Analysis of Per/Polyfluoroalkyl Substances (PFAS) in Drinking Water Using the Agilent Ultivo Triple Quadrupole LC/MS Presentation Title 11/29/2018 Confidentiality Label Terri Sosienski, Ph.D. LC/MS Applications Scientist Agilent Technologies, Santa Clara, CA Tarun Anumol, Ph.D. Agilent Technologies, Wilmington, DE Dorothy Yang, Ph.D.

Per/Poly Fluoroalkyl Substances (PFAS) in Water Presentation Title 11/29/201811/29/2018 Confidentiality Label PFASs widely used and can be sourced from: Fire-fighting foams Non-stick cookware Food packaging Manufacturing USEPA drinking water health advisories for PFOA and PFOS 70 ng/L in drinking water Individual US State guidelines 13 – 32 ng/L PFOS and PFOA in water. US EPA Method 537 14 PFAS compounds PFAS are very common compounds which aren’t limited to their use as surfactants fire retardants, and even in non-stick cookware. They are consistently found in environmental waters, as they are very stable compounds which do not break down nor degrade easily. There are some regulatory guidelines for PFOA and PFOS levels in drinking water set at 70 nanograms per liter 11/29/2018 NEMC 2018

Overview Presentation Title 11/29/201811/29/2018 Confidentiality Label Two methods for detecting PFAS compounds in drinking water using the Ultivo LC/TQ Method 1 17 PFAS Compounds EPA 537 sample preparation Method 2 51 Trace Organic Contaminants Including 10 PFAS compounds Online SPE PFAS are very common compounds which aren’t limited to their use as surfactants fire retardants, and even in non-stick cookware. They are consistently found in environmental waters, as they are very stable compounds which do not break down nor degrade easily. There are some regulatory guidelines for PFOA and PFOS levels in drinking water set at 70 nanograms per liter 11/29/2018 NEMC 2018

The New Ultivo Triple Quad LC/MS Presentation Title 11/29/2018 Confidentiality Label 11/29/2018 NEMC 2018

Ultivo: Space-saving, Sensitive, Robust & Powerful Presentation Title 11/29/2018 Confidentiality Label HED Detector (accessible in stack) VacShield (no-vent maintenance) Vortex Collision Cell (twisted hexapole) Cyclone Ion Optics (dual twisted hexapoles) Agilent Jet Stream Ion Source Hyperbolic Quadrupoles Virtual DC Filters (ultra thin) 11/29/2018 NEMC 2018

Ultivo – Reduced Downtime Presentation Title 11/29/2018 Confidentiality Label Fast Autotune <10 minutes! Vac Shield: Change capillary without venting Accessible maintenance features Some of the other innovations included in ULTIVO were made to reduce the instrument downtime. We have a new swarm tune that can autotuned the instrument in less than 10 minutes! Our new vac shield technology allows the user to change the capillary without venting the instrument. Here we have a rendering of the ion injector installed with the vac shield open, and the ion injector removed with the vac shield closed, maintaining the vacuum pressure in the instrument. Since Ultivo is designed to be in the LC stack, we needed to make sure that the maintenance features were accessible from the sides of the instrument. So we have pictures our easy change detector assembly, which simply pulls out from the side panel of the instrument. New Vac Shield Technology Easy Change Detector Assembly 11/29/2018 NEMC 2018

Software Features for High Throughput Presentation Title 11/29/2018 Confidentiality Label Real time diagnostics Capillary health Spray stability Source Cleanliness Clearer callouts for entry errors We didn’t stop with hardware, we also made some changes to our software because it’s the way that users actually interact with the instrument. Our acquisition software has a fresh look and feel We also incorporated clearer callouts so you know when there was an entry error, so errors can be fixed quickly. We now have real time diagnostics which let the user know the health of the capillary, the stability of the spray and the cleanliness of the source. We also made changes to our quantitative analysis software and we are introducing quant my way, which is a customizable version of our quant software. This version allows an administrator to create different versions or “flavors” of quant for a specific method or workflow, ultimately eliminating errors and streamlining the analysis process. 11/29/2018 NEMC 2018

