EPA/600/R-16/115 I September 2016
www.epa.gov/homeland-security-research
United States
Environmental Protection
Agency
oEPA
Analytical Protocol for
Cyclohexyl Sarin, Sarin, Soman and
Sulfur Mustard Using Gas
Chromatography/Mass Spectrometry
Office of Research and Development
Homeland Security Research Program
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EPA/600/R-16/115
September 2016
Analytical Protocol for
Cyclohexyl Sarin, Sarin, Soman and Sulfur
Mustard Using Gas Chromatography/Mass
Spectrometry
United States Environmental Protection Agency
Office of Research and Development
Homeland Security Research Program
Cincinnati, Ohio 45268
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Acknowledgments
This method is based on procedures developed by Lawrence Livermore National Laboratory (LLNL)
under Interagency Agreement (IAG) DW89922616-01-0 with the U.S. Environmental Protection Agency
(EPA). EPA's Homeland Security Research Program (HSRP) and Office of Land and Emergency
Management managed and funded laboratory testing of the procedures for analysis of water, soil, and
wipe samples in a multi-laboratory study. Laboratories participating in the study and providing technical
support include EPA Regions 1, 3, 6, 9, and 10; EPA's Portable High Throughput Integrated Laboratory
Identification System (PHILIS) Unit mobile laboratory in Castle Rock, Colorado; the Virginia Division
of Consolidated Laboratories; the Florida Department of Environmental Protection; and LLNL.
Technical support, study coordination and data evaluation were provided by CSGov (formerly CSC).
Disclaimer
The U.S. Environmental Protection Agency through its Office of Research and Development funded and
managed the research described herein under EPA Contract No. EP-C-10-060 to CSGov (formerly CSC).
It has been reviewed by the Agency but does not necessarily reflect the Agency's views. No official
endorsement should be inferred. EPA does not endorse the purchase or sale of any commercial products
or services.
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.romv@,epa.gov
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Table of Contents
Section Page
I.0 SCOPE AND APPLICATION 1
2.0 SUMMARY OF PROTOCOL 1
3.0 ACRONYMS, ABBREVIATIONS AND DEFINITIONS 1
3.1 Acronyms and Abbreviations 1
3.2 Definitions 2
4.0 INTERFERENCES 5
5.0 SAFETY 5
6.0 EQUIPMENT AND SUPPLIES 6
6.1 General Equipment 6
6.2 Microscale Extraction Apparatus 7
6.3 Gas Chromatograph/Mass Spectrometer (GC/MS) System 8
7.0 REAGENTS AND STANDARDS 10
7.1 Reagents 10
7.2 Standards 10
8.0 SAMPLE PRESERVATION, STORAGE, AND TECHNICAL HOLDING TIMES 12
8.1 Sample Preservation 12
8.2 Sample Storage 12
8.3 Sample Extract Storage 12
8.4 Technical Holding Times 13
9.0 QUALITY CONTROL (QC) 13
9.1 Initial Demonstration of Capability (IDC) 14
9.2 Initial Precision and Recovery (IPR) Determination 14
9.3 Method Blanks 15
9.4 Matrix Spike and Matrix Spike Duplicate (MS/MSD) 17
9.5 Laboratory Control Sample (LCS) 20
9.6 Instrument Detection Limit (IDL) Determination 20
9.7 Method Detection Limit (MDL) Determination 21
9.8 Quantitation Limit (QL) Determination 22
10.0 CALIBRATION AND STANDARDIZATION 22
10.1 Instrument Operating Conditions 22
10.2 GC/MS Mass Calibration (Tuning) and Ion Abundance 24
10.3 Initial Calibration 25
10.4 Continuing Calibration Verification (CCV) 27
10.5 Instrument Blank 29
II.0 ANALYTICAL PROCEDURE 30
11.1 Sample Preparation - General 30
11.2 Preparation of Water Samples Using Microscale Extraction 30
11.3 Preparation of Solid Samples Using Microscale Extraction 31
11.4 Preparation of Wipe Samples by Microscale Extraction 33
11.5 Final Concentration of Extract - Nitrogen Evaporation Technique (RapidVap® or
EQUIVALENT) FOR SOLID AND WIPE SAMPLES 33
11.6 Extract Analysis by GC/MS 34
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
12.0 CALCULATIONS AND DATA ANALYSIS 35
12.1 Qualitative Identification of Target Compounds 35
12.2 Data Analysis and Calculations of Target Compounds 36
12.3 Technical Acceptance Criteria for Sample Analysis 40
12.4 Corrective Action for Sample Analysis 40
13.0 ANALYTICAL PROCEDURE PERFORMANCE 42
14.0 POLLUTION PREVENTION 42
15.0 WASTE MANAGEMENT 43
16.0 REFERENCES 43
17.0 TABLES AND FIGURES 45
LIST OF TABLES
Table 1. Instrument Detection Limits (IDLs) and Method Detection Limits (MDLs) Based on Multi-
Laboratory Evaluation 45
Table 2a. Example Multi-Laboratory Results for Reagent Water Samples Spiked at Levels
Corresponding to Laboratory Low-Calibration Standards 46
Table 2b. Example Multi-Laboratory Results for Ottawa Sand Samples Spiked at Levels Corresponding
to Laboratory Low-Calibration Standards 46
Table 2c. Example Multi-Laboratory Results for Wipes Spiked at Levels Corresponding to Laboratory
Low-Calibration Standards 47
Table 3. Decafluorotriphenylphosphine (DFTPP) Key Ions and Ion Abundance 47
Table 4. Internal Standards and Surrogates 48
Table 5. Example Retention Times (RTs), Relative Retention Times (RRTs) and Quantitation Ions for
Target Compounds, Surrogate Compounds, and Internal Standards 48
Table 6. Example Multi-Laboratory Precision and Bias in Reference Matrices at Mid-Calibration
Levels 49
Table 7. Surrogate Recovery 50
Table 8. Example Calibration Standard Concentrations ((.ig/niL) Used During Multi-Laboratory Study
51
Table 9a. Example Multi-laboratory Precision and Bias in Water Using GC - Full-Scan Quadrupole
MS 52
Table 9b. Example Multi-laboratory Precision and Recovery in Water Using GC - TOF MS 53
Table 10a. Example Multi-laboratory Precision and Recovery in Soils Using GC - Full-Scan Quadrupole
MS 54
Table 10b. Example Multi-laboratory Precision and Recovery in Soils Using GC - TOF MS 55
Table 11. Multi-Laboratory Study Water Matrices Characterization Data 56
Table 12. Multi-Laboratory Study Soil Matrix Characterization Data 56
LIST OF FIGURES
Figure 1. Example chromatogram for a calibration standard on full-scan quadrupole MS 57
Figure 2. Example chromatogram for a midpoint calibration standard (Cal 5) on time-of-flight MS 58
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
1.0 SCOPE AND APPLICATION
1.1 The U.S. Environmental Protection Agency (EPA) Homeland Security Research Program
(HSRP) and Office of Land and Emergency Management (OLEM), in collaboration with
experts from across EPA and other federal agencies, have identified analytical methods
to be used for the analysis of extractable semi-volatile chemical agents in response to a
homeland security incident. This protocol is to be applied by the national network of
laboratories that has been recruited to the EPA-established Environmental Response
Laboratory Network (ERLN) so that their analytical results are consistent and
comparable. Summaries of these methods are provided in EPA's Selected Analytical
Methods for Environmental Remediation and Recovery (SAM) (Reference 16.1). HSRP
is using the SAM methods (based on the methods listed in Section 1.2) to develop
analytical protocols for laboratory identification and measurement of target agents during
site remediation.
1.2 This document is for the determination and measurement of the chemical warfare agents
(CWAs) listed in the table below and in Table 1, Section 17. The protocol is based on
EPA Methods 8270D (Reference 16.2), 3511 (Reference 16.3), 3570 (Reference 16.4),
and EPA Method 1613 (Reference 16.5) for preparation and analysis of solid, wipe, and
water samples. The procedures have been multi-laboratory tested for analysis of the
analytes listed below in soil, wipe, and water samples.
Contaminant
Cyclohexyl methylphosphonofluoridate - Cyclohexyl sarin (GF)
329-99-7
Bis(2-chloroethyl) sulfide - Sulfur mustard (HD)
505-60-2
(RS)-Propan-2-yl methylphosphonofluoridate - Sarin (GB)
107-44-8
3-Dimethylbutan-2-yl methylphosphonofluoridate - Soman (GD)
96-64-0
* Chemical Abstracts Service (CAS) Registry Number
1.3 Procedures in this protocol have been tested for the target analytes listed in Section 1.2 in
reference matrices (i.e., reagent water, Ottawa sand, and wipes), drinking water from two
sources, and two dried and homogenized soils. Results of laboratory testing are provided
in Sections 13.0 and 17.0.
2.0 SUMMARY OF PROTOCOL
This analytical protocol involves microscale extraction, followed by gas chromatography/mass
spectrometry (GC/MS) analysis to identify and measure target semi-volatile CWAs. Soil and
wipe extracts also might require a concentration step using nitrogen evaporation to achieve
appropriate levels of quantitation.
3.0 ACRONYMS, ABBREVIATIONS and DEFINITIONS
3.1 Acronyms and Abbreviations
%Recovery Percent recovery
%RSD Percent relative standard deviation
amu Atomic mass unit
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
ASTM
CAS RN
CCV
CWA
DCM
DF
DFTPP
EI
EICP
EPA
FC-43
GB
GD
GF
GC/MS
HD
HSRP
IDC
IDL
IS
IPR
LCS
MDL
MS/MSD
OLEM
OSHA
PD
PE
PFK
PPE
PTFE
QC
QL
RPD
rpm
RRF
RRT
RSD
RT
SAM
SDS
S:N
svoc
TOF
VOA
ASTM International (formerly American Society for Testing and
Materials)
Chemical Abstracts Service Registry Number
Continuing calibration verification
Chemical warfare agent
Methylene chloride (dichloromethane)
Dilution factor
Decafluorotriphenylphosphine
Electron ionization
Extracted ion current profile
U.S. Environmental Protection Agency
Perfluoro-tri-«-butylamine
Sarin
Soman
Cyclohexyl sarin
Gas chromatograph/mass spectrometer (gas chromatography/mass
spectrometry)
Sulfur mustard
Homeland Security Research Program
Initial demonstration of capability
Instrument detection limit
Internal Standard
Initial precision and recovery
Laboratory control sample
Method detection limit
Matrix spike/matrix spike duplicate
Office of Land and Emergency Management
U.S. Occupational Safety and Health Administration
Percent drift
Performance evaluation
Perfluorokerosene
Personal protective equipment
Polytetrafluoroethylene (Teflon®)
Quality control
Quantitation limit
Relative percent difference
Revolution(s) per minute
Relative response factor
Relative retention time
Relative standard deviation
Retention time
Selected Analytical Methods for Environmental Remediation and
Recovery, http://www.epa.gov/homeland-securitv-research/sam/
(accessed 05/31/2016)
Safety Data Sheet
Signal-to-noise ratio
Semi-volatile organic compound
Time-of-flight
Volatile organic analysis
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
3.2 Definitions
Aliquot - A measured portion of a field sample, standard, or solution taken for
sample preparation and/or analysis.
Analytical Batch - A set of samples that is analyzed on the same instrument during a
24-hour period of operation or the analysis of 20 samples (whichever comes first). The
analytical batch begins and ends with the analysis of the appropriate Continuing
Calibration Verification (CCV) standards.
Calibration Standard - A solution prepared from the stock standard solution(s) and
the internal standards and surrogate analytes. The calibration standards are used to
calibrate instrument response with respect to analyte concentration.
Continuing Calibration Verification (CCV) - A calibration standard containing the
target analytes, which is analyzed periodically to verify the accuracy of the existing
calibration for those analytes.
Extracted Ion Current Profile (EICP) - A plot of ion abundance versus time (or
scan number) for ion(s) of specified mass(es).
Holding Time - The time elapsed from sample collection until sample extraction
or analysis.
Initial Calibration - Analysis of calibration standards for a series of different
specified concentrations; used to define the quantitative response, linearity, and
dynamic range of the instrument for target analytes.
Initial Demonstration of Capability (IDC) - The IDC is performed prior to use of
the analytical procedures and is used to evaluate the capability of the laboratory in
terms of analytical precision, bias and sensitivity pertaining to the target analytes.
Initial Precision and Recovery (IPR) - A set of four aliquots of a clean reference
matrix (i.e., reagent water, Ottawa sand, clean wipe) to which known quantities of the
target analytes are added. The IPR aliquots are processed and analyzed exactly like a
sample and analyzed prior to the analysis of field samples as part of the IDC. Their
purpose is to determine whether the laboratory is capable of making accurate and
precise measurements.
Instrument Detection Limit (IDL) - The minimum concentration of an analyte
that, when injected into the gas chromatograph/mass spectrometer (GC/MS),
produces an average signal-to-noise ratio (S:N) between 3:1 and 5:1 for at least three
replicate injections.
Instrument Performance Check Solution - A solution of one or more
instrument tuning compounds used to evaluate the performance of the instrument
system with respect to a defined set of method criteria.
Internal Standard (IS) - Analyte added to an extract or standard solution in a known
amount and used to measure the relative responses of target analytes and surrogates.
The internal standard must be an analyte that is not a sample component.
Laboratory Control Sample (LCS) - An aliquot of a clean reference matrix (i.e.,
reagent water, Ottawa sand, clean wipe) to which known quantities of the target
analytes
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
are added. The LCS is processed and analyzed exactly like a sample. Its purpose is to
determine whether the analytical process is in control.
Matrix - The predominant material of which the sample to be analyzed is composed.
For the purpose of this protocol, a sample matrix is either aqueous/water,
soil/sediment/sand, or wipe. Matrix is not synonymous with phase (e.g., liquid or solid).
Matrix Spike (MS) - An aliquot of a field sample to which known quantities of target
analytes are added. The MS is processed and analyzed exactly like the corresponding
sample. Its purpose is to determine whether the sample matrix contributes bias to the
analytical results. Background concentrations of the analytes must be determined in a
separate aliquot.
Matrix Spike Duplicate (MSD) - A second aliquot of the field sample used to prepare
the MS, which is fortified, extracted and analyzed exactly like the MS. The MSD is used
to assess matrix effects on analytical precision and bias.
Method Blank - An aliquot of a clean reference matrix (i.e., reagent water, Ottawa sand,
clean wipe) that is treated exactly as a sample, including exposure to all glassware,
equipment, solvents, reagents, sample preservatives, internal standards, and surrogates
that are used in the extraction batch. The method blank is used to determine whether
target analytes or interferences are present in the laboratory environment, reagents or
equipment.
Method Detection Limit (MDL) - The minimum concentration of an analyte that can be
identified, measured and reported with 99 percent confidence that the analyte
concentration is greater than zero. The MDL is a statistical determination (Section 9.7),
and accurate quantitation at this level is not expected.
Percent Difference - The difference between two values divided by one of the values.
Used in this protocol to compare two relative response factor (RRF) values from
calibration.
Percent Drift (PD) - The difference between the calculated and theoretical value divided
by the theoretical value. Used in this protocol to compare calculated and theoretical
values for calibration by regression techniques.
Quantitation Limit (QL) - The minimum level of quantitation. This concentration must
meet the criteria defined in Section 9.8.
Reagent Water - Water in which an interferent is not observed at or above the low-level
calibration standard for each analyte of interest. The purity of this water must be
equivalent to ASTM International (ASTM) Type II reagent water of Specification
D1193-06, "Standard Specification for Reagent Water" (Reference 16.6).
Relative Percent Difference (RPD) - The difference between two values divided by the
mean of the values. RPD is reported as an absolute value (i.e., always expressed as a
positive number or zero).
Relative Response Factor (RRF) - A measure of the relative mass spectral response of
an analyte compared to its internal standard. RRFs are determined by analysis of
standards and are used in calculating the concentrations of analytes in samples.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Retention Time (RT) - The time an analyte is retained on a GC column before elution.
The RT is dependent on the analyte, nature of the column's stationary phase, the
column's diameter, temperature, carrier gas flow rate, and other column parameters.
Relative Retention Time (RRT) - The ratio of the RT of a compound to the RT of a
corresponding internal standard.
Safety Data Sheet (SDS) - Written information provided by vendors concerning a
chemical's toxicity, health hazards, physical properties, flammability, and reactivity data
including storage, spill, and handling precautions.
Stock Standard Solution - A concentrated solution containing one or more target
analytes prepared in the laboratory using assayed reference materials or materials
purchased from a reputable commercial source.
Surrogate - Analyte that is unlikely to be found in any sample to be analyzed.
Surrogates are added to a sample aliquot in a known amount before extraction or other
processing. Surrogates are measured with the same procedures used to measure other
sample components. The purpose of the surrogate is to monitor method performance
with each sample.
Working Standard Solution - A solution containing target analytes prepared from stock
standard solutions. Working standard solutions are diluted as needed to prepare
calibration and spiking solutions.
