EPA/600/R-16/116 I September 2016 www.epa.gov/homeland-security-research United States Environmental Protection Agency oEPA Analytical Protocol for VX Using Gas Chromatography/Mass Spectrometry (GC/MS) Office of Research and Development Homeland Security Research Program ------- This page left intentionally blank ------- EPA/600/R-16/116 September 2016 Analytical Protocol for VX Using Gas Chromatography/Mass Spectrometry (GC/MS) United States Environmental Protection Agency Office of Research and Development Homeland Security Research Program Cincinnati, Ohio 45268 ------- Acknowledgments This method is based on procedures developed by Lawrence Livermore National Laboratory (LLNL) under Interagency Agreement (IAG) DW89922616-01-0 with the U.S. Environmental Protection Agency (EPA). EPA's Homeland Security Research Program (HSRP) and Office of Emergency Management managed and funded multiple-laboratory testing of the procedures for analysis of water, soil, and wipe samples in a multi-laboratory study. Laboratories participating in the study and providing technical support include EPA Regions 1, 3, 6, 9 and 10; EPA's Portable High Throughput Integrated Laboratory Identification System (PHILIS) Unit mobile laboratory in Castle Rock, Colorado; the Virginia Division of Consolidated Laboratories; the Florida Department of Environmental Protection; and LLNL. Technical support and data evaluation were provided by CSGov (formerly CSC). Disclaimer The U.S. Environmental Protection Agency through its Office of Research and Development funded and managed the research described herein under EPA Contract No. EP-C-10-060 to CSGov (formerly CSC). It has been reviewed by the Agency but does not necessarily reflect the Agency's views. No official endorsement should be inferred. EPA does not endorse the purchase or sale of any commercial products or services. Questions concerning this document or its application should be addressed to: Romy Campisano U.S. Environmental Protection Agency Office of Research and Development National Homeland Security Research Center (NG16) 26 West Martin Luther King Drive Cincinnati, OH 45268 (513) 569-7016 campisano.romv@,epa.gov ------- Analytical Protocol for VX Using GC/MS Table of Contents Section Page I.0 SCOPE AND APPLICATION 1 2.0 SUMMARY OF PROTOCOL 1 3.0 ACRONYMS, ABBREVIATIONS AND DEFINITIONS 1 3.1 Acronyms and Abbreviations 1 3.2 Definitions 2 4.0 INTERFERENCES 5 5.0 SAFETY 5 6.0 EQUIPMENT AND SUPPLIES 6 6.1 General Equipment 6 6.2 Microscale Extraction Apparatus 7 6.3 Gas Chromatograph/Mass Spectrometer (GC/MS) System 8 7.0 REAGENTS AND STANDARDS 10 7.1 Reagents 10 7.2 Standards 10 8.0 SAMPLE PRESERVATION, STORAGE, AND TECHNICAL HOLDING TIMES 12 8.1 Sample Preservation 12 8.2 Sample Storage 12 8.3 Sample Extract Storage 12 8.4 Technical Holding Times 13 9.0 QUALITY CONTROL (QC) 13 9.1 Initial Demonstration of Capability (IDC) 14 9.2 Initial Precision and Recovery (IPR) Determination 14 9.3 Method Blanks 15 9.4 Matrix Spike and Matrix Spike Duplicate (MS/MSD) 17 9.5 Laboratory Control Sample (LCS) 19 9.6 Instrument Detection Limit (IDL) Determination 20 9.7 Method Detection Limit (MDL) Determination 21 9.8 Quantitation Limit (QL) Determination 21 10.0 CALIBRATION AND STANDARDIZATION 22 10.1 Instrument Operating Conditions 22 10.2 GC/MS Mass Calibration (Tuning) and Ion Abundance 23 10.3 Initial Calibration 25 10.4 Continuing Calibration Verification (CCV) 27 10.5 Instrument Blank 29 II.0 ANALYTICAL PROCEDURE 30 11.1 Sample Preparation - General 30 11.2 Preparation of Water Samples Using Microscale Extraction 30 11.3 Preparation of Solid Samples Using Microscale Extraction 31 11.4 Preparation of Wipe Samples by Microscale Extraction 33 11.5 Final Concentration of Extract - Nitrogen Evaporation Technique for solid and wipe SAMPLES 34 11.6 Extract Analysis by GC/MS 34 / ------- Analytical Protocol for VX Using GC/MS 12.0 CALCULATIONS AND DATA ANALYSIS 35 12.1 Qualitative Identification of Target Compounds 35 12.2 Data Analysis and Calculations of Target Compounds 36 12.3 Technical Acceptance Criteria for Sample Analysis 40 12.4 Corrective Action for Sample Analysis 41 13.0 ANALYTICAL PROCEDURE PERFORMANCE 42 14.0 POLLUTION PREVENTION 42 15.0 WASTE MANAGEMENT 43 16.0 REFERENCES 43 17.0 TABLES AND FIGURES 46 LIST OF TABLES Table 1. Instrument Detection Limits (IDL) and Method Detection Limits (MDL) Based on Multi- Laboratory Evaluation 46 Table 2a. Example Multi-Laboratory Results for Reference Matrix Samples Analyzed Using GC- Quadrupole MS 47 Table 2b. Example Multi-Laboratory Results for Reference Matrix Samples Analyzed Using GC-TOF 47 Table 3. Decafluorotriphenylphosphine (DFTPP) Key Ions and Ion Abundance Criteria 48 Table 4. Internal Standard and Surrogates 48 Table 5. Example Retention Times, Relative Retention Times and Quantitation Ions for Target Compounds, Surrogate Compounds, and Internal Standard 48 Table 6. Example Multi-Laboratory Precision and Bias in Reference Matrices at Mid-Calibration Levels 49 Table 7. Surrogate Recovery 49 Table 8. Example Calibration Standard Concentrations ((.ig/niL) Used During Multi-Laboratory Method Validation Study 50 Table 9a. Example Multi-Laboratory Precision and Recovery in Water - Using GC-full scan 51 Table 9b. Example Multi-Laboratory Precision and Recovery in Water - Using GC-TOF 51 Table 10a. Example Multi-Laboratory Precision and Recovery in Soils - Using GC-full scan 52 Table 10b. Example Multi-Laboratory Precision and Recovery in Soils - Using GC-TOF 52 Table 11. Multi-Laboratory Study Water Matrices Characterization Data 53 Table 12. Multi-laboratory Study Soil Matrix Characterization Data 53 LIST OF FIGURES Figure 1. Example chromatogram for a calibration standard on full-scan quadrupole MS 54 Figure 2. Example chromatogram for a midpoint calibration standard (Cal5) on time-of-flight MS 55 ii ------- Analytical Protocol for VX Using GC/MS 1.0 SCOPE AND APPLICATION 1.1 The U.S. Environmental Protection Agency (EPA) Homeland Security Research Program (HSRP) and Office of Emergency Management (OEM), in collaboration with experts from across EPA and other federal agencies, have identified analytical methods to be used for the analysis of extractable semi-volatile chemical agents in response to a homeland security incident. This protocol is to be applied by the national network of laboratories that has been recruited to the EPA-established Environmental Response Laboratory Network (ERLN) so that their analytical results are consistent and comparable. Summaries of these methods are provided in EPA's Selected Analytical Methods for Environmental Remediation and Recovery (SAM) (Reference 16.1). HSRP is using the SAM methods (based on the methods listed in Section 1.2) to develop analytical protocols for laboratory identification and measurement of target agents during site remediation. 1.2 This document is for the determination of the chemical warfare agent 0-ethyl-S-(2- diisopropylaminoethyl)methyl-phosphonothiolate (VX). The protocol is based on EPA Methods 8270D, 3511, 3570, and 1613 (References 16.2, 16.3, 16.4 and 16.5), and a journal article (Reference 16.6). The procedures have been multi-laboratory tested for preparation and analysis of VX in soil, wipe, and water samples. Contaminant 0-ethyl-S-(2-diisopropylaminoethyl)methyl-phosphonothiolate (VX) 50782-69-9 * Chemical Abstracts Service (CAS) Registry Number 1.3 Procedures in this protocol have been tested for the target analyte listed in Section 1.2 in reference matrices (i.e., reagent water, Ottawa sand, and wipes), drinking water from two sources, and two dried and homogenized soils. Results of laboratory testing are provided in Sections 13.0 and 17.0. 2.0 SUMMARY OF PROTOCOL This analytical protocol involves microscale extraction, followed by gas chromatography/mass spectrometry (GC/MS) analysis to identify and measure VX. Soil and wipe extracts also might require a concentration step using nitrogen evaporation to achieve appropriate levels of quantitation. 3.0 ACRONYMS, ABBREVIATIONS and DEFINITIONS 3.1 Acronyms and Abbreviations %Recovery Percent recovery %RSD Percent relative standard deviation amu Atomic mass unit ASTM ASTM International (formerly American Society for Testing and Materials) CAS RN Chemical Abstracts Service Registry Number 1 September 2016 ------- Analytical Protocol for VX Using GC/MS ccv DCM DF DFTPP EI EICP EPA FC-43 GC/MS HSRP IDC IDL IPR IS LCS MDL MS/MSD MSDS OEM OSHA OW PD PE PPE PTFE QC QL RPD rpm RRF RRT RSD RT SAM S:N svoc TOF VOA VX Continuing calibration verification Methylene chloride (dichloromethane) Dilution factor Decafluorotriphenylphosphine Electron ionization Extracted ion current profile U.S. Environmental Protection Agency Perfluoro-tri-n-butylamine Gas chromatograph/mass spectrometer (gas chromatography/mass spectrometry) Homeland Security Research Program Initial demonstration of capability Instrument detection limit Initial precision and recovery Internal standard Laboratory control sample Method detection limit Matrix spike/matrix spike duplicate Material Safety Data Sheet Office of Emergency Management U.S. Occupational Safety and Health Administration Office of Water Percent drift Performance evaluation Personal protective equipment Polytetrafluoroethylene (Teflon®) Quality control Quantitation limit Relative percent difference revolution(s) per minute Relative response factor Relative retention time Relative standard deviation Retention time Selected Analytical Methods for Environmental Remediation and Recovery (SAM), https://www.epa.gov/homeland-securitv- research/sam (accessed 02/04/2015) Signal-to-noise ratio Semivolatile organic compound Time of flight Volatile organic analysis 0-ethyl-S-(2-diisopropylaminoethyl)methyl-phosphonothiolate 3.2 Definitions Aliquot - A measured portion of a field sample, standard, or solution taken for sample preparation and/or analysis. Analytical Batch - A set of samples that is analyzed on the same instrument during a 24- hour period of operation or the analysis of 20 samples (whichever comes first). The 2 September 2016 ------- Analytical Protocol for VX Using GC/MS analytical batch begins and ends with the analysis of the appropriate Continuing Calibration Verification (CCV) standards. Calibration Standard - A solution prepared from the stock standard solution(s) and the internal standards and surrogate analytes. The calibration standards are used to calibrate instrument response with respect to analyte concentration. Continuing Calibration Verification (CCV) - A calibration standard containing the target analytes, which is analyzed periodically to verify the accuracy of the existing calibration for those analytes. Extracted Ion Current Profile (EICP) - A plot of ion abundance versus time (or scan number) for ion(s) of specified mass(es). Holding Time - The time elapsed from sample collection until sample extraction or analysis. Initial Calibration - Analysis of calibration standards for a series of different specified concentrations; used to define the quantitative response, linearity, and dynamic range of the instrument for target analytes. Initial Demonstration of Capability (IDC) - The IDC is performed prior to use of the analytical procedures and is used to evaluate the capability of the laboratory in terms of analytical precision, bias and sensitivity pertaining to the target analytes. Initial Precision and Recovery (IPR) - A set of four aliquots of a clean reference matrix (i.e., reagent water, Ottawa sand, clean wipe) to which known quantities of the target analytes are added. The IPR aliquots are processed and analyzed exactly like a sample and analyzed prior to the analysis of field samples as part of the IDC. Their purpose is to determine whether the laboratory is capable of making accurate and precise measurements. Instrument Detection Limit (IDL) - The minimum concentration of an analyte that, when injected into the gas chromatograph/mass spectrometer (GC/MS), produces an average signal-to-noise ratio (S:N) between 3:1 and 5:1 for at least three replicate injections. Instrument Performance Check Solution - A solution of one or more instrument tuning compounds used to evaluate the performance of the instrument system with respect to a defined set of method criteria. Internal Standard (IS) - Analyte added to an extract or standard solution in a known amount and used to measure the relative responses of target analytes and surrogates. The internal standard must be an analyte that is not a sample component. Laboratory Control Sample (LCS) - An aliquot of a clean reference matrix (i.e., reagent water, Ottawa sand, clean wipe) to which known quantities of the target analytes are added. The LCS is processed and analyzed exactly like a sample. Its purpose is to determine whether the analytical process is in control. 3 September 2016 ------- Analytical Protocol for VX Using GC/MS Safety Data Sheet (SDS) - Written information provided by vendors concerning a chemical's toxicity, health hazards, physical properties, flammability, and reactivity data including storage, spill, and handling precautions. Matrix - The predominant material of which the sample to be analyzed is composed. For the purpose of this protocol, a sample matrix is either aqueous/water, soil/sediment/sand, or wipe. Matrix is not synonymous with phase (e.g., liquid or solid). Matrix Spike (MS) - An aliquot of a field sample to which known quantities of target analytes are added. The MS is processed and analyzed exactly like the corresponding sample. Its purpose is to determine whether the sample matrix contributes bias to the analytical results. Background concentrations of the analytes must be determined in a separate aliquot. Matrix Spike Duplicate (MSD) - A second aliquot of the field sample used to prepare the MS, which is fortified, extracted and analyzed exactly like the MS. The MSD is used to assess matrix effects on analytical precision and bias. Method Blank - An aliquot of a clean reference matrix (i.e., reagent water, Ottawa sand, clean wipe) that is treated exactly as a sample, including exposure to all glassware, equipment, solvents, reagents, sample preservatives, internal standards, and surrogates that are used in the extraction batch. The method blank is used to determine whether target analytes or interferences are present in the laboratory environment, reagents or equipment. Method Detection Limit (MDL) - The minimum concentration of an analyte that can be identified, measured and reported with 99 percent confidence that the analyte concentration is greater than zero. The MDL is a statistical determination (Section 9.7), and accurate quantitation at this level is not expected. Percent Difference - The difference between two values divided by one of the values. Used in this protocol to compare two relative response factor (RRF) values from calibration. Percent Drift (PD) - The difference between the calculated and theoretical value divided by the theoretical value. Used in this protocol to compare calculated and theoretical values for calibration by regression techniques. Quantitation Limit (QL) - The minimum level of quantitation. This concentration must meet the criteria defined in Section 9.8. Reagent Water - Water in which an interferent is not observed at or above the low-level calibration standard for each analyte of interest. The purity of this water must be equivalent to ASTM International (ASTM) Type II reagent water of Specification D1193-06, "Standard Specification for Reagent Water." Relative Percent Difference (RPD) - The difference between two values divided by the mean of the values. RPD is reported as an absolute value (i.e., always expressed as a positive number or zero). 