Quantitative Analysis
of PFAS in Drinking
Water Using Liquid
Chromatography
Tandem Mass
Spectrometry
Emily Parry and Tarun Anumol, Agilent Technologies, Inc., Little Falls, Delaware, USA

science photo/stock.adobe.com

Per- and polyfluoroalkyl substances (PFAS) are chemicals found in
fire-fighting foams and consumer products requiring water-resistant andstain-repellent properties. As a result of their unique chemical properties
and long-term widespread usage, these chemicals are an emerging humanhealth concern. US Environmental Protection Agency (EPA) released analyticalmethods for PFAS measurement in 2009 and most recently in Novemberof 2018. In this article, data generated using these methods with allowed
analytical modifications is presented and demonstrates robustness andreproducibility while achieving low level detection limits in drinking water.

9Per- and polyfluoroalkyl substances (PFAS)
are a class of man-made compounds
widely used in industry and manufacturing

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because of their uniquely desirable
chemical properties. These compounds
are used in non-stick cookware, foodews
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Figure 1: The average spike recoveries of PFAS in ultrapure and finished drinking water using
SPE.
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contact materials, fire-fighting foams,surfactants, and many other applications.
Their chemistry makes these compounds
extremely persistent, bioaccumulative, andpotentially toxic to animals and humans(1). As a result of their widespread usage
over the last few decades, they are now
ubiquitous in the environment.
There are more than 4500 PFAS
commercially manufactured, but only
very few have been monitored in theenvironment. The most commonlymeasured PFAS classes in the environment

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are the perfluorocarboxylic acids (PFCAs),such as perfluorooctanoic acid (PFOA), andperfluorosulfonic acids (PFSAs), such as
perfluorooctanesulfonate (PFOS). Some ofthese PFAS compounds are currently thesubject of regulation and much public andresearch attention (2).
US Environmental Protection Agency(EPA) indicates a drinking water healthguidance for PFOA and PFOS at a
combined 70 ng/L, while several US states
have guidelines for PFOA, PFOS, and other
PFAS (PFNA, GenX) at low ng/L levels.

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Figure 2: Chromatogram of EPA 537.1 analytes with the addition of PFBA andPFPeA.
x10 3
1.05
10.95
0.9
0.85
0.8
0.75
0.7
0.65
0.6
0.55
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1

PFTA

NMeFOSAAPFUnA

11CI-PF3OUdS

NEtFOSAA

Adona

PFHpA

PFOA
PFTrDA

PFPeA

PFBA

PFBS

GenX
PFHxS

0.05
045

4.5

6

5.5

6.5

7.5

7

PFNA
9CI- PFDA
PF3NOS
11.167
PFOS

PFHxA

88.5

9.5

91010.5

11

PFDoA

11.5

1212.5

13

Counts vs. Acquisition Time (min)Figure 3: Linear calibration curves for PFOA and PFOS; 7-point calibration curve in duplicate(14 points) from 0.1–50 ppb in the extract.
PFOS - 7 Levels, 7 Levels Used, 14 Points, 14 Points Used, 0 QCs
x10 3 y = 147.003489 * x + 20.563484
R^2 = 0.99890136
3 Type:Linear, Origin:lgnore, Weight:None

PFOA - 7 Levels, 7 Levels Used, 14 Points, 14 Points Used, 0 QCs
x10 4 y = 3046.084833 * x + 1326.038272
R^2 = 0.99894706
Type:Linear, Origin:lgnore, Weight:None
62.82.6

5.5

2.4

5

2.2
4.5

24
Responses

Responses

1.8
1.6
1.4
1.2

3
2.5

12

PFOS
(0.1–50 ng/mL)

0.8
0.6
0.4

1.5

PFOA
(0.1–50 ng/mL)

1

0.2

0.5

00-0.2

-0.5
-1

0

12

3

4

5

6

7

8

9

10

11

12

13

14

15

16

1718

19

20

21

Concentration (ng/mL)

In Europe, the drinking water directive
recommends levels of lower than 0.1 μg/L
for individual PFAS, and 0.5 μg/L for total
PFAS, while several member countries haveguidelines for PFAS in the ng/L range indrinking water. PFOS and its salts have

11113.5

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-101234567891011 1213141516 1718 192021

Concentration (ng/mL)

been listed as priority pollutants to bephased out from use under the Stockholm
Convention. With PFOA and PFOS banned
or in the process of being phased out
by manufacturers globally, alternative
compounds are being used resulting in“emerging” classes of PFAS now being
detected in the environment.
The measurement of these compounds
at ng/L levels is quite challenging.
Therefore, the need for standard methods
to measure them in the environmentis critical for establishing baselines
and future regulatory decisions. In2009, the US EPA established EPA
Method 537 for the quantificationof 14 PFAS in drinking water, using
solid-phase extraction (SPE) and liquid
chromatography (LC) coupled with tandem
mass spectrometry (MS/MS) (3). In late
2018, the US EPA revised this method(EPA 537.1) to include four emerging
PFAS including hexafluoropropyleneoxide dimer acid (HFPO-DA aka GenX),
ADONA, 9Cl-PF3ONS, and 11Cl-PF3ONS(components of F-53B; replacement forPFOS) (4).
This article aims to provide a simple SPE
procedure for the extraction of PFAS indrinking water analyzed in EPA Method
537, along with an LC–MS/MS method forthe analysis of PFAS listed in EPA Method
537.1 to achieve the required low ng/Llevels in drinking water.

