v>EPA United States Environmental Protection Agency EPA/600/R-15/039 Feasibility Research on Alternative Approaches for Sampling and Extraction Methods in the T0-4A Method for Pesticides in Ambient Air with Analysis by GC/MS and LC/MS/MS Office of Research and Development National Exposure Research Laboratory ------- ------- *>EPA United States Environmental Protection, Agency EPA/600/R-15/039 Prepared by Elin Ulrich and James Starr Tim Slagle National Exposure Research Laboratory Office of Research and Development U.S. Environmental Protection Agency Research Triangle Park, NC Science and Ecosystems Support Division Region 4 U.S. Environmental Protection Agency Athens, GA ------- Disclaimer The research in this document has been funded by the U.S. Environmental Protection Agency (EPA). It has been subjected to the Agency's peer and administrative review and has been approved for publication as an EPA document. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. Acknowledgments This research was conducted by the U.S. Environmental Protection Agency's Office of Research and Development (ORD) and Region 4. Dr. James Starr and Dr. Elin Ulrich led the ORD efforts with support from Environmental Career Organization interns Travis Cummings and Candice Morrison and National Caucus and Center on Black Aged's Senior Environmental Employee Program grantee Anthony Gemma. ORD performed method development work, including matrix cleanup, extraction, sample cleanup, quantitation, and method evaluation. ORD also developed the Standard Operating Procedures (SOPs) included in this compendium of methods. Mr. Tim Slagle led the Region 4 efforts with contracted support from the University of Georgia's Machine Shop to implement the cell/sampler design. Region 4 acquired, modified, and operated air sampling equipment. Numerous ORD and Region 4 staff assisted with essential technical, quality, and administrative support for this project. This research could not have been successful without the support of everyone involved. ii ------- Table of Contents Disclaimer ii Acknowledgments ii Abstract vii Executive Summary ix Background ix Objectives ix Organization ix Approach ix Media Preparation ix Sampling Device ix Sample Collection ix Sample Extraction x Sample Cleanup x Sample Analysis by GC/MS x Sample Analysis by LC/MS/MS x Conclusions x 1.0 Introduction 1 1.1 Background 1 1.2 Strengths and Weaknesses of TO-4A 2 1.3 What was Needed to Improve TO-4A and Goals for this Project 2 2.0 Methods 3 2.1 Media Preparation 3 2.2 Sampler Preparation 3 2.3 Air Sampling 3 2.4 Sample Extraction 3 2.5 Sample Cleanup 3 2.6 Sample Analysis Using GC/MS 3 2.7 Sample Analysis Using LC/MS/MS 4 3.0 Results and Discussion 5 3.1 Compounds Removed 5 3.2 Research Quality Objectives, Limit of Detection (LOD) 5 3.3 Data Quality Objectives, Recovery 6 Bibliography 9 Appendix A A-l 1 Method Summary A-l 2 Scope and Application A-l 3 Personnel Qualifications A-l 4 Health and Safety A-l 5 Definitions, Acronyms, and Abbreviations A-l 6 Equipment and Supplies A-l 7 Procedure A-l 7.1 Cell assembly A-l 7.2 Filling the cells with XAD-2 A-2 7.3 Preparing the instrument A-2 7.4 Preparing the method A-3 7.5 Preparing the schedule A-3 7.6 After a schedule is complete A-3 7.7 After all schedules are complete A-3 8 Data and Records Management A-4 9 Quality Control and Quality Assurance A-4 iii ------- 10 Waste Management A-4 11 References A-4 Appendix B B-l 1 Method Summary B-l 2 Scope and Application B-l 3 Personnel Qualifications B-l 4 Health and Safety B-l 5 Definitions, Acronyms, and Abbreviations B-l 6 Equipment and Supplies B-l 7 Procedure B-l 7.1 Assembling snap rings #1, #2, and #3 B-l 7.2 Packing the XAD resin B-2 7.3 Inserting snap ring #4 B-3 7.4 Reusing cells B-3 8 Data and Records Management B-4 9 Quality Control and Quality Assurance B-4 10 Waste Management B-4 11 References B-4 Appendix C C-l 1 Method Summary C-l 2 Scope and Application C-l 3 Personnel Qualifications C-l 4 Health and Safety C-l 5 Definitions, Acronyms, and Abbreviations C-2 6 Equipment and Supplies C-2 7 Procedure C-2 7.1 Preparation of the sampling cells C-2 7.2 Preparation of the spiking solutions C-2 7.3 Preparation of sampler C-2 7.4 Air sampling C-3 8 Data and Records Management C-4 9 Quality Control and Quality Assurance C-4 10 Waste Management C-4 11 References C-4 Appendix D D-l 1 Method Summary D-l 2 Scope and Application D-l 3 Personnel Qualifications D-l 4 Health and Safety D-l 5 Definitions, Acronyms, and Abbreviations D-2 6 Equipment and Supplies D-2 7 Procedure D-2 7.1 Preparation of the surrogate spiking solution D-2 7.2 Extraction of the sample cells D-2 7.3 Rotary evaporation D-3 8 Data and Records Management D-3 9 Quality Control and Quality Assurance D-3 10 Waste Management D-3 11 References D-3 Appendix E E-l 1 Method Summary E-l 2 Scope and Application E-l 3 Personnel Qualifications E-l 4 Health and Safety E-l 5 Definitions, Acronyms, and Abbreviations E-2 6 Equipment and Supplies E-2 7 Procedure E-2 iv ------- 7.1 Sample and column sorbent preparation E-2 7.2 Column preparation E-2 7.3 Eluant preparation E-3 7.4 Confirming Florisil elution pattern E-3 7.5 Sample cleanup E-4 7.6 Sample splitting E-4 8 Data and Records Management E-6 9 Quality Control and Quality Assurance E-6 10 Waste Management E-6 11 References E-6 Appendix F F-l 1 Method Summary F-l 2 Scope and Application F-l 3 Personnel Qualifications F-2 4 Health and Safety F-2 5 Definitions, Acronyms, and Abbreviations F-2 6 Equipment and Supplies F-2 7 Procedure F-2 7.1 Sample preparation F-2 7.2 Calibration standard preparation F-3 7.3 GC/MS instrument setup F-3 7.4 GC/MS calibration F-4 7.5 GC/MS quantitation F-5 8 Data and Records Management F-5 9 Quality Control and Quality Assurance F-5 10 Waste Management F-6 11 References F-6 Appendix G G-l 1 Method Summary G-l 2 Scope and Application G-l 3 Personnel Qualifications G-l 4 Health and Safety G-l 5 Definitions, Acronyms, and Abbreviations G-2 6 Equipment and Supplies G-2 7 Procedures G-2 7.1 Initial LC/MS/MS setup G-2 7.2 LC/MS/MS internal standard calibration G-3 7.3 LC/MS/MS analysis sequence G-3 7.4 LC/MS/MS data processing G-4 7.5 Extract storage G-4 8 Data and Records Management G-4 9 Quality Control and Quality Assurance G-4 10 Waste Management G-4 11 References G-4 v ------- List of Figures Figure Al. Dionex 300 system: (A) PC with AutoASE software, (B) solvent controller, (C) solvent reservoir, (D) keypad and display, (E) 100 inL cells, and (F) collection bottle A-2 Figure B-l. Linear view of modified ASE cell, snap rings, and retaining screens. Blue grooves are numbered based on the recommended snap-ring-insertion order. B-3 Figure B-2. End view of modified ASE cell showing location of grooves #2 and #4 B-3 Figure B-3. Schematic for insertion of snap ring 1. Ring should be oriented vertically with the axis of the cell. B-3 Figure B-4. Filling the ASE cell using the modified sampling mount, vacuum pump, and cleaned XAD-2 resin B-3 Figure CI. Air sampling unit assembly C-3 Figure C2. Front panel of controller power supply C-3 Figure El. Florisil column setup E-3 Figure E2. Column packing E-3 List of Tables Table 1-1. TO-4A target analyte list with suggested analysis method 1 Table 2-1. GC/MS target analytes, surrogates, and internal standards 4 Table 2-2. LC/MS/MS target analytes and internal standards 4 Table 3-1. TO-4 A analytes removed from consideration in new method 5 Table 3-2. Limit of detection (LOD) 5 Table 3-3. Time of attended and unattended operations for pesticide analysis 6 Table 3-4. Volume of solvent used for pesticide analysis 6 Table 3-5. Comparative recoveries of spiked GC pesticides (percent recovery ± relative standard deviation) 7 Table 3-6. Comparative recoveries of spiked LC pesticides (percent recovery ± relative standard deviation) 8 Table CI. GC and LC target analytes C-l Table Dl. GC and LC target analytes and GC surrogates D-l Table El. Target analytes and surrogate compounds analyzed by this method E-l Table E2. Solvent type and volume for six Florisil column eluants E-3 Table Fl. Target analytes, surrogate compounds, and internal standards for this method F-l Table F2. Preparation of calibration standards F-3 Table F3. Instrumental selected ion monitoring parameters and ion windows F-4 Table Gl. LC target analytes and internal standards G-l Table G2. Analyte-specific LC/MS/MS parameters G-3 vi ------- Abstract This compilation of methods is the result of a Regional Methods project between the U.S. Enviromnental Protection Agency (EPA) Region 4 and the EPA's Office of Research and Development. The research leading to these methods was conducted in response to a need to update an EPA compendium method to use the best currently available technology and incorporate green chemistry concepts. The purpose of this research was to evaluate the feasibility of a new procedure for sampling and analysis of pesticides in ambient air. This work assessed alternatives for the collection, extraction, and analytical methods described in compendium method TO-4A "Determination of Pesticides and Poly chlorinated Biphenyls in Ambient Air Using High Volume Polyurethane Foam Sampling Followed by Gas Chromatographic/Multi- Detector Detection."111 The TO-4A method uses polyurethane foam (PUF) sampling media and requires separate assemblies for sampling, shipping, and extraction. The analytes are stripped from the PUF in Soxhlet extractors. The resulting extract is reduced in volume and then cleaned with alumina or silica. Analysis is by gas and liquid chromatography with one of several relatively nonspecific detectors. The methods described here use integrated devices and automated processes for the determination of the pesticides listed in TO-4A, thereby simplifying it and reducing solvent requirements and the need to handle the sampling media. A modified, reusable, stainless steel accelerated solvent extraction cell, packed with a pre-cleaned resin, functions as the sampling device, shipping container, and extraction vessel. Finally, mass spectrometers replace the electron capture, flame photometric, nitrogen-phosphorus, and ultraviolet absorption detector listed in method TO-4A. To evaluate the effectiveness of the overall process, the work was divided into component methods that could be optimized individually. Standard operating procedures (SOPs) were written for each of the final procedures. Although these SOPs are somewhat specific to this particular EPA research situation, they can be used as a starting point for further development outside their original intended use. The components of the method are (1) media and sampler preparation, (2) sample collection, (3) extraction, (4) cleanup, and (5) analysis. The accuracy and precision of the finalized procedures were assessed separately and as a whole method. Analytical sensitivity for these pesticides matched or exceeded that achieved using the detectors listed in the TO- 4A method. The proposed method was found to have some limitations compared to the TO-4A. The highest achievable air flow rates through the XAD-2 sorbent cartridge were less than those possible with TO-4A. Of the 54 pesticides listed in the method, 48 could be analyzed using the current generation analytical instruments. Difficulties in precision also were encountered; in many instances the relative standard deviations of recoveries from replicate trials was higher than 20%. The marginal loss in sensitivity and precision are offset by gains in specificity, efficiency, and potential reductions in solvent use. vii ------- ------- Executive Summary Background Compendium method TO-4 was first published as a U.S. Environmental Protection Agency (EPA) report in 1984. The report was one of a series of air toxic methods in "Compendium of Methods for the Determination of Toxic Organic Compounds in Ambient Air," (EPA 600/4-89- 018). In 1999, TO-4 was updated to incorporate current technologies, and the revision was published as method TO-4A, "Determination of Pesticides and Polychlorinated Biphenyls in Ambient Air Using High Volume Polyurethane Foam (PUF) Sampling Followed by Gas Chromatographic/ Multi-Detector Detection (GC/MD)," in the second edition of the compendium.111 Briefly, TO-4A describes procedures to sample ambient air and determine concentrations of selected common pesticides and polychlorinated biphenyls (PCBs). Air sampling is via a high-volume pump with polyurethane foam in a glass cartridge as the collection media. The analytes are extracted from the PUF in Soxhlet extractors, and the resulting sample is cleaned up using alumina or silica. Analysis is by GC in conjunction with one of several detectors or by high- performance liquid chromatography (HPLC) with a UV or electrochemical detector. In 2002, a collaborative Regional Methods project was proposed by scientists in Region 4 to work with the Office of Research and Development in evaluating alternative methods of sampling and analysis for possible inclusion in TO-4 A. The project, "Demonstration and regional field testing of sampling and analytical method for sampling pesticides and semi-volatile organic compounds using an adsorbent resin sampling system, accelerated solvent extraction, and GC/MS analysis," proposed replacing the sampling and extraction procedures in TO-4A with methods using an integrated system. Ultimately, the project was expanded to replace the electron capture, flame photometric, and nitrogen-phosphorus detectors (ECD, FPD, and NPD, respectively) and the ultraviolet absorption (UV) and fluorescence (Flor) detector listed in method TO-4 A with current-generation single and tandem mass spectrometers (MS, MS/MS). Objectives This work provides alternatives to the sampling, extraction and analysis methods described in EPA compendium method TO-4 A. Currently, the compendium methods require a PUF sampling media and separate assemblies for sampling, shipping, and extraction. The proposed methods use integrated devices and automated procedures for the determination of these compounds, thereby simplifying the methods and reducing the need to handle the media. The method uses accelerated solvent extraction (ASE) cells packed with XAD-2 resin to function both as a sampling device and an extraction vessel. Since the same durable vessel is used for sampling and extraction, the following benefits are expected: • less analyte lost during sample transport and processing, • fewer samples lost due to glass breakage, • reduction in use of organic solvents for rinsing, and • simplification of sampling and extraction procedures. Organization This compilation of methods (Appendices A through G) contains seven SOPs that detail the steps of the final approach: (1) media preparation, (2) sampler preparation, (3) air sampling, (4) sample extraction (5) sample cleanup, (6) GC/MS analysis, and (7) LC/MS/MS analysis. It is important to note that these SOPs are specific to the work conducted for this study. They provide an excellent starting place for modification, enabling more general use. Approach The SOPs presented here are to be used in conjunction with those for more general laboratory procedures, such as the preparation of standards and balances. Local procedures for laboratory operations, health and safety, and waste disposal must be considered and followed. Media Preparation Amberlite XAD-2 resin was selected as a sorbent material. The resin was cleaned using a solvent series (in order of decreasing polarity), and cleanliness was verified using ECD. Instructions for cleaning of XAD-2 prior to reuse are included in the SOP in Appendix A. Sampling Device The ASE cells purchased from the manufacturer were modified with two sets of grooves to allow for a snap ring and mesh screen to hold 4 in of XAD between them. A fabricated adapter enabled connection of a 100-mL stainless steel ASE cell to a high-vacuum pump with flow controller. Setup of this device is described in the SOP in Appendix B. Sample Collection Gast vacuum pumps were turned on and run for 16 to 24 h at a predetermined flow rate. Once the sampling period was complete, the cells were unscrewed from the sampler, recapped, and placed directly on the extraction device for automated extraction. This procedure is detailed in the SOP in Appendix C. ix ------- Sample Extraction An automated ASE was used for sample extraction, as outlined in the SOP in Appendix D. Surrogate solutions were added through the screen onto the sorbent material. The samples were extracted using 20% acetone in hexane heated to 75 °C at 1500 psi. The cell can be reused for further air sampling without disassembly or replacement of the sorbent material. Sample Cleanup Samples were concentrated using solvent evaporation, then loaded onto a Florisil (10 g) column. The column was then eluted with progressively more polar combinations of ethyl acetate (EtOAc) in hexane, which were split for analysis using GC and LC. This procedure is explained in the SOP in Appendix E. Additional solvent and time savings could be realized using a similar procedure adapted to solid-phase extraction cartridges. Sample Analysis by GC/MS Appendix F contains the SOP for GC/MS analysis of 36 pesticides as identified in compendium method TO-4A. Three internal standards and four surrogate standards are added for quality assurance and quality control (QA/QC) purposes. Optimal GC/MS settings are described along with retention times and fragmentation patterns for each compound. Sample Analysis by LC/MS/MS Appendix G contains the SOP for LC/MS/MS analysis of 23 pesticides as identified in compendium method TO-4A. Four internal standards and four surrogate standards are added for QA/QC purposes. Optimal LC/MS/MS settings are described along with retention times and product ions for each compound. Conclusions The SOPs that follow provide the details of the final methods produced from this research. This research demonstrated that it is feasible to replace the current sampling assembly described in method TO-4A with an alternative that uses a modified stainless steel ASE cell to function both as the sampling device and the extraction vessel. XAD resin, used as the collection media, appeared effective in trapping most of the pesticides for which it was tested. However, the highest achievable air flow rates through the XAD were less than used by TO-4A. Automated ASE provided acceptable recoveries of the majority of the analytes and reduced solvent and time requirements compared to Soxhlet extraction. Additionally, same cell use for sampling and extraction proved very efficient for sample processing and recordkeeping by eliminating the transfer between multiple vessels. ------- 1.0 Introduction 1.1 Background Pesticide usage and environmental distribution are common to rural and urban areas of the United States. The application of pesticides can cause adverse health effects in humans by contaminating soil, water, air, plants, and animal life. Polychlorinated biphenyls (PCBs) are less widely used because of extensive restrictions, but still cause problems due to their presence in various electrical products. Many pesticides and PCBs exhibit bioaccumulative, chronic health effects; therefore, monitoring the presence of these compounds in ambient air is of great importance.12 3 The relatively low levels of such compounds in the enviromnent require the use of high-volume sampling techniques to acquire sufficient samples for analysis. However, the volatility of these compounds prevents efficient collection on filter media. Consequently, compendium method TO-4A utilizes both a particle filter and a polyurethane foam backup cartridge that provides for efficient collection of most common pesticides, PCBs, and other organics within the same volatility range. Moreover, modifications to this method have been applied successfully to measurement of common pesticides and PCBs in outdoor and indoor air and for personal respiratory exposure monitoring. The TO-4A method covers sampling and analysis of a variety of common pesticides and PCBs in ambient air (Table E2. Solvent type and volume for six Florisil column eluants). The procedure is based on the adsorption of chemicals from ambient air on PUF using a high-volume sampler. The high-volume PUF sampling procedure is applicable to multicomponent atmospheres containing common pesticide concentrations from 0.001 to 50 |_ig/m3 over 4- to 24-h sampling periods. The limits of detection will depend on the nature of the analyte and the length of the sampling period. The analytical methodology described in compendium method TO-4A is currently employed by laboratories throughout the United States and typically uses GC and/or HPLC. Table 1-1. TO-4A target analyte list with suggested analysis method'11 Recommended Recommended Compound analysis3 Compound analysis3 Alachlor GC/ECD Folpet GC/ECD Aldrin GC/ECD Heptachlor GC/ECD Allethrin HPLC/UV Heptachlor epoxide GC/ECD Aroclor 1242 GC/ECD Hexachlorobenzene GC/ECD Aroclor 1254 GC/ECD Lindane (g-BHC) GC/ECD Aroclor 1260 GC/ECD Linuron HPLC/UV Atrazine GC/NPD Malathion GC/NPD or FPD Bendiocarb HPLC/UV Methyl parathion GC/NPD or FPD BHC (a- and b- hexachlorocyclohexanes) GC/ECD Methoxychlor GC/FCD Captan GC/ECD Metolachlor GC/ECD Carbaryl HPLC/UV Mexacarbate GC/FCD Carbofuran HPLC/UV Mi rex GC/ECD Chlordane, technical GC/ECD Monuron HPLC/UV Chlorothalonil GC/ECD Trans-nonachlor GC/ECD Chlorotoluron HPLC/UV Oxychlordane GC/ECD Chlorpyrifos GC/ECD Pentachlorobenzene GC/ECD 2,4-D esters and salts GC/ECD Pentachlophenol GC/ECD Dacthal GC/ECD Permethrin (cis- and trans-) HPLC/UV p,p'-DDT GC/ECD o-Phenylphenol HPLC/UV p,p'-DDE GC/ECD Phorate GC/NPD or FPD Diazinon GC/NPD or FPD Propazine GC/NPD Dicloran GC/ECD Propoxur (Baygon) HPLC/UV ------- Compound Recommended analysis3 Compound Recommended analysis3 Dieldrin Dicofol Dicrotophos Diuron Ethyl parathion Fenvalerate Fluometuron GC/ECD GC/ECD HPLC/UV HPLC/UV GC/NPD or FPD HPLC/UV HPLC/UV Pyrethrin Resmethrin Ronnel Simazine Terbuthiuron Trifluralin HPLC/UV HPLC/UV GC/ECD HPLC/UV HPLC/UV GC/ECD aGC = gas chromatography, ECD = electron capture detector, FPD = flame photometric detector, HPLC = high-performance liquid chromatography, NPD = nitrogen-phosphorus detector, UV = ultraviolet absorption detector 1.2 Strengths and Weaknesses of TO-4A The strengths of the TO-4A method are that it can detect environmentally relevant concentrations of a variety of pollutants and current-use pesticides. It has been registered with ASTM International and is used by many scientists with readily accessible analytical techniques. The weaknesses of the TO-4A method are that it uses a fragile glass sampler cartridge and large, noisy pumps for sample collection. Sample cartridges are comprised of two different sorbent materials, which must be transferred prior to extraction. Air must be collected for a 24-h period to accumulate detectable levels of target analytes. Large quantities of solvent and time are required for thorough extraction of all analytes and several GC detectors are required for adequate detection of all analytes. 1.3 What was Needed to Improve TO-4A and Goals for this Project The SOPs presented here proposes methods to replace TO-4A with newer technology. The overall goals are to address the weaknesses listed above, and specifically to • reduce the size and noise level of the sampler; • find a sturdier material, such as stainless steel, to replace the glass sampling cartridge; • replace methylene chloride with a less toxic solvent; • reduce the extraction time per sample; • reduce the volume of solvent required to process a sample; and • match or improve the measurement quality objectives for o sensitivity, o selectivity, o analyte recovery, o reproducibility, o accuracy, and o precision. 2 ------- 2.0 Methods The final procedures used for each process in this report are detailed in Appendices A through G. 2.1 Media Preparation The SOP for media preparation (XAD-2 cleaning procedure. Using the Dionex Accelerated Solvent Extractor 300) is provided in Appendix A. XAD-2 resin was selected as the sorbent of choice for this project. XAD is often used in environmental sampling for air and water and is sometimes used in a sandwich fashion (between PUF plugs) for the TO- 4A and ASTM 4861 methods. XAD was selected for prior performance with these compounds, stability, availability of "clean" materials, and adsorption characteristics. Prior to air sampling, 100-mL accelerated solvent extraction (ASE) cells were packed with XAD-2 resin and precleaned on a Dionex 300 using sequential extractions with methanol, acetone, dichloromethane, hexane, and 20% acetone in hexane. The 20% acetone in hexane extracts were checked for a clean background before further use. To insure that all compounds from previous experiments were removed, reused XAD cartridges were extracted with acetone and checked for a clean background. 2.2 Sampler Preparation The SOP for preparation and packing ASE cells (Preparation and Packing of ASE Cells for Air Sampling with XAD-2) is provided in Appendix B. The TO-4A method requires a high-volume sampler capable of pulling ambient air through the filter/adsorbent cartridge at a flow rate of -0.225 m7 min to obtain a total sample volume of greater than 300 m3 over a 24-h period. To meet research objectives, a Gast vacuum pump (model 0523-101Q-SG588DX) was selected in conjunction with a Teledyne Hastings Instruments flow controller (model THPS-100). This flow controller is capable of setting the flow from 0 to 0.1 m3/min with a maximum sample of -150 m3 over a 24-h period. An adapter was fabricated to enable a stainless steel, 100-mL ASE cell to be directly attached to the unit. The glass sampling cartridge in the T04A method was replaced with a modified 100-mL stainless steel ASE cell. The ASE cells were modified so that they could be unscrewed from the sampler, recapped, and placed directly on the ASE for automated extraction. The ASE cells were also modified by etching two sets of grooves to allow for a snap ring and mesh screen to hold 4 in of XAD between them. 2.3 Air Sampling The SOP for air sampling using ASE cells (Air Sampling with ASE Cells) is provided in Appendix C. Prior to air sampling, the XAD in each cell was spiked with 1 mL of the target analytes in ethyl acetate (EtOAc; 400 ng/mL) or 1 mL of EtOAc only (blanks). The cells were attached to the pumps, and the flow rates were set to either zero flow or 30 L/min. Ambient air was pulled through the pumps for 19, 24, or 48 h. 2.4 Sample Extraction The SOP for sample extraction from XAD in ASE cells (Extracting TO-4A Air Samples with the Dionex ASE 300) is provided in Appendix D. The ASE 300 was used for all extraction experiments. Using spiked XAD in ASE cells, the extraction efficiency of three solvent systems (hexane, 10% acetone in hexane, and 20% acetone in hexane) was compared. Overall, 20% acetone in hexane gave the best recovery, and recovery for the TO-4A compounds that were tested ranged from 66% to 112%. 2.5 Sample Cleanup The SOP for sample cleanup of TO-4A pesticides extracted from XAD (Florisil Column Cleanup for TO-4A Pesticides in Air Samples) is provided in Appendix E. Four cleanup procedures were evaluated. Three used Florisil (1 or 10 g), and one used silica (0.5 g). Elutropic solvent combinations, including hexane, toluene, and EtOAc, were evaluated. In each case, the compounds were loaded onto the cleanup column or cartridge in hexane. The procedure giving the most effective cleanup and recovery was • 1 mm ID column with Florisil (10 g), • 125 mL 6% EtOAc in hexane, • 125 mL 15% EtOAc in hexane, • 125 mL 50% EtOAc in hexane, and • 125 mL 100% EtOAc. This procedure was run in triplicate and gave an average recovery of 86% (all trials and compounds combined) and was the cleanup procedure used for sample analysis. 2.6 Sample Analysis Using GC/MS GC/MS was used to analyze the relevant pesticides as identified in Compendium Method TO-4A. The SOP for GC/ MS analysis of TO-4A pesticides is provided in Appendix F. Surrogate and internal standards were added as a part of the QC, with the surrogates added to the XAD prior to extraction, and the internal standards added to the sample extract just before injection. The pesticides analyzed via GC/ MS and their surrogate and internal standards are provided in Table 2-1. GC/MS target analytes, surrogates, and internal standards 2-1. 3 ------- Table 2-1. GC/MS target analytes, surrogates, and internal standards GC target analytes Alachlorb Aldrinb Atrazine3 Captanc c/'s-Chlordanec frans-Chlordanec Chlorothalonilb Chlorpyrifosb 2,4-D ethyl ester3 2,4-D methyl ester® Dactholb p,p'-DDEc o,p'-DDP pp'-DDP Diazinonb Dicloran (Botran)3 Dieldrinc Ethyl parathionb Fenchlorphos (Ronnel)b Folpetc a-HCHa b-HCHa Heptachlorb Heptachlor epoxide Bc Hexachlorobenzene3 Lindane (g-HCH)b Malathionb Methyl parathionb Methoxychlor0 Metolachlorb Mirexc frans-Nonachlor0 Oxychlordanec Propazine3 Simazine3 Trifluralin3 GC surrogate standard compounds 13C Atrazine3 D Chlorpyrifosb D Diazinonb Heptachlor epoxide Ac GC internal standard compounds TCmX 13C6 d-HCH DCBP aTarget analytes and surrogates using TCmX as an internal standard "Target analytes and surrogates using 13C6 d-HCH as an internal standard Target analytes and surrogates using DCBP as an internal standard 2.7 Sample Analysis Using LC/MS/MS LC/MS/MS was used to analyze the relevant "HPLC" pesticides as identified in Compendium Method TO-4A. The SOP for LC/MS/MS analysis of TO-4A pesticides is provided in Appendix G. The pesticides analyzed via LC/MS/ MS and their surrogate and internal standards are provided in Table 2-1. GC/MS target analytes, surrogates, and internal standards2-2. Table 2-2. LC/MS/MS target analytes and internal standards LC target analytes Allethrind Atrazine3 Bendiocarb3 Carbaryl3 Carbofuran3 Chlortoluron3 Cinerin ld & llb Diazinonb Diuron3 Dicrotophos3 Fenvalerated Fluometuron3 Jasmolin ld & llb Linuron3 Monuron3 c/'s-Permethrinc frans-PermethrirV-1 Propazine3 Propoxur (Baygon)3 Pyrethrin ld &llb Resmethrind Simazine3 Tebuthiuron3 LC internal standard compounds 13C3 Atrazine D10 Diazinon 13C6 c/'s-permethrin 13C6 frans-permethrin aTarget analytes using 13C3 Atrazine as an internal standard Target analytes using D10 Diazinon as an internal standard Target analytes using 13C6 c/s-Permethrin as an internal standard Target analytes using 13C6 frans-Permethrin as an internal standard 4 ------- 3.0 Results and Discussion 3.1 Compounds Removed To simplify the evaluation of the new methods, the Aroclor PCB mixtures listed in TO-4A were excluded from the spiking mixture, and only the TO-4A pesticides were used. Several pesticides were removed from the analyte list during method development because the measurement quality objectives could not be met. Details are provided in Table 3 1. Table 3-1. TO-4A analytes removed from consideration in new method Compound Reason for removal Dicofol Breakdown occurred in standards, poor chromatography Mexacarbate Poor sensitivity/recovery Pentachlorobenzene Inconsistent recovery/sensitivity Pentachlorophenol Did not elute from column/chromatography issues, <20% recovery Phorate Inconsistent and <35% recovery o-Phenylphenol Poor sensitivity PCBs Method evaluation restricted to pesticides 3.2 Research Quality Objectives, Limit of Detection (LOD) The measurement quality objectives for LOD are detailed in Table 3 2. In all but two compound classes (organic compounds [OC's] and carbamates), the TO-4ALOD was matched or improved. Table 3-2. Limit of detection (LOD) Compound class Detector TO-4Aa LOD (ng/mL) GC/MSb LC/MS-MSb OCs ECD 1-50 50 50 OPs FPD/NPD 50-500 50 50 Triazine, carbamate, urea NPD/GC-MS 50-200 50 50 Triazine, carbamate, urea HPLC-UV 1000-5000 50 50 Carbamates HPLC-Flor 10-100 50 50 Carbamate, urea, pyrethroid, phenolic HPLC-UV 200-10,000 50 50 aTO-4A LOD as listed in the compendium method111 bGC/MS and LC/MS-MS LOD is defined as the concentration of the least concentrated calibration standard. ------- Table 3-3. Time of attended and unattended operations for pesticide analysis TO-4A New method Process Attended (h) Unattended (h) Attended (h) Unattended (h) Matrix cleaning, initial 0.5 16a 0.5 1.5 Matrix cleaning, reuse 0.5 16 <0.1 0.25 Media prepb 0.5 — <0.1 — Sampler prepc 1.0 — <0.1 0.5 Extraction 0.25 16 <0.1 0.25 Sample cleanup 0.5 — 6d — Total, initial 3.25 32 6.8 2.25 Total, reuse 3.25 32 6.4 1.0 a16 h is required for the PUF cleaning procedure; 5 h is required for baking the glass fiber filter, which can take place during the PUF cleaning procedure. Filters for the separate collection of particulates were not considered in the new method. blncludes media preparation and cartridge assembly includes flow calibration and flow controller warm-up times dThis procedure should be transferred to SPE cartridges, which require significantly less time (<1 h) Two of the primary reasons for undertaking this method update were to decrease both the time of operations and volume of solvent required. Table 3 3 details the amount of time required for both the TO-4A and new methods. The same amount of time (35.25 h) is required for sample collection and cleanup with the TO-4A method, regardless of whether it is the initial or reuse of the sampling matrix. By reusing the matrix in the new method, about 1.5 h of processing is saved compared with using fresh matrix. The time for unattended operations is significantly less with the new method (2.25 h versus 32 h). The sample cleanup in the new method is the longest step, requiring 6 h. If this step is transferred to solid phase extraction (SPE) cartridges, it will take less than 1 h, making the new method shorter than the TO-4A method. Table 3 4 details the volume of solvent required for taking one sample through the entire method. Dichloromethane was eliminated in all steps, except initial matrix cleaning. If the solvent needed to initially clean the XAD is included, the TO-4A method uses 1450 mL of solvent, whereas the new method uses 1720 mL. If the matrix already has been used, the TO-4A method uses the same 1450 mL of solvent, whereas the new method uses 970 mL, saving nearly 500 mL of solvent. It is important to note that the new method is using large quantities of solvent for the sample cleanup. Table 3-4. Volume of solvent used for pesticide analysisw Process TO-4A New method Matrix cleaning, initial 700 mL 900 mL Matrix cleaning, reuse 700 mL 150 mL Extraction 700 mL 150 mL Sample cleanup 50 mL 670 mLa Total, initial 1450 mL 1720 mL Total, reuse 1450 mL 970 mL "This procedure could be transferred to SPE cartridges, which require significantly less solvent (<50 mL) which can be reduced significantly by transferring the cleanup method to SPE cartridges for an additional 600 mL solvent savings. 