A rapid method for the analysis of perfluorinated alkyl substances in serum by hybrid solid-phase extraction
Masato Honda,AMorgan RobinsonAand Kurunthachalam KannanA,B,C,D
AWadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY 12201-0509, USA.
BDepartment of Environmental Health Sciences, School of Public Health, State University of New York at Albany, Empire State Plaza, PO Box 509, Albany, NY 12201-0509, USA.
CBiochemistry Department, Faculty of Science and Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
DCorresponding author. Email: [email protected]
Environmental context. Although the environmental occurrence of perfluoroalkyl substances was first reported almost 20 years ago, there are continuing concerns about human exposure to these potentially toxic chemicals. Such concerns have necessitated the development of reliable methods for rapid determination of perfluoroalkyl substances in human serum. This article describes a rapid and sensitive analytical method suitable for monitoring human exposure to perfluoroalkyl substances.
Abstract. A method for the analysis of 13 perfluorinated alkyl substances (PFASs) in human serum was developed based on hybrid solid-phase extraction (hybrid-SPE) and ultrahigh-performance liquid chromatography–tandem mass spec- trometry (UPLC-MS/MS). Serum PFASs were extracted using hybrid-SPE-phospholipid cartridge after precipitating proteins and other endogenous biological interferences with 1 % ammonium formate in methanol. The average intra-day accuracy (measured as percent recoveries from fortified samples) and precision of the method (measured as relative standard deviation [RSD, %] between analyses) were 88.7–117 % and 1.0–13.4 %, respectively. The average inter-day precision was 2.8–6.9 %. The method was sensitive, with limits of quantification (LOQs) in the range of 0.05 to 0.09 ng mL1 for all 13 PFASs. The applicability of this method was tested by analysing serum-certified standard reference material and proficiency test samples. In an hour, 100 samples can be processed by hybrid-SPE, and the instrumental run time is 5 min per sample. The developed method is rapid, inexpensive, accurate, precise, and extremely sensitive for the analysis of PFASs in human serum.
Additional keywords: biomonitoring, children’s exposure, UPLC, MS/MS, PFOS.
Received 2 November 2017, accepted 22 January 2018, published online
Introduction
Perfluorinated alkyl substances (PFASs) have been widely used in the past five decades for several commercial applications as surfactants, levelling agents, aqueous film-forming foams, and surface protectors.[1]Following their widespread use, PFASs, especially perfluorooctane sulfonate (PFOS) and per- fluorooctanoic acid (PFOA), have emerged as ubiquitous pol- lutants in the environment[2–4]and in humans.[5–11]In the USA, the Centers for Disease Control and Prevention (CDC) monitor exposure to several PFASs in the general population;[12]serum concentrations of PFOS ranged from 4.99 to 30.4 mg L1 (geometric mean) during 1999–2014. Similarly, Health Canada reported serum PFOS concentrations in Canadian population to range from 6.8 to 9.1mg L1(median) during 2007–2011.[13]
Owing to the toxic potential of PFASs in areas such as immu- notoxicity, hepatotoxicity, and endocrine disruption,[14–16]
exposure to this class of chemicals is a human health concern.
Elevated concentrations of PFASs, particularly PFOS and PFOA, have been identified in the USA in ground water near military bases, airports, landfills, wastewater treatment
facilities, and industrial sites. Furthermore, drinking water supplies for 6 million USA residents exceed the Environmental Protection Agency’s (EPA) lifetime health advisory level (70 ng L1) for PFOS and PFOA.[17]Studies suggest the need for lower analytical reporting limits,[17]and biomonitoring of individuals exposed to PFASs would assist in further identifying sources and risks. Standard methods (developed by the ISO and the USEPA) to measure PFASs in water have been reported.[18,19] Nevertheless, standard methods to measure PFASs in other matrices including serum are not available.
For human biomonitoring surveys of PFASs, serum is the most commonly used sample matrix.[5,6,12,13]Serum is a com- plex matrix containing many ions, lipids, sugars, and pro- teins,[20]and effective removal of these endogenous molecules is a critical step during sample preparation for PFAS analysis.[21]
Whereas ion-pair extraction has been traditionally used,[22]
solid-phase extraction (SPE) methods have gained populari- ty[23–27]because they are less laborious[9]and provide greater selectivity and precision. The most commonly used method of detection of PFASs is liquid chromatography (LC) coupled with Environ. Chem.
https://doi.org/10.1071/EN17192
Journal compilationCSIRO 2018 A www.publish.csiro.au/journals/env
Research Paper
8 May 2018
tandem mass spectrometry (MS/MS).[5,23,28]Analytical meth- ods involving on-line SPE connected to LC-MS/MS[28,29]were shown to minimise background contamination, while improving accuracy, sensitivity and precision. However, the methods reported for analysing PFASs in serum often involve multi- stage sample preparation protocols.[30]A simple, reliable and high-throughput method is required for analysing the many samples resulting from large-scale population-based human biomonitoring studies.
