EPA 600/R-13/027 | March 2013 | www.epa.gov/ord
United States
Environmental Protection
Agency
High Throughput Determination
of VX in Drinking Water by
Immunomagnetic Separation and
Isotope Dilution High Performance
Liquid Chromatography Tandem
Mass Spectrometry (HPLC/MS/MS)
Office of Research and Development
National Homeland Security Research Center
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EPA/600/R-13/027
March 2013
HIGH THROUGHPUT DETERMINATION OF VX IN DRINKING WATER BY
IMMUNOMAGNETIC SEPARATION AND ISOTOPE DILUTION HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY
(HPLC/MS/MS)
Version 1.0
Centers for Disease Control and Prevention
Atlanta, GA 30333
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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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 Interagency
Agreement (IA) # DW75-92259701 with the Centers for Disease Control and Prevention
(CDC). The VX standard provided for this research was provided by Lawrence Livermore
National Laboratory (LLNL) under EPA IA#DW89-92261601 and a material transfer agreement
between LLNL and CDC. This content has been peer and administratively reviewed and has
been approved for publication as a joint EPA and CDC document. Approval does not signify that
the contents necessarily reflect the views of the EPA, LLNL, the CDC, the Public Health
Service, or the U.S. Department of Health and Human Services. Reference herein to any specific
commercial product, process, or service by trade name, trademark, manufacturer, or otherwise
does not constitute or imply its endorsement, recommendation, or favoring by the United States
government. The views and opinions expressed herein do not necessarily state or reflect those of
the United States government and shall not be used for advertising or product endorsement
purposes.
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
Jennifer Knaack, PhD
Mercer University
3001 Mercer University Drive
Atlanta, GA 30341
(678) 547-6737
knaackJ s@mercer. edu
Rudolph Johnson, PhD
Centers for Disease Control and Prevention
4770 Buford Highway, MS F-44
Atlanta, GA 30341
770-488-3543
Rmi6@cdc.gov
11
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Acknowledgments
The following researchers were critical to the development of the method and preparation of the
procedure:
Centers for Disease Control and Prevention, National Center for Environmental Health
Jennifer S. Knaack (currently at Mercer University)
Rudolph Johnson
The following individuals served as members and technical advisors of the Project Team:
U.S. Environmental Protection Agency (EPA), Office of Research and Development,
National Homeland Security Research Center
Matthew Magnuson (EPA Technical Lead)
Erin Silvestri (EPA Project Officer)
in
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Table of Contents
Disclaimer ii
Acknowledgments iii
List of Tables iv
List of Acronyms v
Executive Summary viii
CHAPTERS
1. Scope and Application 1
2. Summary of Method 3
3. Definition 3
4. Interferences 5
5. Safety 6
6. Equipment and Supplies 7
7. Reagents and Standards 10
8. Sample Collection, Preservation, and Storage 16
9. Quality Control 18
10. Calibration and Standardization 24
11. Procedure 27
12. Data Analysis and Calculations 33
13. Method Performance 34
14. Pollution Prevention 35
15. Waste Management 36
16. References 37
List of Tables
Table 3-1. Ion Transitions Monitored for VX-BuChE Peptide and Internal Standard 5
Table 6-1. High Performance Liquid Chromatograph (HPLC) Parameters 9
Table 6-2. Tandem Mass Spectrometer (MS/MS) Parameters 10
Table 7-1. Calibration Standard Stock Solution Volumes 15
Table 8-1. Preservative Concentrations and Purposes of Preservatives 17
Table 13-1. Method Performance 34
Table 13-2. Single Lab Precision and Accuracy Data 35
Table 13-3. Percent Recovery of VX for Several Tap Water Matrices with Sodium Omadine and Sodium
Thiosulfate 35
iv
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Acronyms
BuChE butyrylcholinesterase
CAS chemical abstract service
CCC continuing calibration check
CDC Centers for Disease Control and Prevention
CR confirmation ratio
CTMDL Chemical Terrorism Methods Development Laboratory
DMP dimethylpimelimidate dihydrochloride
DL detection limit
El electron ionization
EPA U.S. Environmental Protection Agency
FD field duplicate
LC liquid chromatography
LCtso median lethal concentration in time
HRpir half range for the predicted interval of results
HPLC high performance liquid chromatography
i.d inside diameter
IDC initial demonstration of capability
IMS immunomagnetic separation
IS internal standard
LDso median lethal dose
LFB laboratory fortified blank
LFSM laboratory fortified sample matrix
LFSMD laboratory fortified sample matrix duplicate
LLNL Lawrence Livermore National Laboratory
LRB laboratory reagent blank
MRL minimum reporting level
MRM multiple reaction monitoring
MS/MS tandem mass spectrometer
MSDS Material Safety Data Sheet
OSHA Occupational Safety and Health Administration
PAL provisional advisory level
PBS phosphate buffered saline
PBST phosphate buffered saline with Tween-20®
PIR mean prediction interval of result ± half range for the predicted interval of results
QC quality control
QCS quality control sample
RBC risk based criteria
RPD relative percent difference
Sect section
TBS tris buffered saline
TOC total organic carbon
VX O-Ethyl S-2-Diisopropylamino-Ethyl Methylphosphonothioate
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Executive Summary
This document provides the standard operating procedure for determination of the chemical
warfare agent VX (O-Ethyl S-2-Diisopropylamino-Ethyl Methylphosphonothioate) in drinking
water by isotope dilution liquid chromatography tandem mass spectrometer (LC/MS/MS). This
method was adapted from one that was initially developed by the Centers for Disease Control
and Prevention, in the National Center for Environmental Health for the determination and
quantitation of VX in aqueous matrices. This method is designed to support site characterization
and to inform site-specific cleanup goals of environmental remediation activities following a
homeland security incident involving this analyte.
In this method, magnetic beads coated with butyrylcholinesterase (BuChE), a serum protein
target of VX, form stable covalent adducts with VX that can be digested into peptides and
analyzed for VX. First, a 50-mL water sample is collected, and preserved with sodium
thiosulfate (80 mg/L) (a dechlorinating agent) and sodium omadine (64 mg/L) (an antimicrobial
preservative). The sample is buffered and aliquotted into a 96-well plate containing magnetic
beads that have been conjugated to antibodies against BuChE and further conjugated to BuChE
from human serum. The sample is incubated with the magnetic beads for two hours allowing
VX-BuChE adducts to form. After washing the beads to remove residual sample, the extracted
proteins are enzymatically digested to convert the protein adducts into smaller peptide adducts.