3.3 Data Quality Objectives, Recovery Using the final preparation, extraction, and cleanup procedures, a series of trials using spiked XAD was performed to evaluate the complete method. Duplicate sampling trains were attached to pumps that were set to draw air through the XAD at the maximal rate possible (~50 m3 in 24 h). After turning on the pumps, the XAD was spiked through the retaining screen with the target analytes and, usually, the GC surrogate standards. Additionally, one control matrix spike was prepared for each trial for comparison. This sample was spiked with the target analytes but was not put on a sampler and no air was collected. The average recovery and relative standard deviation (RSD) from method evaluation experiments are detailed in Table 3 5 for GC compounds and in Table 3 6 for LC compounds. For comparison in method TO-4A, the RSDs range from 5% to 30%, and the acceptable recoveries range from 65% to 125%. The acceptable recovery range for the new methods is 75% to 120%, and the variability target is RSD <20%. The reproducibility is similar for the new GC method and higher for the new LC method. Typically, the new methods are in the acceptable range, but there have been instances of unreasonably low or high recovery for specific compounds, which remains unexplained. It is important to note that most compounds met the measurement quality objectives, but there is still room to improve both the accuracy and precision of the method. 6 ------- Table 3-5. Comparative recoveries of spiked GC pesticides (percent recovery ± relative standard deviation) Compound No airflow (n = 7) With air flow3 (n = 11) 13C3 Atrazine (surrogate) 125 ±32 120 ±26 13Cg 6-HCH (surrogate) 99 ± 17 107 ±6 2,4-D Ethyl Ester 60 ±45 86 ±32 2,4-D Methyl Ester 62 ±53 103 ±8 Alachlor 143 ± 17 157 ± 24 Aldrin 73 ± 33 93 ± 20 a-HCH 58 ±45 89 ± 15 Atrazine (GC) 82 ± 17 95 ±32 b-HCH 90 ± 11 195 ±57 Captan 94 ±43 108 ± 64 Chlorothalonil 134 ± 33 193 ±38 Chlorpyrifos 75 ± 28 76 ±50 c/'s-Chlordane 80 ± 10 101 ±35 D10 Chlorpyrifos (surrogate) 62 ± 24 71 ±40 D10 Diazonon (surrogate) 61 ±30 72 ±35 Dacthal 97 ± 12 108 ± 13 Diazinon (GC) 78 ± 36 74 ±22 Dicloran (Botran) 105 ± 21 127 ±25 Dieldrin 101 ± 28 120 ±28 Ethyl Parathion 92 ± 22 85 ± 15 Fenchlorphos (Ronnel) 102 ± 17 96 ± 25 Folpet 126 ± 65 100 ±22 g-HCH (Lindane) 77 ±27 97 ± 25 Heptachlor 79 ± 29 67 ±38 Heptachlor Epoxide A 78 ± 17 71 ± 11 Heptachlor Epoxide B 75 ± 17 92 ±33 Hexachlorobenzene 36 ±67 77 ± 24 Malathion 116 ± 22 128 ± 19 Methoxychlor 101 ±7 143 ± 36 Methyl Parathion 94 ± 30 96 ± 13 Metolachlor 104 ± 11 119 ± 16 Mi rex 96 ± 19 115 ±41 o,p'- DDT 89 ±7 118 ±40 Oxychlordane 96 ± 17 117 ± 16 p,p'-DDE 99 ± 22 118 ±38 p,p'-DDT -Q 00 00 176 ± 28 Propazine (GC) 99 ± 17 109 ± 24 Simazine (GC) 109 ± 26 130 ±23 frans-Chlordane 79 ± 10 98 ±36 frans-Nonachlor 103 ± 22 128 ± 16 Trifluralin 64 ±45 87 ± 28 aSamples were attached to pumps that ran between 18 and 48 h. bn = 1 for this compound, therefore no standard deviation is reported. ------- Table 3-6. Comparative recoveries of spiked LC pesticides (percent recovery ± relative standard deviation) Compound Without air flow (n = 8) With air flow3 (n = 16) Allethrin 92 ± 22 84 ± 31 Atrazine 117 ± 24 108 ± 25 Bendiocarb 118 ±41 94 ± 20 Carbaryl 100 ± 25 85 ± 20 Carbofuran 74 ±42 64 ± 32 Chlorotoluron 98 ± 20b 74 ± 16° Cinerin I 92 ± 19 90 ±37 Cinerin II 84 ±23 75 ± 30 Diazinon 131 ±35 117 ±36 Dicrotophos 63 ±33b 56 ±31° Diuron 85 ± 24 74 ± 23 Esfenvalerate 96 ± 22 88 ± 23 Fluometuron 129 ±16b 89 ± 27° Jasmolin I 83 ± 24 73 ± 27 Jasmolin II 82 ± 23 71 ± 28 Linuron 96 ± 16 87 ± 13 Monuron 101±19b 73 ± 15° C/'s- and Trons-Permethrin 94 ± 15 91 ± 28 Propazine 127 ± 25 117 ± 28 Propoxur(Baygon) 90 ±38 87 ±31 Pyrethrin I 72 ±41 58 ±45 Pyrethrin II 68 ±47 54 ±47 Resmethrin 67 ±38 48 ± 55 Simazine 138 ±32 120 ± 34 Tebuthiuron 108 ± 28b 87 ± 20° 'Samples were attached to pumps that ran between 18 and 48 h. bn = 3 cn = 10' ------- Bibliography [1IW.T. Winberry Jr., R. Riggin, R.G. Lewis, Compendium Method TO-4A: Determination of Pesticides and Polychlorinated Biphenyls in Ambient Air Using High Volume Polyurethane Foam (PUF) Sampling Followed by Gas Chromatographic/Multi-Detector Detection (GC/MD), U.S. Enviromnental Protection Agency, Cincinnati, OH, 1999. PIT. Colborn, F.S. vom Saal, A.M. Soto, Environ. Health Persp. 101 (1993) 378. [3ID. MacKay, A. Fraser, Environ. Pollut. 110 (2000) 375. ------- ------- Appendix A Standard Operating Procedure: XAD-2 Cleaning Procedure Using the Dionex Accelerated Solvent Extractor 300 1 Method Summary This method describes the procedures used to clean Amberlite XAD-2 resin using five solvents sequentially in the Dionex Accelerated Solvent Extractor 300. 2 Scope and Application This SOP is part of an effort to update the TO-4A method, "Determination of pesticides and PCBs in ambient air using high volume PUF sampling followed by gas chromatographic/multi-detector detection," to utilize improved instrumentation. This method details the cleaning procedure for XAD-2 resin (Supelco/Aldrich Cat. no. 10357 or similar) to be used for sampling semivolatile organic compounds from enviromnental matrices, such as air and water. This SOP assumes that the ASE and peripheral devices are properly installed and functioning. 3 Personnel Qualifications This SOP is written for users who have experience keeping a laboratory notebook and operating the Dionex 300. The operator should have a background in science with laboratory experience and must be trained by experienced personnel before undertaking these techniques. The operator must be able to understand the information in the operating manuals and the SOPs related to this procedure. 4 Health and Safety Standard laboratory protective clothing is required at all times during chemical operations in accordance with a health and safety research protocol. Operations involving the handling of solvents should be performed under a fume hood. Use caution when removing ASE cells from the carousel after extraction, as they may be hot. 5 Definitions, Acronyms, and Abbreviations ASE accelerated solvent extractor ECD electron capture detector GC gas chromatograph L liter 1 to r left to right min minute mL milliliter MS mass spectrometry PCB polychlorinated biphenyl PEEK polyetheretherketone psi pounds per square inch PUF polyurethane foam s second SOP standard operating procedure 6 Equipment and Supplies Filter insertion tool Aluminum funnel Scoopula 2 L Dionex solvent bottles Sample logbook Glass fiber filters (Part # 056781 or similar) Large, flat-bottomed flask Large beaker Laboratory notebook Chemical fume hood Dionex instruction manual Dionex 300 with solvent controller and peripheral devices Personal computer with AutoASE software 100 mL ASE cells, end caps, and components (Part # 068101) 250 mL Dionex collection bottles with lids and septa Blank matrix cleaning notebook Pesticide grade solvents: acetone, hexane, methanol, and dichloromethane Clean amber jars with Teflon lined lids Rotary or nitrogen evaporator Personal protective equipment (gloves, safety glasses or goggles, and lab coat) XAD-2 resin 20-60 mesh, 1.02 g/mL density, 90 A mean pore size, 300 m2/g surface area (Supelco/Al- drich Cat. #10357 or similar) 7 Procedure NOTE: It is important for all users to read the entire SOP before beginning any of the procedure and to ask questions if any of the instructions are unclear. XAD-2 resin will be placed into clean 100 mL ASE cells and cleaned using the following the procedure. 7.1 Cell assembly It is assumed that the end caps have already been assembled. For additional information on assembly and filling of Dionex 300 cells please refer to the instruction manual or an operation SOP for this instrument. This procedure should be performed while wearing nitrile gloves. Select the desired number of 100 mL ASE cells for filling with XAD-2. A-l ------- 1. Check the PEEK seals (orange rings inside endcap) and 0-rings (small white at entrance/exit of end cap) for excessive wear and replace if necessary. 2. Screw bottom end cap in place to hand tight. (The bottom of the cell is the end without the Dionex symbol and cell ID number.) 3. Insert the glass fiber filter with the filter insertion tool. 7.2 Filling the cells with XAD-2 NOTE: No more than twelve cells can be loaded onto the instrument at one time. 1. Insert the aluminum funnel in the top of the cell. 2. Using a scoopula, add 2 to 3 scoopfuls of XAD to the cell. NOTE: Be very cautious during this step. If any XAD spills into the bottom endcap, it may prevent proper sealing and cause the cell to leak during extraction. 3. Tamp down the XAD in the cell by gently tapping the cell on a counter top. 4. Repeat steps 3 and 4 until the cell becomes half full. 5. Use the black filter insertion tool to tamp down the XAD. Continue to add XAD and tamp it down with the insertion tool until the level of XAD is approximately 1-2 in from the top of the cell. 6. Remove the aluminum funnel and insert a glass fiber filter on top of the XAD. 7. Screw the top endcap in place to hand tight. (The top of the cell is the end with the Dionex symbol and cell ID number.) 8. Check the endcaps and outside of the cell for extraneous XAD. If present, thoroughly remove it. 9. Repeat steps 1 through 9 until all cells are filled. 7.3 Preparing the instrument 1. Check air and nitrogen gas tank ON/OFF state and pressure. (Tanks should be on and pressure >200 psi.) 2. Check system pressure gauges (1 to r). a. Solvent bottle 5-10 psi b. Air pressure 50 psi c. Oven compression 130 psi 5. Check solvent levels in all solvent bottles and fill if needed (see Figure E2. Column packing. 4. Empty waste bottle and dispose of waste in accordance with waste management procedures. 5. Empty collection rinse bottle and replace septa if necessaty. The septa should be changed every two to three runs. 6. Check hydrocarbon sensor. a. On the front panel of the Dionex 300, proceed to the "Menu of Screens." b. Push 7 for the "Diagnostic menu." c. Push 4 for the "Hy drocarbon calibration." d. Reading should be between 1000 and 4000. If not, refer to the Dionex 300 manual for adjustment. e. When finished, push the menu button to exit. Do not push "Enter " to calibrate. 7. Perform rinse to check solvent flow. Carefully listen to the pump to insure it is operating correctly. If the pump sounds abnormal, try performing additional rinses until the solvent lines have filled and the pump sounds normal. If the sound does not return to normal, do not use the instrument until it has been checked by a Dionex Service Representative and is fixed. Figure Al. Dionex 300 system: (A) PC with AutoASE software, (B) solvent controller, (C) solvent reservoir, (D) keypad and display, (E) 100 mL cells, and (F) collection bottle. A-2 ------- 8. Check the cells and cell caps for gouges, worn seals, and XAD particles. 9. Place the ASE cells into the top carousel. Use caution when rotating the tray. Make sure it is unlocked and moves freely. 10. Place the same number of clean bottles as cells into the bottom carousel. 11. Record the cell numbers, contents, method name, date, etc., in the sample logbook. 12. Record the activities in the Blank Matrix Cleaning notebook. 7.4 Preparing the method Using the instructions in the Dionex 300 manual or an ASE operation SOP, enter the following method parameters in the Auto ASE software. Pressure: 1500 psi (preset at 1500 psi) Temperature: 75 °C Preheat time: 0 min Purge during preheat: Off Heat time: 5 min Static time: 5 min Flush volume: 50% (percent of total cell volume) Purge time: 90 s Static cycles: 1 Solvent composition: varies (see below) One method needs to be prepared and saved with a unique name for each solvent/mixture listed below. Solvents: Method 1 - 100% Methanol Method 2 - 100% Acetone Method 3 - 100% Dichloromethane Method 4 - 100% Hexane Method 5 - 20% Acetone, 80% Hexane 7.5 Preparing the schedule Using the instructions in the Dionex 300 manual or an ASE operation SOP, enter the schedule conditions in the Auto ASE software. 1. Go to "EDIT" on the menu bar, then open "Schedule File." At this point, an existing schedule may be edited or a new schedule created. Set the parameters for the schedule as follows. a. BOTTLE: The order is preset from 1 to 12. b. CELL: The cell positions are labeled from 1 to 12. Up to 12 cells filled with XAD may be cleaned with each solvent. If the flush volume is changed to >50%, it is recommended that a maximum of 10 cells be used. c. METHOD FILE: In this box, place the name of the method to be used to extract the corresponding cell (e.g.. Method 1, Method 2, etc.). Double-clicking on the box will reveal the method files. Double-click on the method to be used and it will be placed in the schedule box. d. RINSE: Off. The system is not rinsed after each cell is extracted. e. VOLUME: 0. The system is not rinsed after each cell is extracted. f. SAMPLE ID: In this space, include the sample name or code number. 2. Once the schedule has been completed, click "SAVE AS" and give the schedule a name. After the schedule has been named, click "EXIT". 3. Go to "FILE" on the menu bar, then to "Load Schedule." 4. Select the schedule just created from the list and either double-click on the name or click "OK". The schedule is now loaded. 5. Go to "RUN" on the menu bar and select "START". The Dionex 300 will automatically run the schedule after a preprogrammed rinse. Nine (9) mL of solvent will be used to rinse the system prior to the first cell being extracted. 7.6 After a schedule is complete 1. Check solvent levels in all solvent bottles and fill if needed. 2. After each extraction cycle, hand tighten the endcaps on each cell. Use caution when handling cells, as they will be hot from the oven. Allow 10-15 min for the cells to cool. 3. Check the cells and cell caps for XAD particles and remove them if present. 4. Replace the cells into the same location on the top carousel. 5. Empty the solvent collection and rinse bottles into an appropriate waste container. 6. Check the septa on the collection bottles and replace them every two extractions/schedules. Check the septa on the rinse bottle and replace it every five to six extractions/schedules. 7. Replace the solvent collection bottles in the bottom carousel. 8. Using Auto ASE, load the schedule for the next method. 9. Repeat steps 1 through 8 until all five methods are complete. 7.7 After all schedules are complete 1. The Method 5 extracts should be reduced in volume by rotary and/or nitrogen ev aporation. A-3 ------- 2. The resulting extracts should be treated as samples and analyzed by GC/ECD (or GC/MS) using the same method by which samples are analyzed. 3. Non-quantifiable levels of the analytes of interest are required. The exact concentration of non-quantifiable levels will vary depending on the methods and instrumentation used for analysis. Record this analysis in the appropriate laboratory notebook and the Blank Matrix Cleaning notebook. 4. The XAD just cleaned must be dried before storage by one of the methods below. Dry XAD will be free flowing and no longer smell of solvents. a. Pour clean XAD into a large beaker, label the beaker, and place it in a fume hood overnight. b. Pour clean XAD into a large flat-bottom flask and proceed with roto-vapping until no more solvent is being removed (watch for drips into the collection vessel under the condenser). 5. Once dried, the XAD should be placed in amber jars, capped, labeled, and stored at room temperature until used. 8 Data and Records Management Data and records management issues are discussed throughout the procedure. Data will be recorded promptly, legibly, and in permanent ink in the logbook and in laboratory notebooks designated for each instrument and project. Electronic data files related to the project should be noted in the laboratory notebook. A logbook containing pertinent information is kept to record samples extracted on the Dionex 300. A separate notebook is used to record the activities pertaining to the cleaning of matrices that are to be used as matrix blanks. 9 Quality Control and Quality Assurance Quality assurance and control issues are discussed throughout the procedure. Following XAD cleaning procedure, the last extracts will be analyzed as samples. Non-quantifiable levels of the compounds of interest are required for the batch's acceptance as clean XAD. 10 Waste Management Solid, liquid, and glass waste are disposed of in separate containers. Solvents and samples used in this procedure should be disposed according to health and safety regulations and with appropriate labeling and recordkeeping. 11 References Dionex 300 Accelerated Solvent Extractor Operator's Manual. Dionex Corporation, 2000. Revision 01. A-4 ------- Appendix B Standard Operating Procedure: Preparation and Packing of ASE Cells for Air Sampling with XAD-2 1 Method Summary This method describes the procedures needed for preparing stainless steel accelerated solvent extraction (ASE) cells for air sampling. This includes instructions for inserting the snap rings, retaining screens, and XAD-2 resin. 