1. 12/1/2018
Emerging Per/Polyfluoroalkyl Substance (PFAS) Analysis in Water, Soil & Biota NEMC Conference 2018
Thank you for the kind introduction and hello everyone. To begin, I am going to discuss the different analytical techniques, workflows and strategies that can be implemented for the analysis of per and poly (PFAS) using different sample preparation and and LC/MS/MS. After this Dr. Pierri will show you real world data including robustness, accuracy and QA/QC from his commercial lab that analysis 1000s of PFAS samples in water, soil, sludge, egg, milk, meat and other food matrices.
Tarun Anumol, Ph.D.
Global Environmental Industry Manager
Agilent, Wilmington, DE, USA

2. Perfluoroalkyl substance
Terminology
12/1/2018
Perfluoroalkyl substance
ALL H atoms linked to C in alkyl chain are substituted with F
A PER……. Examples would include the 8- carbon chain perfluoro octance sulfonic acid or PFOS and
While a POLY iss………………. Example would include the 6:2 fluorotelomer sulfonate
Polyfluoroalkyl substance
SOME (but not all) H atoms linked to C in alkyl chain are substituted with F
1 December 2018
NEMC DRAFT PENDING REVIEW

3. Per/Polyfluoroalkyl substances Unique Properties
12/1/2018
Thermal & Chemical stability: grease-proof food packaging, stain repellents
Zwitterionic properties: surfactants
Surface-tension lowering: fire-fighting foams
These compounds have many unique properties that make their use in commerce and manufacturing desirable…
Their Thermal and Chemical stability finds them uses in food packaging and stain repellents.
1 December 2018
NEMC DRAFT PENDING REVIEW

4. Contamination in Food and Water
12/1/2018
Because of their widespread use these compounds find their way into the environment and foodchain.
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There have been several reports around the world of PFAS contaminating water supply at concentrations of 100s of ng/L.
Another reason they can enter the water supply is because of their use in fire-fighting foams. An example of a recent spill of this fire fighting foam in Australian airport is shown here. Because of this they can also enter the foodchain through exposure to seafood and other marine based animals.
Finally, like I mentioned earlier these fluorinated compounds are also used in food packaging material and can be a source through this route for human exposure.
Confidentiality Label
1 December 2018
1 December 2018
NEMC DRAFT PENDING REVIEW 4

5. Regulatory Guidance for PFAS
Compound
Agency/State
Concentration (ng/L)
Year
PFOA + PFOS
EPA DWHA 70
2016
PFOA MN 35
2017
PFOS 27 NJ
14 (40)
2017 (2007)
GenX NC
140
PFOA/PFOS UK
300 (Reg. 10)
2009
PFOS/PFHxS
AUS
VIC EPA
0.23
1 December 2018
NEMC DRAFT PENDING REVIEW

6. Standard Methods EPA Method 537 ASTM Method 7979 DRINKING WATER
12/1/2018
EPA Method 537
ASTM Method 7979
DRINKING WATER
250 mL sample
Solid Phase Extraction
10 µL injection (96/4: MeOH/Water)
14 analytes
37 min run time
MDLs: 0.5 – 6.5 ng/L
SURFACE & WASTEWATER, SLUDGE
5 mL sample
LLE with 5 mL MEOH + pH adjustment
30 µL injection (50/50: MeOH/Water)
21 analytes
21 min run time
MDLs: 1.3 – 107 ng/L ( ng/L for 537)
For the most part, PFAS analysis is performed using modifications of the EPA or ASTM method.
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The EPA method 537 was created for the analysis of PFAS in Drinking water only! However several lab use a modified EPA 537 method to analyze other matrices as well. This method used 250 ml of sample and solid phase extraction followe by evap to a 1 ml extract as 96/4 MeOH/Water. The method analyzes 14 analytes including 9 PFCAs , 3 PFSAs and 2 FOSAA. MDLs vary beyween 0.5 – 6.5 ng/L
The ASTM method 7979 deals with wastewater, SW and sludge. The method uses a simpler extractiin process with a 5 ml sample mixed with 5 ml of methanol in a PP tube and a pH adjusmtnet before vortexing and centrifugation.
A 50/50 extract is then injected into the LC/MS after filtration with a PP filter.
This method analyzes 21 analytes with MDLs ranging from ng/L. The ASTM also has a method for soils that Dr. Pierri will discuss later on.
Both methods do not use isotope dilution and this is frequently a modification that Is applied by many labs running this method today.
1 December 2018
NEMC DRAFT PENDING REVIEW