4.0 INTERFERENCES
4.1 Contaminants in solvents, reagents, glassware, and other sample processing hardware can
cause method interferences such as discrete artifacts and/or elevated baselines in the
extracted ion current profiles (EICPs). All of these materials must be routinely
demonstrated to be free from interferences under the conditions of the analysis by running
method blanks. Matrix interferences can be caused by contaminants that are co-extracted
from the sample. The extent of matrix interferences will vary considerably from source to
source and is evaluated using the results from the analysis of matrix spike and matrix spike
duplicate samples (MS/MSDs).
4.2 This protocol includes conditions for collecting mass spectral data using both quadrupole
mass spectrometers in full-scan mode and time-of-flight (TOF) mass spectrometers.
5.0 SAFETY
WARNING: The toxicity of CWAs presents hazards unfamiliar to most experienced laboratory
personnel. Special techniques and precautions must be used even for the simplest procedures
involving these agents. Because CWAs are target analytes for this protocol, laboratory personnel
must be thoroughly trained in appropriate safety procedures prior to using this method.
5.1 Operations with CWAs have specific safety requirements. The laboratory must have these
requirements included in a Chemical Hygiene Plan prior to conducting the analytical
procedures described in this protocol.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
5.2 At a minimum, personal protective equipment (PPE) requirements include safety glasses,
lab coats, and protective gloves. The availability of emergency response equipment and
support personnel should be as indicated in a laboratory Chemical Hygiene Plan.
5.3 Exposure to chemical agent material is possible from contact, and risk is primarily
associated with compromise of PPE. Respiratory exposure can result from spills or
improper use of ventilation controls and PPE.
5.4 At concentrations of CWAs that are within the calibration range of this method, likelihood
of an exposure causing adverse health effects is extremely low (Reference 16.7).
Note: This method does not address all safety issues associated with its use. The
laboratory is responsible for maintaining a safe work environment and a current file of
Occupational Safety and Health Administration (OSHA) regulations regarding the safe
handling of the chemicals (including all solvents, reagents, and standards). A reference
file of safety data sheets (SDSs) must be available to all personnel involved in these
analyses, chemical handling, and contaminated area cleaning, or who might potentially
come in contact with the materials in their workplace.
6.0 EQUIPMENT AND SUPPLIES
Brand names, suppliers, catalog, and part numbers are for illustrative purposes only. No
endorsement is implied. Equivalent performance can be achieved using equipment and supplies
other than those specified; however, laboratories must document use of alternative equipment or
supplies and provide a demonstration of equivalent performance meeting the requirements of this
protocol.
6.1 General Equipment
6.1.1 Vials - Clear or amber glass, with polytetrafluoroethylene (PTFE)-lined screw
or crimp top (2.0 mL capacity for GC auto sampler) (Sigma Aldrich Catalog
No.SU860033, Sigma-Aldrich. St. Louis, MO) or equivalent. Glass inserts can
be used to minimize sample volumes.
6.1.2 Syringes - Contaminant-free, 1.0 mL, 2.0 mL, 10 |iL. 25 |iL. 500 |iL
6.1.3 Pasteur glass pipettes - 1.0 mL, disposable (Fisher Scientific Catalog No.
NC0541803, Thermo Fisher Scientific, Westminster, MD) or equivalent.
6.1.4 Balances - Analytical, capable of accurately weighing ±0.0001 gram, and one
capable of weighing 100 grams (±0.01 grams)
6.1.5 Spatula - Stainless steel or PTFE
6.1.6 Nitrogen evaporation device - Equipped with temperature control that can be
maintained at 35 - 40 °C, a RapidVap® (Labconco Corporation, Kansas City,
MO) or equivalent. To prevent the release of solvent fumes into the laboratory,
this device must be used with suitable engineering controls.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
6.1.7 Water bath (for nitrogen evaporation devices that do not have a heating
apparatus) - Heated, with concentric ring cover capable of heating to 80 °C and
maintaining temperature control (±5 °C). The bath should be used with
appropriate engineering controls.
6.1.8 Glass funnel (Fisher Scientific Catalog No. CG172305, Thermo Fisher Scientific,
Westminster, MD) or equivalent - Used in filtering soil samples that fail to settle
out with centrifugation.
6.1.9 Borosilicate glass wool - Oven-cleaned (muffled) or solvent-rinsed (using
extraction solvent); used in filtering soil samples that fail to settle out with
centrifugation.
6.1.10 pH paper - Including narrow range capable of measuring a pH of 2.0.
6.1.11 pH meter - With a combination glass electrode. Calibrate prior to each use
according to manufacturer's instructions.
6.1.12 Ottawa sand - Held at 450 °C for four hours in a 500-mL wide-mouthed amber
bottle.
6.1.13 Vortexer - VWR (VWR Corporation, Radnor, PA) or equivalent, capable of
accommodating 40 - 60-mL vials and 50-mL centrifuge tubes.
6.1.14 Shaker table
6.1.14.1 Glas-Col Large Capacity Mixer (Part # 099A LC1012, Glas-Col Inc.,
Terre Haute, IN), Glas-Col Digital Pulse Mixer (Part # 099A
DPMI2) or equivalent
6.1.14.2 Foam pad for 40-mL volatile organic analysis (VOA) vials (Part
#099A VC4014, Glas-Col Inc., Terre Haute, IN) or equivalent
6.1.14.3 Foam pad for 60-mL VOA vials (Part #099A VC6014, Glas-Col
Inc., Terre Haute, IN) or equivalent
6.2 Microscale Extraction Apparatus
6.2.1 Solid samples
6.2.1.1 VOA vials - 40-mL capacity, disposable, pre-cleaned with PTFE-
lined caps (Fisher Scientific Catalog No. 05-719-400, Thermo Fisher
Scientific, Westminster, MD) or equivalent. If pre-cleaned vials are
not available, appropriate cleaning procedures are provided in EPA
Methods 525.3 (Reference 16.8) and 1668C (Reference 16.9) and in
SW-846 Chapter 4 (Reference 16.10).
6.2.1.2 Sonicator - Branson 3510 (Branson Ultrasonics Corp., Danbury, CT)
or equivalent
6.2.1.3 Apparatus for determining percent dry weight
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
6.2.1.3.1 Drying oven - Capable of maintaining 103 - 105 °C
6.2.1.3.2 Desiccator
6.2.1.3.3 Crucibles - Disposable aluminum
6.2.1.4 Glass beads - Solvent-rinsed, baked in 400 °C oven for approximately
1 hour (Fisher Scientific Catalog No. S80024 or equivalent)
6.2.1.5 Centrifuge - Capable of at least 500 G-force units and
accommodating 40- or 50-mL vials, Accuspin™ Model 400 (Thermo
Fisher Scientific, Westminster, MD) or equivalent. CAUTION:
Different centrifuge makes and models have different maximum
centrifuge speeds that are recommended for safe operation. The
maximum safe handling speed of each centrifuge will depend, in part,
on the vials used and should be determined prior to centrifuging
samples.
6.2.1.6 Pasteur pipettes - 1.0-mL glass, disposable or re-pipettes/autopipettes
with disposal tips
6.2.2 Water samples
6.2.2.1 Conical bottom, glass screw-top tube, 50-mL or 60-mL vials, pre-
cleaned with PTFE-lined caps. If pre-cleaned vials are not available,
appropriate cleaning procedures are provided in EPA Methods 525.3
(Reference 16.8) and 1668C (Reference 16.9), and in SW-846
Chapter 4 (Reference 16.10).
6.2.2.2 Beakers - 400 mL
6.2.2.3 Class A graduated cylinder - 100 mL
6.2.2.4 Class A volumetric flasks - 10 mL
6.2.3 Wipe samples - Kendall Curity® Gauze Sponges (, USP Type VII Gauze, cotton,
12 ply 3 in x 3 in, Tyco Healthcare Group, Covidien, Mansfield, MA) or
equivalent; pre-cleaned by extracting with methylene chloride (DCM) using an
appropriate extraction method (e.g., pressurized fluid extraction,
Soxhlet extraction, soaking in DCM).
Note: Wipes used for field or laboratory quality control (QC) samples should
be pre-wetted with DCM prior to use.
6.3 Gas Chromatograph/Mass Spectrometer (GC/MS) System
6.3.1 Gas chromatograph - The GC system must be capable of temperature
programming and have a flow controller that maintains a constant column
carrier gas flow rate throughout the temperature program operations. The
system must be suitable for splitless injection and have all required accessories
including syringes, analytical columns, and gases. All GC carrier gas lines must
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
be constructed from stainless steel or copper tubing. Non-PTFE thread
sealants or flow controllers with rubber components are not to be used.
Note: Due to the potential hazards associated with the analysis of CWAs, it is
recommended that a split vent trap be used for the GC system (Agilent
RD-1020 Universal/External Split Vent Trap, Agilent Technologies Inc., Santa
Clara, CA, or equivalent). For additional safety measures, the split and purge
vent lines can be vented to the hood ventilation system.
6.3.2 GC column - Recommended length 30 m X 0.25 mm inner diameter (ID) (or 0.32
mm) bonded phase silicon coated fused silica capillary: DB-5 (J&W Scientific,
Agilent Technologies, Santa Clara, CA); DB-5MS; RTX®-5 (Restek Corp.,
Bellefonte, PA), RTX®-5MS (Restek Corp., Bellefonte, PA), RTX-5Sil MS
(Restek Corp., Bellefonte, PA); Zebron® ZB-5(Phenomenex, Phenomenex Inc.,
Torrance, CA); SPB®-5 (Supelco, Sigma-Aldrich. St. Louis, MO); AT-5
(Alltech, Grace, Columbia, MD); HP-5 (Agilent, Agilent Technologies, Santa
Clara, CA); HP-5MS or HP-5MS UI (Agilent Technologies, Santa Clara, CA,),
CP-Sil 8 CB (Chrompack Raritan, NJ); 007-2 (Quadrex, Quadrex Corp.,
Bethany, CT); BP-5 (SGE, Trajan Scientific Americas, Inc., Austin, TX);
Zebron® ZB-5MS (Phenomenex, Phenomenex Inc., Torrance, CA) or equivalent.
Columns used to generate the example data provided in Section 17.0 include:
Agilent HP-5 MS, RTX® - 5sil, RTX®-5MS, RTX®-5Sil MS, Zebron® ZB-5
MS, DB-5MS.
Although a film thickness of 1.0 micron might be desirable because of its
larger capacity, film thicknesses of 0.25 micron and 0.05 micron were used by
laboratories generating the example data presented in Section 17.0. A
description of the GC column used for analysis shall be provided in the data
narrative. A capillary column is considered equivalent if:
• The column does not introduce contaminants that interfere with the
identification and quantification of the compounds listed in Table 1, Section
17.
• The analytical results generated using the column meet the initial
calibration and CCV technical acceptance criteria listed in the protocol and
the quantitation levels determined as described in Section 9.8.
• The column provides equal or better resolution of the compounds listed
in Table 1, Section 17, when compared to columns listed above.
6.3.3 MS - Must be capable of recording a spectrum from 35 - 500 atomic mass units
(amu) every second or less, using 70 electron volts (nominal) in the electron
ionization (EI) mode, and producing a mass spectrum that meets the tuning
acceptance criteria when 50 ng or less of decafluorotriphenylphosphine
(DFTPP) is injected through the GC inlet. The instrument must be vented to
prevent the release of contaminants into the instrument room.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
7.0 REAGENTS AND STANDARDS
7.1 Reagents
7.1.1 Organic-free reagent water - Water in which an interferent is not observed at or
above the quantitation limit (QL) for each analyte of interest. ASTM Type II
reagent water of Specification D1193-06, "Standard Specification for Reagent
Water," (Reference 16.6) or equivalent.
7.1.2 Acetone, DCM, and toluene - pesticide residue analysis grade or equivalent.
DCM must contain a non-methanol olefinic stabilizer (i.e., amylene or 2-
pentene). Solvents must be anhydrous or pre-dried by adding sodium sulfate
until no clumping occurs and the solution becomes clear (not cloudy).
7.1.3 Sodium sulfate - Powdered or granular anhydrous reagent grade, heated at
400 °C for four hours in a shallow tray, cooled in a desiccator, and stored
in a glass bottle
7.1.4 Sodium chloride - Anhydrous reagent grade, >99 %
7.1.5 Sodium thiosulfate - Reagent grade, 10 % sodium thiosulfate solution in reagent
water. Prepare fresh with each use.
7.2 Standards
The laboratory must be able to verify that stock standard solutions are certified.
Manufacturers' certificates of analysis must be retained by the laboratory and presented
upon request. Stock standard solutions provided in sealed glass ampules may be
retained and used within six months of the preparation date. Solutions used for
calibration verification ideally are prepared from a separate source other than the source
used to prepare calibration standards. Due to the nature of the analytes addressed in this
protocol, identification of a secondary source might be difficult.
7.2.1 Stock standard solutions
Stock standard solutions used to produce working standards may contain
individual target compounds or mixtures of target compounds.
7.2.2 Working standards
7.2.2.1 Surrogate standard spiking solution - Prepare a surrogate standard
spiking solution in DCM or other suitable solvent that contains
appropriate surrogates for the target compounds. A concentration of
25 (ig/mL is recommended for each surrogate. Surrogate standards
are added to all samples and calibration solutions.
Note: Table 4, Section 17 provides a list of surrogates used
during laboratory studies testing this protocol, based on surrogates
typically used for semi-volatile organic compounds (SVOC)
analyses. Alternative surrogates might be more representative of
the analytes targeted in this method and may be used instead of or
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
in addition to those listed in Table 4, provided the surrogates
meet the criteria in Table 7, Section 17.
7.2.2.2 Matrix spiking solution - This solution is prepared in DCM and
should contain all target analytes.
7.2.2.3 Instrument performance check solution - Prepare a solution of
DFTPP in DCM such that a 1 -|_iL injection will contain 50 ng or less
of DFTPP. The DFTPP may also be included in the calibration
standards at this level.
7.2.2.4 Initial and continuing calibration solutions
7.2.2.4.1 Prepare calibration standards in DCM at a minimum of
five concentration levels. Each calibration standard
should contain each target compound of interest,
associated surrogate, and internal standard.
Note 1: All samples analyzed must be injected at the
same volume (e.g., 1.0 or 2.0 (.iL) as the calibration
standard.
Note 2: The concentrations listed in Table 8, Section 17,
provide an example calibration range used during
laboratory evaluation of this protocol. For most
analytes, the low calibration standard is set at the
expected QL (determined in Section 9.8). The
remaining calibration standards should be prepared at
concentrations that meet the specifications in Section
10.3.4.
7.2.2.4.2 The CCV standard is prepared in DCM at or near the
midpoint of the calibration curve.
7.2.2.5 Internal standard solution - An internal standard solution can be
prepared by dissolving 100 mg of each of the following compounds
in 100 mL of DCM: l,4-dichlorobenzene-d4 and naphthalene-ds,
resulting in a concentration of 1.0 mg/mL of each internal standard
solution. A sufficient portion of this solution will be added to each
sample extract just prior to analysis by GC/MS to result in a
concentration of 0.5 ng/(.iL (for both full-scan quadrupole and TOF
mass spectrometers). Alternatively, internal standard solutions can
be purchased from commercial sources (e.g., Supelco part number
861238 or equivalent).
7.2.3 Storage of standard solutions
7.2.3.1 Store unopened ampules of stock standard solutions at <6 °C. If no
manufacturer's expiration date is provided, unopened ampuled
standard solutions may be retained and used for up to six months
after the preparation date (Reference 16.11). Store opened stock
standard solutions at <6 °C in PTFE-lined screw-cap amber bottles.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Fresh standards should be provided every twelve months (for
solutions containing a single target compound) or six months (for
solutions containing a mixture of target compounds), or sooner if
the expiration date has elapsed.
7.2.3.2 Store the working standards at < 6 °C in containers with PTFE-lined
caps. Certain analytes may degrade in as little as two weeks;
therefore, the working standard solution should be checked against
CCV standards at least weekly for stability. If stored as single-
analyte solutions, standards containing soman (GD) or cyclohexyl
sarin (GF) should be stable to up to 12 months; standards containing
sulfur mustard (HD) should be stable for up to six months; and
standards containing sarin (GB) should be stable for up to five
months. Multi-component working standards should be replaced
after five months. Working standard solutions must be replaced if
the stock standard solutions have expired or if comparison with CCV
samples indicates a problem.
7.2.3.3 Protect all standards from light. Samples, sample extracts, and
standards must be stored separately.
7.2.3.4 The laboratory is responsible for maintaining the integrity of
standard solutions and verifying the solution prior to use. The
standards must be brought to room temperature prior to use, checked
for losses, and checked to ensure that all components have remained
in solution. Guidance on standard verification procedures can be
found in EPA's Superfund Analytical Services / Contract
Laboratory Program, Multi-Media, Multi-Concentration Organics
Analysis, SOM02.3, Exhibit E, Section 4 (Reference 16.12).