4 September 2016 ------- Analytical Protocol for VX Using GC/MS Relative Response Factor (RRF) - A measure of the relative mass spectral response of an analyte compared to its internal standard. RRFs are determined by analysis of standards and are used in calculating the concentrations of analytes in samples. Retention Time (RT) - The time an analyte is retained on a GC column before elution. The RT is dependent on the analyte, nature of the column's stationary phase, the column's diameter, temperature, carrier gas flow rate, and other column parameters. Relative Retention Time (RRT) - The ratio of the RT of a compound to the RT of a corresponding internal standard. Stock Standard Solution - A concentrated solution containing one or more target analytes prepared in the laboratory using assayed reference materials or materials purchased from a reputable commercial source. Surrogate - Analyte that is unlikely to be found in any sample to be analyzed. Surrogates are added to a sample aliquot in a known amount before extraction or other processing. Surrogates are measured with the same procedures used to measure other sample components. The purpose of the surrogate is to monitor method performance with each sample. Working Standard Solution - A solution containing target analytes prepared from stock standard solutions. Working standard solutions are diluted as needed to prepare calibration and spiking solutions. 4.0 INTERFERENCES 4.1 Contaminants in solvents, reagents, glassware, and other sample processing hardware can cause method interferences such as discrete artifacts and/or elevated baselines in the extracted ion current profiles (EICPs). All of these materials must be routinely demonstrated to be free from interferences under the conditions of the analysis by running method blanks. Matrix interferences can be caused by contaminants that are co- extracted from the sample. The extent of matrix interferences will vary considerably from source to source and is evaluated using the results from the analysis of matrix spike and matrix spike duplicate samples. In addition to the interferences mentioned above, VX is subject to matrix enhancement effects reported frequently for gas chromatographic analysis of various compounds, potentially resulting in high unexpected recoveries in some samples. 4.2 This protocol includes conditions for collecting mass spectral data using both quadrupole mass spectrometers in full-scan mode and time-of-flight (TOF) mass spectrometers. 5.0 SAFETY WARNING: The toxicity of VX presents hazards unfamiliar to most experienced laboratory personnel. Special techniques and precautions must be used even for the simplest procedures involving these agents. Because VX is the target analyte for this protocol, laboratory personnel must be thoroughly trained in appropriate safety procedures prior to using this method. 5 September 2016 ------- Analytical Protocol for VX Using GC/MS 5.1 Operations with VX have specific safety requirements. The laboratory must have these requirements included in a Chemical Hygiene Plan prior to conducting the analytical procedures described in this protocol. 5.2 At a minimum, personal protective equipment (PPE) requirements include safety glasses, lab coats, and protective gloves. The availability of emergency response equipment and support personnel should be as indicated in a laboratory Chemical Hygiene Plan. 5.3 Exposure to chemical agent material is possible from contact, and risk is primarily associated with compromise of PPE. Respiratory exposure can result from spills or improper use of ventilation controls and PPE. 5.4 At concentrations of VX that are within the calibration range of this method, the likelihood of an exposure causing adverse health effects is extremely low (Reference 16.7). Note: This method does not address all safety issues associated with its use. The laboratory is responsible for maintaining a safe work environment and a current file of Occupational Safety and Health Administration (OSHA) regulations regarding the safe handling of the chemicals (including all solvents, reagents, and standards). A reference file of safety data sheets (SDSs) must be available to all personnel involved in these analyses, chemical handling, and contaminated area cleaning, or who might potentially come in contact with the materials in their work place. 6.0 EQUIPMENT AND SUPPLIES Brand names, suppliers, catalog, and part numbers are for illustrative purposes only. No endorsement is implied. Equivalent performance can be achieved using equipment and supplies other than those specified; however, laboratories must document use of the alternatives and provide a demonstration of equivalent performance meeting the requirements of this protocol. 6.1 General Equipment 6.1.1 Vials - Clear or amber glass, with polytetrafluoroethylene (PTFE)-lined screw or crimp top (2.0-mL capacity for GC auto sampler) (Sigma Aldrich Catalog No.SU860033, Sigma-Aldrich. St. Louis, MO) or equivalent. Glass inserts can be used to minimize sample volumes. 6.1.2 Syringes - Contaminant-free, 1.0 mL, 2.0 mL, 10 |iL. 25 (jL, 500 jjL 6.1.3 Pasteur glass pipettes - 1.0 mL, disposable (Fisher Scientific Catalog No. NC0541803, Thermo Fisher Scientific, Westminster, MD) or equivalent 6.1.4 Balances - Analytical, capable of accurately weighing ±0.0001 gram, and one capable of weighing 100 grams (±0.01 gram). 6.1.5 Spatula - Stainless steel or PTFE 6.1.6 Nitrogen evaporation device - Equipped with temperature control that can be maintained at 35 - 40°C, a RapidVap® evaporator (Labconco Corporation, Kansas City, MO) or equivalent. To prevent the release of solvent fumes into 6 September 2016 ------- Analytical Protocol for VX Using GC/MS the laboratory, this device must be used with suitable engineering controls. Water bath (for nitrogen evaporation devices that do not have a heating apparatus) - Heated, with concentric ring cover capable of heating to 80 °C and maintaining temperature control (±5 °C). The bath should be used with suitable engineering controls. Glass funnel (Fisher Scientific Catalog No. CG172305, Thermo Fisher Scientific, Westminster, MD) or equivalent - Used in filtering soil samples that fail to settle out with centrifugation. Borosilicate glass wool - Oven-cleaned (muffled) or solvent rinsed (using extraction solvent); used in filtering soil samples that fail to settle out with centrifugation. pH paper - Including narrow range capable of measuring a pH of 2.0. pH meter - With a combination glass electrode. Calibrate prior to each use according to manufacturer's instructions. Ottawa sand - Held at 450 °C for four hours in a 500-mL wide-mouthed amber bottle. Vortexer - VWR™ vortexer (VWR Corporation, Radnor, PA) or equivalent, capable of accommodating 40 - 60-mL vials and 50-mL centrifuge tubes. Shaker table 6.1.14.1 Glas-Col Large Capacity Mixer (Part # 099A LC1012, Glas-Col Inc., Terre Haute, IN); or Glas-Col Digital Pulse Mixer (Part # 099A DPMI2) or equivalent 6.1.14.2 Foam pad for 40-mL volatile organic analysis (VOA) vials (Part #099A VC4014, Glas-Col Inc., Terre Haute, IN or equivalent) 6.1.14.3 Foam pad for 60-mL VOA vials (Part #099A VC6014, Glas-Col Inc., Terre Haute, IN) or equivalent 6.2 Microscale Extraction Apparatus 6.2.1 Solid samples 6.2.1.1 VOA vials - 40 mL capacity, disposable, pre-cleaned with PTFE- lined caps (Fisher Scientific Catalog No. 05-719-400,Thermo Fisher Scientific, Westminster, MD or equivalent). If pre-cleaned vials are not available, appropriate cleaning procedures are provided in EPA Methods 525.3 (Reference 16.8) and 1668C (Reference 16.9), and in SW-846 Chapter 4 (Reference 16.10). 6.2.1.2 Sonicator - Branson 3510 sonicator (Branson Ultrasonics Corp., Danbury, CT) or equivalent 6.1.7 6.1.8 6.1.9 6.1.10 6.1.11 6.1.12 6.1.13 6.1.14 7 September 2016 ------- Analytical Protocol for VX Using GC/MS 6.2.1.3 Apparatus for determining percent dry weight 6.2.1.3.1 Drying oven - Capable of maintaining 103 - 105 °C 6.2.1.3.2 Desiccator 6.2.1.3.3 Crucibles - Disposable aluminum 6.2.1.4 Glass beads - Solvent-rinsed, baked in 400 °C oven for approximately 1 hour (Fisher Scientific Catalog No. S80024 or equivalent) 6.2.1.5 Centrifuge - Capable of at least 500 G-force units and accommodating 40- or 50-mL vials, Accuspin™ Model 400 centrifuge (Thermo Fisher Scientific, Westminster, MD) or equivalent. CAUTION: Different centrifuge makes and models have different maximum centrifuge speeds that are recommended for safe operation. The maximum safe handling speed of each centrifuge will depend, in part, on the vials used and should be determined prior to centrifuging samples. 6.2.1.6 Pasteur pipettes - 1.0-mL glass, disposable orre- pipettes/autopipettes with disposal tips 6.2.2 Water Samples 6.2.2.1 Conical bottom glass screw-top tube, 50-mL or 60-mL vials, pre- cleaned with PTFE-lined caps. If pre-cleaned vials are not available, appropriate cleaning procedures are provided in EPA Methods 525.3 (Reference 16.8) and 1668C (Reference 16.9), and in SW-846 Chapter 4 (Reference 16.10). 6.2.2.2 Beakers - 400 mL 6.2.2.3 Class A graduated cylinder - 100 mL 6.2.2.4 Class A volumetric flasks - 10 mL 6.2.3 Wipe samples - Kendall Curity® gauze sponges (USP Type VII Gauze, cotton, 12 ply 3 in x 3 in, Tyco Healthcare Group, Covidien, Mansfield, MA) or equivalent; pre-cleaned by extracting with methylene chloride (DCM) using an appropriate extraction method (e.g., pressurized fluid extraction, Soxhlet extraction, soaking in DCM). Note: Wipes used for field or laboratory quality control (QC) samples should be pre-wetted with DCM prior to use. 6.3 Gas Chromatograph/Mass Spectrometer (GC/MS) System 6.3.1 Gas chromatograph - The GC system must be capable of temperature programming and have a flow controller that maintains a constant column carrier 8 September 2016 ------- Analytical Protocol for VX Using GC/MS gas flow rate throughout the temperature program operations. The system must be suitable for splitless injection and have all required accessories including syringes, analytical columns, and gases. All GC carrier gas lines must be constructed from stainless steel or copper tubing. Non-PTFE thread sealants or flow controllers with rubber components are not to be used. Note: Due to the potential hazards associated with the analysis of VX, it is recommended that a split vent trap be used for every GC system (Agilent® RD- 1020 Universal/External Split Vent Trap (Agilent Technologies, Inc., Santa Clara, CA or equivalent). For additional safety measures, the split and purge vent lines can be vented to the hood ventilation system. 6.3.2 GC column - Recommended length 30 m x 0.25 mm internal diameter (ID) (or 0.32 mm) bonded phase silicon coated fused silica capillary column DB-5 (J&W Scientific, Agilent Technologies, Santa Clara, CA); DB-5MS; RTX®-5 (Restek Corp., Bellefonte, PA), RTX-5MS (Restek Corp., Bellefonte, PA), RTX-5Sil MS (Restek Corp., Bellefonte, PA); Zebron® ZB-5 (Phenomenex®, Phenomenex Inc., Torrance, CA); SPB-5 (Supelco®, Sigma-Aldrich. St. Louis, MO); AT™-5 (Alltech®, Grace, Columbia, MD); HP-5 (Agilent Technologies, Santa Clara, CA); HP-5MS or HP-5MS UI (Agilent Technologies, Santa Clara, CA), CP-Sil 8 CB (Chrompack, Raritan, NJ); 007-2 (Quadrex®, Quadrex Corp., Bethany, CT); BP-5 (SGE, Trajan Scientific Americas, Inc., Austin, TX); Zebron ZB-5MS (Phenomenex®, Phenomenex Inc., Torrance, CA) or equivalent. Columns used to generate the example data provided in Section 17 include: Agilent HP-5 MS, RTX - 5sil, RTX-5MS, RTX-5Sil MS, Zebron ZB-5 MS, DB-5MS. Although a film thickness of 1.0 micron might be desirable because of its larger capacity, film thicknesses of 0.25 micron and 0.05 micron were used by laboratories generating the example data presented in Section 17. A description of the GC column used for analysis shall be provided in the data narrative. A capillary column is considered equivalent if: • The column does not introduce contaminants that interfere with the identification and quantification of the compounds listed in Table 1, Section 17. • The analytical results generated using the column meet the initial CCV technical acceptance criteria listed in the protocol and the quantitation levels determined as described in Section 9.8. • The column provides equal or better resolution of the compounds listed in Table 1, Section 17 when compared to columns listed above. 6.3.3 MS - Must be capable of recording a spectrum from 35 - 500 atomic mass units (amu) every second or less, using 70 electron volts (nominal) in the electron ionization (EI) mode, and producing a mass spectrum that meets the tuning acceptance criteria when 50 ng or less of decafluorotriphenylphosphine (DFTPP) is injected through the GC inlet. The instrument must be vented to prevent the release of contaminants into the instrument room. 9 September 2016 ------- Analytical Protocol for VX Using GC/MS 7.0 REAGENTS AND STANDARDS 7.1 Reagents 7.1.1 Organic-free reagent water - Water in which an interferent is not observed at or above the quantitation limit (QL) for each analyte of interest. ASTM Type II reagent water of Specification D1193-06, "Standard Specification for Reagent Water" (Reference 16.11) or equivalent. 7.1.2 Acetone, DCM, and toluene - pesticide residue analysis grade or equivalent. DCM must contain a non-methanol olefinic stabilizer (i.e., amylene or 2- pentene). Solvents must be anhydrous or pre-dried by adding sodium sulfate until no clumping occurs and the solution becomes clear (not cloudy). 7.1.3 Sodium sulfate - Powdered or granular anhydrous reagent grade, heat at 400°C for four hours in a shallow tray, cool in a desiccator, and store in a glass bottle. 7.1.4 Tris-buffer (for soil extraction of VX) - 2 L of Tris-buffer is prepared as a solution containing 0.2 M tris(hydroxymethyl)aminomethane (Trizma® base, Product # T1503, Sigma Aldrich or equivalent, Sigma-Aldrich, St. Louis, MO) and 32 mL of 1 M hydrochloric acid (HC1). The pH of the solution should be in the range of 8.5 - 9.0. If necessary, adjust accordingly with additional tris(hydroxymethyl)aminomethane to raise the pH, or HC1 to lower it. 7.1.5 Sodium chloride - Anhydrous reagent grade, >99 %. 7.1.6 Sodium thiosulfate - Reagent grade, 10 % sodium thiosulfate solution in reagent water. Prepared fresh with each use. 7.2 Standards The laboratory must be able to verify that stock standard solutions are certified. Manufacturers' certificates of analysis must be retained by the laboratory and presented upon request. Stock standard solutions provided in sealed glass ampules may be retained and used within six months of the preparation date. Solutions used for calibration verification ideally are prepared from a separate source other than the source used to prepare calibration standards. Due to the nature of the analyte addressed in this protocol, identification of a secondary source might be difficult. 7.2.1 Stock standard solutions Stock standard solutions, used to produce working standards, may contain individual target compounds or mixtures of target compounds. 7.2.2 Working standards 7.2.2.1 Surrogate standard spiking solution - Prepare a surrogate standard spiking solution in DCM or other suitable solvent that contains appropriate surrogates for the target compound. A concentration of 25 (ig/mL is recommended for each surrogate. Surrogate standards are added to all samples and calibration solutions. 