Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tfac20 Food Additives & Contaminants: Part A ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tfac20 Method development and evaluation for the determination of perfluoroalkyl and polyfluoroalkyl substances in multiple food matrices Dorothea F. K. Rawn, Cathie Ménard & Sherry Yu Feng To cite this article: Dorothea F. K. Rawn, Cathie Ménard & Sherry Yu Feng (2022) Method development and evaluation for the determination of perfluoroalkyl and polyfluoroalkyl substances in multiple food matrices, Food Additives & Contaminants: Part A, 39:4, 752-776, DOI: 10.1080/19440049.2021.2020913 To link to this article: https://doi.org/10.1080/19440049.2021.2020913 © 2022 The Author(s). Published with license by Taylor & Francis Group, LLC. View supplementary material Published online: 04 Feb 2022. 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K. Rawn, Cathie Ménard, and Sherry Yu Feng Food Research Division, Bureau of Chemical Safety, Health Canada, Ottawa, Ontario, Canada ABSTRACT A method for the determination of 21 perfluorinated and 10 polyfluorinated alkyl substances (PFAS) was developed for application in different food matrices. Acetonitrile was used as the extraction solvent with solid phase extraction weak anion-exchange (SPE-WAX) clean up, with LC-MS/MS analysis using both surrogate and performance standards to correct for losses during sample preparation and matrix effects. The method has been evaluated in four different matrices (fish, pizza, chicken nuggets and spinach). Originally, the focus was to develop a method for foods commonly thought to be a source of PFASs (e.g. fish). It was expanded to include foods where PFAS exposure would be possible through their presence in grease-proof food packaging (e.g. pizza, chicken nuggets). Vegetables (lettuce) and fruit (tomato) have recently been considered as part of proficiency testing programmes, so the inclusion of some testing in a vegetable matrix (i.e. spinach) was also added to the testing. Limits of quantification ranged from 0.018 ng g−1 (L-PFDS) to 5.28 ng g−1 (FHEA), although method quantification limits for PFBA (12.4 ng g−1), 6:2 PAP (8.96 ng g−1) and 8:2 PAP (3.49 ng g−1) were elevated above instrumental limits owing to their consistent detection in reagent blank samples. PFAS analyses were strongly impacted by matrix, therefore the use of isotopically labelled internal stan- dards was critical to the development of accurate results. The accuracy of the method using numerous proficiency testing schemes or interlaboratory comparison studies has shown the developed method to be successful with z-scores for all concerned analytes in all test matrices remaining within ±2.0, with the exception of PFBA in wheat flour which was −2.4. ARTICLE HISTORY Received 19 October 2021 Accepted 6 December 2021 KEYWORDS Perfluorinated substances; PFAS; method evaluation; food matrices; LC-MS/MS Introduction Perfluoroalkyl and polyfluoroalkyl substances (PFASs) encompass an extensive group of alkyl com- pounds with fluorine substitution of all (perfluoro-) or some (polyfluoro-) of the hydrogens on the car- bon chain (OECD 2018). The vast numbers of per- and polyfluoro-substances arise from partially fluori- nated compounds, fluorinated polymers and reac- tion products, with >9,000 PFASs listed on the United States Environmental Protection Agency master list of PFAS substances (US EPA 2021). In addition to the strong, stable backbone, PFASs may have terminal carboxylic acids, sulfonic acid, alcohol, amide or phosphate groups (Buck et al. 2011). This structure provides PFASs with unusual characteris- tics in that they are both chemically stable and lipo- philic, in addition to having hydrophilic terminal groups. Owing to the combination of these proper- ties, they have been used in many applications for more than 50 years (OECD 2018). Their use on non- stick pans and utensils for cooking as well as grease- proofing paper used by the fast food industry was widely discussed in the early 2000s. In addition to these applications, they are present in many other products including paints, cosmetics, as firefighting foams and processing aids in the manufacture of polymers (Domingo 2012; Ayala-Cabrera et al. 2016; OECD 2018; EFSA 2020). PFAS research studies have been undertaken following recognition of the widespread use of PFASs in industrial and consumer applications and understanding the implications associated with stable organohalogen compounds in the environment (Houde et al. 2006). The presence of PFASs has been confirmed in abiotic envir- onmental compartments (e.g. air, water, dust) (Barber et al. 2007; Cornelis et al. 2012; Gellrich and Knepper 2012; Liu et al. 2021; Rodríguez- CONTACT Dorothea F. K. Rawn thea.rawn@hc-sc.gc.ca Food Research Division, Bureau of Chemical Safety, Health Products and Food Branch, Health Canada, 251 Sir Frederick Banting Driveway, Address Locator: 2203C, Tunney’s Pasture, Ottawa, ON K1A 0K9, Canada Supplemental data for this article can be accessed on the publisher’s website. https://doi.org/10.1080/19440049.2021.2020913 © 2022 The Author(s). Published with license by Taylor & Francis Group, LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc- nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way. FOOD ADDITIVES & CONTAMINANTS: PART A 2022, VOL. 39, NO. 4, 752–776 https://doi.org/10.1080/19440049.2021.2020913 http://www.tandfonline.com https://crossmark.crossref.org/dialog/?doi=10.1080/19440049.2021.2020913&domain=pdf&date_stamp=2022-02-03 Varela et al. 2021), and also in biological organ- isms (Vorkamp et al. 2019; Pereira et al. 2021; Wood et al. 2021), including humans (Bjermo et al. 2013; Cariou et al. 2015; Papadopoulou et al. 2015; Vélez et al. 2015; Averina et al. 2018). Detectable concentrations of PFASs have been observed in fish and shellfish globally (Ericson et al. 2008; Noorlander et al. 2011; Stahl et al. 2014; Fujii et al. 2015). PFAS use in consumer products also has resulted in studies to examine levels in indoor environments relative to the outdoors and confirmed that PFASs are elevated in indoor environments (Goosey and Harrad 2012). Unlike the traditional persistent environmental organic contaminants (e.g. poly- chlorinated dibenzo-p-dioxins) for which food consumption is clearly the dominant pathway for human exposure, PFAS exposure is multi-faceted. Important pathways of PFAS exposure include water and food consumption, dust ingestion, inha- lation and dermal exposure. Despite the many sources of human exposure to PFASs, diet was considered to be the major route of exposure for most of the population in a recent evaluation of these compounds (EFSA 2020). The recent EFSA assessment on PFASs in food examined occurrence data from different food groups (e.g. fish, meat, eggs, fruits/fruit products, vegetables/vegetable products) to examine the overall dietary exposure, rather than the many studies that focus on specific food types (EFSA 2020). The complete assessment was performed for the well-known PFASs; PFOS and PFOA, although PFNA and PFHxS were also assessed (EFSA 2020). Although perfluoro- and polyfluoroalkyl sub- stances bioaccumulate in the tissues of living organ- isms, they do not bioaccumulate in the lipid tissues, but rather in tissues rich in protein, and are detected in blood, liver and kidneys (Domingo 2012; Bjermo et al. 2013; Ali et al. 2021). Accurate determination in food matrices of different protein content is impor- tant for proper analysis and exposure estimation. Toxicological studies of perfluoroalkyl com- pounds, particularly perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS), have identified linkages between expo- sure and thyroid function and immunosuppres- sion (Alsen et al. 2021). PFOA exposure has been found to suppress immune function in mice (Dewitt et al. 2016). Ou and coworkers (Ou et al. 2021) identified a relationship between gestational PFAS exposure and conge- nital heart defects in humans. An association between PFAS exposure and negative pregnancy outcomes (e.g. miscarriage, stillbirth, etc.) has been indicated following a global review of relevant studies (Deji et al. 2021). Toxicological data associated with PFAS expo- sure has been the focus of a thorough review of PFASs (EFSA 2020). A wide variety of