We investigated a new method for the analysis of PFASs in human serum incorporating a simplified sample-preparation protocol based on hybrid-solid-phase extraction (hybrid-SPE) tailored to measurement by ultra-performance liquid chromatog- raphy (UPLC) coupled to tandem mass spectrometry. The hybrid- SPE phospholipid technique is a new approach to preparing serum samples for trace chemical analysis,[31,32] whereupon, after the addition of a protein-precipitating solvent, the serum sample is centrifuged to remove proteins, and the supernatant is loaded directly onto the cartridge to remove phospholipids and other interferences from the serum matrix. We demonstrate the applicability of the method for the rapid, accurate, and sensitive determination of 13 PFASs in human serum.
Experimental
Chemicals and reagents
Native standards and13C-labelled standards of perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS), per- fluorooctane sulfonate (PFOS), perfluorodecane sulfonate (PFDS), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluo- rononanoic acid (PFNA), perfluorodecanoic acid (PFDA), per- fluoroundecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoDA), n-methylperfluoro-1-octanesulfonamidoacetic acid (N-MeFOSAA), and perfluorooctane sulfonamide (PFOSA) were purchased from Wellington Laboratories (Guelph, Ontario, Canada). Human serum was purchased from Sigma- Aldrich (Sheboygan Falls, WI, USA). Fortified human-serum standard reference material (SRM 1958) was purchased from the National Institute of Standards & Technology (NIST, Gai- thersburg, MD, USA). Hybrid SPE-phospholipid cartridges (30 mg, 1 mL) were purchased from Supelco (Bellefonte, PA, USA). All reagents and chemicals were of analytical grade.
Sample preparation
Serum samples were stored at208C until analysis. After being thawed at room temperature, 250mL of serum was transferred to a 15 mL polypropylene (PP) tube, and spiked with 50 mL of isotope-labelled internal standard mixture (5.00 ng of each compound). To this mixture, 700mL of methanol (MeOH) con- taining 1 % ammonium formate (w v1) was added, and the mixture was shaken for 30 s. The supernatant was separated by centrifuging at 4330 g for 5 min at room temperature (centrifuge from Eppendorf 5804; Barkhausenweg, Hamburg, Germany).
Hybrid-SPE cartridges were prewashed with 1 mL of MeOH containing 1 % ammonium formate (w v1) to remove any con- tamination that may arise from the cartridge. The supernatant was quantitatively transferred to the cartridge and the eluate was collected and transferred in a 2 mL vial for instrumental analysis.
Instrumental analysis
The chromatographic separation was carried out by using ultra- performance liquid chromatography (UPLC) (Acquity I Class;
Waters, Milford, MA, USA) coupled with electrospray triple- quadrupole tandem mass spectrometry (ESI-MS/MS) (API 5500; AB SCIEX, Framingham, MA, USA). The target analytes were separated by an Acquity UPLC BEH C18 (1.7 mm, 502.1 mm) column (Waters). The mobile phase for UPLC comprised 100 % MeOH for solvent A and 0.1 % ammonium acetate in water (w v1) for solvent B. The flow rate was set at 0.3 mL min1, the mobile phase starting at 10 % MeOH, held for 0.75 min, increasing to 70 % over 0.25 min and then to 75 % over 1 min, increasing to 100 % over 1.2 min and holding for 0.9 min, decreasing to 10 % over 0.1 min and holding for 0.8 min (total run time: 5 min). Injection volume was 3mL. Quantification of PFASs was based on the isotope-dilution method. Target analytes were monitored by multiple reaction monitoring mode under negative ionisation (Table 1, S1, Supplementary material).
Method development
Five different solvents were compared for protein-precipitation efficiency and instrumental response in the sample preparation.
Solvent 1: 1 % formic acid in acetonitrile (ACN, v v1); solvent 2: 1 % formic acid in MeOH (v v1); solvent 3: 0.5 % citric acid in ACN (w v1); solvent 4: 0.5 % citric acid in MeOH (w v1);
solvent 5: 1 % ammonium formate in MeOH (w v1). During these experiments, we assessed procedural background con- tamination, relative recoveries, and matrix effects. To assess relative recoveries for each precipitation solvent, we fortified serum with 5.00 ng of native PFAS standards before processing the mixture as described above. To assess matrix effects from each precipitation solvent, post-spiked samples (5.00 ng of native standards were fortified in matrix after extraction but prior to instrumental analysis) were prepared and compared with the response of standards prepared in MeOH. The electrospray ionisation parameters, ion-fragmentation parameters, and mobile-phase gradients were optimised to obtain the maximum sensitivity and separation of target analytes (Table 1).
Method validation
Instrumental calibration range was verified by matrix-matched calibration standards using a 13-point calibration curve within the range 0.01–100 ng mL1. Quadratic fitting and weighting with ‘1 x1’ was used to calculate the regression equation of the calibration curve (Analyst 1.6 Software; AB SCIEX) in order to minimise the effect of instrumental ion saturation. At each point of the calibration curve, a 20 % error from back-calculated concentration was set as the acceptable limit. The limit of detection (LOD) and the limit of quantitation (LOQ) were cal- culated as 3 and 10 times (respectively) the standard deviation of the lowest acceptable point (a calibration point was deemed acceptable if the calculated values were within 80–120 % of the theoretical values) in the calibration curve (n¼5).
