Dioxin analysis in water by Isotope Dilution using Triple Quadrupole GC/MS Presentation Title 11/28/2018 Confidentiality label Regulatory statement (if applicable)) Diana Wong, Ph.D. GC/MS Scientist National Environmental Monitoring Conference New Orleans, Louisiana August 6, 2018

Overview Method Introduction and Project Outline Presentation Title 11/28/2018 Confidentiality label Regulatory statement (if applicable)) Current EPA Method 1613 Revision B Requires GC/HRMS to achieve a high resolution of ≥10,000 Performance based method Investigate GC/MS/MS for dioxin analysis per Method 1613B Replace HRMS with MS/MS to simplify analysis and cost of operation/maintenance Goal Isomer specificity Calibration Linearity Calibration Verification (VER) Relative Retention Time Initial Precision and Accuracy (IPR) Instrument Detection Limit (IDL) Analysis of spiked real water sample Equivalency Requirements

Nomenclature and Isomers Presentation Title 11/28/2018 Confidentiality label Regulatory statement (if applicable)) Dioxins Polychlorinated dibenzodioxin (CDDs) Furans Polychlorinated dibenzofurans (CDFs) Cl Atoms CDD Isomers CDF Isomers 1 2 4 10 16 3 14 28 22 38 5 6 7 8 Total 75 135 cv Cl Cl cv Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl 2,3,7,8 – TCDD (2,3,7,8-Tetrachlorinated dibenzodioxin) 2,3,4,7,8 – PCDF (2,3,4,7,8-Pentachlorinated dibenzofurans) 17 toxic isomer 75 total CDD isomers 135 total CDF isomers

Toxic Equivalent Factors (TEF) Presentation Title 11/28/2018 17 Toxic CDDs/CDFs Investigated Confidentiality label Regulatory statement (if applicable)) Toxic CDDs I-TEF WHO2005-TEF 2,3,7,8-TCDD 1 1,2,3,7,8-PCDD 0.5 1,2,3,4,7,8-HxCDD 0.1 1,2,3,6,7,8-HxCDD 1,2,3,7,8,9-HxCDD 1,2,3,4,6,7,8-HpCDD 0.01 1,2,3,4,6,7,8,9-OCDD 0.001 0.0003 Toxic CDFs I-TEF WHO2005-TEF 2,3,7,8-TCDF 0.1 1,2,3,7,8-PCDF 0.05 0.03 2,3,4,7,8-PCDF 0.5 0.3 1,2,3,4,7,8-HxCDF 1,2,3,6,7,8-HxCDF 1,2,3,7,8,9-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,4,6,7,8-HpCDF 0.01 1,2,3,4,7,8,9-HpCDF 1,2,3,4,6,7,8,9-OCDF 0.001 0.0003 TEQ = 𝒊=𝟏 𝟏𝟕 [𝑪𝑫𝑫/𝑪𝑫𝑭 ] 𝒊 ( 𝒏𝒈 𝑳 )× 𝑻𝑬𝑭 𝒊 ( 𝑻𝑬𝑸 𝒏𝒈 𝑳 )

EPA Method 1613B Sample preparation Presentation Title 11/28/2018 Confidentiality label Regulatory statement (if applicable)) 13C Internal Standard Clean-up standard Injection standard Filtration (~1 L water) Soxlet >16 h (filter paper) Liquid/Liquid Extraction (filtrate) Sample Clean-up (silica, alumina, florisil, or carbon column) GC/HRMS vs GC/MS/MS EPA 1613B is a performance based method

GC/MS/MS System Parameters 11/28/2018 2mm Dimpled, splitless, UI Inlet liner MMI Inlet  MSD Constant Flow Flow 1.1 mL/min GC Parameters 100 °C (2 min) 30 °C/min to 220 °C (16 min) 2 °C/min to 240 °C (5 min) 5 °C/min to 270 °C (4 min) 15 °C/min to 330 °C (6 min) Oven program Automatic Liquid Sampler Pulsed Splitless 1 µL injection 62 °C (0.31 min)  ramp to 330 °C at 600 °C/min Multimode Inlet (MMI) 7890 GC 7010 EI MS/MS DB-5ms UI 60m x 0.25mm ID 0.25 µm film Helium Carrier Gas HES Source Source: 300 °C Quads: 150 °C Transfer Line: 350 °C He Quench Gas: 4 mL/min N2 Collision Gas: 1.5 mL/min Oven

GC Oven Temperature Program Optimization Presentation Title 11/28/2018 Slow Oven Program for Isomeric Separation Confidentiality label Regulatory statement (if applicable)) Optimal Oven Temperature Program >25% valley First eluted 1368-TCDD <25% valley Rate Oven Temperature Hold time (Initial) 100 °C 2 min 30 °C/min 220 °C 2 °C/min 250 °C 10 min 260 °C 5 °C/min 280 °C 15 °C/min 330 °C 5 min Rate Oven Temperature Hold time (Initial) 100 °C 2 min 30 °C/min 220 °C 16 min 2 °C/min 240 °C 5 min 5 °C/min 270 °C 4 min 15 °C/min 330 °C 6 min

