Prepare to Meet the Challenges
of a Regulated PFAS LandscapeA Public Health Crisis
Decades in the Making
Per- and polyfluoroalkyl substances (PFAS) are man-made substances widely used
in industry and manufacturing because of their unique properties. These compounds
have been used for several decades in many applications like nonstick cookware,stain-repellent clothing, food packaging materials, detergents, cleaning products,
and firefighting foams. The widespread use of these compounds has led to theirubiquity in the environment. Studies have indicated that PFAS are also present
in most humans. Research on these compounds has identified them as being
persistent and bioaccumulative (especially PFAS with a carbon chain length >C7).
Toxic effects, including tumors and thyroid disruption, have also been attributed tosome of them.1 This has resulted in regulatory guidance for water and soil, as wellas accelerated monitoring and identification of these compounds.
US EPA. 2020. Basic Information On PFAS | US EPA.

1

Firefighting foams often contain PFAS to assist in dousing flames.

3Regulatory Overview

There are >4,000 known PFAS compounds that have been created for commercial use. Regulatory guidance
and restrictions exist for just a few of them, leading to unrestricted use for the rest.
– In 2009, perfluorooctanesulfonic acid (PFOS) and its salts were listed as persistent organic pollutants(POPs) under the Stockholm Convention. All parties are required to eliminate the release of thesecompounds into the environment.
– The European Union (EU) Water Framework Directive lists PFOS as a priority hazardous substance that posesa “significant risk to the aquatic environment.” It has established an annual average environmental quality
standard (AA-EQS) of 0.65 ng/L in inland surface waters, and an AA-EQS of 0.13 ng/L for other surface
waters (Directive 2013/39/EU). The EU drinking water directive aims to have routine monitoring of up to 20
PFAS compounds in drinking water from 2021.
– The UK Chemical Investigation Program requires measurement of PFOS and perfluorooctanoic acid (PFOA)
down to 0.09 ng/L. Similarly, in October 2018, the EU Parliament approved a proposal to recast its Drinking
Water Directive with revised limits for monitoring PFOS and PFOA in drinking water. The new limits are
100 ng/L, with combined PFAS concentrations not to exceed 500 ng/L (COM (2017) 753 1.2.2018).
– Several European countries, including those in the Nordic region, have guidance levels for PFAS in drinking
water and surface water. In Sweden, recommended levels for a sum of 11 PFAS in drinking water should
not exceed 90 ng/L.
– In the United States, the US EPA has established a drinking water health advisory level for PFOS and PFOA
at a combined 70 ppt (ng/L). Several states have their own advisories for PFOA, PFOS, and other PFAS—
such as perfluorononanoic acid (PFNA) and GenX—at the low ppt range. Other initiatives are also in theworks. These include the PFAS Action Act (Jan 2019), the US EPA PFAS Plan (Feb 2019), and the US EPA
Commitment to PFAS Drinking Water Standards (Feb 2019).
– Australia, China, and several other countries are implementing restrictions (or establishing maximum
amounts) for drinking water and receiving water regulations for PFOA, PFOS, and newer PFAS detected
at low ng/L to pg/L levels.
As newer PFAS are identified in the environment, and as more toxicological information becomes available,
further guidelines and regulations are almost certain.

4

Europe

USA

Japan
China

Australia

Worldwide regions where strict PFAS monitoring regulations are being enacted.

Current Standards and Consensus methods for PFAS analysis in the environment.

MethodMatrix Tested

No. ofAnalytes

Sample Preparation
Procedure

Quantification Technique

EPA 533

Drinking water

25

Solid phase extraction

Isotope dilution

EPA 537

Drinking water

14

Solid phase extraction

Internal standard correction

EPA 537.1

Drinking water

18

Solid phase extraction

Internal standard correction

EPA 8327 (draft)

Surface water, groundwater,
wastewater influent and effluent

24

Dilute and shootExternal calibration (isotope dilution also
allowed)ASTM 7979

Surface water, groundwater,
wastewater influent and effluent

21

Dilute and shootExternal calibration (isotope dilution also
allowed)ASTM 7968

Soil and solids

21

Organic extraction
with MeOH

External calibration

ISO/DIS 21675

Drinking water, sea water,
fresh water, wastewater (<0.2% solids)

30

Solid phase extraction

Internal standard correction

5

Sample Preparation Techniques to Maximize
PFAS Recoveries and Minimize Contamination
Due to their widespread use in clothing, protective gear, and consumer products, PFAS have beenfound in human and environmental samples in all regions of the world. Use these best practices
to avoid contaminating your sample with PFAS during collection and storage.

6

Do:– Wear well-washed lab coats and nitrile lab gloves.
– Use high-density polyethylene (HDPE) or polypropylene (PP) containers and caps as recommended
in US EPA and ASTM methods.
– Refrigerate samples below 6 ºC during storage.