To evaluate the intra-day accuracy and precision of the method, a commercial serum sample (with five different lot numbers from Sigma-Aldrich to include sample difference on method validation) was spiked with 13 PFASs at three concen- trations: low 0.50 ng, medium 5.00 ng and high 20.0 ng (n¼5).
To evaluate inter-day precision, a commercial serum was forti- fied with 5.00 ng of a native standard mixture and analysed over four different days (n ¼ 2–6). Precision was calculated as relative standard deviation (RSD, %). The SRM (1958, fortified human serum) was analysed as a quality-control sample. Addi- tional serum samples provided by the Biomonitoring Quality B
Assurance Support Program (eight samples: 2015 to 2017) from the CDC (Atlanta, GA, USA) were analysed. Moreover, samples from the Arctic Monitoring and Assessment Program (AMAP) Ring Test for Persistent Organic Pollutants in Human Serum (nine samples: 2015 to 2017) supplied by the Centre de tox- icologie du Quebec (Quebec, Canada) were analysed to further validate the method.
Results and Discussion
Background levels of PFASs in SPE cartridges and precipitation solvents
Although hybrid-SPE cartridges do not require any prewash step before loading the samples, potential PFAS contamination can be removed by including a prewash step. In fact, PFHxA was detected in hybrid-SPE cartridges at up to 11.3 ng mL1(Fig.
S1, Supplementary material) in prewash solvent, and the level varied depending on the lot number of the hybrid-SPE car- tridges. Therefore, a prewash step with the precipitation solvent was necessary, before loading the samples, to eliminate back- ground contamination arising from the cartridges.
Optimisation of precipitation solvent
Among five different precipitation solvents tested, 1 % formic acid in ACN (solvent 1) showed the highest procedural back- ground contamination for PFASs (range: nd–6.93 ng mL1), especially for PFHxA (Fig. 1, S1). The background PFHxA concentration was even higher than that reported for the general
USA population.[33] Other solvent mixtures (solvents 2–5) showed much lower background PFAS levels (nd–
0.07 ng mL1), except for PFHxA (0.10–2.21 ng mL1).
Therefore, prewashing of the cartridge with freshly-opened/
prepared solvents was deemed necessary to minimise contam- ination from cartridge and old solvents. Among five solvent mixtures, 1 % ammonium formate in MeOH (solvent 5) dem- onstrated the lowest background for the 13 PFASs (nd–
0.10 ng mL1).
All precipitation solvents showed relatively acceptable recoveries (100–120 %) that were tested at the fortification level of 5.00 ng (Fig. 2). However, 1 % formic acid in ACN and 0.5 % citric acid in MeOH (solvents 1 and 4) demonstrated higher RSD values (30.6 and 34.9 %, respectively) for PFHxA, because of the existence of a high background signal. Solvents 2, 3, and 5 had low RSD values for PFHxA (2.9–8.1 %) and other PFASs (1.1–6.0 %). Overall, 1 % formic acid in MeOH, 0.5 % citric acid in ACN and 1 % ammonium formate in MeOH (solvents 2, 3 and 5) showed excellent accuracy and reproducibility.
The matrix effect was tested for all five precipitation sol- vents: 1 % formic acid in MeOH (solvent 2) demonstrated the highest signal suppression (13.8 to26.1 %) (Fig. 3), while the mixture of 1 % formic acid in ACN (solvent 1) demonstrated the highest signal enhancement (5.8 to 17.8 %) for all PFASs except for PFHxA (16.1 %). Solvents 3, 4, and 5 had relatively low matrix effects (10.6 to 12.2 %), while 1 % ammonium formate in MeOH had the lowest matrix effects (2.4 to 8.3 %).