CHIRAL TECHNOLOGIESCONGRATULATES OUR
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Dr. Yoshio Okamoto, Professor Emeritus at Nagoya
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representatives, and other distinguished guests.

Experimental
Chemicals: Standards were purchasedfrom Wellington Laboratories, Inc. andews
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TECHNOLOGIES

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Table 1: PFAS compound optimized transitions and estimated limit of detection on the LC-TQ system
AnalytePFBA1PFPeA1

RTTransition

Collision
EnergyEstimated Instrument
Level of Detection(pg on column)

213 > 168.9
60
60.1
263 > 218.9
60
60.06
34298.9 > 80.1
PFBS
7.6
100
0.11298.9 > 99.1
22312.9 > 119.1
14PFHxA
8.5
700.7
312.9 > 269
6HFPO-DA
8.9285.1 > 169
100
00.44362.9 > 169
9PFHpA
9.5
720.18362.9 > 319
0398.9 > 80.1
100
37PFHxS2
9.50.31398.9 > 99.1
7034377.1 > 251.1
0ADONA9.65950.04377.1 > 84.9
30412.9 > 169
13PFOA
10.2
690.08412.9 > 369
3498.9 > 80.1
38
PFOS2
10.8
100
1.30498.9 > 99.1
38462.9 > 169
13PFNA
10.9
66
0.26
462.9 > 418.9
39Cl- PF3ONS
11.2531 > 351.1
9020
1.35512.9 > 219
100
3
1.51
PFDA
11.4
512.9 > 468.9
8112570 > 482.9
15
NMeFOSAA
11.7
115
0.47570 > 418.9
12562.9 > 219
10015PFUnA
11.6
1.17562.9 > 519
734584 > 525.9
15NEtFOSAA
11.9
115
1.01584 > 418.9
1511CI-P3OUdS
12.0631 > 451
7030
1.32612.9 > 269
100
150.50
PFDoA
12.2
612.9 > 568.9
794662.9 > 169
100
23PFTrDA
12.6
0.18662.9 > 618.99177712.9 > 669
PFTA
12.8
100
0.11712.9 > 169
23
1Not included in EPA Method 537 or EPA Method 537.1
2EPA Method 537.1 requires that the 80 m/z product ion must be used to reduce bias between linear
and branched isomers

12125.4
7.3

Fragmentor

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calibration standards diluted to a desiredconcentration in 96:4 methanol–water.
Instrumental: Five μL of the standard–sample were introduced for analysis
into the LC–MS/MS system. Instrument
sensitivity allowed for the reduction of
10 μL cited in the EPA 537 method. LC
separation was performed on an Agilent
1260 Infinity II Prime LC system with a
3.0 × 50 mm, 1.8-μm Zorbax Eclipse
Plus C18 column (Agilent). A 4.6 ×50 mm, 3.5-μm Zorbax Eclipse Plus C18
delay column (Agilent) was used after thebinary pump to separate background PFAS
introduced from the solvent, tubing, andthe degasser from the desired analytes.
The Agilent Jet Stream Technology
Ion Source (AJS) was used for maximum
ionization. Source parameters were thesame as can be seen in reference 5, withthe exception of the increase of drying gas
flow to 7 L/min. The Agilent Ultivo Triple
Quadrupole LC/MS (LC-TQ) was operated
in dynamic multiple reaction monitoring(MRM) mode to optimize sensitivity
through maximizing dwell time. For mostanalytes, two transitions were acquired
to provide quantitation and qualification
ratios. MRM parameters are noted inTable 1. EPA Method 537.1 now requires
the use of 80 mass-to-charge ratio(m/z) for PFHxS and PFOS to reduce bias

between linear and branched isomers andthis was implemented.
Solid-Phase Extraction: Six replicatesof 250-mL ultrapure water and finisheddrinking water samples were spiked
at 4 ng/L for each PFAS. The samples
were then extracted using a weak anion
exchange (WAX, 150 mg, 6 cc) SPE
cartridge (Agilent) as in the proceduredescribed in EPA Method 537. Details forthe specific SPE procedure can be foundin reference 6. The eluate was evaporated
to a final volume of 1 mL constituting~96:4 methanol–water. Figure 1 showsthat the extraction recoveries of all PFAS
compounds were 70–130% and ranging
from 79–112% in both ultrapure anddrinking water. The relative standard
deviations (RSDs) for all compounds was<15% too—within acceptable parameters
for the EPA method—demonstrating thatthe cartridge is effective at extracting
low-level PFAS from drinking water
samples with high efficiency.

Results and Discussion
Background Contamination Elimination:
In this study, a delay column was installed in
between the pump mixer and the injection
port to time resolve any background PFAS
coming from the solvents or the tubing of
the LC system itself.

