Versatile TOP Assay Surrogates for Evaluating Oxidation Efficiency in Complex Environmental Samples

Abstract

Accurate assessment of total per- and polyfluoroalkyl substance (PFAS) contamination remains limited because conventional targeted analyses do not capture unknown precursor compounds. The total oxidizable precursor (TOP) assay has been widely used to address this gap; however, previous studies have shown that matrix effects can cause incomplete oxidation of precursors, which can be detected with a stable-isotope labeled oxidation surrogate. Yet, current surrogates are incompatible with EPA Method 1633A. Here, we introduce a new isotopically labeled surrogate system that integrates isotopically labeled surrogates - ¹³C₃-PFHpSA (oxidation surrogate), ¹³C₃-PFHpA (transformation surrogate), and ¹³C₃-PFNA, a stable extraction surrogate that enables end-to-end tracking of method recovery across the TOP assay workflow and designed to be fully compatible with EPA Method 1633A without analytical interference. Across multiple laboratories and selected matrices, including aqueous film-forming foam (AFFF) and landfill leachate, we observed that disappearance of the oxidation surrogate does not always correspond to quantitative formation of terminal products, consistent with previously reported incidents of incomplete oxidation in complex matrices. Transformation surrogate recovery provides an additional indicator evaluating oxidation performance and potential matrix effects. These results suggest that incorporating surrogate-based quality controls can improve the performance of the TOP assay interpretation, particularly in complex environmental matrices.

Graphical Abstract

1. Introduction

Per- and polyfluoroalkyl substances (PFAS) have been used for decades in firefighting foams, surface coatings and a wide array of consumer products.^1–3 Due to their extreme persistence and mobility, they contaminate water, soil and biota worldwide; however, the full extent of PFAS burden in these environments remains incompletely understood.^4–6 Routine monitoring relies on targeted liquid chromatography/tandem mass spectrometry (LC-MS/MS) methods that quantify a relatively small subset of perfluoroalkyl carboxylates (PFCAs), sulfonates (PFSAs) and selected precursor compounds using certified standards.⁷ The U.S. EPA Method 1633A (EPA 1633A),⁸ for example, measures 40 PFAS compounds, including 13 precursor compounds, and expansion of targeted analytical coverage remains challenging because most PFAS lack authentic and isotopically labeled standards. Additionally, quantitative targeted analyses are currently limited to commercially available standards (e.g., approximately 120 out of thousands of PFAS used in commerce).^9–12 Consequently, important uncertainty remains regarding the overall burden and composition of PFAS present in complex environmental samples.

To address these analytical limitations, the total oxidizable precursor (TOP) assay was developed to estimate the burden of oxidizable PFAS precursors in contaminated samples by subjecting samples to oxidation through the continuous generation of hydroxyl radicals from persulfate under highly alkaline conditions, activated by heat or UV light.^13–19 During the TOP assay, hydroxyl radicals transform many polyfluorinated compounds into measurable terminal perfluoroalkyl acids (PFAAs). Comparison of PFAA concentrations before and after oxidation provides an estimate of oxidizable precursor content that may not be captured by conventional targeted analysis alone. Therefore, the assay relies on standard LC-MS/MS for PFAS measurement, it is accessible to laboratories that lack advanced instrumentation such as high-resolution mass spectrometry. The TOP assay has been applied to diverse matrices (e.g. drinking water, solids, textiles, tissue, etc.) highlighting its utility as a practical screening tool for assessing potential PFAS precursor loading.^13,20

There has been increasing attention and effort to improve the TOP assay and address its limitations; background organic and/or inorganic constituents can compete for hydroxyl radicals or react directly with persulfate, leading to incomplete oxidation of PFAS precursors.^13,15,21 Further, the chain length distribution of resulting transformation products of some PFAS are not identical to the chain length of the parent compound. For example, fluorotelomer-based precursors commonly yield a series of shorter-chain PFCAs rather than a single terminal PFCA.²² These complexities, along with the potential formation of ultrashort-chain acids (<C4) that are not routinely analyzed in standard targeted PFAS methods,^15,17,23 may underestimate PFAS-precursor concentrations.

