^Yes CJNd
Column 21 Back Inlet Gas Saver Fto* ( mL-'min i
2D
Column 21 Back. Inlet Gas Saver Time (minutes ):
1
Back Inlet tempasiurefsjc
# | Rate fC/min)
i i lelTernpfC)
Duralion frnin)
*• 1 Initial
| 250.00
Entire Run
'¦i
No problems detected with oven tempefsfaiies.
0.5
Note. Alt oven temperature ramps ( except the secondary oven
wilt have the same duration.. Thrs is accomplished By extending
die final hold time
# 1 Rate ("Cmini
j Target temp fC)
Duration frrtin)
" 1 Initial
j 55.00
0.50
T 120.00
1100.00
10.00
3 J 40.00
200. DO
2 75
AM
Remove
Coolant to Column Oven O On ®Qff
H En able Secondary Oven
P 1 Rati f C/roinl
Target Temp fCJ
I Duration (min)
J j Initial
moo
16-50
2 [20.00
115.00
0.00
3* [ 40.00
205.00
(2.75
I
Add
Remove
38
-------
Appendix C: GC Method for LECO GC/TOF-MS, setup for 1-D instrument
LLNL's GC/TOF-MS system is capable of performing 2-D GC separations, in which two
different GC columns are used to achieve separation of analytes. Thus, in configuring methods
(such as the GC method shown in Appendix B), the installation of two GC columns was
considered. However, several of the EPA laboratories do not have GC/TOF-MS systems that
offer the ability to perform 2-D separations. In order to assist these laboratories in their
implementation of GC/TOF-MS protocols for the separation and detection of CWAs, several
analyses were performed with a single GC column so that recommended GC operating
conditions could be developed.
A summary of the recommended, and LLNL-tested, operating procedures for
GC/TOF-MS used in 1-D mode (i.e., when only one column is installed) are listed below.
Detailed screen shots of the set-up of the GC/TOF-MS software are presented at the end of this
appendix (note: this information was shared with EPA's GC/TOF-MS working group by
Heather Mulcahy on February 8, 2010).
Injection size:
Injection type:
Pulse pressure:
Purge time:
Carrier gas:
GC injection port:
GC column:
GC oven (primary):
GC transfer line:
1 |iL
pulsed-splitless
40 psi for 0.5 min
35 sec at 30 mL/min
He with constant flow of 1.2 mL/min
250 °C
20 m x 0.18 mm id x 0.18 |im film thickness HP5-MS UI (Agilent
Technologies, Inc, Santa Clara, CA)
50 °C held for 0.5 min, 20 °C/min to 110 °C, 40 °C/min to 170 °C,
45 °C/min to 300 °C, held for 2.11 min
290 °C
The following MS conditions were used for detection.
MS filament delay: 110 sec
MS scan range: 35-500, at an acquisition rate of 15 spectra/sec
MS source: 250 °C
Electron energy: 70 eV
Figure C-l shows a representative chromatogram for a 1 |iL injection when one column
is installed and the GC/TOF-MS is operated in 1-D mode. Using the previously described
conditions, the analyses were shown to be reproducible, with respect to both retention time
stability (relative standard deviations (RSDs) for all analytes were less than or equal to 2%) and
peak areas (RSDs for all analytes were less than or equal to 5%, with the exception of VX, which
had an RSD of 8%); see Table C-l. Note that the analysis time is decreased when using a single
GC column. When two columns are used, as previously described in Appendix B, analysis time
is increased due to the modulator and secondary oven, which are limited to a maximum
39
-------
temperature ramp rate of 40 °C/min and a maximum of only three temperature ramps. Despite
this limitation, 1-D experiments with two columns installed were performed because this
configuration offered the flexibility of performing 2-D experiments without the instrument
down-time associated with venting the instrument. Thus, both 1-D and 2-D experiments could
be performed using a single autosampler sequence.
—
iu
•6
8
2
-X
CL
6
-£
i:
150
200
250
300
350
; AIC
•100
450
500
550
Figure C-l. Total ion chromatogram obtained when separating 0.5 ng of each CWA and 1 ng of each
surrogate standard by GC/TOF-MS with a single GC column installed.
