Environ. Sci. Technol.
2009, 43, 79027908
Branched Perfluorooctane Sulfonate
Isomer Quantification and
Characterization in Blood Serum
Samples by HPLC/ESI-MS(/MS)
N I C O L E R I D D E L L , * ,
GILLES ARSENAULT,
JONATHAN P. BENSKIN,
BROCK CHITTIM,
JONATHAN W. MARTIN,
ALAN MCALEES, AND
ROBERT MCCRINDLE
Wellington Laboratories Inc., Guelph, Ontario, N1G 3M5,
Canada, Department of Laboratory Medicine and Pathology,
University of Alberta, Edmonton, Alberta, T6G 2G3, Canada,
and Department of Chemistry, University of Guelph, Guelph,
Ontario, N1G 2W1, Canada
Received April 28, 2009. Revised manuscript received
August 7, 2009. Accepted August 31, 2009.
Perfluorooctane sulfonate (PFOS) is a global contaminant
and is currently among the most prominent contaminants in
human blood and wildlife samples. Although total PFOS
(PFOS) analytical methods continue to be the most commonly
used for quantification, recent analytical method developments
have made it possible to resolve the various isomers of
PFOS by HPLC-MS/MS. Characterized technical PFOS standards
(i.e., containing a mixture of PFOS isomers) are now available
that enable isomer specific quantification of PFOS, however
the advantages of such an analysis have not yet been examined
systematically. Herein, PFOS isomers have been individually
quantified for the first time in real samples and the results are
compared to a traditional PFOS method; the influence of
analytical standards and isomer specific electrospray and MS/
MS behavior were also investigated. The two human
serum standard reference materials chosen for analysis
contained dramatically different PFOS isomer profiles (30-50%
total branched isomers) emphasizing that isomer patterns
should not be ignored and may provide useful information on
exposure sources (i.e., direct exposure to PFOS vs indirect
exposure from PFOS-precursors). Depending on the sample
and the particular MS/MS transition chosen for PFOS analysis
(i.e., 499f80 or 499f99), PFOS concentrations may be
over- or underestimated compared to the isomer specific
analysis. Differences in the extent of in-source fragmentation
and MS/MS dissociation contributed to the systematic
analytical bias. It was also shown that PFOS data are prone
to interlaboratory variation due to various choices of PFOS
standards and instrumental conditions used. In the future, for
either PFOS or isomer specific PFOS analyses, we suggest
* Corresponding author phone: +1 519 822 2436; fax: +1 519 822
2849; e-mail: nicole@well-labs.com.
Wellington Laboratories Inc.
University of Alberta.
University of Guelph.
7902 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 20, 2009
that accuracy can be maximized and interlaboratory
discrepancies minimized by using a common chemically
pure technical PFOS standard characterized by 19F NMR.
Introduction
Interest in the widespread distribution of perfluoroalkyl
chemicals, including perfluorooctanesulfonate (PFOS,
C8F17SO3-), in the environment (1-6) and their accumulation
in humans has been steadily increasing (7-15). Quantifica-
tion of PFOS in human plasma, blood serum, and whole
blood is not trivial. A recent study addressed the biases
associated with matrix effects in LC/MS-MS quantification
methods (15). However, the difficulties associated with
analyzing for PFOS in various matrices is compounded by
the fact that its production from linear alkyl precursors using
electrochemical fluorination is not a clean process but,
instead, gives complex mixtures. For example, the commercial
PFOS formerly produced by 3 M was a mixture of ca. 70%
linear and ca. 30% branched isomers as measured by 19F
NMR spectroscopy (16-18). Interest in the presence of the
branched isomers of PFOS in the environment, particularly
in biological samples, has also been growing due to issues
relating to the quantification of total PFOS (19, 20) as well
as questions about exposure sources and differences in
bioaccumulation potential (19) and toxicity (21) among the
various isomers. Unfortunately, although qualitative stan-
dards for many of the PFOS branched isomers are com-
mercially available, quantitative standards are not.