Software Features for High Throughput Presentation Title 11/29/2018 Confidentiality Label Quant-My-Way: customize the user interface for data analysis. We didn’t stop with hardware, we also made some changes to our software because it’s the way that users actually interact with the instrument. Our acquisition software has a fresh look and feel We also incorporated clearer callouts so you know when there was an entry error, so errors can be fixed quickly. We now have real time diagnostics which let the user know the health of the capillary, the stability of the spray and the cleanliness of the source. We also made changes to our quantitative analysis software and we are introducing quant my way, which is a customizable version of our quant software. This version allows an administrator to create different versions or “flavors” of quant for a specific method or workflow, ultimately eliminating errors and streamlining the analysis process. Faster, easier, error-free data analysis, review and reporting by novice operators. 11/29/2018 NEMC 2018

Quant-My-Way Presentation Title 11/29/2018 Confidentiality Label We didn’t stop with hardware, we also made some changes to our software because it’s the way that users actually interact with the instrument. Our acquisition software has a fresh look and feel We also incorporated clearer callouts so you know when there was an entry error, so errors can be fixed quickly. We now have real time diagnostics which let the user know the health of the capillary, the stability of the spray and the cleanliness of the source. We also made changes to our quantitative analysis software and we are introducing quant my way, which is a customizable version of our quant software. This version allows an administrator to create different versions or “flavors” of quant for a specific method or workflow, ultimately eliminating errors and streamlining the analysis process. 11/29/2018 NEMC 2018

Presentation Title 11/29/2018 Confidentiality Label Analysis of Per/Polyfluoroalkyl Substances (PFAS) in Drinking Water Using the Agilent Ultivo Triple Quadrupole LC/MS Application Note: 5991-8969EN We wanted to show how great Ultivo will be for environmental applications, and how detection limits can be enhanced with agilents innovate sample prep and automated sample pre-concentration products. 11/29/2018 NEMC 2018

Analysis Method 17 PFAS compounds in drinking water. Presentation Title 11/29/201811/29/2018 Confidentiality Label 17 PFAS compounds in drinking water. All 14 from USEPA 537 method. Additional compounds PFDoA, PFPeA, and PFBA Full list included in Application note 5991-8969EN 250mL water samples Extracted with Agilent Weak Anion Exchange (WAX) cartridges. Similar to US EPA 537 Method Conditions Extracts concentrated to dryness with nitrogen Reconstituted w/ 96:4 (v/v) Methanol/Water IS added For this application we analyzed 17 pfas in drinking water, which included all 14 from the USEPA 537 method. 250 mL of water was extracted using agilent extraction kits. The total runtime for the method was 19 minutes using the agilent 1290 Infinity II HPLC, and the Ultivo Triple Quad MS. 11/29/2018 NEMC 2018

Instrumentation 1290 Infinity II LC Analytical Column: Zorbax Eclipse plus C18 3.0x50mm, 1.8µm Mobile Phase A:H2O and Mobile Phase B: 95:5 MeOH/H2O, 5mM Ammonium Acetate Flow Rate: 0.4 ml/min Injection Volume: 5µL Ultivo Triple Quadrupole MS in negative ionization mode. Dynamic MRM 11/29/2018 NEMC 2018

Overcoming Potential Contamination Presentation Title 11/29/201811/29/2018 Confidentiality Label PFASs could potentially be used in LC and component manufacturing processes Changes to the system: Delay Column: Zorbax Eclipse plus C18 4.6x50mm, 3.5µm Vials: 1 mL polypropylene vials Caps: Snap top polypropylene caps For this application we analyzed 17 pfas in drinking water, which included all 14 from the USEPA 537 method. 250 mL of water was extracted using agilent extraction kits. The total runtime for the method was 19 minutes using the agilent 1290 Infinity II HPLC, and the Ultivo Triple Quad MS. 11/29/2018 NEMC 2018

PFAS Chromatography 1 ppb standard solution of PFAS in water. Good chromatographic separation and S/N with 1 ppb standard solution. EPA Method 537  35 to 37 min analytical run time This method is less than 22 minutes. 11/29/2018 NEMC 2018

Recovery of PFAS Compounds in Water Presentation Title 11/29/201811/29/2018 Confidentiality Label Recovery 70-120% for all compounds at 1 µg/L and 5 µg/L spiking levels RSD%: 0.3-10.8% for all compounds at both spiking levels (5 replicates). We achieved recoveries of 0-120% for all compounds at both 1ug/l and 5ug.l levels Along with excellent sensitivity and linearity at a range of 0.1-20ug/L with R2 values greater than 0.99 Great precision, with rsd% between 0.3 and 10.8% for all compounds in the method. 11/29/2018 NEMC 2018

