Guide to PFAS Testing Methods

Per- and polyfluoroalkyl substances (PFAS) represent a vast class of more than 3,000 synthetic compounds containing perfluorinated chains (with the general formula CnF₂n+₁COOH), commonly utilized in a wide range of consumer products and industrial processes.

The unique structure of PFAS, characterized by multiple carbon-fluorine (C-F) bonds, contributes to their exceptional chemical stability and resistance to degradation through environmental or biological processes. This durability makes them some of the most persistent pollutants in the environment. Their resistance to water and oil has led to widespread use in diverse applications, including water- and stain-repellent coatings for textiles, paints, personal care items, firefighting foams, fluoropolymer-based lubricants and additives, as well as grease- and water-resistant food packaging. Due to their extensive use, PFAS have been found in various environmental and biological samples, such as food, drinking water, wastewater, and human blood.

Like many other chemicals, PFAS can cause various harmful health effects, depending on factors such as the level, duration, and route of exposure, as well as individual characteristics like age, sex, ethnicity, overall health, and genetic background. PFAS exposure has been linked to various health effects in both sexes, with some impacts supported by high certainty evidence—such as thyroid disease, liver damage, kidney cancer, and testicular cancer.

In females, PFAS may also affect pregnancy outcomes and fetal development, including increased miscarriage risk, reduced birth weight, and delayed mammary gland development. Because of their toxic nature, these compounds have been classified as “persistent organic pollutants” by the Stockholm Convention (including PFOS, PFOA, PFHxS, and their salts) and by the European Commission (including PFOA, PFOS, PFNA, and PFHxA). Their durability, chemical stability, harmful effects, and tendency to build up in living organisms make them a serious risk to both environmental and human health; hence, PFAS testing is crucial for monitoring and mitigating these threats.

PFAS testing employs advanced techniques to detect, identify, and quantify both known and emerging compounds across diverse environmental matrices.

Liquid Chromatography-Mass Spectrometry (LC-MS/MS)

LC-MS is a commonly used PFAS testing method. Samples are first processed using suitable preparation techniques, such as solid phase extraction (SPE). The resulting extracted samples are then examined using LC/MS/MS with calibration performed via standards. Target analytes are identified with help of isotopically labeled surrogate standards. . Additionally, product ion ratios are compared with those from standards to support identification. Once confirmed, the concentration of each identified target compound is determined based on the response of its main product ion using external standard calibration.

Non targeted analysis of PFAS has largely been dominated by LC-MS methods, although GC-MS can access a complementary chemical space, especially for volatile and semivolatile PFAS. Studies show that LC‑MS covers most ionic PFAS; GC‑MS captures neutral/volatile precursors less amenable to LC, highlighting the need for GC-based approaches. GC is particularly valuable for identifying novel PFAS and transformation products from sources like manufacturing waste, incineration, wastewater, and consumer products.

Total Organic Fluorine (TOF) analysis is an indirect method used to estimate the overall burden of per- and polyfluoroalkyl substances (PFAS) and other organofluorine compounds in a sample. Instead of targeting individual PFAS molecules, TOF methods quantify the sum of organically bound fluorine.

Because this process measures the organic fluorine present within a sample, it provides a helpful bulk measurement of fluorine from all PFAS present, including those which may not be covered by targeted LC-MS/MS methods.

HRMS is increasingly used to detect emerging PFAS, with two main strategies applied for identification: suspect screening (SS) and non-target screening (NTS). SS is a more direct method, particularly effective when tandem mass spectrometry (MS/MS) reference spectra are available. In contrast, NTS is critical for uncovering new PFAS compounds in the absence of a suspect list, using fragment-based or homologue-based techniques.

HRMS contributes significantly by offering precise mass measurements, which are essential for identifying unknown substances. Since PFAS often form homologous series, patterns in accurate mass, retention time, and common fragments (such as CF₂, C₂F₄, and CF₂O) help in their recognition.

C-MS/MS is the primary technique used for targeted PFAS analysis due to its high sensitivity and accuracy. It is validated by the U.S. EPA through established methods such as 1633, 533, and 537.1 for detecting PFAS in various environmental and drinking water samples. For laboratories seeking reliable and compliant workflows, choosing a comprehensive PFAS analysis solution that aligns with these regulatory methods is essential for accurate detection and quantification.

• EPA Method 1633 is designed for use under the Clean Water Act (CWA) to measure the levels of 40 different PFAS in various sample types including water, soil, biosolids, sediment, and biological tissue, using LC-MS/MS. Weak anion exchange solid phase extraction (WAX-SPE) is employed for extracting various sample matrix types, and PFAS compounds are quantified through calibration with isotopically labeled internal standards.
   
• EPA Method 533 utilizes weak anion exchange solid phase extraction (WAX-SPE) followed by LC-MS/MS to detect 25 specific PFAS in drinking water, including short chain PFAs. This method employs tandem mass spectrometry and isotope dilution to improve analytical selectivity.
   
• Method 537.1 involves extraction via SDBL reversed phase SPE combined with LC-MS/MS for detection of 18 specific PFAs compounds, including the novel GenX compound. This method involves PFAS detection in drinking water samples.