2 Scope and Application This SOP is part of an effort to update the TO-4A method, "Determination of pesticides and PCBs in ambient air using high volume PUF sampling followed by gas chromatographic/multi-detector detection." Updates include stainless steel sampling cartridges that can be extracted by an automated ASE, XAD-2 resin sorption bed, and mass spectrometry. This document describes how to prepare modified 100 mL cells for air sampling based on the TO- 4A method. The cells must have been modified previously with four grooves to retain 4 in of XAD-2 resin with screens and snap rings. XAD-2 resin must be cleaned prior to cell assembly. After assembly, the cells will be ready for air sampling. 3 Personnel Qualifications This SOP is written for users who have experience keeping a laboratory notebook and operating the Dionex 300 and sampling apparatus. The operator should have a background in science with laboratory experience and must be trained by experienced personnel before undertaking these techniques. The operator must be able to understand the information in operating manuals and the SOPs related to this procedure. It is important for all users to read the entire SOP before beginning any of the procedure and to ask questions if any of the instructions are unclear. 4 Health and Safety Standard laboratory protective clothing is required at all times during chemical operations in accordance with a health and safety research protocol. Use caution when manipulating the snap rings, as they may suddenly release a large amount of tension and cause injury. Safety glasses are essential during this procedure. Secure loose personal items (especially long hair) and clothing when working around the vacuum pumps to prevent injury. If present, remove the Teledyne Hastings flow controller from the sampling train during this operation to ensure that XAD resin does not enter and damage the equipment. 5 Definitions, Acronyms, and Abbreviations ASE accelerated solvent extraction mL milliliter g gram PCB polychlorinated biphenyl PUF polyurethane foam 6 Equipment and Supplies • 100 mL ASE cells with two sets of grooves machined 4 in apart • Two ASE cell endcaps per cell • Two retaining screens per cell • Four snap rings per cell • Adjustable spanner wrenches • Dionex 300 with solvent controller and peripherals • Dionex 300 cell funnel or equivalent • Dionex 300 insertion tool or equivalent plunger • Flat-bladed screw driver • Gast vacuum pump Model 0523-101Q-SG588DX or equivalent • High-purity acetone • Modified sampling mount (University of Georgia Machine Shop, see Figure B4) • Personal protective equipment (gloves, safety glasses or goggles, and lab coat) • Precleaned XAD-2 resin (~25 g per cell) • SOP 'XAD-2 cleaning procedure utilizing the Dionex Accelerated Solvent Extractor 300" 7 Procedure 7.1 Assembling snap rings # 1, #2, and #3 Collect all necessary equipment for cell assembly. Figure B1 shows the components of the ASE cell for reference. Figure B2 shows the inside of a cell with one set of grooves for the snap rings. B-l ------- Retaining Screens 4 inches between grooves Figure B-L Linear view of modified ASE cell, snap rings, and retaining screens. Blue grooves are numbered based on the recommended snap-ring- insertion order. 10. Lower it past groove #2 in the perpendicular orientation and then slowly pull it back towards the groove. The snap ring should turn and slide into the groove. 11. Place a screen flat on top of snap ring # 1, noting the orientation of the open section of the snap ring. The filter insertion tool may need to be used to make sure the screen is flat on top of the snap ring. 12. Once the screen is in place, insert snap ring #3 perpendicularly into the cell and turn the open section opposite the open section of snap ring #1. The idea is to make sure that the two open portions DO NOT align. 13. Using your fingers or the filter insertion tool, push the snap ring into groove #3. Use the flat-bladed screwdriver to change the orientation of the open portion of the snap ring if necessary. 7.2 Packing the XAD resin 1. Orient the cell so that the screen between grooves #1 and #3 is on the bottom, and screw the cell onto the modified sampling mount. 2. If necessary, remove the flow controller from the sampling train using adjustable wrenches. 3. Attach the modified sampling mount and cell onto the sampler and tighten the associated fittings. See Figure B4 for proper setup. Figure B-2. End view of modified ASE cell showing location of grooves #2 and #4. Following the diagram in Figure B3, place the filter insertion tool in the cell just below groove #1. 6. Turn snap ring #1 perpendicular to the grooves, and insert it into the cell. Lower the snap ring past groove #1. 7. Using your fingers, a flat-bladed screwdriver, and/or the insertion tool, slowly turn the snap ring so it fits into groove #1. 8. Remove the filter insertion tool from the cell. 9. Place snap ring #2 into the cell perpendicular to the grooves. 4. Plug in the Gast vacuum pump, which will turn on immediately. 5. Place the cell funnel into the open end of the ASE cell. B-2 ------- Filter insertion tool Figure B-3. Schematic for insertion of snap ring 1. Ring should be oriented vertically with the axis of the cell. 6. Slowly pour the pre-cleaned XAD-2 resin into the funnel. This operation should be done near an open fume hood, as the XAD will likely give off acetone fumes. Continue to fill the cell until the level of XAD is just below snap ring #2. Gentle tapping may be required to level the XAD. 7. Pack the cell as close to the ring as possible. Ensure that the snap ring is free of XAD particles, as this will provide a poor seal with the screen and may cause leaking of XAD. Fingers, a brush, or laboratory wipe can be used to clean the snap ring. 8. Unplug the vacuum pump to turn it off. 9. After the last cell is filled, replace the flow controller in the sampling train using adjustable wrenches. 7.3 Inserting snap ring §4 1. Place a screen flat on top of snap ring #2, noting the orientation of the open section of the snap ring. The filter insertion tool may be needed to make sure the screen is flat on top of the snap ring. 2. Once the screen is in place, insert snap ring #4 perpendicularly into the cell and turn the open section opposite the open section of snap ring #2. The idea is to make sure that the two open portions do NOT line up. 3. Using your fingers or the filter insertion tool, push the snap ring into groove #4. Use the flat-bladed screwdriver to change the orientation of the open portion of the snap ring if necessary. 4. Screw endcaps onto both ends of the ASE cell for storage. 5. Record these actions in a laboratory or project notebook. Repeat the entire procedure for each cell to be packed. 7.4 Reusing cells 1. Once the cells have been packed with XAD-2 resin, it is generally not necessary to replace the XAD between samples if proper extraction and cleanup steps are followed. 2. After extraction of the cartridges and just prior to the next sampling event, each cell must be extracted by the Dionex 300 with 100% acetone. This step is detailed in the SOP "XAD-2 Cleaning Procedure Utilizing the Dionex Accelerated Solvent Extractor 300." B-3 Cleaned XAD-2 acuum pump Modified mpling mount Figure B-4. Filling the ASE cell using the modified sampling mount, vacuum pump, and cleaned XAD-2 resin. ------- 3. The extract from this 100% acetone extraction must be analyzed prior to the next sampling event. If detectable concentrations of analytes are found, it may be time to pack the cell with new XAD-2 resin. 8 Data and Records Management Data and records management issues are discussed throughout the procedure. Data will be recorded promptly, legibly, and in permanent ink in the logbook and in laboratory notebooks designated for each instrument and project. Record the cell number and packing date in a laboratory notebook. A logbook containing pertinent information is kept to record samples extracted on the Dionex 300. A separate notebook is used to record the activities pertaining to the cleaning of matrices that are to be used as matrix blanks. 9 Quality Control and Quality Assurance QA/QC is discussed throughout this document, but additional parameters are listed below. At a minimum, one sampling blank (with air, no spiking solutions), one sample duplicate (with air, no spiking solutions), and one QA spike (no air, GC and LC spiking solutions added) are included for each sampling event and should be analyzed together. The procedures in this SOP are in preparation for a sampling event, so enough cells must be packed to accommodate required QC samples. 10 Waste Management Generally, waste is not generated by this procedure. Solid, liquid, and glass waste are disposed of in separate containers. Solvents and samples used in this procedure should be disposed according to health and safety regulations, and with appropriate labeling and record keeping. 11 References SOP "XAD-2 Cleaning Procedure Utilizing the Dionex Accelerated Solvent Extractor 300." ------- Appendix C Standard Operating Procedure: Air Sampling with ASE Cells 1 Method Summary This method describes the procedures needed for collecting air samples using stainless steel accelerated solvent extraction (ASE) cells packed with XAD-2 resin. It includes instructions on the last cleanup step for the XAD, sampler setup, and the use of flow controllers during the sampling period. 2 Scope and Application This SOP is part of an effort to update the TO-4A method, "Determination of pesticides and PCBs in ambient air using high volume PUF sampling followed by gas chromatographic/multi-detector detection." Updates include stainless steel sampling cartridges that can be extracted by an automated ASE, XAD-2 resin sorption bed, and mass spectrometry. This SOP is followed when collecting air samples using modified ASE cells with XAD-2 resin packing material. It is assumed that the cells have already been assembled and packed with XAD-2 resin and that the ASE and peripheral devices are properly installed and functioning. The compounds listed in Table CI. GC and LC target analytes are the target analytes for this method. 3 Personnel Qualifications This SOP is written for users who have experience keeping a laboratory notebook and operating the Dionex 300 and sampling apparatus. The operator should have a background in science with laboratory experience and must be trained by experienced personnel before undertaking these techniques. The operator must be able to understand the information in operating manuals and the SOPs related to this procedure. It is important for all users to read the entire SOP before beginning any of the procedure and to ask questions if any of the instructions are unclear. 4 Health and Safety Standard laboratory protective clothing is required at all times during chemical operations in accordance with a health and safety research protocol. Use caution when manipulating the snap rings, as they may suddenly release a large amount of tension and cause injury. Safety glasses are essential during this procedure. Secure loose personal items (especially long hair) and clothing when working around the vacuum pumps to prevent injury. The toxicity or carcinogencity of each reagent used in this method lias not been precisely defined; however, each chemical compound should be treated as a potential health hazard, as most are pesticides. The following compounds are on the "P" list and should be handled only in a fume hood with proper personal protective equipment: aldrin dieldrin, heptachlor, and methyl parathion. "P" coded compounds (acutely toxic) must also be disposed of separately. Please check with laboratory or safety personnel on proper disposal of these compounds. Table CI. GC and LC target analytes GC target analytes Alachlor Aldrin Atrazine Captan c/'s-Chlordane frans-Chlordane Chlorothalonil Chlorpyrifos 2,4-D ethyl ester 2,4-D methyl ester Dacthol p,p'-DDE o,p'- DDT p,p'-DDT Diazinon Dicloran (Botran) Dieldrin Ethyl parathion Fenchlorphos (Ronnel) Folpet a-HCH b-HCH Heptachlor Heptachlor epoxide B Hexachlorobenzene Lindane (g-HCH) Malathion Methyl parathion Methoxychlor Metolachlor Mi rex frans-Nonachlor Oxychlordane Propazine Simazine Trifluralin LC target analytes Allethrin Atrazine Bendiocarb Carbaryl Carbofuran Chlortoluron Cinerin 1 & II Diazinon Diuron Dicrotophos Fenvalerate Fluometuron Jasmolin 1 & II Linuron Monuron o-Phenylphenol c/'s-Permethrin frans-Permethrin Propazine Propoxur(Baygon) Pyrethrin 1 &II Resmethrin Simazine Tebuthiuron ------- 5 Definitions, Acronyms, and Abbreviations ng nanogram mL milliliter min minute h hour m3 cubic meter |llL microliter s second |u,g microgram PUF polyurethane foam L liter LC liquid chromatography GC gas chromatography ASE accelerated solvent extraction SOP standard operating procedure QA/QC quality assurance/quality control psi pounds per square inch PCB polychlorinated biphenyl HCH hexachlorocyclohexane DDT dichlorodiphenyltrichloroethane DDE dichlorodiphenyldichloroethylene 6 Equipment and Supplies • 100 |-ig/mL stock solutions of target analytes (see Table CI. GC and LC target analytes for analyte list) • 100 inL modified ASE cells packed with cleaned XAD-2 resin • Two ASE cell endcaps per cell • 25 inL volumetric flask and stopper • Dionex 300 with solvent controller and peripherals • Clock • Eppendorf pipet 100-1000 mL with tips • Gast vacuum pump Model 0523-101Q-SG588DX or equivalent • High-purity (pesticide grade or better) ethyl acetate and acetone • Laboratory notebook • Mass flow controller • Modified sampling mount • Personal computer with Auto ASE software • Personal protective equipment (gloves, safety glasses or goggles, and lab coat) • SOP "XAD-2 cleaning procedure utilizing the Dionex Accelerated Solvent Extractor 300" • THPS-100 Power supply and readout 7 Procedure 7.1 Preparation of the sampling cells Use SOP "XAD-2 cleaning procedure utilizing the Dionex Accelerated Solvent Extractor 300" as reference materials for the following procedure. 1. Load/create method in the Auto ASE software with the following parameters. 2. Using a sequence, run this cleaning method for each cell that will be used during the sampling event. Be sure to include enough cells for QA/QC samples. The minimum requirement per sampling event is one blank, one duplicate, and one spike. 3. Dispose of the acetone waste in an appropriately labeled container and record the addition on the waste log sheet. 7.2 Preparation of the spiking solutions 1. It is not necessary to make new spiking solutions for each sampling event. If the spiking solutions are already prepared, remove them from freezer storage and allow them to reach room temperature. 2. If new spiking solutions need to be made, prepare one spiking solution for all GC target analytes in ethyl acetate (400 ng/mL). This can be accomplished for a variety of final volumes, but the suggested method follows. Pressure: 1500 psi (preset at 1500 psi) Temperature: 75 °C Preheat time: 0 min Purge during preheat: Off Heat time: 5 min Static time: 5 min Flush volume: 50% Purge time: 180 s Static cycles: 1 Solvent composition: 100% acetone ¦ Prepare a solution of all GC target analytes by diluting 100 mL of each 100 |ag/mL stock standard to 25 mL with ethyl acetate. 3. Prepare one spiking solution for all LC target analytes in ethyl acetate (400 ng/mL). This can be accomplished for a variety of final volumes, but the suggested method follows. ¦ Prepare a solution of all LC target analytes by diluting 100 mL of each 100 |ag/mL stock standard to 25 mL with ethyl acetate. 4. Label spiking solutions with the solution name, concentration, solvent, date made, expiration date (1 year from date made), and preparers initials. 7.3 Preparation of sampler 1. The sampler (vacuum pump, flow controller, controller power supply, and modified sampler mount) should be assembled as displayed in Figure E2. Column packing.. ------- 2. Screw the XAD-2 packed cell onto the modified sampling mount with the screen closest to the cell threads pointing up and the Dionex symbol and cell number at the bottom. 3. For each sampler unit, plug in the controller power supply and allow it to warm up for at least 30 min. 4. Label each sampling cell with the date, analyst's initials, and sample identification. 5. After the waiting period, check the display on the controller power supply (see Figure C2. Front panel of controller power supply). The display should read zero. If it does not read zero, press and hold the "zero" button until it resets (~5 s). 7.4 Air sampling This procedure has been used only in a laboratory setting during method development activities. Adjustments will be required if this method and procedure are used in a field setting. Specifically, the sampling time and volume of air must be considered. The TO-4A method calls for a 24-h sample of 300 m3 of air. This sampling apparatus will sample 40-60 m3 of air per day, so one must decide to match the time (24 h) or the volume of air sampled (~6 days). 1. Be sure to include QA/QC samples, such as blanks, duplicates, and spikes. At a minimum, one sampling blank (with air. no spiking solutions), one sample duplicate (with air, no spiking solutions), and one QA spike (no air, GC and LC spiking solutions added) are included for each sampling event. 2. Plug in the vacuum pumps. They should start immediately. 