United States
Environmental Protection
Agency
EPA 600/R-11/091 | September 2011 | www.epa.gov/ord
High Throughput Determination
of Tetramine in Drinking Water
by Solid Phase Extraction
and Isotope Dilution Gas
Chromatography/Mass
Spectrometry (GC/MS)
VERSION 1
Office of Research and Development
National Homeland Security Research Center
Centers for Disease Control and Prevention
Atlanta, Georgia
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EPA/600/R-11/091
HIGH THROUGHPUT DETERMINATION OF TETRAMINE IN DRINKING WATER
BY SOLID PHASE EXTRACTION AND ISOTOPE DILUTION GAS
CHROMATOGRAPHY/MASS SPECTROMETRY (GC/MS)
Version 1.0
September 2011
Developed by:
Centers for Disease Control and Prevention
In Support of:
U.S. Environmental Protection Agency
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Disclaimer
The U.S. Environmental Protection Agency (EPA) through its Office of Research and
Development funded and managed the research described herein under EPA IA# DW75-
92259701 with the Centers for Disease Control and Prevention. 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:
Erin Silvestri, MPH (EPA Project Officer)
U.S. Environmental Protection Agency
National Homeland Security Research Center
26 W. Martin Luther King Drive, MS NG16
Cincinnati, OH 45268
513-569-7619
Silvestri.Erin@epa.gov
Matthew Magnuson, PhD (EPA Technical Lead)
U.S. Environmental Protection Agency
National Homeland Security Research Center
26 W. Martin Luther King Drive, MS NG16
Cincinnati, OH 45268
513-569-7321
Magnuson.Matthew@epa.gov
Rudolph Johnson, PhD
Centers for Disease Control and Prevention
4770 Buford Highway
MS F-44
Atlanta, GA 30341
770-488-3543
Rmj6@cdc.gov
Jennifer Links, PhD
Centers for Disease Control and Prevention
4770 Buford Highway
MS F-44
Atlanta, GA 30341
770-488-4311
idr9@cdc.gov
11
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Table of Contents
Disclaimer ii
List of Tables iii
Foreword iv
List of Acronyms v
Acknowledgments vi
CHAPTERS
1. Scope and Application 1
2. Summary of Method 2
3. Definitions 3
4. Interferences 4
5. Safety 5
6. Equipment and Supplies 6
7. Reagents and Standards 9
8. Sample Collection, Preservation, and Storage 13
9. Quality Control 16
10. Calibration and Standardization 22
11. Procedure 25
12. Data Analysis and Calculations 28
13. Method Performance 29
14. Pollution Prevention 31
15. Waste Management 31
16. References 16
List of Tables
Table 6-1. Gas Chromatograph (GC) Parameters 8
Table 7-1. Calibration Standard Stock Solution Volumes 12
Table 8-1. Recoveries of Tetramine in Preservatives over Time 14
Table 8-2. Preservative Concentrations and Purposes of Preservatives 15
Table 13-1. Method Performance 29
Table 13-2. Single Lab Precision and Accuracy Data 29
Table 13-3. Percent Recovery of Tetramine for Several Tap Water Matrices and Residual
Disinfectants 30
in
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Foreword
The National Homeland Security Research Center (NHSRC), part of the U.S. Environmental
Protection Agency's (EPA's) Office of Research and Development, is focused on developing
and delivering scientifically sound, reliable, and responsive products. These products are
designed to address homeland security information gaps and research needs that support the
Agency's mission of protecting public health and the environment. A portion of NHSRC's
research is directed at decontamination of indoor surfaces, outdoor areas, and water
infrastructure. This research is conducted as part of EPA's response to chemical, biological, and
radiological (CBR) contamination incidents. NHSRC has been charged with delivering tools and
methodologies (e.g. sampling and analytical methods, sample collection protocols) that enable
the rapid characterization of indoor and outdoor areas, and water systems following terrorist
attacks, and more broadly, natural and manmade disasters.
The Selected Analytical Methods for Environmental Remediation and Recovery (SAM), formerly
referred to as the Standardized Analytical Methods for Environmental Restoration Following
Homeland Security Events, is a compendium of methods that informs sample collection and
analysis during the response to an incident. SAM can be used by public and private laboratories
which are analyzing a large number of samples associated with chemical, biological, or
radiological contamination. Even though some of the analytes in SAM already have existing
analytical methods, others are in need of improvements that enhance analytical capability and
meet more rigorous performance criteria. Furthermore, not all of the analytical methods listed in
the SAM document address all possible matrices (e.g., water, soil, air, glass) encountered in
sample collection following an incident. Some of the analytical methods in SAM have been
verified in a single laboratory, but most still need to undergo verification with respect to a
specific contaminant in association with a specific matrix.
The sampling and analytical procedure (SAP) presented herein, describes a single laboratory
developed method for the high throughput determination of tetramethylene disulfotetramine in
drinking water by solid phase extraction and isotope dilution gas chromatography/mass
spectrometry. Performance data for this method have been generated in a single lab but the
method has not been studied jointly or independently by multiple labs. This method, which will
be included in the SAM, is expected to provide the Water Laboratory Alliance, as part of EPA's
Environmental Response Laboratory Network, with a more reliable and faster means of analyte
collection and measurement.
Jonathan Herrmann,
Director, National Homeland Security Research Center
IV
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Acronyms
CAS chemical abstract service
CCC continuing calibration check
CDC Centers for Disease Control and Prevention
CR confirmation ratio
CTMDL Chemical Terrorism Methods Development Laboratory
DL detection limit
El electron ionization
FD field duplicate
GC gas chromatography
HRpir half range for the predicted interval of results
HPLC high performance liquid chromatography
i.d inside diameter
IDC initial demonstration of capability
IS standard
ISTD internal standard primary dilution standard
LD50 median lethal dose
LFB laboratory fortified blank
LFSM laboratory fortified sample matrix
LRB laboratory reagent blank
MRL minimum reporting level
MS mass spectrometer
MSDS Material Safety Data Sheet
m/z mass to charge ratio
NHSRC National Homeland Security Research Center
OSHA Occupational Safety and Health Administration
PIR mean prediction interval of result ± half range for the predicted interval of results
QC quality control
RPD relative percent difference
SAM Selected Analytical Methods for Environmental Remediation and Recovery
Restoration Following Homeland Security Events
SAP Sampling and analytical procedure
Sect section
SIM selective ion monitoring
SPE solid phase extraction
SS standard solution
TETS tetramine
TOC Total Organic Carbon
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Acknowledgments
The following researchers were critical to the development of the method and preparation of the
procedure and are acknowledged:
Centers for Disease Control and Prevention, National Center for Environmental Health
Rudy Johnson
Jennifer Links
The following individuals served as members and technical advisors of the Project Team and are
acknowledged:
U.S. Environmental Protection Agency (EPA), Office of Research and Development (ORD),
National Homeland Security Research Center (NHSRC)
Matthew Magnuson (EPA Technical Lead)
Sanjiv Shah
Erin Silvestri (EPA Project Officer)
VI
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HIGH THROUGHPUT DETERMINATION OF TETRAMINE IN DRINKING WATER
1. SCOPE AND APPLICATION
1.1. This is a single laboratory developed isotope dilution gas chromatography/mass
spectrometer (GC/MS) method for the determination of tetramethylene
disulfotetramine (tetramine, TETS, Chemical Abstract Services Registry Number
80-12-6). This method, including QC requirements, is designed to support site
specific clean-up goals of environmental restoration activities following a
homeland security incident involving this analyte.
1.2. Significance: Although banned in the United States, an accidental tetramine
poisoning has been reported in New York City and several intentional poisonings
have been reported in other countries, primarily in China [1,2]. Low levels of
exposure can be deadly and the human oral LDso (median lethal dose) has been
reported to be as low as 0.1 mg/kg [2, 3]. Because tetramine is an odorless,
tasteless white powder that easily dissolves in water but not absorbed through
skin, the most common route of tetramine exposure is by ingestion [2].
Symptoms of mild tetramine poisoning may include headache, dizziness, fatigue,
weakness, lethargy, nausea, vomiting, perioral paresthesias (numbness around the
mouth), and anorexia while high levels of exposures are characterized by seizures,
coma and death [2]. Symptoms may begin 0.5-13 hours post exposure [2].
1.3. The use of 96-well plates for the solid phase extraction (SPE) produces two key
benefits. The 96-well plates allow for extensive automation of the method,
thereby enabling high throughput of samples, as might be required during
environmental restoration. Additionally, the use of this format results in the
ability to perform isotope dilution by enabling the economical addition of
isotopically labeled tetramine as an internal standard to the sample prior to
extraction.
1.4. Isotopically labeled tetramine is added equally to all unknowns, quality controls,
and calibration standards. In addition to enabling accurate quantitation,
isotopically labeled tetramine also accounts for and resolves some of the QC
issues surrounding analysis, including analysis efficiency and sample loss, in the
intended use of this analyte. The overall QC approach utilizing quantitation and
confirmation ions as well as an isotopically labeled analyte greatly increases
confidence that tetramine, and not another molecule with similar fragmentation
patterns, is being quantitated during analysis.