These values were remarkably lower than previous reports:
Table 1. ESI-negative-MS/MS parameters used in the analysis of perfluoroalkyl substances (PFASs)
Compounds Precursor
ion (m/z)
Fragment ion (m/z)
Declustering potential
Entrance potential
Collision energy (V)
Collision cell exit potential
Exit lens
PFBS 299 80 70 10 65 8 120
PFHxS 399 80 100 13 65 9 120
18O2-PFHxS 403 84 100 13 65 9 120
PFOS 499 99 85 13 70 10 120
13C4-PFOS 503 99 85 13 70 10 120
PFDS 599 80 180 7.5 76 10 120
PFBA 213 169 40 7 13 15 100
13C3-PFBA 216 172 40 7 13 15 100
PFPeA 263 219 35 10 12 12 100
13C3-PFPeA 266 222 35 10 12 12 100
PFHxA 313 269 45 7.5 13 12 100
13C2-PFHxA 315 270 45 7.5 13 12 100
PFHpA 363 319 40 7 15 15 100
13C4-PFHpA 367 322 40 7 15 15 100
PFOA 413 369 60 5 15 15 100
13C4-PFOA 417 372 60 5 15 15 100
PFNA 463 419 70 5 15 15 100
13C5-PFNA 468 423 70 5 15 15 100
PFDA 513 469 80 5 17 10 100
13C2-PFDA 515 470 80 5 17 10 100
PFUnDA 563 519 80 5 18 15 100
13C2-PFUnDA 565 520 80 5 18 15 100
PFDoDA 613 569 80 5 19 15 100
13C2-PFDoDA 615 570 80 5 19 15 100
N-MeFOSAA 570 419 130 5 29 15 100
D3-N- MeFOSAA
573 419 130 5 29 15 100
PFOSA 498 78 75 12 70 8 120
13C8-PFOSA 506 78 75 12 70 8 120
C
50 % 60 % 70 % 80 % 90 % 100 % 110 % 120 % 130 % 140 % 150 %
PFBS PFHxS PFOS PFDS PFHxA PFHpA PFOA PFNA PFDA PFUnDA PFDoDA N-MeFOSAA PFOSA
Relative recovery (%)
1 % formic acid in acetonitrile 1 % formic acid in methanol 0.5 % citric acid in acetonitrile 0.5 % citric acid in methanol 1 % ammonium acetate in methanol
Fig. 2. Relative recoveries of PFASs in fortified serum (5.00 ng mL1) using five different protein-precipitation solvents.
⫺40 %
⫺30 %
⫺20 %
⫺10 % 0 % 10 % 20 % 30 %
Matrix effect
1 % formic acid in acetonitrile 1 % formic acid in methanol 0.5 % citric acid in acetonitrile 0.5 % citric acid in methanol 1 % ammonium acetate in methanol
PFBS PFHxS PFOS PFDS PFHxA PFHpA PFOA PFNA PFDA
PFUnDA PFDoDA
N-MeFOSAA PFOSA
Fig. 3. Matrix effects of PFASs in five different protein-precipitation solvents.
0.00001
PFBS PFHxS 0.0001
0.001 0.01 0.1 1 10
PFASs concentration (ng mL⫺1)
1 % formic acid in acetonitrile 1 % formic acid in methanol 0.5 % citric acid in acetonitrile 0.5 % citric acid in methanol
1 % ammonium acetate in methanol
PFOS PFDS PFHxA PFHpA PFOA PFNA PFDA
PFUnDA PFDoDA N-MeFOSAA
PFOSA
Fig. 1. Procedural background levels of PFASs in protein-precipitation (extraction) solvents passed through hybrid-SPE cartridges.
D
Ka¨rrman et al.,[25]47 to 45 %; Kuklenyik et al.,[27]70 to 12 %. The LC retention times of the PFASs were not signifi- cantly affected by any of the precipitation solvents (,0.5 % variability). Based on these results, we considered 1 % ammo- nium formate in MeOH to be the optimal protein-precipitation solvent, demonstrating low background and matrix effects relative to previous studies, while contributing to the good accuracy and reproducibility of the PFAS analysis.
Validation of the method
The matrix-matched calibration calculated with quadratic cali- bration curves showed excellent correlation coefficients (r .0.998) and sensitivity (method LOQ: 0.05–0.09 ng mL1) (Table 2). These LOQ values compare favourably with those reported previously; Kim et al.[24]0.1–0.69 ng mL1, Gump et al.[34]0.2–1.0 ng mL1, Inoue et al.[29]0.5–1.0 ng mL1, Lee et al.[35]0.11–0.81 ng mL1. The instrumental calibration range includes detectable concentration ranges of PFASs reported in previous surveys of the general populations.[6,12]
Matrix spiking tests performed at three concentrations (0.50, 5.00, 20.0 ng mL1, n¼5,Table 3) showed recoveries of 88.7–
117 % (at a fortification level of 0.50 ng mL1), 101–117 % (5.00 ng mL1), and 90.6–112 % (20.0 ng mL1).
Intra-day and inter-day precision were evaluated by using fortified serum analysed by our method. Excluding the 0.50 ng mL1 fortification level, all intra-day and inter-day RSD values were below 7 %. At the 0.50 ng mL1fortification level, due to high initial/background concentration of PFHxS, PFOS and PFHxA in the commercial serum (0.33, 1.00 and 0.05 ng mL1, respectively), relatively high RSD values (8.0, 9.6, 13.4 %, respectively) were obtained. Haug et al.[36]devel- oped an on-line SPE column-switching method and reported RSD values at 0.4–19 % at a 0.20 ng mL1PFAS fortification level. Figure 4 is a chromatogram of 0.50 ng mL1 spiked human serum; clear peaks of the target analytes were observed and no interferences were detected around the target peaks.