10 September 2016 ------- Analytical Protocol for VX Using GC/MS Note: Table 4 provides a list of surrogates used during laboratory studies testing this protocol, based on surrogates typically used for semivolatile organic compounds (SVOC) analyses. Alternative surrogates might be more representative of the analyte targeted in this method, and may be used instead of or in addition to those listed in Table 4, Section 17, provided the surrogates meet the criteria in Table 7, Section 17. 7.2.2.2 Matrix spiking solution - This solution is prepared in DCM and should contain the target analyte. 7.2.2.3 Instrument performance check solution - Prepare a solution of DFTPP in DCM such that a 1 -|_iL injection will contain 50 ng or less of DFTPP. The DFTPP may also be included in the calibration standards at this level. 7.2.2.4 Initial and continuing calibration solutions 7.2.2.4.1 Prepare calibration standards in DCM at a minimum of five concentration levels. Each calibration standard should contain VX, associated surrogates, and internal standard. Note 1: All samples analyzed must be injected at the same volume (e.g., 1.0 or 2.0 (.iL) as the calibration standards. Note 2: The concentrations listed in Table 8, Section 17, provide an example calibration range used during laboratory evaluation of this protocol. The low calibration standard is set at the expected QL (determined in Section 9.8). The remaining calibration standards should be prepared at concentrations that meet the specifications in Section 10.3.4. 7.2.2.4.2 The CCV standard is prepared in DCM at or near the midpoint of the calibration curve. 7.2.2.5 Internal standard solution - An internal standard solution can be prepared by dissolving 100 mg of phenanthrene-dio in 100 mL of DCM, resulting in a concentration of 1.0 mg/mL. A sufficient portion of this solution will be added to each sample extract just prior to analysis by GC/MS to result in a concentration of 0.5 ng/(.iL (for both full-scan quadrupole and TOF mass spectrometers). Alternatively, internal standard solutions can be purchased from commercial sources (e.g., Supelco part numbers 861238 or 48710-U, or equivalent, [Supelco®, Sigma-Aldrich. St. Louis, MO]). 11 September 2016 ------- Analytical Protocol for VX Using GC/MS 7.2.3 Storage of standard solutions 7.2.3.1 Store unopened ampules of stock standard solutions at <6 °C. If no manufacturer's expiration date is provided, unopened ampules of standard solutions may be retained and used for up to twelve months after the preparation date (Reference 16.12). Store opened stock standard solutions at <6 °C in PTFE-lined screw-cap amber bottles. Opened stock standards containing only VX are stable for up to 6 months; opened stock standards containing chemical warfare agents in addition to VX (i.e., sarin, soman, cyclohexyl sarin and/or sulfur mustard) are stable for up to six days. 7.2.3.2 Store the working standards at < 6 °C in containers with PTFE-lined caps. Working standard solutions should be checked against CCV standards at least weekly for stability, and must be replaced after six months, or sooner, if the stock standard solutions have expired or if comparison with CCV samples indicates a problem. 7.2.3.3 Protect all standards from light. Samples, sample extracts, and standards must be stored separately. 7.2.3.4 The laboratory is responsible for maintaining the integrity of standard solutions and verifying the solutions prior to use. The standards must be brought to room temperature prior to use, checked for losses, and checked to ensure that all components have remained in solution. Guidance on standard verification procedures can be found in EPA's Superfund Analytical Services / Contract Laboratory Program, Multi- Media, Multi-Concentration Organics Analysis, SOM02.3, Exhibit E, Section 4 (Reference 16.13). 8.0 SAMPLE PRESERVATION, STORAGE, AND TECHNICAL HOLDING TIMES 8.1 Sample Preservation Samples must be stored on ice or refrigerated at 4 °C (± 2 °C) immediately after collection until receipt in the laboratory. If chlorine is suspected to be present in water samples (e.g., treated drinking water or wastewater) that are to be measured for VX, add approximately four drops (-0.2 mL) of a 10 % solution of sodium thiosulfate per 35-mL sample. If clouding results, add less sodium thiosulfate to a fresh sample aliquot. If sodium thiosulfate is not added during sample collection, it should be added immediately upon sample receipt in the laboratory, prior to sample analysis or extraction. 8.2 Sample Storage Samples must be protected from light and refrigerated at 4 °C (± 2 °C) from the time of receipt until extraction. 8.3 Sample Extract Storage 8.3.1 Sample extracts must be protected from light and stored at <6 °C. 12 September 2016 ------- Analytical Protocol for VX Using GC/MS 8.3.2 Samples, sample extracts, and standards must be stored separately. 8.4 Technical Holding Times 8.4.1 Soil and wipe samples must be extracted within seven days from receipt at the laboratory. Aqueous samples must be extracted within 14 days from receipt at the laboratory. Note: Holding times for soils and wipes are based on holding times listed in guidance documents and similar analytical methods for SVOCs (e.g., EPA Methods 525.2 [Reference 16.14] and 525.3 [Reference 16.8], CLP Method SOM 2.3 [Reference 16.15], SW-846 Chapter 4 [Reference 16.10]). Holding times for water samples were evaluated in a single laboratory using this protocol. 8.4.2 Extracts must be analyzed within 14 days following extraction. 9.0 QUALITY CONTROL (QC) QC requirements for this protocol include the following: Quality Control (QC) Analyses Requirement Section Frequency Instrument Detection Limit (IDL) Determination Section 9.6 Optional. Performed prior to Method Detection Limit (MDL) study Method Detection Limit (MDL) Determination Section 9.7 Performed once, prior to first performing the protocol procedures and with each significant change as part of the Initial Demonstration of Capability (IDC) Initial Precision and Recovery (IPR) Determination Section 9.2 Quantitation Limit (QL) Determination Section 9.8 Method Blanks Section 9.3 At least one per batch of <20 samples of the same matrix Instrument Blank Section 10.5 Following an analysis with suspected carry-over or following analysis of samples containing high concentrations Matrix Spike / Matrix Spike Duplicate (MS/MSD) Section 9.4 One per each batch of <20 samples of the same matrix Laboratory Control Sample (LCS) Section 9.5 At least one per batch of <20 samples of the same matrix Continuing Calibration Verification (CCV) Section 10.4 Prior to the analysis of samples, and after instrument performance check. Analyzed at the beginning and at the end of each analytical batch of <20 injections Precision and bias criteria for data generated using this method are currently set at 50 - 150 % recovery and < 30 % precision (as relative standard deviation [RSD] or relative percent difference [RPD]). These criteria may change as more laboratory data become available. In cases where analyses of difficult sample matrices generate results outside these criteria, data should be flagged, and laboratories should collect additional data to support development of laboratory- and matrix-specific criteria. Example precision and bias results obtained from multiple laboratories analyzing spiked reference matrix samples (reagent water, Ottawa sand, and wipes) and field 13 September 2016 ------- Analytical Protocol for VX Using GC/MS samples (water and soil) are provided in Section 17. 9.1 Initial Demonstration of Capability (IDC) An IDC shall be performed prior to the analysis of any samples and with each significant change in instrument type, detection technique, personnel or method. An IDC consists of the following: • An IPR determination (Section 9.2) • An MDL determination (Section 9.7) • A QL determination (Section 9.8) on a clean matrix (reagent water, Ottawa sand, pre- cleaned wipe) The IPR consists of four replicate samples of a clean matrix spiked with VX around the midpoint of the calibration curve and carried through the entire analytical process. Prior to performing the IDC, a valid initial calibration (Section 10.3) must be established. 9.2 Initial Precision and Recovery (IPR) Determination 9.2.1 Preparation and analysis of IPR samples 9.2.1.1 Water samples Prepare four replicate samples from about 35 mL of reagent water. Add a sufficient amount of surrogate standard spiking solution and matrix spiking solution to result in the addition of 1.0 (.ig of each surrogate (add 40 |_iL if prepared as in Section 7.2.2.1) and a concentration at the mid-point of the calibration range of VX. Extract and analyze according to the procedures for water samples (Sections 11.2 and 11.6). The total volume of DCM added will be slightly greater than the 2 mL needed for extraction, and includes the volumes added for spiking VX and the surrogate. 9.2.1.2 Ottawa sand Prepare four replicate samples consisting of 10 grams of Ottawa sand. Add a sufficient amount of the surrogate standard spiking solution and the matrix spiking solution to result in the addition of 0.5 |ag of each surrogate (add 20 (.iL if prepared as in Section 7.2.2.1) and a concentration at the mid-point calibration range of VX, and follow the appropriate extraction procedure in Section 11.3. Extract, concentrate and analyze according to procedures for solid samples. 9.2.1.3 Pre-cleaned wipes Prepare four replicate samples consisting of pre-cleaned wipes. Pre- wet the wipes with DCM prior to use. Add a sufficient amount of the surrogate standard spiking solution and the matrix spiking solution to result in the addition of 0.5 |_ig of each surrogate (add 20 (.iL if prepared as in Section 7.2.2.1) and a concentration at the mid- point calibration range of VX, and follow the appropriate extraction procedure in Section 11.4. Extract, concentrate and analyze 14 September 2016 ------- Analytical Protocol for VX Using GC/MS according to procedures for wipe samples. 9.2.2 Calculations for IPR 9.2.2.1 Calculate the percent recovery (%Recovery) of each compound in the IPR sample using Eq. 11 (Section 12.2.9.2). Calculate an average %Recovery for each compound. 9.2.2.2 Calculate a percent relative standard deviation (%RSD) for each compound in the IPR samples. 9.2.3 Technical acceptance criteria for IPR 9.2.3.1 The average percent recovery of each compound in the IPR should be within the 50 - 150 %. 9.2.3.2 The %RSD of each compound in the IPR should be less than or equal to 30. 9.2.4 Corrective action for IPR If the technical acceptance criteria in Section 9.2.3 are not met, inspect the system for problems and take corrective actions to achieve the acceptance criteria. 9.3 Method Blanks A method blank is a volume of a clean reference matrix (e.g., reagent water for water samples, clean inert sand for solid samples, or clean pre-wetted wipes for wipe samples) spiked with a sufficient amount of surrogate standard spiking solution (Section 7.2.2.1) such that the same amount of surrogate is added as for the associated samples and carried through the entire analytical procedure. Internal standard solution is added just prior to analysis by GC/MS to give an internal standard concentration of 0.5 ng/|_iL for both full- scan and TOF modes. The volume or weight of the method blank must be approximately equal to the volume or weight of the samples associated with the blank. 9.3.1 Frequency of method blanks A method blank must be extracted each time samples are extracted. The number of samples extracted with each method blank should not exceed 20 field samples (excluding MS/MSDs and Performance Evaluation [PE] samples). In addition, a method blank is: • Extracted by the same procedure used to extract samples • Analyzed on each GC/MS system used to analyze associated samples and conditions (i.e., GC/MS settings) 9.3.2 Method blank preparation 9.3.2.1 A method blank for water samples consists of a 35-mL volume of reagent water spiked with a sufficient amount of the surrogate 15 September 2016 ------- Analytical Protocol for VX Using GC/MS standard spiking solution to result in the addition of 1.0 (ig of surrogate (add 40 |_iL if prepared as in Section 7.2.2.1). The final volume of the extracts will be approximately 2.0 mL; therefore, the concentration of the surrogate in the extract is expected to be approximately 0.50 |_ig/mL. For solid samples, a method blank consists of 10 grams of clean inert sand spiked with a sufficient amount of the surrogate spiking solution to result in the addition of 0.5 (.ig of surrogate (add 20 |_iL if prepared as in Section 7.2.2.1). The final volume of the extracts will be 1.0 mL; therefore, the concentration of the surrogate in the extract is expected to be 0.50 (ig/mL. A method blank for wipe samples consists of a clean unused wipe spiked with a sufficient amount of the surrogate standard spiking solution to result in the addition of 0.5 (ig of surrogate (add 20 |_iL if prepared as in Section 7.2.2.1). The final volume of the extracts will be 1.0 mL; therefore, the concentration of the surrogate in the extract is expected to be 0.50 |_ig/mL. Extract, concentrate, and analyze the blank according to procedures. 9.3.2.2 Under no circumstances should method blanks be analyzed at a dilution. 9.3.3 Technical acceptance criteria for method blank analysis 9.3.3.1 All blanks should be extracted and analyzed at the frequency described in Section 9.3.1 on a GC/MS system meeting the DFTPP tuning criteria in Section 10.2.4 and Table 3, Section 17, initial calibration in Section 10.3, and CCV technical acceptance criteria in Section 10.4.5. 9.3.3.2 The %Recovery of each of the surrogates in the blank must be within the acceptance limits listed in Table 7, Section 17. 9.3.3.3 The blank must meet the sample analysis acceptance criteria listed in Section 12.3. 9.3.3.4 A method blank for solid, water, and wipe samples must contain a concentration less than the MDL of VX. In cases where a blank has detects above the QL, but associated samples have detects greater than 10 times the blank, consult the agency to determine if re- extraction is required. In cases where a method blank fails to meet technical acceptance criteria, but all samples had non-detects for the target analyte, then no re-extraction or qualification of data is necessary. 9.3.4 Corrective action for method blanks 9.3.4.1 If a method blank does not meet the technical acceptance criteria for method blank analysis, the analytical system is considered to be out of control. 9.3.4.2 If contamination is the problem, then the source of the contamination 16 September 2016 ------- Analytical Protocol for VX Using GC/MS should be investigated and appropriate corrective measures taken before further sample analysis proceeds. It is the laboratory's responsibility to ensure that interferences caused by contaminants in solvents, reagents, glassware, and sample storage and processing hardware that lead to discrete artifacts and/or elevated baselines in the GC/MS have been eliminated. If possible, an aliquot of any sample associated with the contaminated blank should be re-extracted and re- analyzed. 9.3.4.3 If surrogate recovery in the method blank does not meet the acceptance criteria listed in Table 7, first reanalyze the method blank. If the surrogate recovery does not meet the acceptance criteria after reanalysis, the method blank and an aliquot of any sample associated with that method blank should be re-extracted, if possible, and re- analyzed. If the surrogate recovery is high and all corresponding samples had non-detects for VX, sample re-extraction and re-analysis are not required. 