1.5. This method was adapted from one that was initially developed by the Centers for
Disease Control and Prevention (CDC), in the National Center for Environmental
Health (NCEH), Division of Laboratory Sciences (DLS), Emergency Response
and Air Toxicants Branch, in the Chemical Terrorism Methods Development
Laboratory (CTMDL) for the determination and quantitation of tetramine in
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human urine. For the adapted method, accuracy and precision data have been
generated in reagent water, and in finished ground and surface waters that use
chlorine and/or chloramine as residual disinfectants.
1.6. The QC approach in this method conforms to CTMDL standards for clinical
samples, and is presented here in terms more familiar to drinking water
laboratories. Methods developed by CTMDL are distributed to the CDC's
laboratory network, and the QC approach included in these methods, while single
lab verified by the CTMDL lab, is designed to be sufficiently rigorous that
network labs can successfully perform the method.
1.7. The minimum reporting level (MRL) is the lowest analyte concentration that
meets data quality objectives for the intended use of the method, e.g., to meet site-
specific remediation goals. Laboratories will need to demonstrate that their
laboratory MRL meets the requirements described in Section 9.2.4.
1.8. Determining the detection limit (DL) is optional (Sect. 9.2.6). Detection limit is
defined as the statistically calculated minimum concentration that can be
measured with 99% confidence that the reported value is greater than zero.
1.9. This method is intended for use by analysts skilled in the performance of solid
phase extractions, the operation of GC/MS instruments, and the interpretation of
the associated data.
1.10. This method has been verified using only the conditions and equipment specified
in the method. Alteration of this method is not recommended.
2. SUMMARY OF METHOD
2.1. A 50-mL water sample is collected, and a preservative and/or dechlorinating
agent are optionally added as required by site-specific conditions. (The data in
Table 8.1 suggest that the presence or choice of the additive does not affect the
results.) An aliquot is pipetted into a well of a preconditioned 96-well solid phase
extraction plate, and the isotopically labeled tetramine is added. Following a
wash step, tetramine is then eluted in acetonitrile. The extract is concentrated to
dryness under nitrogen and heat, and then adjusted to a 100 uL volume in
acetonitrile. Tetramine is separated from the sample matrix and identified by
GC/MS analysis, operated in SIM mode or equivalent. Analyte identification is
accomplished by comparing the acquired mass spectra, including ion ratios, and
retention times to reference spectra and retention times for calibration standards
acquired under identical GC/MS conditions. Quantitation is performed using the
internal standard technique. Utilization of an isotopically labeled internal standard
provides a high degree of accuracy and precision for sample quantitation by
accounting for analyte recovery and analytical efficiency.
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2.2. Compared to some drinking water methods (e.g. certain EPA 500 series methods),
the initial laboratory demonstration of capability (IDC) is lengthier than some
drinking water methods, the frequency of the on-going calibration is shorter, and
the number of continuing calibration checks (CCC) is higher. Based on
experience in the developer's lab, this QC approach ensures successful long-term
implementation of the method in other labs, particularly when these methods are
used infrequently (e.g. in emergency situations). Due to site-specific
circumstances during an environmental remediation activity, e.g. in which sample
throughput requirements exceed available lab capacity, a shorter initial
demonstration of capability (IDC), changes to the on-going calibration frequency,
and number of CCCs may be necessary and appropriate.. However, initial and
ongoing QC requirements and acceptance criteria (see Section 9) should not be
changed. Adopting steps, such as a replacing on-going recalibration with a
calibration check only, to save time may result in higher QC failure rates and
perhaps less accurate quantitation. Labs should discuss these increased risks with
sample submitters before taking such steps.
3. DEFINITIONS
3.1. ANALYSIS BATCH - a sequence of samples, analyzed within a 24-hour period,
including no more than 20 field samples in addition to all of the required QC
samples (Sect. 9.3)
3.2. CALIBRATION STANDARD (CAL) - A solution prepared from the primary
dilution standard solution and/or stock standard solution and the internal standard.
The CAL solutions are used to calibrate the instrument response with respect to
analyte concentration.
3.3. CONFIRMATION ION - for this method, the second most abundant tetramine
ion (See Confirmation Ratio, Sect. 3.4, below). The confirmation ion is used to
calculate the confirmation ratio (Sect. 3.4)
3.4. CONFIRMATION RATIO (CR) - peak area produced by the confirmation ion
divided by the peak area produced by the quantitation ion which serves as an
additional QC measure of analyte selectivity
3.5. CONTINUING CALIBRATION CHECK (CCC) SOLUTION - a calibration
solution containing the method analyte(s), which is extracted in the same manner
as the samples and analyzed periodically to verify the accuracy of the existing
calibration for those analyte(s)
3.6. DETECTION LIMIT (DL) - the minimum concentration of an analyte that can be
identified, measured, and reported to be greater than zero with 99% confidence
3.7. FIELD DUPLICATES (FD1 and FD2) - two separate samples collected at the
same time and place under identical circumstances, and treated exactly the same
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throughout field and laboratory procedures to provide check the precision
associated with sample collection, preservation, storage, and laboratory
procedures
3.8. ISOTOPICALLY-LABELED INTERNAL STANDARD - a pure chemical added
to an extract or to a standard solution in a known amount(s) and used to measure
the relative response of other method analytes and surrogates that are components
of the same solution.
3.9. LABORATORY FORTIFIED BLANK (LFB) - a volume of reagent water or
other blank matrix to which known quantities of the method analytes and all the
preservation reagents are added in the laboratory (Sect. 7.3.5.2) The LFB is
analyzed exactly like a sample and its purpose is to determine whether the
methodology is in control and whether the laboratory is capable of making
accurate and precise measurements.
3.10. LABORATORY REAGENT BLANK (LRB) - an aliquot of reagent water that is
treated exactly as a sample and used to determine if method analytes or other
interferences are present in the laboratory environment, the reagents, or the
apparatus
3.11. MATERIAL SAFETY DATA SHEET (MSDS) - written information provided
by vendors concerning a chemical's toxicity, health hazards, physical properties,
fire, and reactivity data including storage, spill, and handling precautions
3.12. MINIMUM REPORTING LEVEL (MRL) - the minimum concentration qualified
to be reported as a quantitated value for a method analyte in a sample following
analysis (Sect. 9.2.4. for MRL verification procedure)
3.13. PRIMARY DILUTION STANDARD SOLUTION - a solution containing the
analytes prepared in the laboratory from stock standard solutions and diluted as
needed to prepare calibration solutions and other needed analyte solutions
3.14. QUANTITATION ION -for this method, the quantitation ion is the parent
tetramine ion with a mass to charge ratio (m/z) of 240 (See Confirmation Ratio,
Sect. 3.4, above)
3.15. SECOND SOURCE QUALITY CONTROL SAMPLES - materials obtained
from a source different than the original and used to verify the accuracy of the
existing calibration for those analytes
4. INTERFERENCES
4.1 Method interferences that can lead to discrete artifacts and/or elevated baselines
in the chromatograms may be caused by contaminants in solvents, reagents
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(including reagent water), sample bottles and caps, and other sample processing
hardware. All such items must be routinely demonstrated to be free from
interferences under the conditions of the analysis by analyzing laboratory reagent
blanks. Subtracting blank values from sample results is not permitted.
4.2 Matrix interferences may be caused by contaminants that are co-extracted from
the sample. The extent of matrix interferences will vary considerably from source
to source, depending upon the nature of the water.
4.3 Relatively high concentrations, in the mg/L range, of preservatives, antimicrobial
agents, or dechlorinating agents might be added to sample collection vessels
(Section 8.1.2). The potential exists for trace-level organic contaminants in these
reagents. Interferences from these sources should be monitored by analysis of
laboratory reagent blanks particularly when new lots of reagents are acquired.
4.4 Due to the nature of the matrix analyzed in this procedure, occasional
interferences from unknown substances might be encountered. Interfering
compounds can be recognized by deviations in the sample
quantitation/conformation ratios from the calibration standard ratios and can also
be monitored using appropriate LRBs. Any interference that results in QC failure
(Sect. 9) results in rejection of the entire analysis batch. If repeating the analysis
does not remove the interference with the reference standard, the results for that
analyte are not reportable.
4.5 All glassware should be chemically cleaned before running this method. Wash
glassware thoroughly with reagent-grade water followed by acetonitrile. Allow
glass to dry completely before use. If the laboratory wishes to use a muffle oven
for decontamination then the appropriate measures should be taken to assure that
the muffle oven conditions are suitable to remove all traces of tetramine and other
interferences.
4.6 Care should be taken at all times to prevent contamination of QC materials,
standards, and samples.
4.7 Chromatographic separation of the analyte should be carefully monitored for
unknown interferences. See Section 11.2.5 for analyte confirmation.
5. SAFETY
5.1 The toxicity or carcinogenicity of each reagent used in this method has not been
precisely defined. Each chemical should be treated as a potential health hazard,
and exposure to these chemicals should be minimized. Each laboratory is
responsible for maintaining an awareness of OSHA regulations regarding safe
handling of chemicals used in this method. A reference file of MSDSs should be
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made available to all personnel involved in the chemical analyses. Additional
references to laboratory safety are available [4-6].