Order of Isomer Specificity Presentation Title 11/28/2018 Percent Valley must be less than 25% between toxic 2378-TCDD and the closest eluted isomers Confidentiality label Regulatory statement (if applicable)) DB-5 Column EPA Method 1613B DB-5ms UI Column Current Study 1,2,3,9-TCDD 2,3,7,8-TCDD 1,2,3,7/1,2,3,8-TCDD <25% Valley <25% Valley (EPA Method 1613B, 1994) (Fishman et al., 2004 and 2011; Wilken et al., 2008)

MRM Transition and Collision Energy Optimization Presentation Title 11/28/2018 RRT, Ion Ratios, and S/N meet Method 1613B Criteria Confidentiality label Regulatory statement (if applicable)) Calibration Level 3 Method 1613B Criteria 13C-2,3,7,8-TCDD 331.9267.9 CE 26 333.9269.9 CE 26 RT: 31.77 min 2,3,7,8-TCDD 319.9256.9 CE 26 321.9258.9 CE 26 RT: 31.80 min m/z Two transitions RRT = 1.001 (Criteria 0.999-1.002) Method 1613B Limits Relative Retention Time (RRT) QC limits of ±15% Ion Abundance Ratio 2378-TCDD Ion Ratio = 104.8 Criteria (88.9-120.2) 13C-2378-TCDD Ion Ratio = 106.1 Criteria (90.2-122.0) Sample >2.5 Calibration >10 Signal to Noise (S/N) s/n 543 s/n 2569

Retention Time Window Defining Solution Presentation Title 11/28/2018 Method 1613B requires all isomers to fall within first and last eluters Confidentiality label Regulatory statement (if applicable)) 1,2,3,7,8,9 - EPA Method 1613B (1994): Window Defining Solution defines the beginning (first eluted) and ending (last eluted) retention times for dioxin and furan isomers to demonstrate isomer specificity. Standards must contain compounds listed in this order

Tetrachlorinated dibenzodioxins (TCDD) Presentation Title 11/28/2018 Peaks correlate and are well separate on GC/MS/MS and GC/HRMS Confidentiality label Regulatory statement (if applicable)) GC/MS/MS Abundance 321.9258.9 Toxic 2378-TCDD First eluted 1368-TCDD 1239-TCDD Last eluted 1289-TCDD 𝑥+𝑦=4 22 isomers 1 toxic <25% valley 321.88-321.91 GC/HRMS Abundance First eluted 1368-TCDD 1239-TCDD Toxic 2378-TCDD Last eluted 1289-TCDD <25% valley

Tetrachlorinated dibenzofurans (TCDFs) Presentation Title 11/28/2018 Peaks correlate and are well separate on GC/MS/MS and GC/HRMS Confidentiality label Regulatory statement (if applicable)) 305.9242.9 GC/MS/MS Abundance First eluted 1368-TCDF Toxic 2378-TCDF Last eluted 1289-TCDF 𝑥+𝑦=4 38 isomers 1 toxic Toxic 2378-TCDF 305.88-305.91 GC/HRMS Abundance First eluted 1368-TCDF Last eluted 1289-TCDF

Hexachlorinated dibenzofurans (HxCDFs) Presentation Title 11/28/2018 Peaks correlate and are well separated on GC/MS/MS and GC/HRMS Confidentiality label Regulatory statement (if applicable)) Last eluted 123489-HxCDF Toxic 123789-HxCDF Split peaks between toxic and non-toxic HxCDF GC/MS/MS Abundance 373.8310.9 First eluted 123468-HxCDF Toxic 123478-HxCDF Toxic 123678-HxCDF Toxic 234678-HxCDF 373.80-373.84 Toxic 123678-HxCDF GC/HRMS Abundance Toxic 234678-HxCDF Last eluted 123489-HxCDF 𝑥+𝑦=6 16 isomers 4 toxic First eluted 123468-HxCDF Toxic 123478-HxCDF Toxic 123789-HxCDF

Hexachlorinated dibenzodioxins (HxCDDs) Presentation Title 11/28/2018 Peaks correlate and are well separate on GC/MS/MS and GC/HRMS Confidentiality label Regulatory statement (if applicable)) GC/MS/MS Abundance 391.8328.8 First eluted 124679-HxCDD Toxic 123678-HxCDD Last eluted and toxic 123789-HxCDD Toxic 123478-HxCDD 𝑥+𝑦=6 10 isomers 3 toxic 391.79-391.83 Toxic 123678-HxCDD GC/HRMS Abundance First eluted 124679-HxCDD Last eluted and toxic 123789-HxCDD Toxic 123478-HxCDD