The generation of accurate quantitative data for PFOS is
important for addressing environmental fate and toxicity
questions, and ultimately for enabling accurate risk assess-
ments by chemical regulators. Deviations from design values
in round-robin interlaboratory studies demonstrate some of
the difficulties associated with the analysis of PFOS in
biological samples (22). The most common analytical method
used for the analysis of PFOS is HPLC-MS/MS (23, 24). Despite
the fact that multiple PFOS isomers are thought to be present
in any environmental sample, traditional HPLC methods
often avoid their separation so that the entire broadened
peak, or unresolved group of peaks, may be easily integrated.
This will be referred to henceforth as a PFOS method. It
has already been acknowledged that PFOS methods can
lead to a systematic quantification bias of unknown propor-
tions, since the various branched isomers are expected to
have different ionization and fragmentation efficiencies
during electrospray ionization (23, 25) and distinct collision
induced dissociation (CID) patterns in MS/MS (25, 26).
However, the extent of this bias has not previously been
quantified, and quantitative isomer specific PFOS analysis
has not yet been demonstrated as an alternative for any
sample.
In the current work, isomer specific quantification of PFOS
is demonstrated in human serum for the first time and the
cumulative impact of isomer specific response factors and
various standards is discussed by comparison with a PFOS
quantification method.
Materials and Methods
Chemicals, Standards, and Serum Samples. The major
branched isomers of PFOS were previously synthesized,
isolated, and characterized as sulfonamide derivatives (17),
and these were converted to the respective sulfonates (Figure
1) using proprietary methods at Wellington Laboratories
(Guelph, ON) and are available as qualitative standards. HPLC
grade methanol (MeOH) and water were purchased from
10.1021/es901261v CCC: $40.75 2009 American Chemical Society
Published on Web 09/17/2009
FIGURE 1. Structures of the prevalent PFOS isomers in technical mixtures and in samples.
Caledon (Georgetown, ON). Reference standards of linear
PFOS [13C4-MPFOS (MPFOS) and LPFOS] and a mixture of
linear and branched isomers of PFOS (brPFOSK) were
obtained from Wellington Laboratories (Guelph, ON). A
sample of technical PFOS was purchased from TCI America
(Portland, OR).
Freeze-dried human serum samples A and B repre-
senting 10.0 and 10.7 mL, respectively, of reconstituted
human serum were acquired through participation in the
2006 Worldwide PFC intercalibration study (22). Human
Serum Sample A (NIST SRM 1589a) was a freeze-dried
standard reference material (SRM) collected by NIST
consisting of 50 pooled blood samples of 5 mL from donors
who consumed fish caught around the Great Lakes. Human
Serum Sample B (NIST SRM 1957) was a freeze-dried SRM
also collected by NIST. This sample was prepared from a
serum pool of 200 L across the United States. Detailed
information on both SRMs and certificates of analysis can
be found at www.nist.gov.
LC/ESI-MS and LC/ESI-MS/MS. Separation, identifica-
tion, and determination of the response factors of the PFOS
isomers were completed using HPLC/ESI-MS and HPLC/
ESI-MS/MS experiments on a Waters Acquity Ultra Perfor-
mance LC (Waters, Milford, MA) interfaced to a Micromass
Quattro micro atmospheric pressure ionization (API) mass
spectrometer (Micromass, Manchester, UK). Chromatogra-
phy was performed on an Acquity UPLC BEH Shield RP18
column (1.7 m, 2.1 100 mm) with gradient elution. The
gradient started at 46% 80:20 MeOH/ACN and 54% water
(both containing 10 mM ammonium acetate) at a flow rate
of 350 L/min. The program was ramped to 49% 80:20 MeOH/
ACN by 6 min, held for 17 min, then ramped again to 90%
80:20 MeOH/ACN over 0.50 min, held for 1 min, and returned
to initial conditions. The mass spectrometer was set up in
negative-ion electrospray mode with the following conditions:
capillary voltage 2.00 kV, source temperature 110 C, cone
gas 60 L/h, desolvation gas flow 750 L/Hr, desolvation gas
temperature 350 C, cone voltage 60 V, collision energy 40
eV, and gas cell pressure 3.5e-3 mbar. The MS tune
parameters for the individual branched isomers were op-
timized, however essentially no difference in their response
factors was observed. Reduction in the cone voltage did not
reduce the in-source fragmentation observed for isomers 2,
9, 10, and 11. Optimization of MS/MS tune parameters for
isomer specific transitions using the individual isomers also
did not result in a significant increase in response for any of
the isomers (see Supporting Information (SI) for further
details and Table S1).