5.2 Tetramine is highly toxic, and the human oral LDso has been reported to be as low
as 0.1 mg/kg [2, 3]. Ingestion is the primary reported route of exposure, but all
other routes of exposure (e.g. inhalation, dermal contact, and eye exposure)
should be avoided. Follow universal safety precautions when performing this
procedure, including the use of a lab coat, safety glasses, appropriate gloves, and
a high quality-ventilated chemical fume hood and/or biological safety cabinet.
5.3 Avoid inhalation or dermal exposure to acetonitrile, which is used in the sample
preparation steps.
5.4 Mechanical hazards when performing this procedure using standard safety
practices are minimal. Read and follow the manufacturer's information regarding
safe operation of the equipment. Avoid direct contact with the mechanical and
electronic components of the gas chromatograph and mass spectrometer, unless
all power to the instrument is off. Generally, maintenance and repair of
mechanical and electronic components should be performed only by qualified
technicians.
6. EQUIPMENT AND SUPPLIES (It is important to note that specific brands or catalog
numbers included in this section are examples only and do not imply endorsement of
these particular products. These specific products were used during the validation of this
method.)
6.1 MICRODISPENSERS - with adjustable volume (5-100 uL, 100-1000 uL)
(Eppendorf Co., Westbury, NY or equivalent)
6.2 REPEATER PIPETTE - 4780 (Eppendorf Co., Westbury, NY or equivalent)
6.3 CONICAL AUTOSAMPLER VIALS - 300-uL vials (must be compatible with GC
autosampler) especially for use as an alternative to 96 well plates
6.4 ANALYTICAL BALANCE - Capable of weighing to the nearest 0.0001 g
6.5 SOLID PHASE EXTRACTION (SPE) APPARATUS WITH 96 WELL PLATES
6.5.1 96-WELL SPE PLATE - Strata X 60-mg / 6-mL (PN# 8E-S100-UGB),
available from Phenomenex (Torrence, CA) or equivalent
6.5.2 PLATE SHAKER (ThermoFisher Scientific, Waltham, MA or equivalent)
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6.5.3 96-WELL LIQUID HANDLER - Use a 96-well liquid handler equipped
with a solid-phase extraction manifold and vacuum system. These
systems must be calibrated prior to use, according to vendor or laboratory
specifications. In addition, these liquid handlers must be used during
laboratory-method validation. The liquid handlers that have been used
with this method in different laboratories have included the Tomtec®
Quadra 3 SPE (Tomtec, Inc. Hamden, CT), the Caliper Zephyr, and the
Caliper ilOOO (Caliper Life Sciences, Hopkinton, MA). The selection of
these liquid handlers has typically been based on cost and required sample
throughput.
6.6 96-WELL NUNC DEEP WELL PLATE - 2000 mL plate (Nunc PN# 278752 or
equivalent). Must be compatible with 96-well liquid handler described in Section
6.5.3.
6.7 EXTRACT CONCENTRATION SYSTEM. The 96-well plate requires a
compatible dry-down step for sample pre-concentration following extraction. The
TurboVap 96 concentrator evaporator workstation (Zymark® Corp., Hopkinton,
MA) has proven to be well suited for this application, but other evaporator systems
which result in equivalent method performance could be used instead.
6.8 GAS CHROMATOGRAPHY ELECTRON IONIZATION MASS
SPECTROMETRY SYSTEM (GC/MS)
6.8.1 GC COLUMN - 30 m x 0.25-mm inside diameter (i.d.) fused silica capillary
(5%-Phenyl)-methylpolysiloxane column coated with a 0.25um bonded film
(Agilent HP-5ms [Agilent Technologies, Santa Clara, CA] or equivalent). A
nonpolar, low-bleed column designed for GC/MS applications is
recommended for use with this method to provide adequate chromatography
and minimize column bleed.
6.8.2 GC SYSTEM - The GC system (e.g., Agilent 6890N GC or equivalent)
must be equipped with an autosampler and injector and must provide
consistent sample injection volumes. The system should also be capable of
performing linear temperature gradients at a constant flow rate. The GC
should be capable of being configured exactly as stated below:
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Table 6-1. Gas Chromatograph (GC) Parameters
Parameter
GC Method
Column type
Injection Volume
Inlet liner
Inlet Temperature
Injection mode
Autosampler Tray
Temperature
Oven Program
Typical retention time
MS Scan Mode
lonization Type
Dwell Time
Setting
Constant flow at 1 mL/min
Initial pressure: 10.5 psi
Carrier Gas: Helium
HP5-ms (5% phenyl methyl siloxane), 30 m x 0.25 mm x 0.25 um
1 jiL
Splitless liner double taper, unpacked
250°C
Splitless injection; purge flow to split vent 100 mL/min at 1 min; gas saver
at 20 mL/min at 3 min
Room temperature
Initial temperature 100°C
Ramp 8°C/min to 200°C
Ramp50°C/minto300°C
Hold 3 00°C for 1.7 min
Tetramine =11.6 min
Selected ion monitoring (SIM)
Electron ionization (El)
100 msec per ion
MASS SPECTROMETER (MS) - The MS (Agilent 5973 Mass Selective
Detector, Palo Alto, CA, or equivalent) must be capable of performing electron
impact ionization with positive ion detection and must be configured for selected
ion monitoring (SIM, or equivalent depending on MS type) with a dwell time of
100 msec per ion. The SIM ions monitored for this method should be set exactly
as stated below:
Analyte Ion (m/z)
Tetramine quantification ion 240
Tetramine confirmation ion 212
Tetramine internal standard 244
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7. REAGENTS AND STANDARDS (These reagents were used during the validation of
the method, and only these or their equivalent are acceptable for use.)
7. 1 GASES, REAGENTS, AND SOLVENTS - Reagent grade or better chemicals should be
used. Unless otherwise indicated, it is intended that all reagents shall conform to the
specifications of the Committee on Analytical Reagents of the American Chemical
Society, where such specifications are available. Other grades may be used, provided it is
first determined that the reagent is of sufficiently high purity to permit its use without
lessening the quality of the determination.
7.1.1 HELIUM - 99.9999% pure or better, GC carrier gas
7. 1 .2 REAGENT WATER - purified, deionized water which does not contain any
measurable quantities of the method analyte or interfering compounds ([Tedia,
Fairfield, OH] ® HPLC or equivalent grade water)
7. 1 .3 METHANOL - (CH3OH, CAS#: 67-56-1) - high purity, demonstrated to be free
of analytes and interferences (Tedia HPLC or equivalent)
7. 1 .4 ACETONITRILE - (CH3CN, CAS#: 75-05-8) - high purity, demonstrated to be
free of analytes and interferences (Tedia HPLC or equivalent)
7.1.5 SAMPLE PRESERVATION REAGENTS - One of the following sample
preservation reagents may be required by site specific conditions:
7.1.5.1 AMMONIUM CHLORIDE (NH4C1, CAS#: 12125-02-9) - an additive
used in sample collection (Sigma-Aldrich ACS grade or equivalent)
7.1.5.2 SODIUM THIOSULFATE (Na2S2O3, CAS#: 7772-98-7) - an additive
used in sample collection (Sigma-Aldrich ACS grade or equivalent)
7.1.5.3 SODIUM SULFITE (Na2SO3, CAS#: 7757-83-7) - an additive used in
sample collection (Sigma-Aldrich ACS grade or equivalent)
7.1.5.4 ASCORBIC ACID (C6H8O6, CAS#: 50-81-7) - an additive used in sample
collection (Sigma-Aldrich ACS grade or equivalent)
7.1.5.5 AMMONIUM ACETATE (CH3CO2NH4, CAS#: 631-61-8) - An additive
used in sample collection (Sigma-Aldrich ACS grade or equivalent)
7.1.5.6 CITRIC ACID (HOC(COOH)(CH2COOH)2, CAS#: 77-92-9) - an
additive used in sample collection (Sigma-Aldrich ACS grade or
equivalent)
7.1.5.7 DIAZOLIDINYL UREA (CgH^N^T, CAS#: 78491-02-8) - an additive
used for sample collection (Sigma-Aldrich ACS grade or equivalent)
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7.2 REAGENT PREPARATION
7.2.1 5% METHANOL IN WATER - A 5%/95% methanol/water solution is prepared
through volumetric dilution with HPLC grade deionized water. Measure 10 mL
of methanol using an appropriate pipette, volumetric flask, or graduated cylinder
and pour into a clean, dry container with a capacity of 250 mL or more. Measure
190 mL of HPLC grade deionized water with a volumetric flask or graduated
cylinder and pour into the same container with the methanol. Mix the solution
well.
7.3 STANDARDS SOLUTIONS - When a compound purity is assayed to be 96% or
greater, the weight can be used without correction to calculate the concentration of the
stock standard. Solution concentrations listed in this section were used to develop this
method and are included as an example. Standards for sample fortification generally
should be prepared in the smallest volume that can be accurately measured to minimize
the addition of excess organic solvent to aqueous samples. Store all calibration and
control materials at either -20±5°C when not in use. Even though stability times for
standard solutions are suggested in the following sections, laboratories should use
standard QC practices to determine when their standards need to be replaced.