Table C-l. Average Retention Times (± Standard Deviations) and Average Analyte Responses (±
Standard Deviations) for Seven Replicate Injections of 0.5 ng of Chemical Warfare Agents Into the
GC/TOF-MS. Data Were Collected With Only a Single GC Column Installed (ID Operation)
Analyte
Average
Retention
Time (sec)
Average Analyte Response
(peak area in arbitrary
units)
GB
127.97 ±0.25
674428 ± 27895
GD 1
219.75 ±0.22
142379 ±6703
GD 2
221.70 ±0.23
115211 ±5734
GF
279.56 ± 0.22
426259 ±21011
HD
270.40 ±0.17
527628 ±27016
YX
386.75 ± 0.06
42527 ± 3268
40
-------
Appendix C, cont'd: GC Method for LECO GC/TOF-MS, setup for 1-D instrument
*~
Hardware contrai
U Agilent® 7890
® AgBenUB 8800
O Shiimaiai© GC-2D10
O Generic
O Direct Intel
~ MACH/LTM Oven
~ LECO©GCxGC
Agiteni© 7890 Gas Cbromaiograph
AgiienS© 6690 Gas C^romatograph
Shimadzu© GC-2015
Generic Gas Chromatograph
D;rect Inlet to Calibration Compound
£y
Capilia ry Configuration.
# I Type
on Lsntj#i{ffl) I Int. Diameterf.j;
Capillar/
Back
GC Oven 19.800 180.0C
Ma* Temp
350 0
Film Thickness. Phase
0.18
HP5MS
Jleei Masse?.
73149 207 281
Caalllary
Detector
Detector c
TOF
0.200
180-0C
325.0
0.18
HPSM5
7314020? 281
Add
Delete
~ Enable Flow Path 2
Promote
Demote
Copy
Mass Selection for Auto Mass Defect Tracking
® Excluded Masses in Auto Mass Defect Modi
Deled Mode
O Inducted Masses to Auto
( Set Auto Mass Defect Mode In MS method. Select
masses between 130 to 384 inclusive.}
( For general unknown analyses. Select column bleed
matrix, interfering, and non-target masses. >
{ Generally for target analyses. Select significant masses of
target analytes. rn.nimum of 2 masses required., i
Carne'' Gas.
41
-------
Appendix C: GC Method for LECO GC/TOF-MS, 1-D instrument, continued.
haEBidrfl:
T ¦"¦¦¦¦
E j:k Irlrt Tyse:
S: it •"
Met r/ode
Pulsed Spltless
-ttive .-lie; Locator
O Front
Ti-e ret m js: 3s presort tie cac;:;j'y ccr-fjguratic
~ Corrected constant flow via pressure ramps
¦ee:t the co ...ri i "rode:
LCilitait F :
k'o, rtci i's 3 c.*restart " :*• rate of cj"ier 525 v :he ccljnrr
throug.nc.ut :ns rur f. t'~e coIi-tp resistance chan;?® 2te to a
teri3erau.r& p-og.'sni. the co „rn need r^s&ure is adjusted to kee
the ,rw.' .'ate c-M-r-tj-n
:olurn 2 Buck Inlet "1;'iV'0i
n ! Ra:o :TL-'nn"'; llano;
I |lrii!ia»
T><
Entire Run
tkurr 2 B.icK Irirt F.rge T ->ie 1 -:e:
35
1 he ::rn ate.' trw :: eg nn nc of tre m. wren :rs pjnge -...v-ve A'i:i
1:1. !" 2 / BjcK Inlet P-Jrge F-c.v 1
•iv ~ ¦ r—
It .71"
cc-.j"" is net c-e-'p-eci
2 .• BjcK Inlet ! ota' i-:o-„v ; '¦¦.L.rvr i hi-:- t ie c.ctL.a- "1c-a to t ie hie: "i nrg 2 P,*e-Ki. t arc d;ri\g
i: 1 i\.n cetae p_rge t.-ie.
-------
Appendix C: GC Method for LECO GC/TOF-MS, 1-D instrument, continued.
:_-0iL-TI" J MCK im*T H J Si HttSSStR i "S "
40
Cul;,;rn 2 ' Edcl- Irlet Pu>:.r L l "c-: or
D.5
'-oli-m 2 Back r.let t-5.er
•
CoL'rn 2 5:iC-K lie: Gas Saver F:ow i •'¦iL'n r- ,
lolu'rr ; ¦ Eock lies Gss iaver T.-na . r. • r->:
lrlet «,nstr»ti.re.e'
= | R;--:e ! i Targa: Terp; Ci J Duration (min)
t* |Initial 1250.00 lEnilreRun
-¦i
N? problem; ceiMted with o-,en lerrperKures
?>e-i Ec.i I i nie i rii
0.5
£ntcr oven ts mp© mtu ib fsiinp t?ctow~
120.00
110,00
0.50
0,00
40,00
170,80
0,00
145.00
300 00
2.11
Cc-o^r: Col urn Cvsr.