Linearity of PFAS Compounds Excellent Linearity for 0.2 – 100 µg/L calibration curves. R2 > 0.99 for all compounds, based on average of 3 calibration curves run over 1 month period. Linearity over 1 month highly stable. Compound R2 CCV RSD (%) PFBA 0.9965 2.1 PFPeA 0.9996 2.7 PFBS 0.9964 3.1 PFHxA 0.9988 3.0 PFHpA 0.9985 3.4 PFHxS 0.9995 1.7 PFOA 0.9971 1.2 PFOS 0.9991 1.9 PFNA 0.9811 3.7 PFDA 0.9941 3.6 N-MeFOSAA 0.9999 PFDS 2.3 PFUdA 0.9940 N-EtFOSAA 5.2 PFDoA 0.9974 PFTrDA 0.9984 3.5 PFTeDA 0.9986 3.2 11/29/2018 NEMC 2018

Continuous Calibration Verification Calibration sample run after every ten samples - required by US EPA 537. CCV samples at 1 ppb level were calculated for a 26 hour batch. 94% of Analytes have less than 5% RSD at 1ppb. Very stable over 26 hour experiment.   11/29/2018 NEMC 2018

Analysis of Real Water Samples Presentation Title 11/29/2018 Confidentiality label Regulatory statement (if applicable)) PFBS, PFOS, PFOA, PFNA were detected at 10, 4, 0.6, and 8, ng/L Drinking water sample sourced from California 4 PFAS compounds detected 11/29/2018 NEMC 2018

Conclusions Presentation Title 11/29/2018 Confidentiality label Regulatory statement (if applicable)) Ultivo can exceed the sensitivity requirements for PFAS detection in drinking water. This method is robust both in standard curve and check sample evaluations. Shorter runtime and less sample volume than traditional methods. PFBS, PFOS, PFOA, PFNA were detected at 10, 4, 0.6, and 8, ng/L 11/29/2018 NEMC 2018

Presentation Title 11/29/2018 Confidentiality Label Sensitive Detection of Trace Organic Contaminants in Water Using Agilent 1290 Infinity FlexCube Online SPE and Agilent Ultivo Triple Quadrupole LC/MS Application Note: 5991-8767EN We wanted to show how great Ultivo will be for environmental applications, and how detection limits can be enhanced with agilents innovate sample prep and automated sample pre-concentration products. 11/29/2018 NEMC 2018

Ultivo: Even more powerful with Flex Cube Online SPE Presentation Title 11/29/2018 Confidentiality Label Automated sample pre-concentration Fits neatly into LC Stack Operated using Agilent MassHunter Software Many phases compatible with Online SPE format With this method that we are about to present, we are using the Agilent flex cube online SPE, which is a tool that we use to automatically preconcentrate our sample. The Flex cube fits right into the LC stack and is operated using our agilent masshunter software so it fits seamlessly into the LC/MS process. We also have many phases of preconcentration columns which are compatible with the online SPE workflow. Using the flexcube for sample preconcentration is not new, and we have lots of experience applying this process to various methods. Check out agilent’s application portfolio for more methods using our flex cube. 11/29/2018 NEMC 2018

Comparing Methods: Online SPE Wins: Sample volume Number of steps Presentation Title Conventional SPE: 11/29/2018 Confidentiality Label 250-500 mL Water sample Evaporation w/ Nitrogen Benchtop SPE Re-constitution Analysis w/ LC/TQ Online SPE can make your sample preparation more efficient Here is the typical process for conventional SPE: Now for the online SPE process: Online SPE wins with sample volume, Number of steps And when you increase your number of steps, then you introduce more places for error in your process. Online SPE can reduce the cost of materials And ultimately it saves you time. There are many active steps with the traditional SPE, but with the Flex cube online SPE you can sit back, relax (or do other tasks) , and let your instrument do the work for you. Online SPE: Online SPE Wins: Sample volume Number of steps Potential error Cost Your time 900 µL Water sample Online SPE Coupled w/ LC/TQ Analysis 11/29/2018 NEMC 2018

Valve System Presentation Title 11/29/2018 NEMC 2018 11/29/2018 Confidentiality Label 11/29/2018 NEMC 2018

Excellent Reproducibility of Dual Cartridge System Presentation Title 11/29/2018 Confidentiality Label Chromatographic overlay of 6 injections at 20 ng/L with alternating enrichment cartridges 11/29/2018 NEMC 2018