Table 4shows the results of various proficiency test serum samples and the SRM analysed by the method developed in this
Table 2. Limit of detection (LOD), limit of quantification (LOQ), upper limit of quantification (ULOQ) and calibrations (n55) of the analytical method
Equation of calibration curve is given as Y¼a2X2þa1Xþa0. LOD and LOQ are calculated as 3 and 10 times of standard deviation of the lowest acceptable calibration point
Compounds LOD LOQ ULOQ a0 a1 a2 r
(ng mL1)
PFBS 0.02 0.07 80 0.000223 0.121 0.000742 0.9996
PFHxS 0.01 0.05 80 0.005368 0.206 0.000984 0.9999
PFOS 0.02 0.07 200 0.000513 0.172 0.000119 0.9988 PFDS 0.03 0.09 80 0.000295 0.372 0.001111 0.9998
PFHxA 0.02 0.05 200 0.003694 0.205 0.001208 0.9996
PFHpA 0.02 0.07 80 0.001200 0.188 0.000162 0.9996
PFOA 0.02 0.08 200 0.008090 0.212 0.000518 0.9992
PFNA 0.02 0.08 80 0.000879 0.165 0.001267 0.9998
PFDA 0.01 0.02 80 0.000640 0.191 0.001061 0.9998
PFUnDA 0.03 0.09 80 0.002870 0.203 0.000804 0.9997
PFDoDA 0.02 0.06 80 0.000704 0.171 0.001019 0.9999
N-MeFOSAA 0.02 0.06 80 0.000117 0.225 0.000764 0.9995
PFOSA 0.02 0.05 80 0.000344 0.218 0.000657 0.9991
Table 3. Procedural background, relative recoveries and precision of analysis of serum samples spiked with three different PFAS concentrations using 1 % ammonium formate in methanol as the precipitation solvent
Precision was calculated as relative standard deviation (RSD) at 5.00 ng fortification level
Compounds Background 0.50 ng mL1 5.00 ng mL1 20.0 ng mL1 Precision (%)
Average (%) RSD (%) Average (%) RSD (%) Average (%) RSD (%) Intra-day Inter-day
PFBS 0.09 88.7 4.1 101.2 4.9 90.6 5.6 4.9 5.4
PFHxS 0.01 103.9 8.0 110.0 3.2 111.4 4.7 3.2 5.1
PFOS 0.02 92.1 9.6 112.6 4.5 103.0 7.3 4.5 6.6
PFDS 0.01 108.2 3.7 115.4 2.4 100.9 6.1 2.4 6.4
PFHxA 0.07 117.0 13.4 116.8 2.0 110.9 1.1 2.0 4.4
PFHpA 0.01 105.2 2.9 111.5 2.2 107.5 2.2 2.2 2.9
PFOA 0.02 94.3 6.9 103.6 3.4 105.1 2.4 3.4 3.6
PFNA 0.01 103.7 4.6 109.9 3.1 105.7 1.7 3.1 3.7
PFDA 0.00 111.0 6.0 111.2 4.1 103.0 2.3 4.1 2.8
PFUnDA 0.01 101.5 5.3 113.1 2.9 112.3 1.0 2.9 4.2
PFDoDA 0.01 107.4 1.9 109.4 3.2 103.4 2.5 3.2 3.7
N-MeFOSAA 0.02 99.6 5.0 113.9 5.4 104.7 5.3 5.4 6.9
PFOSA 0.02 106.6 1.4 112.0 1.3 106.5 2.8 1.3 4.8
E
study. The method was applied to two proficiency test samples:
CDC, which has target values for seven PFASs and AMAP, which has target values for six PFASs. The recoveries of PFASs were 88–106 % for CDC, and 90–108 % for AMAP. These recoveries were well within the acceptable error range of the analysis (30 %), with better relative recoveries/RSD values than other methods.[36]Additionally, analysis of four PFASs in certified serum SRM showed recoveries that ranged from 86 to 98 % with RSDs that ranged from 4.4 to 6.2 % (n¼20). The current hybrid-SPE method demands little time and only one precipitation solvent, which is different from the traditional SPE methods or liquid–liquid extraction methods[25,34]which usual- ly require multiple steps involving several reagents/solvents, hours to complete extraction (SPE cartridges require condition- ing and other preparatory steps), and are labour-intensive. Thus, the current method uses less reagents/solvents and consumables than other methods.
In summary, we report a method for the determination of PFASs in human serum using hybrid-SPE and UPLC-MS/MS.
Our method provides excellent sensitivity (method LOQ: 0.05–
0.09 ng mL1), repeatability (inter-day RSD: 2.8–6.9 %), accu- racy (spiking test recovery: 88.7–117 %), precision (intra-day RSD: 1.3–5.4 %), and a good quantification range (0.05–0.09 to 80–200 ng/mL). A strength of this method is that it greatly reduces sample preparation time, and requires fewer consum- ables. Typically, the method takes less than 1 h to prepare 100 samples, while the run time is just 5 min per sample for UPLC- MS/MS detection. In short, this method could conceivably be adapted for large studies monitoring human exposure to per- fluoroalkyl substances.
Supplementary material
A figure showing UPLC-MS/MS chromatograms of PFHxA found at background levels in SPE cartridges and protein-pre- cipitation solvents and a table showing UPLC and MS/MS conditions used for PFAS analyses are available on the Journal’s website.
