Analyses of Per- and Polyfluoroalkyl Substances in Water Using Ion Exchange
Solid-Phase Extraction and LC MS/MS with an Activated-Carbon Delay Column
Reika Takahara, Takumi Kunieda, Kazuyuki Ishii, Manabu Takayanagi, Hiroshi Hayashida,
GL Sciences Inc., Tokyo, Japan

Introduction

Results

The phrase per- and polyfluoroalkyl substances (PFAS) is a general term used for organofluorine compounds.
These substances are known to degrade slowly; therefore, they persist in the environment for a long time.
Their toxicity and the environmental pollution they cause have attracted worldwide attention, and research
to mitigate these effects continues. The methods of solid-phase extraction (SPE) and liquid chromatography
tandem mass spectrometry (LC MS/MS) have been used to analyze PFAS in drinking water under EPA
methods 537.1 and 533. Further, some PFAS are known to elute from the materials of laboratory equipment
and can cause contamination to the sample. Therefore, precautions must be taken to accurately quantify
PFAS. In particular, care must be taken to minimize the effects of PFAS background and contamination eluting
from fluorinated resins such as polytetrafluoroethylene (PTFE), which are commonly used as components inLC systems. A known countermeasure for such effects is to delay the elution time of the blank peak byconnecting a delay column packed with a C18 (ODS) material before the autosampler and shift the retention
time relative to the peak derived from the sample. However, it is difficult to increase the difference between
the two retention times sufficiently using a conventional C18 column. The column sizes are limited becauseof the relationship between the rise in pressure and the gradient delay time. Therefore, to perform stable
PFAS analysis, we have developed a new delay column that is packed with high-purity activated-carbon beads.
Furthermore, two SPE cartridges of different sizes, 250 mg and 150 mg, were used and reference standard
addition recovery tests were conducted for each cartridge.

Using our delay column packed with high-purity activated carbon to analyze the PFAS, we confirmed that thepeak to be analyzed and the blank peak were separated sufficiently with respect to the retention time.
Further, we confirmed that the PFAS 21 component in water can be extracted using SPE cartridge, InertSep
MA-2. As a result of the recovery test and the use of extracted tap-water samples, we found the linearity ofthe measurements as 0.99 or better in the range 1 20 ng/L and the repeatability at 5 ng/L was 16%. Whenusing a 150 mg SPE cartridge, the volume of sample water and the eluting solvent could be reduced, theevaporating operation of the elution solvent could be omitted, and the sample preparation time wasshortened.
Background of PFOS and PFOAPFOADelayColumn
For PFAS

Table.3 Pressure Comparison of Delay Columns
PFOS0246810

Analytical column
1214161820

Delay Column

PressureNone

19.8 MPa

Delay Column for PFAS
(30 3.0 mm)

19.8 MPa

C18 (ODS) column(50 2.1 mm, 3 µm)

23 MPa22

Background of PFOS and PFOA
PFOA

C18 (ODS)
columnInertSustain C18-HP(150 2.1 mm, 3 µm)

PFOS02468101214161820

22

Fig.3 Comparison chromatogram of Delay columns

Methods
For LC MS/MS, we used a 4000 QTRAP (AB SCIEX LLC, MA USA) instrument. For the delay column, we packedhigh-purity, spherical, activated carbon in the LC column hardware and installed in the HPLC system. We used
an InertSustain C18 HP 150 mm
2.1 mm analysis column with 3- -particle size (GL Sciences Inc., TokyoJapan). We prepared a standard sample by diluting a PFAS 21 mixture standard PFAC-MXC (Wellington
Laboratories, Ontario, Canada) and adding it to the sample water. We used 13 mixtures of MPFAC-C-ES(Wellington Laboratories, Ontario, Canada) as external standards. For sample preparation, we used SPE
cartridge InertSep MA-2 250-mg (GL Sciences, Inc., Tokyo Japan) packed with a methacrylate polymer
comprising a weak anion-exchange group (diethyl amine). We performed all the operations fromconditioning of the SPE cartridge to the evaporation of the elution solvent using the automated SPE
instrument AquaTrace ASPE899 (GL Sciences, Inc, Tokyo Japan). We passed a 500-mL sample through the SPE
cartridge and then eluted it using 5 mL of 0.1% ammonia methanol. Subsequently, we heated the sample,
exposed it to nitrogen gas, and concentrated it to a volume of 0.5 mL. For realizing a rapid SPE method, weused an InertSep MA-2 150-mg cartridge. We passed 30 mL of sample water through the cartridge and thenused 1 mL of the eluting solvent. We did not distill off the solvent after elution. We added the mixture ofstandard MPFAC-C-IS (Wellington Laboratories, Ontario, Canada) to the eluate as an injection standard. To
avoid contamination of the PFAS, we used a high-purity polypropylene vial in the autosampler and applied analuminum foil and silicon septum cap on the vial. Before use, we soaked and washed all the glassware andpipette tips using methanol (Kanto Chemical Co., Inc, Japan). We also automatically washed the AquaTrace
sample line tube with methanol.
Autosampler Oven