7.3.1 ISOTOPICALLY LABELLED INTERNAL STANDARD SOLUTIONS The
internal standard used in this method is 13C4-tetramine (Cambridge Isotopes, MA;
catalog #CLM-8146-0). Note that in this method, the internal standard is a
chemical that is structurally identical to the method analyte, but is substituted with
13C. The isotopically-labeled internal standard has no potential to be present in
water samples, and is not a method analyte. The internal standard is added to all
samples, standards, and QC solutions as described in Section 11.1.3.
7.3.2 Prepare or purchase the internal standard at a concentration of 500 ng/mL. Steps
for the preparation of this mixture are described below:
7.3.2.1 INTERNAL STANDARD STOCK SOLUTION - Accurately weigh
approximately 20.1 mg of 13C4-tetramine in a weigh boat and then transfer
into a 200 mL volumetric flask. Add 100 mL of acetonitrile and mix well
until dissolved. Dilute to the 200 mL mark with additional acetonitrile
and mix well. The stock solution is stable for at least one year when
stored at -20 ±5°C.
7.3.2.2 INTERNAL STANDARD PRIMARY DILUTION STANDARD (ISTD)
(500 ng/mL) - Combine 50 jiL of the internal standard stock solution with
9.95 mL of deionized water in a 15 mL polypropylene centrifuge tube
(BD, Franklin Lakes, NJ) or equivalent. The stock solution is stable for at
least one year when stored at -20±5°C.
10
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7.3.3 ANALYTE STOCK STANDARD SOLUTIONS. Prepare or purchase three
stock solutions using a reliable source of tetramine (Cambridge Isotopes, 50
Frontage Road, Andover, MA 01810. CAS #:80-12-6; unlabeled material product
#ULM-8147 and labeled material product # CLM-8146 (13C4 label)). These
stock solutions are stable for at least one year when stored at -20±5°C.
7.3.3.1 ANALYTE STOCK STANDARD SOLUTION 1 (SSI, 82.4 mg/L) -
Accurately weigh approximately 20.6 mg of tetramine to a weigh boat and
then into a 250 mL volumetric flask. Add 100 mL of acetonitrile and mix
well until dissolved. Dilute to the 250 mL with additional acetonitrile and
mix well.
7.3.3.2 ANALYTE STOCK STANDARD SOLUTION 2 (SS2, 206 ug/L) -
Accurately transfer 25 uL of SSI into a 10 mL volumetric flask. Dilute
with acetonitrile to 10 mL mark and mix well.
7.3.3.3 ANALYTE STOCK STANDARD SOLUTION 3 (SS3, 8.24 mg/L) --
Accurately transfer 1 mL of SSI into a 10 mL volumetric flask. Dilute
with acetonitrile to the 10 mL mark and mix well.
7.3.4 CALIBRATION STANDARD STOCK SOLUTIONS - Prepare the calibration
standard stock solutions from dilutions of the analyte stock solutions in reagent
water containing any preservatives required by site-specific circumstances (See
Sects 2.2 and 8.1.3). For this purpose, a Falcon polypropylene 50 mL centrifuge
tube (BD, Franklin Lakes, NJ) may be used by quantitatively transferring the
volumes of the respective solution listed in the table below to the tube, diluting to
the 40 mL mark, and mixing well. (Note: Diluting to the 40 mL mark provided
sufficient accuracy in the developer's lab. Other labs may wish to utilize alternate
polypropylene vessels if they experience dilution related inaccuracies.) The
calibration curve is composed of at least six concentrations. These calibration
standard solutions are stable for at least one year when stored at -20±5°C.
7.3.4.1 PREPARATION OF CALIBRATION STANDARD STOCK
SOLUTIONS - Calibrations standard stock solutions may be prepared
using the volumes listed in Table 7-1 below. The concentrations, along
with the numbers of solutions, are for illustration purposes only. Other
concentrations may be required in practice to meet performance and QC
goal. (See Sect. 10.3 for the number of calibration solutions required for
calibration.)
11
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Table 7-1. Calibration Standard Stock Solution Volumes
Concentration
(ng/mL)
Total
Volume
(mL)
Stock Stock „, . „ . .
„ . „ . . Stock Solution
Solution 3 Solution 2 i r T ^
(uL) (uL)
0
0.5
1
2
5
10
15
25
50
75
100
250
40
40
40
40
40
40
40
40
40
40
40
97
194
388
24
49
73
121
24
36
49
121
7.3.5 QUALITY CONTROL SOLUTIONS - There are several types of quality control
solutions, some of which are identical in composition but serve different QC
functions and hence may be referred to by different names in Section 9.
7.3.5.1 SECOND SOURCE QUALITY CONTROL SAMPLE - These samples
are used to verify the accuracy of the calibration standard solutions
(7.3.4) and are prepared the same way as the calibration standards. They
are prepared from an analyte source different than the calibration
standard solutions as described more completely in Section 9.3.7.
7.3.5.2 LABORATORY FORTIFIED BLANKS (LFBs) - LFBs are used
throughout this method for various purposes. The LFB is analyzed
exactly like a sample, and its purpose is to verify that the methodology is
be competently replicated, and that the laboratory has the capability to
make accurate and precise measurements. The two specific LFBs are
required in this method are referred to as LFB-low and LFB-high, which
relate to initial and ongoing QC. For the demonstration of the method in
the developer's laboratory, the LFB-low and -high are 5 and 75 ng/mL,
respectively, prepared as indicated in Table 7.1, in Section 7.3.4. In a
particular lab, the LFBs should be selected from similar points in their
calibration range (e.g., LFB-low should be around 10 times the MRL
(Sect. 9.2.4) and LFB-high should be around 150 times the MRL.
The LFBs are inherently calibration standards and can be used to
construct the calibration curve. However, the LFBs are specifically used
12
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to develop QC criteria during the initial demonstration of capability
(Sect. 9.2), and serve as an additional QC function during each analysis
batch. The LFBs serve a similar, but generally more stringent, QC
function as continuous calibration checks (Sect. 10.3).
7.3.5.3 LABORATORY REAGENT BLANK. This blank is prepared as a LFB
with no analyte added (i.e., the 0 ng/mL in Table 7-1).
8. SAMPLE COLLECTION, PRESERVATION, AND STORAGE
8.1 SAMPLE VESSEL PREPARATION COLLECTION
8.1.1 Samples can be collected in a 50-mL polypropylene vessel fitted with a flat-top
polyethylene screw-cap (e.g., BD Falcon 50 mL centrifuge tube or equivalent).
8.1.2 The performance data for the method presented in Section 13 are presented
without addition of preservatives. This is based on the stability of tetramine in the
presence of preservatives suggested in Table 8-1, which suggests the analyte does
not require sample preservation up to 28 days, particularly if tetramine is the sole
analyte of interest in the sample.
8.1.3 However, vessels should be prepared before sample collection with appropriate
preservative(s) (Table 8-2) required by site-specific circumstances, e.g., to fulfill
the purpose(s) listed in the Table 8-2. Preservation through binding free chlorine
or dechlorination may also be necessary if analytical artifacts are observed in the
samples but not the LFBs. All initial and on-going QC requirements should be
demonstrated for the preservatives added to the sample, particularly if added in
combination. If tetramine is the only analyte determined, necessity of
preservatives is expected to be a very rare event.
13
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Table 8-1. Recoveries of Tetramine in Preservatives over Time (n=3)
Water Type
Deionized
Chlorine
Monochloramine
Chlorine
Chlorine
Monochloramine
Chlorine
Chlorine
Chlorine
Deionized Water
Deionized Water
Preservative
-
-
-
Ammonium Chloride (0.1 g/L)
Sodium Thiosulfate (0.08 g/L)
Sodium Thiosulfate (0.08 g/L)
Sodium Sulfite (0.05 g/L)
Ascorbic Acid (0.1 g/L)
Ammonium Acetate (1.5 g/L)
Citric Acid (9.3 g/L)
Diazolidinyl Urea (1 g/L)
DayO
4°C
109 ±3
104 ±7
108 ±7
106 ±7
107 ±7
109 ±9
112±17
111±8
101 ±8
109 ±8
100 ±26
25°C
102 ±6
105 ±5
101±9
106 ±5
99 ±3
103 ±4
105 ±5
103 ±6
99 ±0
108 ±6
96 ±5
Day?
4°C
105 ± 14
107 ±9
100 ±2
104 ±5
96 ±2
99 ±6
110±6
101±7
116±6
104 ±1
102 ±6
25°C
112±4
111±5
108 ±4
113±8
111±5
111±5
105 ±5
111±10
111±3
110±5
103 ±2
Day 14
4°C
101±2
102 ± 1
101±4
116±4
100 ±2
113±2
101±2
104 ±3
114±2
95 ±2
99 ± 1
25°C
103 ±2
105 ±2
104 ±1
103 ±1
96 ±2
102 ±2
105 ±1
114±3
106 ±3
112±4
98 ± 10
Day 28
4°C
107 ±4
113±4
110±4
124 ±6
111±4
115±2
111±4
114± 11
107 ±2
114±4
109 ±8
25°C
108 ±4
118±2
112±10
112±5
115±9
113±4
109 ±10
111±4
110±3
110±2
110±6
14
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Table 8-2. Preservative Concentrations and Purposes of Preservatives
Compound
Ammonium
chloride
Ammonium
acetate
Sodium
thiosulfate
Sodium sulfite
Ascorbic acid
Citric acid
Diazolidinyl
urea
Mass added to
sample (mg)
5
75
4
2.5
5
465
50
Concentration in
sample (g/L)
0.1
1.5
0.08
0.05
0.1
9.3
1
Purpose
Binds free chlorine
Binds free chlorine
Dechlorinates free
chlorine and chloramine
Dechlorinates free
chlorine and chloramine
Dechlorinates free
chlorine and chloramine
pH adjustment
Microbial inhibitor
8.2 SAMPLE COLLECTION - When sampling from a water tap, samplers should
request guidance about how long to flush the tap, if at all. Depending on site
specific goals, incident managers may request that the tap not be flushed to
minimize loss of contaminant. If incident managers do not specify a shorter time,
flush until the water temperature has stabilized (approximately 3-5 minutes).