9.3.4.4 If the method blank does not meet the internal standard response requirements in Section 12.3.5, follow the corrective action procedure in Section 12.4.4.1. Resolve and document problem resolution before proceeding with sample analysis. 9.3.4.5 If the method blank does not meet the retention time (RT) requirements for the internal standard (Section 12.3.6), check the instrument for malfunction and recalibrate. Reanalyze the method blank. 9.3.4.6 Samples that are analyzed with corresponding method blanks that do not meet any of the criteria listed in Sections 9.3.4.2 - 9.3.4.5 should be reanalyzed. If the method does not meet the criteria, then all corresponding sample data should be flagged. 9.4 Matrix Spike and Matrix Spike Duplicate (MS/MSD) To evaluate the potential effects of the sample matrix on analyses, VX must be spiked into two additional aliquots of a water or solid sample and analyzed in accordance with the appropriate method. VX should be spiked at a concentration near the midpoint of the calibration range. 9.4.1 Frequency of MS/MSD analyses 9.4.1.1 An MS/MSD pair is analyzed with each batch of <20 samples of each water or solid matrix type. MS/MSDs are not performed on wipe samples. 9.4.1.2 For quality assurance purposes, water rinsate samples and/or field blanks (field QC) or PE samples may accompany solid, water, and/or wipe samples that are delivered to the laboratory for analysis. These field QC or PE samples are not used for MS/MSD analyses. 17 September 2016 ------- Analytical Protocol for VX Using GC/MS 9.4.1.3 If the agency requesting the analyses designates a sample to be used as an MS/MSD, then that sample must be used. If there is insufficient sample remaining to perform an MS/MSD, then the laboratory shall choose another sample on which to perform an MS/MSD analysis. At the time the selection is made, the laboratory should notify the agency that insufficient sample was received and identify the sample selected for the MS/MSD analysis. 9.4.1.4 If there is insufficient sample remaining in any of the samples in a batch to perform the required MS/MSD, the laboratory will report this in the data narrative. 9.4.2 Procedure for Preparing MS/MSD 9.4.2.1 Water samples Prepare two additional aliquots of the sample chosen for spiking. The volume should be equal to that of the associated samples. Add a sufficient amount of surrogate standard spiking solution and matrix spiking solution to each aliquot to result in the addition of 1.0 (ig of each surrogate (add 40 |_iL if prepared as in Section 7.2.2.1) and a concentration at the mid-point of the calibration range of VX. Extract, clean up, and analyze the MS/MSD according to the procedures for water samples (Section 11.2). The total volume of DCM added will be slightly greater than the 2 mL needed for extraction, and includes volumes added for spiking VX and the surrogate. 9.4.2.2 Solid samples Prepare two additional aliquots of the sample chosen for spiking in two 40-mL VOA vials with PTFE-lined caps. The amount chosen should be equal to that of the associated sample. Mix well. Add a sufficient amount of the surrogate standard spiking solution and the matrix spiking solution to each aliquot to result in the addition of 0.5 |_ig of each surrogate (add 20 |_iL if prepared as in Section 7.2.2.1) and a concentration at the mid-point of VX, and follow the appropriate extraction procedure in Section 11.3. Extract, concentrate, clean up, and analyze the MS/MSD according to procedures for solid samples. 9.4.3 Dilution of MS/MSD Before any MS/MSD analysis, analyze the original sample, then analyze the MS/MSD at the same concentration as the most concentrated extract for which the original sample results will be reported. 9.4.4 Calculations for MS/MSD 9.4.4.1 Calculate the %Recovery of each matrix spike compound in the 18 September 2016 ------- Analytical Protocol for VX Using GC/MS MS/MSD sample (see Eq. 11 in Section 12.2.9). 9.4.4.2 Calculate the Relative Percent Difference (RPD) of the concentrations of each compound in the MS/MSD using Eq. 1. Concentrations of the matrix spike compounds are calculated using the sample equations used for target compounds (Eq. 6 for water samples and Eq. 7 for solid samples in Section 12.2.6). Eq. 1 Relative Percent Difference Calculation RPD = }C' ~Cl * x 100 ct+c2j where: Ci = Measured concentration of the first sample aliquot C2 = Measured concentration of the second sample aliquot 9.4.5 Technical acceptance criteria for MS/MSD 9.4.5.1 All MS/MSDs must be analyzed on a GC/MS system meeting DFTPP and initial CCV technical acceptance criteria, as well as the method blank technical acceptance criteria. 9.4.5.2 The MS/MSD must be extracted and analyzed within the technical holding time (Section 8.4). 9.4.5.3 The RT shift for the internal standard must be within ± 0.50 minutes (30 seconds) between the MS/MSD sample and the most recent CCV standard analysis. 9.4.5.4 The limits for matrix spike compound recovery and RPD are given 50 - 150 % and < 30 % respectively. For difficult matrices, laboratories are encouraged to collect sufficient data to support development of laboratory-specific criteria. 9.4.6 Corrective action for MS/MSD If recovery or RPD limits are not met and the LCS, CCV and method blank are within acceptable limits, this might be an indication of matrix interferences. If sufficient sample is available, an MS/MSD should be reanalyzed, along with all appropriate QC samples. If, after reanalysis, MS/MSD recovery limits are not met, flag the results of the associated sample. 9.5 Laboratory Control Sample (LCS) An LCS consists of an aliquot of clean reference matrix, of the same weight or volume as the corresponding field samples and spiked with the same compounds at the same concentrations used to spike the MS/MSD. The nominal volume of DCM added to water samples is 2.0 mL. Because target compound and surrogate spiking solutions also 19 September 2016 ------- Analytical Protocol for VX Using GC/MS contain DCM, the total volume of DCM added may be slightly greater than 2.0 mL. The actual total volume of DCM must be used in the calculation in Section 12.2. When the results of the MS/MSD analysis indicate matrix interference might be present, the LCS results are used to verify that the interferences are due to the sample matrix and not from artifacts introduced in the laboratory. 9.5.1 Preparation of LCS Extract and analyze the LCS according to the procedures in Sections 11.2 (for water samples), Sections 11.3 for (solid samples), or Section 11.4 (for wipe samples). 9.5.2 Frequency of LCS analyses One LCS should be prepared, extracted, analyzed, and reported for every 20 or fewer field samples extracted in a batch of a similar matrix. The LCS must be extracted and analyzed concurrently with the samples, using the same extraction procedure, cleanup procedure (if required), and instrumentation. 9.5.3 Calculations for LCS Calculate the recovery of each compound in the LCS using Eq. 11 (Section 12.2.8.2). 9.5.4 Technical acceptance criteria for LCS analysis 9.5.4.1 All LCSs should be extracted and analyzed at the frequency described in Section 9.5.2 on a GC/MS system meeting the tuning, initial calibration, CCV, and method blank technical acceptance criteria. 9.5.4.2 The limits for LCS compound recovery limits are 50 - 150 %. 9.5.5 Corrective action for LCS 9.5.5.1 If LCS recovery limits are not met, inspect the system for problems and take corrective actions to achieve the acceptance criteria. 9.5.5.2 If LCS recovery limits cannot be met, flag all associated sample and blank data accordingly. 9.6 Instrument Detection Limit (IDL) Determination Laboratories may determine an IDL on each instrument used for analysis. While determining IDLs is not required, IDL results can be helpful in determining an appropriate spike level for use in determining the MDL (Section 9.7), as well as instrument sensitivity to the target analyte. It is recommended that IDLs be verified annually thereafter, or after major instrument maintenance. Major instrument maintenance includes, but is not limited to: cleaning or replacement of the mass spectrometer source, mass filters, or electron multiplier, or installing a different GC column type. An IDL is instrument-specific and independent of sample matrices. 20 September 2016 ------- Analytical Protocol for VX Using GC/MS 9.6.1 An IDL is determined as the concentration that produces an average signal-to- noise ratio (S:N) of between 3:1 and 5:1 for at least three replicate injections. 9.6.2 All documentation for the IDL determination shall be maintained at the laboratory and provided to the reference agency or the data user upon request. 9.7 Method Detection Limit (MDL) Determination Before any field samples are analyzed, a laboratory MDL should be determined in appropriate reference matrices (i.e., reagent water, Ottawa sand, or clean wipes), using the sample preparation and analytical procedures described in this protocol for each specific matrix, and following the instructions and requirements described in 40 CFR Part 136, Appendix B. 9.7.1 The laboratory must use full method procedures to prepare and analyze at least seven replicates. 9.7.2 Spike each replicate sample at concentrations of 1 - 5 times the IDL concentration and analyze the samples following protocol procedures. The total volume of DCM added to water samples will be slightly greater than the 2 mL needed for extraction, and includes the volumes added for spiking VX and surrogates. 9.7.3 To determine an MDL, the following equation is applied to the analytical results (Student's t-factor depends on the number of replicates used; a factor of 3.14 assumes seven replicates): Eq. 2 Method Detection Limit (MLD) MDL = 3 .14 xsd where: sd = the standard deviation for the analytical results, and 3.14 = the Student's t-value for seven replicate samples 9.7.4 The MDL result calculated using the equation in Section 9.7.3 must meet the following requirements as well as all other requirements specified in 40 CFR Part 136, Appendix B: • MDL result must not be greater than the spiking level used for the MDL determination. • MDL result must not be less than 0.10 times the spiking level used for the MDL determination. If either requirement is not met, the laboratory must adjust their spiking level appropriately and repeat the MDL determination. 9.8 Quantitation Limit (QL) Determination A QL determination is recommended for each laboratory/technician performing the method for the first time, or in cases where new or repaired instrumentation is being used. 21 September 2016 ------- Analytical Protocol for VX Using GC/MS Laboratory QLs are determined by first assessing at least four samples containing VX at the level of the lowest calibration standard, against the criteria listed below. If any of these criteria are not met, samples are assessed at concentrations of the next (second lowest) calibration standard. These criteria are provided as guidance. If the criteria cannot be met, the laboratory should consult analytical data requestor to determine if the QL is sufficient to address project needs: • Results from spikes at the QL should be above the MDL. • The QL should be at or above the lowest calibration level. • The QL should be at least two times the MDL. • The RSD of results from spikes at the QL should be less than 2 0%. • The mean recovery of spikes at the QL should be within 50 - 150 %. 10.0 CALIBRATION AND STANDARDIZATION 10.1 Instrument Operating Conditions 10.1.1 GC The following GC analytical conditions were used during laboratory studies and are provided for guidance. Other conditions may be used, provided that all technical acceptance criteria are met. Optimize GC conditions for analyte separation and sensitivity. Once optimized, the same GC conditions must be used for the analysis of all standards, samples, blanks, and MS/MSDs. 10.1.1.1 GC - Full-scan quadrupole Initial column temperature: Column temperature program: Final column temperature hold: Injector temperature: Injection mode: Sample injection volume: GC column: Column dimensions: Carrier gas: 10.1.1.2 GC-TOF Initial oven temperature: Column temperature program: Final column temperature hold: 40 °C for 3 minutes 40 - 150 °C at 10 °C/minute 150 - 280 °C at 25 °C/minute 280 °C; 10.8 minutes after the last compound (triphenyl phosphate) has eluted 250 °C Grob-type, splitless for 0.75 minutes 1.0 jaL Bonded phase silicon coated fused silica capillary (see Section 6.4.2) 30 m x 0.25 mm (or 0.32 mm) x 0.25 (im Helium at 32 cm/second 55 °C for 0.5 minutes 20 °C/minute to 100 °C (0 minute), 40 °C/minute to 280 280 °C (2.75 minutes) 22 September 2016 ------- Analytical Protocol for VX Using GC/MS Inj ector temperature: Injection mode: Sample injection volume: GC column: Column dimensions: Carrier gas: 250 °C Grob-type, splitless 1.0 jiL HP-5MS UI (Agilent) or equivalent 15m X 0.18mm X 0.18(im Helium at 1.2 mL/minute 10.1.2 MS The following MS analytical conditions were used during laboratory studies and are provided for guidance. Other conditions may be used, provided that all technical acceptance criteria are met. Optimize MS conditions for analyte separation and sensitivity. Once optimized, the same MS conditions must be used for the analysis of all standards, samples, blanks, and MS/MSDs. 10.1.2.1 MS - Full-scan quadrupole MS transfer line temperature: Source temperature: MS quadrupole temperature: Electron energy: Scan range: Ionization mode: Scan time: Library searching: 10.1.2.2 MS-TOF MS transfer line temperature: Source temperature: Electron energy: Scan range: Ionization mode: Scan time: 280 °C 230 °C or according to manufacturer's specifications 150 °C 70 eV (nominal) 35 to 500 m/z Electron Ionization (EI), positive 3.15 scan/sec (minimum of 3 scans/ second) NIST 05 Mass Spectral Data Base 295 °C 250 °C or according to manufacturer's specifications 70 eV (nominal) 35 to 500 m/z Electron Ionization (EI), positive 15 scans/second 10.2 GC/MS Mass Calibration (Tuning) and Ion Abundance 10.2.1 Summary of GC/MS instrument performance check The GC/MS system must be tuned to meet the manufacturer's specifications, using a suitable calibration compound such as perfluoro-tri-«-butylamine (FC-43) or perfluorokerosene. The mass calibration and resolution of the GC/MS system are verified by the analysis of the instrument performance check solution (Section 7.2.2.3). Prior to the analysis of any samples, including MS/MSDs, blanks, or calibration standards, the laboratory must establish that the GC/MS system meets the mass spectral ion abundance criteria for the instrument performance check solution (Table 3, Section 17) containing DFTPP. 23 June 2016 ------- Analytical Protocol for VX Using GC/MS 10.2.2 Frequency of GC/MS instrument performance check - The instrument performance check solution must be injected once at the beginning of each 24- hour period, during which samples, blanks, or standards are to be analyzed. The 24-hour period begins at the moment of injection of the DFTPP solution. The time period ends after 24 hours have elapsed according to the system clock. 10.2.3 GC/MS instrument performance check The analysis of the instrument performance check solution may be performed as an injection of 50 ng or less of DFTPP into the GC/MS or by adding a sufficient amount of DFTPP to the calibration standards to result in an on-column amount of 50 ng or less of DFTPP (Section 7.2.2.3) and analyzing the calibration standard. 