Table 1 Liquid Chromatograph Conditions
ColumnInertSustain C18 (GL Sciences Inc, Japan)
3 m HP, 150 2.1 mm I.D.

Delay Column

Delay Column for PFAS (GL Sciences Inc, Japan)
30
3.0 mm I.D.

Mobile Phase A

10 mmol/L Ammonium acetate

Mobile Phase B

Acetonitrile

Flow Rate

0.3 mL/min

Column Temp

40Injection Vol

1 µL

Gradient (A/B)

80/20 2 min - 80/20 13 min - 0/100 2 min 100/0-0.1 min - 80/20 6 min - 80/20

PFBAPFPeAPFHxA
PFHpA
PFOA
PFNA
PFDA
PFUnDA
PFDoDA
PFTrDA
PFTeDAPFHxDA
PFOcDA
PFBS
PFPeS
PFHxS
PFHpS
PFOS
PFNS
PFDS
PFDoS

H2O 4 mL

80706050402mL

3mL

Pump B
EluentFig.1 Delay column installation position

DPEP

CE

CXP

-45-50-50-55-45-65-65-65-40-50-50-65-65-80-100-80-100-90-105-80-115-10-10-10-10-10-10-10-10-10-10-10-10-10-10-10-10-10-10-10-10-10-14-11-15-14-14-16-14-16-17-19-15-17-17-62-70-80-104-95-116-80-126-9-9-9-9-9-9-9-9-9-9-9-9-12-3-13-3-15-3-13-3-13

No pH adjustment required.

13C-PFOS/ 13C-PFOA mix 50 µL

5 mL/minExtraction
Standard

Transition
Q1/Q3DP

EP

CE

CXP

C4-PFBA
C5-PFPeA
C5-PFHxA
13
C4-PFHpA
13
C8-PFOA
13
C9-PFNA
13
C6-PFDA
13
C7-PFUdA
13
C2-PFDoA
13
C2-PFTeDA
13
C3-PFBS13
C3-PFHxS
13
C8-PFOS

217/172
268/223
318/273367/322421/376472/427
519/474
570/525
615/570715/670
302/80402/80507/80-30-25-30-30-30-30-40-60-40-45-75-75-110-10-10-10-10-10-10-10-10-10-10-10-10-10-14-12-14-14-14-14-16-16-18-18-70-84-90-31-11-47-19-9-11-13-7-15-17-13-13-13

Injection
Standard

Transition
Q1/Q3

DPEPCE

CXP

13216/172-30-10-14-19

13415/370-30-10-14-913515/470-35-10-16-35

13503/80-105-10-120-131313
13

C3-PFBA
C2-PFOAC2-PFDA
C4-PFOS

Conditioning

Sample 30 mL

H 2O 2 mL

No pH adjustment required.

13C-PFAS-ES mix 1 µL

0.1% NH4OH in MeOH 2 mL
MeOH 2 mL

InertSep MA-2250 mg

5 mL/min

InertSep MA-2150mg/3mL
Wash

H 2O 5 mL

H2O 1 mLSPE Dry

10 min10 minElute
0.1% NH4OH in MeOH 5 mL

Elute
0.1% NH 4OH in MeOH 1 mL

Evaporate

LC/MS/MS
Adjustment
MeOH 0.5 mL
LC/MS/MS

Fig.2 Solid phase extraction method

4mL

5mL

Table.4 Repeatability, Linearity, and Recovery

Pump AWashGL Sciences Inc.
Mar 29, 2022
Doc No.: GL-SM210001R416182022PFPeS-1
PFNS-1PFPeA-1
PFNA-1
PFTrDA-1
HFPO-DA1PFHxS-1PFDS-1
PFHxA-1
PFDA-1PFTeDA-1