7. PFAS Analysis – LC Instrument Setup Eliminate Background Contamination
12/1/2018
Potential Contamination Sources
Solvents
Filtration apparatus
Teflon lined tubing
Before we can talk about the analysis of PFAS. We have to discuss the elimination of background contamination of these compounds. Because of their unique properties fluoropolymers are also used in the manufacturing LC and the impurities present in these FPs can contain PFAS of interest.
Thus we need to “dePFAS” the LC for doin ultra trace levels analysis of PFAS on the LC-MS
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Additionally, Potential sources of PFAS contamination include the solvents, the filtration apparatus and filters containing PTFE lining, and Teflon lined tubing in the SPE process.
At Agilent we have taken a systematic approach to identifying sources of fluoropolymer use in our instruments and have identified replacement parts to eliminate the PFAS contamination that may occur.
For detailed information on how to de-PFAS our LC please contact your local sales rep or download the application note cited here.
Anumol et al., Recommended Plumbing Configurations for Reduction in Per/Polyfluoroalkyl Substance Background with Agilent 1260/1290 Infinity (II) LC’s, Agilent Application Note ( EN)
1 December 2018
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8. Solid Phase Extraction USEPA 537 vs Modified
12/1/2018
However, with the use of this WAX cartridge and a small modification to the extraction procedure used by the EPA method we can also get good recoveries for an additional 16 analytes than span 5 more classes of PFAS.
The only class the had lower recoveries of around 50% were the fluorotelomer alcohol-ethanoic acids.
Nonetheless this data shows that with a small change and minimal additional effort you can extract and analyze several other classes of PFAS.
EPA 537 Analytes
Additional PFAS including ASTM
1 December 2018
NEMC DRAFT PENDING REVIEW

9. Experimental: Procedure: Phospholipid Removal Evaluation:
Protein Precipitation:
Captiva EMR-Lipid:
Add 400 µL of Acetonitrile with 1% Formic Acid into a test tube
Add 100 µL of blank plasma, pre-spun
Vortex on a Heidolph Multi Reax® at rpm, 5 minutes
Centrifuge at 5000 rpm, 5 min
Pipette the supernant to an autosampler vial for analysis
Inject directly into LC/MSMS
Add 400 µL of Acetonitrile with 1% Formic Acid to Captiva EMR-Lipid 1 mL cartridge
Add 100 µL of blank plasma, pre-spun
In-well mixing. Allow PPT.
Pull low vacuum, 2-4 psi, 1 drop/3-5 sec
Collect in polypropylene test tubes
Inject directly into LC/MSMS, using polypropylene* autosampler vials
The protocol we followed is on the right hand side of this slide enclosed within the red line.
This protocol was also compared with the Standard Protein Precipitation protocol and evaluated on lipid removal capacity. I´ll show the results of this evaluation later on.
400ul of acetonitrile with 1% formic acid were added to a Captiva EMR-Lipid 1 mL cartridge. On the evaluation , we performed the Protein precipitación adding the same 400 µL of Acetonitrile with 1% Formic Acid into a test tube.
After we added 100ul of blank plasma, previously centrifuged.
This is a proportion 4:1. A total of 500ul, where the 20% (the 100ul plasma) is water. The protocol states that proportion of organic/plasma which is a water sample could go from 3:1 to 5:1
Then Mixed in the cartridge (in-well mixing) allowing the protein precipitation to occur on about 5 minutes.
On the only Protein Precipitation protocol we mixed on a Vortex for 5 minutes.
We then used a vacuum manifold, pulling a low vacuum of 2-4 psi, which results on a speed of 1 drop every 3-5 seconds. It can be also done using positive pressure.
On the only Protein Precipitation protocol we centrifuged at 5000 rpm during 5 minutes
Finally we collected the eluent in polypropylene test tubes and transfer to polypropylene autosampler vials since PFASs would stick to glass, and inject directly into the LCMSMS
* Polypropylene collection tubes and autosampler vials are recommended since PFASs will stick to glass
1 December 2018
NEMC DRAFT PENDING REVIEW

