Novel Fluorinated Surfactants Tentatively Identified in Firefighters

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Novel fluorinated surfactants tentatively identified in firefighters using LC-QTOF-MS/MS and a case-control approach Anna Rotander, Anna Kärrman, Leisa Toms, Margaret Kay, Jochen F. Mueller, and María José Gómez Ramos Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es503653n • Publication Date (Web): 22 Jan 2015 Downloaded from http://pubs.acs.org on February 4, 2015

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Environmental Science & Technology

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Novel fluorinated surfactants tentatively identified in

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firefighters using LC-QTOF-MS/MS and a case-control

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approach

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Anna Rotander1, Anna Kärrman2, Leisa-Maree L. Toms3, Margaret Kay4, Jochen Mueller1, Maria Jose

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Gomez Ramos*1

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National Research Centre for Environmental Toxicology (Entox), The University of Queensland, QLD

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4108, Australia 2

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Man-Technology-Environment (MTM) Research Centre, Örebro University, SE-701 82 Örebro,

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Sweden 3

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School of Clinical Sciences and Institute of Health and Biomedical Innovation, Faculty of Health,

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Queensland University of Technology, QUT, 4001, Australia 4

The University of Queensland, Discipline of General Practice, Royal Brisbane and Women's Hospital

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Complex, QLD, 4029, Australia

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*Corresponding author:

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Maria Jose Gomez Ramos

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National Research Centre for Environmental Toxicology, Entox

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The University of Queensland

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QLD 4108, Australia

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Phone: +61 0437 511 813

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Fax: +61 7 3274 9003

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Email: [email protected] ACS Paragon Plus Environment

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RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required

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according to the journal that you are submitting your paper to)

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Title Running Head: Novel fluorinated surfactants discovered in blood serum from people exposed to

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AFFF

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Key words: Aqueous film forming foams, AFFF, exposure, firefighters, LC-QTOF-MS/MS, unknown

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PFAS, PFOS, PFHxS

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Abstract

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Fluorinated surfactant-based aqueous film–forming foams (AFFF) are made up of per- and

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polyfluorinated alkyl substances (PFAS), and are used to extinguish fires involving highly flammable

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liquids. The use of perfluorooctanesulfonic acid (PFOS) and other perfluoroalkyl acids (PFAAs) in

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some AFFF formulations has been linked to substantial environmental contamination. Recent studies

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have identified a large number of novel and infrequently reported fluorinated surfactants in different

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AFFF formulations. In this study, a strategy based on a case-control approach, using quadrupole time of

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flight tandem mass spectrometry (QTOF-MS/MS) and advanced statistical methods has been used to

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extract and identify known and unknown PFAS in human serum, associated with AFFF exposed

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firefighters.

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(perfluoropentanesulfonic

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perfluorononanesulfonic acid (PFNS), and four unknown sulfonic acids (Cl-PFOS, ketone-PFOS, ether-

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PFHxS, and Cl-PFHxS) were exclusively or significantly more frequently detected at higher levels, in

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firefighters compared to controls.

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Two

target

sulfonic acid

acids

(PFPeS),

(PFOS

and

PFHxS),

perfluoroheptanesulfonic

three acid

non-target

acids

(PFHpS)

and

The application of this strategy has allowed for identification of previously unreported fluorinated chemicals in a timely and cost efficient way.

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Introduction

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Aqueous film–forming foams (AFFFs) are complex mixtures containing fluorocarbon- and

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hydrocarbon- based surfactants used to extinguish fires involving highly flammable liquids. Fluorinated

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surfactants in AFFF are made up of per- and polyfluorinated alkyl substances (PFAS), which are both

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hydrophobic and oleophobic as opposed to hydrocarbon surfactants that are merely hydrophobic in

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nature1,2. AFFF have been produced either by electrochemical fluorination (ECF) or telomerization. In

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the ECF process, as opposed to telomerization that exclusively yields an even number of fluorinated

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carbon chains, a significant amount of cyclic, cleaved, and branched compounds of different

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perfluoroalkyl chain lengths are formed in a complex mixture3. The old generation AFFFs, produced by

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ECF and based on perfluorooctanesulfonic acid (PFOS), were phased out after 2002 when 3M

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voluntarily discontinued its production of PFOS based products4. The use of PFOS and other

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perfluoroalkyl acids (PFAAs) in AFFF formulations has been linked to environmental contamination

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related to handling, storage and usage1,5. Substantially elevated levels of PFOS have been reported in

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water and biological samples, such as molluscs6, turtles5, wild mink7 and fish8, downstream from

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airports with a history of firefighting training activities.