8.0 SAMPLE PRESERVATION, STORAGE, AND TECHNICAL HOLDING TIMES
8.1 Sample Preservation
Samples must be stored on ice or refrigerated at 4 °C (± 2 °C) immediately after
collection until receipt in the laboratory. The presence of chlorine may increase the
degradation rate of G-agents in water. If chlorine is suspected to be present in water
samples (e.g., treated drinking water or wastewater) that are to be measured for G-agents,
add approximately four drops (-0.2 mL) of a 10 % solution of sodium thiosulfate per 35-
mL sample. If clouding results, add less sodium thiosulfate to a fresh sample aliquot. If
sodium thiosulfate is not added during sample collection, it should be added immediately
upon sample receipt in the laboratory, prior to sample analysis or extraction.
8.2 Sample Storage
Samples must be protected from light and refrigerated at 4 °C (± 2 °C) from the time of
receipt until extraction.
8.3 Sample Extract Storage
8.3.1 Sample extracts must be protected from light and stored at <6 °C.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
8.3.2 Samples, sample extracts, and standards must be stored separately.
8.4 Technical Holding Times
8.4.1 Soil and wipe samples must be extracted within seven days of receipt at the
laboratory. Aqueous samples should be analyzed as soon as possible upon
receipt in the laboratory. At a minimum, DCM must be added to aqueous
samples within 48 hours of receipt (DCM should be added immediately to
aqueous samples that will be analyzed for HD), and samples must be completely
extracted within seven days of receipt.
Note: Holding times for soils and wipes are based on holding times listed in
guidance documents and similar analytical methods for SVOCs (e.g., EPA
Methods 525.2 [Reference 16.13] and 525.3 [Reference 16.8], CLP Method
SOM2.1 [Reference 16.14], SW-846 Chapter 4 [Reference 16.10]). Holding
times for water samples were evaluated in a single laboratory using this
protocol. Although the addition of DCM has not been evaluated, these
CWA agents are likely to partition into the organic phase where they would
be less likely to hydrolyze. Laboratories may wish to verify this holding
time for the specific samples being analyzed.
8.4.2 Extracts must be analyzed within 14 days following extraction.
9.0 QUALITY CONTROL (QC)
QC requirements for this protocol include the following:
Quality Control (QC) Analyses
Requirement
Section
Frequency
Instrument Detection Limit (IDL)
Determination
Section 9.6
Optional. Performed prior to Method Detection
Limit (MDL) Study
Method Detection Limit (MDL)
Determination
Section 9.7
Performed once, prior to first performing the
protocol procedures and with each significant
change as part of the Initial Demonstration of
Capability (IDC)
Initial Precision and Recovery (IPR)
Determination
Section 9.2
Quantitation Limit (QL) Determination
Section 9.8
Method Blanks
Section 9.3
At least one per batch of <20 samples of the same
matrix
Instrument Blank
Section 10.5
Following an analysis with suspected carry-over
or following analysis of samples containing high
concentrations
Matrix Spike and Matrix Spike
Duplicate (MS/MSD)
Section 9.4
One per each batch of <20 samples of the same
matrix
Laboratory Control Sample (LCS)
Section 9.5
At least one per batch of <20 samples of the same
matrix
Continuing Calibration Verification
(CCV)
Section 10.4
Prior to the analysis of samples, and after
instrument performance check. Analyzed at the
beginning and at the end of each analytical batch
of <20 injections
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Precision and bias criteria for data generated using this method are currently set at 50 - 150 %
recovery and < 30 % precision (as relative standard deviation [RSD] or relative percent difference
[RPD]). These criteria may change as more laboratory data become available. In cases where
analyses of difficult sample matrices generate results outside these criteria, data should be
flagged, and laboratories should collect additional data to support development of laboratory- and
matrix-specific criteria. Example precision and bias results obtained from multiple laboratories
analyzing spiked reference matrix samples (reagent water, Ottawa sand, and wipes) and field
samples (water and soil) are provided in Section 17.
9.1 Initial Demonstration of Capability (IDC)
An IDC shall be performed prior to the analysis of any samples and with each significant
change in instrument type, different detection technique, personnel or method. An IDC
consists of the following:
• An IPR determination (Section 9.2),
• An MDL determination (Section 9.7), and
• A QL determination (Section 9.8) on a clean matrix (reagent water, Ottawa sand, pre-
cleaned wipe).
The IPR consists of four replicate samples of a clean matrix spiked with CWAs around
the midpoint of the calibration curve and carried through the entire analytical process.
Prior to performing the IDC, a valid initial calibration (Section 10.3) must be established.
9.2 Initial Precision and Recovery (IPR) Determination
9.2.1 Preparation and analysis of IPR samples
9.2.1.1 Water samples
Prepare four replicate samples, each consisting of 35 mL of reagent
water. Add a sufficient amount of surrogate standard spiking
solution and matrix spiking solution to result in the addition of 1.0
(.ig of each surrogate (add 40 |_iL if prepared as in Section 7.2.2.1)
and a concentration at the mid-point of the calibration range of
each matrix spike compound. Extract and analyze according to the
procedures for water samples (Sections 11.2 and 11.6). The total
volume of DCM added will be slightly greater than the 2 mL
needed for extraction, and includes the volumes added for spiking
target compounds, and surrogates.
9.2.1.2 Ottawa sand
Prepare four replicate samples consisting of 10 grams of Ottawa sand
and 2.5 grams of sodium sulfate. Add a sufficient amount of the
surrogate standard spiking solution and the matrix spiking solution to
result in the addition of 0.5 (.ig of each surrogate (add 20 |_iL if
prepared as in Section 7.2.2.1) and a concentration at the mid-point
calibration range of each matrix spike compound, and follow the
appropriate extraction procedure in Section 11.3. Extract,
concentrate and analyze according to procedures for solid samples.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
9.2.1.3 Pre-cleaned wipes
Prepare four replicate samples consisting of pre-cleaned wipes. Pre-
wet the wipes with DCM prior to use. Add a sufficient amount of
the surrogate standard spiking solution and the matrix spiking
solution to result in the addition of 0.5 (.ig of each surrogate (add 20
l_iL if prepared as in Section 7.2.2.1) and a concentration at the mid-
point calibration range of each matrix spike compound, and follow
the appropriate extraction procedure in Section 11.4. Extract,
concentrate and analyze according to procedures for wipe samples.
9.2.2 Calculations for IPR
9.2.2.1 Calculate the percent recovery (%Recovery) of each compound in
each IPR sample using Eq. 11 (Section 12.2.9.2). Calculate an
average %Recovery for each compound.
9.2.2.2 Calculate a percent relative standard deviation (%RSD) for each
compound in the IPR samples.
9.2.3 Technical acceptance criteria for IPR
9.2.3.1 The average recovery of each compound in the IPR should be within
50- 150 %.
9.2.3.2 The % RSD of each compound in the IPR should be less than or
equal to 30.
9.2.4 Corrective Action for IPR
If the technical acceptance criteria in Section 9.2.3 are not met, inspect the
system for problems and take corrective actions to achieve the acceptance
criteria.
9.3 Method Blanks
A method blank is a volume of a clean reference matrix (e.g., reagent water for water
samples, clean inert sand along with purified sodium sulfate for solid samples, or clean
pre-wetted wipes for wipe samples) spiked with a sufficient amount of surrogate standard
spiking solution (Section 7.2.2.1) such that the same amount of surrogate is added as for
the associated samples and carried through the entire analytical procedure. Internal
standard solution is added just prior to analysis by GC/MS to give a concentration of 0.5
ng/(.iL for each internal standard for both quadrupole and TOF modes. The volume or
weight of the method blank must be approximately equal to the volume or weight of the
samples associated with the blank.
9.3.1 Frequency of method blanks
A method blank must be extracted each time samples are extracted. The number
of samples extracted with each method blank should not exceed 20 field samples
(excluding MS/MSDs and Performance Evaluation [PE] samples). In addition, a
method blank is:
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
• Extracted by the same procedure used to extract samples
• Analyzed on each GC/MS system used to analyze associated samples and
conditions (i.e., GC/MS settings)
9.3.2 Method blank preparation
9.3.2.1 A method blank for water samples consists of a 35-mL volume of
reagent water spiked with a sufficient amount of the surrogate
standard spiking solution to result in the addition of 1.0 (.ig of each
surrogate (add 40 |_iL if prepared as in Section 7.2.2.1). The final
volume of the extracts will be approximately 2.0 mL; therefore, the
concentration of the surrogates in the extract is expected to be
approximately 0.50 (ig/mL. For solid samples, a method blank
consists of 10 grams of clean inert sand and 2.5 grams of sodium
sulfate spiked with a sufficient amount of the surrogate spiking
solution to result in the addition of 0.5 (.ig of each surrogate (add 20
l_iL if prepared as in Section 7.2.2.1). The final volume of the
extracts will be 1.0 mL; therefore, the concentration of the surrogates
in the extract is expected to be 0.50 (ig/mL. A method blank for
wipe samples consists of a clean unused wipe spiked with a
sufficient amount of the surrogate standard spiking solution to result
in the addition of 0.5 (.ig of each surrogate (add 20 |_iL if prepared as
in Section 7.2.2.1). The final volume of the extracts will be 1.0 mL;
therefore, the concentration of the surrogates in the extract is
expected to be 0.50 (ig/mL. Extract, concentrate, and analyze the
blank according to procedures.
9.3.2.2 Under no circumstances should method blanks be analyzed at a
dilution.
9.3.3 Technical acceptance criteria for method blank analysis
9.3.3.1 All blanks should be extracted and analyzed at the frequency
described in Section 9.3.1 on a GC/MS system meeting the DFTPP
tuning criteria in Section 10.2.4 and Table 3, Section 17, initial
calibration in Section 10.3, and CCV technical acceptance criteria in
Section 10.4.5.
9.3.3.2 The % Recovery of each of the surrogates in the blank must be within
the acceptance limits listed in Table 7, Section 17.
9.3.3.3 The blank must meet the sample analysis acceptance criteria listed in
Section 12.3.
9.3.3.4 A method blank for solid, water, and wipe samples must contain a
concentration less than the MDL for all target compounds. In cases
where a blank has detects above the MDL, but associated samples
have detects greater than 10 times the blank, consult the agency to
determine if re-extraction is required. In cases where a method blank
fails to meet technical acceptance criteria but all samples had non-
detects for all target analytes, no re-extraction or qualification of data
is necessary.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
9.3.4 Corrective action for method blanks
9.3.4.1 If a method blank does not meet the technical acceptance criteria for
method blank analysis, the analytical system is considered to be out
of control.
9.3.4.2 If contamination is the problem, then the source of the
contamination should be investigated and appropriate corrective
measures taken before further sample analysis proceeds. It is the
laboratory's responsibility to ensure that interferences caused by
contaminants in solvents, reagents, glassware, and sample storage
and processing hardware that lead to discrete artifacts and/or
elevated baselines in the GC/MS have been eliminated. If
possible, an aliquot of any sample associated with the
contaminated blank should be re-extracted and reanalyzed.
9.3.4.3 If surrogate recoveries in the method blank do not meet the
acceptance criteria listed in Table 7, Section 17, first reanalyze the
method blank. If the surrogate recoveries do not meet the
acceptance criteria after reanalysis, the method blank and an
aliquot of any sample associated with that method blank should be
re-extracted, if possible, and reanalyzed. If a surrogate recovery is
high and all corresponding samples had non-detects for the
associated target compounds, sample re-extraction and reanalysis
are not required.
9.3.4.4 If the method blank does not meet the internal standard response
requirements in Section 12.3.5, follow the corrective action
procedure in Section 12.4.4.1. Resolve and document problem
resolution before proceeding with sample analysis.
9.3.4.5 If the method blank does not meet the retention time (RT)
requirements for internal standards (Section 12.3.6), check the
instrument for malfunction and recalibrate. Reanalyze the method
blank.
9.3.4.6 Samples that are analyzed with corresponding method blanks that do
not meet any of the criteria listed in Sections 9.3.4.2 - 9.3.4.5 should
be reanalyzed. If the method blank does not meet the criteria, then
all corresponding sample data should be flagged.
9.4 Matrix Spike and Matrix Spike Duplicate (MS/MSD)
To evaluate the potential effects of the sample matrix on analyses, a mixture of target
compounds must be spiked into two additional aliquots of a water or solid sample and
analyzed in accordance with the appropriate method. Mixtures should be spiked at a
concentration near the midpoint of the calibration range.
9.4.1 Frequency of MS/MSD analyses
9.4.1.1 An MS/MSD pair is analyzed with each batch of < 20 samples of
each water or solid matrix type. MS/MSDs are not performed on
wipe samples.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
9.4.1.2 For quality assurance purposes, water rinsate samples and/or field
blanks (field QC) or PE samples may accompany solid, water, and/or
wipe samples that are delivered to the laboratory for analysis. These
field QC or PE samples are not used for MS/MSD analyses.
9.4.1.3 If the agency requesting the analysis designates a sample to be used
as an MS/MSD, then that sample must be used. If there is
insufficient sample remaining to perform an MS/MSD, then the
laboratory shall choose another sample on which to perform an
MS/MSD analysis. At the time the selection is made, the laboratory
should notify the agency that insufficient sample was received and
identify the agency sample selected for the MS/MSD analysis.
9.4.1.4 If there is insufficient sample remaining in any of the samples in a
batch to perform the required MS/MSD, the laboratory will report
this in the data narrative.
9.4.2 Procedure for Preparing MS/MSD
9.4.2.1 Water samples
Prepare two additional aliquots of the sample chosen for spiking.
The volume should be equal to that of the associated samples. Add
a sufficient amount of surrogate standard spiking solution and
matrix spiking solution to each aliquot to result in the addition of
1.0 (.ig of each surrogate (add 40 |_iL if prepared as in Section
7.2.2.1) and a concentration at the mid-point of the calibration
range of each matrix spike compound. Extract, clean up, and
analyze the MS/MSD according to the procedures for water
samples (Sections 11.2 and 11.6). The total volume of DCM added
will be slightly greater than the 2 mL needed for extraction, and
includes the volumes added for spiking target compounds, and
surrogates.
9.4.2.2 Solid samples
Prepare two additional aliquots of the sample chosen for spiking in
two 40-mL VOA vials with PTFE-lined caps. The amount chosen
should be equal to that of the associated sample. If the sample
contains moisture, add 2.5 grams of sodium sulfate for every 10
grams of sample.
Mix well. Add a sufficient amount of the surrogate standard
spiking solution and the matrix spiking solution to each aliquot to
result in the addition of 0.5 |_ig of each surrogate (add 20 (.iL if
prepared as in Section 7.2.2.1) and a concentration at the mid-point
of each matrix spike compound, and follow the appropriate
extraction procedure in Section 11.3. Extract, concentrate, clean
up, and analyze the MS/MSD according to procedures for solid
samples.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
9.4.3 Dilution of MS/MSD
Before any MS/MSD analysis, analyze the original sample, then analyze the
MS/MSD at the same concentration as the most concentrated extract for which
the original sample results will be reported.
9.4.4 Calculations for MS/MSD
9.4.4.1 Calculate the % Recovery of each matrix spike compound in
the MS/MSD sample (see Eq. 11 in Section 12.2.9).
9.4.4.2 Calculate the RPD of the concentrations of each compound in the
MS/MSD using Eq. 1. Concentrations of each compound in the
MS/MSD are calculated using the same equations as used for
target compounds (Eq. 6 for water samples and Eq. 7 for solid
samples in Section 12.2.6).
Eq. 1 Relative Percent Difference Calculation
RPD = [Cl ~Cl * x 100
C1+C2
where:
Ci = Measured concentration of the first sample aliquot
C2 = Measured concentration of the second sample aliquot
9.4.5 Technical acceptance criteria for MS/MSD
9.4.5.1 All MS/MSDs must be analyzed on a GC/MS system meeting
DFTPP and initial calibration and CCV technical acceptance criteria,
as well as the method blank technical acceptance criteria.
9.4.5.2 The MS/MSD must be extracted and analyzed within the technical
holding time (Section 8.4).
9.4.5.3 The RT shift for each of the internal standards must be within ±
0.50 minutes (30 seconds) between the MS/MSD sample and the
most recent CCV standard analysis.
9.4.5.4 The limits for matrix spike compound recovery and RPD are 50 -
150 % and < 30 %, respectively. For difficult matrices, laboratories
are encouraged to collect sufficient data to support development of
laboratory-specific criteria.
9.4.6 Corrective action for MS/MSD
If recovery or RPD limits are not met and the LCS, CCV, and method blank are
within acceptable limits, this might be an indication of matrix interferences. If
sufficient sample is available, an MS/MSD should be reanalyzed, along with all
appropriate QC samples. If, after reanalysis, MS/MSD recovery limits are not
met, flag the results of the associated sample.