Serum samples A and B were extracted in triplicate and
analyzed using an isomer specific analytical method (19).
Briefly, 1 mL of serum was diluted with 0.1 M formic acid
containing MPFOS as the internal standard and sonicated
for 20 min. A 200 mg Oasis HLB cartridge (Waters) was
conditioned with HPLC grade methanol (6 mL) and 0.1 M
formic acid (6 mL). The samples were loaded onto the
cartridge and slowly passed through under vacuum. The
cartridges were rinsed with 15 mL of 0.1 M formic acid, 6 mL
of 50% 0.1 M formic acid/50% methanol, and 1 mL of 1%
NH4OH in water. The cartridge was then purged with air and
the analytes were eluted with 6 mL of methanol (1% NH4OH)
into 15-mL centrifuge tubes. The extracts were evaporated
under N2 to 0.5 mL and transferred to methanol rinsed
polypropylene microvials (Fisher), with polyethylene caps
(Supelco). Analysis by HPLC-MS/MS involved 10-L injec-
tions of extract onto a FluoroSep RP Octyl column (3 100
, 15 cm 2.1 mm, ES Industries, West Berlin, NJ). Gradient
elution conditions were 200 L/min, and starting conditions
were 40% A (water adjusted to pH 4.0 with ammonium
formate) and 60% B (100% methanol). Initial conditions were
held for 0.3 min, ramped to 64% B by 1.9 min, increased to
66% B by 5.9 min, 70% B by 7.9 min, 78% B by 40 min, 88%
B by 42 min, 100% B by 55 min, returning to initial conditions
by 60 min, and allowing 30 min for equilibration. Mass
spectral data were collected using a hybrid triple-quadrupole
linear ion trap mass spectrometer (4000QTRAP, MDS Sciex,
Concord, ON) equipped with an electrospray interface
operating in the negative ion mode. The MS/MS tune
parameters utilized for quantification were optimized for
isomer specific transitions using the brPFOSK standard (Table
S1). Chromatograms were recorded by selected reaction
monitoring in MS/MS mode.
Results and Discussion
Comparison of PFOS Quantification by Total and Isomer
Specific Methods. Quantification biases were investigated
in serum samples by first generating chromatograms using
previously developed isomer specific methods (19). These
were subsequently analyzed using three distinct standard
curve quantification techniques: (i) PFOS quantification
using m/z 499f99 and m/z 499f80 transitions and summing
the response of all isomers relative to the technical brPFOSK
standard, (ii) quantification of linear and total branched
isomers separately using m/z 499f99 and m/z 499f80
transitions relative to a LPFOS and brPFOSK standard,
respectively, and (iii) the linear and each of the major
perfluoromonomethyl PFOS isomers were quantified indi-
vidually using separate calibration curves generated from
the known isomer composition of brPFOSK (determined by
19
F NMR) and isomer specific transitions (Table S1 and Figure
S1). For each quantification technique, six-point calibration
curves (each curve the average of standards run before and
after serum samples) were employed using concentrations
ranging from 0.5 to 50 ng/mL total PFOS. All of these methods
were applied to two human serum samples that many
laboratories have in their possession following the second
Worldwide Interlaboratory Study on PFCs.