Don’t:– Wear personal care products (such as sunscreen and hand creams) during sampling.
– Wear waterproof clothing or shoes that may be lined with PFAS or stain-repellent material.
– Use sample collection apparatus that may contain polytetrafluoroethylene (PTFE) or otherplastics containing PFAS.
– Use aluminum foil to cover openings of sample containers, as PFAS can be transferred from foil.

Proper sample cleanup and concentration
are essential to robust, accurate, and reliable
analysis. As the world’s chromatography leader,
Agilent supports your efforts with innovative LC
columns, solid phase extraction (SPE) cartridges,
vials, and filters manufactured to demanding
specifications. All are tested under strict
conditions for reliable analysis of PFAS.

7

Extracting PFAS from water
Several regulatory methods, including EPA 537 and533, call for extracting PFAS from drinking waterusing SPE cartridges followed by LC/TQ analysis.
Typically, a weak anion exchange (WAX) cartridge issuggested due to its ability to extract both shorterand longer-chain PFAS with good recoveries as
done in EPA 533 and ISO methods. EPA 537 uses an
Agilent Bond Elut LMS cartridge, which provides highrecoveries for medium- and long-chain PFAS.
From instruments, columns, and supplies, tofast, worldwide delivery, to decades of method
development expertise—Agilent supports yourentire workflow for PFAS testing.

Name

LC Grade Water
Average
RecoveryLC Water
RSD (%)

Tap Water
Average
RecoveryTap Water
RSD (%)

85

14

99

1
6

EPA 537 Compounds
PFBS
PFDA

101

5

95

PFDoA

86

3

88

2

PFHpA

105

10

101

3

PFHxS

97

15

102

1

PFHxA

104

8

107

2

PFNA

100

5

104

3

PFOS

92

13

94

3

PFOA

102

10

106

2

PFTrDA

91

3

103

15

PFUdA

100

6

102

3

PFTrDA

91

3

103

15

N-MeFOSAA

84

11

79

10

N-EtFOSAA

84

9

89

3

Additional Compounds

Average recovery is between 40% and 70%
RSD is greater than 19%
All 30 PFAS were recovered, and US EPA 537 compounds had recoveries
between 70 and 130% with RSD <15% for both water qualities. These
results comply with US EPA method QA/QC requirements. In addition,
only 4 of the 30 compounds had recoveries below 70%, but all 4 wereabove 40%. The 14 compounds in the US EPA method produced
acceptable recovery using the Agilent WAX SPE cartridge. The 16 other
PFAS, including compounds on the ASTM list, also had good recoveries
and can be analyzed with this method.

PFDoA

86

3

88

2

PFTeDA

96

10

86

8

FOSA

57

14

67

20

FHEA

107

6

90

8

FOEA

61

13

52

14

FDEA

56

21

58

15

PFHpPA

47

27

46

14

4-2 FTS

91

10

91

13

6-2 FTS

87

16

100

6

8-2 FTS

104

10

101

10

6-2 FTUA

118

8

106

3

8-2 FTUA

96

11

78

13

PFPeS

97

15

104

2

PFHpS

83

11

83

3

PFNS

93

12

91

8

PFDS

85

4

81

6

Want a closer look?
Read application note 5994-0250EN: Extraction of Per/Polyfluoroalkyl Substances in Water Using Agilent Offline
Solid Phase Extraction8Extracting PFAS in biota

SPE and supported liquid extraction (SLE) can betime consuming and complicated to perform on
biological samples. Agilent Captiva EMR—Lipid
makes it easy to remove interferences, particularly
phospholipids, in minutes without PFAS loss. Its
pass-through format is fast, repeatable, and delivers
a clean extract with minimal ion suppression,
extending column life and reducing the frequencyof MS cleaning.

125

Recovery (%)

100

75

50

25

0
PF
6: BA
2
FT
PF A
Pe
PF A
H
xA
PF
B
8: S
2
FT
PF A
H
pA
PF
OA
PF
H
xS
PF
10 NA
:2
FT
A
N
M PF
eF DA
N OS
E t AA
FO
SA
A
PF
OS
PF
Ud
PF A
Do
PF A
Tr
PF DA
Te
DA

PFAS compounds are readily absorbed into animaland human tissue. Since these substances havebeen used in industry for many years, their existencein human blood and serum—as well as presencein fish, salmon, and other wildlife—is pervasive.
Research has shown that longer chain PFAS (>C7)
have the potential to bioaccumulate, increasing boththe need and urgency to test and analyze for PFAS
levels in biota and biological fluids.

5 µg/L spike
20 µg/L spike

PFAS recovery fell between 70 and 130% with most compounds having
an extraction recovery of >90%. The full procedure for extraction with
Agilent Captiva EMR—Lipid is detailed in application note 5991-8656EN,
and shows how this simple technique can be performed in minutes.

Protein precipitation

Relative abundance

EMR-Lipid cleanup

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

Acquisition time (min)Chromatographic baseline of EMR—Lipid phospholipid cleanup
compared to protein precipitation. We reduced MS cleaning andincreased column life without sacrificing PFAS recovery.