0E⫹00 5.0E⫹03 1.0E⫹04
0 2 4
Time (min) N-MeFOSAA 0E⫹00
1.0E⫹05 2.0E⫹05
0 2 4
Time (min) PFBS
0E⫹00 5.0E⫹04
0 2 4
Time (min) PFDA
0E⫹00 5.0E+04 1.0E+05
0 1 2 3 4 5
Time (min) PFDoDA
0E⫹00 5.0E⫹04 1.0E⫹05
0 1 2 3 4 5
Time (min) PFDS
0E⫹00 5.0E⫹04 1.0E⫹05
0 1 2 3 4 5
Time (min) PFHpA
0E⫹00 1.0E⫹05 2.0E⫹05
0 2 4
Relative abundance (count)
Time (min) PFHxA
0E⫹00 1.0E⫹05 2.0E⫹05
0 1 2 3 4 5
Time (min) PFHxS
0E⫹00 5.0E⫹04
0 1 2 3 4 5
Time (min) PFNA
0E⫹00 1.0E⫹05 2.0E⫹05
0 1 2 3 4 5
Time (min) PFOA 0E⫹00
2.0E⫹04 4.0E⫹04
0 1 2 3 4 5
Time (min) PFOS
0E⫹00 5.0E⫹04 1.0E⫹05
0 1 2 3 4 5
Time (min) PFOSA 0E⫹00
5.0E⫹04
0 1 2 3 4 5
Time (min) PFUnDA
Fig. 4. Chromatograms of PFASs in fortified serum sample (0.50 ng mL1) separated by Acquity UPLC BEH C-18 column within 5 min.
Table 4. Results of PFAS analysis for serum samples obtained from proficiency test programs and certified serum SRM (CDC:n58, AMAP:n59, SRM:n520)
Compounds CDC AMAP SRM
Average (%) RSD (%) Average (%) RSD (%) Average (%) RSD (%)
PFBS – – – – – –
PFHxS 102.1 8.0 101.3 5.3 95.3 5.0
PFOS 99.4 11.2 104.3 5.6 87.9 4.4
PFDS – – – – – –
PFHxA – – 89.7 10.9 – –
PFHpA – – – – – –
PFOA 102.4 7.3 108.4 3.1 97.9 5.7
PFNA 105.9 16.5 101.7 14.6 85.9 6.2
PFDA 87.7 14.3 – – – –
PFUnDA 105.7 11.3 103.9 8.7 – –
PFDoDA – – – – – –
N-MeFOSAA 91.1 13.9 – – – –
PFOSA – – – – – –
F
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
Research reported in this publication was supported in part by the National Institute of Environmental Health Sciences of the National Institutes of Health under Award Number U2CES026542-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
References
[1] J. P. Giesy, K. Kannan, Peer reviewed: Perfluorochemical surfactants in the environment. Environ. Sci. Technol. 2002, 36, 146A. doi:10.
1021/ES022253T
[2] K. Kannan, J. Koistinen, K. Beckmen, T. Evans, J. F. Gorzelany, K. J.
Hansen, P. D. Jones, E. Helle, M. Nyman, J. P. Giesy, Accumulation of perfluorooctane sulfonate in marine mammals. Environ. Sci. Technol.
2001, 35, 1593. doi:10.1021/ES001873W
[3] N. Yamashita, K. Kannan, S. Taniyasu, Y. Horii, G. Petrick, T. Gamo, A global survey of perfluorinated acids in oceans. Mar. Pollut. Bull.
2005, 51, 658. doi:10.1016/J.MARPOLBUL.2005.04.026
[4] K. Kannan, L. Tao, E. Sinclair, S. D. Pastva, D. J. Jude, J. P. Giesy, Perfluorinated compounds in aquatic organisms at various trophic levels in a Great Lakes food chain. Arch. Environ. Contam. Toxicol.
2005, 48, 559. doi:10.1007/S00244-004-0133-X
[5] K. Kannan, S. Corsolini, J. Falandysz, G. Fillmann, K. S. Kumar, B. G.
Loganathan, M. A. Mohd, J. Olivero, N. Van Wouwe, J. H. Yang, K. M. Aldous, Perfluorooctanesulfonate and related fluorochemicals in human blood from several countries. Environ. Sci. Technol. 2004, 38, 4489. doi:10.1021/ES0493446
[6] A. M. Calafat, L. Wong, Z. Kuklenyik, J. A. Reidy, L. L. Needham, Polyfluoroalkyl chemicals in the U.S. population: Data from the National Health and Nutrition Examination Survey (NHANES) 2003–2004 and comparisons with NHANES 1999–2000. Environ.