An interesting result was the striking difference in the
branched isomer profiles of human serum samples A and B
(Figure 2). The HPLC-MS/MS chromatograms of these
samples qualitatively showed that each had a substantially
different ratio of linear:branched isomers, and the relative
profile of the branched isomers also varied. PFOS exposure
sources are not well characterized, thus the current state of
knowledge provides no explanation for these different isomer
profiles. Although limited to only two samples, these findings
illustrate that PFOS exposure sources can vary and, irrespec-
VOL. 43, NO. 20, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7903

FIGURE 2. HPLC-MS/MS chromatograms produced using the
Benskin et al. method (19) showing the PFOS isomer profiles in
(a) serum A, (b) serum B, and (c) the brPFOSK standard on the
FluoroSep RP Octyl column tracking m/z 499f80 (red), m/z
499f99 (green), and m/z 499f130 (blue).
tive of improvements to accuracy, isomer specific analytical
methods could be a useful tool for exposure tracking in
biomonitoring studies. Nevertheless, the chromatograms of
neither human serum sample looked exactly like the brPFOSK
standard, thus some systematic analytical bias should be
anticipated from traditional PFOS methods.
Quantification of PFOS in the serum samples relative to
brPFOSK, using either the m/z 499f80 or m/z 499f99
transition, produced lower concentrations than the reference
values generated in the associated interlaboratory study (22)
(Table S2). It is notable that a pure linear standard (LPFOS)
was provided for this interlaboratory study, however, its use
was not made mandatory. Differences were also evident
between PFOS quantification with m/z 80 and m/z 99, and
this depended on the sample. Specifically, the m/z 99 product
ion produced a higher PFOS concentration than m/z 80 for
serum A (two-tailed students t test, p ) 0.006), whereas in
serum B the m/z 99 product ion produced a lower concen-
tration relative to m/z 80 (p ) 0.003, Table 1). It is important
to note that the m/z 99 product ion is the most commonly
used PFOS quantification ion due to endogenous interfer-
ences associated with the m/z 80 product ion (19), however
these interferences are chromatographically separated by
the perfluorooctyl stationary phase used here and thus do
not contribute to the reported variation.
The linear PFOS isomer was then quantified using both
m/z 80 and m/z 99 product ions, and LPFOS and brPFOSK
standards. The calibration curve for linear PFOS in the
brPFOSK standard was produced by simply adjusting the
calibrated mass of linear PFOS in the mixture to 78.8% of
the total mass based on 19F NMR spectroscopy data (supplied
with the standard). Regardless of the product ion (m/z 80 or
m/z 99) chosen, or the standard used, the resulting con-
centrations of linear PFOS were indistinguishable in both
serum A and B (Table S3). This result was anticipated, and
it furthermore validates the 19F NMR spectral data provided
7904 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 20, 2009
with brPFOSK. For simultaneous quantification of all of the
branched isomers (hereafter referred to as total branched
isomers) using brPFOSK, a calibration curve was constructed
by adjusting the concentration of the branched PFOS isomers
in each calibration standard to 21.2% of the total concentra-
tion based on 19F NMR spectroscopy data (supplied with the
standard). Quantification was achieved using both m/z
499f80 and 499f99 transitions (Table 1). Good agreement
was observed for the data obtained using the m/z 80 and
m/z 99 product ions for quantification of total branched
isomers (determined to not be significantly different using
a two-tailed students t test, p ) 0.32).
Finally, individual perfluoromonomethyl isomers were
quantified by building calibration curves for the individual
isomers using isomer specific transitions (Figure S1 and Table
1) after adjusting for their composition in the brPFOSK
standard. This can only be done for the PFOS isomers that
have been characterized by 19F-NMR. Utilizing the brPFOSK
standard, we were able to detect and quantify five perfluo-
romonomethyl isomers. The rank order of individual isomers
differed between the two samples, but the general rank order
was isomer 7 > 5 > 64 > 2 (Table 1). The sum concentration
of individual branched PFOS isomers is not a true total,
because some minor isomers are not accounted for, which
explains why the sum of all individual isomers was somewhat
less than that estimated by the total branched PFOS analysis
(Table 1). However, since the levels of perfluorodimethyl
isomers observed in the serum samples were lower than those
in the brPFOSK standard, any effect on the accuracy of the
data resulting from the exclusion of the dimethyl branched
isomers during isomer specific quantification is expected to
be minimal; albeit this cannot necessarily be assumed for
other human matrices or other organisms. It is also possible
that quantification of total branched isomers by summing
all branched peaks in the 499f80 transition may cause over-
reporting because all but 3 isomers have relative response
factors greater than 100% in this transition (Table 2).
