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Materials and Methods
Sample Preparation
PFC Standard Solutions
Standards for perfluoro-1-butanesulfonate (PFBS), perfluoro-
1-hexanesulfonate (PFHxS), perfluoro-n-heptanoic acid
(PFHpA), perfuoro-1-decanesulfonate (PFDS), perfluoro-n-
undecanoic acid (PFUnA), and perfluoro-n-dodecanoic acid
(PFDoA) were obtained from a proprietary source. A stock
solution of a mixture of these six PFCs was prepared at
a concentration of 1 mg/L. Calibration solutions, with
concentrations of 0.04-2.5 ng/mL (ppb), were prepared
by serial dilution of the stock solution in 60:40 (v/v)
methanol/water. Two internal standards, m-PFUnA and
m-PFHxS, were added into each calibration solution and
sample at 2 ng/mL (ppb) concentration.
Milk Matrix A
A 2 g human breast milk sample, obtained from a proprietary
source, was diluted in acetonitrile to precipitate proteins.
Weak anion exchange solid-phase extraction was performed
and the resulting PFC extract was eluted using 2%
ammonium hydroxide in methanol, evaporated to dryness
and reconstituted in 60% methanol/water (0.6 mL).
Milk Matrix B
The six PFCs were spiked into Matrix A at concentrations
of 0.1 ng/mL to generate Matrix B.
Milk Matrix C
To generate Matrix C, six PFCs were spiked into Matrix A
at concentrations of 0.3 ng/mL.
Milk Matrix D
Matrix D was prepared by spiking the six PFCs into
Matrix A at concentrations of 1.0 ng/mL.
LC/MS Analysis
Instrumentation
LC/MS analysis was performed on a PFC-free Accela 600 LC
system and PAL autosampler coupled to a TSQ Vantage
triple-stage quadrupole mass spectrometer. The PFC-free
Accela pump was equipped with a pre-cleaned PFC-free
degasser and all Teflon tubing was replaced with PEEK tubing.
LC Parameters
Column:
Thermo Fisher Scientific Hypersil GOLD PFP
column (100 x 3 mm, 1.9 µm particle size)
Mobile Phase:
A: 5 mM ammonium acetate and 10%
methanol/water
B: 2 mM ammonium acetate/99% methanol
Flow Rate:
see gradient
Column Temperature:
ambient
Sample Injection Volume:
10 µL
Gradient:
Time (min)
A% B% Flow rate (µL/min)
0.0
70
30
400
0.5
70
30
400
1.0
54
46
400
4.0
30
70
400
9.0
12
88
400
9.4
12
88
400
9.6
0
100
400
9.7
0
100
500
11.0
0
100
500
11.1
70
30
500
14.5
70
30
500
15.0
70
30
400
MS Parameters
Negative Ion Mode Ionization with HESI Probe
Heated Electrospray Ionization Source Conditions:
Spray Voltage:
3500 V
Capillary Temperature:
300 °C
Sheath Gas:
60 au
Auxiliary Gas:
15 au
Vaporizer Temperature:
400 °C
Resolution for SRM Setup:
Q1, Q3 = Unit [0.7 Da. FWHM]
Resolution for H-SRM Setup: Q1 = 0.2 Da. FWHM; Q3 = 0.7 Da. FWHM
#
Parent
Product
Collision Energy
RT Start
RT End
S-Lens
Name
1
299.0
80.2
43
3.15
4.15
115
PFBS
2
299.0
99.2
34
3.15
4.15
115
PFBS
3
299.0
169.0
23
3.15
4.15
115
PFBS
4
399.0
80.2
45
4.7
5.7
89
PFHxS
5
399.0
99.2
35
4.7
5.7
89
PFHxS
6
399.0
169.1
29
4.7
5.7
89
PFHxS
7
403.0
84.2
43
4.7
5.7
89
m-PFHxS
8
403.0
103.2
37
4.7
5.7
89
m-PFHxS
9
363.0
169.0
10
5.0
6.0
51
PFHpA
10
363.0
319.0
17
5.0
6.0
51
PFHpA
11
598.9
99.1
47
7.1
8.1
128
PFDS
12
598.9
230.1
50
7.1
8.1
128
PFDS
13
598.9
80.3
47
7.1
8.1
128
PFDS
14
562.9
269.0
18
7.75
8.75
62
PFUnA
15
562.9
519.0
12
7.75
8.75
62
PFUnA
16
564.9
520.0
18
7.75
8.75
64
m-PFUnA
17
612.9
169.0
25
8.4
9.4
78
PFDoA
18
612.9
569.0
12
8.4
9.4
78
PFDoA
Table 1: SRM transitions monitored for the detection of PFBS, PFHxS, PFHpA, PFDS, PFUnA and PFDoA.