• Off C-.c!.»i l-Y.-K;.' ' 1 12
..r.T TeTpera:j'e Eau:i t.'a'.c-n in e ••••••:
T'-v-o'er _,r<3 Teirpe'ccjre i "C
AckJ
Remove
290
~ Specif Additional De'ec: I ary Pneumatics
43
-------
Appendix D: MS Method for LECO GC/TOF-MS
Use GC method total time for MS memod total time:
® Yes O No
Acquisition desay
™ ®Sec. The lencth of time from injection untN ti* data system
o Min will start storing data from the mass spectrometer.
) in the grid below
Filament
Add
Remove
Required Disk Space
IMA
Enter Ene mass spectrometer settings:
Start Mass f u)
35
Acquisition Rate ( spectra / second )
15
DeiectorVtftafe
1650
tlectror. Energy (Volts)
-70
Mass defect mode
O Auto {Se-ecc masses for ailomaic tracking in column infonmaiion section of GC method.)
& Manual
H Verify offset before crttecfcg data
Mass Defect ( mu l 100 u )
0
-------
Appendix D: MS Method for LECO GC/TOF-MS, continued.
Set the temperature for the Ion Source.
Jon Source {*C )
250
H Wat for ion source temperatures to reach set. point before staring acquisiicm
Source Temperature Equ*lib~ation Time (Seconds)
0
Enter me masses to display dunng acquisit'on
Examoles
t
69,131
69+131
Masses 69 and 131
Sum of masses 69 and
-------
Appendix E: Data Analysis Method for LECO GC/TOF-MS
"*0
C;e-sc.t me tasiv or tasks yc-j ^is!" to ->esforns from the list below.
V Baseline - computes baseline
V Peak Find - finds peaks above the basel ine
v' Library Search - identifies all peaks found
V Calculate Area / Height - computes the area and height of peaks w ithout a calibration
Retention Index Method
Classifications
V Apply Caiibration(s) - computes the absolute concentration of peaks based upon a cali bration
Apply References) - computes the relative concentration of peaks with respect to a reference
Semi Quantification - computes concentration based on another arialytes calibration curve
i Tone Check
Tailing Factor Check - checks to see if the arialytes have an acceptable peak shape
Calibration Check
Blank Check - checks to make sure none of the analytes exceed their blank concentration
EPA Method - selects the EPA Method
Report - prints selected reports for each sample
Export peak information in ASCII CSV format
Export data in Andi MS format (.cdf)
I ! Export data file
l 'i'i-i iraok"Xj "ir :virw
<* 1 Start ; End j Mode _ ! | Add
\ End of Run Default " |
Remove
Enter the baseline offset below ( 0.8-3,0 ]
10.8
Examples;
0.5 Through the middle of the noise
1.0 Just above the noise
Enter the number of data points that should be averaged for smoothing below:
I Auto !
- • - • * ' " - * is below: {as measured from Baseline to baseline)
Peak widths broaden throughout the chromatographic run
Peak Width Retention Time
1.5
times,
these two points.
1 at two
100
Keep False Peaks
",J> .,c p. TlS.xSiHu >nTo b^ioo
46
-------
Appendix E: Data Analysis Method for LECO GC-TOF-MS, continued.
#
IgjjkHwd l.S/1 [ Waggs IiB»terof»pxi«fl Masse
laartrfRun I End of Run iOit jit,I ; i2
Add
Remove
Common masses in cferivatized products:
1 GCxGC Parameters
Library identity Search Mode:
Normal O Quick
Library Search Mode:
(P> Forward O Reverse
Enter the number of library hits to return:
50
Enter the masses to library search below:
Examples
*
31:99
31:99,200:211 masses 31:
i:211
Mass Threshold {Relative abundance of base ion { 0 - 998))
jlO |
before name is assigned {0 - 999}
500
''w' i'ip 1 h'.?r
mainlib
repltb
EPA
LLNL_CW
[ Add...
I Remove
Promote
Demote
Rrxw-ifv Aririitirmal I ihrarv Rewrch C.ritpria
-------
Appendix E: Data Analysis Method for LECO GC/TOF-MS, continued.