Previous studies have demonstrated that incomplete oxidation and matrix effects can influence TOP assay results.^14,15 Specifically, matrix radical scavengers lead to a systematic underestimation of precursor concentrations through incomplete conversion of precursors into terminal carboxylates, whereas MS ionization effects (e.g., differential ion suppression or enhancement of surrogates) can distort recovery corrections, leading to potential over- or underestimates precursor concentrations. However, quantitative tools to directly diagnose these effects remain limited. As a result, measured increases in terminal PFAAs are often assumed to reflect total precursor conversion, despite evidence that matrix effects can arrest oxidation at intermediate stages. This constraint reduces the ability to interpret TOP assay results and may lead to potential bias in precursor estimation under certain conditions. Isotopically labelled internal standards (IS) added prior to instrument analysis internally normalize for instrument response but provide no information regarding incomplete oxidation or sample preparation. Tsou et al. advanced TOP assay versatility by adding a pre-oxidation, mass-labeled oxidation-efficiency indicator (¹³C₈-PFOSA) and quantifying its conversion to (¹³C₈-PFOA) to confirm oxidation completion and assess the extent of radical scavenging by natural organic matter, minerals, and other solutes.¹⁵ Using ¹³C₈-PFOSA, it was demonstrated that oxidation can fail under strong radical-scavenging conditions (e.g., no ¹³C₈-PFOA detected at 4 mM butyl carbitol and in undiluted primary effluent at 250 mg-C/L dissolved organic carbon (DOC)), with improved ¹³C₈-PFOA recoveries (>79%) restored by dilution. Tsou et al. also demonstrated that TOP assay can underestimate precursors in some aqueous film-forming foam (AFFF) when ultrashort-chain products are not measured, with known fluorotelomer precursor recoveries improving from 28% to 82–108% when trifluoroacetic acid (TFA) and perfluoropropanoic acid (PFPrA) were included. Similarly, other studies have also evaluated different oxidation surrogates (e.g., native n:2 FTS and ¹³C₂-4:2 FTS) to improve TOP assay versatility.^22,24 However, native n:2 FTS may be difficult to differentiate from fluorotelomer-derived compounds already present in environmental samples, while the release of EPA Method 1633A has introduced additional challenges because some surrogate compounds and/or their oxidation products overlap with analytes included in the method target list.²³ These limitations complicate data interpretation and broader implementation of TOP assay surrogates.

To further support ongoing efforts to improve TOP assay quality assurance and reproducibility, we developed and evaluated a new set of isotopically labelled oxidation surrogate standards that serve as internal quality controls without compromising routine PFAS analyses (e.g., EPA 1633A).²⁵ These surrogates, which are currently commercially available and broadly accessible, were designed so that (i) the oxidation surrogate (¹³C₃-PFHpSA) undergoes oxidation to a transformation surrogate (¹³C₃-PFHpA), providing a practical indicator of oxidation performance, (ii) the oxidation surrogate and transformation surrogate are not included among the labeled or target analytes listed in EPA 1633A, thereby minimizing interferences with existing target methods, and (iii) a stable extraction surrogate (¹³C₃-PFNA), which does not undergo oxidation, provides extraction traceability and an additional measure of overall method performance. We evaluated the new surrogate system under both UV-activated and thermally activated TOP protocols across diverse aqueous matrices, including reagent water, dilute AFFF, and landfill leachate. These matrices were selected as representative test cases to assess surrogate performance under controlled yet compositionally distinct conditions. Collectively, our findings provide an additional quality-control framework for assessing oxidation completeness and improving confidence in TOP assay interpretation across complex environmental samples.

