1. EPA method 521 by direct-inject LC-MS/MS
Agustin Pierri, PhD
NEMC 2017
Washington DC

2. Background

3. Polyfluoroalkyl Substances
Polyfluoroalkyl Substances (PFASs)
Aliphatic backbone—from C4 on up
Typically per-fluorinated
Acid functional group
Unique environmental chemistry 
Persistent
Lipophilic
Bioaccumulative
Chemically stable: low reactivity, resistant to heat, oil, water, acid, almost anything!

4. Polyfluoroalkyl Substances (PFASs)
Uses?
Repellents
Performance chemicals
Consumer and industrial goods

5. Polyfluoroalkyl Substances (PFASs)
Routes of exposure
Direct contact with food products
Environmental exposure
EPA established a limit of 70 ppt in water to minimize lifetime exposure

6. Target compounds Carboxylic acids Sulfonic acids Sulfonamides
6:2 FTS
8:2 FTS
EtFOSA
MeFOSA
FOSA
EtFOSE
MeFOSE
PFBA
PFBS
PFDA
PFDS
PFHpA
PFHpS
PFHxA
PFHxS
PFNA
PFOA
PFOS
PFPeA
PFDoA
PFTeDA
PFTrDA
PFUdA
Carboxylic acids
Sulfonic acids
Sulfonamides
Sulfonamido-ethanols

7. Sample prep

8. Sample prep approach SPE: QuEChERS: Only suitable for liquid samples
Weak anion exchange offline SPE
QuEChERS:
Suitable for non-liquid samples

9. QuEChERS Screening: C18 d-SPE C18 d-SPE + hexanes 👎
PSA d-SPE + hexanes
C18/GCB d-SPE
C18/GCB d-SPE + hexanes
PSA d-SPE 👎 👎 👎 👎
Semi isotope dilution methods 👎 👌👏

10. QuEChERS procedure Add 1.0 grams homogenized sample to 50 mL tube
Add 10 mL H2O and 10 mL MeCN
Add 4 g MgSO4 and 1 g NaCl
Vortex 3 minutes, centrifuge 5 minutes
Transfer 1 mL aliquot to tube with 150 mg MgSO4 and 50 mg PSA
Semi isotope dilution methods
Transfer 200 µL to conical vial for analysis

11. Secondary cleanup QuEChERS followed by SPE QuEChERS: 1.0 g  10 mL
WAX SPE: 500 µL  500 µL

12. QuEChERS + SPE procedure
Add 1.0 grams homogenized sample to 50 mL tube
Add 4 g MgSO4 and 1 g NaCl
Vortex 3 minutes, centrifuge 5 minutes
Transfer 1 mL aliquot to tube with 150 mg MgSO4 and 50 mg PSA
Transfer 500 µL, dilute to ~15 mL with H2O for SPE
Add 10 mL H2O and 10 mL MeCN
Condition Strata-XAW 200 mg/3 mL SPE cartridge with 0.3% NH4OH/MeCN
Load diluted QuEChERS extract, wash with 5 mL H2O
Elute with 4 mL 0.3% NH4OH/MeCN
Evaporate to 500 µL, transfer to conical vial for analysis
This extra cleanup affords a 20 uL injection, which gets me to a 10x lower MRL
Transfer 200 µL to conical vial for analysis

13. Analysis: LC/MS

14. PFAS by LC/MS Unique analytical challenges Low levels
Relative polarities
Isomers
Contamination

15. Sensitivity 1290 LC, 6460 QQQ Choosing the right mobile phase modifier
UHPLC with the 1290 coupled with the 6460 gives us plenty of sensitivity for these.

16. Mobile phase modifiers for ESI
Negative mode 
Ammonium salts
Ammonium acetate
Ammonium formate
Ammonium fluoride—the secret sauce (usually)

17. Mobile phase choice 👍 Separation/run time: Ammonium acetate
Acetic acid
Ammonium formate
PFAS peak shape with different mobile phases
Ammonium fluoride

18. Chromatographic conditions
Column: Phenomenex Luna Omega PS C18 50 x μm Injection volume: 2 μL or 20 μL Mobile phase: A: Water with 5 mM ammonium acetate B: 100% Acetonitrile Flow rate: mL/min
Time %B
0.0 40
0.5
3.0 90
3.1
100 4

19. Mid level chromatogram (0.5 ppb)
Mid level CCV (0.5 ppb), equivalent to 5 ng/g in the sample

20. Low level (0.05 ppb) 👍 Low levels:
Low level CCV (0.05 ppb in the standard), equivalent to 0.5 ng/g in the sample 👍
Low levels:

21. Solvent blank
Solvent blank 👍
No instrument contamination:

22. Branched vs. linear
PFBA
PFHxS
PFOS
Solvent blank 👍
Isobars:

23. Dynamic range Calibrates up to 0.05 ppb – 1000 ppb
PFPeA: 0.05 ppb to 5 ppb, but it is capable of going up to 1000 ppb.
Calibrates up to 0.05 ppb – 1000 ppb

24. Method performance

25. Extracts
Blank
Butter
Cheese
Egg
Milk
Fish

26. QuEChERS recoveries, milk
n=4, spiked at 2x the MRL: 100 ppt
n=4, 1 ng/g

27. QuEChERS recoveries, egg
n=4, spiked at 2x the MRL: 100 ppt
n=4, 1 ng/g

28. QuEChERS recoveries, butter
n=4, spiked at 2x the MRL: 100 ppt
n=4, 1 ng/g

29. QuEChERS recoveries, tuna
n=4, spiked at 2x the MRL: 100 ppt
n=4, 1 ng/g

30. QuEChERS recoveries, cheese
n=4, spiked at 2x the MRL: 100 ppt
n=4, 1 ng/g

31. QuEChERS + SPE recoveries, egg
n=4, spiked at 2x the MRL: 10 ppt
n=4, 0.1 ng/g

32. Robust and versatile method for PFAS in food
Challenges unique to PFAS Trace levels, MRL at 0.5 ng/g No instrument contamination Resolution Handle complicated matrix
0.05 ng/g

33. Agustin Pierri, PhD agustin.pierri@wecklabs.com
Questions?
Agustin Pierri, PhD
Weck Laboratories, Inc.
Industry, CA 91745