approaches to the extraction, clean up and analysis of PFAS in food have been reported in the literature. Alkaline digestion with sodium hydroxide in advance of extraction has been reported by Jogsten et al. and Haug et al. (Jogsten et al. 2009; Haug et al. 2010). The use of tetrabutylammonium (TBA) hydrogen sulfate solu- tion with sodium carbonate buffer in advance of solvent extraction has also been described in the literature (Guerranti et al. 2013). Another pre- extraction technique employed has been the precipi- tation of proteins in milk samples (Cariou et al. 2015). In addition to using solvents employed for traditional POP analysis (e.g. acetone: hexane), methyl-tert-butyl ether, methanol, tetrahydrofuran: water and acetonitrile have been used for PFAS extraction from food (Farré et al. 2012; Herzke et al. 2013). Tetrahydrofuran: water (75:25) has been reported to effectively extract PFAS of varying chain lengths (4–14) from food matrices (Ballesteros-Gómez et al. 2010). A variety of sorbents have been used for clean up, including Florisil, weak anion exchange resin and carbon (Farré et al. 2012). LC-MS/MS generally has been used for the determi- nation of PFAS in all matrices (Kuklenyik et al. 2004; Ericson et al. 2008; Chung and Lam 2014; Tang et al. 2014; Manzano-Salgado et al. 2015), although gas chromatography-mass spectrometry also has been reported (Fujii et al. 2015). In the mid-2000s, our group developed a method for the determination of seven perfluorocarboxylic acids ranging in carbon chain length from seven to 14, and one perfluorosulfonic acid in food using liquid chromatography–tandem mass spectrometry (Tittlemier et al. 2007). The original method devel- oped in our laboratories utilized methanol for extrac- tion with vortex, rotary mixing and centrifugation in advance of analysis (Tittlemier et al. 2007). Since that FOOD ADDITIVES & CONTAMINANTS: PART A 753 time, regulatory action has been taken on perfluor- ooctanoic acid, perfluorooctanesulfonic acid, their salts and related compounds (Stockholm Convention 2019). Following regulatory action and voluntary restrictions on the use of longer chain PFASs, use of shorter chain analogues has increased as replacements (Brendel et al. 2018). As a result, our group was tasked with adapting the methodology developed for longer chain compounds to also include shorter chain analogues, in multiple different food matrices. The analytes considered for this method were selected based on availability of analytical stan- dards, are consistent with the PFASs reported in environmental samples and considered as part of the recently published assessment on PFASs (EFSA 2020). Telomer acids were added to the list of analytes to determine whether the method would capture these compounds, with individual compounds added based on standard availability. The developed method employs the use of stable isotope correction for the determination of 13 perfluorocarboxylic acids ranging in chain length from four to 18 carbons, eight sulfonates (PFOS is determined as linear and branched isomers) with carbon chains extending from four to 12, two phosphate esters, two dipho- sphate esters and six telomer acids. Materials and methods Chemicals and reagents Analytical standards (>98%) of perfluoro-n-butanoic acid (PFBA), perfluoro-n-pentanoic acid (PFPeA), perfluoro-n-hexanoic acid (PFHxA), perfluoro- n-heptanoic acid (PFHpA), perfluoro-n-octanoic acid (PFOA), perfluoro-n-nonanoic acid (PFNA), perfluoro-n-decanoic acid (PFDA), perfluoro- n-undecanoic acid (PFUdA), perfluoro-n-dodecanoic acid (PFDoA), perfluoro-n-tridecanoic acid (PFTrDA), perfluoro-n-tetradecanoic acid (PFTeDA), perfluoro-n-hexadecanoic acid (PFHxDA) and per- fluoro-n-octadecanoic acid (PFODA) in methanol were purchased from Wellington Laboratories, (Guelph, ON, Canada). Analytical standards of per- fluorosulfonates were purchased as potassium or sodium salts (>98% salt). Perfluoro-1-butanesulfonate (L-PFBS) as the potassium salt, perfluoro- 1-pentanesulfonate (L-PFPeS), perfluoro-1-hexane- sulfonate (L-PFHxS), perfluoro-1-heptanesulfonate (L-PFHpS) as sodium salts in methanol were pur- chased from Wellington Laboratories. A mixture of linear and branched isomers of perfluorooctanesulfo- nate (br-PFOS (total)) present as potassium salts in methanol was purchased, while perfluoro-1-nonane- sulfonate (L-PFNS), perfluoro-1-decanesulfonate (L-PFDS) and perfluoro-1-dodecanesulfonate (L-PFDoS) were purchased as sodium salts in metha- nol, from Wellington Laboratories. Telomer acids (>98%) prepared in isopropanol were purchased from Wellington Laboratories, including 2H-perfluoro-2-octenoic acid (FHUEA), 2H-perfluoro-2-decenoic acid (FOUEA), 2H- perfluoro-2-dodecenoic acid (FDUEA), 2-perfluor- odecyl ethanoic acid (FDEA), 2-perfluorohexyl ethanoic acid (FHEA) and 2-perfluorooctyl etha- noic acid (FOEA). In addition, four polyfluoroalk- ylphosphate esters (PAPs) (>98%) were purchased as sodium salts in methanol: 1H,1H,2H,2H-per- fluorooctylphosphate (6:2 PAP), 1H,1H,2H,2H- perfluorodecylphosphate (8:2 PAP), bis (1H,1H,2H,2H-perfluorooctyl)phosphate (6:2 diPAP) and bis(1H,1H,2H,2H-perfluorodecyl)phos- phate (8:2 diPAP) from Wellington Laboratories. Stable isotope (13C) analogues of the perfluorocar- boxylic acids: 13C4 PFBA, 13C5 PFPeA, 13C5 PFHxA, 13 C4 PFHpA, 13C4 PFOA, 13C2 PFUdA, 13C2 PFDA, 13 C9 PFNA, 13C2 PFDoA and 13C2 PFHxDA in metha- nol were purchased from Wellington Laboratories, surrogate standard isotopic purity was >99% for all compounds. Similarly, stable isotope analogues of the perfluorosulfonates were purchased from Wellington Laboratories including: 18O2 PFHxS and 13C4 PFOS as sodium salts with >99% isotopic purity. Stable isotope analogues (13C2) of the telomer acids (FHUEA, FDUEA, FDEA and FHEA) and phosphate esters (6:2 PAP, 8:2 PAP and 8:2 diPAP) also were purchased as sodium salts from Wellington Laboratories and were similarly of >99% isotopic purity. In addition to the surrogates, 13C5 PFNA, 13C2 PFTeDA, 13C2 FOEA, 13C2 FOUEA and 13C2 6:2 diPAP were purchased from Wellington Laboratories as performance stan- dards (>99% isotopic purity), while 13C8 PFOA (99% isotopic purity) was purchased from Cambridge Isotope Laboratories Inc. (Tewksbury, MA, USA) 754 D. F. K. RAWN ET AL. and 18O2-ammonium–PFOS (>99% labelled) was pur- chased from RTI International (Research Triangle Park, NC, USA). Optima LC/MS grade acetonitrile, water, 2-propanol and methanol were purchased from Fisher Scientific (Ottawa, ON, Canada). Optima LC/MS grade formic acid, ammonium formate and ammonium acetate were similarly purchased from Fisher Scientific while ammonium hydro- xide was purchased from either Fisher Scientific or Sigma-Aldrich (Oakville, ON, Canada). Nitrogen gas (ultra-pure) was purchased from Linde Canada Inc. (Ottawa, ON, Canada) and purified water was obtained by passing de- ionised water through a Millipore system (18.2 Ω cm, Sigma-Aldrich, Oakville, ON, Canada). Calibration standards Six-point calibration curves above the zero concen- tration point (2-propanol with isotope-labelled compounds) were developed for each analyte (Table 1). The calibration range varied between compounds, based on relative sensitivity and 3 µL injection volumes were used for all calibrants. The calibration point with no analytes added was gen- erally free of all compounds except the added sur- rogates and performance standards throughout the work. If the presence of analytes was observed in the ‘zero concentration’ point, it was used as a trigger to clean the instrument. Branched-PFOS (isomer 3–11) was the most sensitive analyte and the calibration range extended from 0.05 pg µL−1 to 12.4 pg µL−1 (Table 1). The calibration standards for PFHxA, PFNA, PFDA, PFUdA, PFDoA and Table 1. Analyte information including transitions monitored, surrogate standard selection and linearity of response established using LC-MS/MS. Analyte Retention time (min) Transition monitored Surrogate standard Cone voltage (V) Collision energy (eV) Calibration concentration (pg µL−1) Correlation coefficient (r2) PFBA 9.65 213 > 169 13C4-PFBA 20 8 1, 2.5, 5, 10, 25, 50 0.999578 PFPeA 10.27 263 > 219 13C5-PFPeA 20 8 1, 2.5, 5, 10, 25, 50 0.999590 L-PFBS 10.53 299 > 80 18O2-PFHxS 45 30 0.1, 0.25, 0.5, 2.5, 10, 25 0.996507 PFHxA 10.63 313 > 269 13C5-PFHxA 