Collect samples from the flowing stream. It may be convenient to collect a bulk
sample in a polypropylene vessel from which to generate individual 50 mL
samples. Keep samples sealed from collection time until analysis. When
sampling from an open body of water, fill the sample container with water from a
representative area. Sampling equipment, including automatic samplers, should be
free of plastic tubing, gaskets, and other parts that may leach interfering analytes
into the water sample.
8.3 SAMPLE SHIPMENT AND STORAGE - Results of the sample storage stability
study (Table 8-1) suggest that storage at 25°C produces results similar to reduced
temperatures. As a matter of practice to ensure that the samples do not experience
excessive temperature outside the stability range investigated, it is recommended
that all samples be iced, frozen (-20°C), or refrigerated (4°C) from the time of
collection until extraction. During method development, no significant differences
were observed between standards that were frozen or refrigerated.
8.4 SAMPLE AND EXTRACT HOLDING TIMES - Results of the sample storage
stability study (Table 8-1, n=3) suggest that tetramine has adequate stability for at
least 28 days when collected, preserved, shipped, and stored as described in
Sections 8.1, 8.2, and 8.3. As matter of practice, water samples should be
15
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extracted as soon as possible but must be extracted within 28 days. Data generated
during this study indicates that extracts are stable for at least 28 days when stored
at 0 °C or lower. As matter of practice, analysis should occur as soon as possible.
9. QUALITY CONTROL
9.1 QC requirements include the initial demonstration of capability (IDC) and
ongoing QC requirements that must be met when preparing and analyzing field
samples. This section describes the QC parameters, their required frequencies,
and the performance criteria that must be met in order to meet typical EPA quality
objectives for drinking water analysis, although these objectives will be site
specific during a remediation activity. These QC requirements are considered the
minimum acceptable QC criteria in particular for this method which utilizes an
isotopically labeled internal standard. Laboratories are encouraged to institute
additional QC practices to meet specific needs [7].
9.2 INITIAL DEMONSTRATION OF CAPABILITY (IDC) - The IDC must be
successfully performed prior to analyzing any field samples. Prior to conducting
the IDC, the analyst must first generate an acceptable initial calibration following
the procedure outlined in Section 10.2. It should be noted that the IDC is
lengthier than some drinking water methods, but based on experience in the
developer's lab, the IDC helps ensure successful long-term implementation of the
method in a variety of other labs. Due to site-specific conditions during a
environmental remediation activity, a shorter IDC may be necessary and
appropriate. For example, a more minimal IDC could consist of: a)
demonstration of low system background (Sect. 9.2.1); b) 4-7 same-day replicates
fortified near the midrange of the initial calibration curve for precision and
accuracy demonstration, combined with c) the MRL estimation described in
Section 9.2.4. However, QC acceptance requirements, both initial (Sect. 9.2.1-
9.2.4) and ongoing (Sect. 9.3) should not be changed, and a shorter IDC may
result in higher QC failure rates and less accurate quantitation in some
concentration ranges. Labs should consider these risks before choosing a shorter
IDC.
9.2.1 INITIAL DEMONSTRATION OF LOW SYSTEM BACKGROUND -
Any time a new lot of solvents, reagents, and autosampler vials/plates are
used, it must be demonstrated that an LRB is reasonably free of
contamination and that the criteria in Section 9.3.1 are met.
9.2.2 INITIAL DEMONSTRATION OF PRECISION - Prepare and analyze at
least twenty replicates of both laboratory fortified blanks (LFB-high and
LFB-low, see Sect. 7.3.5.2) over the course of at least 10 days. Any
sample preservative, as described in Section 8.1.2, must be added to these
samples. For the initial demonstration of precision, the coefficient of
16
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variation for the concentrations of the replicate analyses must be less than
20%.
9.2.3 INITIAL DEMONSTRATION OF ACCURACY - Using the same set of
replicate data generated for Section 9.2.2, calculate the mean recovery. For
the initial demonstration of accuracy, the mean recovery of the replicate
values must be within ± 30% of the true value.
9.2.4 MINIMUM REPORTING LEVEL (MRL) ESTIMATION - Because
clean-up goals will be site specific, laboratories need to estimate a
minimum reporting level so that incident managers can understand a
specific laboratory's capabilities and can distribute samples to appropriate
laboratories. Establishing the MRL concentration too low may cause
repeated failure of ongoing QC requirements. If the IDC procedure (Sect.
9.2.1-9.2.3) is followed explicitly, establishing the MRL as the lowest
standard is expected to ensure compliance with QC requirements, This is a
result of the rigor of the QC requirements in the lengthy IDC (Sect. 9.2.1-
9.2.3), especially those associated with the LFBs (see Sect. 10.3.3). If a
shorter IDC is required by site specific conditions (see Sect. 2.2), the MRL
should be confirmed with the procedure below.
9.2.4.1 Fortify and analyze seven replicate LFBs at the proposed MRL
concentration. These LFBs must contain all method
preservatives described in Section 8.1.2. Calculate the mean
measured concentration (Mean) and standard deviation for the
method analytes in these replicates. Determine the half range for
the prediction interval of results (HRpiR) for each analyte using
the equation below:
HRPIR = 3.9635
where s is the standard deviation and 3.963 is a constant value
for seven replicates.
9.2.4.2 Confirm that the upper and lower limits for the prediction
interval of result (PIR = Mean ± HRPIR) meet the upper and
lower recovery limits as shown below:
The Upper PIR Limit must be <150% recovery.
HRP1R
FortifiedConcentration
The Lower PIR Limit must be > 50% recovery.
17
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FortifiedConcentration
lo0o/0>
9.2.4.3 The MRL is validated if both the upper and lower PIR limits
meet the criteria described above (Sect. 9.2.4.2). If these criteria
are not met, the MRL has been set too low and must be
confirmed again at a higher concentration.
9.2.5 CALIBRATION CONFIRMATION - The calibration is confirmed by
analysis of a second source quality control sample as described in Section
9.3.5
9.2.6 DETECTION LIMIT (DL). This is a statistical determination of precision
and accurate quantitation is not expected at this level.Replicate analyses
for this procedure should be done over at least three days (i.e., both the
sample preparation and the LC/MS/MS analyses should be done over at
least three days). At least seven replicate LFBs should be analyzed during
this time period. The concentration may be estimated by selecting a
concentration at two to five times the noise level.). The appropriate
fortification concentrations will be dependent upon the sensitivity of the
GC/MS system used. Any preservation reagents added in Section 8.1.2
must also be added to these samples. Note that the concentration for some
IDC steps may be appropriate for DL determination, in which case the
IDC data may be used to calculated the DL. (For example, for the results
presented in Section 13, twenty replicate LFBs were analyzed over 10
days, e.g., three LFBs individually fortified on day one, two LFBs
individually fortified on day two, and two LFBs individually fortified on
day three, etc). Analyze the replicates through all steps of Section 11.
Calculate the DL from the equation: DL = sxt(n.\)
where:
s = standard deviation of replicate analysis, without subtraction of
values of analyte free blanks
t = Student's t value for the 99% confidence level with n-l degrees
of freedom
n = number of replicates
9.3 ONGOING QC REQUIREMENTS - This section summarizes the ongoing QC
criteria that must be followed when processing and analyzing field samples. The
required QC samples for an analysis batch include the laboratory reagent blank
(LRB) and four continuing calibration check (CCC) solutions.
18
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9.3.1 LABORATORY REAGENT BLANK (LRB) - An LRB is required with
each analysis batch (Sect. 3.1) to confirm that potential background
contaminants are not interfering with the identification or quantitation of
method analytes. Running the LRB first may prevent unnecessary
analysis if the LRB is invalid. Preparation of the LRB is described in
Section 7.3.5. If the LRB produces a peak within the retention time
window of the analyte that would prevent the determination of the analyte,
determine the source of contamination and eliminate the interference
before processing samples. Background contamination must be reduced to
an acceptable level before proceeding. Background from method analyte
or other contaminants that interfere with the measurement of method
analyte must be below 1/3 of the MRL. Blank contamination is estimated
by extrapolation, if the concentration is below the lowest calibration
standard. This extrapolation procedure is not allowed for sample results as
it may not meet data quality objectives. If the method analytes are detected
in the LRB at concentrations equal to or greater than 1/3 the MRL, then all
data for the problem analyte(s) must be considered invalid for all samples
in the analysis batch.