Following digestion, the beads are removed from digest solution, isotopically-labeled peptide
internal standard is added, and the sample is filtered to remove residual beads and undigested
proteins. Filtered samples are then separated by high performance liquid chromatography and
analyzed by tandem mass spectrometry (HPLC/MS/MS) for VX-BuChE peptide adducts using
multiple reaction monitoring. 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 LC/MS/MS conditions. Quantitation is performed
using the internal standard technique. Utilization of an isotopically labeled internal standard VX-
BuChE adducts provides a high degree of accuracy and precision for sample quantitation by
accounting for analyte recovery from sample filtration and analytical efficiency.
Accuracy and precision data have been generated in reagent water, and in finished ground and
surface waters containing residual chlorine or chloramine that have been used as disinfectants.
VI
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HIGH THROUGHPUT DETERMINATION OF VX IN DRINKING WATER BY
IMMUNOMAGNETIC SEPARATION AND ISOTOPE DILUTION HIGH
PERFORMANCE LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY
(HPLC/MS/MS)
1. SCOPE AND APPLICATION
1.1 This report describes an isotope dilution liquid chromatography tandem mass
spectrometer (LC/MS/MS) method for the determination of the
organophosphorous nerve agent, VX (O-Ethyl S-2-Diisopropylamino-Ethyl
Methylphosphonothioate; Chemical Abstract Services [CAS] Registry Number®
50782-69-9) in the drinking water matrix. This method, including quality control
(QC) requirements, is designed to support site characterization and to inform site-
specific cleanup goals of environmental remediation activities following a
homeland security incident involving this analyte. VX is not a chemical regulated
in drinking water under the Safe Drinking Water Act (as amended), so there is no
maximum contaminant level for VX in drinking water that has been set by federal
regulation [1].
1.2 Significance: VX is a highly toxic synthetic organophosphorous nerve agent,
which is used solely as a chemical weapon. The estimated oral LDso (median
lethal dose) for liquid VX in rats has been reported at 100 ug/kg and 66 ug/kg [2].
Risk-based criteria (RBC) have been identified from existing health benchmarks
to serve as analytical targets when developing analytical methods for various
chemicals. RBCs reflect a lifetime exposure and are expected to generally be
lower than minimum reporting levels (MRLs) required for most site-specific
objectives. The RBC for VX in water is 0.021 ug/L for the general public for 30
days [3]. VX is persistent in the environment and less volatile than other nerve
agents such as sarin [2,4]. Subchronic exposure to VX has been shown to induce
behavioral effects in rats [5] so monitoring for low levels of VX is necessary to
protect the environment and public health.
1.3 Samples are extracted by immunomagnetic separation (IMS), which provides
increased selectivity and sensitivity for VX in aqueous matrices. Magnetic beads
coated with butyrylcholinesterase (BuChE), a serum protein target of VX, form
stable covalent adducts with VX that can be digested into peptides and analyzed
for VX.
1.4 Whether performed manually or with automation, the use of 96-well plates for the
IMS procedure provides a key benefit to sample extraction throughput. The 96-
well plates allow for extensive automation of the method, thereby enabling high
throughput of samples, as might be required during environmental remediation.
1.5 Isotopically labeled peptides corresponding to VX adducts to
butyrylcholinesterase (BuChE) serve as an internal standard. The internal standard
1
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is added equally to all unknowns, quality controls, and calibration standards after
sample extraction and before sample filtration. In addition to enabling accurate
quantitation of samples, calibrators, and QC samples by tandem mass
spectrometry (MS/MS), internal standards also account for and resolve some of
the issues surrounding analysis including analysis efficiency and sample loss
during filtration. The overall QC approach utilizing quantitation and confirmation
ions, as well as an isotopically labeled internal standard, greatly increases
confidence that VX, and not another molecule with similar fragmentation
patterns, is being quantitated during analysis.
1.6 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, Division of Laboratory Sciences, Emergency Response Branch, in the
Chemical Terrorism Methods Development Laboratory (CTMDL) for the
determination and quantitation of VX in aqueous matrices [6]. For the adapted
method, accuracy and precision data have been generated in reagent water, and in
finished ground and surface waters that contain residual chlorine or chloramine
that have been used as disinfectants.
1.7 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.
1.8 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.
1.9 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.
1.10 This method is intended for use by analysts skilled in the performance of IMS
extractions, the operation of high performance liquid chromatography tandem
mass spectrometer (HPLC/MS/MS) instruments, and the interpretation of the
associated data.
1.11 This method has been verified using only the conditions and equipment specified
in the method. Alteration of this method is not recommended.
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2. SUMMARY OF METHOD
2.1 A 50-mL water sample is collected, and preserved with sodium thiosulfate (80
mg/L) (a dechlorinating agent) and sodium omadine (64 mg/L) (an antimicrobial
preservative). Magnetic beads are conjugated to antibodies against BuChE and
further conjugated to BuChE from human serum. The sample is buffered and
dispensed as aliquots into a 96-well plate containing the prepared magnetic beads.
The sample is incubated with the magnetic beads for two hours in order to allow
VX-BuChE adducts to form. After washing the beads to remove residual sample,
the proteins are enzymatically digested to convert the protein adducts into smaller
peptide adducts. Following digestion, the beads are removed from digest solution,
isotopically-labeled peptide internal standard is added, and the sample is filtered
to remove residual beads and undigested proteins. Filtered samples are then
separated by high performance liquid chromatography and analyzed by tandem
mass spectrometry (HPLC/MS/MS) for VX-BuChE peptide adducts using
multiple reaction monitoring. 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 LC/MS/MS conditions. Quantitation is performed using the internal
standard technique. Utilization of an isotopically-labeled internal standard VX-
BuChE adduct peptide provides a high degree of accuracy and precision for
sample quantitation by accounting for analyte recovery from sample filtration and
analytical efficiency.
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, 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).
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3.2 CALIBRATION STANDARD STOCK SOLUTION- a solution prepared from
the primary dilution standard solution(s) and/or stock standard solution(s) and the
internal standard(s). The calibration standard stock solutions are used to calibrate
the instrument response with respect to analyte concentration.
3.3 CONFIRMATION ION TRANSITION - the second most abundant ion transition
for the VX-BuChE peptide adduct analyte (see Confirmation Ratio, Sect. 3.4,
below). The confirmation ion transition for this peptide is listed in Table 3-1. The
confirmation ion transition is used to calculate the confirmation ratio (Sect. 3.4).
3.4 CONFIRMATION RATIO (CR) - peak area produced by the confirmation ion
transition divided by the peak area produced by the quantitation ion transition
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
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. The internal standard ion transition monitored in this method
is listed in Table 3-1.
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 (Sect. 7.3.5.3).
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3.11 MATERIAL SAFETY DATA SHEET (MSDS) - written information provided
by vendors detailing a chemical's toxicity, health hazards, physical properties, fire
and reactivity data, and including precautions for storage, spill, and handling.
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 TRANSITION - the most abundant ion transition for
each analyte and internal standard as shown below (See Confirmation Ratio, Sect.
3.4, above). Only quantitation ion transitions are monitored for internal standards.