Establishing Method Equivalency Presentation Title 11/28/2018 Confidentiality label Regulatory statement (if applicable)) Calibration linearity Calibration verification Relative retention time Initial precision and recovery Ongoing precision and recovery Labeled compound recovery Method detection limit demonstration (Instrument detection limit) Performed Analysis of a performance testing (PT) on real samples Ongoing

Calibration (CAL), Linear Range, and Relative Retention Time Presentation Title 11/28/2018 Met Method 1613B criteria Confidentiality label Regulatory statement (if applicable)) Level Cal (ppt) L1 200 L2 500 L3 1000 L4 4000 L5 10000 L6 50000 L7 250000 L8 1000000 L9 2500000 Analyte RF (Avg) (CS1) RF Difference (CS1-Avg) RF RSD S/N RRT 1613B RRT Criteria Pass/Fail 2378-TCDD 1.123 1.004 -11% 6 25 1.002 0.999-1.002 Pass 2378-TCDF 0.97 0.943 -3% 2.9 50 1.001 0.999-1.003 12378-PCDD 0.985 0.994 1% 3.5 42 12378-PCDF 0.991 1.025 3% 2.8 54 23478-PCDF 1.007 0.997 -1% 2.1 63 1.000 123478-HxCDD 0.999 4.2 21 0.999-1.001 123478-HxCDF 0.924 0.921 0% 4.4 33 0.998-1.004 123678-HxCDD 0.929 0.917 3.6 1.000-1.019 123678-HxCDF 0.908 0.877 4.5 43 123789-HxCDD 1.027 5.3 0.997-1.005 123789-HxCDF 0.912 0.902 5.2 38 234678-HxCDF 0.983 2% 4.1 48 1234678-HpCDD 1.008 1.033 4 83 1234678-HpCDF 92 1234789-HpCDF 0.948 5% 90 OCDD 1.056 1.040 2.4 150 OCDF 0.913 0.940 148 0.999-1.008 Note: RF = 𝑨 𝟐,𝟑,𝟕,𝟖−𝐓𝐂𝐃𝐃,𝑺𝒕𝒅 𝑨 𝟏𝟑𝐂,𝑺𝒕𝒅 × 𝑴 𝟏𝟑𝐂,𝑺𝒕𝒅 (𝒏𝒈) 𝑴 𝟐,𝟑,𝟕,𝟖−𝐓𝐂𝐃𝐃,𝑺𝒕𝒅 (𝒏𝒈) CS1 Range RF difference -11% to 5% RF RSD: 2.1 to 6 S/N: 21 to 15 EPA 1613B Cal Range 1613B Criteria: RF Difference < 15%; Avg RSD < 10%; S/N (CS1) > 10; Relative Retention Time (RRT) must meet RRT criteria

Verification Standard Recoveries Presentation Title 11/28/2018 Calibration is verified and passed Method1613B criteria Confidentiality label Regulatory statement (if applicable))

Initial Precision and Accuracy (IPR) Presentation Title 11/28/2018 All native and labeled recoveries are within Method 1613B Criteria Confidentiality label Regulatory statement (if applicable))

Spiked Water Sample (GC/HRMS vs GC/MS/MS) Presentation Title 11/28/2018 Similar concentration detected in water sample on both systems! Confidentiality label Regulatory statement (if applicable)) 1234678-H7CDD

Low working range and sensitivity Presentation Title 11/28/2018 50 femtogram of 2378-TCDD can be detected by GC/MS/MS Confidentiality label Regulatory statement (if applicable)) Primary Native 2378-TCDD S/N = 5.8 Secondary Native 2378-TCDD S/N = 4.6 Method 1613B Criteria > 2.5 signal to noise ratio The relative ion intensities is within 15% difference to the calibration average

Isomer specificity and Separation Passed Method1613B Performance Summary Presentation Title 11/28/2018 GC/MS/MS meets Method 1613B Criteria in water sample Confidentiality label Regulatory statement (if applicable)) HRMS to MS/MS Good correlation Detected similar concentration in spiked water samples Isomer specificity and Separation < 25% valley between toxic 2378-TCDD and closest isomer Isomer Specificity observed Analytes elute within the defined retention time window Passed Method1613B Performance RF RSD CC S/N RRT VER IPR

Acknowledgements Project Collaborators Hui Lin, Ph.D. Jessica Betz Jim Dunn Greg Martin David Somerville, Ph.D. Brian Pike Chuck Sueper Agustin Pierri Agilent Technologies Craig Marvin Dale Walker Tarun Anumol, Ph.D. Jeff Nutter Matt Leyden Tom Barrett 7890 Gas Chromatograph 7010 Triple Quadrupole Mass Spectrometer

Thank you NEMC! Diana Wong, Ph.D. GC/MS Scientist diana.wong@agilent.com Agilent Booth #16 and #17