10.2.4 Technical acceptance criteria for GC/MS instrument performance check 10.2.4.1 The instrument performance check solution must be analyzed at the frequency described in Section 10.2.2. 10.2.4.2 Abundance criteria are listed in Table 3, Section 17 for guidance. The mass spectrum of DFTPP must be acquired in the following manner: three scans (the peak apex scan and the scans immediately preceding and following the apex) are acquired and averaged. Background subtraction is required, and must be accomplished using a single scan acquired no more than 20 scans prior to the elution of DFTPP. The background subtraction should be designed only to eliminate column bleed or instrument background ions. Do not subtract part of the DFTPP peak. Note 1: All subsequent standards, samples, MS/MSDs, and blanks associated with a DFTPP analysis must use the identical GC/MS instrument run conditions. Note 2: The above tuning criteria are suggested when using DFTPP. If alternative tuning methods are used, consult the method or manufacturer notes for guidance on criteria. 10.2.5 Corrective action for GC/MS instrument performance check The following corrective actions are minimum procedures. The analyst may try other corrective action procedures to meet criteria. 10.2.5.1 If the GC/MS instrument performance check technical acceptance criteria are not met, re-tune the GC/MS system. It may be necessary to perform maintenance to achieve the technical acceptance criteria. 10.2.5.2 The instrument performance check technical acceptance criteria in Section 10.2.4 must be met before any standards, samples, including MS/MSDs, or required blanks are analyzed. 24 September 2016 ------- Analytical Protocol for VX Using GC/MS 10.3 Initial Calibration Prior to sample analysis and after instrument performance check technical acceptance criteria have been met, each GC/MS system must be calibrated at a minimum of five concentrations (Section 7.2.2.4.1 and Table 8, Section 17) to determine instrument sensitivity and the linearity of GC/MS response for the target and surrogate compounds. If the RSD criteria cannot be met, a linear or quadratic curve may be used. Each initial calibration standard contains the target compound, surrogate, and internal standard. 10.3.1 Frequency of initial calibration 10.3.1.1 Each GC/MS should be calibrated whenever the laboratory takes corrective action that might change or affect the initial calibration criteria, or if the CCV technical acceptance criteria are not met. 10.3.1.2 If time remains in the 24-hour period after meeting initial calibration acceptance criteria, samples may be analyzed. In this case, it is not necessary to analyze an opening CCV standard prior to sample analysis. 10.3.2 Procedure for initial calibration 10.3.2.1 Prepare at least five calibration standards containing the detected target compound and associated surrogate. Example concentrations for the calibration standards are provided in Section 7.2.2.4.1 and Table 8, Section 17. 10.3.2.2 Add a sufficient amount of internal standard solution (Section 7.2.2.5) to aliquots of calibration standards to result in 0.5 ng/(.iL of the internal standard. Standards specified in Section 7.2.2.4 should permit the target compound to have a relative retention time (RRT) of approximately 0.60 to 1.70, using the internal standard listed in Table 4, Section 17. 10.3.2.3 Analyze each calibration standard by injecting 1.0 |_iL of standard. The same injection volume must be used for all standards, samples, and blanks. 10.3.3 Calculations for initial calibration 10.3.3.1 Calculate the relative response factors (RRFs) for VX and the surrogate using Eq. 3 and the primary characteristic ions found in Table 5, Section 17. For the internal standard, use the primary ion listed in Table 5 unless interferences are present (e.g., peak overlap, co-elution). Unless otherwise stated, the area response of the primary characteristic ion is the quantitation ion. Eq. 3 RRF Calculation A C RRF = —— x —— A is Cx 25 September 2016 ------- Analytical Protocol for VX Using GC/MS where: Ax = Area of the characteristic ion for the compound to be measured (Table 5, Section 17) Ais = Area of the characteristic ion for specific internal standard (Table 5) Cis = Amount of the internal standard injected (ng) Cx = Amount of the target compound or surrogate injected (ng) 10.3.3.2 The Mean RRF ( RRF) for the Initial Calibration RRFs and mean RRFs must be calculated for all compounds. Calculate the %RSD of the RRF values for the initial calibration. 10.3.4 Technical acceptance criteria for initial calibration 10.3.4.1 An initial calibration should be performed at the frequency described in Section 10.3.1 on a GC/MS system meeting the instrument performance check technical acceptance criteria (Section 10.2.4). 10.3.4.2 The RRFs for VX and the surrogate should be greater than or equal to 0.01. 10.3.4.3 The %RSD of the RRFs over the initial calibration range for VX and each surrogate must be less than or equal to 20. If %RSD cannot meet this criterion, curve fitting by linear or quadratic regression may be used provided the R2 value is greater than or equal to 0.99 (linear) or 0.995 (quadratic). Refer to Section 12.2.7 if linear regression is used; refer to SW-846 Method 8000C (Reference 16.16), if quadratic curve fitting is needed. If regression curve fitting is used, percent drift (PD) (as calculated using Eq. 4, Section 10.4.4.2) for each standard must be less than or equal to ±50. 10.3.5 Corrective action for initial calibration The following corrective actions are minimum procedures. The analyst may try other corrective action procedures to meet criteria. 10.3.5.1 If technical acceptance criteria using at least one of the three optional approaches to initial calibration (%RSD of the RRFs, linear regression, or quadratic regression) are not met, inspect the system for problems, take corrective actions, remake standards and re-calibrate. If criteria are not met with re-calibration, remake the calibration standards and repeat. If the criteria are still not met, the laboratory will flag all data associated with the calibration. Note: If technical acceptance criteria for the initial calibration are not met and the initial calibration contains more than five calibration levels, the laboratory may remove calibration point(s) from either extreme end of the calibration range and reassess the calibration. Data points from within a calibration range must not be removed. 10.3.5.2 Initial calibration technical acceptance criteria must be met before any 26 September 2016 ------- Analytical Protocol for VX Using GC/MS samples, including MS/MSDs or required blanks are analyzed and reported without data qualification. 10.4 Continuing Calibration Verification (CCV) 10.4.1 Summary of CCV Prior to the analysis of samples and after instrument performance check and initial calibration technical acceptance criteria have been met, each GC/MS system must be routinely checked by analyzing a CCV standard to ensure that the instrument continues to meet the instrument sensitivity and linearity requirements. The CCV standard contains VX, surrogate, and internal standard. 10.4.2 Frequency of CCV - Each GC/MS used for analysis must be checked at the beginning and at the end of each analytical batch of <20 injections, excluding instrument blanks. When subsequent analytical batches are run within a single 24-hour period, the closing CCV may be used as the opening CCV for a new analytical batch, provided that the closing CCV meets all technical acceptance criteria for an opening CCV (see Section 10.4.5). 10.4.3 Procedure for CCV 10.4.3.1 Add a sufficient amount of internal standard solution (Section 7.2.2.5) to an aliquot of CCV standard to result in a concentration of 0.5 ng/(.iL for both quadrupole and TOF analyses. 10.4.3.2 Analyze the CCV standard by injecting 1.0 |_iL of standard. 10.4.4 Calculations for CCV Calibration verification involves calculation of the PD (Eq. 4a) or the percent difference of the RRFs between the initial calibration and each subsequent CCV (Eq. 4b). The CCV approach will depend on the how the initial calibration was performed. If a regression technique (linear or quadratic) was used, then Eq. 4a is used to determine a percent drift. If the RRF approach is used, then Eq. 4b is used to determine the percent difference of the RRFs. 10.4.4.1 If regression techniques are used for initial calibration, the CCV must be evaluated in terms of PD, which is calculated using concentrations (see Eq. 3a). Eq. 4a Percent Drift (PD) Calculation for CCV Calculated Concentration-Theoretical Concentration lJL) = xl00% Theoretical Concentration 10.4.4.2 Calculate an RRF for the target compound and surrogate using Eq. 3 and the primary quantitation ions found in Table 5, Section 17. If regression techniques are used for the initial calibration, proceed to Section 10.4.4.3. 27 September 2016 ------- Analytical Protocol for VX Using GC/MS 10.4.4.3 Calculate the Percent Difference (%Difference) between the RRF of the most recent initial calibration and the continuing calibration verification RRF for each target compound and surrogate using Eq. 4b. Eq. 4b Relative Response Factor (RRF) % Difference Calculation RRF — RRF %DifferenceRRF = 0 —L x 100 RRF RRF, where: RRFi= Mean RRF from the most recent initial calibration meeting technical acceptance criteria. RRFC = RRF from CCV standard. 10.4.5 Technical acceptance criteria for CCV 10.4.5.1 The CCV standard should be analyzed at or near the mid-point concentration level, at the frequency described in Section 10.4.2, on a GC/MS system meeting the instrument performance check and the initial calibration technical acceptance criteria. 10.4.5.2 The RRF for VX and the surrogate must be greater than or equal to 0.01. 10.4.5.3 For the opening CCV, the PD or %Difference of RRFs for the target compound and surrogate should be within the range of ±40. 10.4.5.4 For the closing CCV, the PD or %Difference of RRFs for the target compound and surrogate should be within the range of ±50. 10.4.5.5 Excluding those ions in the solvent front, no quantitation ion may saturate the detector. 10.4.6 Corrective action for CCV The following corrective actions are minimum procedures. The analyst may try other corrective action procedures to meet criteria. 10.4.6.1 If the CCV technical acceptance criteria in Section 10.4.5 are not met, recalibrate the GC/MS instrument according to Section 10.3. 10.4.6.2 CCV technical acceptance criteria should be met before any samples MS/MSDs, or required blanks are analyzed. If CCV criteria are not met, flag associated samples and blanks accordingly. 28 September2016 ------- Analytical Protocol for VX Using GC/MS 10.5 Instrument Blank 10.5.1 Summary of instrument blank An instrument blank is comprised of DCM spiked with internal standard at the same concentration used for associated samples. The purpose of the instrument blank is to investigate the impact of carry-over. 10.5.2 Frequency of instrument blank An instrument blank is recommended for analysis following suspected carry-over or during analysis of samples containing suspected high concentrations. 10.5.3 Procedure for instrument blank analysis Add sufficient amount of internal standard solution (Section 7.2.2.5) to an aliquot of the solvent used to prepare calibration standards and sample extracts to result in a concentration of 0.5 ng/|_iL. Analyze each instrument blank by injecting a volume of 1.0 (iL. 10.5.4 Calculations for instrument blank Calculate the concentrations of any observed target analyte using Eq. 6 (Section 12.2.6.1), setting Vt, V0, and dilution factor (DF) all equal to 1. 10.5.5 Technical acceptance criteria for instrument blank If an instrument blank is analyzed, the concentration of VX in the instrument blank should be less than the concentration of VX in the low calibration standard. The area response of the internal standard should be within 50 - 150 % of the associated CCV or mid-level concentration of the initial calibration. 10.5.6 Corrective action for instrument blank If an instrument blank is analyzed and the instrument blank technical acceptance criteria are not met, analyze an additional instrument blank. If the problem persists, inspect the system for problems and take corrective actions to achieve the acceptance criteria. Instrument blank technical acceptance criteria should be met before samples are analyzed. Samples that are analyzed with corresponding instrument blanks that do not meet the instrument blank criteria should be re- analyzed, or the corresponding data should be flagged. 29 September 2016 ------- Analytical Protocol for VX Using GC/MS 11.0 ANALYTICAL PROCEDURE 11.1 Sample Preparation - General 11.1.1 If less than the specified sample amount is received, the laboratory should use a reduced sample size for the analysis and adjust the calculation accordingly. For the purposes of this method, it is recommended that some sample be retained (if possible) for potential future evidentiary use. 11.1.2 If multi-phase samples (e.g., a two-phase liquid sample, oily, sludge/sandy soil sample) are received by the laboratory, the laboratory shall contact the agency to apprise them of the type of sample received. If some or all phases of the sample are amenable to analysis, the agency requesting the analyses may require the laboratory to do any of the following: • Mix the sample and analyze an aliquot from the homogenized sample. • Separate the phases of the sample and analyze each phase separately. • Separate the phases and analyze one or more of the phases, but not all of the phases. • Do not analyze the sample. 11.2 Preparation of Water Samples Using Microscale Extraction 11.2.1 Approximately 35 mL of a water sample is required for this extraction. If extraction is to be performed in the sample receipt vial, remove any excess sample such that a total sample volume of 35 mL is obtained and recap the vial. Weigh the capped vial. Record the weight to the nearest 0.1 gram. Alternatively, 35.0 mL of sample can be transferred by pipette into the vial and the weighing step can be eliminated. Note: The conical bottoms of centrifuge vials allow the DCM layer to be removed more easily than the VOA vials. 11.2.2 pH determination - If pH determination is requested, transfer a separate aliquot of the water sample to a beaker, and determine the pH of the sample with a pH meter or wide-range pH paper. Document the pH in the data narrative. Discard the sample aliquot. 11.2.3 For GC/MS full-scan and TOF analysis, spike l .0 |ig of each surrogate (add 40 (.iL if prepared as in Section 7.2.2.1) into each sample, blank, etc. The final volume of the extracts will be 2.0 mL; therefore, the concentration of the surrogates in the extract is expected to be 0.50 |_ig/mL. 11.2.4 Add -8.8 grams of sodium chloride and shake vigorously, or vortex each vial for two minutes or until the sodium chloride dissolves completely. 11.2.5 Add 2.00 mL of DCM, using a Class A volumetric pipette or syringe. Cap tightly and agitate the contents vigorously for approximately two minutes, either by hand or using a vortex mixer or shaker table. 