The quantitation ion for this method is listed in Table 3-1.
Table 3-1. Ion Transitions Monitored for VX-BuChE Peptide and Internal Standard
Analyte
VX-BuChE Peptide Analyte
Internal Standard
Precursor Ion
[M + H]+
902
913
Quantitation Ion
[M + H - nerve
agent adduct - OH]+
778
785
Confirmation Ion
[M + H - nerve agent
adduct - OH - serine]+
673
Not Monitored
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 can be caused by contaminants in solvents, reagents
(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
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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 bleach and reagent-grade water followed by
acetonitrile. Allow glass to dry completely before use. It is important that all
residual bleach is removed from glassware that will be used in this method as
bleach will degrade VX. An oven can be used to dry glassware thoroughly, but
should not be used for decontamination purposes.
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 11.3.5 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 complying with OSHA regulations regarding safe handling of
chemicals used in this method. A reference file of MSDSs must be made available
to all personnel involved in sampling handling or chemical analyses. Additional
references to laboratory safety are available [5, 7, 8].
5.2 VX is highly toxic and all routes of exposure (dermal, inhalation, ocular, etc.)
should be avoided. The estimated LD50 value for VX is 10 mg for dermal
exposure and the estimated LCtso (median lethal concentration in time) value for
VX exposure to vapors is 10 mg • min/m3 [7]. The oral LDso value is estimated
between 66-100 |j,g /kg [2] with a provisional advisory level (PAL) value for
ingestion of 0.22 |J,g/L [3]. The concentration of solutions of VX used in this
method should never exceed 10 |j,g/mL. Universal safety precautions should be
used including the use of a lab coat, safety glasses, appropriate gloves, and a high
quality-ventilated chemical fume hood and/or biological safety cabinet. In
addition, all work areas should be thoroughly cleaned with a 5% hypochlorite
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solution before and immediately after work with VX is performed in these areas.
All materials that come in contact with VX should be decontaminated in a
container of 5% solution of hypochlorite. An additional container of same
solution should be available during any work with VX and used to decontaminate
spills, equipment, and other work areas.
5.3 Avoid inhalation or dermal exposure to acetonitrile, methanol, and formic acid,
which are used in the sample preparation and analysis steps.
5.4 Mechanical hazards when performing this procedure using standard safety
practices are minimal. Read and follow the manufacturers' information regarding
safe operation of the equipment. Avoid direct contact with the mechanical and
electronic components of the liquid 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 verification 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 - Model 4780 (Eppendorf Co., Westbury, NY) or
equivalent
6.3 ANALYTICAL BALANCE - Capable of weighing to the nearest 0.0001 g
6.4 IMMUNOMAGNETIC SEPARATION EQUIPMENT 96-WELL MAGNET OR
MAGNETIC BEAD AUTOMATION EQUIPMENT WITH 96 WELL PLATES
6.4.1 IMS can be performed manually or using automated equipment.
6.4.2 Manual extractions require magnets compatible with 96-well formatting.
Equipment required for manual extractions:
6.4.2.1 DynaMag® 2 magnetic bead separator (PN# 12321D, available
from Invitrogen, Grand Island, NY) or equivalent
6.4.2.2 Dynal® sample mixer (PN# 947-01, available from Invitrogen,
Grand Island, NY) or equivalent
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6.4.3 Automated extraction can be performed using automation instrumentation
such as the KingFisher® Flex Magnetic Particle Processor with deep-well
head (Thermo Fisher Scientific, Waltham, MA; PN# 95041-912 available
from VWR, St. Louis, MO). Consumables for use with the KingFisher
instrument include:
6.4.3.1 Eppendorf® MixMate® plate mixer (Eppendorf AG, Hamburg,
Germany; PN#14900-548 available from VWR, St. Louis, MO) or
equivalent
6.4.3.2 96-well deep V-bottom KingFisher microplates, 2mL (PN#11388-
566 available from VWR, St. Louis, MO) or equivalent
6.4.3.3 KingFisher 96-well plates, 200 |iL (PN# 83007-596 available from
VWR, St. Louis, MO) or equivalent
6 A3 A KingFisher tip comb for 96-well deep well magnets (PN# 83007-
594 available from VWR, St. Louis, MO) or equivalent
6.4.4 All extractions require the following additional instrumentation:
6.4.4.1 A water bath capable of maintaining 37°C such as the Precision
water bath (Winchester, VA) or equivalent
6.4.4.2 Heat sealer (PN# AB0384/110, available from ABgene House,
Surrey, UK) or equivalent
6 A A3 Easy Pierce 20 um heat sealing foil (Thermo Fisher Scientific,
Waltham, MA; PN# AB1720, available from ABgene House,
Surrey, UK) or equivalent
6.4.4.4 Eppendorf adhesive foils (PN# 0030127820, available from Fisher
Scientific, Fair Lawn, NJ) or equivalent
6.4.4.5 96-well PCR plate (ABgene product, Thermo Fisher Scientific,
Waltham, MA; sold by Advion, Inc., Ithaca, NY, PN# 1002611) or
equivalent
6.4.4.6 Multiscreen Ultracel®-10 filter plates (PN# MAUF01010 available
from Fisher Scientific, Pittsburgh, PA) or equivalent
6.4.4.7 MixMate plate mixer (PN# 53532G807598 available from
Eppendorf, Hamburg, Germany) or equivalent
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6.5 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ELECTRON
IONIZATION TANDEM MASS SPECTROMETRY SYSTEM (HPLC/MS/MS)
6.5.1 HPLC COLUMN - Aquasil® CIS column, 50 x 1 mm inside diameter
(i.d.), 3 um particle size (Aquachemi Inc., Missouri, TX; PN# 77503-
051030 available from Fisher Scientific, Pittsburgh, PA) or equivalent
6.5.2 HPLC SYSTEM - The LC system (Waters nanoAcquity HPLC, Waters
Technology Inc., Milford, MA; or equivalent) should be equipped with an
autosampler and injector and should provide consistent sample injection
volumes. Mobile phases should be connected to an inline degasser that
runs consistently during sample analysis. The HPLC should be capable of
being configured exactly as stated in Table 6-1.