Therefore, if laboratories undertake an isomer-specific
analysis, there may still be a benefit to reporting results from
an accompanying total branched analysis.
Relative Response Factors of Branched PFOS Isomers.
In an effort to understand the above analytical biases, the
relative response factors of all PFOS isomers under electro-
spray and MS/MS conditions were investigated using a more
rapid UPLC-MS(/MS) method. The preparation and puri-
fication of 10 different branched PFOS isomers (Figure 1)
permitted the relative response factor of each to be studied,
the determination of their elution order on a UPLC BEH
Shield RP18 column (Figure 3), and hence the assignment of
the signals in various other technical PFOS mixtures. The
elution order differed substantially from that previously
observed using a perfluorophenyl (25) or the perfluorooctyl
(19) stationary phase used above. For example, on the UPLC
Shield RP18 column the elution order was (in order of
increasing retention time) 9 < 10 < 8 < 11 < 6 < 5 < 4 < 7
< 3 < 2 < 1, whereas on the perfluorophenyl column the
order was 8-11 < 4 < 5 < 2 < 3 < 6 < 7 < 1, and on perfluorooctyl
it was 8-11 < 2 < 4 < 3 < 5 < 6 < 7 < 1. This highlights the
importance of carefully identifying each isomer peak using
isolated standards or CID patterns when undertaking an
isomer-specific analysis, rather than relying on elution orders
reported in the literature. This UPLC method is more rapid
than existing isomer specific methods, however in our
experience it had limited applicability for biological samples
since it did not adequately separate endogenous steroid
interferences (11, 19). However, this UPLC method may be
very useful for abiotic samples such as water, air, or dust. It
is also important to point out that, even with this highly
resolved method, not all branched isomers were baseline
separated from each other or the linear isomer. Specifically,
TABLE 1. Quantification of PFOS (ng/mL) in Human Serum A and B by the Traditional PFOS Method, a Total Branched Method,
and an Isomer Specific Method (All Methods Used the brPFOSK Technical Standard; All Data Were Collected in Selected
Reaction Monitoring Mode Using m/z 499 as the Precursor Ion (m/z Values in the Table Are Product Ions))
PFOS and total branched PFOS isomer specific analysis
PFOS a
PFOS b
total branched PFOS linear 7 6 5 4 2
sum of branched sum of all
m/z 99 80 99 80 80 80 130 330 130 419
isomers isomers
Serum A
mean 3.72 2.85 1.15 0.92 1.8 0.29 0.07 0.19 0.12 0.06 0.7 2.5
95% CI (0.53 (0.82 (0.85 (0.22 (0.7 (0.02 (0.04 (0.09 (0.03 (0.05 (0.13
Serum B
mean 18.1 21.1 9.6 9.5 10.6 3.58 1.06 1.44 0.82 0.39 7.3 17.9
95% CI (4.20 (4.89 (2.31 (2.54 (2.86 (0.80 (0.18 (0.31 (0.12 (0.14 (1.41
a b
Quantification by summing the areas of the m/z 80 product ions for all of the peaks. Quantification by summing the
areas of the m/z 99 product ions for all of the peaks.

TABLE 2. MS and MS/MS Relative Response Factors (%) for the Various PFOS Isomers Relative to Linear PFOS (Isomer 1)
(Uncertainty of All Measurements is 16%)
isomer 1 2 3 4 5 6 7 8 9 10 11
MS relative response factor for m/z 499 using electrospray ionization
100 40 106 80 101 109 91 84 69 73 74
In-source fragmentation (% ) 100 A/B)a
3.0 77 6 4.6 1.7 1.7 1.8 7.3 44 38 22
MS/MS relative response factors
m/z 499 f m/z 99 100 117 97 49 39 43 78 10 0 0 19
m/z 499 f m/z 80 100 0 78 135 241 142 123 113 118 220 90
a
A is the sum of the signals from the different channels set at m/z 80, 99, 130, 180, 230, 280, 330, 169, 219, 269, 419,
and B is equal to A + signal at m/z 499.