2. Materials and Methods

2.1. Materials.

Isotopically labeled oxidation surrogates (¹³C₃-PFNA, ¹³C₃-PFHpSA, and ¹³C₃-PFHpA) were synthesized in collaboration with Cambridge Isotope Laboratories (Tewksbury, MA) and are commercially available.²⁵ Concentrated working stock solutions were prepared in PFAS-Free water—rather than methanol—to prevent trace residual solvent from quenching radicals during the TOP assay. Solutions were stored in glass containers for up to two weeks. Native PFAS standards, extracted internal standards (EIS), and non-extracted internal standards (NIS) were purchased independently by participating laboratories from Wellington Laboratories (Guelph, ON). All isotopically labeled surrogates possessed an isotopic purity of >99% (per manufacturer certificates of analysis), and potential interferences from standard impurities were determined to be negligible. Working standard solutions were prepared by each participating laboratory (See SI, Text S1 in SI). Additional chemicals and solvents were purchased from Fisher Scientific (Hampton, NH) and Sigma-Aldrich (St. Louis, MO). All solvents were HPLC grade or higher. The AFFF formulation used in this study was a PFSA-dominant mixture (“Peterson Bucket”) provided to participating laboratories by UC Berkeley.^7,20,26 The baseline PFAS composition and major ion matrix are summarized in Tables S4 and S5, respectively.

2.2. Oxidation Surrogate Performance in the TOP Assay.

The stability and solubility of the oxidation surrogates were evaluated following equilibration in reagent water and landfill leachate. Experiments were conducted at elevated temperatures in glass (Methods A and C), polypropylene (PP; Method B), and both glass and polypropylene containers (Method D), with and without oxidants. Surrogate performance was assessed across three participating laboratories using different TOP assay protocols. Four sample sets were prepared: (1) reagent water pre-TOP, (2) reagent water post-TOP, (3) Peterson Bucket AFFF diluted 10⁴-, 10⁵-, 10⁶-fold in tap water, and (4) landfill leachate samples collected from a site withheld under a non-disclosure agreement. Landfill leachate baseline PFAS levels and chemical characteristics are provided in Tables S6 and S7, respectively.

The oxidation surrogate (¹³C₃-PFHpSA) and extraction surrogate (¹³C₃-PFNA) were added prior to oxidation. The transformation surrogate (¹³C₃-PFHpA) was not spiked because it is formed through oxidation of ¹³C₃-PFHpSA. This study was conducted as a multi-laboratory evaluation of various TOP assay approaches, in which participating groups applied different protocols to assess method performance; it was not designed as a formal interlaboratory comparison. Briefly, the evaluated protocols included (Figure 1): Method A, a conventional thermally activated TOP assay followed by the EPA 1633A SPE procedure;²⁷ Method B, SPE using WAX/GCB and SCX cleanup followed by sample evaporation, aqueous reconstitution and thermal oxidation;^14,16 Method C, a UV-activated TOP assay followed by the EPA 1633A SPE procedure;¹⁹ Method D, a thermally activated TOP assay employing a lower persulfate dosage.¹⁸ Method A–C were evaluated using sample sets 1–3, whereas Method D was applied only to landfill leachate samples because of limited sample volume availability (Method details in SI. Text S1). Each participating laboratory independently performed the ¹³C₃-PFHpSA and ¹³C₃-PFNA spiking before experimentation.

Figure 1.

Schematic representing experimental design. (A) Conventional thermally activated TOP assay with EPA 1633A SPE; (B) SPE cleanup (WAX/GCB and SCX) followed by thermally activated TOP assay; (C) UV-activated TOP assay; and (D) thermally activated TOP assay with reduced persulfate dosage.

Acceptance criteria for the surrogate framework were established as 50–150% recovery for ¹³C₃-PFNA (extraction surrogate), 0–10% remaining ¹³C₃-PFHpSA (oxidation surrogate), and 50–150% recovery for ¹³C₃-PFHpA (transformation surrogate). Under sufficiently oxidative conditions, ¹³C₃-PFHpSA is expected to transform to ¹³C₃-PFHpA with near-quantitative molar yield. Surrogates and target PFAS were quantified using a multi-point linear calibration curve (0.025–5 ng/mL on-column, corresponding to 1.25–25 ng/L sample-equivalent concentration), yielding a limit of quantitation (LOQ) of 2 ng/L. Method detection limits (MDL) for the oxidation surrogates were comparable to those of native PFAS analytes (approximately 0.5 ppt). Prior to oxidation, ¹³C₃-PFHpSA and ¹³C₃-PFNA were fortified at a nominal concentration of 100 ng/L. For Methods A and C, aqueous samples (125 mL) were processed via solid-phase extraction (SPE) following EPA Method 1633A protocols to a final extract volume of 5 mL (25× concentration), whereas Methods B and D were prepared via direct aqueous sample dilution (10⁴-10⁶-fold) prior to LC-MS/MS injection. Signal suppression from co-eluting PFAS compounds (e.g., ¹³C₃-PFNA: PFNA, ¹³C₅-PFNA, ¹³C₉-PFNA) was negligible.