Table 6-1. High Performance Liquid Chromatograph (HPLC) Parameters
Parameter
LC Method
Column type
Injection Volume
Autosampler Tray Temperature
Column Temperature
Injection Settings
Needle Rinse Settings
Sample Loop Option
Typical retention time for VX-
BuChE
Setting
Gradient:
Reservoir A = 0. 1% Formic Acid in HPLC-Grade Water
Reservoir B = 0. 1% Formic Acid in Acetonitrile
Time(min) %A %B Flow Rate (jiL/min)
0 98 2
0.1 98 2
1.4 65 35
2.2 65 35
2.5 55 45
4.5 55 45
4.51 98 2
5.0 98 2
75
75
75
75
75
75
75
75
Aquasil C18 3(im column, 1.0x50 mm, 3 um particle size
10 (iL
4°C
25±5°C
Draw Speed: 200 uL/min
Eject Speed: 200 uL/min
Injection Mode: Standard
Weak Solvent Wash: Water, 600 uL
Strong Solvent Wash: 50%Methanol
ML)
Partial Loop (Loop Offline Disabled)
1.87 minutes
in HPLC-Grade Water (200
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6.5.3 MASS SPECTROMETER (MS) - The MS/MS (Applied Biosystems API
4000 quadrupole ion trap mass spectrometer, Foster City, CA; or
equivalent) should be capable of performing electrospray ionization with
both positive and negative ion detection and must be configured for
multiple reaction monitoring (MRM) with a dwell time of 100 msec per
ion. Tandem mass spectrometer (MS/MS) parameters used during method
verification are shown in Table 6-2.
Table 6-2. Tandem Mass Spectrometer (MS/MS) Parameters used During Method
Verification
Parameter
MS Scan Mode
Ionization Type
Dwell Time
Curtain Gas
Source Temperature
Ion Source Gas 1
Ion Source Gas 2
Collision Gas
Ion Spray Voltage
Entrance Potential
Collision Energy
Cell Exit Potential
Setting
Multiple Reaction Monitoring
Electrospray ionization
35 msec per channel
5
250 (interface heater ON)
40
50
11
5500
12
41
20
7. REAGENTS AND STANDARDS (These reagents were used during the verification of
the method, and only these or their equivalent are acceptable for use. No endorsement of
any supplier or organization should be inferred.)
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 NITROGEN - 99.9999% pure or better, MS/MS collision cell gas
7.1.2 REAGENT WATER - purified, deionized water which does not contain
any measurable quantities of the method analyte or interfering compounds,
HPLC or equivalent grade water (available from Tedia, Fairfield, OH and
other commercial sources)
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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 (available from Tedia HPLC and
other commercial sources)
7.1.5 FORMIC ACID - (HCOOH, CAS#: 64-18-6) - reagent grade >95%
purity, demonstrated to be free of analytes and interferences (available
from Sigma-Aldrich, St. Louis, MO and other commercial sources)
7.1.6 1 Ox PHOSPHATE BUFFERED SALINE WITH TWEEN-20® , pH 7.4
(PBST) - demonstrated to be free of analytes and interferences (available
from Sigma-Aldrich and other commercial sources)
7.1.7 TRIETHANOLAMINE BUFFER SOLUTION - demonstrated to be free
of analytes and interferences (PN# T0449-120ML from Sigma-Aldrich, or
equivalent)
7.1.8 PHOSPHATE BUFFERED SALINE lOx - Phosphate buffered saline lOx
concentrate demonstrated to be free of analytes and interferences
(available from Sigma-Aldrich and other commercial sources)
7.1.9 DIMETHYLPIMELIMIDATE DfflYDROCHLORIDE (DMP) -
demonstrated to be free of analytes and interferences (available from
Sigma-Aldrich and other commercial sources)
7.1.10 0.2M TRIS BUFFERED SALINE, lOx solution - demonstrated to be free
of analytes and interferences (available from Sigma-Aldrich and other
commercial sources)
7.1.11 PEPSIN FROM PORCINE GASTRIC MUCOS A - demonstrated to be
free of analytes and interferences (available from Sigma-Aldrich and other
commercial sources)
7.1.12 BuChE ANTIBODIES FROM CLONE SEA - Product number
HAH0020102 (Fisher Scientific, Fair Lawn, NJ) or equivalent
7.1.13 PROTEIN G DYNABEADS® magnetic beads - Product number 10003D
(from Invitrogen, Grand Island, NY) or equivalent
7.1.14 POOLED HUMAN SERUM - Pooled human serum preserved with
potassium EDTA (ethylenediaminetetraacetic acid) from Tennessee Blood
Services (Memphis, Tennessee) or equivalent. Serum should be verified as
11
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free from interferences by extracting blank samples as described in Sect.
11.
7.1.15 SYNTHETIC UNLABELED AND ISOTOPICALLY-LABELED
PEPTIDES CORRESPONDING TO VX-BuChE ADDUCTS -custom
synthesized at Los Alamos National Laboratory (Los Alamos, NM)
7.1.16 VX IN ISOPROPANOL, 10 ppm - Obtained from Lawrence Livermore
National Laboratory through an agreement with the EPA (Interagency
Agreement #DW89-92261601)
7.1.17 SAMPLE PRESERVATION REAGENTS - the following sample
preservation reagents are required for this method:
7.1.17.1 SODIUM THIOSULFATE (Na2S2O3, CAS#: 7772-98-7) - an
additive used in sample collection (Sigma-Aldrich ACS grade or
equivalent)
7.1.17.2 SODIUM OMADINE (C5H4NNaOS, CAS#: 3811-73-2) - an
additive used for sample collection (Sigma-Aldrich > 96% pure or
equivalent)
7.2 REAGENT PREPARATION
7.2.1 PHOSPHATE BUFFERED SALINE IX (PBS) - Dilute 100 mL of lOx
phosphate buffered saline with 900 mL of HPLC-grade water and mix
well.
7.2.2 PHOSPHATE BUFFERED SALINE WITH TWEEN-20 IX (PBST) -
Dilute 100 mL of lOx phosphate buffered saline with 900 mL of HPLC-
grade water and mix well.
7.2.3 TRIS BUFFERED SALINE IX (TBS) - Dilute 100 mL of lOx tris
buffered saline with 900 mL of HPLC-grade water and mix well.
7.2.4 0.1% FORMIC ACID IN WATER (MOBILE PHASE A) - Prepare a
0.1% solution of formic acid through dilution with HPLC-grade water. For
example, add 500 jiL formic acid to 500 mL HPLC-grade water in a
volumetric container.
7.2.5 0.1% FORMIC ACID IN ACETONITRILE (MOBILE PHASE B) -
Prepare a 0.1% solution of formic acid through dilution with acetonitrile.
For example, add 500 jiL formic acid to 500 mL HPLC-grade water in a
volumetric container.
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7.2.6 0.6% FORMIC ACID IN WATER - Prepare a 0.6% solution of formic
acid through dilution with HPLC-grade water. For example add 600 uL
formic acid to 100 mL HPLC-grade water in a volumetric container.
7.2.7 DMP SOLUTION - Prepare a 5.4 mg/niL solution of DMP in
triethanolamine buffer. For example, weigh out 27 mg DMP and dissolve
in 4 mL triethanolamine buffer.
7.2.8 PEPSIN SOLUTION - Prepare a 2 mg/mL solution of pepsin in 5%
formic acid in water. For example, weigh out 20 mg of pepsin and
dissolve in 9.5 mL HPLC-grade water with 0.5 mL formic acid and mix
thoroughly. This solution should be prepared 30 minutes prior to use.