15 8 0.2, 0.5, 1, 5, 20, 50 0.999251 L-PFPeS 10.86 349 > 80 18O2-L-PFHxS 50 35 0.1, 0.25, 0.5, 2.5, 10, 25 0.999046 FHUEA 10.93 357 > 293 13C2-FHUEA 20 10 0.2, 0.5, 1, 2.5, 10, 20 0.999545 PFHpA 10.98 363 > 319 13C4-PFHpA 20 8 0.1, 0.25, 0.5, 2.5, 10, 25 0.999262 FHEA 10.94 377 > 293 13C2-FHEA 20 12 2, 5, 10, 25, 100, 200 0.996909 L-PFHxS 11.28 399 > 80 18O2-L-PFHxS 40 40 0.1, 0.25, 0.5, 2.5, 10, 25 0.999209 PFOA 11.50 413 > 369 13C4-PFOA 20 8 0.1, 0.25, 0.5, 2.5, 10, 25 0.999270 L-PFHpS 11.90 449 > 80 13C4-PFOS (fish) 18O2-L-PFHxS 40 45 0.1, 0.25, 0.5, 2.5, 10, 25 0.998508 FOUEA 12.17 457 > 393 13C2-FHUEA 20 10 0.2, 0.5, 1, 2.5, 10, 20 0.997255 PFNA 12.21 463 > 419 13C9-PFNA 20 10 0.2, 0.5, 1, 5, 20, 50 0.999403 FOEA 12.19 477 > 393 13C2-FHEA 20 15 2, 5, 10, 25, 100, 200 0.996630 br-PFOS (isomer 3–11) 12.46 499 > 80 13C4-PFOS 65 45 0.05, 0.12, 0.25, 1.24, 4.98, 12.4 0.997613 L-PFOS + isomer 2 12.68 499 > 80 13C4-PFOS 65 45 0.2, 0.5, 1, 5, 20, 50 0.999168 PFDA 13.08 513 > 469 13C2-PFDA 20 10 0.2, 0.5, 1, 5, 20, 50 0.999448 L-PFNS 13.58 549 > 80 13C4-PFOS 50 50 0.1, 0.25, 0.5, 2.5, 10, 25 0.999032 FDUEA 14.05 557 > 493 13C2-FDUEA 20 10 0.2, 0.5, 1, 2.5, 10, 20 0.999448 PFUdA 14.04 563 > 519 13C2-PFUdA 15 10 0.2, 0.5, 1, 5, 20, 50 0.999239 FDEA 14.08 577 > 493 13C2-FDEA 20 12 2, 5, 10, 25, 100, 200 0.995151 L-PFDS 14.52 599 > 80 13C4-PFOS 30 50 0.1, 0.25, 0.5, 2.5, 10, 25 0.998479 PFDoA 15.01 613 > 569 13C2-PFDoA 20 10 0.2, 0.5, 1, 5, 20, 50 0.999247 PFTrDA 15.95 663 > 619 13C2-PFHxDA 20 12 0.1, 0.25, 0.5, 2.5, 10, 25 0.997546 L-PFDoS 16.31 699 > 80 13C2-PFHxDA 70 60 0.1, 0.25, 0.5, 2.5, 10, 25 0.964784 PFTeDA 16.82 713 > 669 13C2-PFHxDA 30 12 0.1, 0.25, 0.5, 2.5, 10, 25 0.998431 PFHxDA 18.33 813 > 769 13C2-PFHxDA 30 15 0.1, 0.25, 0.5, 2.5, 10, 25 0.999068 PFODA 19.51 913 > 869 13C2-PFHxDA 30 15 0.1, 0.25, 0.5, 2.5, 10, 25 0.996746 6:2 PAP 5.06 443 > 97 13C2-6:2 PAP 20 15 0.5, 1, 2.5, 5, 10, 25 0.993862 8:2 PAP 5.48 543 > 97 13C2-8:2 PAP 20 20 0.5, 1, 2.5, 5, 10, 25 0.990141 6:2 diPAP 5.90 789 > 97 789 > 443 13C2-8:2 diPAP 1 13C2-8:2 diPAP 2 25 25 30 17 0.1, 0.2, 0.5, 1, 2, 5 0.998351 0.998492 8:2 diPAP 6.04 989 > 97 989 > 543 13C2-8:2 diPAP 1 13C2-8:2 diPAP 2 30 30 35 20 0.1, 0.2, 0.5, 1, 2, 5 0.998243 0.998596 FOOD ADDITIVES & CONTAMINANTS: PART A 755 L-PFOS + isomer 2 were between 0.2 and 50 pg µL−1, while the majority of compounds (car- boxylic acids: PFHpA, PFOA, PFTrDA, PFTeDA, PFHxDA, PFODA, sulfonates: L-PFBS, L-PFPeS, L-PFHxS and L-PFHpS, L-PFNS, L-PFDS and L-PFDoS) ranged from 0.1 to 25 pg µL−1. The calibrants for 6:2 diPAP and 8:2 diPAP ranged in concentration from 0.1 to 5 pg µL−1, although 6:2 PAP and 8:2 PAP, which were less sensitive, were prepared in calibration curves extending from 0.5 to 25 pg µL−1. PFBA and PFPeA calibration stan- dards ranged from 1 to 50 pg µL−1. Telomer acids (FHUEA, FOUEA and FDUEA) ranged from 0.2 to 20 pg µL−1 and the remaining telomer acids (FHEA, FOEA and FDEA) ranged from 2 to 200 pg µL−1. All calibration curves followed a linear regression with correlation coefficients of ≥0.99, with the exception of L-PFDoS (Table 1). Deviation from anticipated concentrations was generally <10%, with average deviations ranging from 1.11% to 2.84%. All surrogate standards were present in calibration solutions at 10 pg µL−1 with a few exceptions, includ- ing 13C2 FHEA and 13C2 FDEA which were present at 100 pg µL−1 (Table 2). 13C2 6:2 and 8:2 PAPs were present in calibration standards at 2.5 pg µL−1 while 13C2 8:2 diPAP was present at 1 pg µL−1. Performance standards were similar for most PFAS, present at 10 pg µL−1 with the exception of 13C2 FOEA (100 pg µL−1). 13C2 6:2 diPAP was used as a perfor- mance standard for the PAPs and diPAPs (1 pg µL−1). Representative isotope-labelled compounds were selected as surrogate standards by class (carboxylic acid, sulfonate, etc.) to align similar chemicals and hence optimizing the efficacy of the surrogates. Performance standard selection was identified simi- larly, to ensure that the method utilized the isotope- labelled compounds most effectively. Evaluation Evaluation studies were performed using archived composite samples collected and prepared as part of the Canadian Total Diet Study (Government of Canada, n.d.). The protocol for sample collection as part of the Canadian Total Diet Study involved the collection of equivalent food samples from four different stores in the city of collection (Fish: Montréal, QC; Pizza: Ottawa, ON). Tuna and sal- mon (3:1) comprised the canned fish composites Table 2. List of surrogates and performance standards. Compound Retention time (min) Transition monitored Performance standard Cone voltage (V) Collision energy (eV) Surrogate standard 13C4-PFBA 9.65 217 > 172 13C8-PFOA 20 8 13C5-PFPeA 10.27 268 > 223 13C8-PFOA 20 8 13C5-PFHxA 10.62 318 > 273 13C8-PFOA 15 8 13C2-FHUEA 10.93 359 > 294 13C2-FOUEA 20 10 13C4-PFHpA 10.98 367 > 322 13C8-PFOA 20 8 13C2-FHEA 10.94 379 > 294 13C2-FOEA 20 12 18O2-L-PFHxS 11.28 403 > 84 18O2-PFOS 40 40 13C4-PFOA 11.50 417 > 372 13C8-PFOA 20 8 13C9-PFNA 12.21 472 > 427 13C5-PFNA 20 10 13C4-PFOS 12.68 503 > 80 18O2-PFOS 65 45 13C2-PFDA 13.08 515 > 470 13C5-PFNA 20 10 13C2-FDUEA 14.05 559 > 494 13C2-FOUEA 20 10 13C2-PFUdA 14.04 565 > 520 13C5-PFNA 15 10 13C2-FDEA 14.08 579 > 494 13C2-FOEA 20 12 13C2-PFDoA 15.01 615 > 570 13C5-PFNA 20 10 13C2-PFHxDA 18.33 815 > 770 13C2-PFTeDA 30 15 13C2-6:2 PAP 5.06 445 > 97 13C2-6:2 diPAP 1 20 15 13C2-8:2 PAP 5.48 545 > 97 13C2-6:2 diPAP 1 20 20 13C2-8:2 diPAP 1 13C2-8:2 diPAP 2 6.04 6.04 993 > 97 993 > 545 13C2-6:2 diPAP 1 13C2-6:2 diPAP 2 30 30 35 20 Performance standard 13C8-PFOA 11.50 421 > 376 – 20 8 13C2-FOUEA 12.17 459 > 394 – 20 10 13C5-PFNA 12.21 468 > 423 – 20 10 13C2-FOEA 12.19 479 > 394 – 20 15 18O2-PFOS 12.68 503 > 84 – 65 45 13C2-PFTeDA 16.82 715 > 670 – 30 12 PAPs method 13C2-6:2 diPAP 1 13C2-6:2 diPAP 2 5.90 6.04 793 > 97 793 > 445 – – 25 25 30 17 756 D. F. K. RAWN ET AL. and mixed topping pizzas from fast food outlets obtained in grease-proofed cardboard were used in this work. The samples were shipped to the laboratory where the foods (e.g. pizza) from each store were combined and homogenised thoroughly, followed by the preparation of individual aliquots (~50 g) for analysis. Uncooked food items were cooked following long-used recipes. The cooked foods were then thoroughly homogenised prior to aliquot preparation. Aliquots of the homogenised foods were used for the method testing and PFAS present in the Total Diet Study samples were sub- tracted from the concentrations determined in for- tified samples. Evaluation was performed over three fortification levels in two matrices with five repli- cates at each level of fortification (see supplemen- tary information Tables S1–S2). Initial evaluation of the method focused on the perfluorocarboxylic acids and perfluorosulfonates with the telomer acids and phosphate esters being added later. The two primary matrices, archived Canadian Total Diet Study samples of canned fish (2013) and commercial pizza (2011) were selected for evaluation because they are very different in nature, yet both relevant in terms of possible PFAS detection (e.g. fish: due to bioaccumulation from environmental contamination, pizza: impacted by grease-proofing present in food packaging). Fish and shellfish have been widely reported to have detectable levels of PFAS globally and are thought to be an important route of exposure for PFAS in humans (Chung and Lam 2014; Pérez et al. 2014; Stahl et al. 2014; Ali et al. 2021). Follow-up work was performed using archived chicken nuggets from the Canadian Total Diet Study (2013; Ottawa, ON) over three fortification levels. Chicken nugget samples were homogenised composites of samples collected from fast food res- taurants (see supplementary information Table S3), similar to the pizza collection. Archived spinach composites from samples obtained in 2013, made from raw spinach and spinach boiled in tap water for 1 min (1:1) were tested at two fortification levels (supplementary Table S4). Method development was performed by one analyst, although two analysts evaluated the method via participation in interlaboratory com- parison studies and proficiency test sample analysis. Quality assurance testing With each set of samples tested, a reagent blank sample was subject to