isomer 2 coeluted with linear PFOS (Figure 3), however
quantification by using their unique product ions in MS/MS
(m/z 419 and 80, respectively) allowed for their resolution
and accurate quantification.
The relative response factors for the branched PFOS
isomers under electrospray ionization were determined in
separate injections by UPLC-MS using MPFOS [13C4-PFOS]
as an internal standard under selected ion monitoring
conditions optimized for linear PFOS (1). In the selected ion
monitoring mode (m/z 499), isomers 2, 9, 10, and 11 showed
a substantially lower response (<75%) compared to the linear
isomer (Table 2). The full scan spectra of these particular
isomers confirmed considerable in-source fragmentation
which largely explained the reduction in signal strength
observed for m/z 499 (Figure S2). As an example, the full
scan spectrum of isomer 2 gave fragments at m/z 99, 169,
219, and 419. The cumulative signal count for these four
fragments largely accounted for the 60% reduction in signal
response for m/z 499 (Table 1). These results indicate that
in-source fragmentation can be a major contributor to
analytical bias in PFOS methods where it is an inherent
assumption that all isomers behave similarly. Branched
isomers exhibiting lower response factors (isomers 2, 4, 9,
10, and 11) have been observed to vary in amounts present
(range of between 3 and 10%) in technical PFOS samples
(27). The overall bias will depend on the tuning parameters
of the instrument.
The relative response factors for the branched PFOS
isomers (to the linear isomer) were then determined by UPLC-
MS/MS using MPFOS [13C4-PFOS] as an internal standard
and MS/MS conditions optimized for linear PFOS (1): only
the m/z 499f99 and 499f80 transitions were monitored for
native PFOS (Table 2). Isomers 9 and 10 did not fragment
to m/z 99, while isomer 2 did not fragment to m/z 80. PFOS
quantification is typically performed under MS/MS condi-
tions using the m/z 499f99 or m/z 499f80 transitions (23),
thus certain branched PFOS isomers can be missed entirely,
and furthermore the two transitions will never yield the same
quantitative result in a PFOS analysis. The MS/MS differ-
ences appear to be due to some of the branched PFOS isomers
preferring alternate CID pathways as discussed below. It
should also be noted that the isomers produced vastly
different m/z 80:99 product ion ratios (Table 2), and this
factor will therefore contribute to systematic analytical bias
when PFOS or total branched PFOS concentrations are
reported without an isomer specific analysis.
Preferential CID Pathways for the Branched PFOS
Isomers. Careful examination of the product ion mass spectra
of the branched PFOS isomers revealed interesting details
concerning preferential CID pathways. As expected, isomer
1 fragmented mainly to m/z 80 ([SO3]-) and m/z 99 ([SO3F]-).
However, the preferred CID pathways for some of the
branched isomers can be very different from that of 1 (19, 25).
For example, isomer 2 fragmented through the perfluoroalkyl
route to give the 9-series ions (25) and m/z 99, however,
m/z 80 was totally absent from the mass spectrum. It appears,
in this case, that the primary CID pathway involved the loss
of SO3 with the charge being retained by the perfluoroalkyl
fragment m/z 419 [CF3(CF2)7]- instead of the m/z 80 [SO3]-
fragment. The perfluoroalkyl fragment (m/z 419) then
underwent secondary fragmentation to give the expected
fragments at m/z 119, 169, 219, and 269 (26). This observation
was explained by the formation of more stable secondary
perfluoroalkyl anions after primary CID. This pathway was
not seen for the linear isomer 1 since loss of SO3 would have
led to a much less stable linear primary perfluoroalkyl anion,
therefore, the charge preferred to stay on the SO3 fragment.
Some of the isomers also produced perfluoroalkyl fragments,
for example, isomer 9 fragmented to produce both the 9-
series ([CF3(CF2)n]-) and 0-series ([CF2(CF2)nSO3]-) frag-

VOL. 43, NO. 20, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 7905
FIGURE 3. Chromatograms of the individual PFOS isomers and brPFOSK obtained in SIM mode (m/z 499) on a Waters Acquity UPLC
using a Shield RP18 column at a concentration of 500 ng/mL. Compounds in brackets are impurities in the sample.
ment ions (25) while isomers 10 and 11 only produced the
0-series ions.