3. Results and Discussion

3.1. Evaluation of Oxidation Performance Using New Surrogates

The surrogate framework enabled assessment of oxidation performance and transformation product formation during the TOP assay across diverse aqueous matrices (Figure 2). Oxidation surrogates previously incorporated into TOP assay workflows are often either (1) extracted internal standards (EIS) in EPA Method 1633A (Table S1; e.g. ¹³C₈-FOSA and ¹³C₂-n:2 FTS) or (2) transformed into products that overlap with target analytes used in routine PFAS quantification (e.g. complete oxidation of ¹³C₂-4:2 FTS). In contrast, the oxidation surrogate ¹³C₃-PFHpSA and its transformation product ¹³C₃-PFHpA do not interfere with the EPA 1633A quantitation scheme or overlap with target analytes included in the method. The oxidation surrogate ¹³C₃-PFHpSA was added immediately prior to oxidation and was expected to transform to ¹³C₃-PFHpA with near-quantitative molar yield under sufficiently oxidative conditions.

Figure 2.

Chemical structures of (A) ¹³C₃-PFHpSA and (B) ¹³C₃-PFHpA, and corresponding recovery efficiencies measured using three analytical methods for five different sample types: pre-TOP samples; post-TOP samples in DI water; and post-TOP samples in AFFF diluted at the factors of 10⁴, 10⁵, and 10⁶. Dilution factors of 10⁵ and 10⁶ are only valid for Method B. n =3 samples per test condition.

In post-TOP analysis of reagent water blanks, the oxidation surrogate ¹³C₃-PFHpSA exhibited recoveries below 10% across all evaluated methods (Figure 2A): 0±0% (Method A), 0±0% (Method B), and 2±1% (Method C). Similarly, in diluted AFFF samples, ¹³C₃-PFHpSA recoveries remained below 6%, although variability among methods was slightly greater. These results are consistent with near-complete transformation of the oxidation surrogate under the tested conditions, analogous to Tsou et al.’s indicator approach where successful oxidation was confirmed by converting ¹³C₈-PFOSA to ¹³C₈-PFOA.¹⁵

However, recovery of the transformation surrogate ¹³C₃-PFHpA revealed substantial variability in transformation product formation across methods and sample matrices (Figure 2B). In Method A, ¹³C₃-PFHpA recovery was 64±7% in reagent water blanks and 104±9% in diluted AFFF. In Method B, ¹³C₃-PFHpA recovery was 128±5% in reagent water blanks, decreasing to 39±2% in 10⁴-fold diluted AFFF but increasing to 135±9% and 143±4% at higher dilution factors (10⁵- and 10⁶-fold dilution, respectively). Similarly, in Method C, recovery decreased from 68±6% in reagent water blanks to 40±3% in 10⁴-fold diluted AFFF.

Heat-activated persulfate oxidation under alkaline conditions generates hydroxyl radicals (OH^•) that rapidly attack the sulfonamide headgroup of electrochemical fluorination (ECF)-derived precursors such as ¹³C₃-PFHpSA. In the absence of OH^• scavengers, cleavage of the headgroup yields full conversion to the corresponding perfluorinated carboxylate (¹³C₃-PFHpA). Under standard TOP assay conditions (85°C, alkaline pH values), OH^• radical attack is not accompanied by the formation of measurable amounts of transient intermediate species (Houtz and Sedlak, 2012). When oxidation is incomplete, primarily due to radical scavenging by co-occurring organic matrix components, the unreacted oxidation surrogate remains mostly in the form of ¹³C₃-PFHpSA rather than converting into stable intermediate products, presumably because the intermediates are more reactive with OH^• or decompose in the presence of water and oxygen. Because both the initial precursor (¹³C₃-PFHpSA) and the terminal transformation product (¹³C₃-PFHpA) are directly monitored via LC-MS/MS, tracking the loss of the precursor alongside the recovery of the product provides an explicit, quantitative measure of transformation efficiency.