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9.5 Laboratory Control Sample (LCS)
An LCS consists of an aliquot of clean reference matrix of the same weight or volume as
the corresponding field samples and spiked with the same compounds at the same
concentrations used to spike the MS/MSD. The nominal volume of DCM added to water
samples is 2.0 mL. Because target compound and surrogate spiking solutions also
contain DCM, the total volume of DCM added may be slightly greater than 2.0 mL. The
actual total volume of DCM must be used in the calculations in Section 12.2. When the
results of the MS/MSD analysis indicate matrix interference might be present, the LCS
results are used to verify that the interferences are due to the sample matrix and not from
artifacts introduced in the laboratory.
9.5.1 Preparation of LCS
Extract and analyze the LCS according to the procedures in Section 11.2
(for water samples), Section 11.3 (for solid samples), or Section 11.4 (for
wipe samples).
9.5.2 Frequency of LCS analyses
One LCS should be prepared, extracted, analyzed, and reported for every 20 or
fewer field samples extracted in a batch of a similar matrix. The LCS must be
extracted and analyzed concurrently with the samples, using the same
extraction procedure, cleanup procedure (if required), and instrumentation.
9.5.3 Calculations for LCS
Calculate the recovery of each compound in the LCS using Eq. 11 (Section
12.2.9.2.
9.5.4 Technical acceptance criteria for LCS analysis
9.5.4.1 All LCSs must be extracted and analyzed at the frequency described
in Section 9.5.2 on a GC/MS system meeting the tuning, initial
calibration and CCV, and method blank technical acceptance
criteria.
9.5.4.2 The limits for LCS compound recovery are 50 - 150 %.
9.5.5 Corrective action for LCS
9.5.5.1 If LCS recovery limits are not met, inspect the system for problems
and take corrective actions to achieve the acceptance criteria.
9.5.5.2 If LCS recovery limits cannot be met, flag all associated sample and
blank data accordingly.
9.6 Instrument Detection Limit (IDL) Determination
Laboratories may determine an IDL for each target compound on each instrument used
for analysis. While determining IDLs is not required, IDL results can be helpful in
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
determining an appropriate spike level for use in determining the MDL (Section 9.7), as
well as instrument sensitivity to the target analytes. It is recommended that IDLs be
verified annually thereafter, or after major instrument maintenance. Major instrument
maintenance includes, but is not limited to: cleaning or replacement of the mass
spectrometer source, mass filters, or electron multiplier; or installing a different GC
column type. An IDL is instrument-specific and independent of sample matrices.
9.6.1 An IDL is determined for each compound as the concentration that produces an
average signal-to-noise ratio (S:N) of between 3:1 and 5:1 for at least three
replicate injections.
9.6.2 All documentation for the IDL determination shall be maintained at the
laboratory and provided to the agency or the data user upon request.
9.7 Method Detection Limit (MDL) Determination
Before any field samples are analyzed, laboratory MDLs should be determined for each
target analyte in appropriate reference matrices (i.e., reagent water, Ottawa sand, or clean
wipes), using the sample preparation and analytical procedures described in this protocol
for each specific matrix, and following the instructions and requirements described in 40
CFR Part 136, Appendix B.
9.7.1 The laboratory must use full method procedures to prepare and analyze at least
seven replicates.
9.7.2 Spike each replicate sample at concentrations of 1-5 times the IDL concentration
for each analyte and analyze the samples following protocol procedures. The
total volume of DCM added to water samples will be slightly greater than the 2
mL needed for extraction, and includes the volumes added for spiking target
compounds and surrogates.
9.7.3 To determine analyte MDLs, the following equation is applied to the analytical
results (Student's t-factor depends on the number of replicates used; a factor of
3.14 assumes seven replicates):
Eq. 2 MDL Determination
MDL = 3 .14 xsd
where:
sd = the standard deviation for the analytical results, and
3.14 = the Student's t-value for seven replicate samples
9.7.4 The MDL results calculated using the equation in Section 9.7.3 must meet the
following requirements as well as all other requirements specified in 40 CFR Part
136, Appendix B:
• MDL result must not be greater than the spiking level used for the MDL
determination.
• MDL result must not be less than 0.10 times the spiking level used for
the MDL determination.
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If either requirement is not met, the laboratory must adjust their spiking level
appropriately and repeat the MDL determination.
9.8 Quantitation Limit (QL) Determination
A QL determination is recommended for each laboratory/technician performing the
method for the first time, or in cases where new or repaired instrumentation is being used.
Laboratory QLs are determined by first assessing at least four samples containing
concentrations of target analytes at the levels of the lowest calibration standard, against
the criteria listed below. If any of these criteria are not met, samples are assessed at
concentrations of the next (second lowest) calibration standard. These criteria are
provided as guidance. If the criteria cannot be met, the laboratory should consult the
analytical data requestor to determine if the QL is sufficient to address project needs:
• Results from spikes at the QL should be above the MDL.
• The QL should be at or above the lowest calibration level.
• The QL should be at least two times the MDL.
• The RSD of results from spikes at the QL should be less than 30 %.
• The mean recovery of spikes at the QL should be within 50 - 150 %.
The following GC analytical conditions were used during laboratory studies
and are provided for guidance. Other conditions may be used, provided that
all technical acceptance criteria are met. Optimize GC conditions for analyte
separation and sensitivity. Once optimized, the same GC conditions must be
used for the analysis of all standards, samples, blanks, and MS/MSDs.
10.1.1.1 GC - Full-scan quadrupole
10.0 CALIBRATION AND STANDARDIZATION
10.1 Instrument Operating Conditions
10.1.1 GC
Initial column temperature:
Column temperature program:
40 °C for 3 minutes
40 - 150 °C at 10 °C/minute
150 - 280 °C at 25 °C/minute
280 °C; 10.8 minutes after the last
compound (triphenyl phosphate)
has eluted
250 °C
Final column temperature hold:
Inj ector temperature:
Injection mode:
Grob-type, splitless for 0.75
minutes
1.0 (iL
30 m X 0.25 mm ID (or 0.32 mm)
bonded phase silicon coated fused
silica capillary (see Section 6.4.2)
30 m X 0.25 mm X 0.25 (im
Helium at 32 cm/second
Sample injection volume:
GC column:
Column dimensions:
Carrier gas:
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10.1.1.2 GC-TOF
Initial oven temperature:
Column temperature program:
Final column temperature hold:
Inj ector temperature:
Injection mode:
Sample injection volume:
GC column:
Column dimensions:
Carrier gas:
55 °C for 0.5 minutes
20 °C/minute to 100 °C (0
minute), 40 °C/minute to 280 °C
280 °C (2.75 minutes)
250 °C
Grob-type, splitless
1.0 jaL
HP-5MS UI or equivalent
15m X 0.18mm X 0.18(im
Helium at 1.2 mL/minute
10.1.2 MS
The following MS analytical conditions were used during laboratory studies and
are provided for guidance. Other conditions may be used, provided that all
technical acceptance criteria are met. Optimize MS conditions for analyte
separation and sensitivity. Once optimized, the same MS conditions must be
used for the analysis of all standards, samples, blanks, and MS/MSDs.
10.1.2.1 MS - Full-scan quadrupole
MS transfer line temperature:
Source temperature:
MS quadrupole temperature:
Electron energy:
Scan range:
Ionization mode:
Scan time:
Library searching:
280 °C
230 °C or according to
manufacturer's specifications
150 °C
70 eV (nominal)
35 to 500 m/z
Electron Ionization (EI),
positive
3.15 scan/sec (minimum of 3
scans/second)
NIST 05 Mass Spectral Data
Base
10.1.2.2 MS-TOF
MS transfer line temperature:
Source temperature:
Electron energy:
Scan range:
Ionization mode:
Scan time:
295 °C
250 °C or according to
manufacturer's specifications
70 eV (nominal)
35 to 500 m/z
Electron Ionization (EI),
positive
15 scans/second
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
10.2 GC/MS Mass Calibration (Tuning) and Ion Abundance
10.2.1 Summary of GC/MS instrument performance check
The GC/MS system must be tuned to meet the manufacturer's specifications,
using a suitable calibration compound such as perfluoro-tri-«-butylamine (FC-43)
or perfluorokerosene (PFK). The mass calibration and resolution of the GC/MS
system are verified by the analysis of the instrument performance check solution
(Section 7.2.2.3). Prior to the analysis of any samples, including MS/MSDs,
blanks, or calibration standards, the laboratory must establish that the GC/MS
system meets the mass spectral ion abundance criteria for the instrument
performance check solution (Table 3, Section 17) containing DFTPP.
10.2.2 Frequency of GC/MS instrument performance check - The instrument
performance check solution must be injected once at the beginning of each
24-hour period, during which samples, blanks, or standards are to be
analyzed. The 24-hour period begins at the moment of injection of the
DFTPP solution. The time period ends after 24 hours have elapsed according
to the system clock.
10.2.3 GC/MS instrument performance check
The analysis of the instrument performance check solution may be performed as
an injection of 50 ng or less of DFTPP into the GC/MS or by adding a sufficient
amount of DFTPP to the calibration standards to result in an on-column amount
of 50 ng or less of DFTPP (Section 7.2.2.3) and analyzing the calibration
standard.
10.2.4 Technical acceptance criteria for GC/MS instrument performance check
10.2.4.1 The instrument performance check solution must be analyzed at the
frequency described in Section 10.2.2.
10.2.4.2 Abundance criteria are listed in Table 3, Section 17 for guidance.
The mass spectrum of DFTPP must be acquired in the following
manner: three scans (the peak apex scan and the scans immediately
preceding and following the apex) are acquired and averaged.
Background subtraction is required, and must be accomplished using
a single scan acquired no more than 20 scans prior to the elution of
DFTPP. The background subtraction should be designed only to
eliminate column bleed or instrument background ions. Do not
subtract part of the DFTPP peak.
Note 1: All subsequent standards, samples, MS/MSDs, and blanks
associated with a DFTPP analysis must use the identical GC/MS
instrument run conditions.
Note 2: The above tuning criteria are suggested when using DFTPP.
If alternative tuning methods are used, consult the method or
manufacturer notes for guidance on criteria.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
10.2.5 Corrective action for GC/MS instrument performance check
The following corrective actions are minimum procedures. The analyst may try
other corrective action procedures to meet criteria.
10.2.5.1 If the GC/MS instrument performance check technical acceptance
criteria are not met, re-tune the GC/MS system. It may be
necessary to perform maintenance to achieve the technical
acceptance criteria.
10.2.5.2 The instrument performance check technical acceptance criteria in
Section 10.2.4 must be met before any standards, samples, including
MS/MSDs, or required blanks are analyzed.
10.3 Initial Calibration
Prior to sample analysis and after instrument performance check technical acceptance
criteria have been met, each GC/MS system must be calibrated at a minimum of five
concentrations (Section 7.2.2.4.1 and Table 8, Section 17) to determine instrument
sensitivity and the linearity of GC/MS response for the target and surrogate compounds.
If the RSD criteria cannot be met, a linear or quadratic curve may be used. Each initial
calibration standard contains all the target compounds, surrogates, and internal standards.
10.3.1 Frequency of initial calibration
10.3.1.1 Each GC/MS should be calibrated whenever the laboratory takes
corrective action that might change or affect the initial calibration
criteria, or if the CCV technical acceptance criteria are not met.
10.3.1.2 If time remains in the 24-hour period after meeting initial calibration
acceptance criteria, samples may be analyzed. In this case, it is not
necessary to analyze an opening CCV standard prior to sample
analysis.
10.3.2 Procedure for initial calibration
10.3.2.1 Prepare at least five calibration standards containing the
detected target compounds and associated surrogates. Example
concentrations for the calibration standards are provided in
Section 7.2.2.4.1 and Table 8, Section 17.
10.3.2.2 Add a sufficient amount of internal standard solution (Section
7.2.2.5) to aliquots of calibration standards to result in 0.5 ng/(.iL of
each internal standard. Standards specified in Section 7.2.2.4
should permit most of the target compounds to have relative
retention times (RRTs) of approximately 0.60 to 1.70, using the
assignments of internal standards given in Table 4, Section 17.
10.3.2.3 Analyze each calibration standard by injecting 1.0 |_iL of standard.
The same injection volume must be used for all standards, samples,
and blanks.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
10.3.3 Calculations for initial calibration
10.3.3.1 Calculate the RRFs for each analyte and surrogate using Eq. 3 and
the primary characteristic ions found in Table 5, Section 17. Assign
target compounds and surrogates to internal standards according to
Table 4, Section 17. For internal standards, use the primary ion
listed in Table 5, Section 17 unless interferences are present (e.g.,
peak overlap, co-elution). Unless otherwise stated, the area response
of the primary characteristic ion is the quantitation ion.
Eq. 3 RRF Calculation
A C
RRF = —— x ——
A, Cx
where:
Ax = Area of the characteristic ion for the compound to be measured
(Table 5, Section 17)
Ais = Area of the characteristic ion for specific internal standard
(Table 5, Section 17)
Cis = Amount of the internal standard injected (ng)
Cx = Amount of the target compound or surrogate injected (ng)
10.3.3.2 The Mean RRF ( RRF) for the Initial Calibration RRFs and mean
RRFs must be calculated for all compounds. Calculate the %RSD of
the RRF values for the initial calibration.
10.3.4 Technical acceptance criteria for initial calibration
10.3.4.1 An initial calibration should be performed at the frequency described
in Section 10.3.1 on a GC/MS system meeting the instrument
performance check technical acceptance criteria (Section 10.2.4).
10.3.4.2 The RRF for each target compound and surrogate should be greater
than or equal to 0.01.
10.3.4.3 The % RSD of the RRFs over the initial calibration range for
each target compound and surrogate must be less than or equal to
20. If % RSD for a target analyte or surrogate cannot meet this
acceptance criterion, curve fitting by linear or quadratic regression
may be used provided the R2 value is greater than or equal to 0.99
(linear) or 0.995 (quadratic). Refer to Section 12.2.7 if linear
regression is used; refer to SW-846 Method 8000C (Reference
16.15) if quadratic curve fitting is needed. If regression curve
fitting is used, percent drift (PD), as calculated using Eq. 4a, Section
10.4.4.1 for each standard, should be less than or equal to 40.
10.3.5 Corrective action for initial calibration
The following corrective actions are minimum procedures. The analyst may try
other corrective action procedures to meet criteria.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
10.3.5.1 If technical acceptance criteria using at least one of the three optional
approaches to initial calibration (% RSD of the RRFs, linear
regression, or quadratic regression) are not met, inspect the system
for problems, take corrective actions, remake standards and re-
calibrate. If criteria are not met with re-calibration, remake the
calibration standards and repeat. If criteria still are not met, the
laboratory will flag all data associated with the calibration.
Note: If technical acceptance criteria for the initial calibration are
not met and the initial calibration contains more than five calibration
levels, the laboratory may remove calibration point(s) from either
extreme end of the calibration range and reassess the calibration.
Data points from within a calibration range must not be removed.
10.3.5.2 Initial calibration technical acceptance criteria must be met before
any samples, including MS/MSDs or required blanks, are analyzed
and reported without data qualification.
10.4 Continuing Calibration Verification (CCV)
10.4.1 Summary of CCV
Prior to the analysis of samples and after instrument performance check and
initial calibration technical acceptance criteria have been met, each GC/MS
system must be routinely checked by analyzing a CCV standard to ensure that the
instrument continues to meet the instrument sensitivity and linearity
requirements. The CCV standard contains all the target compounds, surrogates,
and internal standards.
10.4.2 Frequency of CCV - Each GC/MS used for analysis must be checked at the
beginning and at the end of each analytical batch of < 20 injections, excluding
instrument blanks. When subsequent analytical batches are run within a single
24-hour period, the closing CCV may be used as the opening CCV for a new
analytical batch, provided the closing CCV meets all technical acceptance criteria
for an opening CCV (see Section 10.4.5).
10.4.3 Procedure for CCV
10.4.3.1 Add a sufficient amount of internal standard solution (Section
7.2.2.5) to an aliquot of CCV standard to result in a
concentration of 0.5 ng/(.iL for both quadrupole and TOF
analyses.
10.4.3.2 Analyze the CCV standard by injecting 1.0 (.iL of standard.
10.4.4 Calculations for CCV
Calibration verification involves calculation of the percent drift (PD) (Eq. 4a)
or the percent difference of the RRFs between the initial calibration and each
subsequent CCV (Eq. 4b). The CCV approach will depend on the how the
initial calibration was performed. If a regression technique (linear or quadratic)
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
was used, then Eq.4a is used to determine a PD. If the RRF approach is used,
then Eq. 4b is used to determine the percent difference of the RRFs.
10.4.4.1 If regression techniques are used for initial calibration, the
CCV must be evaluated in terms of PD, which is calculated
using concentrations (see Eq. 4a).
Eq. 4a Percent Drift (PD) Calculation for CCV
Calculated Concentration-Theoretical Concentration
lJL) = xl00%
Theoretical Concentration
10.4.4.2 Calculate an RRF for each target compound and surrogate
using Eq. 3 in Section 10.3.3.1, and the primary quantitation
ions found in Table 5, Section 17. If regression techniques are
used for the initial calibration, proceed to Section 10.4.4.3.