Want a closer look?
Read application note 5991-8656EN: Analysis of Per- and Polyfluoroalkyl Substances (PFAS) in Biological Fluid Using a Novel
Lipid Removing Sorbent and LC/TQ
For Research Use Only. Not for use in diagnostic procedures.

9Configure Your LC for Success:
PFAS Elimination
Fluoropolymers are commonly found in many industrial products, including those used in the lab foranalysis, extraction, and cleanup. Lab supplies such as solvents, syringes, pipettes, and SPE devices
can contain trace PFAS levels that may contaminate your samples and interfere with your results.
Some LC instrument parts can also contribute to trace-level contamination.

InfinityLab LC instruments:
Efficiency for any application and budget
Agilent InfinityLab HPLC and UHPLC systems feature thelatest technology while maintaining full compatibility withlegacy LC instrumentation.
– The Agilent 1260 Infinity II LC is the flexible choice for
operational efficiency.
– The Agilent 1290 Infinity II LC is the next generation ofliquid chromatography—delivering ultrahigh performancefor superior analytical results.

Is PFC background noise hurting your PFAS analysis?
The InfinityLab PFC-free HPLC conversion kit for the Agilent 1290
Infinity II LC helps keep your flow path free from perfluorinatedcompounds, so you can minimize PFAS background noise andmeet stringent regulations.
To request your kit, simply use our easy ordering guide that links
to a prefilled shopping cart at the Agilent online store.

10

Consumables solutions for analyzing trace-level PFAS with confidence
Agilent’s new InfinityLab PFC-free HPLC conversion kit includes everything needed to ensure that the1290 Infinity II HPLC systems and high-speed pumps are free of PFAS contamination. This includes
various replacement parts, tubing, inline filters, solvent bottle assembly, and an InfinityLab Delaycolumn with Quick Connect HPLC fitting.
Several regulatory methods, including EPA 537 and 533, call for extracting PFAS from drinking waterusing SPE cartridges followed by LC/MS/MS analysis. Agilent’s SPE cartridge portfolio includes both
ultraclean divinylbenzene sorbent for EPA 537, as well as the weak anion exchange or WAX cartridge
required for analyzing shorter-chain and emerging PFAS compounds. Captiva syringe filters withregenerated cellulose (RC) membranes are ideal for PFAS analysis described in EPA 8327 and ASTM
D7979 methods for non-potable waters.
Agilent ZORBAX RRHD columns, based on a fully porous particle, increase resistance to strong
sample injection solvents and allow for high-volume injections, making them ideal for analyzing PFAS
compounds in water.

×102
5

Blank std
LC setup(isomers
separated)

Without
delay column

Counts4

Injection of 100 fg
of sample on column
PFC-free LC setup

With
delay column

Blank
std LC setup

Blank
PFC-freeLC setupBlank
PFC-free LC setup

3

2

1

0

3.5

3.6

3.7

3.8

3.9

4.0

4.1

4.2

4.3

4.4

4.5

4.6

4.7

4.8

4.9

5.0

5.1

5.2

PFHpA blank and sample
chromatograms fromdifferent LC system setups.
We tested Agilent PFCfree conversion kits using
38 analytes representing
standard and regulatory
methods—including US EPA
533, 537.1, 8327, and ISO
21675. No background was
detected for 36 out of the
38 analytes.

Acquisition time (min)Want a closer look?
Read these application notes:
5994-2291EN: Reduce PFAS Background with the Agilent PFC-Free HPLC Conversion Kit
5994-2151EN: Filtration for the Analysis of Per- and Polyfluoroalkyl Substances in Environmental Extracts
5994-0250EN: Extraction of Per/Polyfluoroalkyl Substances in Water Using Agilent Offline Solid Phase Extraction11

Meet Regulatory Guidance Levels
with Robust Results

The sensitivity, robustness, and reliability of Agilent
triple quadrupole LC/MS systems (LC/TQ) make them
ideal for analyzing general and regulated PFAS targets.
An Agilent Ultivo can quantify low levels (ppt) of PFOA
and PFAS in water and soil using WAX SPE cartridge
concentration and cleanup.
Here, we used Agilent WAX SPE cartridges to extract
PFOA and PFOS from water and soil.
The Agilent InfinityLab Poroshell 120 EC-C18(2.1 x 100 mm, 2.7 μm) column allows for effective
separation of linear and branched isomers for PFOA.
Recoveries between 80 and 120% for both compounds
in surface water, wastewater (2.5, 40, and 200 ng/L),
soil, and sediment (0.5, 5, and 20 ng/g) were achievedat different spiking levels.