These findings indicate that disappearance of the oxidation surrogate alone does not necessarily correspond to quantitative formation of the terminal product in the presence of co-occurring organic constituents likely due to matrix effects, including radical scavenging. Baseline recoveries of ~64–68% in reagent water blanks represent conversion yields in the absence of OH^• scavengers along with routine analytical processing losses (e.g., SPE extraction efficiency, nitrogen blowdown concentration). Consequently, ¹³C₃-PFHpA recovery serves as a relative empirical performance indicator within an established 50–150% acceptance window rather than a rigid measure of absolute mass balance. The lower product recoveries in complex matrices (e.g., 39±2% in 10⁴-fold diluted AFFF) demonstrate matrix-induced suppression relative to this baseline. Increased dilution likely decreases radical scavenging and promotes more complete transformation. These observations are consistent with previous findings reported by Tsou et al.,¹⁵ in which transformation of ¹³C₈-PFOSA to ¹³C₈-PFOA reached 80–96% in samples containing ≤125 mg-C/L wastewater effluent or ≤1.5 mM butyl carbitol in water, whereas no ¹³C₈-PFOA was detected at higher matrix concentrations (250 mg-C/L wastewater effluent and 4.1 mM butyl carbitol).

In addition, part of the apparent recovery behavior may reflect isotopic overlap rather than reaction performance alone. Specifically, ¹³C₃-PFHpA differs by only one mass unit from the ¹³C₄-PFHpA extracted internal standard (EIS) used in EPA Method 1633. Based on a certified >99% per-position isotopic purity (p>0.99), binomial calculations indicate that isotopic impurities in the ¹³C₄-PFHpA EIS contribute a minor baseline signal (~3.9%) to the monitored ¹³C₃ channel (M+3), while contributions to the native PFHpA channel (M+0) are negligible (<0.001%). Conversely, natural isotope abundance contributes a minor signal overlap from the A+1 isotopologue of ¹³C₃-PFHpA into the ¹³C₄-PFHpA channel. Collectively, these observations confirm that while isotopic overlap modestly influences recovery variability, matrix effects and transformation dynamics remain the primary drivers of observed performance.

Since ¹³C₃-PFHpA is generated in situ during oxidation rather than directly spiked pre-extraction, its measured recovery represents an integrated performance metric reflecting the combined outcomes of transformation yield, matrix interactions, and downstream sample processing. Downstream analytical biases are accounted for via method internal standards (¹³C₄-PFHpA), making surrogate yield a cumulative indicator of overall assay performance.

Finally, Method C, which employed UV irradiation in open vessels, exhibited comparatively lower transformation efficiency in complex matrices. Volatilization losses and reduced radical availability associated with pH shifts during oxidation may have contributed to these observations.^7,14 The observed discrepancies for UV Method C may also be attributable to its implementation in open vessels, rather than an inherent limitation of the UV-TOP approach. These findings suggest that additional optimization of UV-activated TOP protocols may be beneficial, particularly for large-volume or high-organic-content samples.

More broadly, the results highlight the value of tracking transformation surrogate recovery as an additional indicator of oxidation performance and potential uncertainty in TOP assay interpretation.

3.2. Extraction Traceability Using ¹³C₃-PFNA

To enable end-to-end recovery tracking throughout the TOP workflow, ¹³C₃-PFNA was used as a stable extraction surrogate and added prior to sample pre-treatment or oxidation. Because ¹³C₃-PFNA does not undergo measurable transformation during the TOP assay, its recovery reflects the overall transfer efficiency of the analytical workflow, including pre-treatment, oxidation, sample preparation, extraction, and instrumental analysis (Figure 3). Recoveries of ¹³C₃-PFNA remained within the established acceptance criteria (50–150%) across all tested samples and methods. In post-TOP reagent water blanks, recoveries were 85±7% (Method A), 122±7% (Method B), and 88±7% (Method C). In post-TOP diluted AFFF samples, recoveries were 104±11% (Method A), 99±4% (Method B), and 67±4% (Method C). These results demonstrate that ¹³C₃-PFNA remained stable across multiple TOP assay workflows and was not substantially affected by oxidation, degradation, or sorptive losses under the tested conditions. Accordingly, ¹³C₃-PFNA serves as a useful extraction and process surrogate for monitoring overall method performance during the TOP assay without interfering with the EPA 1633A quantitation scheme.

