1. Douglas Stevens Kari Organtini Waters Corporation
Direct Injection Method for LC-MS/MS Detection of Polyfluoroalkyl Substances in Water
Douglas Stevens
Kari Organtini
Waters Corporation

2. Drinking water analysis
Overview
PFAS Overview
Drinking water analysis
SPE sample prep
Other water type analyses
Large volume direct injection (LVI/DI)

3. Perfluoroalkylated Substances (PFAS)
Widespread applications
Surface treatments, surfactants, firefighting foam AFFF’s
Polymerization aid for polytetrafluoroethylene (PTFE) and other fluoropolymers
Stable and persistent in the environment (POP)
Bio-accumulative
Identified in environmental samples worldwide
Found in arctic polar bears
Many Americans have ~5 ppb of PFOA in their blood!
Worldwide interest in PFC analysis
PFOS
Perfluorinated compounds such as PFOA and PFOS have been used for over 50 years in various applications including surface coating, surfactants, fire fighting foam and as polymerization aid in making polymers. They are extremely stable and persistent in the environment and are bio-accumulative. Perfluorinated compounds have been identified in environmental samples around the world. They were found in polar bears. Many Americans have about 5 ppb of PFOA in their blood. Increasing concerns about the potential impact of perfluorinated compounds on human health and the environment have resulted in the growing interest in PFC analysis worldwide. It’s also worth noting here that Method 537 was developed on a Waters column, LC and TQ.

4. UCMR3 - PFASs 40 ppt 20 ppt 20 ppt 30 ppt 10 ppt
The analytes included in UCMR monitoring. Suitable for occurrence studies but not necessarily for source apportionment. Still, if the purpose is to monitor for the presence of PFASs in drinking water then this list is fit for purpose as they represent the major components of products used across many application areas from AFFFs, to paints and coatings as well as non-stick surface coatings.
10 ppt
90 ppt

5. Unregulated Contaminant Monitoring Rule
Contributing to the recent interest in PFASs occurrence is the recent completion of the monitoring stage of UCMR3. The link at the top of the slide leads to a presentation given in January 2016 by the EPA and provides and excellent explanation of the UCMR program. Complete coverage of UCMR is beyond the scope of this talk. In brief though, this is the process in the United States by which emerging environmental contaminants are investigated for the potential need for regulation.

6. PFAS’s in the Literature
Hu, Xindi C., et al. "Detection of Poly-and Perfluoroalkyl Substances (PFASs) in US Drinking Water Linked to Industrial Sites, Military Fire Training Areas, and Wastewater Treatment Plants." Environmental Science & Technology Letters (2016)
A recent publication showing occurrence is widespread in the USA.
This is based on data from UCMR 3.
Not all occurrences indicated are due to AFFFs in this data. Note though that AFFF sources are called out in the lower right hand side of the slide.
Environmental releases of AFFFs are of international concern. As one example….
Åse Høisæter of Norwegian Geotechnical Institute reported, in a talk at the Spring 2016 ACS National Meeting (ENVR234), that a firefighter training area at an airport near Oslo was contaminated with 150kg of PFOS in just three years prior to the banning of it’s use in training. There are of course many other PFASs contaminating that site and do note that the 150kg is for PFOS alone. That site was used for training of firefighters working at the airport as well as those from surrounding cities.

7. PFAS Analysis Sensitivity Requirements
SPE enrichment
Sample prep allows for use of low- to mid-level sensitivity for mass spectrometer
Large Volume Injection
High sensitivity TQ required
Xevo TQD OR
Xevo TQ-S micro
Increasing Sensitivity

8. Comparison of analysis methods
EPA Method 537
ISO
21675
ASTM
Sample Prep
SPE
Dilute and Filter aka No Prep
Injection Volume
10 µL
30 µL
Number of compounds 14 27 38

9. UPLC/TQD Conditions System: ACQUITY UPLC/TQD with PFC Kit
Analytical column: ACQUITY UPLC BEH C18 2.1x 50 mm
Mobile Phase A: 2mM ammonium acetate in water/MeOH [95:5]
Mobile Phase B: MeOH
Column Temp: 50 C
Injection volume: 10 µL (full loop)
Time (min)
Flow (mL/min) %B
Curve
0.0
0.40 25
0.5 6
5.0 85
5.1
100
5.6
7.0
0.55 1
9.0
5% methanol solution with 2mM ammonium acetate was used as the weak eluent and methanol was used as the strong eluent. The column cleaning up and equilibration steps are included in the gradient method. The injection cycle time is 9 minutes. With the isolator column and UPLC C18 column in series, the maximum system pressure is 11,000 psi.
537

10. PFAS’s Interferences/Contamination
100 ppt
PFOA standard
Blank injection
Background
PFOA
463>369
463>369
PFC contaminants cause a significant problem for quantification. Shown here are two MRM chromatograms. The top one is an injection of 100ppt PFOA standard and the bottom one is a blank injection. The data show that the background PFOA peak elutes at the same retention time as the analyte PFOA; therefore, for accurate quantification, the background PFC peaks originating from the LC system and mobile phases have to be eliminated from the PFC peaks originating from the injected sample.