10. Results and Discussion:
Recovery and RSD r2
5 and 20 ug/L= 5 and 20 ng/mL = 5 and 20 ppb
The recoveries and RSDs for PFASs were evaluated at 5ppb(red bars) and 20ppb (blue bars) spikes.
The overall recovery was between 75 and 125% for both 5 and 20 ppb spikes.
Relative estándar deviation was between 0.8 and 14% for 5 and 20 ppb
Figure 3. Recovery and RSD for the PFASs evaluated in the study at 5 and 20 ng/mL
The recovery and relative standard deviation for the 22 PFASs were determined at 5 and 20 ng/mL shown in Figure 3. The overall recovery was between % for both 5 and 20 ng/mL. Relative standard deviation was % for 5 and 20 ng/mL.
1 December 2018
NEMC DRAFT PENDING REVIEW

11. Results and Discussion:
Phospholipid Removal by Captiva EMR-Lipid versus Protein Precipitation:
Captiva EMR-Lipid, a novel phospholipid removing sorbent available in a SPE cartridge format allows for in-situ protein precipitation and phospholipid removal as the extract passes through the sorbent during elution.
Figure 4 shows the overall phospholipid removal of Captiva EMR-Lipid when compared to protein precipitation, overlay.
The results of the comparison bewteen the Captiva EMR-Lipid versus Protein Precipitation only protocol for Phospholid removal show a much higher phospholid removal capacity for the Captiva EMR-Lipid protocol.
This is an overlay of the MRM chromatogram after protein precipitation (red área) and Captiva EMR-Lipid clean up (blue área) of 11 phospholipids.
Captiva EMR-Lipid allows for in-situ protein precipitation and phospholipid removal as the extract passes through the sorbent during elution.
Captiva EMR-Lipd removes 99% of the phospholipids based on peak área determined from the LC/MSMS MRM method of the 11 phospholipids.
Figure 4. Overlay of the MRM chromatogram after protein precipitation and Captiva EMR-Lipid clean-up, evaluation of 11 phospholipids
Captiva EMR-Lipid removes ~99% of the phospholipids based on peak area determined from the LC/MSMS MRM method for 11 phospholipids.
1 December 2018
NEMC DRAFT PENDING REVIEW

12. PFOA – 1 µL injection of 24 fg/µL
RSD = 9%
IDL = 6.4 fg
Methanol blank
No inject
1 December 2018
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13. 52 PFAS in one LC-MS/MS method (5 ng/mL chromatogram)
Compound classes:
PFCAs
PFSAs
ADONA
PFPAs
n:2 FTS
FASAs
FASAAs
FASEs
diPAPs
diSAmPAP
PFPis
1 December 2018
NEMC DRAFT PENDING REVIEW

14. Calibration curves PFOA and PFOS
2 µL injection in MeOH
linear (r2 = 0.999)
Range 0.1 to 100 ng/mL
PFOS:
2 µL injection in MeOH
linear (r2 = 0.999)
Range 0.1 to 100 ng/mL
1 December 2018
NEMC DRAFT PENDING REVIEW

15. Calibration curves 6:2 FTS and 6:2 diPAP
2 µL injection in MeOH
linear (r2 = 0.999)
Range 0.1 to 25 ng/mL
6:2 diPAP:
2 µL injection in MeOH
linear (r2 = 0.999)
Range 0.1 to 100 ng/mL
1 December 2018
NEMC DRAFT PENDING REVIEW

16. Fish Extract Recovery & Data
1 December 2018
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17. Fish Extract Recovery & Data
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18. Acknowledgements Agilent Technologies Peter Abrahamsson Sheher Mohsin
Michael Woodman
James Pyke
Courtney Milner
Craig Marvin
RMIT University
Bradley Clarke
Timothy Coggan
1 December 2018
NEMC DRAFT PENDING REVIEW