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 for VX-BuChE is 13C4D615N-labeled and is
custom-synthesized by Los Alamos National Laboratory (Los Alamos,
NM). The internal standard can be custom-synthesized by other companies
including Battelle Memorial Institute (Columbus, OH) or other
appropriate vendors. Note that in this method, the isotopically-labeled
internal standard is structurally identical to the method analyte, but
substituted with 13C4, De, and 15N. Isotopically-labeled internal standards
have no potential to be present in water samples, and are not method
analytes. These internal standards are 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 550 ng/mL.
Steps for the preparation of the mixture are described below:
7.3.2.1 RECONSITUTE INTERNAL STANDARD - Weigh out 19.2 mg
of VX-BuChE internal standard and dilute to 2 mL with 0.6%
formic acid in water using a 2 mL volumetric flask. The
concentration of this solution is 9.6 mg/mL.
7.3.2.2 INTERNAL STANDARD STOCK SOLUTION - Dilute the
reconstituted internal standard solution to 550 ng/mL. To make 10
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mL of this solution, dilute 573 uL of the reconstituted internal
standard (9.6 mg/mL) to 10 mL with 0.6% formic acid in water
using a 10 mL volumetric flask.
7.3.3 ANALYTE STOCK STANDARD SOLUTIONS - Obtain a stock solution
of 10 ppm VX in isopropanol from an appropriate source. 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 -Make 50 uL of
a 1000 ng/mL solution of VX in HPLC-grade water. To make this
solution, dilute 5 uL of 10 ppm VX with 45 uL HPLC-grade water
and mix well. This solution should be prepared fresh for each
analysis.
7.3.3.2 ANALYTE STOCK STANDARD SOLUTION 2 - Make 200 uL
of a 100 ng/mL solution of VX in HPLC-grade water. To make
this solution, dilute 20 uL of analyte stock standard solution 1
(1000 ng/mL) with 180 uL HPLC-grade water and mix well.
7.3.3.3 ANALYTE STOCK STANDARD SOLUTION 3 - Make 1000 uL
of a 1 ng/mL solution of VX in HPLC-grade water. To make this
solution, diluted 10 uL of analyte stock standard solution 2 (100
ng/mL) with 990 uL HPLC-grade water 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 Sections 2.2 and 8.1.3). The calibration curve
is composed of at least five 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.) All
standards should be diluted into HPLC-grade water.
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Table 7-1. Calibration Standard Stock Solution Volumes
entration VX Analyte Stock Analyte Stock
(ng/mL) T6 Solution 2 (uL) Solution 3 (uL)
4 700 28
2 700 14
1.13 700 7.9
0.31 700 217
0.25 700 175
0.09 700 63
0.025 700 17.5
0 700
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 competently replicated, and that the laboratory has
the capability to make accurate and precise measurements. The two
specific LFBs 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 is 0.25 ng/mL VX. The LFB-high for this demonstration
is 2.0 ng/mL VX. LFB-low and LFB-high can be 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 to develop QC criteria during the initial demonstration of
capability (Sect. 9.2) and serve as an additional QC function during
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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
[BD, Franklin Lakes, NJ] or equivalent).
8.1.2 VX is not stable in some finished tap water samples without preservatives
and will rapidly degrade over several days. All samples used in
development of this method, including QC samples, were preserved via
addition of sodium thiosulfate (80 mg/L) (a dechlorinating agent) and
sodium omadine (64 mg/L) (an antimicrobial preservative). The choice of
these preservative is based on the stability of VX in the presence of
chlorine and monochloramine with preservatives tested, as described in
Knaack et al. [8], which suggests VX degradation rate is slowed to the
theoretical minimum by this combination of preservatives. Studies of VX
indicate that hydrolysis can only be slowed and not stopped. Therefore,
any data quality objectives for site-specific remediation plans must take
this degradation into account. Minimizing the time between sample
collection and analysis will maximize analyte signal.
8.1.3 SAMPLE STORAGE STABILITY STUDIES - Stability data were
collected up to 91 days preserved with sodium omadine and sodium
thiosulfate and stored at 4°C. This corresponds to approximately one half-
life of VX degradation in the presence of these preservatives.
8.1.4 Vessels should be prepared before sample collection with sodium
thiosulfate and sodium omadine according to Table 8-1. Preservation
through binding free chlorine or dechlorination is necessary for all
samples that will be analyzed for VX. All initial and on-going QC
requirements should be demonstrated for the preservatives added to the
sample.
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Table 8-1. Preservative Concentrations and Purposes of Preservatives
Compound
Sodium
thiosulfate
Sodium
Omadine
Mass added to
sample (mg)
4
3.2
Concentration in
sample (g/L)
0.08
0.064
Purpose
Dechlorinates free
chlorine and chloramine
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 tubing, gaskets, and other parts that could leach interfering analytes into
the water sample.
8.3 SAMPLE SHIPMENT AND STORAGE - Sample stability was tested at 4°C and
stability measurements are only valid at this temperature. As a matter of practice,
ensure that samples do not experience excessive heat above this temperature. It is
recommended that all samples be iced, frozen (-20±5°C), or refrigerated (4±2°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) suggest that VX stability is best preserved when
samples are collected, preserved, shipped, and stored as described in Sections 8.1,
8.2, and 8.3. VX will hydrolyze in aqueous matrices although adequate detection
is possible after holding samples for 91 days. As matter of practice, water samples
should be extracted as soon as possible but must be extracted within 91 days for
this method. Data generated during this study indicates that extracts are stable for
at least 28 days when preserved and stored at 0°C or lower. As matter of practice,
analysis should occur as soon as possible.
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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 [9].
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 to ensure successful long-term implementation of the method
in a variety of other labs. Due to site-specific conditions during an 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 might 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, magnetic beads, or 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 seven 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 relative standard
deviation for the concentrations of the replicate analyses should 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 should be within ± 30% of the true value.
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9.2.4 MINIMUM REPORTING LEVEL (MRL) ESTIMATION - Because
cleanup 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:
HRpm = 3.963s
where s is the standard deviation and 3.963 is a constant value for
at least 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 should be <150% recovery.
Mean + HRP1R
FortifiedConcentration
The Lower PIR Limit should be > 50% recovery.
Mean-HRPIR
FortifiedConcentration
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 should be confirmed again
at a higher concentration.
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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 can 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
LC/MS/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 calculate the DL. (For example, for the results
presented in Section 13, eight replicate LFBs were analyzed over 10 days,
with two 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 = s x t(n.\)
where:
s = standard deviation of replicate analysis, without blank subtraction
t = Student's t value for the 99% confidence level with n-\ degrees of
freedom
n = number of replicates
9.3 ONGOING QC REQUIREMENTS - This section summarizes the ongoing QC
criteria 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.