9.3.2 ONGOING CALIBRATION. The analytical system in recalibrated at the
beginning of each analysis batch using the same analyte concentrations
determined during the initial calibration. The acceptance criteria for the
ongoing calibration is described in Section 10.2.5, except that removal of
calibration points may result in too few calibration points and therefore an
invalid calibration. The ongoing calibration is performed after the first
two continuing calibration check (CCC) samples (Sec. 9.3.3) to allow for
corrective action if the calibration fails. As mentioned in Sect. 2.2, in
some well considered circumstances and in consultation with the sample
submitter about increased QC and quantitation risk, it may be desirable to
not perform the ongoing calibration (Sect. 9.3.2) and instead rely on CCC
samples (as described in Sect. 9.3.3) to verify ongoing calibration. If so,
the beginning CCC of each analysis batch must be at or below the MRL in
order to verify instrument sensitivity prior to any analyses. Subsequent
CCCs should alternate between a medium and high concentration
calibration standard.
9.3.3 CONTINUING CALIBRATION CHECK (CCC) - CCC standards,
containing the preservatives, if any, are analyzed at the beginning of each
analysis batch, after every 20 field samples. Note that there are up to four
CCCs depending on the IDC appropriate for the site specific circumstance.
In the lengthier IDC described in Sect. 9.2, there are four CCCs: LFB-low
and LFB-high, which are analyzed before the batch, and the lowest and
highest calibration standards from the ongoing calibration (Sect 9.3.2),
which are analyzed after the field samples. If this IDC approach is not
appropriate, then there are at most two CCC standards, i.e. the calibration
standards. Depending on site specific goals and tolerance of QC and
19
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quantitation risk, it may acceptable to only run one of these calibration
standards as the CCC before and after the batch. If so, the beginning CCC
of each analysis batch must be at or below the MRL in order to verify
instrument sensitivity prior to any analyses. Subsequent CCCs should
alternate between a medium and high concentration calibration standard.
See Section 10.3 for acceptance criteria for the various CCCs. Preparation
of the CCCs is described in Section 7.3.5.
9.3.4 LABORATORY FORTIFIED BLANK (LFB) - Since this method utilizes
procedural calibration standards, which are fortified reagent waters, there
is no difference between the LFB and the CCC, except for the order in
which they are run as part of an analysis batch and the corresponding QC
acceptance criteria. The acronym LFB is used for clarity in the IDC.
9.3.5 SECOND SOURCE QUALITY CONTROL SAMPLES (QCS) - As part
of the IDC (Sect. 9.2), each time a new analyte stock standard solution 1
(SSI, Sect. 7.3.3.1) is prepared, and at least quarterly, analyze a QCS
sample from a source different from the source of the calibration
standards. If a second vendor is not available, then a different lot of the
standard should be used. The QCS should be prepared near the midpoint
of the calibration range and analyzed as a CCC. Acceptance criteria for the
QCS are identical to the CCCs; the calculated amount for each analyte
must be ± 30% of the expected value. If measured analyte concentrations
are not of acceptable accuracy, check the entire analytical procedure to
locate and correct the problem.
9.3.6 INTERNAL STANDARD (IS) - The analyst must monitor the peak area
of the IS in all injections during each analysis day. The IS peak area must
meet the criteria in the both following two subsection
9.3.6.1 The internal standard should produce a peak area at least five
times higher than the peak area of the quantitation ion of
tetramine in the lowest concentration calibration solution. If it
does not, the concentration of IS may not be as predicted.
Prepare new calibrations solutions, QC samples, and field
samples with an appropriately increased concentration of IS.
9.3.6.2 The IS response (peak area) in any chromatographic run must not
deviate from the response in the most recent CCC by more than
30%, and must not deviate by more than 50% from the area
measured during initial analyte calibration. If the IS area in a
chromatographic run does not meet these criteria, inject a second
aliquot of that extract.
9.3.6.2.1 If the reinjected aliquot produces an acceptable IS response,
report results for that aliquot.
20
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9.3.6.2.2 If the reinjected aliquot fails the IS criterion, the analyst should
check the calibration by reanalyzing the most recently acceptable
calibration standard. If the calibration standard fails the criteria
of Section 10.3, recalibration is in order per Section 10.2. If the
calibration standard is acceptable, report results obtained from
the reinjected aliquot, but annotate as "suspect/IS recovery."
Alternatively, prepare another aliquot of the sample as specified
in Section 11.2 or collect a new sample and re-analyze.
9.3.7 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) and LFSM
DUPLICATES - The isotopically labeled internal standard in this method
also serves the role of the LFSM, which is used to determine that the
sample matrix does not adversely affect method accuracy. In the context
of application of this method for environmental restoration, it is not
expected that there would be a native tetramine background concentration.
Also, it is likely that the water samples will come from the same drinking
water system, and hence the sample matrices from a single collection time
will be very similar. Further, experience with the automated extraction
equipment used suggests that if most failures in IS QC requirements result
from failure of the automation equipment. This would correspond to
LFSM failure, as well. Accordingly, neither LFSMs or duplicate LFSMs
would be expected to yield additional information about influence of
sample matrix on method accuracy, except for the unlikely case of a
feature of the sampling/restoration plan that produces a co-eluting peak
with identical chromatographic and mass spectral properties as tetramine.
In this case, the lab should discuss with the submitter.
9.3.7.1 If an LFSM and LFSM is deemed necessary, calculate the
relative percent difference (RPD) for duplicate LFSMs (LFSM
and LFSMD) using the equation
\LFSM -LFSMD\
= -± ^
(LFSM + LFSMD} I'2
9.3.7.2 Relative percent difference (RPD) for duplicate LFSMs should
be <30% for samples fortified at or above their native
concentration. Greater variability may be observed when
LFSMs are fortified at analyte concentrations that are within a
factor of two of the MRL. LFSMs fortified at these
concentrations should have RPDs that are <50%. If the RPD of
any analyte falls outside the designated range, and the
laboratory performance for that analyte is shown to be in control
21
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in the CCC, the recovery is judged to be matrix biased. The
result for that analyte in the unfortified sample is labeled
"suspect/matrix" to inform the data user that the results are
suspect due to matrix effects.
9.3.5 FIELD DUPLICATE (FD) - Field duplicates check the precision
associated with sample collection, preservation, storage, and laboratory
procedures. Some of these factors are out of control of the laboratory, and
the rest are covered by other QC checks. Accordingly, results of any field
duplicates requested should be discussed with the sample submitter if they
do not meet the following criteria.
9.3.5.1 Calculate the relative percent difference (RPD) for duplicate
samples (FD1 and FD2) using the equation
FDI-FD2
RPD=-, r—xlOO
FD2}I2
9.3.5.2 RPDs for FDs should be <30%. Greater variability may be
observed when FDs have analyte concentrations that are within a
factor of two of the MRL. At these concentrations, FDs should
have RPDs that are <50%. If the RPD of any analyte falls outside
the designated range, and the laboratory performance for that
analyte is shown to be in control in the CCC, the recovery is
judged to be biased. The result for that analyte in the unfortified
sample is labeled "suspect/field duplicate bias" to inform the data
user that the results are suspect due to field bias. (Note some other
sources of lab bias may also be present.)
10. CALIBRATION AND STANDARDIZATION
10.1 All laboratory equipment should be calibrated according to manufacturer's
protocols and equipment with expired calibrations should not be used.
Demonstration and documentation of acceptable mass spectrometer tune and
initial calibration is required before any samples are analyzed. After the initial
calibration is successful, the instrument is recalibrated using the same conditions
as the initial calibration before each analysis batch. After the batch, the lowest
and highest calibration solutions are run as continuing calibration checks (CCC)
Verification of mass spectrometer tune must be repeated each time a major
instrument modification is made or maintenance is performed, and prior to analyte
calibration.
10.2 INITIAL CALIBRATION
22
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10.2.1 MS TUNE - Calibrate the mass and abundance scales of the MS
with calibration compounds and procedures prescribed by the
manufacturer with any modifications necessary to meet tuning
requirements. For an Agilent MSD, some labs have experienced
better results if following the automatic tune, they perform a
manual tune to set the mass resolution to unit mass, the peak width
to 0.50 ± 0.01 amu and the abundance for the ion at mass 69 to
500,000 ± 50,000 counts. For other instruments, follow
manufacturer's protocols to tune the instrument.
10.2.2 INSTRUMENT CONDITIONS - Operational conditions are
tabulated in Section 6.8.3. Alteration of the conditions is not
recommended and would require redevelopment of QC criteria.
Frequently reported problems can be avoided by: 1) checking that
needle wash solutions are adequately filled and the injection
syringe is functioning properly and 2) changing the septum and
inlet liner as needed.
10.2.3 Prepare six calibration standards as described in Section 7.3.4.
Note that as procedural calibration standards, they are processed
through the procedure in Section 11, in which the isotopically
labeled internal standard is added before extraction. In practice,
the lowest concentration of the calibration standard must be at or
below the MRL (Sect. 9.2.4), which will depend on system
sensitivity. The lowest point on the calibration curve is close to the
reported detection limit and the highest point is above the expected
range of results. The remainder of the points are distributed
between these two extremes, with the majority of points in the
concentration range where most unknowns are expected to fall.