the complete sample prepara- tion and used to assess background levels in the laboratory. In addition, a matrix blank (i.e. an ali- quot of unfortified food) was included with each set to correct for any PFASs present in the samples prior to fortification during method evaluation work. To determine the impact of matrix, an unfor- tified sample was prepared for analysis alongside each sample type being evaluated. Following extraction and clean up, the unfortified sample was fortified at the same level as the samples in each set, to gain insight into the impact of matrix on the results. Proficiency test samples Once the method was tested using fortified samples, our group participated in a number of proficiency test schemes for PFASs and interlaboratory com- parison studies. Most tests included one or two samples and the present method has been primarily tested using fish test materials. Fish samples were named differently by the domestic and interna- tional programmes leading the testing and were identified as fish (n = 5), pike-perch (n = 1), perch (n = 1), freeze-dried fish tissue (n = 1) and fish tissue (n = 1). In addition to fish samples, the method was assessed with prawns (n = 2), wheat flour (n = 2), egg yolk (n = 1), tomato (n = 1) and lettuce (n = 1). Two fish samples were analysed in 2016 (24 and 29 participants), 2017 (29 partici- pants), 2018 (33 participants) and 2019 (25 partici- pants) (Figure 2). The fish tissue was analysed in 2017, similar to the dried fish tissue with 14 labora- tories participating. Pike-perch was tested in 2015 with 17 labs having participated in the comparison testing, whereas only 10 labs reported results for the perch in 2019 (Figure 2). Prawn samples were ana- lysed in 2018 (33 participating labs) and 2020 (21 labs participated). Wheat flour samples were both analysed in 2019 and there were 29 laboratories that participated in the comparison (Figure 3). Egg yolk was tested in 2017 with 25 participants reporting results while tomato and lettuce were the test FOOD ADDITIVES & CONTAMINANTS: PART A 757 https://doi.org/10.1080/19440049.2021.2020913 https://doi.org/10.1080/19440049.2021.2020913 https://doi.org/10.1080/19440049.2021.2020913 matrices in 2019 and 2020, respectively. The num- ber of participants in these studies were 25 and 21, respectively (Figure 3). Extraction To avoid contamination of samples during sample preparation or loss due to glass adsorption, poly- propylene products were used while preparing samples for analysis. Samples were thawed at 4°C in advance of initiating preparation for analysis. Individual samples (2 g) were weighed into 15 mL polypropylene centrifuge tubes that had been rinsed with methanol and allowed to dry under ambient conditions prior to addition of the sample. Water (2 mL) was used in the place of sample for the reagent blank. Each tube containing sample, reagent blank, etc., was fortified with 42 µL of stable isotope-labelled PFAS analogues (250 pg µL−1 [5.25 ng g−1] 13C4 PFBA, 13C4 PFPeA, 13C5 PFHxA, 13C4 PFHpA, 13C4 PFOA, 13C2 PFUdA, 13 C2 PFDA, 13C9 PFNA, 13C2 PFDoA, 13C2 PFHxDA, 18O2 L-PFHxS, 13C4 PFOS, 13C2 FHUEA, 13C2 FDUEA; 2500 pg µL−1 [52.5 ng g−1] 13C2 FDEA, 13 C2 FHEA; 62.5 pg µL−1 [2.63 ng g−1] 13C2 6:2 PAP, 13C2 8:2 PAP; 25 pg µL−1 [0.53 ng g−1] 13C2 8:2 diPAP). The samples were briefly vortexed and allowed to sit at ambient temperature on the laboratory bench for 30 min prior to commencing extraction. Acetonitrile (3 mL) was added to each sample and each tube was vortexed for 30 s, followed by sonication for 15 min at ambient temperature. In a situation where vortex and sonication did not appear to break apart the sample thoroughly (e.g. pizza with small pieces of hard crust), the samples were homogenised using a tissue homogeniser (OMNI-TH with G10-95 stainless steel generator probe, Omni International, Marrietta, GA, USA) for 15 s. The samples were mixed using a vortex mixer for an additional 30 s and rotated with sol- vent using a Roto-torque variable speed rotator (Cole-Parmer, Montreal, QC, Canada) for 5 min. Samples were then centrifuged for 10 min at 12,857 × g at 10°C (Eppendorf, Mississauga, ON, Canada). Supernatants were removed and trans- ferred to a clean 15 mL polypropylene centrifuge tube using a polypropylene pipette. Following removal of the raw extract, samples were re-extracted an additional two times, as described above and the resultant supernatants were combined after each centrifugation. Once combined, extracts were allowed to remain in a refrigerator (4°C) overnight and re-centrifuged in the morning, as described above to remove pre- cipitated material. If a precipitate was observed, the extracts were then transferred to a clean centrifuge tube. Extracts were concentrated to 2 mL (3 mL for reagent blanks) under a gentle stream of nitrogen in a water bath set to 40°C (Meyer N-evap analytical evaporator, Organomation, Model 111, Berlin, MA, USA). Each tube was diluted to 10 mL using pur- ified water and vortexed for 10 s prior to initiation of sample clean up. Clean up Clean up was performed using 6 cc, 150 mg Oasis solid phase extraction–weak anion exchange (SPE- WAX) cartridges (Waters Corporation, Mississauga, ON, Canada). Cartridges were conditioned first with one cartridge volume of methanol, followed by one volume of purified water. If samples appeared cloudy or a precipitate was observed, the extracts were cen- trifuged for 10 min at 12,857 × g to separate and remove precipitates prior to loading extracts onto the cartridges. If centrifugation was not required, the diluted extracts were loaded directly onto the cartridge and eluted by gravity and the effluent was discarded. Prior to elution, cartridges were rinsed with 1 mL aqueous formic acid (2%) followed by 2 mL pur- ified water. Each cartridge was dried under vacuum for 1 min and then washed with methanol (3 mL). The first 1 mL of methanol was discarded while the remaining 2 mL were collected and combined with the analytes and surrogate PFASs that eluted using 3 mL 1% ammonium hydroxide in methanol by gravity, with 5 s vacuum to collect any remaining droplets. Following collection, the eluates were concen- trated just to dryness under a gentle stream of nitro- gen in a water bath held at 40°C to allow more timely analysis of the samples than if they were left to passively evaporate. Analyte recovery was not impacted using this approach. The extracts were diluted to 1 mL in isopropanol and vortexed for 758 D. F. K. RAWN ET AL. 10 s. The final extracts were then transferred into 2 mL propylene microcentrifuge tubes and centri- fuged at 17,000 × g for 10 min using a microcentri- fuge (Fisher Scientific, Ottawa, ON, Canada). From each supernatant, two, 400 µL aliquots were removed and transferred to separate 500 µL polypropylene microvials and prepared for analysis by the addition of performance standards. One aliquot was taken for carboxylic acid, sulfonate and telomer acid analysis while the second aliquot was retained for PAP ana- lysis. Prior to carboxylic acid, sulfonate and telomer acid analysis, the performance standards: 13C8 PFOA, 13C5 PFNA, 13C2 PFTeDA, 13C2 FOUEA, 18O2 PFOS, 13C2 FOEA were added, while 13C2 labelled 6:2 diPAP was added to the extract aliquots planned for analysis of PAPs. Vials were capped using polypropylene caps, mixed by hand and stored at 4°C until ready for analysis. Following completion of the initial method testing with fish and pizza, the method was evaluated in two other matrices. Chicken nuggets and spinach were used for the second phase of evaluation and the method was tested using reduced sample sizes (1 g) in an effort to reduce matrix artefacts. In addition, the cartridges were conditioned first with 10 mL 0.1% ammonium hydroxide in methanol, followed by 10 mL purified water prior to use, then rinsed with 3 mL of 20 mM ammonium acetate: methanol (1:5, v/v) at pH 4, followed by 2 mL methanol:water (1:5, v/v) after the sample extracts were loaded onto the column. Analysis Instrumental analysis of the carboxylic acids and sulfonates was performed using a Waters Acquity ultra-high pressure liquid chromatograph I-Class coupled to a Waters Xevo TQ-XS triple quadrupole mass spectrometer (Waters Corporation, Milford, MA, USA) with electrospray ionization operating in the negative ion detection mode. Given