3.805

2

0
×10 3
Counts (cps )General PFAS analysis with SPE cleanup

×101 A
4
PFOS
499.0 99.0
3 S/N = 120

1

2.0

PFOA
413.0 369.0
S/N = 1,193

1.5
1.0
0.5
0

2.0

2.5

3.0

×10 1 B

3.5
4.0
Retention time (min)

4.5

5.0

4.5

5.0

3.836

PFOS
499.0 99.0
S/N = 226

6
Counts (cps )PFOS and its formulations are listed as priority
substances in the Stockholm Convention. The EPA safe
drinking water guidelines for PFOS and PFOA are not
to exceed 70 ng/L. However, several regulatory andmunicipal bodies require monitoring and detection of
PFAS at low single-digit ng/L levels in water and soil.

4
2
0
×10
8

Counts (cps )PFOS and PFOA are the two most commonlymeasured PFAS and have regulatory guidance levels
in regions including China, Europe, the United States,
Japan, and Australia.

Counts (cps )Global regulations for PFOS and PFOA

2

PFOA
2.791
413.0 369.0
S/N = 1,382

6
4
2
0

2.0

2.5

3.0

3.5
4.0
Retention time (min)Chromatograms for PFOA and PFOS spiked at 2.5 ng/L in water(A) and 0.5 µg/kg in blank soil (B). Note: Only a quantitative ion
chromatogram is shown for each compound.

12

US EPA drinking water methods
US EPA method 537.1 analyzes 18 PFAS, including PFOS and PFOA, in drinking water using SPE
followed by LC/TQ analysis. EPA 537 uses an Agilent Bond Elut LMS solid phase extraction cartridge(p/n 12255021) to extract 500 mL of drinking water. This process is followed by evaporation andcreation of the final extract with ~96% methanol. The following chromatogram shows the separationand detection of PFAS using an Agilent ZORBAX Eclipse Plus C18, 3.0 × 50 mm, 1.8 μm column(p/n 959757-302). Samples were analyzed on the Agilent Ultivo triple quadrupole LC/MS.

x103
1.05
1
0.95
0.85
0.8
0.75
0.7
0.65
0.6
0.75
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

PFTA

NMeFOSAA

PFUnA

11Cl-PF3OUdS

NEtFOSAA

PFHpA

PFPeA

PFBA

4

4.5

5

5.5

6.5

7

7.5

PFOA

GenX
PFHxS
8

8.5

PFTrDA
PFNA

PFHxA

PFBS
6

Adona

9

PFOS
9.5

10

10.5

PFDA
9Cl-PF3NOS
11

11.5

PFDoA

12

12.5

13

Counts vs. acquisition time (min)Want a closer look?
Read application note 5994-0437EN: LC/TQ Determination of PFOS and PFOA in Water and Soil Matrices13

In addition to meeting and exceeding reporting levels required by the US EPA method, you mustalso achieve robustness across a batch of real water samples. Here, we show a relative response
in area counts for 11 continuous calibration water samples run over a 26-hour batch for EPA 537.
The Ultivo LC/TQ system allows you to continuously run routine EPA methods for PFAS with thedesired sensitivity and reproducibility. Its small footprint lets you fit approximately three Ultivo
systems in the space needed for one traditional LC/TQ instrument.

1.6

PFBA
PFHxA
PFOA
PFOS
N-MeFOSAA
PFUdA
PFTrDA

RSD

1.4

1.2%

Relative response

1.2
1.9%

1.0
0.8
0.6

3.5%
2.1%

0.4

3.0%
1.9%

0.2
0
0

3.6%
4

8

12

16

20

24

28

Time (hrs)

Want a closer look?
Read application note 5991-8969EN: Analysis of Per/Polyfluoroalkyl Substances (PFAS) in Drinking Water
Using the Agilent Ultivo Triple Quadrupole LC/MS

14

Eliminating sample preparation for US EPA 537
SPE sample preparation and extraction is a critical step for cleanup and interference removal.
However, it is time consuming and tedious, and can introduce sources of PFAS contamination
into the sample.
For labs looking to monitor the PFAS listed on EPA method 537, the Agilent 6495 LC/TQ system
is an ideal choice. It eliminates the hassle of sample preparation in clean water samples and getsyou from sample collection to data reporting quickly. The 6495 offers excellent sensitivity andincreased throughput without any sacrifice in method reporting levels.
A method developed on the 6495 LC/TQ quantified 14 PFAS present in EPA 537. The analysis
took less than six minutes, with a direct injection of 80 µL of sample diluted slightly with methanol
to achieve detection levels of 0.83 to 3.3 ng/L.
The following graph shows the recovery of these PFAS at two spiking levels.

Mid-level spike
Low-level spike

125

100

75

50

Spike recoveries of PFAS
at low level (10 ng/L; 2
ng/L for PFOS and PFOA)
and mid level (40 ng/L) in
drinking water samples.

25!

=->*0-?@AB

PF
Pe
S

PF
OS

PF
OA

PF
NS

PF
NA

PF
Hx
S

PF
Hx
A

PF
Hp
S

PF
Hp
A

PF
DS

PF
DA

PF
BS

PF
BA

0

CD>E-?@AFTo learn more about the challenges and mitigation strategies in detection of PFAS in water, watch
this on-demand webinar by Dr. Arjun Venkatesan, Stony Brook University.