10.4.4.3 Calculate the Percent Difference (% Difference) between the
RRF of the most recent initial calibration and the CCV RRF for
each target compound and surrogate using Eq. 4b.
Eq. 4b Relative Response Factor (RRF) % Difference
Calculation
RRF — RRF
%DifferenceRRF = c —L x 100
RRF RRF,
where:
RRF, = RRF from CCV standard.
RRFi= Mean RRF from the most recent initial calibration meeting
technical acceptance criteria.
10.4.5 Technical acceptance criteria for CCV
10.4.5.1 The CCV standard should be analyzed at or near the mid-point
concentration level, at the frequency described in Section 10.4.2, on
a GC/MS system meeting the instrument performance check and the
initial calibration technical acceptance criteria.
10.4.5.2 The RRF for each target compound and surrogate must be greater
than or equal to 0.01.
10.4.5.3 For the opening CCV, the PD or percent difference of RRFs for each
target compound should be within the range of ±40.
10.4.5.4 For the closing CCV, the PD or percent difference of RRFs for each
target compound should be within the range of ±50.
10.4.5.5 Excluding those ions in the solvent front, no quantitation ion may
saturate the detector.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
10.4.6 Corrective action for CCV
The following corrective actions are minimum procedures. The analyst may try
other corrective action procedures to meet criteria.
10.4.6.1 If the CCV technical acceptance criteria in Section 10.4.5 are not
met, recalibrate the GC/MS instrument according to Section 10.3.
10.4.6.2 CCV technical acceptance criteria should be met before any samples
MS/MSDs, or required blanks are analyzed. If CCV criteria are not
met, flag associated samples and blanks accordingly.
10.5 Instrument Blank
10.5.1 Summary of instrument blank
An instrument blank is comprised of DCM spiked with internal standards at the
same concentration used for associated samples. The purpose of the instrument
blank is to investigate the impact of carry-over.
10.5.2 Frequency of instrument blank
An instrument blank is recommended for analysis following suspected carry-over
or during analysis of samples containing suspected high concentrations.
10.5.3 Procedure for instrument blank analysis
Add sufficient amount of internal standard solution (Section 7.2.2.5) to an aliquot
of the solvent used to prepare calibration standards and sample extracts to result
in a concentration of 0.5 ng/(.iL. Analyze each instrument blank by injecting a
volume of 1.0 (iL.
10.5.4 Calculations for instrument blank
Calculate the concentrations of any observed target analyte using Eq. 6 (Section
12.2.6.1), setting Vt, V0, and dilution factor (DF) all equal to 1.
10.5.5 Technical acceptance criteria for instrument blank
If an instrument blank is analyzed, the concentration of all target analytes in the
instrument blank should be less than the concentration of the target analytes in
the low calibration standard. The area response of the internal standards should
be within 50 - 150 % of the associated CCV or mid-level concentration of the
initial calibration.
10.5.6 Corrective action for instrument blank
If an instrument blank is analyzed and the instrument blank technical acceptance
criteria are not met, analyze an additional instrument blank. If the problem
persists, inspect the system for problems and take corrective actions to achieve
the acceptance criteria. Instrument blank technical acceptance criteria should be
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
met before samples are analyzed. Samples that are analyzed with corresponding
instrument blanks that do not meet the instrument blank criteria should be
reanalyzed, or the corresponding data should be flagged.
11.0 ANALYTICAL PROCEDURE
11.1 Sample Preparation - General
11.1.1 If less than the specified sample amount is received, the laboratory should
use a reduced sample size for the analysis and adjust the calculation
accordingly. For the purposes of this method, it is recommended that some
sample be retained (if possible) for potential future evidentiary use.
11.1.2 If multi-phase samples (e.g., a two-phase liquid sample, oily, sludge/sandy soil
sample) are received by the laboratory, the laboratory shall contact the agency
requesting the analyses to apprise them of the type of sample received. If some
or all phases of the sample are amenable to analysis, the agency may require the
laboratory to do any of the following:
• Mix the sample and analyze an aliquot from the homogenized sample.
• Separate the phases of the sample and analyze each phase separately.
• Separate the phases and analyze one or more of the phases, but not all of the
phases.
• Do not analyze the sample.
11.2 Preparation of Water Samples Using Microscale Extraction
11.2.1 Approximately 35 mL of a water sample is required for this extraction. If
extraction is to be performed in the sample receipt vial, remove any excess
sample such that a total sample volume of 35 mL is retained and recap the vial.
Weigh the capped vial. Record the weight to the nearest 0.1 gram. Alternatively,
35.0 mL of sample can be transferred by pipette into the vial and the weighing
step can be eliminated.
Note: The conical bottoms of centrifuge vials allow the DCM layer to be
removed more easily than the VOA vials.
11.2.2 For GC/MS full-scan and TOF analysis, spike 1.0 |ig of each surrogate (add 40
(.iL if prepared as in Section 7.2.2.1) into each sample, blank, etc. The final
volume of the extracts will be 2.0 mL; therefore, the concentration of the
surrogates in the extract is expected to be 0.50 (ig/mL.
11.2.3 Add -8.8 grams of sodium chloride and shake vigorously, or vortex each vial for
two minutes or until the sodium chloride dissolves completely.
11.2.4 Add 2.00 mL of DCM, using a Class A volumetric pipette or syringe. Cap
tightly and agitate the contents vigorously for approximately two minutes, either
by hand or using a vortex mixer or shaker table.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
11.2.5 Briefly allow the phases to settle by gravity for ~ five minutes. Centrifugation
[three minutes at 500 revolutions per minute (rpm)] is strongly recommended to
facilitate separation of the phases and affords a greater recovery of the final
sample extract. CAUTION: The maximum safe handling speed of each
centrifuge will depend, in part, on the vials used and should be determined prior
to use.
11.2.6 Using a 2. 0-mL syringe or pipette, transfer approximately 1.0 mL (or as much as
possible) of the DCM (lower) layer to a 2-mL or 4-mL vial with a PTFE-lined
screw cap, taking precautions to exclude any water from the syringe or pipette.
Add a small amount (-50 mg) of anhydrous sodium sulfate to the vial, cap the
vial, and shake vigorously or vortex for two minutes. Make sure that the extract
is sufficiently dry and that some of the sodium sulfate added is free-flowing (i.e.,
not clumped).
11.2.7 Using a 1.0 mL syringe or pipette, transfer 1.0 mL (or a known volume, if less
than 1.0 mL of extract is collected) of the dried extract to a 2.0-mL vial (or
autosampler vial insert) with a PTFE-lined screw cap. Cap the vial.
Note: If stored prior to analysis, extracts must be protected from light and
stored at < 6 °C (Section 8.3).
11.2.8 Discard the remaining contents of the VOA vials according to laboratory waste
disposal guidelines. Shake off the last few drops with short, brisk wrist
movements. If needed, rinse the vial with a water-soluble solvent to ensure that
the extraction solvent is removed. If the vial was pre-weighed (i.e., exact sample
volume used in Section 11.2.1 is unknown), reweigh the capped vial, and record
the weight to the nearest 0.1 gram. The difference between this weight and the
weight determined in Section 11.2.1 is equal to the volume of water extracted, in
milliliters. As the density of water is 1.00 g/mL (at 20 °C), the volume of water
extracted, in milliliters, may be assumed to be equal to the weight of water
extracted.
11.2.9 Proceed to Section 11.6 for sample analysis.
11.3 Preparation of Solid Samples Using Microscale Extraction
Note: The following procedures have been evaluated in a multi-laboratory study using
Ottawa sand and dried soils and have not been evaluated for field samples. Laboratory
results are provided in Section 17.0.
11.3.1 Decant and discard any water layer. Mix samples thoroughly. Discard any
foreign objects such as sticks, leaves, and rocks.
11.3.2 pH determination - If pH determination is requested, transfer a 1:1 (w:w)
ratio of sample:water to a 100-mL beaker and stir for one hour. Determine
the pH of the sample with a pH meter or wide-range pH paper, and document
this value in the data narrative. Discard this portion of the sample.
11.3.3 Percent moisture determination - If percent moisture determination is requested,
immediately after weighing the sample for extraction, weigh 5-10 grams of the
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
sample into a tared vial. Dry overnight at 103 - 105 °C and cool in a
desiccator before weighing. Determine the percent moisture (% Moisture)
using Eq. 5. CAUTION: Due to the high toxicity associated with CWAs
vaporized during this procedure, percent moisture determinations should be
performed only in an oven with appropriate engineering controls.
Eq. 5 Percent Moisture Calculation
„ , . grams of wet sample-grams of dry sample ,
% Moisture = - — x 100
grams of wet sample
11.3.4 Extraction of G-agents and HD from soil samples
11.3.4.1 Weigh 10 g of sample into a tared extraction vial (i.e., 40-mL VOA
vial). Wipe the lip and threads of the vial with a clean cloth (e.g.,
Kimwipe® [Kimberly-Clark Professional, Roswell, GA] or
equivalent). Record weight to the nearest 0.01 gram.
11.3.4.2 For full-scan quadrupole and TOF MS analyses, add 0.5 |ig of each
surrogate standard compound (add 20 |_iL if prepared as in Section
7.2.2.1) in DCM to the vial. The final volume of the extract is 1.0
mL; therefore, the concentration of the surrogates in the extract is
expected to be 0.5 (ig/mL.
11.3.4.3 Add approximately 2.5 grams of anhydrous sodium sulfate to a pre-
cleaned 40-mL VOA vial that has a PTFE-lined screw cap. Also add
5-10 pre-cleaned glass beads. Mix the solids together until
homogenized using the glass beads and/or a metal spatula. Break up
any chunks with a metal spatula, working quickly but gently.
Note: Alternatively, the anhydrous sulfate and glass beads may be
added to the extraction vial prior to the addition of sample. If added
prior to the sample, the anhydrous sulfate and glass beads must be
included in the tared weight of the vial.
11.3.4.4 Add 25 mL of DCM to the vial, and cap tightly.
11.3.4.5 Shake the vial vigorously or vortex for approximately 20 seconds or
until the slurry is free-flowing. Break up any chunks with a metal
spatula, working quickly but gently. Cap immediately when
finished. Add more sodium sulfate and manually mix as necessary
to produce a free flowing, finely divided slurry.
11.3.4.6 Extract the sample by agitating for approximately 15 minutes using a
shaker table or sonicator.
Note: Sonication at high power should be avoided for soils having
high silt content; the resulting fine particles can create problems with
filtering and extracting solvent from the sample.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
11.3.4.7 Vortex each sample for 30 seconds. Add ~ 1 gram anhydrous sodium
sulfate to each sample. Cap and briefly shake or vortex to ensure
thorough mixing. Allow the solids to settle or centrifuge for 1-2
minutes at 1000 rpm. If the solid is still unsettled, repeat the
centrifuge step, but increase the speed to 2500 rpm. CAUTION:
The maximum safe handling speed of each centrifuge will depend, in
part, on the vials used and should be determined prior to use.
Repeat until the solid is completely settled. If, after repeating the
centrifuging steps several times, the solid is still unsettled, proceed to
Section 11.3.4.8. Once the solid has settled, decant or pipette the
solvent layer into a pre-cleaned, 40-mL VOA vial with PTFE-lined
screw cap and proceed to Section 11.3.4.9.
Note: For solids that have difficulty settling, pipetting is
recommended.
11.3.4.8 If solids do not settle by centrifugation (Section 11.3.4.7), filter by
placing a small plug of glass wool into a small glass funnel. Add
anhydrous sodium sulfate to cover the glass wool plug. Wet the
sodium sulfate thoroughly with DCM, and decant the sample solvent
layer into the funnel. Rinse the sodium sulfate with approximately 2
- 3 mL of DCM as soon as the surface is exposed, not allowing it to
dry.
11.3.4.9 Proceed to Section 11.5 for extract concentration. Once the extract is
concentrated, proceed to Section 11.6 for analysis.
11.4 Preparation of Wipe Samples by Microscale Extraction
11.4.1 Place the wipe into an extraction vial (i.e., 40-mL VOA vial). For GC/MS full-
scan and TOF analyses, add 0.5 |ig of each surrogate standard compound (add 20
|_iL if prepared as in Section 7.2.2.1) in DCM directly onto the wipe. The final
volume of the extract is 1.0 mL; therefore, the concentration of the surrogates in
the extract is expected to be 0.5 (ig/mL.
11.4.2 Add 15 mL of DCM to the vial and cap tightly.
11.4.3 Extract the sample by agitating for approximately 15 minutes, using a shaker
table or sonicator.
11.4.4 Remove the vials from the sonicator or shaker table, shake briefly by hand and
allow the solvent layer to settle. Transfer the solvent layer by pipette into a pre-
cleaned, 40-mL, clear glass vial with PTFE-lined screw cap.
11.4.5 Proceed to Section 11.5 for extract concentration. Once the extract is
concentrated, proceed to Section 11.6 for analysis.
11.5 Final Concentration of Extract - Nitrogen Evaporation Technique (RapidVap® or
equivalent) for solid and wipe samples.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
11.5.1 Nitrogen evaporation technique - If using a RapidVap® or TurboVap® N2 system
(Biotage, LLC, Charlotte, NC), follow the manufacturer's guidelines. For the
RapidVap®, a temperature of 40 °C is recommended. If using a water bath,
place the vial in a warm water bath (30-40 °C recommended) and evaporate the
solvent volume to just below 1.0 mL by blowing a gentle stream of clean, dry
nitrogen above the extract. It is recommended that the internal wall of the vial
be rinsed down several times with DCM during the operation. If using a
RapidVap® the solvent rinse, at a minimum, be done during final adjustment of
the extract volume. During evaporation, the tube solvent level must be kept
below the water level of the bath. The extract must never be allowed to become
dry. Adjust the final volume to 1.0 mL with the same solvent used for
extraction. Transfer the extract to a 2-mL autosampler vial, cap, and label the
vial. Store at 4 °C (± 2 °C). CAUTION: Gas lines from the gas source to the
evaporation apparatus should be stainless steel, copper, or PTFE tubing. With
the exception of PTFE, plastic tubing must not be used between the carbon trap
and the sample since it can introduce interferences.
11.5.2 Final extract volumes -The final extract volumes in Sections 11.5.2.1 -
11.5.2.4 are recommended volumes.
11.5.2.1 Water/Liquid -As concentration of the sample extract is not needed
for these sample matrices, no adjustment of the final extract volume
is required. The nominal volume of DCM added to water samples is
2.0 mL. Target compound and surrogate spiking solutions also
contain DCM; therefore, the total volume of DCM added may be
slightly greater than 2.0 mL. The actual total volume of DCM added
should be used in the calculations in Section 12.2.
11.5.2.2 Solid -Adjust the extract to a final volume of 1.0 mL with DCM.
11.5.2.3 Wipe -Adjust the extract to a final volume of 1.0 mL with DCM.
11.5.2.4 If extracts are stored prior to analysis, transfer the extract to a
PTFE-lined screw-cap vial (approximately 2.0 mL), label the
vial, and store at < 6 °C.
11.6 Extract Analysis by GC/MS
11.6.1 Analyze extracts only after the GC/MS system has met the instrument
performance check (Section 10.2.3), initial calibration (Section 10.3.4 and
10.3.5), and CCV technical acceptance requirements (Section 10.4.5). The
same instrument conditions used for calibration must be used for the
analysis of samples.
11.6.2 Add internal standard solution (Section 7.2.2.5) to an accurately measured
aliquot of each extract, cap the vial, and invert several times to mix. For full-
scan quadrupole or TOF MS analyses, add a sufficient amount of internal
standard solution to result in 0.5 ng/(.iL concentration of each internal standard.
11.6.3 If extracts are to be diluted, add internal standards after dilution. Internal
standards must be added to maintain the required 0.5 ng/(.iL (for both full-
scan quadrupole and TOF) of each internal standard in the extract volume.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
11.6.4 Inject 1.0 |_iL of the extract into the GC/MS.
Note: The same injection volume used for calibration standards must be used
for extracts.
11.6.5 Sample extract dilution
11.6.5.1 If the response of any target compound in any sample extract
exceeds the response of the same target compound in the high
standard of the initial calibration, that extract must be diluted. Add
additional internal standard solution such that the concentration in
the diluted extract is 0.5 ng/(.iL for each internal standard, and
analyze the diluted extract.
11.6.5.2 Use the results of the original analysis to determine the approximate
dilution factor (DF) required for the largest analyte peak to fall
within the initial calibration range. The DF chosen must keep the
response of the largest peak for a target compound in the upper half
of the calibration range of the instrument.
12.0 CALCULATIONS AND DATA ANALYSIS
12.1 Qualitative Identification of Target Compounds
12.1.1 The target compounds should be identified by an analyst competent in the
interpretation of mass spectra by comparison of the sample mass spectrum to
the mass spectrum of the standard of the suspected compound. Two criteria
must be satisfied to verify the identifications:
1) Elution of the sample analyte within the GC RRT unit window
established from the 24-hour calibration standard; and
2) Correspondence of the sample analyte and calibration standard
component mass spectra.