that PFASs are used in the manufacture of Teflon non- stick coatings, Teflon coated solvent lines were replaced with polyether ether ketone (PEEK) tub- ing to minimize any PFAS contamination in the chromatographic system. A Phenomenex Gemini – NX C18 column (3 µm, 2 × 150 mm) with a securityGuard ULTRA C18 guard cartridge, AJO- 8782 (Phenomenex, Torrance, CA, USA) was used for analysis. A Phenomenex Kinetex EVO C18 col- umn (5 µm, 100 × 2.1 mm) was installed after the solvent mixer and before the sample injector as a delay column to trap system-related PFAS inter- ferences from co-eluting with analytes from the sample. A gradient elution of the mobile phase was applied (mobile phase A: 5 mM aqueous ammonium formate, mobile phase B: acetonitrile: methanol [1:1, v/v]). Mobile phase A was set to 95% for the first 4 min of each chromatographic run, by 7 min mobile phase A was reduced to 40% and further lowered to 5% by 19 min where it remained until 22 min. By 23 min, mobile phase A was re- established at 95% where it remained until the end of each chromatographic run. The flow rate was held at 0.175 mL min−1 for the complete run, with the exception of the period between 23 and 25.1 min where it was increased to 0.250 mL min−1. The capillary voltage was 2.5 kV. The cone and desolvation gases were nitrogen, set at a flow of 170 L h−1 and 1,000 L h−1, respectively. The collision gas was argon set to a flow rate of 0.15 mL min−1 and resolution was established using unit mass resolution. The source temperature was set to 150°C while the desolvation temperature was 400°C. The nebuliser gas (nitrogen) flow was set to 6.0 Bar. Injection volumes were 3 µL. Analysis of PAPs was completed using the same Waters LC-MS/MS system with electrospray ioniza- tion operating in the negative ionization mode. Separation was performed on a Phenomenex Gemini NX C18 column (3 µm, 2 × 150 mm) and the securityGuard ULTRA C18 guard cartridge col- umn was also used for these analyses, although a delay column was not required. Optimization of the PAPs analysis required a different mobile phase than was suitable for the carboxylic acids, sulfonates and telomer acids, requiring separate analyses. Mobile phase A was 2 mM ammonium acetate and 10 mM ammonium hydroxide in water, adjusted to pH 9–9.5 and mobile phase B was LC/MS grade methanol. A gradient elution began with 80% mobile phase A where it remained until 2 min, and by 4 min mobile phase A was reduced to 5% where it remained until 10 min. By 11 min the ratio of mobile A to B was returned to the initial setting (80% mobile phase A). The flow rate was maintained at 0.175 mL min−1 with the exception of the period between 11.10 and 16.00 min when it was FOOD ADDITIVES & CONTAMINANTS: PART A 759 0.200 mL min−1. As with the previous instrumental method, all injections were 3 µL. The capillary vol- tage was 3.0 kV. Nitrogen was used as both cone and desolvation gases, set at a flow of 300 L h−1 and 1,000 L h−1, respectively. The collision gas was argon set to a flow rate of 0.15 mL min−1 and resolu- tion was established using unit mass resolution. The source temperature was set to 150°C while the des- olvation temperature was 500°C. The nebuliser gas (nitrogen) flow was set to 6.0 Bar. Instrumental analysis of both fractions for indi- vidual sets of samples was able to be completed in a single day, although more frequently, multiple sets of extracts were analysed for carboxylic acids/ sulfonates at one time. Similarly, a series of multiple sets of samples for PAP determination were ana- lysed collectively to optimize productivity. Results and discussion Method development Extraction using solvents of different polarity (e.g. acetone, hexane) initially had been evaluated, but the recoveries were unsatisfactory. The method first reported from our laboratory for the determination of PFAS used methanol as the extraction solvent followed by limited clean up (i.e. centrifugation) was found to be inadequate as the number of analytes was expanded to meet the new requirements. For exam- ple, the recoveries of the longer chain compounds were consistently poor (<10%) using the original method. The inclusion of charcoal (untreated powder 100–400 mesh) in the extraction tube with methanol was found to improve recoveries of the long chain PFAS (84–104%). Unfortunately, PFODA recoveries were extremely high (>500%) using this approach. In addition, extraction using tetrahydrofuran: water was evaluated and although several carboxylic acids and sulfonates were recovered (69–136%), longer chain length compounds had poor recovery (1–30%). Additionally, the telomer acids were not recovered using this solvent system. The selection of acetonitrile resulted in improved recoveries; however, clean up of extracts was impor- tant in addressing the matrix artefacts present in the raw extracts. Our testing included the evaluation of charcoal, size exclusion/hydrophobic interaction cartridges, SPE Si-fluorochrom and SPE-WAX. Results obtained using SPE-WAX clean up provided the most consistent recoveries for analytes belonging to all the PFAS sub-groups (e.g. carboxylic acids, sulfonates, telomer acids, PAPs) while other clean up methods resulted in inconsistent recoveries among the PFAS sub-groups of interest (Supplementary Table S5). The developed method was assessed based on the following general method characteristics: linearity of instrument response, applicable range, limits of detection, accuracy and preci- sion of method, and matrix impacts. Despite PFASs having similar structural backbones, analytes investigated had variable terminal ends, including carboxylic acids, sulfonic acids and phosphate esters. As a result, instrument sensitivity varied considerably between analyte calibration curves. Analyte linearity was gener- ally determined over two orders of magnitude and extended beyond the concentration antici- pated to be present in food samples. PFOS and PFOA have been observed in food samples at concentrations <10 ng g−1 whole weight more frequently than other PFASs (e.g. PFHpA, PFNA) which, when detected similarly, remained <10 ng g−1 ww (Tittlemier et al. 2007; Ericson et al. 2008; Haug et al. 2010). In all but one case, the correlation coefficients (r2) were ≥0.99 (Table 1). Instrumental approach While the ideal situation involves the determi- nation of two transitions for analyte confirma- tion, in order to maintain >10–15 data points for a well-defined LC peak shape, the MRM cycle time had to be short to accommodate the narrow LC peaks observed. Much of the method development work for the method described was performed using a Waters Premier XE using the two most sensitive MRM transitions for each analyte. As the num- ber of analytes, corresponding surrogates, and performance standards were added, the method was not able to sustain so many transitions. As a result, it was decided to use a single MRM transition for each compound because it was determined that if two transitions were retained for all the compounds in the expanded method, 760 D. F. K. RAWN ET AL. https://doi.org/10.1080/19440049.2021.2020913 the shared dwell time for each peak would be insufficient to provide enough data points to allow for quantitative results. In the present method, a comprehensive sample clean up was performed, good chromatographic separation was achieved, standard calibration curves had good linearity and a strong QC protocol was followed using both reagent and matrix blanks throughout each sequence. Isotope-labelled standards have been used for both recovery and performance correction so both impacts of sample preparation and instrument matrix effects are taken into account in the method. When considering the complete method from sample preparation to instrumental analysis, it was determined that a single transition would be acceptable to overcome potential interfer- ence issues. Other researchers have reported successful PFAS methods similarly using single transitions (Genualdi and deJager 2019; Shoemaker and Tettenhorst 2020; Pierri et al. 2020). Limits of detection/quantification Instrumental detection limits were generally low for all compounds, ranging from 0.006 ng g−1 (L-PFDS) to 1.76 ng g−1 (FHEA), with correspond- ing limits of quantification (LOQ) of 0.018 ng g−1 to 5.28 ng g−1, respectively (Table 