Trace Organic Compounds in Drinking Water (51 Analytes) Presentation Title 11/29/2018 Confidentiality Label Pesticides/Metabolites 3-Hydroxy Carbofuran Alachlor DEET Disulfoton-Sulfoxide Ethion Fenamiphos Indoxacarb Metalaxyl Mevinphos (Cis) Methomyl Oxadixyl Propoxur Vamidothion sulfoxide Blue=EPA 540 Pharmaceuticals Carbamazepine Diclofenac Dilitiazem Gemfibrozil Hydrocortisone Ibuprofen Norgestrel Primidone Propanolol Simazine Sulfamethoxazole Testosterone Triclocarban Trimethoprim Other Chemicals Caffeine Benzotriazole Diphenylhydramine Propylparaben TCEP PFASs (Neg ESI) PFBS PFDA PFDoA PFHpA PFHxA PFHxS PFNA PFOA PFPeA PFUdA 11/29/2018 NEMC 2018

Trace Organic Compounds in Drinking Water Presentation Title 11/29/2018 51 representative compounds 1260 Infinity II HPLC Column: Poroshell 120 EC C18, 3.0 x 50 mm, 2.7 μm (p/n 699975-302) Mobile Phases: H2O (1mM NH4OAc + 0.1mM NH4F + 0.01% HOAc) and ACN (0.01% HOAc) Flow Rate: 0.4 ml/min Flex Cube Online SPE 900uL injection volume. No bench-top sample pretreatment. SPE Cartridge: PLRP, 2.1x12.5mm Load: H2O, 0.1% Formic Acid Clean: 1:1 ACN/IPA Fast 21 min total (including post time)! Confidentiality Label AJS Source and MS Parameters Drying Gas Temp 250oC Drying gas Flow 11L/min Sheath Gas Temp 375oC Sheath Gas Flow 12L/min Nebulizer Pressure 45 psi Nozzle Voltage (V) 500 (+); 500 (-) Capillary Voltage (V) 4000 (+); 3500 (-) 11/29/2018 NEMC 2018

Excellent Signal Response Presentation Title 11/29/2018 Confidentiality Label Signal response at 10 ppt with 900 uL injection 11/29/2018 NEMC 2018

Excellent Dynamic Range (1.25 ng/L to 500 ng/L) Presentation Title 11/29/2018 Confidentiality Label 3-Hydroxy Carbofuran 100 200 300 400 500 4 x10 2 6 R^2 = 0.999 Disulfoton-Sulfoxide 5 1 3 Methomyl PFOA 0.5 Concentration (pg/ml) Ibuprofen R^2 = 0.997 Testosterone 0.2 0.6 R^2 = 0.995 11/29/2018 NEMC 2018

Detection Sensitivity Presentation Title 11/29/2018 Confidentiality Label Distribution of the limits of quantitation for 51 contaminants (accuracy 80-120%) Analyte LOQ (ng/L) PFBS 5 PFDA 20 PFDoA 10 PFHpA 1.25 PFHxA 2.5 PFHxS PFNA PFOA PFPeA PFUdA 50 11/29/2018 NEMC 2018

%RSD at the lowest concentration for accurate quantitation (n=6) Detection Precision Presentation Title 11/29/2018 Confidentiality Label %RSD at the lowest concentration for accurate quantitation (n=6) 11/29/2018 NEMC 2018

Conclusions PFAS compounds included in TOrC method. Presentation Title 11/29/2018 Confidentiality label Regulatory statement (if applicable)) PFAS compounds included in TOrC method. Online SPE is fast, and can streamline your laboratory workflows. Only 900ul of sample required. Sensitive and reproducible results. Quantitation limits below 50 ppt – many in single digit ppt range PFBS, PFOS, PFOA, PFNA were detected at 10, 4, 0.6, and 8, ng/L 11/29/2018 NEMC 2018

UNBELIEVABLY POWERFUL REMARKABLY SMALL Ultivo Triple Quad LC/MS Presentation Title 11/29/2018 Confidentiality Label UNBELIEVABLY POWERFUL REMARKABLY SMALL Ultivo Triple Quad LC/MS Fit for Purpose!

Thank you Tarun Anumol and Dorothy Yang References Presentation Title 11/29/2018 Confidentiality Label 5991-8969EN 5991-8767EN Thank you Tarun Anumol and Dorothy Yang 11/29/2018 NEMC 2018