Health Perspect. 2007, 115, 1596. doi:10.1289/EHP.10598 [7] G. W. Olsen, K. J. Hansen, L. A. Stevenson, J. M. Burris, J. H. Mandel,
Human donor liver and serum concentrations of perfluorooctanesul- fonate and other perfluorochemicals. Environ. Sci. Technol. 2003, 37, 888. doi:10.1021/ES020955C
[8] L. S. Haug, C. Thomsen, G. Becher, Time trends and the influence of age and gender on serum concentrations of perfluorinated compounds in archived human samples. Environ. Sci. Technol. 2009, 43, 2131.
doi:10.1021/ES802827U
[9] L. W. Y. Yeung, M. K. So, G. Jiang, S. Taniyasu, N. Yamashita, M. Song, Y. Wu, J. Li, J. P. Giesy, K. S. Guruge, P. K. S. Lam, Perfluorooctanesulfonate and related fluorochemicals in human blood samples from China. Environ. Sci. Technol. 2006, 40, 715.
doi:10.1021/ES052067Y
[10] L. Tao, K. Kannan, K. M. Aldous, M. P. Mauer, G. A. Eadon, Biomonitoring of perfluorochemicals in plasma of New York State personnel responding to the World Trade Center Disaster. Environ.
Sci. Technol. 2008, 42, 3472. doi:10.1021/ES8000079
[11] L. Trasande, T. T. Koshy, J. Gilbert, L. K. Burdine, T. M. Attina, A. Ghassabian, M. Honda, M. Marmor, D. B. Chu, X. Han, Serum perfluoroalkyl substances in children exposed to the world trade center disaster. Environ. Res. 2017, 154, 212. doi:10.1016/
J.ENVRES.2017.01.008
[12] CDC Fourth national report on human exposure to environmental chemicals updated tables, January 2017, Volume 1. Available at https://www.cdc.gov/exposurereport/index.html (verified August 2017).
[13] Health Canada second report on human biomonitoring of environ- mental chemicals in Canada. Available at: https://www.canada.ca/en/
health-canada/services/environmental-workplace-health/reports- publications/environmental-contaminants/second-report-human- biomonitoring-environmental-chemicals-canada-health-canada-2013.html (verified August 2017).
[14] X. Fang, G. Gao, H. Xue, X. Zhang, H. Wang, In vitro and in vivo studies of the toxic effects of perfluorononanoic acid on rat hepato- cytes and Kupffer cells. Environ. Toxicol. Pharmacol. 2012, 34, 484.
doi:10.1016/J.ETAP.2012.06.011
[15] E. Corsini, E. Sangiovanni, A. Avogadro, V. Galbiati, B. Viviani, M. Marinovich, C. L. Galli, M. Dell’Agli, D. R. Germolec, In vitro characterization of the immunotoxic potential of several perfluori- nated compounds (PFCs). Toxicol. Appl. Pharmacol. 2012, 258, 248.
doi:10.1016/J.TAAP.2011.11.004
[16] L. S. Kjeldsen, E. C. Bonefeld-Jørgensen, Perfluorinated compounds affect the function of sex hormone receptors. Environ. Sci. Pollut. Res.
Int. 2013, 20, 8031. doi:10.1007/S11356-013-1753-3
[17] X. C. Hu, D. Q. Andrews, A. B. Lindstrom, T. A. Bruton, L. A.
Schaider, P. Grandjean, R. Lohmann, C. C. Carignan, A. Blum, S. A.
Balan, C. P. Higgins, E. M. Sunderland, Detection of poly- and perfluoroalkyl substances (PFASs) in U.S. drinking water linked to industrial sites, military fire training areas, and wastewater treatment plants. Environ. Sci. Technol. Lett. 2016, 3, 344. doi:10.1021/ACS.
ESTLETT.6B00260
[18] S. Taniyasu, K. Kannan, Q. Wu, K. Y. Kwok, L. W. Y. Yeung, P. K. S.
Lam, B. Chittim, T. Kida, T. Takasuga, Y. Tsuchiya, N. Yamashita, Inter-laboratory trials for analysis of perfluorooctanesulfonate and perfluorooctanoate in water samples: Performance and recom- mendations. Anal. Chim. Acta 2013, 770, 111. doi:10.1016/J.ACA.
2013.01.056
[19] E. P. A. Method, 537, https://www.google.com/url?sa=t&rct=j&q=&
esrc=s&source=web&cd=1&ved=0ahUKEwiF2fWGo83VAhUq94 MKHd3TBlgQFggoMAA&url=https%3A%2F%2Fcfpub.epa.gov%
2Fsi%2Fsi_public_file_download.cfm%3Fp_download_id%3D525 468&usg=AFQjCNE_mLI1yaJvbqXO7gNCgJGNensAog (verified August 2017).
[20] E. Cotlove, E. K. Harris, G. Z. Williams, Biological and analytic components of variation in long-term studies of serum constituents in normal subject. Clin. Chem. 1970, 16, 1028.
[21] L. W. Y. Yeung, S. Taniyasu, K. Kannan, D. Z. Y. Xu, K. S. Guruge, P. K. S. Lam, N. Yamashita, An analytical method for the determina- tion of perfluorinated compounds in whole blood using acetonitrile and solid phase extraction methods. J. Chromatogr. A 2009, 1216, 4950. doi:10.1016/J.CHROMA.2009.04.070
[22] K. J. Hansen, L. A. Clemens, M. E. Ellefson, H. O. Johnson, Compound-specific, quantitative characterization of organic fluoro- chemicals in biological matrices. Environ. Sci. Technol. 2001, 35, 766. doi:10.1021/ES001489Z
[23] J. L. Reiner, K. W. Phinney, J. M. Keller, Determination of perfluori- nated compounds in human plasma and serum Standard Reference Materials using independent analytical methods. Anal. Bioanal.