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
the method analyte. Running the LRB first could 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, accurate determination of the analyte will not be
possible. 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
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of method analyte should 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
analyte is detected in the LRB at concentrations greater than 1/3 the MRL,
then all data for VX are considered invalid for all samples in the analysis
batch.
9.3.2 ONGOING CALIBRATION - The analytical system is 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 are described in Section 10.2.5, except that removal of
calibration points could 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 should 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 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 should 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 CCC 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
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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 VX analyte stock standard solution
1 (Sect. 7.3.3.1) is prepared, 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 should 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 should monitor the peak area
of the IS in all injections during each analysis day. The IS peak area must
meet the criteria in the following two subsections (9.3.6.1 and 9.3.6.2).
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 transition of the
corresponding analyte 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.
• If the reinjected aliquot produces an acceptable IS
response, report results for that aliquot.
• 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 or
collect a new sample and re-analyze.
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9.3.7 LABORATORY FORTIFIED SAMPLE MATRIX (LFSM) and LFSM
DUPLICATES (LFMSD) - 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 remediation, it is
not expected that there would be a VX 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 most failures in IS QC requirements result
from failure of the automation equipment. This would correspond to
LFSM failure, as well. Accordingly, neither LFSMs nor 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/remediation plan that produces a co-eluting peak
with identical chromatographic and mass spectral properties as VX. In this
case, the lab should discuss the irregularities with the submitter.
9.3.7.1 If an LFSM and LFSMD are deemed necessary, calculate the
relative percent difference (RPD) for duplicate LFSMs (LFSM and
LFSMD) using the equation
LFSM-LFSMD
RPD = -, r— x 100
(LFSM+LFSMD} 12
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 could 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 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.
9.3.8 FIELD DUPLICATE (FD) - Field duplicates are used to check the
precision associated with sample collection, preservation, storage, and
laboratory procedures. Some of these factors are out of the 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 the results do not meet the following criteria.
23
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9.3.8.1 Calculate the relative percent difference (RPD) for duplicate
samples (FD1 and FD2) using the equation
FD2)/2
xlOO
9.3.8.2 RPDs for FDs should be <30%. Greater variability could 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 should be repeated each time a major
instrument modification is made or maintenance is performed, and prior to analyte
calibration.
10.2 INITIAL CALIBRATION
10.2.1 MS/MS TUNE - Calibrate the mass and abundance scales of the MS/MS
with calibration compounds and procedures prescribed by the
manufacturer with any modifications necessary to meet tuning
requirements. For an Applied Biosystems® API 4000 tandem mass
spectrometer, some labs have experienced better results if following the
automatic tune, they perform a manual tune to fine-tune declustering
potential, collision energy, and collision cell exit potential settings. For
other instruments, follow manufacturer's protocols to tune the instrument.
10.2.2 INSTRUMENT CONDITIONS - Operational conditions for the
instrument used in this verification are tabulated in Section 6.8.3 and
Table 6-2. Alteration of the conditions is not recommended and would
require redevelopment of QC criteria. Frequently reported problems can
24
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be avoided by: (1) checking that needle wash solutions are adequately
filled and the injection needle is functioning properly and (2) changing the
analytical column as needed.
10.2.3 CALIBRATION STANDARDS- 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
isotopically-labeled internal standard is added after sample extraction and
prior to filtration. In practice, the lowest concentration of the calibration
standard should 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 is distributed between these
two extremes, with the majority of points in the concentration range where
contaminated samples are expected to fall.
10.2.4 HPLC/MS/MS CALIBRATION- The HPLC/MS/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 0.980.
Linearity of the standard curve should extend over the entire standard
range. Each calibration point for the analyte should calculate to be 70 to 130
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 r2 value for the curve is < 0.980), 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 should be adjusted accordingly. The
resulting r value should be greater than 0.980.
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.
25
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10.3.1 Inject an aliquot of calibration salutation at the appropriate concentration
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 HPLC/MS/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) 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
should 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
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 solid phase extraction 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 or re-inject standards.
ii. If peak tailing or fronting is a significant issue, replace the
HPLC column.
iii. Ensure the source of the MS/MS is clean.
iv. Clean the mass spectrometer source plate.
v. Flush all tubing on the HPLC/MS/MS instrument with
95% / 5% acetonitrile/water for 15 minutes followed by 5
minutes of equilibration with 5% / 95% acetonitrile/water.
26
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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.
Note: Steps 11.1.4 through 11.1.7 can be performed using an automated magnetic
bead processor or with stationary magnets. Data presented in this document was
collected using an automated magnetic bead processor.
11.1.2 Prepare Magnetic Beads (enough for 20 samples)
11.1.2.1 Resuspend Protein G Dynabeads® magnetic beads and transfer
2 mL to a 15-mL Falcon tube. Apply a DynaMag-2 magnet to
the sample for 30 seconds and then remove the supernatant
without disturbing the beads.
11.1.2.2 Add 4 mL PBS to the beads, mix by vortexing, apply a
DynaMag-2 magnet to sample, and then remove supernatant.
Repeat this step two more times.
11.1.2.3 Dilute 400 ug antibody (400 (jL of a 1 mg/mL solution) in 8 mL
PBST. Add this antibody solution to the bead solution and
vortex the beads.
11.1.2.4 Incubate overnight at room temperature with rotation on the
Dynal sample mixer at a setting of 21.
27
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11.1.2.5 Apply a DynaMag-2 magnet to the sample, wait 30 seconds,
and then remove the supernatant.
11.1.2.6 Add 4 mL triethanolamine buffer to the sample, vortex, apply
magnet, and then remove the supernatant. Repeat.
11.12.1 Add 4 mL DMP/triethanolamine buffer (refer to section 7.2.7)
to sample. Vortex.
11.1.2.8 Incubate for 30 minutes at room temperature using a Dynal
sample mixer at a setting of 21.
11.12.9 Apply a DynaMag-2 magnet to the sample, wait 30 seconds,
and then remove the supernatant.
11.1.2.10 Add 4 mL TBS to sample. Vortex. Incubate at room
temperature for 15 minutes using a Dynal sample mixer at a
setting of 21.
11.1.2.11 Apply a DynaMag-2 magnet to the sample, wait 30 seconds,
and then remove the supernatant.
11.1.2.12 Add 2 mL PBST to sample, vortex, apply magnet, and then
remove supernatant. Repeat twice.
11.1.2.13 Add 10 mL pooled human serum free from interferences (refer
to section 7.1.14) to the beads.
11.1.2.14 Incubate for 2 hours at room temperature using a Dynal sample
mixer at a setting of 21.
11.1.2.15 Apply a DynaMag-2 magnet to the sample, wait 30 seconds,
and then remove the supernatant.