PFHpS-1
PFDoS-1
PFHpA-1
PFUnDA-1
PFHxDA-16mL

7mL

8mL

Fig.5 SPE cartridge InertSep MA-2 250mg elution profile

SPE Dry

www.glsciences.com

14PFBS-1
PFOS-1PFBA-1PFOA-1
PFDoDA-1
PFOcDA-1

90Compounds

MeOH 4 mL

12Time, min

0Degassing unit

Sample 500 mL

10104000 QTRAP (AB SCIEX LLC, MA USA)

Conditioning

8100

Delay Column

Mixer

0.1% NH 3 in MeOH 4 mL

6Fig.4 PFAS 21 chromatogram

1mL

Table 2 Compound and Mass Spectrometer Conditions
Transition
Q1/Q3
213/169
263/219313/269
363/319
413/369
463/419513/469
563/519613/569
663/619713/669
813/769
913/869
299/80349/80399/80449/80499/80549/80599/80699/80430

MS

ColumnNexera UFLC (Shimadzu Corporation, Japan)

Compounds

220

SystemSystem

1.2e5
1.1e5
1.0e5
9.0e4
8.0e4
7.0e4
6.0e4
5.0e4
4.0e4
3.0e4
2.0e4
1.0e4
0.0

Table.5 Repeatability Using a Small SPE (150 mg)

Repeatability CalibrationLinearity
Recovery(CV %, n = 5)
Range
(1 20 ng/L) Rate (%)

R.T(min)

PFBA

131-500.9999

80

4.11

PFPeA

81-500.9999

100

6.69

PFHxA

141-500.9999

96

7.88

PFHpA

71-500.9996

107

8.76

PFOA

101-500.9999

99

9.52

PFNA

101-500.9999

87

10.25

PFDA

71-50

1101

10.95

PFUdA

71-500.9997

104

11.65

PFDoA

51-50

0.999996

12.32

PFTrDA

51-50

0.9997108

12.96

PFTeDA

101-500.9999

88

13.58

PFHxDA

31-500.9999

119

14.67

PFODA

81-100.9999915.5

PFBS

121-500.9998

92

8.15

PFPeS

61-500.9998

95

PFHxS

81-20

0.9996

97

9.97

PFHpS

91-200.999

9310.73

PFOS

161-200.9995

102

11.45

PFNS

91-100.996

95

12.13

PFDS

41-20

0.9992

86

12.77

PFDoS

101-100.999

83

13.95

9.13

Compounds
PFBA
PFPeA
PFHxA
PFHpA
PFOA
PFNA
PFDA
PFUnDA
PFDoDA
PFTrDA
PFTeDAPFHxDA
PFOcDAHFPO-DA
PFBS
PFPeS
PFHxS
PFHpS
PFOS
PFNS
PFDS
PFDoS

Repeatability(CV %, n = 5)
8.4
7.5
8.7
9.2
17.5
1318.219.9
14
10.8
8.4
12.9
10.2
8.8
14.8
11.9
16.6
18.1
18.4
24.3
23.8
15.8

RecoveryRate (%)
96.2
96.8
95.6
106.1
92.7
97.6
83.3
63
40.1
38.8
44.4
64.8
80.1
96.8
103.7
98.2
98.3
90.2
90.4
68.6
52
39.4

Conclusions
Use of a delay column packed with high-purity, spherical, activated-carbon beads reduced both the system
and mobile-phase blanks and the PFAS were analyzed with high accuracy. When we used InertSep MA-2 as
the SPE cartridge which is a weak anion-exchange cartridge without a reverse-phase mode, we obtained astable, high recovery rate.

References
1. EPA method 537.1: Determination of selected per- and polyfluorinated alkyl substances in drinking waterby solid-phase extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS),Version
1.0,November 2018
2. EPA method 533: Determination of per- and polyfluoroalkyl substances in drinking water by isotope
dilution anion-exchange solid phase extraction and liquid chromatography/tandem mass spectrometry
3. ISO 21675: 2019 - Water quality Determination of perfluoroalkyl and polyfluoroalkyl substances (PFAS)
in water Method using solid phase extraction and liquid chromatography tandem mass spectrometry
4. Standard test method in water, Ministry of Health, Labor and Welfare, Japan
5. Water supply test method, 2011 Edition, Japan Water Works Association