Figure 3.

Chemical structure of ¹³C₃-PFNA, and recovery efficiencies in three sample matrices (1) pre-TOP DI water, (2) post-TOP DI water and (3) 10⁴-fold diluted AFFF. Acceptance criteria for ¹³C₃-PFNA were set at 50–150% (shaded area). n =3 samples per test condition.

3.3. Matrix and Container Effects on Surrogate Recovery

To further investigate factors contributing to surrogate recovery variability, we evaluated the effects of container material and sample matrix on surrogate behavior under identical experimental conditions (Figure 4). Initial spike-and-recovery experiments conducted (10 μg/L initial spike concentration across all treatments, conducted in duplicate) in reagent water using polypropylene containers showed substantial differences between the extraction and oxidation surrogates following storage. While the extraction surrogate, ¹³C₃-PFNA remained within the acceptance criteria (~90% recovery), the oxidation surrogate, ¹³C₃-PFHpSA, exhibited substantially lower recovery (~27%). In contrast, when the same experiment was performed in glass containers at the same 10 μg/L spiking level, recoveries increased to 97% for ¹³C₃-PFNA and approximately 120% for ¹³C₃-PFHpSA.

Figure 4.

Impact of container material on the duplicate recovery (n = 2) of ¹³C₃-PFNA and ¹³C₃-PFHpSA spiked at 10 μg/L in (A) DI water and (B) landfill leachate.

When the surrogates were evaluated in landfill leachate at 10 μg/L spike concentration, surrogate recovery trends differed from those observed in reagent water. Recovery of ¹³C₃-PFNA remained relatively stable across matrices and container materials. However, ¹³C₃-PFHpSA exhibited higher recovery in polypropylene (~80%) relative to reagent water (~27%), while recovery in glass containers (~75%) was lower than that observed in reagent water (~120%). Consequently, recovery differences between polypropylene and glass containers became less pronounced in landfill leachate than in reagent water. The transformation surrogate ¹³C₃-PFHpA was not detected in any pre-oxidation matrix stability experiments.

The decreased recovery of the oxidation surrogate (¹³C₃-PFHpSA) may partially reflect sorption to polypropylene (PP) containers, consistent with previous studies reporting stronger PFAS sorption to PP relative to glass, particularly for neutral or less polar species such as sulfonamides.^29,30 However, recovery behavior appears to vary across experimental conditions and analytical workflows. For example, PP containers were used without significant recovery losses in the Method B datasets (Figure 2, 3), whereas lower recoveries were observed in DI water experiments associated with Method D at the 10 μg/L spike level (Figure 4A). These observations suggest that sorption behavior of sulfonamide-series PFAS may depend on the physicochemical characteristics of the PP material itself (e.g., polymer composition, additives, surface treatments), consistent with observations reported by Zenobio et al. (2022).²⁹

Although volatilization of neutral PFAS during heating cannot be excluded,³¹ the matrix-dependent recovery patterns observed in this study, particularly the reduced losses in landfill leachate compared to DI water, are consistent with the possibility that ¹³C₃-PFHpSA associates with suspended particles or dissolved organic matter in compositionally complex matrices. Such interactions may reduce volatilization losses and/or decrease interactions with container surfaces. Overall, these findings demonstrate that container choice must be evaluated in the context of sample matrix complexity. While glass containers remain preferable for clean water matrices (e.g., DI water or low-TOC waters) to minimize wall-sorption of neutral sulfonamide surrogates (¹³C₃-PFHpSA), container selection is less impactful in organic-rich matrices such as landfill leachate, where dissolved matrix components suppress surface interaction differences between polypropylene and glass.