3. Using the buttons on the controller power supply (see Figure C2. Front panel of controller power supply.), set the flow to 100 L/min. Push the "MODE" button, then the 5 "increment" button, and then the4 "step" button (until the first digit is 1). Push the "ZERO" button to exit. 4. The display and flow will begin to increase slowly, reaching a maximum in <1 min. The flow will be approximately 30 L/min with the XAD cartridge attached Record the sampler start time in the laboratory notebook from a laboratory or personal clock. 5. Record the beginning flow rate in the laboratory notebook for each sampler. 6. Prepare the QA spike sample by removing the top endcap (near Dionex symbol and cell number) from a XAD cartridge and add 0.5 ml. each of the 400 ng/ ml. GC and LC spiking solutions with an Eppendorf pipet onto the XAD through the stainless steel screen to produce a 200 ng/sample concentration. Replace the endcap and store this sample near the vacuum pumps. Record these actions in the laboratory notebook. 7. Additional measurements of the flow rate can be recorded in the laboratory notebook if desired. 8. Allow the sampler to run for 24 h. Approximately 40-60 m3 will be collected. Longer time periods may be required for different experimental goals. 9. After the sample has been collected, record the ending flow rate and sampler stop time in the laboratory notebook using the same laboratory or personal clock as in step 4. 10. Unplug the samplers, followed by the controller power supplies. 11. Remove the modified ASE cartridges and screw endcaps on to both ends. 12. Calculate the total volume of air sampled using the equation below and record the results in the laboratory notebook. Display Mode Increment Figure C2. Front panel of controller power supply. XAD-2 acked cell Modified ^ sampling mountfj Flow controller jfc. Vacuum pump^^- . Figure CI. Air sampling unit assembly. Controller power supply Volume(m3) = Fl°wAvg(L) 60(min) Samplmgtime(h) l(/«!) 1000(/.) C-3 ------- 8 Data and Records Management Data and records management issues are discussed throughout the procedure. Data will be recorded promptly, legibly, and in permanent ink in the logbook and in laboratory notebooks designated for each instrument and project. For each sampler, the start and stop times must be recorded in a laboratory notebook. Corresponding flow controller readings should be taken at the beginning and end of the sampling period and recorded along with the ASE cell number. Descriptive information of the sample should also be included (spike, duplicate, date, location, etc.). Electronic data files related to the project should be specified in the laboratory notebook. A logbook containing identifying information (sample name, date, cell number, analyst initials, method, and sequence names) is kept to record samples extracted on the Dionex 300. A separate notebook is used to record the activities pertaining to the cleaning of matrices that are to be used as matrix blanks. 9 Quality Control and Quality Assurance Quality assurance and control issues are discussed throughout the procedure. At a minimum, one sampling blank (with air, no spiking solutions), one sample duplicate (with air, no spiking solutions), and one QA spike (no air, GC and LC spiking solutions added) are included for each sampling event. 10 Waste Management Solid, liquid, and glass waste are disposed of in separate containers. Solvents and solids used in this procedure should be disposed according to health and safety regulations and recorded on waste logsheets. Several compounds used in this method (aldrin, dieldrin heptachlor, and methyl parathion) are to be disposed of separately with "P" coded waste. 11 References Teledyne Hastings Instruments instruction manual Mass flowmeter, controller readout THPS-100, Document Number 161-112005, Revision 05, November 2005. Instruction manual for THPS-100 Power supply and readout. Revision 05, November 2005. SOP "XAD-2 cleaning procedure utilizing the Dionex Accelerated Solvent Extractor 300." ------- Appendix D Standard Operating Procedure: Extracting TO-4A Air Samples with the Dionex ASE 300 1 Method Summary This method describes the procedure for extracting selected TO-4A pesticides from air samples collected in stainless steel accelerated solvent extraction (ASE) cells containing XAD-2 resin including instructions for the adding surrogate standards, ASE extraction, and rotary evaporation. 2 Scope and Application This SOP is part of an effort to update the TO-4A method, "Determination of pesticides and PCBs in ambient air using high volume PUF sampling followed by gas chromatographic/multi-detector detection." Updates include stainless steel sampling cartridges that can be extracted by an automated ASE, XAD-2 resin sorption bed, and mass spectrometry. This SOP is used to extract and reduce air samples after collection using modified ASE cells with XAD- 2 resin packing material. It is assumed that the ASE and peripheral devices are properly installed and functioning. The compounds listed in Table Dl. GC and LC target analytes and GC surrogates are the target analytes and surrogate standards for this method. 3 Personnel Qualifications This standard operating procedure is written for users who have experience keeping a laboratory notebook and operating the Dionex 300 and rotary evaporator. The operator should have a background in science with laboratory experience and must be trained by experienced personnel before undertaking these techniques. The operator must be able to understand the information in operating manuals and the SOPs related to this procedure. 4 Health and Safety Standard laboratory protective clothing is required at all times during chemical operations in accordance with a health and safety research protocol. Operations involving the handling of solvents should be performed under a fume hood. The toxicity or carcinogencity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard, as most are pesticides. The following compounds are on the "P" list and should be handled only in a fume hood with proper personal protective equipment (PPE): aldrin dieldrin, heptachlor, and methyl parathion. "P" coded (acutely toxic) compounds also must be disposed of separately. Please check with laboratory or safety personnel on proper disposal of these compounds. Use caution when removing ASE cells from the carousel after extraction as they may be hot. Table Dl. GC and LC target analytes and GC surrogates GC target analytes Alachlor Aldrin Atrazine Captan c/'s-Chlordane frans-Chlordane Chlorothalonil Chlorpyrifos 2,4-D ethyl ester 2,4-D methyl ester Dacthol p,p'-DDE o,p'- DDT p,p'-DDT Diazinon Dicloran (Botran) Dieldrin Ethyl parathion Fenchlorphos (Ronnel) Folpet a-HCH b-HCH Heptachlor Heptachlor epoxide B Hexachlorobenzene Lindane (g-HCH) Malathion Methyl parathion Methoxychlor Metolachlor Mi rex frans-Nonachlor Oxychlordane Propazine Simazine Triflu ralin GC Surrogate standard compounds 13C Atrazine D10 Chlorpyrifos D10 Diazinon Heptachlor epoxide A LC target analytes Allethrin Atrazine Bendiocarb Carbaryl Carbofuran Chlortoluron Cinerin 1 & II Diazinon Diuron Dicrotophos Fenvalerate Fluometuron Jasmolin 1 & II Linuron Monuron o-Phenylphenol c/'s-Permethrin frans-Permethrin Propazine Propoxur(Baygon) Pyrethrin 1 &II Resmethrin Simazine Tebuthiuron D-l ------- 5 Definitions, Acronyms, and Abbreviations |u,g microgram |llL microliter ASE accelerated solvent extraction DDE dichlorodiphenyldichloroethylene DDT dichlorodiphenyltrichloroethane GC gas chromatography HCH hexachlorocyclohexane LC liquid chromatography min minute mL milliliter ng nanogram PCB polychlorinated biphenyl PPE personal protective equipment psi pounds per square inch PUF polyurethane foam QA/QC quality assurance/quality control s second SOP standard operating procedure 6 Equipment and Supplies • 100 |-ig/mL stock solutions of surrogate standard compounds (see Table Dl. GC and LC target analytes and GC surrogates) • 100 inL ASE cells used for air sampling through XAD-2 resin • Two ASE cell endcaps per cell • 25 mL volumetric flask and stopper • 250 mL ASE collection bottles with lids and septa • 250 mL flat-bottom flasks with stoppers • Dionex 300 with solvent controller • Sample logbook • Eppendorf pipet 100-1000 mL with tips • Heidolph rotary evaporator and associated equipment (or similar) • High-purity (e.g., pesticide grade) ethyl acetate, acetone, and hexane • Laboratory notebook • Personal computer with Auto ASE software • Personal protective equipment (gloves, safety glasses or goggles, and lab coat) • SOP "Air sampling with ASE cells" 7 Procedure NOTE: It is important for all users to read the entire SOP before beginning any of the procedure and to ask questions if any of the instructions are unclear. 7.1 Preparation of the surrogate spiking solution NOTE: Prior to sample analysis for both GC and LC compounds on the same extracts, a decision must be made regarding two standards added to the samples. 13C, Atrazine and D10 Diazinon cannot be used simultaneously as a surrogate for GC compounds and as an internal standard for LC compounds. The addition of these compounds prior to extraction will impact the internal standard quantitation by LC. A suggested compromise is to select one of these compounds as a GC surrogate and the other as an LC internal standard. 1. It is not necessary to make new surrogate spiking solution for each extraction event. If the surrogate spiking solution is already prepared, remove it from freezer storage and allow it to reach room temperature. 2. If a new spiking solution needs to be made, prepare one spiking solution for all GC surrogate standards in ethyl acetate (400 ng/mL). This can be accomplished for a variety of final volumes, but the suggested method follows. ¦ Prepare a solution of all GC surrogate standards by diluting 100 mL of each 100-|j.g/mL stock standard to 25 mL with ethyl acetate. 3. Label spiking solutions with the solution name, concentration, solvent, date made, expiration date (1 year from date made), and preparer's initials. 7.2 Extraction of the sample cells 1. All samples, including QA/QC samples, are treated in the same way as enviromnental samples throughout this procedure. At a minimum, one sampling blank (with air, no spiking solutions), one sample duplicate (with air, no spiking solutions), and one QA spike (no air, GC and LC spiking solutions added) are included for each sampling event and should be extracted together. See SOP 'Air sampling with ASE cells" for additional details. 2. Remove the top endcap from the ASE cell (near Dionex symbol and cell number). 3. Add 0.5 mL of the 400 ng/mL GC surrogate standards with an Eppendorf pipet onto the XAD through the stainless steel screen to produce a 200 ng/sample concentration. 4. Replace the endcap. 5. Repeat steps 2 through 4 for each ASE cell to be extracted. 6. Load the cells into the top carousel with the Dionex symbol at the top. 7. Label one 250 mL ASE collection bottle for each cell with the sampling date, analyst's initials, and sample identification information. Load these bottles into the bottom carousel. 8. Load/create method in the Auto ASE software with the following parameters. ------- Pressure: 1500 psi (preset at 1500 psi) Temperature: 75 °C Preheat time: 0 min Purge during Preheat: Off Heat time: 5 min Static time: 5 min Flush volume: 100% Purge time: 120 s Static cycles: 1 Solvent composition: 20% acetone, 80% hexane 9. Prepare a sequence that will run this method for each sample, including QA/QC samples such as blanks, duplicates, and spikes. Save, load, and run the sequence. 10. Record these actions in both the laboratory notebook and the ASE sample logbook. Include sample identification, cell number, method and sequence name, date, and analyst. 7.3 Rotary evaporation 1. Label a 250 mL flat-bottom flask for each sample with the sampling date, analyst's initials, and sample identification information. 2. Remove the extract bottles from the lower ASE carousel. 3. Pour the extract from each sample into the corresponding fiat-bottom flask. 4. Rinse the sample bottle three times with approximately 5 mL of hexane each time. Add the rinses to the sample's flat-bottom flask. 5. Using a rotary evaporator, evaporate the samples to dryness. Dryness is obtained when the condenser is no longer dripping solvent. NOTE: The rotary evaporator water bath temperature should not exceed 40 °C. 6. Stopper the sample and store at room temperature until Florisil cleanup. If the next procedure will not begin within 1 week, the stoppered sample flasks should be refrigerated. 8 Data and Records Management Data and records management issues are discussed throughout the procedure. Data will be recorded promptly, legibly, and in permanent ink in the logbook and in laboratoiy notebooks designated for each instrument and project. Electronic data files related to the project must be specified in the laboratory notebook. A logbook containing identifying information (sample name, date, cell number, analyst initials, method, and sequence names) is kept to record samples extracted on the 300. 9 Quality Control and Quality Assurance Quality assurance and control issues are discussed throughout the procedure. All samples, including QA/QC samples, are treated in the same way as enviromnental samples throughout this procedure. A solution containing four surrogate standards is added to each sample prior to extraction to quantify the recovery of compounds using this method. 10 Waste Management Solid, liquid, and glass waste are disposed of in separate containers. Solvents and solids used in this procedure should be disposed according to health and safety regulations and recorded on waste logsheets. Several compounds used in this method (aldrin, dieldrin, heptachlor, and methyl parathion) are to be disposed of separately with "P" coded waste. 11 References SOP "Air sampling with ASE cells." D-3 ------- ------- Appendix E Standard Operating Procedure: Florisil Column Cleanup for TO-4A Pesticides in Air Samples 1 Method Summary This SOP describes the cleanup step needed for the analysis of selected TO-4A pesticides. A Florisil cleanup column is loaded with an air sample extract, and the target analytes are eluted with six solvents. Following sample elution each fraction volume is measured and divided in half to accommodate GC and LC analysis. The samples are then evaporated to dryness prior to instrumental analysis. 2 Scope and Application This SOP is part of an effort to update the TO-4A method, "Determination of pesticides and PCBs in ambient air using high volume PUF sampling followed by gas chromatographic/multi-detector detection." Updates include stainless steel sampling cartridges that can be extracted by an automated accelerated solvent extractor (ASE), XAD-2 resin sorption bed, and mass spectrometry. This SOP assumes that the samples have been reduced to dryness prior to this procedure, that the rotary evaporator is operational and ready for use, and that the samples will be split for both GC and LC analysis. This SOP is used to cleanup air extracts using Florisil and six solvent fractions. The samples are collected on Amberlite XAD-2 resin, although this cleanup procedure also may be appropriate for other matrices. All GC and LC target analytes and GC surrogates for this method are listed in Table El. Target analytes and surrogate compounds analyzed by this method. 3 Personnel Qualifications This SOP is written for users who have experience keeping a laboratory notebook and operating the rotary evaporator. The operator should have a background in science with laboratory experience and must be trained by experienced personnel before undertaking these techniques. The operator must be able to understand the information in operating manuals and the SOPs related to this procedure. 4 Health and Safety Standard laboratory protective clothing is required at all times during chemical operations in accordance with a health and safety research protocol. Operations involving the handling of solvents should be performed under the fume hood. The toxicity or carcinogencity of each reagent used in this method has not been precisely defined; however, each chemical compound should be treated as a potential health hazard, as most are pesticides. The following compounds are on the "P" list and should be handled only in a fume hood with proper personal protective equipment: aldrin, dieldrin. Table El. Target analytes and surrogate compounds analyzed by this method GC target analytes Alachlor Aldrin Atrazine Captan c/'s-Chlordane frans-Chlordane Chlorothalonil Chlorpyrifos 2,4-D ethyl ester 2,4-D methyl ester Dacthol p,p'-DDE o,p'- DDT p,p'-DDT Diazinon Dicloran (Botran) Dieldrin Ethyl parathion Fenchlorphos (Ronnel) Folpet a-HCH b-HCH Heptachlor Heptachlor epoxide B Hexachlorobenzene Lindane (g-HCH) Malathion Methyl parathion Methoxychlor Metolachlor Mi rex trans-Nonachlor Oxychlordane Propazine Simazine Triflu ralin GC surrogate standard compounds 13C Atrazine D10 Chlorpyrifos D10 Diazinon Heptachlor epoxide A LC target analytes Allethrin Atrazine Bendiocarb Carbaryl Carbofuran Chlortoluron Cinerin 1 & II Diazinon Diuron Dicrotophos Fenvalerate Fluometuron Jasmolin 1 & II Linuron Monuron o-Phenylphenol c/'s-Permethrin frans-Permethrin Propazine Propoxur(Baygon) Pyrethrin 1 &II Resmethrin Simazine Tebuthiuron ------- heptachlor, and methyl parathion. "P" coded (acutely toxic) compounds must also be disposed of separately. Please check with laboratory or safety personnel on proper disposal of these compounds. The user may choose to wear a dust mask type respirator during the weighing and pouring of Florisil and sodium sulfate to decrease the amount of inhaled particles. 