12.1.2 For establishing correspondence of the GC RRT, the sample component must
compare within ± 0.06 RRT units of the standard component. For samples
analyzed during the same 24-hour time period as the initial calibration
standards, compare the analyte RTs to those from the midpoint initial
calibration standard. Otherwise, use the corresponding CCV standard. If
coelution of interfering components prohibits accurate assignment of the
sample component RRT from the total ion chromatogram, the RRT should be
assigned by using EICPs for ions unique to the component of interest (see
Table 5, Section 17 for appropriate characteristic ions, surrogates and internal
standards).
12.1.3 For comparison of standard and sample component mass spectra, mass spectra
obtained from a calibration standard at a concentration of the target analyte
closest to the concentration of the analyte in the sample are required. Once
obtained, these standard spectra may be used for identification purposes only
if the GC/MS meets the DFTPP instrument performance requirements (see
Section 10.2 for instrument performance check requirements).
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
12.1.4 For TOF MS and full-scan quadrupole MS analyses, all ions present in the
standard mass spectrum at a relative intensity greater than 10 % (the most
abundant ion in the spectrum equaling 100 %) must be present in the sample
spectrum. The relative intensities of ions specified in Table 5, Section 17 must
agree within ± 20 % between the standard and sample spectra (e.g., for an ion
with an abundance of 50 % in the standard spectra, the corresponding sample
ion abundance must be between 30 - 70 %). Ions greater than 10 % in the
sample spectrum, but not present in the standard spectrum, must be considered
and accounted for by the analyst making the comparison. The verification
process should favor false positives. All compounds meeting the identification
criteria must be reported with their spectra. When target compounds are below
QLs but the spectrum meets the identification criteria, report the concentration
with a "J". For example, if the QL is 5.0 |ig/L and concentration of 3.0 |ig/L is
calculated, report as "3.0 J".
For TOF analysis, signals for the quantitation ions in Table 5, Section 17 must
be present and must maximize within a period of two seconds. The S:N for
the GC peak at each ion must be greater than or equal to 2.5 for each target
compound and surrogate detected in a sample extract, and greater than or
equal to 10 for all target compounds and surrogates in the CCV standard.
12.1.5 If a compound cannot be verified by all of the spectral identification criteria in
Sections 12.1.1-12.1.4 but in the technical judgment of the mass spectral
interpretation specialist the identification is correct, the laboratory should
report the identification and proceed with quantitation.
12.2 Data Analysis and Calculations of Target Compounds
12.2.1 Target compounds identified are quantitated by the internal standard method.
The internal standard used shall be the one assigned to that analyte for
quantitation (Table 4, Section 17).
12.2.2 Situations are expected to arise when the automated quantitation procedures in
the GC/MS software provide inappropriate quantitations. These situations
normally occur when there is compound coelution, baseline noise, or matrix
interferences. In these circumstances, the laboratory must perform a manual
quantitation. Manual integrations are performed by integrating the area of the
quantitation ion of the compound. This integration should include only the
area attributable to the specific target compound. The area integrated should
not include baseline background noise. The area integrated must not extend
past the point where the sides of the peak intersect with the baseline noise.
Manual integration is not to be used solely to meet QC criteria, nor is it to be
used as a substitute for corrective action on the chromatographic system.
12.2.3 In some instances, the data system report may have been edited or manual
integration or quantitation may have been performed. In all such instances, the
GC/MS operator should identify such edits or manual procedures by initialing
and dating the changes made to the report, and include the integration scan
range. The GC/MS operator should also mark each integrated area on the
quantitation report.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
12.2.4 The requirements listed in Sections 12.2.1 - 12.2.3 apply to all standards,
samples, and blanks.
12.2.5 The RRF from the initial calibration is used to calculate the concentration in
the sample. Secondary ion quantitation is allowed only when there are sample
interferences with the primary ion. If linear regression is used, a regression
curve should be used to calculate the concentration in samples. Refer to
Section 12.2.7 for calculating sample concentration using linear regression.
12.2.6 Calculate the concentration in the sample using the RRF and Eqs. 6-8.
12.2.6.1 Water
Eq. 6 Concentration of Water Sample
; Av M Ij ii Vt m' 3F i
CoBcentrstionug'L =
where:
A, = Area of the characteristic ion for the target compound
AIS = Area of the characteristic ion for the internal standard
Is = Amount of internal standard injected in ng
V0 = Volume of water extracted in m L
V, = Volume of extract injected in |.iL
V, = Volume of the extract in |.iL
(Extraction of water samples does not include
concentration; V, is equal to the sum of the volumes of
solvent added for extraction and the addition of surrogates,
and any spiked target compounds.)
RRF = Mean RRF determined from the initial calibration
standard
DF = Dilution Factor. If no dilution is performed, DF = 1.0.
The DF for analysis of water samples is defined as:
r^r, ill, most conc. extract used to make dilution + liL clean solvent
DF = L
(iL most conc. extract used to make dilution
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
12.2.6.2 Solid
Eq. 7 Concentration of Solid Sample
Eq. 7 includes a %moisture factor (D) for those cases when data are
to be reported on the basis of dry sample weight. In cases where
results are reported in terms of sample weight, this factor is deleted
from the equation.
(A )(I XV )(DF)
Concentration iig/Kg (Dry weight basis) =
(Ais)(Vi)(RRF)(Ws)(D)
where:
Ax, Is, AIS, V, and RRF are as given for water, above.
V, = Volume of concentrated extract in |.iL
^ 100 - %Moisture
100
%Moisture is as given in EQ. 5
Ws = Weight of sample extracted in grams
RRF = Mean RRF determined from the initial calibration
standard
DF = Dilution Factor
12.2.6.3 Wipes
Eq. 8 Concentration of Wipe Sample
I Ax I; It i, V-I'D? i
C"™«re/c-5= !,4ni:r;:iAre,; [isF;
where:
A, = area response for the compound to be measured, counts AIS
= area response for the internal standard, counts
Is amount of internal standard, |.ig
RRF = the mean RRF from the most recent initial calibration,
dimensionless
Area = area of surface wiped, cm2. If concentration is reported
as ng/wipe, area = 1 wipe.
V, = volume of concentrated extract, |.iL
V, = volume of extract injected, |.iL
DF = dilution factor for the extract. If there was no dilution, DF
equals 1. If the sample was diluted, DF is greater than 1.
12.2.7 Calculate the concentration in the sample using linear regression.
The following procedure is used to calculate analyte concentrations using a linear
regression calibration curve. Refer to SW-846 Method 8000C (Reference 16.15)
if calibration curves were determined using quadratic equations.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
12.2.7.1 Set y = (Peak Area of Target/Peak Area of Internal Standard) and x
= (Theoretical Concentration of Target/Theoretical Concentration of
Internal Standard).
12.2.7.2 Plot (Peak Area of Target/Peak Area of Internal Standard [Y-axis])
vs. (Theoretical Concentration of Target/Theoretical Concentration
of Internal Standard).
12.2.7.3 Determine the slope of the line (m) and the y-intercept (b).
12.2.7.4 Rearrange the line equation to solve for x; x = (y-b)/m.
12.2.7.5 Multiply x by the concentration of the internal standard to get
concentration of target anayte in extract.
12.2.7.6 Multiply the concentration of target analyte in the extract by the
extract volume, and divide by the sample volume to get
concentration of target analyte in sample.
12.2.8 Adjusted QL calculations
Adjusted QLs are used in situations when the laboratory may not have a sample
size that is sufficient for the method as written, or if the prescribed extract
volume was not used or recovered.
12.2.8.1 Water samples
Eq. 9 Aqueous Adjusted QL
Adjusted QL = Method QL x
(V0)(VC)
where:
Vt, DF, and V0 are as given in Eq. 6.
Vx = Recommended method sample volume (35 mL)
Vc = Recommended method concentrated extract volume (2000 jxL)
12.2.8.2 Solid samples
Eq. 10 Solid Adjusted QL
Adjusted QL = Method QL x (Wx)(Vt)(DF)
(WS)(VC)(D)
where:
Vt and DF are as given in Eq. 6.
Ws and D are as given in Eq. 7.
Wx = Recommended method sample weight (10 grams)
Vc = Recommended method concentrated extract volume (1000 jxL)
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
12.2.9 Surrogate recoveries
12.2.9.1 Calculate surrogate recoveries for all samples, blanks, and
MS/MSDs. Determine if recovery is within limits (Table 7, Section
17).
12.2.9.2 Calculate the concentrations of the surrogates using the same
equations as used for the target compounds. Calculate the
recovery of each surrogate using Eq. 11.
Eq. 11 Percent Recovery
Cs
%Recovery = %R = — x 100
where:
Cs = Measured concentration of the spiked sample aliquot.
Cn = Nominal (theoretical) concentration increase that results from
spiking the sample, or the nominal concentration of the spiked
aliquot (for LCS).
12.3 Technical Acceptance Criteria for Sample Analysis
12.3.1 The samples must be analyzed on a GC/MS system meeting the instrument
performance check, initial calibration, CCV, and blank technical acceptance
criteria.
12.3.2 The sample must be extracted and analyzed within the technical holding times.
12.3.3 The sample must have an associated method blank meeting the blank technical
acceptance criteria.
12.3.4 The percent recoveries of the surrogates in a sample should be within the
recovery limits listed in Table 7, Section 17 (see Table 4, Section 17 for analyte
specific surrogates). The surrogate recovery requirements do not apply to
samples that have been diluted.
12.3.5 The instrumental response (EICP area) for each of the internal standards in the
sample must be within the range of 50.0 - 200 % of the response of the internal
standard in the most recent CCV standard analysis.
12.3.6 The RT shift for each internal standard must be within ± 0.50 minute (30
seconds) between the sample and the most recent CCV standard analysis.
12.3.7 Excluding those ions in the solvent front, no ion may saturate the detector. If a
target compound concentration exceeds the upper limit of the initial calibration
range, a more dilute aliquot of the sample extract must also be analyzed.
12.4 Corrective Action for Sample Analysis
12.4.1 The sample technical acceptance criteria must be met before data are reported. If
the corrective actions described in this section did not solve the problem, all
associated sample and blank data must be flagged accordingly.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
12.4.2 Corrective action for failure to meet instrument performance checks and initial
calibration and CCV must be completed before the analysis of samples. If the
corrective actions described in Sections 10.2.5 (for instrument performance
check), 10.3.5 (for initial calibration), or 10.4.6 (for CCV) did not solve the
problem, all associated sample and blank data must be flagged accordingly.
12.4.3 Corrective Action for Surrogate Recoveries that Fail to Meet Their Acceptance
Criteria (Section 12.3.4 and Table 7, Section 17).
12.4.3.1 If the surrogate recoveries in a sample fail to meet the acceptance
criteria specified in Section 12.3.4, check calculations, sample
preparation logs, surrogate standard spiking solutions, and the
instrument operation.
12.4.3.2 If the above actions do not correct the problem, then the problem
might be due to a sample matrix effect. To determine if there was a
matrix effect, take the following corrective actions:
12.4.3.2.1 Reextract (if possible) and reanalyze the sample.
Note: Samples with corresponding MS and MSDs
should be re-extracted and reanalyzed only if
surrogate recoveries in the sample were
considered unacceptable, and the surrogate
recoveries met the acceptance criteria in both the
corresponding MS and MSP.
12.4.3.2.2 If surrogate recoveries meet acceptance criteria in the
reextracted/reanalyzed sample, the problem was within
the laboratory's control.
12.4.3.2.3 If surrogate recoveries are outside the acceptance
criteria in the reanalysis, flag the sample data for the
associated target compounds and submit data from
both analyses. Distinguish between the initial analysis
and the reanalysis on all data.
12.4.4 Corrective action for internal standard compound responses and/or RTs that fail
to meet the acceptance criteria (Sections 12.3.5 and 12.3.6)
12.4.4.1 If the internal standards in a sample fail to meet their acceptance
criteria, check calculations, internal standard solutions, and
instrument operation.
12.4.4.2 If the above actions do not correct the problem, then the problem
might be due to a sample matrix effect. To determine if there was
matrix effect, take the following corrective action steps:
12.4.4.2.1 Reanalyze the sample extract.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Note: Samples with corresponding MS and MSDs
should be re-extracted and reanalyzed only if
internal standard recoveries in the sample were
considered unacceptable, and the internal standard
recoveries met the acceptance criteria in both the
corresponding MS and MSP.
12.4.4.2.2 If the internal standard responses and RTs meet
acceptance criteria in the reanalyzed sample extract, then
the problem was within the laboratory's control.
12.4.4.2.3 If the internal standard responses and RTs do not meet
acceptance criteria in the reanalyzed sample extract, flag
the results of the associated sample.
12.4.4.2.4 Submit data from both analyses. Distinguish between
the initial analysis and the reanalysis on all data.
13.0 ANALYTICAL PROCEDURE PERFORMANCE
Performance of this protocol was evaluated in multiple laboratories for measurement of the target
analytes in water, soil, and wipes. Resulting IDLs and MDLs from the multi-laboratory
evaluation are listed in Table 1, Section 17. Multi-laboratory results of reference samples spiked
at levels corresponding to laboratory low-calibration standards are provided in Tables 2a - 2c
(Section 17). Precision (as RPD and RSD) and bias (as % Recovery) results based on multi-
laboratory data are provided in Table 6 (Section 17). Additional laboratory data for real world
samples are provided in Section 17 Tables 9a - 9b (multi-laboratory data for groundwater and
drinking water), and 10a - 10b (multi-laboratory data for Virginia-a and ASTM soils).
Characterization data for the samples used during the multi-laboratory study are provided in
Tables 11 and 12, Section 17. Figures 1 and 2 provide example chromatograms generated during
a multi-laboratory study using full-scan quadrupole MS and TOF MS, respectively.
14.0 POLLUTION PREVENTION
14.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity
and/or toxicity of waste at the point of generation. Numerous opportunities for pollution
prevention exist in laboratory operation. EPA has established a preferred hierarchy of
environmental management techniques that places pollution prevention as the option of
first choice. Whenever feasible, laboratory personnel should use pollution prevention
techniques to address their waste generation. When wastes cannot be feasibly reduced at
the source, the Agency recommends recycling as the next best option.
14.2 For information about pollution prevention that might be applicable to laboratories and
research institutions consult Less is Better: Laboratory Chemical Management for Waste
Reduction, available from the American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th St., N.W. Washington, D.C. 20036, (202) 872-
4477.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
15.0 WASTE MANAGEMENT
EPA requires that laboratory waste management practices be conducted in a manner consistent
with all applicable rules and regulations. The Agency urges laboratories to protect the air, water,
and land by minimizing and controlling all releases from hoods and bench operations, complying
with the letter and spirit of any sewer discharge permits and regulations, and by complying with
all solid and hazardous waste regulations, particularly the hazardous waste identification rules
and land disposal restrictions. For further information on waste management, consult The Waste
Management Manual for Laboratory Personnel, available from the American Chemical Society
at the address listed in Section 14.2.
Note: It is strongly recommended that all glassware and waste be decontaminated with bleach
containing active chlorine at a concentration of at least 5 %, for at least six hours to provide
effective decontamination of CWAs. Chemical agent decontamination procedures and inventory
records should be consistent with the laboratory's Chemical Hygiene Plan for CWAs.
16.0 REFERENCES
16.1 U.S. Environmental Protection Agency. Selected Analytical Methods for
Environmental Remediation and Recovery (SAM) - 2012. EPA/600/R-12/555.
Cincinnati, OH: U.S. Environmental Protection Agency, Office of Research and
Development, http://www.epa.gov/homeland-securitv-research/sam (accessed
05/31/2016).
16.2 U.S. Environmental Protection Agency. Semivolatile Organic Compounds by Gas
Chromatography/Mass Spectrometry (GC/MS). SW-846 Method 8270D, Revision 4.
February 2007. [In: Test Methods for Evaluating Solid Waste, Physical/Chemical
Methods. EPA Publication SW-846. Washington DC: U.S. Environmental Protection
Agency, Office of Land and Emergency Management (formerly, Office of Solid
Waste and Emergency Response).]
16.3 U.S. Environmental Protection Agency. Organic Compounds in Water by
Microextraction. SW-846 Method 3511, Revision 0. November 2002. Washington
DC: U.S. Environmental Protection Agency, Office of Land and Emergency
Management.
16.4 U.S. Environmental Protection Agency. Microscale Solvent Extraction. SW-
846 Method 3570, Revision 0. November 2002. Washington DC:
U.S. Environmental Protection Agency, Office of Emergency
Management.
16.5 U.S. Environmental Protection Agency. Tetra- through Octa-ChlorinatedDioxins and
Furans by Isotope Dilution HRGC/HRMS. Method 1613. October 1994. Washington DC:
U.S. Environmental Protection Agency, Office of Water.
16.6 ASTM. 2011. Method D1193-06. Standard Specification for Reagent Water. West
Conshohocken, PA: ASTM International, http://www.astm.org/ (accessed 05/31/2016)
16.7 U.S. Army, Marine Corps, Navy and Air Force. Potential Military Chemical/Biological
43
September 2016
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Agents and Compounds. Washington DC: Headquarters, U.S. Dept. of the Army.