11. PFC Kit PFC Isolator Column PEEK tubing PEEK tubing for solvent lines
Stainless steel tubing
Isolator column to injector
PEEK tubing
for autosampler lines
PFC
Isolator Column
Solvent mixer
Coiled
stainless steel tube
Mixer to
PFC isolator column
The PFC kit was developed to eliminate background PFC interference and allow accurate quantification of perfluorinated compounds. The kit provides PEEK tubes for solvent lines and stainless steel tubes to place a PFC Isolator column in-line between the solvent mixer and the injector. The isolator column is designed to temporarily hold-up the PFC contaminants from solvent delivery system during the column equilibration and the early stage of the gradient; eventually, the accumulated PFC contaminants will elute out of the column at the end of each injection cycle when 100% organic mobile phase is applied.

12. MRM Chromatograms of PFOA With PFC Kit Installed
No peaks
Background PFOA contaminant
PFOA
Blank Injection
300 ppt
PFOA standard
0.5
min
Here are two MRM chromatograms obtained with PFC isolator column in-line. The top chromatogram is an injection of PFOA standard. It shows that the analyte PFOA peak elutes first and the background PFOA contaminant elutes 0.5 minutes later as a band of broad peaks. The bottom chromatogram without the analyte PFOA peak is a blank injection, confirming that the background PFOA contaminant has been isolated by PFC isolator column successfully.
537

13. Quanpedia Generates all the necessary methods for routine analysis
Methods -> Sample List -> Data Processing Use a predefined method
UPLC, Xevo TQD, List of pesticides and TargetLynx methods are already set up for the analysis
Create a larger pesticide list
Add compound / pesticides that already exist in Quanpedia to an existing predefined method
Use IntelliStart to automatically tune up a new compound / pesticide to be included into the database and existing predefined method
Develop a new method
Use IntelliStart to automatically tune up new compounds / pesticide to used in a new method

14. Typical Calibration Curve
PFOA calibration curve (0.09 ppb to 9.4 ppb) r2 > 0.997
This is an example of a typical calibration curve of PFC having correlation coefficient greater than
537

15. Recovery Study 537 1 ppt spiked water (n=6) Target 10 PFCs
RSD (%)
PFBuS
105 6
PFHxA
112 11
PFHpA
124 15
PFHxS
103 5
PFOA
122 17
PFNA
107 9
PFOS
104
PFDA
109 8
PFUnA
100
PFDoA 12
Here are the recovery results obtained from 1ppt spiked bottled water samples. The recovery and RSD values for the 10 PFC certainly meet analysis needs.
537

16. PFOS in the bottled water
PFOS in Bottled Water
PFOS in the bottled water
Injection
sample 1
Sample 2
sample 3
sample 4 1
0.42
0.39
0.44
0.29 2
0.37
0.43
0.35 3
0.48
0.52 4
0.46
0.38 5 6
0.36
Average
(ppt)
0.40
RSD (%)
12.7
11.1
7.7
8.1
Mean = 0.41 ppt
7.98e2
Primary transition
Secondary transition
537
In the same water samples, about 0.4 ppt of PFOS was also detected.
This demonstrates fit for purpose detection for EPA Method 537.
Instrument LOQ <25ppt; SPE enrichment factor =250; Method LOQ <0.1ppt

17. Direct (large volume) Injection Approach
ASTM

18. ASTM 7979-17 Direct Injection Method Sample Prep
5 mL MeOH + 5 mL sample in 15mL tube, vortex
Filter: Glass fiber + GHP (polypropylene)
Add 10 ul acetic acid
500 ul to LC vial
Reagent Water
Surface (River) Water
Ground Water
Influent Waste Water
Effluent Waste Water
June 2018 Application Note: EN
Filters washed with 2 x 10 mL ACN and 2 x 10 mL ACN before use
Matrix blanks and spikes - Spiked with 80 ppt
Controls
Prep blank – no spikes
Method blank – 80 ppt of surrogates only
Lab control – surrogates at 80 ppt and native targets (varied conc)
Reporting Limit Control – surrogates at 80 ppt and native targets (at RL)

19. Instrument Methods LC Method Source Parameters Instrument: Xevo TQ-XS
Acquity I-Class with PFC Kit
CSH Phenyl Hexyl 2.1mm x 100 mm, 1.7 um
A: 95:5 H2O:MeOH + 2 mM am ac
B: MeOH + 2 mM am ac
Inj Vol: 30 ul
Gradient:
Source Parameters
Instrument: Xevo TQ-XS
Ion Mode: ESI-
Capillary Voltage: 1.0 kV
Desolvation Temperature: 500°C
Desolvation Flow: 1100 L/hr
Cone Flow: 150 L/hr
Time (min)
Flow (mL/min) %A %B
0.3
100 1 80 20 6 55 45 13 14
0.4 5 95 17 18 22
MS Method
Developed using QuanOptimize
MRMs, CV, CE
Divert flow to waste from 15 – 21 mins