11.1.2.16 Add 2 mL PBST to sample, vortex, apply magnet, and then
remove supernatant. Repeat.
11.1.2.17 Add 2 mL PBST to sample and vortex. This is the final
prepared bead solution. Store at 4°C for up to 8 months.
11.1.3 Prepare 96-wells plates
11.1.3.1 Sample Plate - Label a 96-well deep V-bottom KingFisher
microplate (VWR, St. Louis, MO) as the "Sample Plate" and
add 500 uL of each calibrator, blank, quality control and sample
into individual wells (refer to section 7.3.3).
28
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• Add 55 uL lOx PBST to each well containing a calibrator,
blank, quality control, or sample.
11.1.3.2 Wash Plates - Label three 96-well deep V-bottom KingFisher
microplates (VWR, St. Louis, MO) as "Wash Plate 1," "Wash
Plate 2," and "Wash Plate 3." Add 500 uL of Ix PBST (refer to
section 7.2.2) to each well corresponding to a calibrator, blank,
quality control or sample.
11.1.3.3 Digestion Plate - Label a 200 uL 96-well Kingfisher plate
(VWR, St. Louis, MO) as "Digestion Plate" and set aside.
11.1.3.4 Bead Plate - Label a 200 uL 96-well Kingfisher plate (VWR,
St. Louis, MO) as "Bead Plate" and add 100 uL of the prepared
magnetic beads (refer to section 11.1.2) to each well that
corresponds to a calibrator, blank, quality control or sample.
11.1.3.5 Tip Plate -Place a tip comb into a 200 uL 96-well Kingfisher
plate (VWR, St. Louis, MO) and label the plate "Tip Comb
Plate."
11.1.4 Extract Samples
11.1.4.1 Run KingFisher Flex "Step 1 - Add Beads" Program (See
"KingFisher Flex Magnetic Particle Processor Methods",
Section 11.2.1). The KingFisher Flex particle processor will
transfer the antibody-conjugated beads from the "Bead Plate" to
the "Sample Plate".
11.1.4.2 Seal the "Sample Plate" (now containing magnetic beads) with
an Eppendorf adhesive foil.
11.1.4.3 Incubate "Sample Plate" on a MixMate plate mixer for 2 hours
at 1400 rpm.
11.1.4.4 Prepare pepsin solution. Make a concentrated pepsin solution
30 minutes before samples are finished the 2-hour incubation
period in the magnetic bead solution. It is important to make
this solution exactly 30 minutes before the samples are
finished the 2-hour incubation. Mix thoroughly and then set
aside.
11.1.4.5 When the sample plate has finished mixing for 2 hours, dilute
207 uL of the pepsin solution with 1440 uL of HPLC-grade
water and mix thoroughly.
29
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11.1.4.6 Dispense a 75 uL aliquot of the diluted pepsin solution into the
wells corresponding to calibrators, blanks, quality controls, and
samples in the "Digestion Plate".
11.1.5 Wash Beads using the KingFisher Flex.
11.1.5.1 Remove adhesive PCR foil from "Sample Plate."
11.1.5.2 Run KingFisher Flex: "Step 2 -Bead Washes" program (see
"KingFisher Flex Magnetic Particle Processor Methods", Section
11.2.2).
11.1.5.3 Add "Digestion Plate" plate to the KingFisher processor when
prompted.
11.1.6 Digest Extracts in Pepsin
11.1.6.1 Remove "Digestion Plate" from the KingFisher processor and
cover with an adhesive PCR foil. Carefully place "Digestion
Plate" into a 37°C water bath while being careful to not get
water on top of the plate. The plate will float. Incubate
"Digestion Plate" plate in 37°C water bath for 1.5 hours.
11.1.6.2 Remove "Digestion Plate" plate from 37°C water bath after the
1.5-hour incubation period and flash centrifuge for 3-5 seconds
to collect condensation. Remove PCR foil from the "Digestion
Plate".
11.1.6.3 Run KingFisher Flex: "Step 3 - Remove Beads from Digestion
Plate" program (see "KingFisher Flex Magnetic Particle
Processor Methods", Section 11.2.3) to Remove Beads from the
"Digestion Plate." The "Digestion Plate" contains the extracted
samples.
11.1.7 Add Internal Standard Solution to Extracts and Filter Extracts
11.1.7.1 Add 10 uL internal standard stock solution (refer to section
7.3.2.2) to each calibrator, blank, quality control, and sample
using a single- or multi-channel pipettor, and mix samples with
the pipettor or on a plate shaker.
11.1.7.2 Transfer internal standard-containing extracts from "Digestion
Plate" to a 96-well, 10 kDa filter plate using a single- or multi-
channel pipettor.
30
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11.1.7.3 Place the filter plate directly on top of 96-Well ABgene PCR
Plate and seal with tape around the four sides for maximum
sample recovery.
11.1.7.4 Centrifuge at 3700 rpm for 1.5 hours.
11.1.7.5 Remove the plate assembly from centrifuge and discard the
filter plate. Seal the collection plate with the heat sealer. The
plate is now ready for analysis.
11.2 KingFisher Flex Magnetic Particle Processor Methods. Program the following
methods into the processor.
11.2.1 "Step 1 - Add Beads"
11.2.1.1 Pick up plate "Tip Plate"
11.2.1.2 Collect Beads from "Bead Plate"
• Collect count 1
• Collect time [s] 30
11.2.1.3 Release beads in "Sample Plate"
• Release time [hh:mm:ss] 00:00:05
• Release speed Medium
11.2.2 "Step 2-Bead Washes"
11.2.2.1 Pick up plate "Tip Plate"
11.2.2.2 Collect beads from "Sample Plate"
• Collect count 1
• Collect time [s] 30
11.2.2.3 Wash 1
• Release beads into "Wash Plate 1"
• Mixing time [hh:mm:ss] 00:00:30
• Mixing speed Slow
• Collect count 3
• Collect time [s] 1
11.2.2.4 Wash 2
11.2.2.5 Release beads into "Wash Plate 2"
• Mixing time [hh:mm:ss] 00:00:30
• Mixing speed Slow
• Collect count 3
• Collect time [s] 1
11.2.2.6 Wash 3
11.2.2.7 Release beads into "Wash Plate 3"
• Mixing time [hh:mm:ss] 00:01:00
• Mixing speed Slow
• Pause with the message "Add Digestion Plate"
• Collect count 1
• Collect time [s] 30
31
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11.2.2.8 Begin Digestion
• Release beads into "Digestion Plate"
• Release time [hh:mm:ss] 00:00:05
• Release speed Fast
11.2.3 "Step 3 - Remove Beads from Digestion Plate"
11.2.3.1 Pick up plate "Tip Plate"
11.2.3.2 Collect beads from "Digestion Plate"
• Collect count 1
• Collect time [s] 30
11.2.3.3 Release beads into "Wash Plate 1"
• Release time [hh:mm:ss] 00:00:05
• Release speed Fast
11.3 ANALYSIS OF SAMPLE EXTRACTS
11.3.1 Establish operating conditions for the instrument as described in Section
10.2.2.