30 September 2016 ------- Analytical Protocol for VX Using GC/MS Briefly allow the phases to settle by gravity for ~ five minutes. Centrifugation (three minutes at 500 revolutions per minute [rpm]) is strongly recommended to facilitate separation of the phases and affords a greater recovery of the final sample extract. CAUTION: The maximum safe handling speed of each centrifuge will depend, in part, on the vials used and should be determined prior to use. Using a 2.0-mL syringe or pipette, transfer approximately 1.0 mL (or as much as possible) of the DCM (lower) layer to a 2-mL or 4-mL vial with a PTFE-lined screw cap, taking precautions to exclude any water from the syringe or pipette. Add a small amount (-50 mg) of anhydrous sodium sulfate to the vial, then cap the vial, and shake vigorously or vortex for two minutes. Make sure that the extract is sufficiently dry and that some of the sodium sulfate added is free- flowing (i.e., not clumped). Using a 1.0 mL syringe or pipette, transfer 1.0 mL (or a known volume, if less than 1.0 mL of extract is collected) of the dried extract to a 2.0-mL vial (or autosampler vial insert) with a PTFE-lined screw cap. Cap the vial. Note: If stored prior to analysis, extracts must be protected from light and stored at<6°C (Section 8.3). 11.2.9 Discard the remaining contents of the VOA vials according to laboratory waste disposal guidelines. Shake off the last few drops with short, brisk wrist movements. If needed, rinse the vial with a water-soluble solvent to ensure that the extraction solvent is removed. If the vial was pre-weighed (i.e., exact sample volume used in Section 11.2.1 is unknown), reweigh the capped vial, and record the weight to the nearest 0.1 grams. The difference between this weight, and the weight determined in Section 11.2.1 is equal to the volume of water extracted, in milliliters. As the density of water is 1.00 g/mL (at 20°C), the volume of water extracted, in milliliters, may be assumed to be equal to the weight of water extracted. 11.2.10 Proceed to Section 11.6 for sample analysis. 11.3 Preparation of Solid Samples Using Microscale Extraction Note: The following procedures have been evaluated in a multi-laboratory study using Ottawa sand and dried soils, and have not been evaluated for field samples. Laboratory results are provided in Section 17. 11.3.1 Decant and discard any water layer. Mix samples thoroughly, especially composited samples. Discard any foreign objects such as sticks, leaves, and rocks. 11.3.2 pH determination - If pH determination is requested, transfer a 1:1 (w:w) ratio of sample:water to a 100-mL beaker and stir for one hour. Determine the pH of the sample with a pH meter or wide-range pH paper, and document the pH in the data narrative. Discard this portion of the sample. 11.2.6 11.2.7 11.2.8 31 September 2016 ------- Analytical Protocol for VX Using GC/MS 11.3.3 Percent moisture determination - If percent moisture determination is requested, immediately after weighing the sample for extraction, weigh 5-10 grams of the sample into a tared vial. Dry overnight at 103 - 105 °C, and cool in a desiccator before weighing. Determine the percent moisture (%Moisture) using Eq. 5. CAUTION: Due to the high toxicity associated with VX, percent moisture determinations should be performed only in an oven with appropriate engineering controls. Eq. 5 Percent Moisture Calculation vet sample grams of wet sample %Moistu,e = gramS °f WCt Samp'e " gnmS °f iry' Smlp'e x 100 11.3.4 Extraction of VX from soil samples 11.3.4.1 Weigh 10 grams of sample into a tared extraction vial (i.e., 60-mL VOA vial). Wipe the lip and threads of the vial with a clean cloth (e.g., Kimwipe®[Kimberly-Clark Professional, Roswell, GA] or equivalent). Record weight to the nearest 0.01 gram. 11.3.4.2 For full-scan quadrupole and TOF MS, add 0.5 |ig surrogate (add 20 (.iL if using a 25 |ig/m L surrogate spiking solution) in DCM to the vial. The final volume of the extract is 1.0 mL; therefore, the concentration of surrogates in the extract is expected to be 0.5 |_ig/mL. 11.3.4.3 Add 5-10 pre-cleaned glass beads. Mix until the sample is homogenized using a metal spatula. Break up any chunks with a metal spatula, working quickly but gently. 11.3.4.4 Add 30 mL of Tris buffer (Section 7.1.4) to the vial, and cap tightly. 11.3.4.5 Shake the vial vigorously or vortex for approximately 20 seconds or until the slurry is free-flowing. Break up any chunks with a metal spatula, working quickly but gently. Cap immediately when finished. 11.3.4.6 Extract the sample by agitating for approximately 15 minutes, using a shaker table or sonicator. Note: Sonication at high power should be avoided for soils having high silt content; the resulting fine particles can create problems with filtering and extracting solvent from the sample. 11.3.4.7 Allow the solids to settle or centrifuge for 1 - 2 minutes at 1000 rpm. If the solid is still unsettled, repeat the centrifuge step, but increase speed to 2500 rpm. CAUTION: The maximum safe handling speed of each centrifuge will depend, in part, on the vials used and should be determined prior to use. Repeat until the solid is completely settled. If after repeating the centrifuging steps several times the solid is still unsettled, proceed to Section 11.3.4.8. Once the solid has 32 September 2016 ------- Analytical Protocol for VX Using GC/MS settled, decant or pipette the solvent layer into a pre-cleaned, 60-mL VOA vial with PTFE-lined screw cap and proceed to 11.3.4.9. Note: For solids that have difficulty settling, pipetting is recommended. 11.3.4.8 If solids are not settled out by centrifugation (Section 11.3.4.7), filter by placing a small plug of glass wool into a small glass funnel. Wet the glass wool thoroughly with Tris buffer. Pour the sample (both solid and liquid portions) into the funnel. Rinse with approximately 2 - 3 mLs of Tris buffer as soon as the surface is exposed, not allowing it to dry. 11.3.4.9 Add 5.00 mL of DCM, using a Class A volumetric pipette or syringe. Cap tightly, and agitate by vigorous shaking, vortex mixer, shaker table, or sonication for approximately two minutes. 11.3.4.10 Briefly allow the phases to settle by gravity or by centrifugation for ~5 minutes. Note: Centrifugation (three minutes at 500 rpm) is strongly recommended to facilitate separation of the phases and affords a greater recovery of the final sample extract. 11.3.4.11 Using a syringe or pipette, transfer as much of the DCM (lower) layer as possible to a vial with a PTFE-lined screw cap, taking precautions to exclude any water from the syringe or pipette. Add a small amount (-50 mg) of anhydrous sodium sulfate to the vial, cap the vial, and shake vigorously or vortex for two minutes. Make sure that the extract is sufficiently dry and that some of the sodium sulfate added is free-flowing (e.g., not clumped). 11.3.4.12 Proceed to Section 11.5 for extract concentration. Once the extract is concentrated, proceed to Section 11.6 for analysis. 11.4 Preparation of Wipe Samples by Microscale Extraction 11.4.1 Place the wipe into an extraction vial (i.e., 40-mL VOA vial). For GC/MS full- scan and TOF analyses, add 0.5 |ig of each surrogate standard compound (add 20 (.iL if prepared as in Section 7.2.2.1) in DCM directly onto the wipe. The final volume of the extract is 1.0 mL; therefore, the concentration of the surrogates in the extract is expected to be 0.5 |_ig/mL. 11.4.2 Add 15 mL of DCM to the vial and cap tightly. 11.4.3 Extract the sample by agitating for approximately 15 minutes, using a shaker table or sonicator. 11.4.4 Remove the vials from the sonicator or shaker table, shake briefly by hand and allow the solvent layer to settle. Transfer the solvent layer by pipette into a pre- cleaned, 40-mL, clear glass vial with PTFE-lined screw cap. 33 September 2016 ------- Analytical Protocol for VX Using GC/MS 11.4.5 Proceed to Section 11.5 for extract concentration. Once the extract is concentrated, proceed to Section 11.6 for analysis. 11.5 Final Concentration of Extract - Nitrogen Evaporation Technique (RapidVap® or equivalent) for solid and wipe samples. 11.5.1 Nitrogen evaporation technique - If using a RapidVap® or TurboVap® N2 systems (Biotage, LLC, Charlotte, NC), follow the manufacturer's guidelines. For the RapidVap®, a temperature of 40 °C is recommended. If using a water bath, place the vial in a warm water bath (30-40 °C recommended) and evaporate the solvent volume to just below 1.0 mL by blowing a gentle stream of clean, dry nitrogen above the extract. It is recommended that the internal wall of the vial be rinsed down several times with DCM during the operation. If using a RapidVap® the solvent rinse should, at a minimum, be done during final adjustment of the extract volume. During evaporation, the tube solvent level must be kept below the water level of the bath. The extract must never be allowed to become dry. Adjust the final volume to 1.0 mL with the same solvent used for extraction. Transfer the extract to a 2-mL autosampler vial, cap, and label the vial. Store at 4 °C (± 2 °C). CAUTION: Gas lines from the gas source to the evaporation apparatus should be stainless steel, copper, or PTFE tubing. With the exception of PTFE, plastic tubing must not be used between the carbon trap and the sample since it can introduce interferences. 11.5.2 Final extract volumes - The final extract volumes in Sections 11.5.2.1 through 11.5.2.4 are recommended volumes. 11.5.2.1 Water/Liquid - As concentration of the sample extract is not needed for these sample matrices, no adjustment of the final extract volume is required. The nominal volume of DCM added to water samples is 2.0 mL. The target compound and surrogate spiking solutions also contain DCM; therefore, the total volume of DCM added may be slightly greater than 2.0 mL. The actual total volume of DCM should be used in the calculations in Section 12.2. 11.5.2.2 Solid - Adjust the extract volume to a final volume of 1.0 mL with DCM. 11.5.2.3 Wipe - Adjust the extract volume to a final volume of 1.0 mL with DCM. 11.5.2.4 If extracts are stored prior to analysis, transfer the extract to a PTFE- lined screw-cap vial (approximately 2.0 mL), label the vial, and store at <6 °C. 11.6 Extract Analysis by GC/MS 11.6.1 Analyze extracts only after the GC/MS system has met the instrument 34 September 2016 ------- Analytical Protocol for VX Using GC/MS performance check (Section 10.2.4), initial calibration (Section 10.3.5), and CCV technical acceptance requirements (10.4.5). The same instrument conditions used for calibration must be used for the analysis of samples. 11.6.2 Add internal standard solution (Section 7.2.2.5) to an accurately measured aliquot of each extract, cap the vial, and invert several times to mix the contents. For full-scan quadrupole or TOF MS analyses, add a sufficient amount of internal standard solution to result in 0.5 ng/(.iL concentration of the internal standard. 11.6.3 If extracts are to be diluted, add the internal standard after dilution. Internal standard must be added to maintain the required 0.5 ng/(.iL (for both full-scan quadrupole and TOF) of internal standard in the extract volume. 11.6.4 Inject 1.0 |_iL of the extract into the GC/MS. Note: The same injection volume used for calibration standards must be used for extracts. 11.6.5 Sample extract dilution 11.6.5.1 If the response of the target compound in any extract exceeds the response of the target compound in the high standard of the initial calibration, that extract must be diluted. Add additional internal standard solution such that the concentration in the diluted extract is 0.5 ng/(.iL for each internal standard, and analyze the diluted extract. 11.6.5.2 Use the results of the original analysis to determine the approximate dilution factor (DF) required for the analyte peak to fall within the initial calibration range. The DF chosen must keep the response of the peak for the target compound in the upper half of the calibration range of the instrument. 12.0 CALCULATIONS AND DATA ANALYSIS 12.1 Qualitative Identification of Target Compounds 12.1.1 The target compound should be identified by an analyst competent in the interpretation of mass spectra by comparison of the sample mass spectrum to the mass spectrum of the standard of the suspected compound. Two criteria must be satisfied to verify identification: 1) Elution of the sample analyte within the GC RRT unit window established from the 24-hour calibration standard; and 2) Correspondence of the sample analyte and calibration standard component mass spectra. 12.1.2 For establishing correspondence of the GC RRT, the sample component must compare within ±0.06 RRT units of the standard component. For samples analyzed during the same 24 hour time period as the initial calibration standards, 35 September 2016 ------- Analytical Protocol for VX Using GC/MS compare the analyte RTs to those from the midpoint initial calibration standard. Otherwise, use the corresponding CCV standard. If coelution of interfering components prohibits accurate assignment of the sample component RRT from the total ion chromatogram, the RRT should be assigned by using EICPs for ions unique to the component of interest (see Table 5, Section 17 for appropriate characteristic ions, surrogate, and internal standard). 12.1.3 For comparison of standard and sample component mass spectra, mass spectra obtained from a calibration standard at a concentration of the target analyte closest to the concentration of the analyte in the sample are required. Once obtained, these standard spectra may be used for identification purposes only if the GC/MS meets the DFTPP instrument performance requirements (see Section 10.2 for instrument performance check requirements). 12.1.4 For TOF MS and full-scan quadrupole MS analyses, all ions present in the standard mass spectrum at a relative intensity greater than 10 % (the most abundant ion in the spectrum equaling 100 %) must be present in the sample spectrum. The relative intensities of ions specified in Table 5, Section 17 must agree within ± 20 % between the standard and sample spectra (e.g., for an ion with an abundance of 50 % in the standard spectra, the corresponding sample ion abundance must be between 30 - 70 %). Ions greater than 10 % in the sample spectrum, but not present in the standard spectrum, must be considered and accounted for by the analyst making the comparison. The verification process should favor false positives. All compounds meeting the identification criteria must be reported with their spectra. When target compounds are below QLs but the spectrum meets the identification criteria, report the concentration with a "J". For example, if the QL is 5.0 (ig/L and concentration of 3.0 |ig/L is calculated, report as "3.0 J". For TOF analysis, the signals for the quantitation ions in Table 5, Section 17 must be present and must maximize within a period of two seconds. The S:N for the GC peak at each ion must be greater than or equal to 2.5 for the target compound and surrogate detected in a sample extract, and greater than or equal to 10 for the target compound and surrogate in the CCV standard. 12.1.5 If the compound cannot be verified by all of the spectral identification criteria in Sections 12.1.1-12.1.4, but in the technical judgment of the mass spectral interpretation specialist the identification is correct, then the laboratory should report the identification and proceed with quantitation. 12.2 Data Analysis and Calculations of Target Compounds 12.2.1 The target compound is quantitated by the internal standard method, using the EICP area of primary quantification ions listed in Table 5, Section 17. 