4. Conclusion

The surrogate framework developed in this study integrates traceable extraction recovery and oxidation-performance assessment within the EPA Method 1633A quantitation framework, providing an additional quality-control approach for TOP assay applications. However, the results also demonstrate that surrogate performance is strongly influenced by sample matrix composition. Samples with elevated organic content exhibited greater radical scavenging, reducing transformation efficiency even following extensive pre-treatment. Importantly, strategic sample dilution (a further 10- to 10²-fold increase beyond the initial 10⁴-fold dilution, reaching 10⁵- to 10⁶-fold overall) substantially improved surrogate recoveries, in some cases restoring ¹³C₃-PFHpA recoveries to within the target acceptance range. These findings suggest that dilution optimization should be considered for TOP assay implementation in complex environmental samples. In practice, dilution factors should be selected to achieve acceptable transformation surrogate recovery (e.g., 50–150% ¹³C₃-PFHpA recovery) while maintaining precursor concentrations above analytical detection limits. Although a 10⁵-fold dilution was effective for high-concentration matrices such as AFFF without substantially compromising sensitivity, lower-concentration environmental samples may require more careful optimization to balance matrix suppression effects against the potential loss of detectable precursor signals.

Importantly, transformation surrogate recoveries provide a compound-specific quality control indicator of oxidative conditions within the sample matrix, rather than proof of quantitative transformation across all uncharacterized PFAS precursor classes. Lower ¹³C₃-PFHpA recoveries are consistent with reduced transformation efficiency under certain conditions and could therefore introduce additional uncertainty into interpretation of TOP assay-derived precursor estimates. Such behavior is consistent with incomplete conversion to terminal PFAAs and formation of intermediate products not routinely captured by targeted LC-MS/MS analysis. In practice, transformation surrogate recoveries could help guide data interpretation, identify samples that may benefit from additional dilution or repeat analysis, and support broader assessment of oxidation performance across complex matrices. Conversely, acceptable ¹³C₃-PFHpA recoveries are generally consistent with improved oxidation performance under the tested conditions. Collectively, incorporation of oxidation surrogate performance metrics may improve confidence and transparency in TOP assay interpretation for environmental and site assessment applications.

Beyond surrogate chemistry, TOP assay interpretation could further benefit from broader consideration of ultra-short-chain PFAS (<C4) and neutral intermediates, which may contribute additional uncertainty to mass balance closure in conventional LC-MS/MS workflows. Building on prior studies, including Tsou et al.,¹⁵ which highlighted the contributions of neutral intermediates and ultra-short PFAS during precursor transformation, the reported apparent conversion efficiencies of known precursors (82–108%) and substantial contributions of ultra-short PFAS in AFFFs and environmental samples (up to 50%) collectively suggest that a portion of transformation products may not be routinely captured using standard targeted analytical methods. More systematic inclusion of these species could therefore improve assessment of transformation pathways and support more comprehensive characterization of precursor oxidation products. However, ultrashort PFAS analysis requires additional/separate LC–MS/MS methods, which can increase analytical cost and complexity.

Additionally, the present study evaluated a limited number of matrices and should therefore be interpreted as proof-of-concept rather than a comprehensive assessment across environmental systems. Future interlaboratory validation studies across diverse matrices could help further evaluate robustness, reproducibility, and develop a Standard Operating Procedure (SOP) which can be adopted by all practitioners.

Supplementary Material

Supporting Information. Detailed methods description for tested TOP assay protocols. List of acronyms. Tables with surrogates and the mass-labeled PFAS extraction standard mixture used in this study.

Highlights.

• First study on new TOP Assay oxidation surrogates compatible with EPA 1633A.

• ¹³C₃-PFHpSA converts to ¹³C₃-PFHpA and ¹³C₃-PFNA enables end-to-end method recovery tracking.

• Surrogate recoveries evaluated in DI water, diluted AFFF, and landfill leachate.

• 50–150% recovery of surrogates in thermal and UV TOP tested pre- and post- solid phase extraction.

• Sample dilution improved surrogate recoveries in samples with elevated organic content.