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Figure 4: Raw response deviation for six PFAS in the continuous calibration standards run
across a 26-h batch; the number in brackets is the RSD%.

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10.53 iC5 8mV8.04 UC3 5chromatographic separation and peak
shapes of the early PFAS eluters, eventhough these are not present in the EPAmethod. The mobile phase was 5-mM
ammonium acetate in water and 5-mM
ammonium acetate in 95:5 methanol–water, instead of the 20 mM used in theEPA methods. The EPA’s method flexibility
allows changes in the LC separation.
However, the EPA notes that reduced
RT stability was observed over time withlower concentrations. Reduced stability
at the lower concentration has not been

No more cold or hot spots. Program all elements simultaneously.

8.47 iC4 6
8.68 nC4 7

Chromatographic Separation andMethod Performance: The analysis andseparation of the 18 PFAS in EPA Method
537.1 were performed with all analytes
achieving good peak shapes and peakwidths between 6–10 s. Figure 2 showsa representative chromatogram of the14 analytes in EPA Method 537, fourof the emerging PFAS (GenX, ADONA,9Cl-PF3OUdS, and 11Cl-PF3OUdS)
added to EPA Method 537.1, and the
addition of PFBA and PFPeA. PFBA andPFPeA were added to show the good•

6.30 C3 415SimDis Analysis

C12C7to 1/8” diameter

5.25 Uc2 3

10Fused silica or metal columns- from capillary

3.98 C2 25•

PFNA (3.7%)
N-MeFOSAA (3.6%)

3.29 C1 10

PFOA (1.2%)
PFDA (3.6%)

130

4

68

1012SECONDS

Visit www.vici.com/fast-gc for more information

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observed so far. The gradient run time
was reduced from 37 min in EPA Method
537 to 19.5 min (14-min gradient and a
5.50-min post time).
Figure 3 shows representative calibration
curves for PFOA and PFOS from 0.1–
50 parts per billion (ppb) in the final
extract. Calibration curves were linear with
R2 > 0.99. Complete details of the analyticalmethod including method optimized
parameters and method verification alongwith linearity, robustness, and analysis ofreal-world drinking water samples can befound in reference 5.
Robustness and Reproducibility: US
EPA Method 537 requires sensitive analysisof PFAS and robustness of the data acrosssamples and batches. For example, themethod calls for the injection and analysis
of a continuing calibration standard ina batch every 10 samples to monitor
system performance and variability. In
this study, this method was evaluated byfollowing the raw response of the PFAS
standards run as continuous calibration
standards every 10 samples across a batchof samples over a 26-h worklist. The
standards were prepared in drinking water
extracts at 1 ppb in the vial (~2.5 ng/L insample equivalent). All PFAS analytes had
response variation less than 5%RSD
except N-EtFOSAA (5.6%). Figure 41414amautar
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illustrates the response stability of thecalibration standards across the 26-h batchand shows that the relative response,
uncorrected by internal standards (ISs), wasstable across the 11 CCV samples analyzed
over 26 h.

Water by Solid Phase Extraction and Liquid
Chromatography/Tandem Mass Spectrometry(LC/MS/MS),” Version 1.1. September 2009.
J.A. Shoemaker US EPA, Office of Researchand Development.
4. US EPA, “Method 537.1: Determinationof selected per- and polyfluorinated alkyl

Conclusions

substances in drinking water by solid phase

The analysis of PFAS at extremely low
levels in drinking water is required
for adequate baseline monitoring andregulatory determination. This article
provides a sample extraction protocol for
PFAS in the US EPA method that achieves
high recoveries in the target matrix, anda robust LC–MS/MS method for excellent
separation, low level detection, andreliable and robust quantification of PFAS.

extraction and liquid chromatography/tandem
mass spectrometry (LC/MS/MS),” Version 1.0.
November 2018. J.A. Shoemaker and D.R.
Tettenhorst US EPA, Office of Research andDevelopment.
5. “Analysis of per/polyfluoroalkyl substances(PFASs) in drinking water using the Agilent
Ultivo triple quadrupole LC/MS (59918969EN)”, Agilent Application Note,
6. “Extraction of Per/Polyfluoroalkyl Substances
in Water Using Agilent Offline SolidReferences

Phase Extraction (5994-0250EN)”, Agilent

1.

Application Note,

ATSDR (Agency for Toxic Substances andDisease Registry), “Per- and Polyfluoroalkyl
Substances (PFAS) and Your Health,” (Atlanta,Georgia, USA, Department of Health andHuman Services, ATSDR).

2. S.F. Nakayama, M. Yoshikane, Y. Onoda,
Y. Nishihama, M. Iwai-Shimada, M. Takagi,Emily Parry is an LC–MS Applications
Scientist at Agilent Technologies, Inc.
Tarun Anumol is the Global
Environment Market Manager atAgilent Technologies, Inc.

Y. Kobayashi, and T. Isobe, TrAC Trends inAnalytical Chemistry in press (2019). DOI:
10.1016/j.trac.2019.02.011
3. US EPA, “Method 537: Determination ofE-mail: tarun.anumol@agilent.com
Website: www.agilent.com

Selected Perfluorinated Alkyl Acids in Drinkingews
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