3). Limits of detec- tion (LOD) were routinely re-examined throughout the method development and evaluation work to ensure that the reported limits would be represen- tative of the system under operational conditions (i.e. freshly cleaned, after it had been used for a period, etc.). Presence of perfluorinated com- pounds in the environment has been reported in the literature (Taniyasu et al. 2005), including Table 3. Instrumental limits of detection (LOD) and limits of quantification (LOQ) determined for method analytes. Analyte Determined using 3:1 signal/noise ratio (ng g−1) Frequently detected in reagent blanks (Y/N) Determined using level in reagent blank samples (ng g−1) LOD LOQ LOD LOQ PFBA 0.373 1.12 Y 4.12 12.4 PFPeA 0.402 1.20 Y 0.255 0.764 L-PFBS 0.033 0.100 N – – PFHxA 0.048 0.145 Y 0.220 0.661 L-PFPeS 0.007 0.022 N – – FHUEA 0.087 0.262 N – – PFHpA 0.063 0.190 N – – FHEA 1.76 5.28 N – – L-PFHxS 0.009 0.026 Y 0.151 0.452 PFOA 0.101 0.303 Y 0.516 1.55 L-PFHpS 0.017 0.051 N – – FOUEA 0.057 0.170 N – – PFNA 0.077 0.230 N – – FOEA 0.903 2.71 N – – L-PFOS + isomer 2 0.032 0.097 Y 0.218 0.654 br-PFOS (isomer 3–11) 0.021 0.063 N – – PFDA 0.110 0.330 Y 0.191 0.572 L-PFNS 0.018 0.055 N – – FDUEA 0.068 0.205 N – – PFUdA 0.083 0.249 Y 0.049 0.148 FDEA 1.10 3.29 N – – L-PFDS 0.006 0.018 N – – PFDoA 0.105 0.314 N – – PFTrDA 0.038 0.114 N – – L-PFDoS 0.027 0.082 N – – PFTeDA 0.070 0.211 N – – PFHxDA 0.052 0.156 Y 0.165 0.496 PFODA 0.036 0.107 N – – 6:2 PAP 0.295 0.884 Y 2.99 8.96 8:2 PAP 0.329 0.987 Y 1.16 3.49 6:2 diPAP 1 0.015 0.046 Y 0.242 0.726 6:2 diPAP 2 0.009 0.028 Y 0.250 0.749 8:2 diPAP 1 0.012 0.036 Y 0.020 0.060 8:2 diPAP 2 0.013 0.039 N – – FOOD ADDITIVES & CONTAMINANTS: PART A 761 indoor environments (Goosey and Harrad 2012) and, as a result, blanks were used to assess back- ground concentrations in the laboratory. Background levels of 14 analytes were detected in reagent blank samples, at low concentrations, which indicated laboratory background contami- nation (Table 3). As a result, method detection limits for the analytes present in reagent blanks were established by summing the mean concentra- tion in the reagent blanks plus three times the standard deviation (SD) of the mean values (Table 3), rather than based on instrumental limita- tions, consistent with the AOAC guidelines for determination of detection limits (Shrivastava and Gupta 2011). Limits of quantification were estab- lished by multiplying the LOD by 3. Most com- pounds were present at sufficiently low levels that LOD remained below 0.5 ng g−1, however elevated limits of detection/quantification for PFBA (4.12 ng g−1/12.4 ng g−1), 6:2 PAP (2.99 ng g−1/ 8.96 ng g−1) and 8:2 PAP (1.16 ng g−1/3.49 ng g−1) were observed relative to instrumental detection limits. Consistent with our laboratory protocol, concentrations observed in reagent blanks were subtracted from the sample concentrations throughout the work. Blank subtraction was per- formed by pairing reagents blanks with samples from the corresponding set to ensure that blank subtraction was set specific. For those compounds not observed in reagent blanks, the signal-to-noise ratio, cor- rected for sample weight and final volume, was used to determine the method detection limit. Matrix impacts Fortification of fish and pizza was performed at three levels (low [0.104 ng g−1 branched PFOS (isomer 3–11) – 13.1 ng g−1 FHEA, FOEA, FDEA], medium [0.522–52.5 ng g−1], high [2.10 ng g−1 6:2 and 8:2 diPAPs – 131 ng g−1 FHEA, FOEA, FDEA]) and evaluation was com- pleted with five replicates (Tables 4 and 5, respec- tively). Chicken nuggets were fortified at similar concentrations (Table 6), while spinach, a matrix rich in colour, was evaluated to determine the impact of colour on method performance at the low and mid-level of fortification (Table 7). Although the method testing with fish matrix was largely successful at all fortification levels, pizza having multiple matrices present (e.g. crust, mushrooms, cheese, etc.), provided additional challenges. During method testing with fish, 13C4 PFOS was used as a surrogate for L-PFHpS quite successfully; however, enhancement (132–200%) was observed for this ana- lyte in pizza extracts at all fortification levels using 13C PFOS. The introduction of 18O2 L- PFHxS as a surrogate replacement for 13C PFOS led to improved L-PFHpS recoveries (75.1–106%) in pizza over all levels of fortification. This resulted in the decision to complete the evaluation in all matrices using 18O2 L-PFHxS as the surrogate for L-PFHpS. The adoption of the structurally more appropriate surrogate standard allowed the work to proceed, given that the method is planned for application with many different matrices and developing matrix- matched standards would limit the progress of the work. Pizza extracts, noted to have matrix artefacts, fortified at the lowest levels, had good peak shape and separation, consistent with what was observed in other simpler matrices (Figure 1). Post-extraction fortification Post-extraction fortification of samples provided insight into the effect of each food matrix on PFAS recovery. The matrix impacted PAP recoveries in each of the four matrices evaluated. Inhibition of both transitions monitored for 6:2 diPAP was observed in fortified fish and pizza extracts (Tables 4 and 5) at the low level (0.105 ng g−1). In contrast, enhancement of 6:2 diPAP was noted in chicken nugget extracts for- tified at all levels (115–151%). Both 6:2 PAP and 8:2 PAP were found to have enhancement in the samples fortified post-extraction in the case of both chicken nuggets and spinach (109–156%; Tables 6 and 7). Additionally, L-PFDoS was subject to enhancement in the post-extraction fortification samples of chicken nuggets and spinach (156–182%). Recovery experiments – accuracy, precision Performance/surrogate standards Results of the method were developed using both surrogate standards to correct for sample loss dur- ing sample preparation and performance standards 762 D. F. K. RAWN ET AL. Figure 1. Chromatograms of individual PFASs, note Br-PFOS and L-PFOS appear in the same trace. A and B – carboxylic acids and sulfonate traces, while C shows PAP traces. FOOD ADDITIVES & CONTAMINANTS: PART A 763 Figure 1. (Continued). 764 D. F. K. RAWN ET AL. Figure 1. (Continued). FOOD ADDITIVES & CONTAMINANTS: PART A 765 Table 4. Summary of recovery information for analytes in canned fish samples. Analyte Fortification level (ng g−1) Average recovery (%) ± Standard Deviation (n = 5) Coefficient of variation (%) Fortified extract recovery (%) PFBA 1.05 2.63 13.1 93.3 ± 31 100 ± 10 92.5 ± 3.6 33.5 9.9 3.9 68.5 97.8 90.8 PFPeA 1.05 2.63 13.1 96.5 ± 8.5 94.7 ± 6.3 91.2 ± 1.1 8.8 6.6 1.2 102 104 93.6 L-PFBS 0.525 2.63 13.1 81.4 ± 9.6 95.8 ± 4.7 115 ± 6.0 11.8 4.9 5.2 84.0 91.8 109 PFHxA 0.210 1.05 5.25 51.0 ± 17 82.7 ± 16 94.6 ± 8.2 32.4 19.4 8.6 37.5 82.5 116 L-PFPeS 0.210 1.05 5.26 101 ± 33 95.6 ± 5.5 101 ± 6.8 32.7 5.8 6.8 77.5 92.5 98.0 FHUEA 0.656 2.63 6.56 118 ± 36 103 ± 7.2 120 ± 8.5 30.4 7.0 7.1 96.8 97.8 99.8 PFHpA 0.210 1.05 5.25 79.0 ± 29 89.5 ± 8.0 99.4 ± 7.4 36.5 8.9 7.4 60.0 88.5 108 FHEA 13.1 52.5 131 101 ± 21 89.8 ± 8.7 103 ± 9.0 21.2 9.7 8.8 55.4 102 106 L-PFHxS 0.210 1.05 5.25 110 ± 22 92.1 ± 7.7 103 ± 5.2 19.9 8.4 5.0 70.0 91.0 106 PFOA 0.525 2.63 13.1 104 ± 19 95.2 ± 7.0 99.7 ± 8.0 17.7 7.3 8.0 66.0 100 99.4 L-PFHpS 0.210 1.05 5.25 127 ± 32 90.8 ± 12 104 ± 6.5 25.3 13.4 6.3 97.5 78.5 75.2 FOUEA 0.656 2.63 6.56 49.1 ± 39 86.8 ± 9.8 89.1 ± 9.6 79.6 11.3 10.8 75.2 89.0 106 PFNA 0.525 2.63 13.1 102 ± 39 92.7 ± 15 94.4 ± 9.6 37.8 16.2 10.2 133 97.6 108 FOEA 13.1 52.5 131 105 ± 62 61.1 ± 8.9 83.8 ± 11 59.2 14.6 13.4 91.1 93.4 110 L-PFOS + isomer 2 0.420 2.10 10.5 104 ± 19 103 ± 12 103 ± 4.7 18.6 12.1 4.6 57.5 90.0 108 br-PFOS (isomer 3–11) 0.104 0.522 2.61 98.0 ± 34 83.0 ± 16 89.6 ± 2.9 35.1 19.7 3.3 105 80.0 97.6 PFDA 0.525 2.63 13.1 100 ± 16 91.9 ± 8.5 105 ± 4.6 15.9 9.3 4.4 104 99.0 105 L-PFNS 0.525 2.63 13.1 93.4 ± 12 90.4 ± 12 99.9 ± 7.0 12.4 13.4 7.0 84.0 93.8 105 FDUEA 0.656 2.63 6.56 95.8 ± 32 98.3 ± 18 116 ± 27 33.1 18.6 23.1 105 103 105 PFUdA 0.525 2.63 13.1 116 ± 51 90.2 ± 15 110 ± 9.5 43.8 16.2 8.7 110 105 99.6 FDEA 13.1 52.5 131 99.8 ± 44.1 97.7 ± 8.6 118 ± 16 44.2 8.8 13.7 134 90.3 126 L-PFDS 0.525 2.63 13.1 94.6 ± 26 102 ± 8.4 104 ± 4.2 26.9 8.3 4.0 106 83.2 