5 Definitions, Acronyms, and Abbreviations ASE accelerated solvent extraction cm centimeter DDE dichlorodiphenyldichloroethylene DDT dichlorodiphenyltrichloroethane EtOAc ethyl acetate GC gas chromatography HCH hexachlorocyclohexane ID inner diameter LC liquid chromatography min minute mL milliliter mm millimeter Na2S04 sodium sulfate PCB polychlorinated biphenyl PUF polyurethane foam QA/QC quality assurance/quality control s second SOP standard operating procedure 6 Equipment and Supplies • 100 and 150 mL beakers • 125 and 500 mL flat-bottom flasks and stoppers • 250 mL glass reservoirs • 5, 50, 100 and 250 mL graduated cylinders • 6%, 15%, and 50% mixtures of EtOAc in hexane • Acetone and hexane squirt bottles • Aldrich Florisil reagent grade 60-100 mesh or equivalent, activated at 675 °C by the manufacturer • Burdick & Jackson high purity solvents or equivalent: hexane, ethyl acetate, and acetone • Dust gun or source of dry air • Eppendorf pipet 100-1000 mL with tips • GC target analyte spiking solution, 400 ng/mL in EtOAc (see SOP 'Air sampling with ASE cells" for preparation instructions) • Glass chromatography columns (45 mm x l mm ID) with Teflon stopcock and fitted with a glass frit • Heidolph rotary evaporator and associated equipment • J.T. Baker anhydrous sodium sulfate, granular 12-60 mesh or equivalent • LC target analyte spiking solution 400 ng/mL in EtOAc (see SOP "Air sampling with ASE cells" for preparation instructions) • Organomation nitrogen evaporator system (N-evap) • Pasteur pipettes and bulbs • Personal protective equipment (gloves, safety glasses or goggles, and lab coat) • Ring stands equipped with two-sided burette holders • Scoopula • SOP "Air sampling with ASE cells" • SOP "Analytical methods for the determination of selected TO-4A pesticides by liquid chromatography tandem mass spectrometry (LC/MS/MS)" • SOP "Extracting TO-4A air samples with the Dionex 300" • SOP "Gas chromatography-mass spectrometry method for the analysis of TO-4A pesticides" • Surrogate spiking solution, 400 ng/mL in EtOAc (see SOP "Extracting TO-4A air samples with the Dionex 300" for preparation instructions) • TO-4A air sample extracts 7 Procedure NOTE: It is important for all users to read the entire SOP before beginning any of the procedure and to ask questions if any of the instructions are unclear. It is assumed that samples have been collected, extracted, and reduced to dryness prior to beginning these procedures. Previously extracted QA/QC samples, such as blanks, duplicates, and spikes will be cleaned up and analyzed along with the samples. 7.1 Sample and column sorbentpreparation 1. Reconstitute the samples with 5 mL of hexane. Swirl flask to distribute the solvent to areas where compounds may be stuck to the glass. 2. Florisil and sodium sulfate must be stored in the laboratory at 120 °C for at least 24 h prior to use. 7.2 Column preparation 1. Remove the Florisil and sodium sulfate from the oven and allow them to cool to room temperature on a laboratory bench. 2. Place one chromatography column in the burette holder with the frit at the bottom for each sample. Insert the Teflon stopcock and tighten the retaining nut (see Figure El. Florisil column setup, and Figure E2. Column packing.). Mark each column with a permanent marker to identify the corresponding sample. 3. Using a clean scoopula, fill each column with approximately 10 grams of Florisil. Gently tap each column to settle the Florisil (a permanent marker is a good instrument for tapping). Add 1-2 cm of anhydrous E-2 ------- sodium sulfate to the top of the Florisil (see Figure E2. Column packing.). Gently tap each column to settle the sodium sulfate. 4. Place a 100 or 150 mL beaker under each column. 5. Ensure that the stopcocks are in the closed position. Add 50 mL of hexane to each column prior to loading samples. 6. Turn the stopcocks to the open position and allow the hexane to drain through the column (flow rate unimportant) into the beakers until the level of the solvent is 1-2 cm from the top of the sodium sulfate. 7. Close the stopcocks until the samples are ready to load onto the column. 8. The collected hexane may be discarded in an appropriate waste container. 7.3 EIu ant preparation 1. Prepare 6%, 15%. and 50% v/v mixtures of EtOAc in hexane, enough for the number of samples involved in the cleanup step. 2. Label six 125 mL flat-bottom flasks with ground glass stoppers for each sample (one for each of six fractions) with analyst's initials; sample identification information; fraction number; and, if desired, fraction composition. 3. Using Table E2. Solvent type and volume for six Florisil column eluants and a 50, 100, or 250 ml . graduated cylinder, add the following volume of each solvent to the appropriately labeled flask for each sample. Table E2. Solvent type and volume for six Florisil column eluants Fraction no. 1 2 3 4 5 6 Solvent Hexane 6% EtOAc in hexane 15% EtOAc in hexane 50% EtOAc in hexane EtOAc Acetone Volume (mL) 40 125 125 125 100 100 4. Place a clean glass stopper in each flask. 7.4 Confirming Florisil elution pattern NOTE: Prior to sample analysis for both GC and LC compounds on the same extracts, a decision must be made regarding two standards added to the samples. 13 C, Atrazine and D10 Diazinon cannot be used simultaneously as a surrogate for GC compounds and as an internal standard for LC compounds. The addition of these compounds prior to extraction will impact the internal standard quantitation by LC. A suggested compromise is to select one of these compounds as a GC surrogate and the other as an LC internal standard. 250 mL reservoir Burette holder I \Chromatography ( column Ringstand Figure El. Florisil column setup. r 10 g j Florisil \ 1-2 cm Sodium sulfate Teflon stopcock and nut Figure E2. Column packing. E-3 ------- 1. Because Florisil may vary from batch to batch and among different manufacturers, each new batch or lot of Florisil should be checked using this procedure to confirm the elution pattern of the compounds of interest. 2. Prepare one Florisil column as described in section 9.7.2. 3. Using an Eppendorf pipet, combine 1000 mL of surrogate spiking solution GC target analyte spiking solution, and LC target analyte spiking solution (each 400 ng/mL in EtOAc) in a 150 mL beaker. 4. Evaporate the 3 mL of EtOAc to dryness using a nitrogen evaporator. 5. Reconstitute the sample with 5 mL of hexane. 6. Continue the sample cleanup steps described in section 9.7.5. 7. Split the sample fractions for LC and GC analysis as described in section 9.7.6, but do not combine fractions for GC analysis. 8. Analyze each fraction separately to determine which compounds elute in each fraction and that the collected fractions meet the method's needs. Details for sample analysis are described in SOP "Gas chromatography- mass spectrometry method for the analysis of TO-4A pesticides" and SOP "Analytical methods for the determination of selected TO-4 A pesticides by liquid chromatography tandem mass spectrometry (LC/MS/MS)." 9. Record the percent recovery in table form in the laboratory notebook (fraction number and compound). Include the date of the pattern determination, brand, and lot number of the Florisil. Make sure that the compounds elute with >75% cumulative recovery in the fractions collected. 7.5 Sample cleanup 1. Open the stopcock on one column and allow the previously added hexane to drip out slowly into a beaker. 2. When the solvent level has reached the top of the Na2S04, use a Pasteur pipette to load the sample (in 5 mL hexane) onto the appropriate column. Run the liquid down the side of the glass column to help insure that the Florisil bed is not disturbed during sample loading. The stopcock should remain slightly open during this procedure, with the solvent dripping into a beaker. 3. Add 5 mL of hexane to the sample flask and swirl to remove residues from the sides of the glass. 4. Wait until the solvent level has reached the top of the Na2S04 before adding the rinse to the column with a Pasteur pipette. 5. Place the empty 250 mL reservoir on top of the column. 6. Pour the 40 mL hexane eluant from the Fraction 1 flask into the sample flask. 7. Remove the beaker from beneath the column and quickly replace it with the Fraction 1 flask to collect the first fraction. 8. When the solvent level has reached the top of the Na2S04, pour the 40 mL of hexane eluant from the sample flask through the reservoir and onto the column. 9. Adjust the stopcock so that the drip rate is approximately one drip per second. 10. Repeat steps 1 through 9 for each sample. Be aware of the solvent level in each column and do not allow it to drop below the level of the top of the Na2S04. 11. Pour the 125 mL eluant (6% EtOAc in hexane) from the Fraction 2 flask into the sample flask. 12. When the solvent level has reached the top of the Na2S04, pour the 125 mL of 6% EtOAc in hexane eluant from the sample flask through the reservoir and onto the column. 13. Remove the flask labeled Fraction 1 and stopper this flask. Immediately place the labeled Fraction 2 flask under the column tip to collect the second fraction. 14. Repeat steps 11 through 13 for each sample. 15. Repeat steps 11 through 14 for each eluant fraction (see Table E2. Solvent type and volume for six Florisil column eluants for composition and volume). 16. When the last fraction has reached the top of the Na2S04, open the stopcock fully to collect any remaining drips. 17. When no additional solvent drips off the column, remove the flask labeled Fraction 6 and stopper this flask. 18. Remove the Teflon stopcock and reservoir. 19. After allowing the columns to dry at least overnight, remove the Florisil and Na2S04 by dumping them out the top of the column into a beaker. A dust gun or other source of dry air may be required to eliminate all of the solids from the column. The Florisil and Na2S04 mixture should be disposed in an appropriate solid waste container. 7.6 Sample splitting 1. Get clean 100 and 250 mL graduated cylinders, the sample fractions, hexane and acetone squirt bottles, and additional solvents for each fraction (see Table E2. Solvent type and volume for six Florisil column eluants). 2. Label two 500 mL flat-bottom flasks with ground glass stoppers for each sample (not for each fraction) with the analyst's initials, sample identification information, fraction number (1 through 3 and 4 through 6 for GC analysis), and fraction composition. ------- 3. Split Fraction 1. a. Pour Fraction 1 into the 100 inL graduated cylinder. b. Add hexane to the cylinder until the total volume is easily divisible by two (on a line). Record this volume in the laboratory notebook. c. Pour Fraction 1 back into the flask for mixing purposes. d. Pour half of the volume into the 100 mL graduated cylinder. Stopper the flask containing the remaining LC portion of the fraction. e. Empty the graduated cylinder into the 500 mL flask labeled for GC Fractions 1 through 3. f. Rinse the 100 mL graduated cylinder once with acetone, then hexane. Discard the rinsate. 4. Split Fraction 2. a. Pour Fraction 2 into the 250 mL graduated cylinder. b. Add 6% EtOAc in hexane to the cylinder until the total volume is easily divisible by four (on a line). Record this volume in the laboratory notebook. c. Pour Fraction 2 back into the flask for mixing purposes. d. Pour half of the volume into the 250 mL graduated cylinder. Stopper the flask containing the remaining LC portion of the fraction. e. Empty the graduated cylinder into the 500 mL flask labeled for GC Fractions 1 through 3. f. Rinse the 250 mL graduated cylinder once with acetone, then hexane. Discard the rinsate. 5. Split Fraction 3. a. Pour Fraction 3 into the 250 mL graduated cylinder. b. Add 15% EtOAc in hexane to the cylinder until the total volume is easily divisible by four. Record this volume in the laboratory notebook. c. Pour Fraction 3 back into the flask for mixing purposes. d. Pour half of the volume into the 250 mL graduated cylinder. Stopper the flask containing the remaining LC portion of the fraction. e. Empty the graduated cylinder into the 500 mL flask labeled for GC Fractions 1 through 3. f. Rinse the 250 mL graduated cylinder once with acetone, then hexane. Discard the rinsate. 6. Split Fraction 4. a. Pour Fraction 4 into the 250 mL graduated cylinder. b. Add 50% EtOAc in hexane to the cylinder until the total volume is easily divisible by four. Record this volume in the laboratory notebook. c. Pour Fraction 4 back into the flask for mixing purposes. d. Pour half of the volume into the 250 mL graduated cylinder. Stopper the flask containing the remaining LC portion of the fraction. e. Empty the graduated cylinder into the 500 mL flask labeled for GC Fractions 4 through 6. f. Rinse the 500 mL graduated cylinder once with acetone, then hexane. Discard the rinsate. 7. Split Fraction 5. a. Pour Fraction 5 into the 100 mL graduated cylinder. b. Add EtOAc to the cylinder until the total volume is easily divisible by two. Record this volume in the laboratory notebook. c. Pour Fraction 5 back into the flask for mixing purposes. d. Pour half of the volume into the 100 mL graduated cylinder. Stopper the flask containing the remaining LC portion of the fraction. e. Empty the graduated cylinder into the 500 mL flask labeled for GC Fractions 4 through 6. f. Rinse the 100 mL graduated cylinder once with acetone, then hexane. Discard the rinsate. 8. Split Fraction 6. a. Pour Fraction 6 into the 100 mL graduated cylinder. b. Add acetone to the cylinder until the total volume is easily divisible by two. Record this volume in the laboratory notebook. c. Pour Fraction 6 back into the flask for mixing purposes. d. Pour half of the volume into the 100 mL graduated cylinder. Stopper the flask containing the remaining LC portion of the fraction. e. Empty the graduated cylinder into the 500 mL flask labeled for GC Fractions 4 through 6. f. Rinse the 100 mL graduated cylinder once with acetone, then hexane. Discard the rinsate. 9. Repeat steps 3 through 8 for each sample. Between samples, rinse each graduated cylinder with an additional portion of acetone and hexane to reduce the possibility of cross-contamination. 10. After splitting, Florisil column fractions are evaporated to dryness using a rotary evaporator. Dryness is obtained when the condenser is no longer dripping solvent. NOTE: The rotary evaporator water bath temperature should not exceed 40 °C. 11. Stopper the sample flasks and store at room temperature until instrumental analysis. If the instrumental analysis will not begin within 1 week, the stoppered sample flasks should be refrigerated. E-5 ------- 8 Data and Records Management Data and records management issues are discussed throughout the procedure. Data will be recorded promptly, legibly, and in permanent ink in the logbook and in laboratory notebooks designated for each instrument and project. Electronic data files related to the project should be specified in the laboratory notebook. 9 Quality Control and Quality Assurance QA/QC is discussed throughout this document, but additional parameters are listed below. All samples, including QA/ QC samples, are treated in the same way as environmental samples throughout this procedure. Because Florisil may vary from batch to batch and among different manufacturers, the elution pattern of the compounds of interest should be checked as described in section 9.7.4 for each batch or lot of Florisil. Surrogate standards are employed for the GC compounds that will be monitored for acceptable method performance. 10 Waste Management Solid, liquid, and glass waste are disposed of in separate containers. Solvents and solids used in this procedure should be disposed according to health and safety regulations and with appropriate labeling and record keeping. Several compounds used in this method (aldrin, dieldrin, heptachlor, and methyl parathion) are to be disposed of separately with "P" coded waste. 