January 2005. http://fas.org/irp/doddir/army/fm3-ll-9.pdf (accessed 05/23/2016)
16.8 U.S. Environmental Protection Agency. Semivolatile Organic Compounds in Drinking
Water by Solid-Phase Extraction and Capillary Column Gas Chromatography/Mass
Spectrometry (GC/MS), Version 1.0. Method 525.3. Cincinnati, OH: U.S. Environmental
Protection Agency, Office of Research and Development. EPA/600/R-12/010. February
2012.
16.9 U.S. Environmental Protection Agency. Method 1668C: ChlorinatedBiphenyl Congeners
in Water, Soil, Sediment, Biosolids, and Tissue by HRGC/HRMS. Method 1668C. April
2010. Washington DC: U.S. Environmental Protection Agency, Office of Water. EPA-
820-R-10-005.
16.10 U.S. Environmental Protection Agency. "Organic Analytes." Chapter 4 in Test Methods
for Evaluating Solid Waste, Physical/Chemical Methods. EPA publication SW-846.
Washington DC: U.S. Environmental Protection Agency, Office of Land and Emergency
Management..
16.11 U.S. Environmental Protection Agency. Stability Study for Ultra-Dilute Chemical
Warfare Agent Standards. Cincinnati, OH: U.S. Environmental Protection Agency,
Office of Research and Development, National Homeland Security Research Center.
EPA 600/R-13/044. May 2013.
16.12 U.S. Environmental Protection Agency. Analytical Standards Requirements. Exhibit E,
Section 4 [In: EPA Contract Laboratory Program Statement of Work for Organic
Superfund Methods Multi-Media, Multi-Concentration SOM02.3, September 2015.
https://www.epa.gov/clp/epa-contract-laboratory-program-statement-work-organic-
superfund-methods-multi-media-multi-0 (accessed 05/23/2016).]
16.13 U.S. Environmental Protection Agency. Determination of Organic Compounds in
Drinking Water by Liquid-Solid Extraction and Capillary Column Gas
Chromatography/Mass Spectrometry, Method 525.2, Revision 2.0. 1995. Cincinnati, OH:
U.S. Environmental Protection Agency, Office of Research and Development.
16.14 Superfund Analytical Services/Contract Laboratory Program (CLP) Multi-Media, Multi-
Concentration Organics Analysis, SOM02.3. September 2015. Exhibit D: Analytical
Method for the Analysis of Semivolatile Organic Compounds.
http://www.epa.gov/sites/production/files/2015-10/documents/som23d.pdf (accessed
05/31/16).
16.15 U.S. Environmental Protection Agency. Determinative Chromatographic Separations.
Revision 3. SW-846 Method 8000C. March 2003. Washington DC: U.S. Environmental
Protection Agency, Office of Land and Emergency Management.
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
17.0 TABLES and FIGURES
Table 1.
Instrument Detection Limits (IDLs) and Method Detection Limits (MDLs)
Based on Multi-Laboratory Evaluation
Note: IDLs and MDLs were determined in 3-6 laboratories during a multi-laboratory study. IDLs were determined as the concentration necessary
to achieve a S:N ratio of at least 3:1. MDLs were determined following Section 9.7 of this protocol, using samples spiked at levels corresponding
to the lowest calibration standards.
Analyte
Full-Scan Quadrupole MS
Full-Scan TOF MS
Reagent Water
#
Labs
IDL Range
(ng/pL)
S:N1 Range
MDL Range
(H9/L)
Pooled MDL2
(H9'L)
#
Labs
MDL Range
(H9'L)
Pooled
MDL2
(H9/L)
Cyclohexyl sarin
6
0.04-0.2
-3"
I
CM
CO
0.510-1.71
0.840
6
0.0590-0.241
0.110
Sarin
6
0.03-0.2
LO
I
o
CO
0.534-2.11
1.03
4
0.0788-0.21
0.113
Soman
6
0.04-0.1
CD
LO
I
CO
0.428- 1.35
0.690
5
0.0511 -0.47
0.0813
Sulfur mustard
6
0.02-0.1
LO
CD
I
0.343-2.29
0.831
6
0.0140-0.164
0.0805
Sand
#
Labs
IDL
(ng/pL)
S:N1 Range
MDL Range
(|jg/kg)
Pooled MDL2
(|jg/kg)
#
Labs
MDL Range
(|jg/kg)
Pooled
MDL2
(Mg/kg)
Cyclohexyl Sarin
4
0.04-0.2
-3"
I
CM
CO
2.25-4.99
3.05
5
0.150-0.200
0.171
Sarin
4
0.03-0.2
LO
I
o
CO
1.89-3.38
2.10
4
0.104-0.261
0.164
Soman
4
0.04-0.1
CD
LO
I
CO
0.650-2.46
1.30
3
0.0860-0.244
0.116
Sulfur mustard
4
0.02-0.1
LO
CD
I
0.810-1.32
1.50
5
0.0617-0.147
0.0923
Wipes
#
Labs
IDL
(ng/pL)
S:N1 Range
MDL Range
(|jg/wipe)
Pooled MDL2
(|jg/wipe)
#
Labs
MDL Range
(|jg/wipe)
Pooled
MDL2
(|jg/wipe)
Cyclohexyl Sarin
5
0.04-0.2
3.2-7.4
0.0196-0.0560
0.0321
4
0.00154-0.00762
0.00374
Sarin
5
0.03-0.2
3.0-5.1
0.00699-0.0400
0.0253
4
0.00162-0.00395
0.00261
Soman
5
0.04-0.1
CD
LO
I
CO
0.00801 -0.0285
0.0150
3
0.000590-0.00235
0.00142
Sulfur mustard
4
0.02-0.1
4.4-6.5
0.0150-0.0495
0.0232
5
0.000830-0.00177
0.00123
1 S:N value from the weaker of two secondary quantitation ions (see Table 5). S:N values were determined by measuring peak-to-peak noise using Agilent
Chemstation software.
2 Pooled MDLs are calculated by taking the square root of the sum of the squares of each lab's MDL divided by the total number of MDL values and multiplied by a
weighting factor based on the degrees of freedom (e.g., for three MDL values, the weighting factor is 0.81).
45
September 2016
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Tables 2a-2c: Multi-Laboratory Results of Reference Matrix Samples Spiked at Levels
Corresponding to Laboratory Low Calibration Standards
Note: Tables 2a through 2c provide summary results of reference matrix samples spiked at levels
corresponding to the lowest calibration standards used by laboratories participating in a multi-laboratory
study. The last column of these tables provides spike levels adjusted based on the lowest recovery
result, for comparison to pooled MDLs generated using study data. With the exceptions noted in table
footnotes, adjusted spike levels are above the pooled MDLs in all cases.
Table 2a.
Example Multi-Laboratory Results for Reagent Water Samples
Spiked at Levels Corresponding to Laboratory Low-Calibration Standards
# of
labs
Analyte
n
Spike
Level
(mq/l)
% Recovery
Range
Pooled
RSD
Pooled
MDL
(mq/l)
Spike Level adjusted
for recovery
(mq/l)
Full-Scan Quadrupole MS
6
Cyclohexyl Sarin
42
5.71
42.6-117
11.0
0.840
2.44
6
Sarin
42
5.71
34.5-112
9.61
1.03
1.97
6
Soman
42
2.86
62.4-152
10.2
0.690
1.78
6
Sulfur mustard
42
2.86
35.4-167
9.51
0.831
1.01
Full-Scan TOF MS
6
Cyclohexyl Sarin
49
0.571
67.2-155
7.72
0.110
0.384
6
Sarin
48
0.571
43.7-124
8.46
0.113
0.250
6
Soman
49
0.286
54.2-128
12.8
0.0813
0.155
5
Sulfur mustard
42
0.286
55.2-126
14.1
0.0805
0.158
n = number of replicates; MDL = method detection limit; RSD = relative standard deviation
Table 2b.
Example Multi-Laboratory Results for Ottawa Sand Samples
Spiked at Levels Corresponding to Laboratory Low-Calibration Standards
# of
labs
Analyte
n
Spike
Level
(MQ/kg)
% Recovery
Range
Pooled
RSD
Pooled
MDL
(MQ/kg)
Spike Level adjusted
for recovery
(Mg/kg)
Full-Scan Quadrupole MS
4
Cyclohexyl Sarin
27
10.0
46.7-105
14.3
3.05
4.67
4
Sarin
28
10.0
48.0-87.5
12.9
2.10
4.80
4
Soman
28
5.00
64.2-130
11.3
1.30
3.21
4
Sulfur mustard
28
5.00
43.2-190
10.5
1.50
2.16
Full-Scan TOF MS
5
Cyclohexyl Sarin
35
1.00
41.3-177
7.71
0.171
0.413
5
Sarin
35
1.00
40.8-129
11.5
0.164
0.408
3
Soman
21
0.500
81.4-284
9.41
0.16
0.407
5
Sulfur mustard
35
0.500
29.6-83.4
11.9
0.0923
0.148
n = number of replicates; MDL = method detection limit; RSD = relative standard deviation
46
September 2016
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Table 2c.
Example Multi-Laboratory Results for Wipes
Spiked at Levels Corresponding to Laboratory Low-Calibration Standards
# of
labs
Analyte
n
Spike
Level
(Mg/wipe)
% Recovery
Range
Pooled
RSD
Pooled
MDL
(|jg/wipe)
Spike Level
adjusted for
recovery
(Mg/wipe)
Full-Scan Quadrupole MS
5
Cyclohexyl Sarin
35
0.100
54.6-110
15.2
0.0321
0.0546
5
Sarin
35
0.100
45.9-88.4
15.4
0.0253
0.0459
5
Soman
35
0.0500
59.0-122
12.7
0.0150
0.030
4
Sulfur mustard
27
0.0500
52.1 - 128
14.3
0.0232
0.0260
Full-Scan TOF MS
5
Cyclohexyl Sarin
35
0.0100
62.0-187
11.0
0.00374
0.0062
5
Sarin
35
0.0100
38.9-105
14.7
0.00261
0.0039
3
Soman
21
0.00500
53.2-111
14.2
0.00142
0.0027
5
Sulfur mustard
34
0.00500
40.2-86.8
14.1
0.00123
0.00201
n, number of replicates; MDL, method detection limit; RSD, relative standard deviation
Table 3.
Decafluorotriphenylphosphine (DFTPP) Key Ions and Ion Abundance
Note: All ion abundances MUST be normalized to m/z 198.
Mass
Ion Abundance Criteria
Quadrupole
Time of Flight (TOF)
51
10.0 - 80.0 % of mass 198
10.0 - 85.0 % of mass 198
68
Less than 2.0 % of mass 69
Less than 2.0 % of mass 69
69
Present
Not used
70
Less than 2.0 % of mass 69
Less than 2.0 % of mass 69
127
10.0 - 80.0 % of mass 198
10.0 - 80.0 % of mass 198
197
Less than 2.0 % of mass 198
Less than 2.0 % of mass 198
198
Base peak 100 % relative abundance
Base peak 100 % relative abundance
199
5.0-9.0 % of mass 198
5.0-9.0 % of mass 198
275
10.0 - 60.0 % of mass 198
10.0 - 60.0 % of mass 198
365
Greater than 1.0 % of mass 198
Greater than 0.5 % of mass 198
441
Present but less than mass 443
Less than 150 % of mass 443
442
Greater than 50.0 % of mass 198
Greater than 30.0 %
443
15.0-24.0% of mass 442
15.0-24.0% of mass 442
47
September 2016
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Table 4.
Internal Standards and Surrogates
Note: Table 4 provides a list of surrogates used during laboratory studies testing this protocol, based on
surrogates typically used for SVOC analyses. Alternative surrogates might be more representative of the
analytes targeted in this method and may be used or added at the laboratory's discretion provided the
surrogates meet the criteria in Table 7.
Analyte
Surrogate Compounds
Internal Standards
Cyclohexyl Sarin
Triphenyl phosphate or p-Terphenyl-d14
Naphthalene-ds
Sarin
Nitrobenzene-ds
1,4-Dichlorobenzene-d4
Soman - GD1
Nitrobenzene-ds
1,4-Dichlorobenzene-d4
Soman - GD2
Nitrobenzene-ds
1,4-Dichlorobenzene-d4
Sulfur mustard
Triphenyl phosphate or p-Terphenyl-d-M
Naphthalene-ds
Table 5.
Example Retention Times (RTs), Relative Retention Times (RRTs) and Quantitation Ions
for Target Compounds, Surrogate Compounds, and Internal Standards
Note: Bold quantitation ions indicate the secondary quantitation ions used during single-laboratory
testing.
Analyte
Retention Time
(minutes)
Relative
Retention Time
Primary
Quantitation
Ion
Secondary
Quantitation
Ions
Sarin
6.14-6.17
0.63
99
125, 81
1,4-Dichlorobenzene-d4 (IS)
9.70
-
152
150, 115, 78
Soman - GD1
10.15-10.21
1.05
99
126, 82, 69
Soman - GD2
10.22-10.29
1.06
99
126, 82, 69
Nitrobenzene-ds (S)
10.98-11.01
1.13
82
128, 54, 98
Sulfur Mustard
12.36-12.39
0.98
109
158, 160, 63, 111
Naphthalene-ds (IS)
12.55-12.58
-
136
68, 108
Cyclohexyl sarin
12.79-12.84
1.02
99
67, 81, 137, 82
p-Terphenyl-d14 (S)
19.75-19.77
1.57
244
122
Triphenyl phosphate (S)
20.68
1.64
326
325, 215
(S) = Surrogate
(IS) = Internal Standard
48
September 2016
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Table 6.
Example Multi-Laboratory Precision and Bias in Reference Matrices
at Mid-Calibration Levels
Full-Scan Quadrupole MS
Initial Precision and
Recovery (IPR)
Laboratory Control
Sample (LCS)
Analyte
#
Labs
n
% Recovery
Pooled
% RSD
% Recovery
Rea<
gent Water1
Cyclohexyl sarin
5
20
72.0-132
6.79
72.0-132
Sarin
5
20
68.9-110
9.19
68.9-110
Soman
5
20
82.9-125
6.00
82.9-125
Sulfur mustard
5
20
83.3-136
5.84
83.3-136
Ottawa Sand2
Cyclohexyl sarin
4
16
56.0-101
5.40
56.0-101
Sarin
4
16
52.0-98.8
5.60
52.0-98.8
Soman
4
16
56.0-115
6.90
56.0-115
Sulfur mustard
4
16
60.0-123
5.15
60.0-123
Wipes3
Cyclohexyl sarin
4
16
54.1 - 111
12.0
54.1 - 111
Sarin
4
16
52.3-80.0
12.1
52.3-80.0
Soman
4
16
53.8-94.0
12.5
53.8-94.0
Sulfur mustard
4
16
50.8-104
12.5
50.8-104
Full-Scan TOF MS
Initial Precision and
Recovery (IPR)
Laboratory Control
Sample (LCS)
Analyte
#
Labs
n
% Recovery
Pooled
% RSD
% Recovery
Rea<
gent Water1
Cyclohexyl sarin
5
20
63.3-119
6.4
63.3-119
Sarin
5
20
37.0-98.9
6.5
37.0-98.9
Soman
5
20
60.2-126
8.0
60.2-126
Sulfur mustard
5
20
11.7-127
10.4
11.7-127
Ottawa Sand2
Cyclohexyl sarin
5
20
43.8-92.3
11.0
43.8-92.3
Sarin
5
20
42.3-82.1
13.2
42.3-82.1
Soman
5
20
38.3-108
14.4
38.3-108
Sulfur mustard
5
20
42.8-82.5
11.0
42.8-82.5
Wipes3
Cyclohexyl sarin
4
16
26.7-105
27.2
26.7-105
Sarin
4
16
23.6-97.3
23.1
23.6-97.3
Soman
4
16
29.2-110
24.5
29.2-110
Sulfur mustard
4
16
29.3-82.0
21.3
29.3-82.0
1 Reagent water spike levels: 45.7 |jg/L (sarin and cyclohexyl sarin); 22.8 |jg/L (soman and sulfur mustard)
2 Soil spike levels: 80.0 [jg/kg (sarin and cyclohexyl sarin); 40.0 [jg/kg (soman and sulfur mustard)
3 Wipe spike levels: 0.1 pg/wipe (sarin and cyclohexyl sarin); 0.05 pg/wipe (soman and sulfur mustard)
49
September 2016
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Table 7.
Surrogate Recovery
Note: Table 7 lists surrogates used during laboratory studies testing this protocol. Alternative surrogates
might be more representative of the analytes targeted in this method, and may be used or added at the
laboratory's discretion, provided the surrogates meet the criteria listed in this table.