12.2.2 It is expected that situations will arise when the automated quantitation procedures in the GC/MS software provide inappropriate quantitations. This normally occurs when there is compound coelution, baseline noise, or matrix interferences. In these circumstances, the laboratory must perform a manual quantitation. Manual integrations are performed by integrating the area of the quantitation ion of the compound. This integration should include the area 36 September 2016 ------- Analytical Protocol for VX Using GC/MS attributable to the specific target compound. The area integrated must not include baseline background noise. The area integrated should not extend past the point where the sides of the peak intersect with the baseline noise. Manual integration is not to be used solely to meet QC criteria, nor is it to be used as a substitute for corrective action on the chromatographic system. 12.2.3 In some instances, the data system report may have been edited or manual integration or quantitation may have been performed. In all such instances, the GC/MS operator should identify such edits or manual procedures by initialing and dating the changes made to the report, and include the integration scan range. The GC/MS operator should also mark each integrated area on the quantitation report. 12.2.4 The requirements listed in Sections 12.2.1 - 12.2.3 apply to all standards, samples, and blanks. 12.2.5 The RRF from the initial calibration is used to calculate the concentration in the sample. Secondary ion quantitation is allowed only when there are sample interferences with the primary ion. If linear regression is used, a regression curve should be used to calculate the concentration in samples. Refer to Section 12.2.7 for calculating sample concentration using linear regression. 12.2.6 Calculate the concentration in the sample using the RRF and Eq. 6-8. 12.2.6.1 Water Eq. 6 Concentration of Water Sample Concentration (|ig/L) = (A ) (I ) (V,) (DF) (Ais) (RRF) (V0) (Vi) where: Ax = Area of the characteristic ion for the target compound A1S = Area of the characteristic ion for the internal standard Is = Amount of internal standard injected in ng V0 = Volume of water extracted in mL V, = Volume of extract injected in |_iL Vt = Volume of the extract in |aL (Extraction of water samples does not include concentration; Vtis equal to the sum of the volumes of solvent added for extraction and the addition of surrogates and any spiked target compound.) RRF = Mean RRF determined from the initial calibration standard DF = Dilution Factor. If no dilution is performed, DF = 1.0. The DF for analysis of water samples is defined as: (jL most conc. extract used to make dilution + jxL clean solvent Dr — (jL most conc. extract used to make dilution 37 September 2016 ------- Analytical Protocol for VX Using GC/MS 12.2.6.2 Solid Eq. 7 Concentration of Solid Sample Eq. 7 includes a %moisture factor (D) for those cases when data is to be reported on the basis of dry sample weight. In cases where results are reported in terms of sample weight, this factor is deleted from the equation. (A )(I )(V )(DF) Concentration ug/Kg (Dry weight basis) = ===== (Ais)(Vi)(RRF)(Ws)(D) where: Ax, Is, Ais, V, and RRF are as given for water, above. V, = Volume of concentrated extract in |.iL ^ 100 - %Moisture 100 %Moisture is as given in EQ. 5 Ws = Weight of sample extracted in grams RRF = Mean RRF determined from the initial calibration standard DF = Dilution Factor 12.2.6.3 Wipes Eq. 8 Concentration of Wipe Sample (Ax)(ls)(V<)(PF) Concentration „gW - ,a) ( RgJ ) where: Ax = area response for the compound to be measured, counts A1S = area response for the internal standard, counts Is = amount of internal standard, |.ig RRF = the mean RRF from the most recent initial calibration, dimensionless Area = area of surface wiped, cm2. If concentration is reported as ng/wipe, area = 1 wipe. V, = volume of concentrated extract, |.iL V, = volume of extract injected, |.iL DF = dilution factor for the extract. If there was no dilution, DF equals 1. If the sample was diluted, DF is greater than 1. 38 September 2016 ------- Analytical Protocol for VX Using GC/MS 12.2.7 Calculate the concentration in the sample using linear regression. The following procedure is used to calculate analyte concentrations using linear regression calibration curves. Refer to SW-846 Method 8000C (Reference 16.16) if calibration curves were determined using quadratic equations. 12.2.7.1 Set y = (Peak Area of Target/Peak Area of Internal Standard) and x = (Theoretical Concentration of Target/Theoretical Concentration of Internal Standard). 12.2.7.2 Plot (Peak Area of Target/Peak Area of Internal Standard [Y-axis]) vs. (Theoretical Concentration of Target/Theoretical Concentration of Internal Standard). 12.2.7.3 Determine the slope of the line (m) and the y-intercept (b). 12.2.7.4 Rearrange the line equation to solve for x: x = (y-b)/m. 12.2.7.5 Multiply x by the concentration of the internal standard to get concentration of target analyte in extract. 12.2.7.6 Multiply the concentration of target analyte in the extract by the extract volume, and divide by the sample volume to get concentration of target analyte in sample. 12.2.8 Adjusted QL calculations Adjusted QLs are used in situations when the laboratory may not have a sample size that is sufficient for the method as written, or if the prescribed extract volume was not used or recovered. 12.2.8.1 Water samples Eq. 9 Aqueous Adjusted QL Adjusted QL = Method QL x (Vx)(Vt)(DF) (Vo)(vc) where: Vt, DF, and V0 are as given in Eq. 6. Vx = Recommended sample volume (35 mL) Vc = Recommended concentrated extract volume (2000 jxL) 12.2.8.2 Solid samples Eq. 10 Solid Adjusted QL Adjusted QL = Method QL x (Wx)(Vt)(DF) (WS)(VC)(D) where: Vt and DF are as given in Eq. 6. Ws and D are as given in Eq. 7. 39 September 2016 ------- Analytical Protocol for VX Using GC/MS Wx = Method sample weight (10 grams) Vc = Method concentrated extract volume (1000 |iL) 12.2.9 Surrogate recoveries 12.2.9.1 Calculate surrogate recoveries for all samples, blanks, and MS/MSDs. Determine if recovery is within limits (Table 7, Section 17). 12.2.9.2 Calculate the concentrations of the surrogates using the same equations used for VX. Calculate the recovery of each surrogate using Eq. 11. Eq. 11 Percent Recovery Cs Recovery = %R = — x 100 where: Cs = Measured concentration of the spiked sample aliquot. Cn = Nominal (theoretical) concentration increase that results from spiking the sample, or the nominal concentration of the spiked aliquot (for LCS). 12.3 Technical Acceptance Criteria for Sample Analysis 12.3.1 The samples must be analyzed on a GC/MS system meeting the instrument performance check, initial calibration, CCV, and blank technical acceptance criteria. 12.3.2 The sample must be extracted and analyzed within the technical holding times. 12.3.3 The sample must have an associated method blank meeting the blank technical acceptance criteria. 12.3.4 The percent recoveries of the surrogates in a sample should be within the recovery limits listed in Table 7, Section 17 (see Table 4, Section 17 for analyte specific surrogates). The surrogate recovery requirements do not apply to samples that have been diluted. 12.3.5 The instrumental response (EICP area) for the internal standard in the sample must be within the range of 50.0 - 200 % of the response of the internal standard in the most recent CCV standard analysis. 12.3.6 The RT shift for the internal standard must be within ± 0.50 minute (30 seconds) between the sample and the most recent CCV standard analysis. 12.3.7 Excluding those ions in the solvent front, no ion may saturate the detector. If a target compound concentration exceeds the upper limit of the initial calibration range, a more dilute aliquot of the sample extract must also be analyzed. 40 September 2016 ------- Analytical Protocol for VX Using GC/MS 12.4 Corrective Action for Sample Analysis 12.4.1 The sample technical acceptance criteria must be met before data are reported. If the corrective actions described in this section did not solve the problem, all associated sample and blank data must be flagged accordingly. 12.4.2 Corrective action for failure to meet instrument performance checks and initial and continuing calibration verification must be completed before the analysis of samples. If the corrective actions described in Sections 10.2.5 (for instrument performance check), 10.3.5 (for initial calibration), or 10.4.6 (for CCV) did not solve the problem, all associated sample and blank data must be flagged accordingly. 12.4.3 Corrective Action for Surrogate Recoveries that Fail to Meet Their Acceptance Criteria (Section 12.3.4 and Table 7, Section 17). 12.4.3.1 If the surrogate recoveries in a sample fail to meet the acceptance criteria specified in Section 12.3.4, then check calculations, sample preparation logs, surrogate standard spiking solutions, and the instrument operation. 12.4.3.2 If the above actions do not correct the problem, then the problem might be due to a sample matrix effect. To determine if there was matrix effect, take the following corrective action steps: 12.4.3.2.1 Re-extract (if possible) and reanalyze the sample. Note: Samples with corresponding MS and MSDs should be re-extracted and reanalyzed only if surrogate recoveries in the sample were considered unacceptable, and the surrogate recoveries met the acceptance criteria in both the corresponding MS and MSD. 12.4.3.2.2 If the surrogate recoveries meet acceptance criteria in the re-extracted/re-analyzed sample, then the problem was within the laboratory's control. 12.4.3.2.3 If surrogate recoveries are outside the acceptance criteria in the reanalysis, flag the sample data for the associated target compounds and submit data from both analyses. Distinguish between the initial analysis and the reanalysis on all data. 12.4.4 Corrective action for internal standard compound responses and/or RTs that fail to meet the acceptance criteria (Sections 12.3.5 and 12.3.6) 12.4.4.1 If the internal standard in a sample fails to meet the acceptance criteria, check calculations, the internal standard solution, and the instrument operation. 41 September 2016 ------- Analytical Protocol for VX Using GC/MS 12.4.4.2 If the above actions do not correct the problem, then the problem might be due to a sample matrix effect. To determine if there was matrix effect, take the following corrective action steps: 12.4.4.2.1 Reanalyze the sample extract. Note: Samples with corresponding MS and MSDs should be re-extracted and reanalyzed only if internal standard recoveries in the sample were considered unacceptable, and the internal standard recoveries met the acceptance criteria in both the corresponding MS and MSD. 12.4.4.2.2 If the internal standard responses and RTs meet acceptance criteria in the re-analyzed sample extract, then the problem was within the laboratory's control. 12.4.4.2.3 If the internal standard responses and RTs do not meet acceptance criteria in the re-analyzed sample extract, flag the results of the associated sample. 12.4.4.2.4 Submit data from both analyses. Distinguish between the initial analysis and the reanalysis on all data. 13.0 ANALYTICAL PROCEDURE PERFORMANCE Performance of this protocol was evaluated in multiple laboratories for measurement of VX in water, soil, and wipe matrices. Resulting IDLs and MDLs from multi-laboratory evaluation are listed in Table 1, Section 17. Multi-laboratory results of reference matrix samples spiked at levels corresponding to laboratory low-calibration standards are provided in Table 2, Section 17. Precision (as RPD and RSD) and bias (as percent recovery) results based on multi-laboratory data are provided in Table 6, Section 17. Additional laboratory data for real-world samples are provided in Tables 9a and 9b, Section 17 (multi-laboratory data for groundwater and drinking water), and 10a - 10b, Section 17 (multi-laboratory data for Virginia-a and ASTM soils). Characterization data for the samples used during the multi-laboratory study are provided in Tables 11 and 12, Section 17. Figures 1 and 2 provide example chromatograms generated during a multi-laboratory study using full-scan quadrupole MS and TOF MS, respectively. 14.0 POLLUTION PREVENTION 14.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity and/or toxicity of waste at the point of generation. Numerous opportunities for pollution prevention exist in laboratory operation. EPA has established a preferred hierarchy of environmental management techniques that places pollution prevention as the management option of first choice. Whenever feasible, laboratory personnel should use pollution prevention techniques to address their waste generation. When wastes cannot 42 September 2016 ------- Analytical Protocol for VX Using GC/MS be feasibly reduced at the source, the Agency recommends recycling as the next best option. 14.2 For information about pollution prevention that might be applicable to laboratories and research institutions consult Less is Better: Laboratory Chemical Management for Waste Reduction, available from the American Chemical Society's Department of Government Relations and Science Policy, 1155 16th St., N.W. Washington, D.C. 20036, (202) 872- 4477. 15.0 WASTE MANAGEMENT EPA requires that laboratory waste management practices be conducted in a manner consistent with all applicable rules and regulations. The Agency urges laboratories to protect the air, water, and land by minimizing and controlling all releases from hoods and bench operations, complying with the letter and spirit of any sewer discharge permits and regulations, and by complying with all solid and hazardous waste regulations, particularly the hazardous waste identification rules and land disposal restrictions. For further information on waste management, consult The Waste Management Manual for Laboratory Personnel, available from the American Chemical Society at the address listed in Section 14.2. Note: It is strongly recommended that all glassware and waste be decontaminated with bleach containing active chlorine at a concentration of at least 5 %, for at least 6 hours to provide effective decontamination of VX. Chemical agent decontamination procedures and inventory records should be consistent with the laboratory's Chemical Hygiene Plan for chemical warfare agents. 16.0 REFERENCES 16.1 U.S. Environmental Protection Agency. Selected Analytical Methods for Environmental Remediation and Recovery (SAM) - 2012. EPA/600/R-12/555. Cincinnati, OH: U.S. Environmental Protection Agency, Office of Research and Development. http://www.epa.gov/homeland-securitv-research/sam (accessed 05/31/2016) 16.2 U.S. Environmental Protection Agency. Semivolatile Organic Compounds by Gas Chromatography/Mass Spectrometry (GC/MS). SW-846 Method 8270D, Revision 4. February 2007. [In: Test Methods for Evaluating Solid Waste, Physical/Chemical Methods. EPA publication SW-846. Washington DC: U.S. Environmental Protection Agency, Office of Land and Emergency Management (formerly, Office of Solid Waste and Emergency Response).] 16.3 U.S. Environmental Protection Agency. Organic Compounds in Water by Micro extraction. SW-846 Method 3511, Revision 0. November 2002. Washington DC: U.S. Environmental Protection Agency, Office of Land and Emergency Management. 16.4 U.S. Environmental Protection Agency. Microscale Solvent Extraction. SW-846 Method 3570, Revision 0. November 2002. Washington DC: U.S. Environmental Protection Agency, Office of Land and Emergency Management. 43 September 2016 ------- Analytical Protocol for VX Using GC/MS 16.5 U.S. Environmental Protection Agency. Tetra-through Octa-ChlorinatedDioxins and Furans by Isotope Dilution HRGC/HRMS. Method 1613. October 1994. 