11 References SOP 'Air sampling with ASE cells" SOP "Extracting TO-4A air samples with the Dionex 300" SOP "Gas chromatography-mass spectrometry method for the analysis of TO-4A pesticides" SOP "Analytical methods for the determination of selected TO-4A pesticides by liquid chromatography tandem mass spectrometry (LC/MS/MS)" ------- Appendix F Standard Operating Procedure: Gas Chromatography-Mass Spectrometry Method for the Analysis of TO-4A Pesticides 1 Method Summary This SOP describes the analysis of selected TO-4A pesticides by electron impact GC/MS in selected ion mode. ADB5-type chromatography column is utilized for the separation portion of the analysis. Once separated, the analytes are ionized in the mass spectrometer where fragments specific to each compound are detected. Three internal standards (tetrachloro- «/-xylene [TCrnX], 13C6 d-HCH, and decachlorobiphenyl [DCBP]) are used to minimize small differences in instrumental conditions during analysis. Additionally, up to four GC surrogate standards (13C atrazine, D10 diazinon, D10 chlorpyrifos, and heptachlor epoxide A) are analyzed to quantify and correct for extraction efficiency. The calibration range for this method is 50-300 pg/mL. 2 Scope and Application This SOP is part of an effort to update the TO-4A method, "Determination of pesticides and PCBs in ambient air using high volume PUF sampling followed by gas chromatographic/multi-detector detection." Updates include stainless steel sampling cartridges that can be extracted by an automated accelerated solvent extractor (ASE), XAD-2 resin sorption bed, and mass spectrometry. This SOP updates TO-4A from multidetector detection for GC amenable compounds (high vapor pressure) to one detection technique. mass spectrometry. This improvement results in a more sensitive and selective method. All analytes, surrogates, and internal standards for this method are listed in Table E2. Solvent type and volume for six Florisil column eluants. This SOP assumes the following. • The instrument has been properly installed according to the manufacturer's specifications; accessories and attachments have been installed and verified as functioning correctly; and it is in its operational location and ready for general use. • An autosampler is used with this system. • The operator has access to the manufacturer's instrument, accessory operating, software, and reference manuals and current laboratory SOPs; see Equipment and Supplies. • The operator is trained and knowledgeable in the use of a GC system. • Internal standard calibration • The samples to be analyzed by this method have been collected, extracted, cleaned up, and reduced to dryness prior to final determination. Table Fl. Target analytes, surrogate compounds, and internal standards for this method Target analytes Alachlor2 Aldrin2 Atrazine1 Captan3 c/'s-Chlordane3 frans-Chlordane3 Chlorothalonil2 Chlorpyrifos2 2,4-D ethyl ester1 2,4-D methyl ester1 Dacthol2 p,p'-DDE3 o,p'-DDT3 p,p'-DDT3 Diazinon2 Dicloran (Botran)1 Fenchlorphos Dieldrin3 Ethyl parathion2 (Ronnel)2 Folpet3 a-HCH1 b-HCH1 Heptachlor2 Heptachlor epoxide B3 Hexachlorobenzene1 Lindane (g-HCH)2 Malathion2 Methoxychlor3 Metolachlor2 Mirex3 Oxychlordane3 Propazine1 Simazine1 Surrogate standard compounds 13C Atrazine1 D10 Chlorpyrifos2 D10 Diazinon2 Internal standard compounds TCmX1 13C6 d-HCH2 DCBP3 1Target analytes and surrogates using TCmX as an internal standard 2Target analytes and surrogates using 13C6 d-HCH as an internal standard 3Target analytes and surrogates using DCBP as an internal standard F-l Methyl parathion2 frans-Nonachlor3 Trifluralin1 Heptachlor epoxide A3 ------- 3 Personnel Qualifications This SOP is written for users who have experience keeping a laboratory notebook and operating a gas chromatographic mass spectrometer and is trained in and can use Microsoft Excel. The operator should have a background in science with laboratory experience and must be trained by experienced personnel before undertaking these techniques. The operator must be able to understand the information in operating manuals and the SOPs related to this procedure. 4 Health and Safety Standard laboratory protective clothing is required at all times during chemical operations in accordance with a health and safety research protocol. Operations involving the handling of solvents should be performed under a fume hood. The toxicity or carcinogencity of each reagent used in this method lias not been precisely defined; however, each chemical compound should be treated as a potential health hazard, as most are pesticides. The following compounds are on the "P" list should be handled only in a fume hood with proper personal protective equipment: aldrin, dieldrin heptachlor, and methyl parathion. "P" coded (acutely toxic) and PCB waste (DCBP) also must be disposed of separately. Please check with laboratory or safety personnel on proper disposal of these compounds. When running a sample using the autosampler or when troubleshooting the autosampler, keep hands away from the syringe needle to avoid puncture wounds. The needle is sharp and may contain hazardous chemicals. The Agilent 6890 is supplied with a three-conductor power cord that provides a protective grounding when plugged in to a properly wired receptacle. Proper receptacle grounding must be verified. Wear safety glasses to prevent possible eye injury from flying particles while handling, cutting, or installing glass or fused-silica capillary columns. Observe caution in handling capillary columns to prevent skin puncture wounds. Many parts of this instrument are kept at a high temperature (>100 °C), including the injector, transfer line, and oven. Use extreme caution when touching these zones and allow them to cool or wear heat resistant gloves when performing maintenance. 5 Definitions, Acronyms, and Abbreviations |u,g microgram (XL microliter |o,m micrometer cm centimeter DCBP decachlorobiphenyl (PCB 209) DDE dichlorodiphenyldichloroethylene DDT dichlorodiphenyltrichloroethane GC/MS gas chromatographic mass spectrometry HCH hexachlorocyclohexane HP Hewlett-Packard ID inner diameter m meter m/z mass to charge ratio min minute mL milliliter mm millimeter msec millisecond ng nanogram PCB polychlorinated biphenyl Pg picogram psi pounds per square inch PUF polyurethane foam QA/QC quality assurance/quality control RSD relative standard deviation s second SIM selected ion monitoring SOP standard operating procedure TCmX tetrachloro-meta-xylene 6 Equipment and Supplies • 100 |-ig/mL stock solutions of 36 target analytes, 4 surrogate standards, and 3 internal standards (see Table E2. Solvent type and volume for six Florisil column eluants) • 100-1000 mL Eppendorf pipet and tips • -20 °C freezer • 25 mL volumetric flask and stopper • Autosampler vials and caps • Helium (99.9999%) research grade with gas purifier • Hewlett-Packard or Agilent GC/MS system with autosampler—with all accessories and in working condition • High-purity ethyl acetate • Laboratory notebook • Personal protective equipment (gloves, safety glasses or goggles, and lab coat) • TO-4A samples • Varian VF5-MS with 10 m easy guard (0.25mm ID, 0.25 |_im film thickness, 30 m length) or similar DB5 type column 7 Procedure NOTE: It is important for all users to read the entire SOP before beginning any of the procedure and to ask questions if any of the instructions are unclear. 7.1 Sample preparation It is assumed that samples have been collected, extracted, cleaned up, and reduced to dryness prior to beginning these procedures. 1. Prepare fresh GC internal standard solution for all GC internal standards in ethyl acetate (150 pg/mL each TCmX, 13C6 d-HCH, and DCBP). This can be accomplished for a variety of final volumes, but the suggested method follows. ------- a. If individual internal standard solutions are available, remove them from freezer storage and allow them to reach room temperature. Otherwise, prepare a 3 |ag/mL solution of each individual internal standard by diluting 300 mL of 100 |ag/mL stock standard to 10 mL with ethyl acetate. ¦ Prepare a 150 pg/mL solution of all three internal standards by diluting 1.25 mL of each 3 |ag/mL solution to 25 mL with ethyl acetate. 2. Label standards with the solution name, concentration, solvent, date made, expiration date (6 months from date made), and preparers initials. 3. Pipet 1 mL of the 150 pg/mL internal standard solution into each sample flask. 4. Stopper and swirl the flask to remove any residues that might be sticking to the sides. 5. Remove the stopper and pipet the solution into a labeled autosampler vial and cap it. 6. Repeat steps 4 through 6 until all samples have been transferred to autosampler vials. 7.2 Calibration standard preparation NOTE: Prior to sample analysis for both GC and LC compounds on the same extracts, a decision must be made regarding two standards added to the samples. 13C, Atrazine and D10 Diazinon cannot be used simultaneously as a surrogate for GC compounds and as an internal standard for LC compounds. The addition of these compounds prior to extraction will impact the internal standard quantitation by LC. A suggested compromise is to select one of these compounds as a GC surrogate and the other as an LC internal standard. Table F2. Preparation of calibration standards 1. If calibration standard solutions are available, remove them from freezer storage and allow them to reach room temperature. 2. Otherwise, prepare six calibration standard solutions containing 36 target analytes, 4 surrogate standards, and 3 internal standards using Table E2. Solvent type and volume for six Florisil column eluants. See Table E2. Solvent type and volume for six Florisil column eluants for the identity of these compounds. These standards cover a concentration range of 50-300 pg/ mL. This procedure assumes that all stock solutions have a 100 |ag/mL concentration, and the final volume of the calibration solutions is 25 mL in each case. 3. Label standards with the solution name, concentration, solvent, date made, expiration date (6 months from date made), and preparer's initials. 4. After each standard is prepared or equilibrated, pipet at least 1 mL of each into a separate labeled autosampler vial. Include the standard's name, concentration date prepared, and preparer's initials on the label. 7.3 GC/MS instrument setup General operation and maintenance of the GC/MS system are not detailed here. The user will follow their laboratory's SOP for GC/MS analysis and manufacturer's recommendations using the specific instrument parameters for this analysis detailed below. Initial GC/MS setup • Ensure that the instrument is not leaking and is tuned. • Ensure that the two rinse vials are filled with ethyl acetate and the two waste vials are empty. Volume of stock Final Concentration Standard # Compound type (mL) (ng/mL or pg/mL) Target analytes 12.5 50 1 Surrogate standards 12.5 50 Internal standards 37.5 150 Target analytes 25 100 2 Surrogate standards 25 100 Internal standards 37.5 150 Target analytes 37.5 150 3 Surrogate standards 37.5 150 Internal standards 37.5 150 Target analytes 50 200 4 Surrogate standards 50 200 Internal standards 37.5 150 Target analytes 62.5 250 5 Surrogate standards 62.5 250 Internal standards 37.5 150 Target analytes 75 300 6 Surrogate standards 75 300 Internal standards 37.5 150 ------- • Use a Varian VF5-MS with 10 m easy guard (0.25 mm ID, 0.25 |_im film thickness, 30 m length) or similar DB5 type column. • Either create and save or load the acquisition method for this analysis. GC/MS method parameters are listed below. GC parameters oMax oven temp 350 °C : Equilibration time 3 0.5 min GC Temperature program o Initial temperature 50 °C hold 2 min oRamp 1 15 °C/minto 150 °C hold 0 min o Ramp 2 5 °C/min to 280 °C hold 0 min oRamp 3 15 °C/minto 300 °C hold 5 min o Total time 41 min Injection parameters : Injection: One sample wash; three sample pumps; 2-mL injection; no Solvent A rinses; three Solvent B rinses; Viscosity Delay 0; Plunger speed fast o Injector temp 230 °C o Injector pressure 18.69-psi helium in constant pressure mode; 1.6 mL/min; 40 cm/s MS parameters o Electron impact ionization mode o Quad temp 150 °C o Source temp 230 °C o Multiplier offset 400, Absolute mode off o Solvent delay 8 min o Selected ion monitoring—see Table E2. Solvent type and volume for six Florisil column eluants for ion windows o Transfer line temp 300 °C NOTE: Prior to sample analysis, the MS windows must be verified. Analyze a standard solution containing all 36 target analytes, 4 surrogate compounds, and 3 internal standard compounds in scan mode. Ensure that the retention times of the compounds and mass spectral windows are correlated to properly capture the eluting peaks. Slight adjustments may be needed after instrument maintenance or column clipping. 7.4 GC/MS calibration General operation and calibration of the GC/MS system are not detailed here. The user will follow their laboratory's SOP for calibration and quantitation utilizing the specific instrument parameters for this analysis detailed below. 1. The internal standard/analyte pairs are listed in Table E2. Solvent type and volume for six Florisil column eluants by superscript. Be sure to set up the calibration compound list so that the correct internal standard will be used for each analyte and surrogate compound. Calibration curves must have an R2 value greater than 0.90. 2. When preparing to analyze the samples and calibration standards, alternate them throughout the sequence to capture any instrument drift in time. Because the instrument is calibrated for each sample set, daily and continuing calibration standards are not necessary, but also may be included throughout the sample sequence. Table F3. Instrumental selected ion monitoring parameters and ion windows Group/start Retention Target Qualifier Backup Dwell tim< time (min) Compound time (min) m/z m/z m/z (ms) 1/8.0 Tet ra ch 1 o ro-m-xy 1 e n e 13.630 207 209 244/242 75 2,4-D methyl ester 13.851 199 234 175 75 2/14.0 Trifluralin 14.251 306 264 290 100 3/14.45 2,4-D ethyl ester 14.872 175 248 40 a-HCH1 15.005 183 109 181/219 40 Hexachlorobenzene 15.094 284 286 249 40 4/15.25 Dicloran (Botran) 15.449 206 208 40 Simazine 15.649 201 186 40 Atrazine 15.793 200 215 40 13C Atrazine 15.793 203 218 40 b-HCH1 15.871 183 181 40 Propazine 15.904 214 229 40 Lindane (g-HCH)1 16.115 183 109 40 5/16.25 D„„ Diazinon 10 16.296 183 314 75 Diazinon 16.426 179 137 75 Chlorothalonil 16.692 266 264 75 6/16.85 13C6 d-HCH 17.092 187 189 40 Methyl parathion 18.301 263 125 40 Alachlor 18.312 160 188 40 Heptachlor 18.534 272 274 40 Fenchlorphos (Ronnel) 18.656 285 287 125 40 F-4 ------- Group/start Retention Target Qualifier Backup Dwell tim< time (min) Compound time (min) m/z m/z m/z (ms) 7/19.0 Malathion 19.511 127 125 40 D10 Chlorpyrifos 19.577 324 99 40 Metolachlor 19.633 162 238 40 Chlorpyrifos 19.721 314 97 40 Aldrin 19.777 263 265 40 Dacthol 19.899 301 299 40 Ethyl parathion 20.021 291 139 40 8/20.6 Oxychlordane 21.175 185 387 45 Heptachlor epoxide B2 21.197 353 81 45 Heptachlor epoxide A2 21.342 353 81 45 Captan 21.590 79 149 45 Folpet 21.798 260 262 45 trans-Chlordane3 22.041 373 375 45 9/22.25 c/s-Chlordane3 22.496 373 375 237 80 trans-Nonach lor 22.596 409 407 237 80 10/22.95 p,p'- DDE 23.339 246 248 318 5 Dieldrin 23.483 263 277 79 75 11/23.9 o,p'- DDT4 24.925 235 237 165 100 p,p'- DDT4 26.196 235 237 165 100 12/27.0 Methoxychlor 28.221 227 228 152 100 13/29.0 Mi rex 29.842 272 274 237 100 14/31.5 DCBP 33.825 498 500 214 100 '"Compounds share fragmentation pattern. 3. Be sure to recap and archive the autosampler vials in a -20 °C freezer after they have been analyzed. 7.5 GC/MS quantitation NOTE: TO-4A samples that have been split for GC and LC analysis will have a concentration twice that calculated. Be sure to add a correction factor before reporting results. Sample calculation OC Verify that calculations are correct by manually deriving the answer for a portion of the data based on the following criteria. • Verifier will not be involved in data generation. • Ten percent of mathematical calculations are checked. • Calculations to be checked are randomly determined. • Initial or sign and date calculations that were verified. laboratory notebooks with tape so long as a line is drawn through the attached page and onto the notebook page with initials and date. 9 Quality Control and Quality Assurance QA/QC is discussed throughout this document, but additional parameters are listed below. Compare ongoing data quality checks with established performance criteria to determine if the results of analyses meet the performance characteristics of the method. At a minimum, one sampling blank (with air, no spiking solutions), one sample duplicate (with air, no spiking solutions), and one QA spike (no air, GC and LC spiking solutions added) are included for each sampling event and should be analyzed together. • Blank samples should not contain residues at a level higher than the lowest calibration standard. 8 Data and Records Management Data and records management issues are discussed throughout the procedure. Data will be recorded promptly, legibly, and in permanent ink in the logbook and in laboratory notebooks designated for each instrument and project. Electronic data files related to the project should be specified in the laboratory notebook. Electronic instrument data must be stored in duplicate at separate locations. This will be accomplished within 4 weeks of data collection or file creation. Compiled data may be printed and attached to • Replicate samples should agree within ±20%. • QA spike samples should have a recovery between 75% and 120%. • Surrogate compounds should have a recovery between 75% and 120%. • If any of these requirements are not met, related samples should be flagged as having QA measures outside of acceptable limits. Corrective actions for future samples should be investigated. • Calibration curves must have an R2 value greater than 0.90. F-5 ------- In addition to quantitative measures of comparison, analysts should evaluate chromatograms and instrument operation. Questions that should be asked include the following. • Are peaks symmetrical? Broad peaks and excessive tailing may indicate a leaky septum, a dirty injection liner, or a dirty column. Also, the column fittings may need to be tightened. • Is the response obtained comparable to the response from previous calibrations? Responses (area counts or peak height) should be within ±20% of previous analyses. • Are nontarget peaks present in calibration standard chromatograms? If so, this may indicate breakdown of one or more standards. A new set of calibration standards should be made. • Are interferences present in the blanks? This may indicate lab or solvent contamination. New standards and blanks should be prepared using fresh solvents. Additional research may be required to determine the source of interferences and eliminate them. • Immediately correct any significant peak tailing, leaks, changes in detector response, and laboratory contamination. • Recalibrate the instrument when the performance changes to the point that the calibration verification acceptance criteria cannot be achieved. In addition, significant maintenance activities or hardware changes also require recalibration. Tuning the GC/MS also requires recalibration. • If the RSD of the response factors is less than or equal to 20% over the calibration range, then linearity through the origin may be assumed, and the average calibration or response factor may be used to determine sample concentrations. 