20. Compounds Included in Method
39 Native Compounds
27 Isotope Labels – Recovery only!

21. Overall Method Summary

22. Spiked Surface Water 7979-17 Sulfonates Carboxylates
4:2 FTS
FHEA
6:2 FTS*
FOEA
8:2 FTS
FDEA
PFBS
PFPeS
PFHpS
PFHxS
PFOS
PFNS
PFDS
PFBA
PFPeA
PFHxA
PFHpA
PFOA
PFNA
PFDA
PFUnDA
PFDoDA
PFTriDA
PFTreDA
FHUEA
FOUEA
FHpPA
FOSA
N-EtFOSAA
N-MeFOSAA
Carboxylates
Sulfonates
Telomer Sulfonates and Telomer Acids
Sulfonamidoacetic acids and
unsaturated telomer acids
All PFAS compounds detected in low concentration spiked surface water sample. PFBA and PFPeA at 300 ng/L; 4:2, 6:2, and 8:2 FTS at 1200 ng/L; all other compounds at 60 ng/L.
PFBA and PFPeA at 300 ng/L; 4:2, 6:2, and 8:2 FTS at 1200 ng/L; all other compounds at 60 ng/L. (*) this compound shown off scale.

23. Full scan data TIC purple
Data Independent Acquisition (DIA) Concurrent MRM + Full Scan (aka RADAR)
Primary Influent
Sample
PFOA
MRM red
PFOS
MRM green 1 2
Full scan data TIC purple

24. Method Detection Limits (MDL)
Below reporting limits described by ASTM 7979

25. Linearity and Sensitivity
PFOS
PFOA
R2 =
412.9 > 368.9
412.9 > 168.9
498.9 > 98.9
498.9 > 79.9
2.5 ng/L injection
(0.5x Reporting Limit)
GET S:N VALUES FOR THESE!!

26. Recovery and Repeatability
n = 20 injections
Matrix
% Recovery PFOS
Reagent Water
92.8
Ground Water
90.6
Surface Water
94.6
Influent Water
94.1
Effluent Water
93.5
Overall Recoveries: 80 – 125%
*PFTreDA, PFTriDA ~ 200% - matrix effects
Overall RSD: < 10 %
Show example of one compound – give overall statement of range of all compounds

27. Water Sample Types and Recoveries
Average Percent Recovery in Matrix
Reagent Water
Ground Water
Surface Water
Influent Water
Effluent Water
Recovery Compound
PFBA
82.7
82.1
80.9
80.8
85.5
13C-PFBA
PFPeA
89.1
87.7
90.2
88.1
91.4
13C5-PFPeA
PFHxA
89.7
90.1
91.7
91.3
93.3
13C5-PFHxA
PFHpA
90.6
89.8
92.6
91.9
13C4-PFHpA
PFOA
92.5 92
94.2
94.7
94.3
13C8-PFOA
PFNA 93
92.2
94.8
95.2
13C9-PFNA
PFBS
94.6
92.1
96.8
93.8
96.1
13C3-PFBS
PFPeS
PFHxS
93.5
13C3-PFHxS
PFHpS
92.8
94.1
13C8-PFOS
PFOS
PFNS
Reagent Water
Surface (River) Water
Ground Water
Influent Waste Water
Effluent Waste Water
Just a few examples of recoveries in the different matrices – this is not full list of analytes

28. Water Sample Reproducibility
Reagent Water
Surface (River) Water
Ground Water
Influent Waste Water
Effluent Waste Water
% RSD in Matrix 
Ground Water
Surface Water
Low spike (n=3)
High spike (n=3)
PFBA
15.6
2.7
2.1
2.8
PFPeA
7.4
1.6
2.2
0.4
PFHxA
6.6
1.7
1.9
PFHpA
4.4
2.4
8.9
PFOA
5.9
1.5
9.5
2.5
PFNA
6.3
8.2
1.0
PFDA
8.7
1.3
5.2
PFBS
6.2
1.2
PFPeS
0.3
1.4
0.1
PFHxS
2.6
4.1
PFHpS
3.8
PFOS
7.9
3.1
7.2
PFNS
4.0
5.6
Just a few examples of %RSD two selected matrices – this is not full list of analytes

29. PFAS’s Summary
Occurrence studies have led to increased PFAS’s testing needs
Drinking water analysis is increasing steadily due to increased public awareness
Other water types (non-drinking water) of increasing interest as well
SPE enrichment allows entry level TQ to achieve ppt detection. HP TQ required for LVI/DI water analysis
MRM database, analyte optimization tool and isolator column facilitate implementation

30. Thank you for your attention!
Questions???