15

Analyzing challenging compounds with EPA 533
EPA method 533 addresses some of the challenging compounds in EPA methods 537 and 537.1 for C4
and C5 acids and sulfonates. It also limits chain length to C12 acids, while adding some emerging PFAS.
Agilent’s sample preparation, consumables, and Ultivo LC/TQ provide the tools needed to achieve thedetection limits required by EPA 533 while offering ultimate robustness and easy-maintenance features
for novice mass spectrometry users too.
An Agilent 6470 LC/TQ with Agilent Jet Stream ESI source delivers sensitivity, precision, and accuracy
exceeding EPA requirements, allowing users to future-proof their lab against dropping regulatory limits
that could be expected. Recoveries were between 87% and 103% for all PFAS in reagent and tap water.

LCMRLs calculated according to EPA for all analytes in EPA 533 with Ultivo and 6470 LC/MS compared to EPA single lab LCMRL.
18
Ultivo LCMRL

6470 LCMRLEPA Single Lab LCMRL

16
14

LCMRL (ng/L)

12
10
8
6
4
2
0

BA

PF

PA

M

PF

s
S
S
S
A
A
xS
eS -DA
xA
pA
SA
OA p
NA
HA FT
NA FT
Pe PFB MB
FP PO
EE
FH
FH PFHPF PFHPF
FD 4:2
DO 6:2
P
F
P
P
N
PF
A
F
P
H

PF

OS

PF

S

9

ON

F3

-P
CI

2F

8:

TS

DA

PF

Read these application notes:
5994-1628EN: EPA Method 533 for Analysis of Per/Polyfluoroalkyl Substances in Drinking Water
Using Agilent 6470 LC/TQ
5994-1920: Analysis of Per/Polyfluoroalkyl Substances (PFAS) in Drinking Water by EPA 537.1 and
EPA 533 Using the Agilent Ultivo Triple Quadrupole LC/MS

S

Ud

3O

F
I-P

C

11

Want a closer look?

16

A

Un

PF

A

Do

PF

EU regulations
The EU has an environmental quality standard of 0.65 ng/L in water bodies for PFOS, andresearchers anticipate similarly low reporting levels for PFOA in water. The high sensitivity of
the Agilent 6495 triple quadrupole LC/TQ lets you achieve these low levels by direct aqueous
injection and run your analysis in a “dilute-and-shoot” manner, so you can skip laborious,time-consuming sample concentration.
We generated these linear calibration curves for PFOS and PFOA standards (0.25, 0.5, 1, 2.5, 5,
10, 25, and 50 ng/L with R2 > 0.99) using direct aqueous injection on the 6495 LC/TQ.

x104
1.2
1.1
1

PFOA — 8 Levels, 8 Levels used, 8 Points, 8 Points used, 0 QCs
y = 233.309613* x + 130.582233
R^2 = 0.99841616
Type: Linear, Origin: Ignore, Weight: 1/x

Responses

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-4 -2

2

4

6

8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54
Concentration (ng/L)

PFOA — 8 Levels, 8 Levels used, 8 Points, 8 Points used, 0 QCs
y = 496.082875* x + 213.285758
R^2 = 0.99971471
Type: Linear, Origin: Ignore, Weight: 1/x

Responses

x104
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
-0.2

0

-4 -2

0

2

4

6

8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54
Concentration (ng/L)

Calibration curves for PFOS and PFOA, standards at 0.25, 0.5, 1, 2.5, 5, 10, 25, and 50 ng/L.

0.5 ng/L Standard
Compound

1.0 ng/L Standard

Average Area

RSD (%)

Average Area

RSD (%)

PFOS

423.3

5.3

668.3

4.9

PFOA

280.1

5.0

387.7

4.4

Enhanced sensitivity allows you to measure PFAS at extremely low levels without any loss in reproducibility
or robustness. The RSD for both PFOS and PFOA at a 0.5 ng/L concentration was <6%.

17

ASTM and EPA 8327 methods
ASTM method 7979-17 analyzes 21 PFAS in nonpotable waters—including surface water,
groundwater, and wastewater. It uses a simplified methanol dilution and large-volume (30 μL)
injection onto an LC/TQ. EPA 8327 follows identical sample preparation and analytical protocols.
Briefly, the method requires 5 mL of sample to be diluted with 5 mL of methanol, acidified, filtered,and injected onto an LC/TQ. This sample preparation technique increases throughput and reduces
contamination sources. Reporting levels for most compounds in this method are 10 ng/L with an
expected calibration range of up to 400 ng/L. Analysis at low ng/L levels demands a sensitive mass
spectrometer with good robustness to deal with different water matrices. It also requires strategies
to eliminate any PFAS contamination present in glass containers and consumables.

