Posted on Feb 7, 2025
Updated March 23, 2026
Author: Our Phenomenex Team
Per- and polyfluoroalkyl substances (PFAS), often referred to as ‘forever chemicals,’ are a large class of fluorinated compounds that can be environmentally persistent and are widely detected in environmental media and in human serum. Epidemiologic studies have reported associations between PFAS exposure and multiple health endpoints (for example, immune and metabolic outcomes), although strength of evidence varies by compound and endpoint. As analytical methods and target lists evolve in biomonitoring programs, laboratories increasingly need extraction workflows that control matrix effects and background contamination to support accurate exposure assessment
Overview of PFAS Extraction Methods from Blood and Serum
PFAS extraction from blood and serum is fundamentally a trace-analysis problem in a highly complex matrix. Proteins (including strong PFAS-binding proteins), lipids/phospholipids, salts, and endogenous small molecules can all contribute to matrix effects and elevated background, so most workflows balance two competing goals: maximize recovery across a chemically diverse PFAS panel while producing an extract clean enough for stable LC–MS/MS quantitation at very low levels.
Protein Precipitation
Protein precipitation methods use organic solvents to denature proteins and release PFAS into solution. These approaches are valued for simplicity and speed. Because manual protein precipitation can introduce variability (pipetting, mixing, timing, and filtration differences), many labs use standardized sample preparation formats that support automation and consistent processing. Cartridge- or plate-based precipitation consumables can reduce operator-to-operator variability and improve reproducibility by fixing key physical parameters (for example, flow path and filtration behavior); dedicated products for this purpose (such as Impact Protein Precipitation) are often used as standardized “hardware” to make protein precipitation workflows more consistent at scale.
WAX SPE
Solid-phase extraction (SPE) remains a mainstay when higher selectivity cleaner extracts, and higher analyte concentrations are needed. For targeted PFAS panels, weak anion exchange (WAX) SPE is widely used because many PFAS are anionic under typical conditions; WAX provides both reversed-phase and ionic-retention mechanisms, which can improve cleanup relative to protein precipitation alone. The benefit is usually concentration of the PFAS analytes, lower matrix interference, and improved robustness for low-level quantitation. The main drawbacks are that SPE can be more method-sensitive (conditioning, wash, and elution choices matter) are difficult to develop, and can show analyte-dependent recovery if conditions are not tuned for both short- and long-chain PFAS. The PFAS WAX extraction may be preceeded by a protein precipitation step.
Stacked SPE
Stacked SPE (WAX + GCB) is a modern cleanup strategy that combines weak anion exchange (WAX) with graphitized carbon black (GCB) in a single cartridge format (WAX/GCB or GCB/WAX, with orientation selected based on the sample matrix). The WAX layer provides broad retention of anionic PFAS, while the GCB layer serves as a polishing step to remove co-extractives that can contribute to LC–MS/MS bias and ion suppression in complex samples. The main practical advantage is workflow control, because integrating retention and carbon cleanup reduces transfers and hands-on steps, which can improve throughput and reduce variability versus sequential WAX followed by dispersive carbon cleanup. The main tradeoff is that carbon can retain some longer-chain PFAS, so contact time and elution conditions must be managed, and recoveries verified across the full target list. A practical example of this stacked approach is Strata PFAS. Nonetheless, this technology is mainly related to environmental PFAS analysis for complex mixtures rather than PFAS extraction from blood and serum.
In practice, the best approach comes down to which constraint is most limiting for your lab. Protein precipitation is typically chosen when speed and simplicity are the priorities. WAX SPE is often selected when you need stronger selectivity and cleaner extracts than PPT can provide, particularly for targeted PFAS panels where matrix effects and background control are limiting, even though it may require more method tuning and additional handling steps. Stacked SPE is generally not used for PFAS serum or blood extraction but highly efficient in highly complex matrices.
Recent Advancements for PFAS Extraction from Serum and Blood
Recent progress in PFAS extraction from blood and serum has focused on improving throughput while keeping low-level LC–MS/MS results stable in a protein- and lipid-rich matrix. Serum remains the primary clinical specimen for PFAS biomonitoring, so workflow changes that reduce matrix effects and background contamination have an outsized impact on data quality.
A major trend is greater use of standardized, automation-friendly sample preparation (for example, plate or in-well precipitation formats) to reduce variability from manual pipetting, mixing, and filtration. These approaches are often paired with steps that reduce lipid-driven ion suppression, since residual phospholipids can destabilize signal over long batches, and performance should be verified across the full PFAS panel to avoid analyte-dependent bias.
Another important direction is the broader adoption and optimization of **online SPE–LC–MS/MS** workflows for serum, which integrate extraction into the instrument to reduce handling and improve repeatability. CDC Method 6304.09 is an established example of online SPE coupled to LC–MS/MS for serum PFAS, and follow-on method-development work has further optimized sorbents and UHPLC conditions while addressing background through strategies such as delay columns and system cleaning.
Finally, as serum PFAS target lists evolve in biomonitoring datasets, contamination control remains a central “advancement” in practice: PFAS-aware consumables, careful control of contact materials, and rigorous blanks are needed because background contributions can be comparable to the concentrations being measured.
Challenges in PFAS Extraction from Blood and Serum
PFAS work in serum is often limited by three practical issues: matrix effects, background contamination, and analyte-dependent recovery. Serum is rich in proteins and lipids, and co-extracted phospholipids can destabilize LC–MS/MS response through ion suppression, especially across long analytical sequences, so cleanup consistency matters as much as recovery.
Background contamination is the other major constraint because PFAS can originate from sample-contact materials and instrument flow paths, creating false positives or elevated blanks at the same order of magnitude as the concentrations being measured. In addition to PFAS-aware lab practices (strict blanks, controlled contact materials, and system mitigation strategies such as delay columns), many laboratories reduce risk by using consumables that are specifically quality-controlled for low PFAS background. “Designed for PFAS” product lines, for example, include consumables that are specifically designed to reduce the PFAS contamination.
Finally, recovery is not uniform across PFAS because chain length and head-group chemistry influence extraction behavior, and many PFAS bind strongly to serum proteins.
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