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The new generation AFFF is made up of varieties of PFAS and it has been reported that mixtures of

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6:2 and 8:2 fluorotelomers predominates9. In environmental samples, for example in seawater10,

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shrimp11, and human serum12, a significant portion of the total organofluorine can be in the form of

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unknown fluorinated chemicals. It has been suggested that this may originate from proprietary

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fluorinated surfactants that may enter the environment through the use of AFFF13. Because many of the

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chemicals included in these new generation foams are unknown, a number of recent studies have

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focused on optimizing analytical methods to identify these unknown fluorinated compounds13-17. This

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has resulted in the identification of a large number of novel and infrequently reported fluorinated

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surfactants in different AFFF formulations. Previous degradation studies have shown that AFFF

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components can break down to short chain (C5-C7) perfluorinated carboxylic acids (PFCAs), and more

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research has been called for to examine these novel surfactants for their potential as PFAA precursors 15, ACS Paragon Plus Environment

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compared to AFFF formulations, suggesting that AFFF precursors were transformed to PFAAs19.

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. The fraction of PFAAs was also found to be higher in contaminated groundwater and solid samples

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Current advances in analytical instrumentation, specifically in accurate mass/high resolution mass

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spectrometry (HRMS), provide new opportunities for the identification of non-target compounds (i.e.

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compounds not targeted by the analytical method) and unknown compounds (i.e. compounds not

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included in any chemical structure database or compounds to date not described in the scientific

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literature). Quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) is increasingly being

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used for this purpose20,21,22. The identification/elucidation of unknown compounds, not present in any

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library, is more complicated than non-target analyses. Non-target analyses usually rely upon the use of

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personal and commercial libraries. Given the high number of compounds that are generated in a QTOF-

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MS/MS analysis, filtering strategies are necessary to focus attention on a specific group of masses of

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interest, enabling their subsequent accurate identification. Filtering strategies using mass defect filtering

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has been recently employed to identify novel fluoropolymer thermal decomposition products22 and

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unknown pefluoroalkyl species in fish21. Filtering strategies based on case-control samples have proved

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to be extremely valuable in the field of clinical chemistry making it possible to identify new biomarkers

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for the early diagnosis of diseases, such as cancer23 or Alzheimer’s disease24. However, this approach

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has to our knowledge not yet been applied for identification of environmental contaminants in humans

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or biota.

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The purpose of this study was to analyze fluorinated surfactants in blood serum of firefighters who

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had previously been exposed to AFFF in their work, with the aim of discovering “previously

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unidentified” persistent PFAS. To carry out this study a case-control approach using LC-QTOF-MS/MS

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and statistical analysis was developed and applied to detect and identify chemicals associated with

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AFFF exposure.

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Materials and Methods


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Study participants. Both firefighters and controls had been recruited for participation in two

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previous studies. For the current study, blood sera of 20 firefighters collected in 2013 with 19-38 years

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of exposure to AFFF in their work were selected. Sera from 20 university students and office workers,

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who had not been exposed to AFFF, had been previously collected in 2011/12. These sera were used as

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controls. Written informed consent was obtained from all participants at recruitment. Ethics approval for

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this study was granted by The University of Queensland Medical Research Ethics Committee (Number

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2014000614).

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Chemicals and Extraction. Details of chemicals and standard compounds used in the extraction are

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available in the Supporting Information. Mass labeled internal standards were added to 0.2 mL serum.

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The analytes were then extracted with 1.5 mL 100% acetonitrile using ultrasonication followed by

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vortex extraction, centrifugation and evaporation to 0.2 mL under a gentle stream of nitrogen.

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Performance standards and 0.3 mL 5 mM ammonium acetate in water were added prior to analysis.