I Specify Additional Library Search Criteria
Enter mass to use for area / height calculation:
U
Examples
U
T
Unique mass
TIC
Apexing masses
m/z 55 used for every peak
A
55
! ! Allow skimming off small riding peaks
i ! Approximate Concentration of Unknowns ( This uses the nearest IS arid a RF of 1 ;
Mass Threshold ( Relative abundance of base ton (0-998))
10 |
/-.Jj: Lie L,al.bfat.Q!,S to USu if. ;L- to t list belOlV
1DCW091609
Promote
Demote
-------
Appendix F: Analysis Method for LECO GC/TOF-MS, Modified ECBC
Conditions.
Initial experiments were performed using the faster temperature ramps of an ECBC
method (5) and a 10 m x 0.18 mm i.d. x 0.18 |im film thickness, HP5-MS UI, GC column.
Separation was accomplished in 6 minutes; see Figure F-l. The parameters used to achieve this
separation are shown below. Screen shots of the GC/TOF-MS setup are shown at the end of this
appendix.
Injection size:
Injection type:
Pulse pressure:
Purge time:
Carrier gas:
GC injection port:
GC column:
GC oven (primary):
GC transfer line:
1 |iL
pulsed-splitless
40 psi for 0.5 min
35 sec at 30 mL/min
He with constant flow of 1.2 mL/min
250 °C
10 m x 0.18 mm i.d. x 0.18 |im film thickness, HP5-MS UI
(Agilent Technologies, Inc, Santa Clara, CA)
30 °C held for 1.25 min, 85 °C/min to 120 °C, 65 °C/min to 180 °C,
45 °C/min to 290 °C, held for 2.35 min
290 °C
The following MS conditions were used for detection.
MS filament delay: 90 sec
MS scan range: 50-500, at an acquisition rate of 35 spectra/sec
MS source: 250 °C
Electron energy: 70 eV
Reproducibility of retention times and analyte responses for seven replicate injections of
a standard containing 0.5 |ig/mL of CWAs were documented and are shown in Table F-l. As
was evident from the data, for seven replicate injections, retention times were stable and varied
by less than 0.1% for all compounds. This stability is consistent with reports that a sample of 32
Agilent 6890 Plus GCs consistently demonstrated relative standard deviation of less than 0.1
percent, with some lower than 0.01 percent (6). All analyte responses, measured as peak areas,
were also reproducible and varied by less than 10%.
49
-------
125
150
175
200
225
250
275
I
300
325
AIC
Figure F-l. Total ion chromatogram (filename = TOF:287) obtained when separating 0.5 ng of
each agent and 1 ng of each surrogate standard by GC/TOF-MS. Note: Naphthalene-ds is the peak
between HD and GF.
Table F-l. Average retention times (± standard deviations) and average analyte responses (±
standard deviations) for seven replicate injections of 0.5 ng of CWAs into the GC/TOF-MS.
Analyte
Average
Retention
Time (sec)
Average Analyte Response
(peak area in arbitrary
units)
GB
102.34 ±0.14
1167925 ±71630
GD 1
136.51 ±0.03
1101851± 109658
GD 2
137.08 ±0.02
1208078 ± 104845
GF
156.89 ±0.09
5799223 ± 564933
HD
153.36 ±0.09
1711803 ± 160493
VX
213.89 ±0.04
402713 ±31773
50
-------
Appendix F: Analysis Method for LECO GC/TOF-MS using Modified ECBC Conditions,
continued.
M
O Agilent© 7890
® Agilent© 6890
O ShimadzuS' GC-20 0
¦ ¦ Generic
Direct Inlet
~ MACH/LTM Oven
~ LECCXS- GC.xGC
Agilent® 7890 Gas Cr-rornatograph
Agilent© 6890 Gas Crsromatograph
Shimadzu^ GC-2010
Generic Gas Chromatograph
Direct Inlet to Calibration Compound
¦I apil aiy Oon' gui afion
No problems detected with column configuration
Flow Path 1:
#(Type
Location |Length{«n
)I int. Diamejtsrfu Hd.ixTfiT-r
Him ¦, hic-snnss? j Phase j Blofc Masses
1 *110161 |
Back
T"
2j Capillary
[GO Oven tio.000
j 180.00 1300..0
0.18 |HP5MS 173 149 207 281
3 (Detector
ITGF 1 i 1
I i
Add
Delete
Promote
Demote
Copy
Paste
~ Enable Flow Path 2
Mass Selection for Auto Mass Defect : racking
& Excluded Masses
-------
Appendix F: Analysis Method for LECO GC/TOF-MS using Modified ECBC Conditions,
continued.
uamer uas
Helium
T
Back infet Type;
Split i Splliess
Back In et '.lode:
P^sec Split-ess
Active Inlet Locaton:
O Front ® Back
The active ir^et must be present in the cavitary configuration.