10.2.4 The GC/MS system is calibrated using the internal standard
technique, as implemented by the data system software. Construct
a calibration curve using at least a six-point curve of response
ratios (i.e., ratio of calibration standard peak area to internal
standard peak area). As the internal standard concentration is
consistent among samples and calibrators, some labs have found it
convenient to set it to a value of one instead of the actual
concentration.
10.2.5 CALIBRATION ACCEPTANCE -- Calculate the slope and
intercept of the calibration curve with 1/x weighting (or other
appropriate weighting) by a linear least squares fit (or other
appropriate calibration function). Evaluate the r2 value for the
curve, which must be greater than 0.990. Linearity of the standard
curve should extend over the entire standard range. The intercept
should not be significantly different from 0; if it is, the source of
23
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the bias should be identified. Each calibration point, except the
lowest point, for the analyte should calculate to be 70 to 130 percent
of its true value. The lowest point should calculate to be 50 to 150
percent of its true value. If these criteria cannot be met, the analyst
will have difficulty meeting ongoing QC criteria. If any standard
is in error and does not fit the standard curve (i.e., the r value for
the curve is < 0.990), it can be removed from the calibration. No
more than one standard may be discarded in any given calibration
curve. If either the high or low standard is dropped, the reporting
limits must be adjusted accordingly. The resulting r2 value must be
greater than 0.990.
10.3 CONTINUING CALIBRATION CHECKS (CCCs). As described in Sect 9.3.3,
up to four CCCs are used in conjunction with each analysis batch. If applicable,
LFB-low and LFB-high are run at the beginning of the batch, and the calibration
solutions are run at the end. The LFBs serve to verify the initial IDC, and the
calibration solutions verify the calibration generated at the start of the analysis.
The LRBs, LFBs, and CCCs are not counted as the 20 samples that constitute an
analysis batch.
10.3.1 Inject an aliquot of the appropriate concentration calibration
solution and analyze with the same conditions used during the
initial calibration.
10.3.2 Acceptance of the calibration solutions is based on the same
criteria as described in Section 10.2.5. Failure to meet these
criteria is a rare occurrence, and suggests maintenance of the
GC/MS system is required.
10.3.3 Acceptance of the results of the LFB-Low and LFB-High is based
on the Quality Control Limits (Sect. 10.3.3.1) established via the
IDC. Acceptability of results for that entire analytical batch is
dependent upon the agreement of the results from these control
materials within established ranges. Quality Control Limits for the
CCCs are based primarily on the standard deviation (on-i, sigma)
of the replicate analysis in the IDC (Sect. 9.2.2). Section 13.3
presents sample values for these parameters obtained in the
developer's laboratory, in which 20 replicate analyses performed
over no less than 10 days are used to establish the LFB-low and -
high limits (Sect. 9.2.2). If the CCC results do not meet the
following criteria, it is "out-of-control," and the cause of the failure
must be determined and corrected. No results from the associated
analytical batch may be reported. These criteria apply to non-zero
analyte concentrations used to make the quality control solutions in
section 7.3.5.1
24
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10.3.3.1 If both of the LFB-Low and LFB-High results are
within 2on-i of the mean determined during the IDC,
then accept the entire analytical batch. Otherwise,
reject the entire analytical batch.
10.3.4 Common remedial actions if the CCCs fails to meet acceptable
criteria
10.3.4.1 LOW ANALYTE RESPONSE - If the signal-to-noise of the
low standard confirmation ion falls below 10, this indicates that
the instrumental sensitivity, or SPE recovery, has fallen below
acceptable limits. The following steps should be taken and the
instrument sensitivity rechecked after each corrective action is
performed. Once sensitivity has been reestablished, further
steps are not necessary.
i. Re-extract the samples.
ii. If tailing is a significant issue, clip the GC column.
iii. Ensure the filament for the MS is still intact.
iv. Clean the mass spectrometer source
v. Clean the gas chromatograph inlet liner
10.3.4.2 Analyte in standards - If an inordinately large amount of
analyte is measured in one of the calibration standards, but this
is not seen in the remainder of the samples, this indicates a
contamination of this particular sample. The source of this
incident should be investigated to prevent repeat occurrences,
but no further action is required. The contaminated calibration
standard should be excluded when developing the calibration
curve.
10.3.4.3 Analyte in all samples - If an inordinately large amount of
analyte is present in all measurements for a particular day, it is
likely that one or more of the spiking solutions are
contaminated. If necessary, prepare new solutions.
11. PROCEDURE
11.1 SAMPLE PREPARATION
11.1.1 Samples are preserved, collected and stored as presented in Section
8. Allow samples to come to room temperature prior to analysis.
11.1.2 If using a TurboVap 96 evaporator system, set it to 65-75°C.
Follow manufacturer's direction for other equipment.
25
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Note: Steps 11.1.3 through 11.1.13 can be performed using an automated
liquid handler or a manual pipettor with a manual 96-well manifold.
However, data presented in this document was collected using an
automated liquid handler.
11.1.3 Fill 96 plate wells.
11.1.3.1 Into each well of the 96-well Nunc deep well plate
(Nalge Nunc International, Rochester, NY), add 50 uL
of the isotopically-labeled internal standard (refer to
section 7.3.2)
11.1.3.2 Into each sample well, add 1000 uL of sample.
11.1.3.3 Into each blank well, add 1000 uL of reagent water (for
the LRB).
11.1.3.4 Into each calibration standard well, add 1000 uL of
tetramine calibration standard stock solutions (refer to
sections 7.3.4)
11.1.3.5 Into each quality control well, add 1000 uL of
appropriate quality control material, (refer to section
7.3.5)
11.1.4 Mix on the plate shaker for 2 min or by other appropriate means.
11.1.5 Plate SPE procedure. For each well on the Nunc plate filled in
Section 11.1.3, perform the following steps and do not let wells go
dry for more than 1 minute:
11.1.5.1 Condition/preclean the selected well on the
Phenomenex® Strata-X 60-mg SPE well plate
(Phenomenex, Torrance, CA) with 1125 uL of 100%
methanol.
11.1.5.2 Condition the SPE plate with 1125 uL of deionized
water.
11.1.5.3 Load 1000 uL from the Nunc plate and draw through the
SPE plate using positive or negative pressure.
11.1.5.4 Wash the SPE plate with 1125 uL of 5% methanol/95%
water (Sect. 7.2.1).
11.1.5.5 Elute the sample with 800 uL of acetonitrile into a 96-
well Nunc deep well plate.
11.1.6 Blow down the sample to dryness using nitrogen gas at 65-75°C.
If using a TurboVap evaporator system, set the flow rate to 45 flow
26
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units until approximately 50% has been evaporated. Then raise the
flow rate to 75 flow units until dry. When using systems other than
the TurboVap, set the flow rate for the blow down gas according to
manufacturer's directions.
11.1.7 Add 100 uL of acetonitrile to reconstitute each sample and vortex.
11.1.8 Transfer the acetonitrile solution into appropriate autosampler
vials or a 96-well autosampler plate.
11.2 ANALYSIS OF SAMPLE EXTRACTS
11.2.1 Establish operating conditions as described in Section 10.2.2.
11.2.2 Establish a valid initial calibration following the procedures
outlined in Section 10.2 or confirm that the calibration is still valid
by running both CCCs as described in Section 10.3. If establishing
an initial calibration for the first time, complete the IDC as
described in Section 9.2.
11.2.3 Set up the available automation equipment and software as
specified by the manufacturer for batch analysis, paying particular
attention to the following frequent stumbling blocks:
11.2.3.1 On the instrument computer, edit the automation
software:
(a) Select the sample type.
(b) Identify the correct vial position.
(c) Name the sample. Due to large number of samples
analyzable with the automation equipment, it is important
that appropriate record keeping (e.g., database, notebooks,
data files, etc.) should be used to track specimens.
(d) Enter information related to particular specimens into
the software manually or by electronic transfer.
(e) Select the instrument control method.
(f) Identify the target path where the data will be stored.
11.2.3.2 Check to be sure that the number and positions of
samples entered on the sequence set-up page correspond
to the samples in the autosampler.
11.2.4 Run the automation sequence to analyze the batch of aliquots of
field and QC samples at appropriate frequencies (Sect. 9, 10.3).
All field, QC, and calibration standards should be run using the
same GC/MS conditions. At the conclusion of data acquisition, use
the same software that was used in the calibration procedure to
27
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identify the peaks in predetermined retention time windows of
interest. Use the data system software to examine the ion
abundances of components of the chromatogram.
11.2.5 COMPOUND IDENTIFICATION - The presumed tetramine peak
in the sample must appear in the same retention time window as
the isotopically-labeled internal standard (around 11.6 min in the
developer's lab) and have similar chromatographic characteristics
such as peak shape. This relies on expert judgment of the analyst
since the retention times reported by the software are not always
reliable. Identification of the peak as tetramine is then confirmed
through calculating the confirmation ratio (CR), i.e., by dividing
the response for m/z 240 by the response for m/z 212 of the
presumed tetramine peak. Using the manufacturer's software or
manually, compare the confirmation ratio of the peak from the
sample with the mean of the CRs measured for the six calibration
standards associated with that batch. The mean CR is the average
CR from the calibration standards only and is batch dependent.