10 Waste Management Solid, liquid, and glass waste are disposed of in separate containers. Solvents and solids used in this procedure should be disposed according to health and safety regulations and with appropriate labeling and record keeping. Several compounds used in this method (aldrin, dieldrin, heptachlor, and methyl parathion DCBP) are to be disposed of separately with "P" coded waste or PCB waste. 11 References Agilent Technologies. Overview of the MSD Productivity ChemStation, 2003. Agilent Technologies. Agilent ChemStation, Understanding Your ChemStation, Part #G2070-91115, June 2003. F-6 ------- Appendix G Standard Operating Procedure: Analytical Methods for the Determination of Selected T0-4A Pesticides by Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS) 1 Method Summary In this procedure, sample analysis is performed via LC/MS/ MS operated with the turbo ion spray/atmospheric pressure ionization (API) source. Positive ionization is used for all pesticides listed in the method. Transition ion pairs and retention times are used to verify the identities of analytes. Isotopically labeled internal and surrogate standards are used for quantitation and quality control. This method is valid for samples containing between 50 and 250 ng of each analyte. 2 Scope and Application Although the SOP is somewhat specific to a particular EPA research situation, it can easily be used as a starting point for further development outside its original use. This SOP describes the method for detection and quantification of selected pesticides by liquid chromatography /tandem mass spectrometry (LC/MS/MS) using target transition ions to determine the presence of analytes in the sample extracts. The relevant pesticides are listed in Table E2. Solvent type and volume for six Florisil column eluants. The SOP is applicable to samples collected on XAD-2 media that lias been pre-cleaned under the relevant NERL SOP. This SOP assumes the following. • The instrument has been properly installed according to the manufacturer's specifications; accessories and attachments have been installed and verified as functioning correctly; and it is in its operational location and ready for general use. Table Gl. LC target analytes and internal standards • An autosampler is used with this system. • The operator has access to the manufacturer's instrument, accessory operating, software, and reference manuals and current laboratory SOPs; see Section 12.6 • The operator is trained and knowledgeable in the use of a LC system. • Internal standard calibration • The samples to be analyzed by this method have been collected, extracted, cleaned up, and reduced to dryness prior to final determination. 3 Personnel Qualifications This SOP is written for users who have experience operating a liquid chromatograph-tandem mass spectrometer (LC/ MS/MS) and a rotary evaporator, and are trained in keeping a laboratory notebook and the use of Microsoft Excel. The operator should have a background in science with laboratory experience, must be trained by experienced personnel before undertaking these techniques, and have completed the required laboratory safety training. The operator must be able to understand the information in the operating manuals and SOPs related to this procedure. 4 Health and Safety Standard laboratory protective clothing, gloves, and eye covering is required when performing chemical operations, such as aliquoting standard solutions into autosampler vials for analysis and recapping standard solutions and sample extracts vials after analysis for storage. LC target analytes Allethrind Atrazine3 Bendiocarb3 Carbaryl3 Carbofuran3 Chlortoluron3 Cinerin ld & llb Diazinonb Diuron3 Dicrotophos3 Fenvalerated Fluometuron3 Jasmolin ld & llb Linuron3 Monuron3 c/'s-Permethrinc fra/7s-Permethrind Propazine3 Propoxur (Baygon)3 Pyrethrin ld &llb Resmethrind Simazine3 Tebuthiuron3 LC internal standard compounds 13C3 Atrazine D10 Diazinon 13Cg c/'s-permethrin 13Cg frans-permethrin aTarget analytes using 13C3 Atrazine as an internal standard "Target analytes using D10 Diazinon as an internal standard Target analytes using 13C6 c/s-Permethrin as an internal standard "Target analytes using 13C6 frans-Permethrin as an internal standard G-l ------- 5 Definitions, Acronyms, and Abbreviations Extract. The sample extract that contains native target analytes, surrogate recovery standard, and internal standard Surrogate Recovery Standard (SRS): The compound used for QA/QC purposes to assess the extraction efficiency obtained for individual samples. The concentration of the SRS is determined by the expected concentration of the pesticide in each tissue sample. The SRS is spiked into the sample prior to extraction and quantified at the time of analysis. The SRS recovery indicates the extraction and recovery efficiency. Acceptable recovery of the SRS is 100% ± 20%. If recovery of the SRS is outside of the acceptable limits, the sample tissue is reanalyzed. The SRS standard(s) to be used for all analysis will be determined by the chemist performing the analysis. Internal Standard (IS): The compound is added to sample extracts prior to LC/MS/MS analysis. The ratio of the instrument response from the analyte relative to the response from the corresponding IS is compared to ratios obtained for calibration curve solutions, where the IS level is fixed and the analyte levels varied. The IS is used to correct for minor run-to-run differences in LC injection, chromatographic behavior, and MS ionization efficiency. The IS to be used for all analysis is determined by its ionization mode and its retention time relative to the analyte of interest (see Table E2. Solvent type and volume for six Florisil column eluants). The included internal standards are 13C6 c/.v-pcrmcthrin. 13C6 /nmv-pcrmcthrin. D diazinon, and 13C6 atrazine. Acquisition Method: Under Analyst Software, the LC/MS/MS conditions used to analyze one or more samples Acquisition Batch: Under Analyst Software, a group name assigned to a set of vials in the autosampler tray so that all samples may be analyzed in order under conditions specified in the method Quantitation Method: Under Analyst Software, the method used to define the integration parameters for a set of samples. This method also defines ion pairs that are IS(s) and assigns analytes to the appropriate IS. |llL microliter CE collision energy CXP collision excitation potential DP declustering potential HPLC high-performance liquid chromatography . . liquid chromatograph/mass spectrometer/ mass spectrometer min minute mM millimolar mm millimeter SOP standard operating procedure T retention time r V volt Quantitation Wizard: Under Analyst Software, the method used to define whether a sample is a standard, blank, unknown, or quality control. Also used to assign concentrations to the standards so that concentrations of the unknown samples may be calculated. 6 Equipment and Supplies • Agilent 1100 high-pressure liquid chromatography system coupled with an Applied Biosystems API 4000 triple quadrupole mass spectrometer • PC with Analyst Software and printer • Analytical Column—Zorbax C18, 150 x 3 mm • 5 mM ammonium acetate in water • Methanol (pesticide grade or better) 7 Procedures LC/MS/MS analysis General operation and maintenance of the LC/MS/MS system are not detailed here. The user will follow their laboratory's SOP for LC/MS/MS and manufacturer's recommendations using the specific instrument parameters for this analysis detailed below. 7.1 Initial LC/MS/MS setup The column is installed in the LC separations module and the mobile phase flow rate is set. LC Parameters • Mobile-phase program: 5 mM ammonium acetate in water (A) and 100% MeOH (B) • Preinjectionequilibration: 5 mint®, 10%A: 90%B • Mobile-phase ramp: 7 min to 4% A: 96% B • Final mobile phase: 1 min a 4% A: 96% B • Column flow rate: 400 |iL/min • Total run time: 8 min • Column oven temp: 30 °C • Injection volume: 2.0 (iL MS/MS Parameters • Positive ion mode • Runtime: 8 min • Column flow rate: 400 |iL/min • Curtain gas setting: 40 • GS1 setting: 40 • GS2 setting: 407 • IS voltage: 5500 • Temperature: 400 °C • Mass spectrometer source: electrospray, positive ion mode • Exit potential: 10 V • Collision cell gas setting: 2 G-2 ------- Table G2. Analyte-specific LC/MS/MS parameters Compound T (min) DP CE Allethrin 3.62 66.0 15.0 Fenvalerate 4.80 51.0 23.0 c/'s-Permethrin 6.89 46.0 25.0 frans-Permethrin 5.94 46.0 25.0 Resmethrin 5.83 41.0 59.0 Pyrethrin I 4.42 66.0 13.0 Pyrethrin II 2.85 36.0 19.0 Jasmolin I 5.35 66.0 15.0 Jasmolin II 3.28 71.0 15.0 Cinerin I 4.45 66.0 25.0 Cinerin II 2.86 71.0 31.0 Bendiocarb 1.82 61.0 13.0 Carbaryl 1.88 31.0 17.0 Propoxur (Baygon) 1.83 51.0 19.0 Carbofuran 1.84 61.0 29.0 Diazinon 2.71 71.0 31.0 Dichrotophos 1.65 61.0 17.0 Chlorotoluron 2.01 66.0 37.0 Diuron 2.01 61.0 35.0 Fluometuron 1.89 66.0 41.0 Linuron 2.21 61.0 25.0 Monuron 1.89 61.0 27.0 Tebuthiuron 1.98 61.0 25.0 Simazine 1.97 66.0 27.0 Propazine 2.26 76.0 31.0 Atrazine 2.09 71.0 25.0 13Cg c/'s-Permethrin 6.88 46.0 23.0 13Cg frans-Permethrin 5.92 46.0 23.0 D10 Diazinon 2.67 76.0 31.0 13C3 Atrazine 2.09 71.0 25.0 • Compound-specific parameters: see Table G2. Analyte- specific LC/MS/MS parameters 7.2 LC/MS/MS internal standard calibration 1. Area counts for each analyte are determined automatically using the Analyst software provided with the API 4000. Each chromatogram is reviewed by the operator to insure proper and consistent integration, and manual correction of inappropriate integrations is performed if necessary. 2. A calibration curve for each analyte will be constructed with a minimum of five concentrations that encompass the relevant calibration range. The internal standards are present at equal concentrations in all samples CXP Transition ions 8.0 303.24/135.31 12.0 437.17/167.24 12.0 408.14/183.24 12.0 408.14/183.24 8.0 356.16/128.17 10.0 329.24/161.35 10.0 390.21/161.41 10.0 331.24/163.53 10.0 375.21/163.40 6.0 317.24/107.13 6.0 361.18/107.16 6.0 224.24/109.09 8.0 219.26/145.04 6.0 210.27/111.06 10.0 222.23/165.31 10.0 305.17/169.27 6.0 238.20/112.02 4.0 213.22/71.85 12.0 233.16/71.82 4.0 233.16/71.82 10.0 249.13/160.09 12.0 199.20/71.84 10.0 229.23/172.20 8.0 202.20/132.01 10.0 230.21/146.00 10.0 216.27/174.05 12.0 414.15/189.40 12.0 414.15/189.40 10.0 315.21/154.23 12.0 219.27/177.02 and standards. Details regarding the specifics of each standard, including actual concentration, will be recorded in the appropriate laboratory notebook. 3. The calibration curve will be generated using the theoretical analyte concentration versus the relative area (analyte area/IS area). The calibration curve may not be forced through the origin. The coefficient of determination (R2) of the curves must be >0.99. 7.3 LC/MS/MS analysis sequence 1. A solvent blank is analyzed first to verify that the LC/ MS/MS system is clean of carry-over or artifacts. The acceptance criterion is an analyte quantitation ion area that is two times lower than the area of the lowest level standard in the previous set. G-3 ------- 2. Five calibration standards are analyzed, followed by up to five sample extracts. 3. A mid-level calibration standard (from the calibration curve standards) is analyzed, followed by a blank. 4. The sample extract, mid-level standard, blank sequence is repeated until all samples in a set have been analyzed. After the final sample has been run the complete set of calibration standards will be rerun. In this way, the calibration curve of standards reflects the condition of the instrument while samples are being analyzed. The percent relative error for recalculation of each calibration standard (including the mid-level control standard) against the initial curve must be <25%. 7.4 LC/MS/MS data processing NOTE: TO-4A samples that have been split for GC and LC analysis will have a concentration twice that calculated. Be sure to add a correction factor before reporting results. 1. A calibration curve will be generated for each analyte and its respective internal standard from the results of the standard analyses. Each data file will be reviewed to ensure that the identification and integration of quantified target peaks are correct. 2. Analyte identification will be based on a retention time of ±0.2 min relative to the retention time of the two standards that bracket the sample in the LC/MS/MS run order. 3. The instrument software will calculate concentration of the analytes and surrogates in the sample based on the calibration curve. A quantitation report will be generated for each sample or standard. 7.5 Extract storage Extracts are stored protected from light at -20 °C except during analysis. 8 Data and Records Management • All operations, and instrument settings are electronically stored in the instrument's Analyst software on the computer used to control the LC/MS/MS. • All original analytical results are located in specific study folders identified as projects. For any sample set, all files (acquisition quantitation etc.) associated with that sample set will have identical names except for the file extension. In addition to other descriptors, all file names will include the actual date of sample analysis. • All compiled data will be reviewed by the analyst. For each analysis set, the calibration curve, results, and analytical sequence will be copied into an excel spreadsheet to include the following information. o Data ID/sample type o Notebook number and pages o Date extracted o Analyst name o Calibration standards concentration o Surrogate standards concentration o Internal standards concentration o Spike concentration o Comments o Sample list and location of each sample in autosampler tray • All data files (to include Analyst files and Excel spreadsheets) are stored on disks for permanent record. The disks are stored permanently in the LC/MS/MS laboratory computer as part of the LC/MS/MS laboratory records. Periodically, these files will be copied and backed up to an additional storage media. This backup will maintain the integrity of the file structure. • Final calculations of the data are performed and/or recorded in the study database and are a responsibility of the analyst. 9 Quality Control and Quality Assurance • The absolute response levels for the internal standard will be recorded for each analysis. If IS areas decrease throughout a sample set or if a difference is observed in the area of the IS in samples and in standards, but the SRS recoveries in samples remain within the acceptance range, then no action will be taken. If IS areas decrease or if a difference is observed in the area of the IS in samples and in standards, and the SRS recoveries in samples do not remain within the acceptance range, then corrective action will be taken. These actions will include cleaning the LC/MS/MS source, cleaning/replacing the ion probe capillary, changing the HPLC precolumn, cleaning the HPLC column, and/or repreparing the mobile phase and reanalyzing the a subset of the samples or the entire sample set. • Samples will be reanalyzed when the calibration curve data cannot be fit to a first-order equation with fit parameter R2 > 0.99 or when the recalculation of the standards against the curve does not meet the tolerances set in sections and . Corrective action, as listed in this section, will be undertaken before samples are reanalyzed. • Surrogate recovery values of 70% to 130% in the actual samples will be deemed acceptable, and no correction to the data will be made. For recoveries less than the minimum goal, the data will be flagged. For recoveries greater than the maximum goal, the concentration of the surrogate spiking solution will be checked against a calibration curve to determine whether inadvertent solvent loss has resulted in higher spike levels. 10 Waste Management Solid, liquid, and glass waste are disposed of in separate containers. Solvents and solids used in this procedure should be disposed according to health and safety regulations and with appropriate labeling and record keeping. 11 References Not applicable. ------- ------- *>EPA 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 ------- |