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LC/ESI-QTOF-MS Analysis. Chromatographic separation of the analytes was carried out using a

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Shimadzu Nexera X2 UHPLC system equipped with a binary pump and a reverse-phase Gemini-NX

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C18 column (3µm x 2.0 mm x 50 mm, Phenomenex). The injection volume was 10 µL. The column unit

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was held at 45 °C and the flow rate was 0.6 mL/min. An isolator column (Phenomenex) was placed

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directly after the mobile phase mixing chamber to delay the elution of solvent derived background.

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Mobile phases A and B were, respectively, 1% methanol in water and 10% water in methanol with

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5mM ammonium acetate in both phases. The initial gradient (10% B) was held for 0.20 min. A gradient

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ramp followed over 6.5 min to 100% B, which was held for 3 min, then returned to the initial

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composition in 0.10 min, followed by equilibrium for 2.20 min. The UHPLC was coupled to a hybrid

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quadrupole time-of-flight mass spectrometer system TripleTOF 5600 System (AB Sciex) with

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electrospray ionization (ESI) interface working in negative ionization mode. The MS was operated in

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full scan TOFMS and MS/MS mode with information dependent acquisition (IDA) in a single run ACS Paragon Plus Environment

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analysis. The data acquisition and processing was carried out using Analyst® TF 1.6, PeakView®, and

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MultiQuantTM software (AB Sciex). MarkerViewTM software (AB Sciex) was used for the

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simultaneous feature finding, alignment, and statistical analysis to highlight compounds of interest.

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Quantification and quality assurance. Quantification of non-target and unknown PFAS was

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performed using labeled standards and response factors for PFOS and perfluorohexanesulfonic acid

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(PFHxS). The method detection limits (MDLs) for PFOS and PFHxS in this study were 0.02 ng/mL and

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0.07 ng/mL, respectively, and the limit of reporting (LORs) were 0.06 ng/mL for PFOS and 0.35 ng/mL

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for PFHxS. The reproducibility of the method was calculated from multiple analyses of a pooled serum

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sample on different days using the internal standard method and mass labeled standards. Other method

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details and the method validation study (repeatability, reproducibility, and accuracy) are provided in the

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Supporting Information.

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Statistical analysis. Statistical data analysis was performed using MarkerView software to highlight

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any groups within the data and identify any trends and unexpected compounds. This software

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automatically aligns mass and retention time to compensate for minor variations ensuring that identical

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compounds in different samples are accurately compared. The principal component analysis was

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performed both without and with normalization of the data using the area of 13C4- PFOS and total area

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sums. Detailed information about the statistical analysis is provided in the Supporting Information.

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Results and Discussion

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The relatively rapid and cost efficient identification of both known and unknown PFAS within the

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sera of workers with occupational exposure to AFFF, was facilitated by an analytical methodology

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based on a case-control filtering strategy, that has not yet been established in environmental studies.

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This approach helps to address some of the challenges that researchers face when attempting to identify

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novel organic chemicals in serum or other biological matrices. To date such research has been

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challenging because the samples being tested contain many compounds at low concentrations. This ACS Paragon Plus Environment

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study provides a data reduction strategy for addressing the large amount of data that is generated using

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non-target QTOF-MS/MS screening analysis. This analytical strategy combines the use of accurate

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mass data and statistical evaluation of sample constituents to extract the most important components for

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further identification. After the multivariate analysis, the compounds of interest were reduced from

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almost 3000 chemicals to a group of about 50 compounds. Figure 1 illustrates the four components of

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the ‘case-control’ strategy that were applied; 1) Selection of case and control samples, 2) Sample

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preparation and analysis, 3) Statistical analysis, and 4) Identification of the extracted compounds. For

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analytical method and statistical analysis details please see Supporting Information.

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All components of the two groups of samples (firefighters and controls) were compared using

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multivariate statistical tools including supervised and unsupervised methods in order to facilitate the

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isolation of the compounds responsible for the discrimination between the groups. The principal

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component analysis (PCA, unsupervised) showed a clear separation between firefighters and controls

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(Fig 1, 3A). This separation was obtained both with and without data normalization. The corresponding

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loading plot indicated that PFOS and PFHxS were strongly associated with the firefighters.