A simple technique for background removal or background delay
To perform the ASTM 7979 efficiently, we eliminated PFAS contamination quickly and easily using
an Agilent delay column and replacing any PTFE-containing tubing with PEEK. The Agilent 6470
LC/TQ achieved the detection limits required for all 21 PFAS in ASTM 7979. It also detected sevenother PFAS, including ADONA and 9Cl-PF3ONS (component in F53-B), that are being monitored
as “emerging” PFAS.
The following table shows recoveries for 28 PFAS, including those in the ASTM 7979 and EPA 8327
methods, at the low limit of quantification (LLOQ) levels of 10 and 20 ng/L with the Agilent 6470
LC/TQ. It includes six replicates with recoveries between 50% and 150%, as stated in the ASTM
method for all compounds. Most compounds showed recoveries between 80% to 120%, and therelative standard deviation (RSD) was below 20% for all compounds.

18

Spike Recovery, % at 10 ng/L

Spike Recovery, % at 20 ng/L

Average

RSD (%)

Average

RSD (%)

11Cl-PF3OUdS

119

1.8

116

3.6

4:2 FTS

115

5.8

96

8.6

8:2 FTS

103

15.2

114

0.8

9Cl-PF3ONS

110

10.5

108

6.8

ADONA

107

11.1

102

4.6

N-EtFOSAA

122

12.3

111

2.7

N-MeFOSAA

118

16.4

117

17.1

PFBA

101

10.7

104

10.6

PFBS

108

8.4

117

3.2

PFDA

110

17.1

107

6.6

PFDoA

101

13.3

106

13.3

PFDS

65

36.0

90

16.7

PFEESA

55

2.3

125

6.1

PFHpA

125

15.4

114

13.6

PFHpS

83

15.5

129

8.7

PFHxA

104

5.0

118

11.7

PFHxS

114

12.6

100

11.3

PFMBA

141

4.2

125

4.6

PFNA

102

18.8

106

9.4

PFNS

100

19.4

106

18.2

PFOA

121

11.1

108

9.1

PFOS

97

9.9

116

16.3

PFOSA

94

11.1

94

10.0

PFPeA

105

3.9

111

2.9

PFPeS

106

9.3

99

15.6

PFTreA

100

8.0

102

8.4

PFTriA

105

10.1

106

6.6

PFUnA

116

12.4

106

1.1

CompoundSpike recoveries and RSD (%) at 10 ng/L and 20 ng/L for all 28 PFAS, including those in ASTM 7979.
Consult application note 5994-0678EN for full method parameters.

Want a closer look?
Read application note 5994-0678EN: Simplified and Fast Analysis of Per- and Polyfluoroalkyl
Substances in Nonpotable Waters

19

Future-Proof Your Lab to Address
the Growing List of PFAS
More than 4,000 PFAS are currently known to have been used, and this list continues to expand.
But current regulatory monitoring lists globally only look at a very limited subset of PFAS. Whileregulatory lists may vary across regions, it is safe to assume that many additional PFAS will bemonitored across the globe in time.
The following table shows method detection and method quantification levels (MDLs and MQLs)of more than 50 PFAS representing 16 different classes of legacy and emerging PFAS analyzed inwater. We used a single analytical method that covered both legacy and emerging PFAS, including
diPAPs, ADONA, and PFESAs (components of F-53B). Samples were analyzed on the 6495 LC/TQ,
making use of its enhanced sensitivity to analyze all these PFAS in one injection withoutcompromising detection levels.
The results can be summarized as follows:– Instrument detection limits (IDLs) ranged from 2.5 to 469 fg on column for all compounds.
– Calculated IDLs were below 10 fg on column for 22 compounds from the classes PFCA, PFSA,
FTS, FOSAA, and Cl-PFAES—plus the compounds FOSA, diSAmPAP, and ADONA.
– MDLs ranged from 0.28 to 18 ng/L. MQLs ranged from 0.35 to 26 ng/L,with 46 PFAS having quantification levels below 5 ng/L.
– Run time was less than 12 minutes.
MDLs for the 53 PFAS were calculated based on US EPA 40 CFR Part 136, Appendix B, Revision 2.
Seven 250 mL aliquots of ultrapure water were spiked at 5 ng/L for all compounds except FTCAs,
FOSEs, and PFDPA, which were spiked at 20 ng/L.