The consequence of these preferred CID pathways on
quantitative data is dependent on the transitions chosen
during selected reaction monitoring analyses. Transitioning
to only the 80 or 99 product ions during MS/MS analysis
could result in the exclusion of some PFOS isomers from
reported findings. This is of importance since these isomers
may possess unique biological properties. For example, the
alpha-PFOS branched isomer (2) is more bioaccumulative
than linear PFOS in rats (28, 29) but will not be quantified
when using the m/z 80 product ion as it does not produce
this ion. Acquiring and analyzing accurate isomer patterns
could also give clues as to the exposure sources and
environmental fate of perfluorinated substances.
Influence of Pure Linear or Technical Calibration
Standards. Even if a technical PFOS sample is chemically
pure (i.e., just branched and linear PFOS), the use of such
material as a reference standard will introduce interlaboratory
data variability because of the significant differences in isomer
7906 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 20, 2009
distribution between the various sources of commercial
technical PFOS (27) and the aforementioned differences in
the response factors of the branched isomers. Under our
experimental conditions it was possible to calculate the
overall relative response factors (RRFs) to the linear isomer
for commercial PFOS standards, which is relevant in a PFOS
analysis. This involves knowing the MS/MS RRFs for the most
abundant PFOS isomers (Table 2) and the isomer content of
several standards as measured by 19F NMR spectroscopy
(Table S4) (27). Table S4 summarizes examples in which the
theoretical and measured RRFs for both the m/z 499f99
and m/z 499f80 transitions were calculated and measured
for brPFOSK and a commercial PFOS sample (TCI America).
In both cases the measured values were higher than the
calculated values, but they are generally in good agreement.
The variation between the calculated and measured values
was larger for brPFOSK compared to the TCI sample, but
this may be due to the presence of other isomers that were
not accounted for in the theoretical calculations. The results
highlight that laboratories using a PFOS method and
quantifying with a linear standard using the m/z 499f99
transition may underestimate the true PFOS concentration
because the RRFs for the branched isomers are lower than
for linear PFOS in this transition. Conversely, only using the
m/z 499f80 transition and a linear PFOS standard would
overestimate PFOS content in a real sample because the RRFs
for the branched isomers are higher than that for linear PFOS.
This is based on our instrument conditions and may be
instrument dependent. The actual difference between labo-
ratories analyzing the same sample will vary, thus making it
difficult to compare interlaboratory data. To limit ambiguity
when comparing data generated by different laboratories,
we suggest that a common standard should be used in all
laboratories so that interlaboratory data can be more easily
compared.
The use of linear PFOS (1) as the reference standard for
PFOS analyses would remove the unknown variable of
branched isomer content. Consistency between analytical
laboratories would probably improve, however accuracy
would remain an issue since no environmental or human
sample, to our knowledge, will contain only linear PFOS.
For any PFOS analysis, unless a standard of exactly the
same isomer composition as the unknown sample is used,
the isomer specific RRFs will always lead to a quantification
bias of unknown proportions. However, any technical PFOS
standard will always more closely resemble a sample, thus
minimizing bias, and if the linear and branched isomers
have been quantified by 19F NMR spectroscopy, it can also
be used for an isomer specific quantitative analysis.
Therefore, until quantitative standards of the branched
PFOS isomers are available, this approach provides an
improvement in the methodology relating to the quan-
tification of total PFOS.
Supporting Information Available
Tables summarizing the optimization of MS/MS tune
parameters for isomer specific transitions, a comparison
of our data to that obtained in the 2nd Worldwide
Interlaboratory Study, the linear PFOS concentrations
obtained using the brPFOSK versus the LPFOS standard,
and the measured versus calculated response factors of
the linear and branched PFOS isomers. Figures illustrating
the chromatograms obtained during isomer specific
quantification and expressing the inverse association of
isomer response factors with in-source fragmentation. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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