104 PFDoA 0.525 2.63 13.1 63.0 ± 5.3 99.2 ± 10 102 ± 7.3 8.4 10.4 7.1 95.0 96.0 116 PFTrDA 0.525 2.63 13.1 86.0 ± 17 79.9 ± 14 113 ± 16 20.1 18.0 13.8 71.0 75.6 106 (Continued) 766 D. F. K. RAWN ET AL. to adjust for poor injections and/or matrix arte- facts. The use of performance standards to adjust for matrix effects and injection issues was particu- larly beneficial given the analyte sensitivity to matrix. Performance standard deviations ranged from −79% (13C2 FOEA) in spinach to 160% (18O2 PFOS) in chicken nuggets. Average surrogate stan- dard recoveries ranged from 26% (13C FDEA) to 80% (13C PFHxA), although a single sample of pizza had <10% recovery of 13C FDUEA, 13C PFUdA, 13C FDEA, 13C PFDoA and 13C PFHxDA. The application of performance standards to compensate for signal enhancement or inhibition due to matrix artefacts aided in the accuracy of analyte concentration measurements while surrogate standards adjusted for analyte loss during sample preparation. Analyte recovery Recoveries of a number of analytes were found to be low in more than one matrix, at low fortification levels. Standard deviations varied most widely at the low fortification level across all matrices tested. Low mean recoveries from fish at the low fortification level were observed for PFHxA, FOUEA, PFDoA and L-PFDoS (Table 4). In addition, PFODA was observed to have low recoveries at all levels of fortifi- cation (mean recovery 56.4–68.4%) of canned fish. Inhibition of the telomer acids FOUEA (average recoveries 63.2% and 54.1%, low and high fortifica- tion, respectively) and FOEA (mean recovery 52.2– 62.0%) was observed in pizza samples at multiple levels of fortification, similar to L-PFNS (mean recov- ery 55.0–59.7%) and L-PFDS (mean recovery 31.4– 40.3%) (Table 5). L-PFDS was observed to have poor recoveries from chicken nuggets (mean recovery 42.0– 56.8%) (Table 6). Low recovery of L-PFHxS (67.4%), and L-PFHpS (65.1%) and 8:2 PAP (46.5%) was achieved from spinach fortified at the low level. 6:2 diPAP (mean 60.7–70.7%) had low recovery from both low and mid-level fortification. In addition to inhibition, some analytes were sub- ject to enhancement resulting in elevated recoveries even with the use of surrogates and performance standards. 6:2 PAP and 8:2 PAP recoveries were ele- vated at the low fortification level (169% and 135%, respectively) in fish samples. Both transitions Table 4. (Continued). Analyte Fortification level (ng g−1) Average recovery (%) ± Standard Deviation (n = 5) Coefficient of variation (%) Fortified extract recovery (%) L-PFDoS 0.525 2.63 13.1 60.2 ± 18 79.1 ± 10 88.7 ± 7.6 29.4 12.8 8.6 52.0 68.8 87.2 PFTeDA 0.525 2.63 13.1 95.2 ± 19 94.1 ± 5.6 116 ± 8.0 19.6 6.0 6.9 68.0 99.0 123 PFHxDA 0.525 2.63 13.1 98.2 ± 12 96.6 ± 4.5 100 ± 6.0 12.0 4.7 6.0 102 91.0 102 PFODA 0.525 2.63 13.1 68.4 ± 15 56.4 ± 11 68.4 ± 8.2 22.6 20.2 12.0 87.0 77.4 109 6:2 PAP 2.63 13.1 52.5 169 ± 26 95.0 ± 11 96.4 ± 6.6 16 11 6.9 128 91.8 106 8:2 PAP 2.63 13.1 52.5 135 ± 17 102 ± 5.5 99.3 ± 7.7 12 5.4 7.8 90.7 107 104 6:2 diPAP 1 0.105 0.525 2.10 198 ± 144 166 ± 68 220 ± 38 73 41 17 −72.5 49.8 49.9 6:2 diPAP 2 0.105 0.525 2.10 180 ± 156 178 ± 65 265 ± 49 86 36 19 −122 45.0 46.5 8:2 diPAP 1 0.105 0.525 2.10 131 ± 18 100 ± 8.6 95.8 ± 6.8 13 8.6 7.1 87.5 102 105 8:2 diPAP 2 0.105 0.525 2.10 133 ± 9.1 113 ± 13 121 ± 19 6.8 12 15 99.5 99.2 102 FOOD ADDITIVES & CONTAMINANTS: PART A 767 Table 5. Summary of recovery information for analytes in pizza. Analyte Fortification level (ng g−1) Average recovery (%) ± Standard Deviation (n = 5) Coefficient of variation (%) Fortified extract recovery (%) PFBA 1.05 2.63 13.1 79.2 ± 4.6 99.7 ± 2.7 102 ± 1.9 5.8 2.7 1.8 96.1 96.2 105 PFPeA 1.05 2.63 13.1 103 ± 3.7 103 ± 3.1 106 ± 1.9 3.6 3.0 1.8 107 100 105 L-PFBS 0.525 2.63 13.1 110 ± 5.7 115 ± 2.7 123 ± 9.2 5.2 2.4 7.5 104 115 106 PFHxA 0.210 1.05 5.25 94.3 ± 6.4 96.4 ± 2.6 107 ± 2.0 6.8 2.7 1.9 97.5 101 105 L-PFPeS 0.210 1.05 5.26 104 ± 5.0 100 ± 4.2 111 ± 5.5 4.8 4.2 5.0 109 98.8 104 FHUEA 0.656 2.63 6.56 105 ± 4.3 95.3 ± 2.2 96.3 ± 1.9 4.0 2.3 1.9 95.9 96.2 90.7 PFHpA 0.210 1.05 5.25 110 ± 4.5 99.8 ± 2.7 111 ± 1.9 4.1 2.7 1.8 107 103 107 FHEA 13.1 52.5 131 125 ± 21 106 ± 4.9 102 ± 5.5 16.4 4.6 5.4 102 106 100 L-PFHxS 0.210 1.05 5.25 102 ± 4.1 95.3 ± 2.0 109 ± 3.3 4.0 2.1 3.0 106 94.7 102 PFOA 0.525 2.63 13.1 95.5 ± 7.3 97.5 ± 2.5 109 ± 3.4 7.6 2.6 3.1 88.1 97.9 104 L-PFHpS 0.210 1.05 5.25 0.210 1.05 5.25 13C PFOS 158 ± 20 139 ± 7.8 158 ± 24 18O2 L-PFHxS 88.9 ± 13 87.9 ± 4.1 93.5 ± 4.5 13 5.6 15 14 4.7 4.8 108 287 99.7 103 99.0 100 FOUEA 0.656 2.63 6.56 63.2 ± 15 70.4 ± 13 54.1 ± 11 23.6 18 20.7 86.7 92.9 83.5 PFNA 0.525 2.63 13.1 103 ± 4.4 102 ± 2.0 111 ± 3.4 4.3 2.5 3.0 103 103 109 FOEA 13.1 52.5 131 60.1 ± 17 62.0 ± 10 52.2 ± 17 28.6 16 32.7 99.1 92.2 87.3 L-PFOS + isomer 2 0.420 2.10 10.5 103 ± 6.5 96.9 ± 3.2 109 ± 4.4 6.3 3.3 4.0 99.9 97.5 107 br-PFOS (isomer 3–11) 0.104 0.522 2.61 145 ± 27 120 ± 7.8 140 ± 13 18.5 6.5 4.0 97.0 89.7 95.7 PFDA 0.525 2.63 13.1 94.5 ± 5.8 102 ± 3.8 113 ± 4.4 6.1 3.7 9.1 92.5 98.9 105 L-PFNS 0.525 2.63 13.1 57.8 ± 8.6 59.7 ± 4.4 55.0 ± 13 14.9 7.4 3.9 104 104 104 FDUEA 0.656 2.63 6.56 103 ± 4.9 103 ± 3.5 99.8 ± 3.4 4.7 3.4 3.5 100 96.4 96.8 PFUdA 0.525 2.63 13.1 104 ± 6.0 108 ± 3.8 115 ± 7.3 5.8 3.5 3.4 103 102 105 FDEA 13.1 52.5 131 96.9 ± 5.3 131 ± 12 120 ± 29 5.4 8.8 24 97.9 87.0 97.6 L-PFDS 0.525 2.63 13.1 35.5 ± 8.0 40.3 ± 5.1 31.4 ± 12 23 13 39 110 101 102 PFDoA 0.525 2.63 13.1 95.0 ± 11 112 ± 5.8 119 ± 7.7 12 5.2 6.4 91.6 102 107 (Continued) 768 D. F. K. RAWN ET AL. Table 5. (Continued). Analyte Fortification level (ng g−1) Average recovery (%) ± Standard Deviation (n = 5) Coefficient of variation (%) Fortified extract recovery (%) PFTrDA 0.525 2.63 13.1 118 ± 27 153 ± 23 136 ± 18 23 15 13 107 97.9 80.0 L-PFDoS 0.525 2.63 13.1 126 ± 35 190 ± 29 161 ± 21 28 16 13 102 104 75.5 PFTeDA 0.525 2.63 13.1 124 ± 25 137 ± 18 120 ± 14 20 13 11 101 98.7 81.5 PFHxDA 0.525 2.63 13.1 90.5 ± 12 119 ± 11 138 ± 10 13 9.6 7.2 101 103 105 PFODA 0.525 2.63 13.1 99.0 ± 26 178 ± 18 245 ± 23 26 10 9.5 109 136 133 6:2 PAP 2.63 13.1 52.5 93.8 ± 6.6 101 ± 4.5 104 ± 5.8 7.1 4.4 5.6 89.5 103 109 8:2 PAP 2.63 13.1 52.5 104 ± 8.7 111 ± 6.8 114 ± 12 8.4 6.1 11 70.4 111 114 6:2 diPAP1 0.105 0.525 2.10 119 ± 15 96.0 ± 12 105 ± 11 13 13 10 52.5 79.4 99.2 6:2 diPAP2 0.105 0.525 2.10 111 ± 43 87.1 ± 7.1 102 ± 7.4 39 8.2 7.2 64.5 81.7 105 8:2 diPAP1 0.105 0.525 2.10 106 ± 20 109 ± 7.7 119 ± 12 19 7.1 9.7 88.5 95.9 92.5 8:2 diPAP2 0.105 0.525 2.10 113 ± 27 101 ± 10 111 ± 9.9 24 10 8.9 93.5 98.0 107 Table 6. Summary of recovery information for analytes in chicken nuggets. Analyte Fortification level (ng g−1) Average recovery (%) ± Standard Deviation (n = 5) Coefficient of variation (%) Fortified extract recovery (%) PFBA 2.03 4.92 25.8 97.8 ± 9.6 103 ± 7.3 98.6 ± 4.1 9.8 7.1 4.2 109 115 103 PFPeA 2.03 4.92 25.8 90.8 ± 1.3 98.6 ± 1.1 96.7 ± 1.3 1.5 1.1 1.4 84.3 97.5 99.3 L-PFBS 0.203 1.97 20.7 114 ± 8.6 103 ± 4.3 98.0 ± 4.9 7.5 4.2 5.0 105 94.6 99.9 PFHxA 0.407 3.94 41.3 106 ± 7.4 99.0 ± 3.3 99.2 ± 1.8 7.0 3.4 1.8 99.0 100 102 L-PFPeS 0.203 1.97 20.7 92.1 ± 15 101 ± 6.7 99.2 ± 3.1 17 6.7 3.1 84.7 106 104 FHUEA 0.254 1.23 15.5 112 ± 6.9 105 ± 3.9 104 ± 2.4 6.1 3.7 2.3 110 109 101 PFHpA 0.203 1.97 20.7 100 ± 5.9 98.2 ± 3.2 97.6 ± 2.8 5.9 3.2 2.8 106 103 100 FHEA 2.54 12.3 155 104 ± 17 104 ± 4.8 101 ± 2.8 17 4.6 2.8 107 110 101 L-PFHxS 0.203 1.97 20.7 84.4 ± 20 95.8 ± 6.2 97.7 ± 4.2 24 6.5 4.3 75.1 102 107 PFOA 0.203 1.97 20.7 87.6 ± 8.0 98.9 ± 1.1 98.8 ± 1.4 9.1 1.1 1.4 81.0 97.1 103 (Continued) FOOD ADDITIVES & CONTAMINANTS: PART A 769 Table 6. (Continued). Analyte Fortification level (ng g−1) Average recovery (%) ± Standard Deviation (n = 5) Coefficient of variation (%) Fortified extract recovery (%) L-PFHpS 0.203 1.97 20.7 112 ± 14 95.4 ± 7.6 91.6 ± 3.9 12 8.0 4.2 109 103 102 FOUEA 0.254 1.23 15.5 97.2 ± 14 112 ± 15 87.6 ± 7.5 14 14 8.5 111 100 89.3 PFNA 0.407 3.94 41.3 90.2 ± 4.3 102 ± 4.1 98.9 ± 3.0 4.7 4.0 3.0 92.9 104 103 FOEA 2.54 12.3 155 86.3 ± 20 108 ± 18 79.2 ± 10 24 17 13 104 106 94.0 L-PFOS + isomer 2 0.402 3.94 41.3 80.5 ± 25 95.3 ± 1.9 97.4 ± 3.5 31 2.0 7.6 95.0 106 100 br-PFOS (isomer 3–11) 0.101 0.980 10.3 105 ± 22 106 ± 5.6 114 ± 8.6 21 5.3 7.6 106 92.0 95.3 PFDA 0.407 3.94 41.3 90.6 ± 8.5 100 ± .4.8 99.5 ± 2.4 9.4 4.8 2.4 89.0 