20

PFAS analysis using SPE extraction and triple quadrupole LC/MS.
Compound

MDL (ng/L)

MQL (ng/L)

Extraction Method
Accuracy (%)

MethodPrecision RSD (%)

PFBA

0.59

0.75

93

4

PFPeA

0.71

0.89

92

5

PFHxA

0.87

1.1

90

6

PFHpA

0.84

1.1

96

6

PFOA

0.28

0.35

93

2

PFNA

0.61

0.77

98

4

PFDA

0.71

0.89

98

4

PFUnA

0.80

1.0

85

6

PFDoA

1.2

1.5

93

8

PFTrA

1.4

1.8

78

12

PFTeA

0.67

0.84

93

5

PFBS

0.49

0.62

89

3

PFPeS

1.2

1.5

100

9

PFHxS

0.69

0.88

91

5

PFHpS

0.79

1.0

99

6

PFOS

0.78

1.0

95

5

PFNS

1.0

1.3

87

7

PFDS

1.1

1.3

83

8

PFDoS

1.4

1.8

72

13

ADONA

0.82

1.0

88

6

6:2 FTCA

13

17

103

16

8:2 FTCA

16

19

92

23

10:2 FTCA

17

21

67

28

6:2 FTUCA

1.7

2.1

121

9

8:2 FTUCA

1.6

2.0

111

10

10:2 FTUCA

2.8

3.6

87

19

3:3 FTCA

1.4

1.7

118

7

Want a closer look?
Read application note 5994-0919EN: Analysis of >50 Legacy and Emerging PFAS in Water
Using the Agilent 6495C LC/TQ

21

Ultrapure water blanks (n=7) were extracted alongside method validation samples. Method accuracy
was expressed as the mean recovery of method validation samples for the expected concentration asa percentage and relative standard deviation.
The versatility, accuracy, and robustness of our LC/TQ systems let you add several PFAS to yourmethod without sacrificing sensitivity or throughput. If you need to add new or novel PFAS to yourmethods, Agilent LC/MS compound optimizer automates the process and provides reliable compoundparameters in minutes.
In addition, Agilent consultants can help make your PFAS analysis hassle free. Our decades ofexperience, combined with our MRM and method data capabilities, can get your lab up to speed quickly.

PFAS analysis using SPE extraction and triple quadrupole LC/MS.
Compound

MDL (ng/L)

MQL (ng/L)

Extraction Method
Accuracy (%)

Method Precision
RSD (%)

5:3 FTCA

1.8

2.3

103

11

7:3 FTCA

2.4

3.1

75

20

PFHxPA

2.9

3.4

104

17

PFOPA

4.6

5.8

100

26

PFDPA

18

26

82

10

6:2 diPAP

1.9

2.4

81

14

6:2/8:2 diPAP

1.9

2.4

123

11

8:2 diPAP

0.83

1.1

93

6

6:2 Cl-PFESA

1.3

1.7

88

9

8:2 Cl-PFESA

1.1

1.4

80

9

4:2 FTS

2.7

3.4

93

16

6:2 FTS

0.56

0.7

90

4

8:2 FTS

1.3

1.7

87

9

10:2 FTS

1.4

1.8

66

13

FOSA

0.76

1.0

70

7

MeFOSA

4.0

5.0

127

18

EtFOSA

2.1

2.7

80

19

FOSAA

3.2

4.0

91

17

MeFOSAA

1.4

1.7

106

8

EtFOSAA

1.5

1.9

93

10

MeFOSE

2.9

3.7

96

5

EtFOSE

4.9

6.2

93

9

6:6 PFPiA

1.2

1.5

74

10

6:8 PFPiA

1.8

2.3

95

12

8:8 PFPiA

3.1

4.0

138

11

diSAmPAP

3.3

3.0

76

19

22

For more information on theanalysis, fate, and removal of
PFAS in water treatment plants
using the 6495C LC/TQ, pleasewatch this on-demand webinar by
Prof. Bradley Clarke, University of
Melbourne, Australia.

23

Automate PFAS sample preparation with online SPE
Solid phase extraction for PFAS can be time consuming and tedious, and offers the potential ofadding contamination while reducing reproducibility of the overall analysis. Automated online SPE
can provide similar detection limits while using a fraction of the water sample while completely
automating sample extraction and analysis which reduces labor time, increases accuracy of
results and drastically improves throughput. Further, it also reduces costs of expensive andenvironmentally unfriendly solvent usage and the amount of isotopically labelled standards needed.
While automation can seem complicated, Agilent’s online SPE setup coupled to the most reliable
and robust Agilent mass spectrometers provide seamless software interface to make the process
much simpler, letting you reduce the loads of doing manual and tiresome offline extractions.

Agilent InfinityLab Online SPE solution for analysis of PFAS and emerging
contaminants in water.

Solvents for LC

Solvents required​for SPE​​ rinsing
and conditioning

1290 Infinity Flexible Cube​
The host of the switching valves

Triple quad MS for quantification24

Linearity, dynamic range (0.1-1000 ng/L), and sensitivity of PFOA analysis using Agilent InfinityLab Online SPE with a 6470 LC/MS/MS.
PFOA - 9 Levels, 9 Levels Used, 9 Points, 9 Points Used, 7 QCs
x10 1 y = 0.046187 * x + 0.025018
4.8
R^2 = 0.99905565
Type:Linear, Origin:Ignore, Weight:1/x
4.6

4.4
4.2
4

0.1 ng/L or 0.1 ppt

3.8
3.6
Counts3.4
3.2
3
Relative responses

2.8
2.6
2.4
2.2
Acquisition time (min)