Surrogate
Surrogate % Recovery
Minimum
Maximum
Full-Scan Quadrupole MS
Water
Nitrobenzene-d5
50.0
150
p-Terphenyl-di4
50.0
150
Triphenyl phosphate
50.0
150
Soil
Nitrobenzene-d5
50.0
150
p-Terphenyl-d-M
50.0
150
Triphenyl phosphate
50.0
150
Wipes
Nitrobenzene-d5
50.0
150
p-Terphenyl-d-M
50.0
150
Triphenyl phosphate
50.0
150
Full-Scan TOF MS
Water
Nitrobenzene-ds
50.0
150
p-Terphenyl-di4
50.0
150
Triphenyl phosphate
50.0
150
Soil
Nitrobenzene-ds
50.0
150
p-Terphenyl-di4
50.0
150
Triphenyl phosphate
50.0
150
Wipes
Nitrobenzene-ds
50.0
150
p-Terphenyl-di4
50.0
150
Triphenyl phosphate
50.0
150
50
September 2016
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Table 8.
Example Calibration Standard Concentrations (|jg/mL)
Used During Multi-Laboratory Study
Full-Scan Quadrupole MS
Analyte
CAS RN
Cal 1
Cal 2
Cal 3
Cal 4
Cal 5
Cal 6
Cyclohexyl Sarin
329-99-7
0.1
0.2
0.4
0.8
1.0
2.0
Sarin
107-44-8
0.1
0.2
0.4
0.8
1.0
2.0
Soman-GD1 and GD2
96-64-0
0.05
0.1
0.2
0.4
0.5
1.0
Sulfur mustard
505-60-2
0.05
0.1
0.2
0.4
0.5
1.0
Nitrobenzene-ds(S)
4165-60-0
0.1
0.2
0.4
0.8
1.0
2.0
p-Terphenyl-di4(S)
1718-51-0
0.2
0.4
0.8
1.0
2.0
2.0
Triphenyl phosphate (S)
115-86-6
0.2
0.4
0.8
1.0
2.0
2.0
1,4-Dichlorobenzene-d4 (IS)
3855-82-1
0.5
0.5
0.5
0.5
0.5
0.5
Naphthalene-ds(IS)
1146-65-2
0.5
0.5
0.5
0.5
0.5
0.5
Full-Scan TOF MS
Analyte
CAS RN
Cal 1
Cal 2
Cal 3
Cal 4
Cal 5
Cal 6
Cal 7
Cyclohexyl Sarin
329-99-7
0.01
0.05
0.08
0.1
0.25
0.5
1.0
Sarin
107-44-8
0.01
0.05
0.08
0.1
0.25
0.5
1.0
Soman-GD1 and GD2
96-64-0
0.005
0.025
0.04
0.05
0.125
0.25
0.5
Sulfur mustard
505-60-2
0.005
0.025
0.04
0.05
0.125
0.25
0.5
Nitrobenzene-ds(S)
4165-60-0
0.01
0.05
0.08
0.1
0.25
0.5
1.0
p-Terphenyl-di4(S)
1718-51-0
0.01
0.05
0.08
0.1
0.25
0.5
1.0
Triphenyl phosphate (S)
115-86-6
0.01
0.05
0.08
0.1
0.25
0.5
1.0
1,4-Dichlorobenzene-d4 (IS)
3855-82-1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Naphthalene-ds(IS)
1146-65-2
0.5
0.5
0.5
0.5
0.5
0.5
0.5
(S) = Surrogate; (IS) = Internal Standard
51
September 2016
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Table 9a.
Example Multi-laboratory Precision and Bias in Water
Using GC Full-Scan Quadrupole MS
Drinking Water
Analyte
# of labs
n
Pooled
% RSD
% Recovery
Range
% RSD
Range
Low-Level Spike Results1
Cyclohexyl sarin
3
28
6.04
76.5-108
5.58-6.22
Sarin
3
21
6.88
58.8-107
6.19-10.4
Soman
3
21
8.86
67.7-122
4.44-14.7
Sulfur mustard
3
21
6.69
69.5-114
4.02-14.7
Mid-Level Spike Results2
Cyclohexyl sarin
3
21
2.97
59.5-115
1.85-5.73
Sarin
3
21
3.57
60.4-102
2.48-5.69
Soman
3
21
3.77
49.6-117
3.17-5.39
Sulfur mustard
3
21
3.99
66.7-115
2.43-7.07
Surrogates3
Nitrobenzene-ds
4
56
13.7
30.0-135
1.91 -27.0
p-Terphenyl-d-M
3
56
7.41
58.0-140
2.14-19.1
Triphenyl phosphate
4
70
5.76
52.0-142
2.01 - 12.6
Groundwater
Analyte
# of labs
n
Pooled
% RSD
% Recovery
Range
% RSD
Range
Low-Level Spike Results1
Cyclohexyl sarin
3
21
5.03
79.6-115
3.07-5.81
Sarin
3
21
8.22
61.1 - 108
6.37-9.66
Soman
3
21
9.92
70.2-123
4.52-14.1
Sulfur mustard
3
21
12.4
47.0-120
2.16-16.7
Mid-Level Spike Results2
Cyclohexyl sarin
3
21
4.22
68.5-138
1.42-6.66
Sarin
3
21
4.63
66.9-117
2.05-6.05
Soman
3
21
4.31
56.1 - 126
3.29-4.91
Sulfur mustard
3
21
5.05
73.7-127
2.33-6.08
Surrogates3
Nitrobenzene-ds
3
42
7.33
34.0-136
1.17-10.5
p-Terphenyl-d-M
3
42
15.0
50.3-147
2.93-26.2
Triphenyl phosphate
3
42
9.45
60.0-156
4.17-18.1
n = number of replicates; MDL = method detection limit; RSD = relative standard deviation
1 Low-level samples were spiked at 5.71 |jg/L (sarin and cyclohexyl sarin), and 2.86 |jg/L (soman and sulfur mustard).
2 Mid-level samples were spiked at 45.7 |jg/L (sarin and cyclohexyl sarin), and 22.8 |jg/L (soman and sulfur mustard).
3 Surrogates were spiked at 28.6 |jg/L.
52
September 2016
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Table 9b.
Example Multi-laboratory Precision and Recovery in Water
Using GC Full-Scan TOF MS
Drinking Water
Analyte
# of labs
n
Pooled
% RSD
% Recovery
Range
% RSD
Range
Low-Level Spike Results1
Cyclohexyl sarin
4
28
3.59
65.1 - 112
3.66-5.02
Sarin
4
28
4.23
44.9-103
2.01 -6.26
Soman
4
28
4.32
50.4-121
3.65-6.55
Sulfur mustard
4
28
10.4
8.65-120
4.08-17.1
Mid-Level Spike Results2
Cyclohexyl sarin
4
28
3.84
63.6-120
1.39-6.66
Sarin
4
28
4.11
52.6-99.5
1.45-7.14
Soman
4
28
5.50
57.0-118
0.978- 11.2
Sulfur mustard
4
28
5.04
56.8-120
2.34-6.94
Surrogates3
Nitrobenzene-ds
4
64
3.39
74.4-128
0.985-7.29
p-Terphenyl-d-M
4
62
10.2
17.9-129
1.87-32.0
Triphenyl phosphate
5
90
6.02
31.1 - 136
2.63-17.0
Groundwater
Analyte
# of labs
n
Pooled
% RSD
% Recovery
Range
% RSD
Range
Low-Level Spike Results1
Cyclohexyl sarin
4
28
6.94
48.2-144
3.10-9.40
Sarin
4
28
7.32
32.6-124
4.28-9.97
Soman
4
28
9.88
41.7-147
4.34-15.4
Sulfur mustard
4
28
12.4
21.6-124
6.77-20.9
Mid-Level Spike Results2
Cyclohexyl sarin
4
25
2.18
82.9-120
1.47-8.08
Sarin
4
25
5.08
65.3-97.2
1.02-8.39
Soman
4
25
4.95
69.7-116
2.48-8.04
Sulfur mustard
4
25
7.14
39.8-132
0.837-9.86
Surrogates3
Nitrobenzene-ds
4
64
4.61
66.7-129
1.68-7.32
p-Terphenyl-d-M
4
64
15.0
26.7-132
2.04-29.6
Triphenyl phosphate
4
64
7.82
36.6-143
2.35-18.6
n = number of replicates; MDL = method detection limit; RSD = relative standard deviation
1 Low-level samples were spiked at 0.571 |jg/L (sarin and cyclohexyl sarin), and 0.286 |jg/L (soman and sulfur mustard).
2 Mid-level samples were spiked at 5.71 |jg/L (sarin and cyclohexyl sarin), and 2.86 |jg/L (soman and sulfur mustard).
3 All surrogates were spiked at 28.6 |jg/L.
53
September 2016
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Table 10a.
Example Multi-laboratory Precision and Recovery in Soils
Using GC Full-Scan Quadrupole MS
Virginia-a Soil
Analyte
# of
labs
n
Pooled
% RSD
% Recovery
Range
% RSD
Range
Low-Level Spike Results1
Cyclohexyl sarin
3
20
7.92
94.9-168
4.36-11.6
Sarin
3
21
9.65
56.9-91.9
6.25-13.0
Soman
3
21
10.9
102-165
10.5-11.6
Sulfur mustard
3
21
13.3
73.2-124
11.8-14.3
Mid-Level Spike Results2
Cyclohexyl sarin
3
21
6.29
108-137
4.32-7.81
Sarin
3
21
8.12
57.7-80.5
6.61 -9.47
Soman
3
21
6.66
86.6-114
4.96-8.22
Sulfur mustard
3
20
8.74
69.2-93.7
3.15-8.22
Surrogates3
Nitrobenzene-ds
3
42
10.7
52.7-98.7
7.18-15.0
p-Terphenyl-d-M
3
42
6.86
87.7-156
3.81 -9.49
Triphenyl phosphate
3
42
8.25
113-202
3.37-12.5
ASTM Soil
Analyte
# of
labs
n
Pooled
% RSD
% Recovery
Range
% RSD
Range
Low-Level Spike Results1
Cyclohexyl sarin
3
21
28.0
10.4-31.2
16.5-39.1
Sarin
3
21
29.2
5.15-30.3
13.6-46.4
Soman
3
21
19.1
24.8-91.6
8.13-24.0
Sulfur mustard
3
21
17.5
38.9-113
10.2-23.2
Mid-Level Spike Results2
Cyclohexyl Sarin
3
21
19.3
15.4-37.4
7.27-30.3
Sarin
3
20
16.2
9.98-22.9
8.07-24.6
Soman
3
21
10.0
31.2-56.5
6.41 - 13.2
Sulfur Mustard
3
20
8.70
59.9-88.3
4.75-10.3
Surrogates3
Nitrobenzene-ds
3
42
10.6
44.0-101
3.97-17.3
p-Terphenyl-d-M
3
42
9.68
59.6-115
4.46-16.6
Triphenyl phosphate
3
42
9.52
59.9-158
5.83-16.6
n = number of replicates; MDL = method detection limit; RSD = relative standard deviation
1 Low-level samples were spiked at 10.0 [jg/kg (sarin and cyclohexyl sarin), and 5.00 [jg/kg (soman and sulfur mustard).
2 Mid-level samples were spiked at 80.0 [jg/kg (sarin and cyclohexyl sarin), and 40.0 [jg/kg (soman and sulfur mustard).
3 All surrogates were spiked at 50.0 [jg/kg.
54
September 2016
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Table 10b.
Example Multi-laboratory Precision and Recovery in Soils Using GC Full-Scan TOF MS
Virginia-a Soil
Analyte
# of
labs
n
Pooled
% RSD
% Recovery
Range
% RSD
Range
Low-Level Spike Results1
Cyclohexyl sarin
3
21
11.0
120-174
10.8-11.1
Sarin
3
21
14.0
56.9-129
8.14-17.8
Soman
3
19
8.40
67.2-167
1.71 - 11.9
Sulfur mustard
3
21
16.7
62.8-141
14.7-17.7
Mid-Level Spike Results2
Cyclohexyl sarin
3
18
9.65
112-158
6.37-12.5
Sarin
3
18
7.24
66.1 - 129
5.14-10.2
Soman
3
18
6.96
130-171
4.95-9.58
Sulfur mustard
3
18
7.87
62.9-100
4.76-11.7
Surrogates3
Nitrobenzene-ds
3
39
9.06
52.0-91.8
3.04-16.2
p-Terphenyl-d-M
3
39
8.76
64.8-137
5.98-12.2
Triphenyl phosphate
3
25
10.9
97.9-155
3.89-18.3
ASTM Soil
Analyte
# of
labs
n
Pooled
% RSD
% Recovery
Range
% RSD
Range
Low-Level Spike Results1
Cyclohexyl sarin
3
21
18.6
26.9-49.3
12.0-24.5
Sarin
2
14
24.8
8.7-35.1
18.2-30.0
Soman
1
7
30.6
29.6-87.2
30.6
Sulfur mustard
3
21
22.2
20.6-118
9.23-34.9
Mid-Level Spike Results2
Sarin
3
18
27.0
6.51 -24.8
11.5-40.8
Cyclohexyl sarin
3
18
28.3
8.61 -36.1
15.3-42.2
Soman
3
17
15.1
30.0-58.7
8.29-19.3
Sulfur mustard
3
18
17.1
28.4-92.4
3.41 -29.1
Surrogates3
Nitrobenzene-ds
3
39
9.08
44.2-89.1
3.07-174
p-Terphenyl-d-M
3
39
8.72
52.6-108
2.24-17 8
Triphenyl phosphate
2
25
10.4
57.4-111
3.45-14 6
n = number of replicates; MDL = method detection limit; RSD = relative standard deviation
1 Low-level samples were spiked at 1.00 [jg/kg (sarin and cyclohexyl sarin), and 0.500 [jg/kg (soman and sulfur mustard).
2 Mid-level samples were spiked at 10.0 [jg/kg (sarin and cyclohexyl sarin), and 5.0 [jg/kg (soman and sulfur mustard).
3 All surrogates were spiked at 50.0 [jg/kg.
55
September 2016
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Analytical Protocol for Cyclohexyl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Table 11.
Multi-Laboratory Study Water Matrices Characterization Data
Drinking Water
Groundwater
Collection Date
12/2011 -07/2012
Collection Date
01/2012-02/2012
pH
CO
cn
I
CO
^1
pH
6-7
Chlorine (free) mg/L
1.16-1.35
Chlorine (free) mg/L
0.02-0.05
Chlorine (total) mg/L
1.20-1.42
Chlorine (total) mg/L
NR
Total Organic Carbon (TOC) (ppm)
0.83-1.14
Total Organic Carbon (TOC)
(ppm)
1.08-1.24
Conductivity (|jS)
294-413
Conductivity (|jS)
0.82-0.87
Oxidation-Reduction Potential (mV)
766 - 770
Oxidation-Reduction Potential
(mV)
NR
Turbidity (NTU)
0.06-0.07
Turbidity (NTU)
CO
o
I
o
Total Hardness (mg/L)
114-131
Total Hardness (mg/L)
37-65
Alkalinity (mg/L)
64-80
Alkalinity (mg/L)
30-36
NR = Not Reported
Table 12.
Multi-Laboratory Study Soil Matrix Characterization Data
Parameter
Matrix
ASTM Soil ML-1
Virginia-a Soil
PH
8.68
4.41
TOC (% C)
< 0.10
2.2
% Solids
98.9
99.1
TOC = total organic carbon
56
September 2016
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Analytical Protocol for Cyclohexvl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
Abundance
3100000
3000000
2900000
2800000
2700000
2600000
2500000
2400000
2300000
2200000
2100000
2000000
1900000
1800000
1700000
1600000
1500000
1400000
1300000
1200000
1100000
1000000
900000
800000
700000
600000
500000
400000
300000
200000
100000
Tirne~> 3 50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.0010.5011.0011.5012.00
Notes: (1) Concentrations of all analytes as described in Table 8, Calibration Level 6 (GC/MS- Full-Scan
Quadrupole MS); (2) Unlabelled peaks represent compounds not specifically targeted by this protocol.
Time units = minutes
T = Target; S = Surrogate; I = Internal standard
Figure 1.
Example chromatogram for a calibration standard on full-scan quadrupole MS.
57
September 2016
-------
Analytical Protocol for Cyclohexvl Sarin, Sarin, Soman and Sulfur Mustard Using GC/MS
2.25e+006
2e+006
1.75e+006
1.5O+006
1.25o+006
1e+006
750000
500000
250000
0
Time (s) 150
§
o
-C
(J
a
•4
m
"O
8
O
2
X
o
D Q
" s?
"O
c
©
£
c
CD
U")
"O
o
>%
8
Q_
LA
¦*T
5
I
>»
C
5
CL
lr
L lAJ
I W
o
-C
CL
200
250
300
350
400
Notes: (1) Concentrations of all analytes as described in Table 8, Calibration Level 4 (GC/MS - TOP); (2) Unlabelled peaks represent compounds not specifically
targeted by this protocol.
T = Target; S = Surrogate; I = Internal standard; (s) = seconds
Figure 2,
Example chromatogram for a midpoint calibration standard (Cal5) on time-of-flight MS.
58
September 2016
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