62 FR 48394. Washington, DC: U.S. Environmental Protection Agency, Office of Water. 16.6 Montauban, C., Begos, A., and Bellier, B., Extraction of Nerve Agent VXfrom Soils, Anal. Chem. 2004, 76, 2791-2797. 16.7 U.S. Army, Marine Corps, Navy and Air Force. Potential Military Chemical/Biological Agents and Compounds. January 2005. http://fas.org/irp/doddir/army/fm3-ll-9.pdf (accessed 05/23/2016) 16.8 U.S. Environmental Protection Agency. Semivolatile Organic Compounds in Drinking Water by Solid-Phase Extraction and Capillary Column (GC/MS), Version 1.0, Method 525.3. February 2012. Cincinnati, OH: U.S. Environmental Protection Agency, Office of Research and Development. EPA/600/R-12/010. 16.9 U.S. Environmental Protection Agency. Method 1668C: ChlorinatedBiphenyl Congeners in Water, Soil, Sediment, Biosolids, and Tissue by HRGC/HRMS. Method 1668C. April 2010. Washington DC: U.S. Environmental Protection Agency, Office of Water. EPA- 820-R-10-005. 16.10 U.S. Environmental Protection Agency. "Organic Analytes." Chapter 4 in Test Methods for Evaluating Solid Waste, Physical/Chemical Methods. EPA publication SW-846. Washington DC: U.S. Environmental Protection Agency, Office of Land and Emergency Management. 16.11 ASTM. 2011. Method D1193-06. Standard Specification for Reagent Water, ASTM International, West Conshohocken, PA. http://www.astm.org (accessed 05/31/2016) 16.12 U.S. Environmental Protection Agency. Stability Study for Ultra-Dilute Chemical Warfare Agent Standards. EPA 600/R-13/044. May 2013. Cincinnati, OH: U.S. Environmental Protection Agency, Office of Research and Development, National Homeland Security Research Center. 16.13 U.S. Environmental Protection Agency. Analytical Standards Requirements. Exhibit E, Section 4 [In: EPA Contract Laboratory Program Statement of Work for Organic Superfund Methods Multi-Media, Multi-Concentration SOM02.3, September 2015. https://www.epa.gov/clp/epa-contract-laboratorv-program-statement-work-organic- superfund-methods-multi-media-multi-0 (accessed 05/23/2016)] 16.14 U.S. Environmental Protection Agency. Determination of Organic Compounds in Drinking Water by Liquid-Solid Extraction and Capillary Column Gas Chromatography/Mass Spectrometry, Method 525.2, Revision 2.0. 1995. Cincinnati, OH: U.S. Environmental Protection Agency, Office of Research and Development. 16.15 U.S. Environmental Protection Agency. Analytical Standards Requirements. Exhibit C, [In: Superfund Analytical Services / Contract Laboratory Program Statement of Work for Multi-Media, Multi-Concentration Organics Analysis (SOM01.2). May 2005. http: //www. epa.gov/sites/production/file s/2015 -10/documents/som23 d .pdf (accessed 05/31/2016)] 44 September 2016 ------- Analytical Protocol for VX Using GC/MS 16.16 U.S. Environmental Protection Agency. Determinative Chromatographic Separations. Revision 3, SW-846 Method 8000C. March 2003. Washington DC: U.S. Environmental Protection Agency, Office of Land and Emergency Management. 45 September 2016 ------- Analytical Protocol for VX Using GC/MS 17.0 TABLES AND FIGURES Table 1. Instrument Detection Limits (IDL) and Method Detection Limits (MDL) Based on Multi- Laboratory Evaluation Note: IDLs and MDLs were determined in six laboratories during a multi-laboratory study. IDLs were determined as the concentration necessary to achieve a S:N (signal-noise) ratio of at least 3:1. MDLs were determined following the procedures in Section 9.7 of this protocol, using spike concentrations corresponding to the lowest calibration standards. Sample Matrix Full-Scan Quadrupole MS Full-Scan TOF MS # labs IDL Range (ng/|jL)( S:N1 Range MDL Range (ra/L) Pooled MDL2 (ng/L) # labs MDL Range (ng/L) Pooled MDL2 (ng/L) Reagent Water 6 0.04-0.2 -3" I cd 0.740-3.06 1.51 6 0.0820-0.44 0.178 Sample Matrix # labs IDL Range (ng/pL) S:N1 Range MDL Range (Hg/kg) Pooled MDL2 (Hg/kg) # labs MDL Range (Hg/kg) Pooled MDL2 (Hg/kg) Ottawa Sand 6 0.04-0.2 -3" I cd 1.30-8.283 4.733 6 0.267-0.589 0.328 Sample Matrix # labs IDL Range (ng/pL) S:N1 Range MDL Range (|jg/wipe) Pooled MDL2 (|jg/wipe) # labs MDL Range (|jg/wipe) Pooled MDL2 (|jg/wipe) Wipes 6 0.04-0.2 -3" I cd 0.0188- 0.0822 0.0457 6 0.00223-0.0129 0.00656 1 S:N value from the weakest of two secondary quantitation ions (see Table 5). S:N values were determined by measuring peak to peak noise using Agilent Chemstation® software. 2 Pooled MDLs are calculated by taking the square root of the sum of the squares of each laboratory's MDL, divided by the total number of MDL values and multiplied by a weighting factor based on the degrees of freedom (e.g., for three MDL values, the weighting factor is 0.81). 3 Includes MDLs resulting from samples spiked at a concentration corresponding to the second lowest calibration standard. 46 September 2016 ------- Analytical Protocol for VX Using GC/MS Tables 2a and 2b: Multi-Laboratory Results of Reference Matrix Samples Spiked at Levels Corresponding to Laboratory Low-Calibration Standards Note: Table 2 provides summary results of reference matrix samples spiked at levels corresponding to the lowest calibration standard used by laboratories participating in the multi-laboratory protocol validation study. The last column of this table provides spike levels adjusted based on the lowest recovery result, for comparison to pooled MDLs generated using study data. With the exception of the analysis of VX in wipes using GC-TOF, the adjusted spike levels are above the pooled MDLs in all cases. Table 2a. Example Multi-Laboratory Results for Reference Matrix Samples Analyzed Using Full-Scan Quadrupole MS # of labs n Spike Level %Recovery Range Pooled RSD Pooled MDL Spike Level adjusted for recovery Reagent Water (|jg/L)()) () 6 42 5.71 53.3 - 264 10.3 1 1.51 3.05 Ottawa Sand (pg/kg) ) 4 28 10.0 62.0-283 13.2 | 4.73* 6.20 Wipes (|jg/wipe) 5 35 0.100 79.0-245 11.8 | 0.457 0.0790 n = number of replicates; MDL = method detection limit; RSD = relative standard deviation includes results of samples spiked at levels corresponding to the second lowest calibration standard Table 2b. Example Multi-Laboratory Results for Reference Matrix Samples Analyzed Using Full-Scan TOF MS # of labs n Spike Level %Recovery Range Pooled Pooled RSD MDL Spike Level adjusted for recovery Reagent Water (|jg/L) 6 42 0.571 60.6-164 10.9 1 0.178 0.346 Ottawa Sand (|jg/kg) 4 28 1.00 61.0-250 9.80 | 0.328 0.610 Wipes (|jg/wipe) 4 28 0.0100 0 -188 87.4 | 0.00656 0* n = number of replicates; MDL = method detection limit; RSD = relative standard deviation *Ten of 28 recoveries produced an adjusted spike level that is lower than the pooled MDL 47 September 2016 ------- Analytical Protocol for VX Using GC/MS Table 3. Decafluorotriphenylphosphine (DFTPP) Key Ions and Ion Abundance Criteria Note: All ion abundances MUST be normalized to m/z 198. Mass Quadrupole Time of Flight (TOF) 51 10.0 - 80.0 % of mass 198 10.0 - 85.0 % of mass 198 68 Less than 2.0 % of mass 69 Less than 2.0 % of mass 69 69 Present Not used 70 Less than 2.0 % of mass 69 Less than 2.0 % of mass 69 127 10.0 - 80.0 % of mass 198 10.0 - 80.0 % of mass 198 197 Less than 2.0 % of mass 198 Less than 2.0 % of mass 198 198 Base peak 100 % relative abundance (see Note above) Base peak 100 % relative abundance 199 5.0 - 9.0 % of mass 198 5.0 - 9.0 % of mass 198 275 10.0 - 60.0 % of mass 198 10.0 - 60.0 % of mass 198 365 Greater than 1.0 % of mass 198 Greater than 0.5 % of mass 198 441 Present but less than mass 443 Less than 150 % of mass 443 442 Greater than 50.0 % of mass 198 Greater than 30.0 % 443 15.0 - 24.0 % of mass 442 15.0 - 24.0 % of mass 442 Table 4. Internal Standard and Surrogates Note: Table 4 provides a list of surrogates used during laboratory studies testing this protocol, based on surrogates typically used for SVOC analyses. Alternative surrogates might be more representative of VX, and may be used or added at the laboratory's discretion provided the surrogates meet the criteria in Table 7. Analyte Surrogate Compound Internal Standard VX Triphenyl phosphate Phenanthrene-dio Table 5. Example Retention Times, Relative Retention Times and Quantitation Ions for Target Compounds, Surrogate Compounds, and Internal Standard Note: Bold quantitation ions indicate the secondary ions used during single-laboratory testing. Analyte Retention Time (min) Relative Retention Time Primary Quantitation Ion Secondary Quantitation Ions VX 17.50-17.51 0.97 114 127, 79, 72 Triphenyl phosphate (S) 20.68 1.64 326 325, 215 Phenanthrene-dio (IS) 18.02-18.03 - 188 94, 80 (S) = Surrogate (IS) = Internal Standard 48 September 2016 ------- Analytical Protocol for VX Using GC/MS Table 6. Example Multi-Laboratory Precision and Bias in Reference Matrices at Mid-Calibration Levels Sample Matrix Initial Precision and Recovery (IPR) Laboratory Control Sample (LCS) Matrix Spike/Matrix Spike Duplicate (MS/MSD) % Recovery Pooled RSD % Recovery % Recovery Pooled RPD Full-Scan Quadrupole MS Water1 61.0-121 10.3 61.0-121 61.0-121 14.6 Soil2 51.8-142 12.1 51.8-142 51.8-142 17.2 Wipes3 45.9-154 13.8 45.9-154 45.9-154 19.5 Full-Scan TOF MS Water1 49.0-154 10.2 49.0-154 49.0-154 14.4 Soil2 30.0-131 22.9 30.0-131 30.0-131 32.44 Wipes3 29.3-125 16.5 29.3-125 29.3-125 23.4 1 Reagent water spike levels: 28.6 - 57.1 |jg/L (Full Scan); 5.71 - 22.86 |jg/L (TOF) 2 Soil spike levels: 40.0 - 80.0 [jg/kg (Full Scan); 10-40 [jg/kg (TOF) 3 Wipe spike levels: 0.4 - 1.0 pg/wipe (Full Scan); 0.1 - 0.4 pg/wipe (TOF) 4 Removal of one of 20 results from this dataset yields a pooled RPD of 21.0 % Table 7. Surrogate Recovery Note: Table 7 list the surrogate used during laboratory studies testing this protocol. Alternative surrogates might be more representative of VX, and may be used or added at the laboratory's discretion, provided the surrogates meet the criteria listed in this table. Sample Matrix Surrogate Surrogate %Recovery Minimum Maximum Full-Scan Quadrupole MS Water Triphenyl phosphate 50.0 150 Soil Triphenyl phosphate 50.0 150 Wipes Triphenyl phosphate 50.0 150 Full-Scan TOF MS Water Triphenyl phosphate 50.0 150 Soil Triphenyl phosphate 50.0 150 Wipes Triphenyl phosphate 50.0 150 49 September 2016 ------- Analytical Protocol for VX Using GC/MS Table 8. Example Calibration Standard Concentrations (|jg/mL) Used During Multi-Laboratory Method Validation Study Full-Scan Quadrupole MS Analyte CAS RN Cal 1 Cal 2 Cal 3 Cal 4 Cal 5 Cal 6 VX 50782-69-9 0.1 0.2 0.4 0.8 1.0 2.0 Triphenyl phosphate (S) 115-86-6 0.1 0.2 0.4 0.8 1.0 2.0 Phenanthrene-dio(IS) 1517-22-2 0.5 0.5 0.5 0.5 0.5 0.5 Full-Scan TOF MS Analyte CAS RN Cal 1 Cal 2 Cal 3 Cal 4 Cal 5 Cal 6 Cal 7 VX 50782-69-9 0.01 0.05 0.08 0.1 0.25 0.5 1.0 Triphenyl phosphate (S) 115-86-6 0.01 0.05 0.08 0.1 0.25 0.5 1.0 Phenanthrene-dio(IS) 1517-22-2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 (S) = Surrogate; (IS) = Internal standard 50 September 2016 ------- Analytical Protocol for VX Using GC/MS Table 9a. Example Multi-laboratory Precision and Recovery in Water Using GC Full-Scan Quadrupole MS Groundwater Analyte # of labs n Pooled %RSD %Recovery Range %RSD Range VX (5.71 |jg/L) 3 21 8.02 58.8-133 4.06-12.4 VX (45.7 |jg/L) 3 21 5.58 62.4-102 3.70-7.65 Surrogate Triphenylphosphate (28.6 pg/L) 3 42 9.45 60.0-156 4.17-18.1 Drinking Water Analyte # of labs n Pooled %RSD %Recovery Range %RSD Range VX (5.71 |jg/L) 3 21 26.9 58.1 - 125 5.77-44.7 VX (45.7 |jg/L) 3 21 5.76 60.8-108 3.65-7.41 Surrogate Triphenylphosphate (28.6 pg/L) 4 70 7.44 52.0-124 2.01 - 12.6 n = number of replicates; RSD = relative standard deviation Table 9b. Example Multi-laboratory Precision and Recovery in Water Using GC Full-Scan TOF MS Groundwater Analyte # of labs n Pooled %RSD %Recovery Range %RSD Range VX (0.571 |jg/L) 3 21 28.4 11.8-106 4.86-48.0 VX (5.71 |jg/L) 3 21 3.38 41.7-122 4.96-23.6 Surrogate Triphenylphosphate (28.6 pg/L) 4 64 7.82 36.6-143 2.35-18.6 Drinking Watter Analyte # of labs n Pooled %RSD % Recovery Range %RSD Range VX (0.571 |jg/L) 3 21 15.9 28.9-107 5.63-23.5 VX (5.71 |jg/L) 3 21 6.74 72.6-117 2.53 -10.6 Surrogate Triphenylphosphate (28.6 pg/L) 4 62 7.23 69.0-136 2.81 - 16.9 n = number of replicates; RSD, relative standard deviation; TOF, time of flight 51 September 2016 ------- Analytical Protocol for VX Using GC/MS Table 10a. Example Multi-laboratory Precision and Recovery in Soils Using GC Full-Scan Quadrupole MS Virginia-a Soil Analyte # of labs n Pooled %RSD %Recovery Range %RSD Range VX(10.0 [jg/kg) 3 21 8.60 80.5-144 1.47-13.8 VX (80 pg/kg) 3 21 13.3 19.2-68.2 9.56-16.8 Surrogate Triphenylphosphate (50 pg/kg) 3 31 29.0 0.516-76.0 6.43-55.8 ASTM Soil Analyte # of labs n Pooled %RSD % Recovery Range %RSD Range VX(10.0 pg/kg) 3 21 2.35 66.8-113 1.71 -2.80 VX (80.0 pg/kg) 3 20 16.1 5.60-42.9 6.17-21.5 Surrogate Triphenylphosphate (50 pg/kg) 3 31 16.1 5.57-194 5.87-27.6 n = number of replicates; RSD, relative standard deviation Table 10b. Example Multi-laboratory Precision and Recovery in Soils Using GC Full-Scan TOF MS Virginia -a Soil Analyte # of labs n Pooled %RSD %Recovery Range %RSD Range VX (1.0 pg/kg) 2 14 11.7 56.9-146 6.14-15.3 VX(10.0 pg/kg) 3 18 8.12 34.0-84.6 7.37-9.11 Surrogate Triphenylphosphate (50 pg/kg) 3 39 14.7 2.32-7.82 7.77-16.1 ASTM Soil Analyte # of labs n Pooled %RSD %Recovery Range %RSD Range VX (1.0 pg/kg) 0 0 NA 0 NA VX(10.0 pg/kg) 3 16 20.7 12.7-34.0 7.74-34.0 Surrogate Triphenylphosphate (50 pg/kg) 3 39 18.8 9.31 -47.6 3.68-30.6 n = number of replicates; RSD = relative standard deviation; TOF = time of flight 52 September ------- Analytical Protocol for VX Using GC/MS Table 11. Multi-Laboratory Study Water Matrices Characterization Data Drinking Water Groundwater Collection Date Dec 2011 - July 2012 Collection Date Jan/Feb 2012 PH 00 cn I 00 PH 6-7 Chlorine (free) mg/L 1.16-1.35 Chlorine (mg/L) 0.02-0.05 Chlorine (total) mg/L 1.20-1.42 Chlorine (total) mg/L NR Total Organic Carbon (TOC) (ppm) 0.83-1.14 Total Organic Carbon (TOC) (ppm) 1.08-1.24 Conductivity (|jS) 294-413 Conductivity (|jS) 0.82-0.87 Oxidation-reduction potential (mV) 766 - 770 Oxidation-reduction potential (mV) NR Turbidity (NTU) 0.06-0.07 Turbidity (NTU) CO o I o Total Hardness (mg/L) 114-131 Total Hardness (mg/L) 37-65 Alkalinity (mg/L) 64-80 Alkalinity (mg/L) 30-36 Table 12. Multi-laboratory Study Soil Matrix Characterization Data Parameter ASTM Soil ML-1 Virginia-a Soil PH 8.68 4.41 TOC (%C) <0.10 2.2 %Solid 98.9 99.1 TOC = total organic carbon 53 September 2016 ------- Analytical Protocol for VX Using GC/MS Abundance 600000 500000 400000 300000 200000 100000 x > ¦o i Time (min) 16.00 V 18.00 r. a. 20.00 22.00 Notes: (1) Concentrations of all analytes as described in Table 8, Calibration Level 6 (GC/MS - Full Scan Quadrupole); (2) Unlabelled peaks represent compounds not specifically targeted by this protocol (min) = minutes I = internal standard S = surrogate T = target Figure 1. Example chromatogram for a calibration standard on full-scan quadrupole MS. 54 September 2016 ------- Analytical Protocol for VX Using GC/MS x > CD •*-> CO £ Q. (/) O >* c CD Aj 175 200 I 225 250 Time(s) 150 Notes: (1) Concentrations of all analytes as described in Table 8, Calibration Level 4 (GC/MS -TOF); (2) Unlabelled peaks represent compounds not specifically targeted by this protocol (s) = seconds 275 300 —t—- 325 375 Figure 2. Example chromatogram for a midpoint calibration standard (Cal5) on time-of-flight MS. 55 September 2016 ------- vvEPA United States Environmental Protection Agency PRESORTED STANDARD POSTAGE & FEES PAID EPA PERMIT NO. G-35 Office of Research and Development (8101R) Washington, DC 20460 Official Business Penalty for Private Use $300 ------- |