104 104 L-PFNS 0.203 1.97 20.7 77.7 ± 19 80.3 ± 2.6 74.7 ± 6.3 24 3.2 8.4 111 98.5 105 FDUEA 0.254 1.23 15.5 113 ± 5.3 103 ± 7.0 102 ± 3.2 4.7 6.8 3.1 97.1 100 101 PFUdA 0.407 3.94 41.3 83.7 ± 7.4 102 ± 5.0 99.7 ± 1.7 8.8 4.9 1.7 96.6 95.9 98.8 FDEA 2.54 12.3 155 94.9 ± 12 98.3 ± 11 113 ± 12 13 12 11 107 102 113 L-PFDS 0.203 1.97 20.7 42.0 ± 15 56.8 ± 3.9 49.2 ± 3.1 35 6.9 6.4 67.4 103 93.5 PFDoA 0.407 3.94 41.3 92.5 ± 7.8 108 ± 4.7 101 ± 2.9 8.4 4.3 2.8 93.4 101 98.0 PFTrDA 0.203 1.97 20.7 120 ± 19 150 ± 9.2 153 ± 18 16 6.1 12 130 127 121 L-PFDoS 0.203 1.97 20.7 98.5 ± 22 204 ± 6.7 221 ± 16 23 3.3 7.4 156 176 182 PFTeDA 0.203 1.97 20.7 108 ± 6.5 120 ± 7.5 116 ± 8.8 6.1 6.3 7.6 96.6 109 105 PFHxDA 0.203 1.97 20.7 103 ± 13 109 ± 4.7 108 ± 4.9 12 4.3 4.6 96.0 102 97.4 PFODA 0.203 1.97 20.7 118 ± 15 126 ± 10 119 ± 1.5 13 8.0 1.3 100 110 95.0 6:2 PAP 1.19 5.17 29.5 89.1 ± 11 94.9 ± 5.1 103 ± 6.4 13 5.4 6.2 109 133 150 8:2 PAP 1.29 5.70 31.7 98.5 ± 13 107 ± 6.5 116 ± 10 14 6.0 8.8 133 151 153 6:2 diPAP 1 0.286 1.97 8.56 158 ± 24 170 ± 9.0 156 ± 10 15 5.3 6.6 151 137 139 6:2 diPAP 2 0.344 1.78 8.02 147 ± 6.4 158 ± 6.4 156 ± 7.2 5.5 4.1 4.6 115 134 145 8:2 diPAP 1 0.193 1.05 5.59 97.2 ± 8.9 102 ± 1.4 102 ± 4.8 9.2 1.4 4.7 96.4 102 101 8:2 diPAP 2 0.249 1.10 5.63 101 ± 10 108 ± 5.6 107 ± 3.2 10 5.2 3.0 91.9 106 107 770 D. F. K. RAWN ET AL. Table 7. Summary of recovery information for analytes in spinach. Analyte Fortification level (ng g−1) Average recovery (%) ± Standard Deviation (n = 4 low level; n = 5 mid-level) Coefficient of variation (%) Fortified extract recovery (%) PFBA 2.03 4.92 252 ± 40 116 ± 26 16 23 81.2 90.5 PFPeA 2.03 4.92 96.9 ± 3.7 104 ± 3.6 3.8 3.5 99.9 94.7 L-PFBS 0.203 1.97 100 ± 6.7 87.0 ± 1.6 6.7 1.9 94.5 99.4 PFHxA 0.407 3.94 106 ± 15 97.7 ± 1.9 14 2.0 97.8 94.7 L-PFPeS 0.203 1.97 88.3 ± 40 88.8 ± 8.1 45 9.2 105 97.1 FHUEA 0.254 1.23 110 ± 13 105 ± 3.5 11 3.4 96.3 98.6 PFHpA 0.203 1.97 101 ± 3.9 98.9 ± 4.1 3.9 4.1 109 99.5 FHEA 2.54 12.3 101 ± 17 108 ± 12 17 11 90.6 91.4 L-PFHxS 0.203 1.97 67.4 ± 33 95.0 ± 14 50 15 102 105 PFOA 0.203 1.97 106 ± 15 101 ± 4.6 14 4.5 80.2 96.6 L-PFHpS 0.203 1.97 65.1 ± 31 93.1 ± 10 48 11 112 91.5 FOUEA 0.254 1.23 94.8 ± 8.2 85.3 ± 12 8.7 14 89.1 85.6 PFNA 0.407 3.94 99.6 ± 5.2 101 ± 5.2 5.2 5.2 94.1 97.4 FOEA 2.54 12.3 105 ± 14 86.1 ± 15 13 17 96.2 89.1 L-PFOS + isomer 2 0.402 3.94 90.0 ± 20 101 ± 5.8 23 5.8 91.2 90.9 br-PFOS (isomer 3–11) 0.101 0.980 96.4 ± 8.9 86.1 ± 8.7 9.2 10 76.4 94.2 PFDA 0.407 3.94 95.6 ± 16 103 ± 2.1 16 21 92.6 101 L-PFNS 0.203 1.97 94.0 ± 7.5 98.2 ± 10 7.9 10 83.5 104 FDUEA 0.254 1.23 105 ± 9.4 103 ± 5.1 9.0 5.0 98.1 101 PFUdA 0.407 3.94 96.7 ± 6.0 102 ± 6.0 6.2 5.9 103 103 FDEA 2.54 12.3 93.4 ± 18 99.8 ± 3.5 19 3.5 99.4 99.8 L-PFDS 0.203 1.97 76.0 ± 22 88.5 ± 5.4 28 6.1 120 85.9 PFDoA 0.407 3.94 99.4 ± 6.5 104 ± 2.4 6.5 2.4 103 97.7 PFTrDA 0.203 1.97 124 ± 8.3 128 ± 4.9 6.7 3.8 120 115 L-PFDoS 0.203 1.97 149 ± 27 174 ± 14 18 7.7 157 180 PFTeDA 0.203 1.97 112 ± 12 112 ± 4.4 10 3.9 105 103 PFHxDA 0.203 1.97 92.5 ± 6.9 102 ± 2.8 7.4 2.7 102 97.4 PFODA 0.203 1.97 88.0 ± 7.6 91.9 ± 5.7 8.7 6.2 95.6 94.7 6:2 PAP 1.04 5.11 124 ± 11 123 ± 36 8.7 29 148 153 8:2 PAP 1.04 5.11 46.5 ± 16 140 ± 6.9 34 4.9 125 156 6:2 diPAP 1 0.207 1.02 70.7 ± 11 63.7 ± 2.1 15 3.2 75.9 77.6 6:2 diPAP 2 0.207 1.02 65.6 ± 3.4 60.7 ± 3.0 5.1 5.0 89.1 73.0 8:2 diPAP 1 0.207 1.02 104 ± 3.2 100 ± 2.4 3.1 2.4 91.6 103 8:2 diPAP 2 0.207 1.02 91.7 ± 6.8 102 ± 2.8 7.4 2.8 91.0 95.7 FOOD ADDITIVES & CONTAMINANTS: PART A 771 monitored for 6:2 diPAP were subject to enhance- ment in fish (mean recoveries 166–265%) and chicken nuggets (147–170%) at all levels of fortification. Enhancement of PFTrDA was observed in pizza, chicken nuggets and spinach at more than one for- tification level, similar to L-PFDoS (Tables 5–7). Enhancement of another late-eluting compound (PFODA) was only observed in pizza at both the mid and high fortification level (178% and 245%, respectively). Proficiency tests In addition to our internal evaluation, our laboratory participated in a number of proficiency test pro- grammes and interlaboratory comparison studies to evaluate our method. We participated in proficiency testing available via the National Measurement Institute (Australia), Norwegian Institute of Public Health’s Interlaboratory Comparison of persistent organic pollutants in food, the Northern Contaminants Program/Arctic Monitoring Assessment Program interlaboratory comparison (Canada) and the European Union Reference Laboratory interlaboratory comparison. Samples in the testing included both incurred and fortified sam- ples to encompass the range of concentrations from near the detection limit extending to the regulatory levels. The majority of testing was based on PFAS determination in fish (n = 9) samples, although additional proficiency test materials have been eval- uated during this work, including egg yolk (n = 1), tomato (n = 1), wheat flour (n = 2) and lettuce (n = 1). Although we participated in numerous pro- grammes to evaluate our method, not all analytes were included in each testing scheme. PFOA and total PFOS have been determined in multiple proficiency testing schemes and interlabora- tory comparisons with the results from our method resulting in z-scores of <2 (graphical abstract). Z-scores are calculated by subtracting the assigned/ consensus value from the result reported by Figure 2. Proficiency test z-scores for PFASs in fish and prawns; note PFOA and ΣPFOS data provided in graphical abstract. 772 D. F. K. RAWN ET AL. participants and dividing the result by the target stan- dard deviation. Z-scores that are ±2.0 are considered satisfactory results. The fish and prawn samples had the greatest number of analytes and the results of the method produced satisfactory results for all com- pounds (Figure 2). In all cases, the present method resulted in z-scores of < ±2.0 regardless of matrix, with one exception, PFBA in wheat flour, where the z-score obtained was −2.4 (Figure 3). All z-scores obtained for proficiency tests and interlaboratory comparisons using fish or shellfish matrices were within the satis- factory range, with a maximum z-score of 1.67 observed for PFTeDA in prawn. Matrix impacts are particularly important in some foods (e.g. pizza), how- ever, the correct choice of surrogate standard aids in addressing enhancement issues. The described method allows for the determi- nation of carboxylic acid, sulfonate, telomer acid and polyfluoroalkylphosphate ester PFASs in complex food matrices. It is recognized that some of the longer chain PFASs (e.g. PFTrDA, L- PFDoS, PFODA) and PAPs are more prone to enhancement of recoveries. Although these obser- vations have been noted, the method is applicable to an expanded suite of analytes relative to our original method. Continued efforts to reduce matrix artefacts will aid in the quantitative deter- minations of all analytes at all concentration levels. Acknowledgments The authors thank Dr. Benjamin P.-Y. Lau for additional insight into the approach for expanding the instrumental method, Amy R. Sadler for participating in the final testing of samples to confirm method applicability and Sue C. Quade for her effort to review the manuscript in detail. Disclosure statement No potential conflict of interest was reported by the authors. 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RAWN ET AL. https://doi.org/10.1016/j.chroma.2014.03.032 https://doi.org/10.1016/j.chroma.2005.07.053 https://doi.org/10.1021/jf0634045 https://comptox.epa.gov/dashboard/chemical_lists/pfasmaster https://comptox.epa.gov/dashboard/chemical_lists/pfasmaster https://doi.org/10.1093/humrep/deu350 https://doi.org/10.1093/humrep/deu350 https://doi.org/10.1016/j.scitotenv.2018.08.090 https://doi.org/10.1016/j.envpol.2021.116875 https://doi.org/10.1016/j.envpol.2021.116875 Abstract Introduction Materials and methods Chemicals and reagents Calibration standards Evaluation Quality assurance testing Proficiency test samples Extraction Clean up Analysis Results and discussion Method development Instrumental approach Limits of detection/quantification Matrix impacts Post-extraction fortification Recovery experiments – accuracy, precision Performance/surrogate standards Analyte recovery Proficiency tests Acknowledgments Disclosure statement Funding References