The CR value for each sample should be within 30% of the mean.
(CR value was 1.74 in the developer's lab for the IDC samples).
12. DATA ANALYSIS AND CALCULATIONS
12.1 Concentrations are calculated using the ions listed in Section 6.8.3.
Use of other ions is not advised. If a particular instrument cannot
produce the fragments listed in section 6.8.3, this instrument should
not be used to run this method.
12.2 Calculate analyte concentrations using the ongoing multipoint
calibration established in Section 9.3.2. Do not perform calibration
using just the CCC or LFB-low and -high data to quantitate analytes
in samples, although these samples might be part of the ongoing
calibration curve.
12.3 All raw data files are quantified using the quantitation capabilities of
the instrument software. The peaks are automatically integrated
using the software-associated integration program, and the
integration of each peak is reviewed and manually corrected as
appropriate. This is particularly important for the calibration
standards. The quality control samples (e.g., CCCs and LFBs) are
quantified and evaluated against the calibration curve, and each field
sample is then quantified against that calibration curve. The run data
can be processed within instrument data analysis software and
exported to external spreadsheets, per laboratory policy, generating
28
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files containing the unknown and QC concentrations, retention
times, standard curves, and other run information.
12.3.1 Results are generally reported to two significant digits. In addition
to analytical measurements of unknowns, statistical results of
measurement of blanks should accompany all results.
12.3.2 Check all sample and analytical data for transcription errors and
overall validity after being entered into the instrument software
database. Back up onto external media both the instrument and
data storage databases according to laboratory guidelines.
29
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13. METHOD PERFORMANCE
13.1 ANALYTICAL IDENTIFICATION-Analyte identification using
the approach described in Section 11.2.5 resulted in no false
positives or negatives for the samples reported below. There was
very low background noise according to the signal-to-noise ratios for
the m/z monitored.
13.2 SINGLE LABORATY MINIMUM REPORTING LEVELS and
DETECTION LIMIT- The reportable range of results for tetramine
is summarized below, along with the DL determined from the IDC
procedure (n=20, >10 days) described previously. The lowest
standard is used as the method reportable limit, and the DL
calculated from the standard deviation of replicate measurements of
that standard (in the case of Table 13-1, 0.059). The highest
reportable limit is based on the highest linear standard.
Table 13-1. Method Performance
Compound
Tetramine
(retention time =
11.6 min)
Minimum reporting
level (ng/mL)
0.5
Highest reportable
limit (ng/mL)
250
Method DL (ng/mL)
0.15
13.3 SINGLE LABORATORY ACCURACY AND PRECISION for
LFBs - Single lab precision and accuracy data is represented in
Table 13-2. Accuracy is defined as the mean of the measured
concentration in the fortified samples divided by the fortification
concentration, expressed as a percentage. Method accuracy was
determined by analyzing LFBs at the two non-zero levels in Section
7.3 (i.e., LBF-low and -high) and twenty analyses for each of the
two concentration levels were completed over a period of 28 days.
The means, standard deviations, and relative standard deviations for
the two LFBs are shown in Table 13-2. The means are less than one
standard deviation from the known concentration.
Table 13-2. Single Lab Precision and Accuracy Data
Analyte
Tetramine
Sample
LFB-low
LFB-
high
Fortified
Concentration
(Mg/L)
5
75
Mean of
IDC
Replicates
(Mg/L)
5.03
75.5
Standard
Deviation
(Mg/L)
0.28
2.8
RSD
(%)
5.6
3.7
Accuracy
of Mean
(%)
100
101
30
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13.4 SINGLE LABORATORY RECOVERY AND PRECISION FOR
TAP WATER MATRICES. Table 13-3 expresses percent mean
recoveries for tetramine in several different chlorinated and
chloraminated tap waters derived from the types of sources (i.e.,
ground or surface water) indicated. Water quality parameters
describing these sources are indicated in the footnotes. Percent
recoveries were determined by dividing the measured concentration
by the spiked concentration (75 ug/L) (n=3 for each day). No
tetramine or interferences were detected in the unspiked samples.
13.5 SAMPLE STORAGE STABILITY STUDIES - Table 13-3 also
presents tetramine storage stability data. Samples were collected and
stored as described in Section 8 and also at room temperature. No
preservatives were added to the samples (See Sect. 8.1.2). The
precision and average recovery of triplicate analyses was conducted
on Days 0, 7, 14 and 28. These data support the maximum 28 day
aqueous holding time specified in Section 8.4.
Table 13-3. Percent Recovery of Tetramine for Several Tap Water Matrices and
Residual Disinfectants
Water Type
Ground Water la
(chlorine)
Surface Water 2b
(chloramine)
Surface Water 3C
(chlorine)
Surface Water 4d
(chloramine)
Surface Water 5e
(chlorine)
DayO
4°C
103 ±8
107 ±8
110±3
102 ±2
102 ±3
25°C
109
100
96
94
99
±3
±1
±0
±4
±7
Day?
4°C
105
99
102
102
117
±3
±3
±3
±1
±4
25°C
113
114
106
107
108
±4
±3
±6
±3
±3
Day 14
4°C
113±4
92 ±3
108 ±6
98 ±7
108 ±6
25°C
104 ±5
105 ±6
107 ±5
98 ±5
108 ±6
Day 28
4°C
122 ±2
119±6
111±2
109 ±2
112±3
25°C
114±7
111±3
107 ±2
114±3
116±8
aTotal organic carbon (TOC) not detected in well-field; pH 7.6; hardness 350 mg/L; Chlorine 0.2-0.4 mg/L;
(monthly averages)
TOC 7.61 mg/L; pH 9.2; hardness 65 mg/L; Monochloramines 2.4 mg/L (monthly averages)
°TOC 2.0 mg/L; pH 7.3; hardness 135 mg/L; Chlorine 1 mg/L (monthly averages)
TOC 2.3; pH 7.4; hardness 190 mg/L; Monochloramine 3.4 mg/L (monthly averages)
eTOC 1.0; pH 8.5; 130 mg/L; Chlorine 0.8 mg/L (monthly averages)
31
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14. POLLUTION PREVENTION
14.1 This method utilizes solid phase extraction to extract analytes from
water. It requires the use of reduced volumes of organic solvent and
very small quantities of pure analytes, thereby minimizing the
potential hazards to both the analyst and the environment as
compared to the use of large volumes of organic solvents in
conventional liquid-liquid extractions.
14.2 For information about pollution prevention that may be applicable to
laboratory operations, consult "Less is Better: Laboratory Chemical
Management for Waste Reduction" available from the American
Chemical Society's Department of Government Relations and
Science Policy on-line at
http ://portal. acs. org/portal/fileFetch/C/WPCP_012290/pdf/WPCP_0
12290.pdf (accessed May 2010).
15. WASTE MANAGEMENT
15.1 Dispose of waste materials in compliance with the laboratory
chemical hygiene plan, as well as federal, state, and local
regulations. Always dispose of solvents and reagents in an
appropriate container clearly marked for waste products and
temporarily store them in a chemical fume hood. Dispose of
tetramine in an appropriate waste stream as well. Tetramine is not
destroyed by autoclaving [8], so wash any other non-disposable
glassware, empty ampoules, and/or apparatus before recycling or
disposing of in an appropriate manner.
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16. REFERENCES
1. Barrueto, F., Jr.; L.S.Nelson; R.S. Hoffman; M.B. Heller; P.M. Furdyna; et al.
Poisoning by an Illegally Imported Chinese Rodenticide Containing
Tetramethylenedisulfotetramine. Morbidity and Mortality Weekly Report
(MMWR). 52(10): p. 199-201.
2. Whitlow, K.S., B. Belson, F. Buarueto, L. Nelson and A.K. Henderson.
Tetramethylenedisulfotetramine: old agent and new terror. Ann Em erg Med,
2005. 45(6): p. 609-13.
3. Chau, C.M., A.K. Leung, and I.K. Tan, Tetraminepoisoning. Hong Kong Med J,
2005. 11(6): p. 511-4.
4. "Carcinogens - Working With Carcinogens," Department of Health, Education,
and Welfare, Public Health Service, Center for Disease Control, National Institute
for Occupational Safety and Health, Publication No. 77-206, August 1977.
5. "OSHA Safety and Health Standards, General Industry," (29CFR1910),
Occupational Safety and Health Administration, OSHA 2206, (Revised, January
1976).
6. "Safety in Academic Chemistry Laboratories," American Chemical Society
Publication, Committee on Chemical Safety, 7th Edition. Information on
obtaining a copy is available at http://membership.acs.org/C/CCS/pub_3.htm
(accessed June, 2009). Also available by request at OSS@acs.org.
7. Taylor, J.K., Quality Assurance of Chemical Measurements. 1987, Boca Raton:
Lewis Publishers.
8. Li, X-h., Z-h Chen, Y-f Lu , X-w. GE, and J. Quo.Safety destruction of
tetramethylene disulfotetramine and its medical waste. Chin J Prev Med, 2005.
39(2): p. 88-90.
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