~ Corrected constant flow via pressure ramps
Use this mode when in SCxGC mode or using
short5 m ) si^qle column or two columns.
Constant Flow
Maintains a constant mass fiow rate of carrier qas jps the column
throughout tf-e run. If the column resistance changes due to a
temperature program. the column head pressure is adjusted to keec
the flow rate constant.
Column 2 / Back Inlet f;ow:si:
if j Rate iml./rr.in';
! T,vr
e'. T;ov. ¦T=Ur|tn)
| Duration (mlri) .
1' ilmtial
|
, Entire Run
Column 2 < Back Infet Purge » me ( sec j. _
Column 2 / Back Infet Purge Fow {
numin ).
he time. afte*- the beginning o* tre run. when the o^rge valve wii
open.
; he flow from the purge vent. I h«s value cannot be used if your
column is not defined
Column 2 / Back Iniet Tota' Flow i mL-'min . his is the actual flow to the inlet coring a Pre-Run and during a
):i run before purge tme.
30 9
¦Vja,«n *"1 K-vUt- Di Dr,-v,-.-i.r.-, /
52
-------
Appendix F: Analysis Method for LECO GC/TOF-MS using Modified ECBC Conditions,
continued.
..'Uiuhhi z ¦ duck I'iifc. r usse rresswwe \ P->i i
15
Column 2 / Back Inlet Pulse Duration f minutes s:
0.5
Column 2 ¦' Back in'et Gas Saver
Yes No
Column 2 ¦¦ Bac=< iivet Gas Saver Flow ( mL'min i
Column 2 Back irvet. Gas Saver Time i minuses r
Back iivet temperaturefsi:
i? ! Rate rC/rnini
I
i
250,00
Entire Run
No problems detected with oven temperatures.
Oven Equilibration T.me ! minutes }: Note: All oven temoerature ramps ; except the secondary oven >
will have the same duration. This is accomplished by extending
the finai hold time.
Enter oven lemperatu'-e ramp oelcw.
i Rate f C/mln)
| Target Temp fC)
¦Initial
f 30.00
] 1.25
J 85.00
J 120.00
j d-66
166.00
(180.00
I b.oo
J45.00
1290,00
fz.SS
Add
Remove
Coolant to Column Oven O Qn $ off
Transfer Line Temperature Ec|u»ib<"at:on Time ( sec i
V.H
10
53
-------
Appendix F: Analysis Method for LECO GC/TOF-MS using Modified ECBC Conditions,
continued.
Transfer Line Temperature i 5C j:
250
~ Specify Additional Detectors & Auxiliary Pneumatics
54
-------
Appendix F: Analysis Method for LECO GC/TOF-MS using Modified ECBC Conditions,
continued.
Jse GC method total time for MS method totes! time:
¦•"Yes :"'IMo
Acqoisit-cm delay
90
'• Sec
' - ¦ Mm
The 'enath of tine from injection unti! tne data system
will sta't storing data *'rom the mass spectrometer.
¦ne(s) when the ^lament snoald be turned oP ( min of 3 sec j in the arid below
c Start: End filament
ya s
End of Run
Off
On
Add
Remove
Enter '~e mars spei trcrieter seiti
Start Mass ( u '1
hnd Massu
500
Acquisition Rate ( spectra second >
35
1850
-70
Mass defect mode
O Auto ('Select masses for automatic tracking in column information section of GC method.
® Manual
0 Verify offset before collecting data
Mass Defect 1 mu 1100 u )
0
55
-------
Appendix F: Analysis Method for LECO GC/TOF-MS using Modified ECBC Conditions,
continued.
Set the temperature for the Son Source,
source ¦: u i
250
B Wait for ion source temperatures to reach set point before starting acquisition
Source Temperature Equitation Time (Seconds)
0
Enter the masses to cisp&y during acquisition Examples
1 T'c
t 6&. -Z' Masses 69 and 131
69- i 31 Sum of masses 69 and 131
56
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