2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

Relative concentration

-0.2
-0.4
0

100

200

300

400

500

600

700

800

900

1000

Concentration (ng/L)

25

Discovering New PFAS Using
High-Resolution LC/Q-TOF
Triple quadrupole LC/MS is a great tool for measuring known PFAS at extremely low levels. However,
several thousand PFAS are used in commerce, and many have yet to be identified in the environment.
This is where a quadrupole time-of-flight (Q-TOF) mass spectrometer shows its value.
LC/Q-TOF instruments allow you to perform untargeted analysis and to identify unknown PFAS.
You can also use libraries to screen for PFAS without the need for analytical reference standards.
In addition, LC/Q-TOF technology lets you acquire data over a wide dynamic range. That means
you can analyze trace sample amounts next to abundant peaks in the same spectrum. Together
with simultaneous high sensitivity and high resolution, Agilent LC/Q-TOF instruments are ideal for
identifying unknown PFAS in environmental samples.

26

In the following spectra, we identified 6:2 FtS with high mass accuracy and isotope fidelity in a
wastewater sample. The compound was later verified with a reference standard to have the same
retention time by LC/Q-TOF.

Identification of PFAS PCDL + Std ID.

104
4

WWTP1 Final Effluent
Cpd 11: 6:2 FTS / C2H4-perfluorooctane sulfonate: -ESI EIC (426.96790, 854.94309, 855.94614) Scan Frag=350.0V ETP FEFF 1.d

3.5

104
2.6
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0

6:2 FTS
Mass: 427.97458
Mass diff: -1.41 ppm
Score: 98.11

3
2.5
2
1.5
1
0.5
0

4.5

5

5.5

6

6.5

7

7.5

8

424

8.5

425

426

9

427
428
429
430
Counts vs. Mass-to-charge (m/z)

9.5

431

432

10

10.5

11

10

10.5

11

Counts vs. acquisition time (min)Standard
Cpd 22: 6:2 FTS / C2H4-perfluorooctane sulfonate: -ESI EIC (426.96790, 854.94309, 855.94614) Scan 6-2 FTS.d
104
2.25
2
1.75
1.5
1.25
1
0.75
0.5
0.25
0
4.5

5

5.5

6

6.5

7

7.5

8

8.5

9

9.5

Counts vs. acquisition time (min)27

Since LC/Q-TOF collects all ionizable species in the analysis, you are able to retroactively mine fordata and identify compounds without sample reinjection. Here, 6:2 FtS samples were identified at
all treatment stages of the wastewater plant samples. Therefore, the abundance of this compound
could be plotted to track efficacy in the wastewater treatment plant.
Alternatively, an LC/Q-TOF system can be used for fingerprinting PFAS sources, or for studying
PFAS composition in aqueous film-forming foams (AFFF).
Mass Profiler Professional (MPP) software can be used to identify new PFAS and performsource appropriation and fingerprinting studies. It includes statistical profiling tools like principal
component analysis (PCA), hierarchical clustering, and Kendrick mass defect plots.

F
A and B:
Compounds removed

AC
C, D, and E:
Compounds formed

F:
Compounds unchanged

D

B

E

Molecular feature abundance
normalized intensity (Log2)

28

Color range
-11.2

0

11.2

Capture, Analyze, and Share Water Quality Data
The advanced data mining and processing tools in the Agilent MassHunter software suite let you
quickly and accurately extract available information from the analytes in your samples.
– Take advantage of powerful data collection, processing, and reporting tools. Breeze throughapplication-specific workflows with comprehensive support of GC, GC/MS, and LC/MS technology.
– Use one software for all Agilent GC, GC/MS, and LC/MS instruments, including single quadrupole,tandem quadrupole, and Q-TOF.
– Analyze complex environmental samples with compound-based analysis and reporting workflows,
using Agilent MassHunter Quantitative Analysis with Quant-My-Way customization.
– Simplify target and suspect screening with highly confident compound identification using Agilent
Personal Database and Libraries.
– Quickly build targeted screening and quantitation acquisition methods using the Agilent PFAS MRM
database for triple quadrupole LC/MS of a few—or a few dozen—PFAS compounds.

29

Agilent CrossLab services
CrossLab is an Agilent capability that integrates services and consumables tosupport workflow success and important outcomes like improved productivity
and operational efficiency. Through CrossLab, Agilent strives to provide insightin every interaction to help you achieve your goals. CrossLab offers method
optimization, flexible service plans, and training for all skill levels. We have manyother products and services to help you manage your instruments and your lab
for best performance.
Learn more about Agilent CrossLab and see examples of insight that leads togreat outcomes at www.agilent.com/crosslab

Learn more:
www.agilent.com/chem/environmental
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www.agilent.com/chem/pfas
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www.agilent.com/chem/contactus
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RA.44141.2106018519
This information is subject to change without notice.
© Agilent Technologies, Inc. 2020
Published in the USA, November 24, 2020
5994-1322EN