11.3.2 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.
11.3.3 Set up the available automation equipment and software as specified by
the manufacturer for batch analysis, paying particular attention to the
following:
11.3.3.1 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) 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.
11.3.3.2 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.
11.3.4 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 HPLC/MS/MS
32
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conditions. At the conclusion of data acquisition, use the same software
that was used in the calibration procedure to 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.
11.3.5 COMPOUND IDENTIFICATION - The presumed peak in the sample
must appear in the same retention time window as the internal standard
corresponding to VX (around 2.2 min in the developer's lab) and have
similar chromatographic characteristics such as peak shape. Relative to the
analyte retention time, the internal standard retention time should be + or -
0.02 minutes. 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 VX (corresponding to VX-BuChE adducts) is
then confirmed through calculating the confirmation ratio (CR), i.e., by
dividing the response for confirmation transition by the response for
quantitation transition of the presumed analyte 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 approximately 0.9 in the developer's lab.)
12. DATA ANALYSIS AND CALCULATIONS
12.1 Concentrations are calculated using the ions listed in Table 3-1. Use of other ions
is not advised. If a particular instrument cannot produce the fragments listed in
Table 3-1, 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 files containing the unknown and QC
concentrations, retention times, standard curves, and other run information.
33
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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 individual laboratory guidelines.
13. METHOD PERFORMANCE
13.1 ANALYTICAL IDENTIFICATION-Analyte identification using the approach
described in Section 11.3.5 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 ion transitions monitored.
13.2 SINGLE LABORATY MINIMUM REPORTING LEVELS and DETECTION
LIMIT- The reportable range of results for VX are summarized below, along with
the DL determined from the IDC procedure described previously. The lowest
standard is used as the minimum reporting level, and the DL calculated from the
standard deviation of replicate measurements of that standard (in the case of Table
13-1, 5.6 ng/mL). The highest reportable limit is based on the highest linear
standard.
Table 13-1. Method Performance
Compound
VX
Minimum Reporting
Level (ng/L)
0.025
Highest Reportable
Limit ((J,g/L)
4.00
Method DL (ng/L)
0.006
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 at least seven replicates for each of the two
concentration levels over a period of 91 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.
34
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Table 13-2. Single Lab Precision and Accuracy Data
Analyte
Sample
LFB-
low
LFB-
high
Fortified
Concentration
(Mg/L)
0.25
2.0
Number of
Replicates
8
8
Mean of
IDC
Replicates
(HS/L)
0.25
2.05
Standard
Deviation
(Mg/L)
0.01
0.12
RSD
(%)
3
6
Accuracy
of Mean
(%)
102
103
13.4 SINGLE LABORATORY RECOVERY AND PRECISION FOR TAP WATER
MATRICES. Table 13-3 expresses percent mean recoveries for VX in several
different chlorinated and chloraminated tap waters derived from the types of
sources (i.e., ground or surface water) indicated. Samples were extracted
immediately after preparation. Water quality parameters describing these sources
are indicated in the footnotes. Percent recoveries were determined by dividing the
measured concentration by the spiked concentration (n=3). No analytes or
interferences were detected in the unspiked samples.
Table 13-3. Percent Recovery of VX from Several Tap Water Matrices with Sodium
Omadine and Sodium Thiosulfate (n=3)
Water Type
Ground Water la
Surface Water 2b
Surface Water 3C
Surface Water 4d
Surface Water 5e
HPLC-Grade Water
% Recovery
81 ± 1
85 ±2
92 ±2
85 ±3
92 ±4
101±2
aTotal organic carbon (TOC) not detected in well-field; pH 7.6; hardness 500 mg/L; Chlorine 0.2-0.4 mg/L
(monthly averages)
bTOC 1.0 mg/L; pH 8.5; hardness 130 mg/L; Chlorine 0.8 mg/L (monthly averages)
°TOC 2.3 mg/L; pH 7.4; hardness 190 mg/L; Monochloramine 3.4 mg/L (monthly averages)
dTOC 7.6 mg/L; pH 9.2; hardness 65 mg/L; Monochloramine 2.4 mg/L (monthly averages)
eTOC 0.3 mg/L; pH 8.9; hardness 17 mg/L; Chlorine 1.3 mg/L (monthly averages)
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
35
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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/CAVPCP_012290/pdfAVPCP_012290.pdf
(accessed May 2010).
15. WASTE MANAGEMENT
15.1 Dispose of waste materials must be in compliance with the individual laboratory's
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, if temporary storage is needed, store them in a chemical fume
hood.
15.2 VX is a highly lethal cholinesterase inhibitor. Dispose of VX in an appropriate
waste stream as well. Unused VX-containing solutions shall be diluted no less
than 3x initial volume with freshly prepared 5% solution of hypochlorite prior to
disposal. Hypochlorite bleach solution should not be more than one week old.
Unused solutions containing VX should then be placed in waste collection
container labeled as corrosive or flammable waste as appropriate. Pipettor tips
used to aspirate VX-containing solution will be fully immersed in freshly
prepared 5% solution of hypochlorite, with bleach solution drawn into pipettor
tip, prior to disposal in an appropriate container.
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16. REFERENCES
[1] U.S. EPA(2012). Drinking water contaminants: National primary drinking water
regulations. US Environmental Protection Agency. Accessed January 15, 2013
http://water.epa. gov/drink/contaminants/index. cfm
[2] N.B. Munro, S.S. Talmage, G.D. Griffin, L.C. Waters, A.P. Watson, J.F. King, V.
Hauschild. (1999). The sources, fate, and toxicity of chemical warfare agent degradation
products. Environmental health perspectives 107 (12): 933-974.
[3] U.S. EPA. (2008). Risk-based criteria to support validation of detection methods for
drinking water and air. U.S. Environmental Protection Agency, Washington, DC.
EPA/600/R-08/021.
[4] N. Munro. (1994). Toxicity of the organophosphate chemical warfare agents GA, GB,
and VX: Implications for public protection. Environmental health perspectives 102 (1):
18-37.
[5] E. Bloch-Shilderman, I. Rabinovitz, I. Egoz, L. Raveh, N. Allon, E. Grauer, E. Gilat,
B.A. Weissman. (2008). Subchronic exposure to low-doses of the nerve agent VX:
physiological, behavioral, histopathological and neurochemical studies. Toxicology and
appliedpharmacology 231 (1): 17-23.
[6] J.S. Knaack, Y. Zhou, C. Abney, S. Prezioso, M. Magnuson, R. Evans, E. M.
Jakubowski, K. Hardy, R. Johnson. High-Throughput Immunomagnetic Scavenging
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