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Interestingly, after removing PFOS and PFHxS and rerunning the PCA the separation of the two groups

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remained, indicating that there were other chemicals present that were responsible for this separation.

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The ions associated with the firefighters were extracted with discriminant analysis (PCA-DA) and T-test

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(Fig 1, 3B). A volcano plot was used to show the significant entities, which are those at the extremes of

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the axis (low p-value and a large fold change). The ions with a log fold-change higher than 0.90 and a p-

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value lower than 0.05 (ions within the red squares in Figure 1, 3B) were selected, which are the ions

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most strongly associated with the firefighters. All the chemicals identified in this study showed a p-

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value lower than 0.001, except for PFPeS that presented a p-value of 0.04. Most of the ions selected

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(ions with fold-change higher than 0.90 and p-value lower than 0.05) presented a mass defect higher

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than 0.90, which is characteristic of PFAS.


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A profile plot was used to explore the content of the identified chemicals across all samples (Fig 1,

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3C). This tool was very useful for confirming that the compounds of interest were almost exclusively

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present or present at significantly higher levels in the firefighters compared to controls. This tool was

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also useful for confirming that these compounds were absent in the blanks and standards. In the example

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from Figure 1 (4A and 4B), 1-chloro-perfluorooctane sulfonic acid (Cl-PFOS; m/z 514.9017,

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C8O3F16SCl-, 2.4 ppm) is used as an example to show the identification process. Fig 1, 3C shows the

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profile plot for this mass in all 40 investigated sample, and demonstrates that this compound is uniquely

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present in the firefighters’ samples.

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For identification of the discriminating components, the masses extracted with statistical analysis

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were linked back to the raw mass spectra. The first step in the identification process was to ensure that

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the signal of interest corresponded to a monoisotopic ion and not to an isotope, adduct or ion product

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generated during the ionization process. The characterization of compounds not present in commercial

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libraries was achieved in two steps (Figure 1, 4). i). The determination of the elemental composition

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from the accurate mass and the use of isotope patterns, mass defect and rings and double bonds (RDB)

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to reduce the list of candidates (Figure 1, 4A). ii). Public libraries (eg., ChemSpider, PubChem) were

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used to elucidate possible structures from the elemental formula and MS/MS fragment information

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(Figure 1, 4B). The non-target compounds (PFPeS, PFHpS and PFNS) were found in ChemSpider.

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Since these chemical databases do not currently have any mass spectra data for these compounds,

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proposed molecular structure was crosschecked with the mass spectral data to verify that primary

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fragments corresponded to realistic chemical compositions. For the unknown compounds (Table 1) the

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consulted on-line chemical databases did not come up with any results. These compounds were

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tentatively identified based on the precise interpretation of its accurate mass spectra, MS/MS

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fragmentation, as well as literature and background information. For confirmation the suspected

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molecular structure was drawn and linked to the experimental MS/MS data (Fig. 1.4.B).

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Target, non-target, and unknown PFAS exclusively present or present at significantly higher levels in

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firefighters compared to controls that were either identified (using native standards) or tentatively

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identified are summarized in Table 1 and shown in the chromatogram in Figure 2. Two target ions (m/z

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498.9302, PFOS and 398.9633, PFHxS), three non-target ions (m/z 348.9398, perfluoropentanesulfonic

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acid (PFPeS); m/z 448.9334, perfluoroheptanesulfonic acid (PFHpS) and m/z 548.9270,

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perfluorononanesulfonic acid (PFNS)), and four unknown ions (m/z 414.9071, m/z 414.9315, m/z

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514.9007 and m/z 476.9283) were strongly associated with AFFF exposure. It should be noted that

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method development was performed with strong acidic fluorosurfactants such as PFOS and PFHxS

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hence several classes of PFAS might not have been extracted and isolated by this analysis. For example,

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telomer alcohols do not ionize under current conditions and can therefore not be analyzed25. Table 1

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summarizes the main parameters obtained when applying the LC-QTOF-MS/MS screening. Mass

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accuracy was

Novel Fluorinated Surfactants Tentatively Identified in Firefighters

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