Chem. 2011, 401, 2899. doi:10.1007/S00216-011-5380-X
[24] S. Kim, K. T. Lee, C. S. Kang, L. Tao, K. Kannan, K. Kim, C. Kim, J. S. Lee, P. S. Park, Y. W. Yoo, J. Y. Ha, Y. Shin, J. Lee, Distribution of perfluorochemicals between sera and milk from the same mothers and implications for prenatal and postnatal exposures. Environ.
Pollut. 2011, 159, 169. doi:10.1016/J.ENVPOL.2010.09.008 [25] A. Ka¨rrman, J. F. Mueller, B. van Bavel, F. Harden, L. L. Toms,
G. Lindstro¨m, Levels of 12 perfluorinated chemicals in pooled Australian serum, collected 2002–2003, in relation to age, gender, and region. Environ. Sci. Technol. 2006, 40, 3742. doi:10.1021/
ES060301U
[26] A. Ka¨rrman, K. H. Harada, K. Inoue, T. Takasuga, E. Ohi, A. Koizumi, Relationship between dietary exposure and serum perfluorochemical (PFC) levels—A case study. Environ. Int. 2009, 35, 712. doi:10.1016/
J.ENVINT.2009.01.010
[27] Z. Kuklenyik, J. A. Reich, J. S. Tully, L. L. Needham, A. M. Calafat, Automated solid-phase extraction and measurement of perfluorinated organic acids and amides in human serum and milk. Environ. Sci.
Technol. 2004, 38, 3698. doi:10.1021/ES040332U
[28] Z. Kuklenyik, L. L. Needham, A. M. Calafat, Measurement of 18 perfluorinated organic acids and amides in human serum using on-line solid-phase extraction. Anal. Chem. 2005, 77, 6085. doi:10.1021/
AC050671L
G
[29] K. Inoue, F. Okada, R. Ito, M. Kawaguchi, N. Okanouchi, H. Nakazawa, Determination of perfluorooctane sulfonate, perfluor- ooctanoate and perfluorooctane sulfonylamide in human plasma by column-switching liquid chromatography–electrospray mass spec- trometry coupled with solid-phase extraction. J. Chromatogr.
B Analyt. Technol. Biomed. Life Sci. 2004, 810, 49. doi:10.1016/
S1570-0232(04)00587-2
[30] W. K. Reagen, M. E. Ellefson, K. Kannan, J. P. Giesy, Comparison of extraction and quantification methods of perfluorinated compounds in human plasma, serum, and whole blood. Anal. Chim. Acta 2008, 628, 214. doi:10.1016/J.ACA.2008.09.029
[31] A. G. Asimakopoulos, N. S. Thomaidis, A. Bisphenol, 4-t-octylphe- nol, and 4-nonylphenol determination in serum by Hybrid Solid Phase Extraction–Precipitation Technology technique tailored to liquid chromatography–tandem mass spectrometry. J. Chromatogr. B Ana- lyt. Technol. Biomed. Life Sci. 2015, 986–987, 85. doi:10.1016/
J.JCHROMB.2015.02.009
[32] H. Son, J. Moon, H. S. Seo, H. H. Kim, B. C. Chung, M. H. Choi, High- temperature GC-MS-based serum cholesterol signatures may reveal sex differences in vasospastic angina. J. Lipid Res. 2014, 55, 155.
doi:10.1194/JLR.D040790
[33] G. W. Olsen, D. C. Mair, C. C. Lange, L. M. Harrington, T. R. Church, C. L. Goldberg, R. M. Herron, H. Hanna, J. B. Nobiletti, J. A. Rios, W. K. Reagen, C. A. Ley, Per- and polyfluoroalkyl substances (PFAS) in American Red Cross adult blood donors, 2000–2015. Environ. Res.
2017, 157, 87. doi:10.1016/J.ENVRES.2017.05.013
[34] B. B. Gump, Q. Wu, A. K. Dumas, K. Kannan, Perfluorochemical (PFC) exposure in children: associations with impaired response inhibition. Environ. Sci. Technol. 2011, 45, 8151. doi:10.1021/
ES103712G
[35] Y. J. Lee, M. Kim, J. Bae, J. Yang, Concentrations of perfluoroalkyl compounds in maternal and umbilical cord sera and birth outcomes in Korea. Chemosphere 2013, 90, 1603. doi:10.1016/J.CHEMO SPHERE.2012.08.035
[36] L. S. Haug, C. Thomsen, G. Becher, A sensitive method for determi- nation of a broad range of perfluorinated compounds in serum suitable for large-scale human biomonitoring. J. Chromatogr. A 2009, 1216, 385. doi:10.1016/J.CHROMA.2008.10.113
Handling Editor: Kevin Francesconi
H
