Per- and polyfluoroalkyl substances (PFAS) are notorious for both their persistence in the environment and complexity in analytical detection. Perfluorobutanoic acid (PFBA) is a particularly notable PFAS compound. Due to its small size, high polarity, and tendency to coelute with other compounds not readily discriminated by mass spectrometry (MS), PFBA analysis is especially difficult.
When a European governmental lab tasked Agilent with developing a more reliable method for PFBA analysis, it was obvious that this was more than a routine project, and innovation would be key. Susanne Soelter, LC-MS Application Engineer, and Stephan Buckenmaier, R&D Principal Scientist, reveal their approach to this unique problem.
Challenge: Proof of Concept for 2D-LC/MS in PFBA Detection
As Soelter explains, a government laboratory specializing in persistent organic pollutants approached Agilent with their challenge. The lab needed a proof-of-concept study to determine whether two-dimensional liquid chromatography coupled with mass spectrometry (2D-LC/MS) could simplify sample preparation, lower detection limits, and minimize false positives when analyzing PFBA.
“In PFAS analysis, people are looking for a variety of compounds, usually in methods which cover more than 20 components,” elaborates Soelter. "PFBA is the smallest in this set, and in complex samples, simple LC/MS/MS methods often lack the selectivity to reliably detect it at low levels. We might see something that looks like PFBA, but we cannot be completely certain.” Soelter adds that PFBA detection is often complicated by coeluting compounds and non-specific MS/MS transitions. The main component of this proof-of-concept challenge was to make the detection results at low levels more reliable and unambiguous.
Soelter reveals that she was keen to embrace this challenge for several reasons, one being that PFAS analysis is an ongoing global issue. “PFAS is not going away,” she remarks, adding that examples of this class of compounds continue to be discovered. “And as the number of global PFAS regulations increases, we need to expand our analytical toolbox.” She also points to the fact that 2D-LC is a relatively new technique within the PFAS space—she and Buckenmaier were eager to explore its potential.
Solution: Innovations in Sensitivity and Workflow
Soelter and Buckenmaier bring extensive experience in environmental and food applications, particularly in developing methods for PFAS analysis. The team utilizes their knowledge of 2D-LC, coupling the technique with a triple quadrupole mass spectrometer to tackle the proof-of-concept challenge.
“Using 2D-LC, we can isolate the PFBA peak from the chromatogram by parking it in a loop, transferring it in an online manner to a second LC column with different selectivity, and separating it from interfering compounds,” Soelter explains. “In traditional 2D-LC with UV detection, we would have two detectors—one for the first dimension and another for the second.” A similar setup is infeasible when using mass spectrometry because this detection technique will destroy the analytes. However, by adding a valve, it’s possible to direct the mass spectrometer to detect analytes from one dimension or the other.
“In this case, we also want to analyze other PFAS compounds eluting from the first dimension,” reports Soelter. “So we park the PFBA peak, analyze the remainder of the run, and transfer the PFBA peak to the second dimension for analysis.” This enables the team to achieve a well-resolved and unambiguous PFBA signal without compromising the analysis of other PFAS compounds in the same run.
The workflow also incorporates a novel feed injection technique that Buckenmaier was inspired to suggest after observing Soelter’s data. This allows for the injection of larger sample volumes without loss of chromatographic performance. “This helps increase the detection sensitivity by about tenfold, a significant boost for low-level PFBA detection,” advises Buckenmaier.
The team also implements practical measures to eliminate potential PFAS contamination from instrument components, chemicals, or other sources. These include using PFAS-free materials throughout the workflow, including within the analytical system itself, and installing a scavenger column to shift any residual PFAS compounds away from the retention times of interest.
Advancing PFBA Detection with 2D-LC/MS
The 2D-LC/MS method resolves PFBA from interferences and results in calibration curves and reproducibility that exceed expectations. “What impresses me most is how well the method performed with minimal adjustments,” says Soelter. “The additional separation achieved in the second dimension effectively reduces ion suppression effects, which could potentially eliminate the need for expensive, isotopically labeled internal standards in the future.”
This project underscores the growing importance of 2D-LC in analytical chemistry. “2D-LC is becoming a go-to technique for resolving complex analytical challenges, especially when coupled with MS,” notes Soelter. “While it has traditionally been paired with high-resolution MS systems such as quadrupole time-of-flight (Q-TOF) mass spectrometers, our work demonstrates its viability with triple quadrupole systems, which opens doors for applications such as mycotoxin and nitrosamine analysis.”
Soelter reveals that the client also sees immediate potential in the method. They are now partnering with Agilent to share the results at external conferences and in the form of a scientific paper. Agilent continues to work closely with customers to expand the capabilities of multidimensional chromatography, ensuring that as regulations around PFAS tighten globally, the analytical community is well-equipped to respond.
Meet the Experts
Susanne Soelter, PhD, Field Application Specialist LC/MS, Agilent Technologies
Susanne Soelter started her career as a product specialist for LC/MS at Varian in 2004. As Field Application Specialist, Susanne develops new methods in the Agilent laboratory in Waldbronn and supports customers on site. Her focus areas are environmental applications and food analysis.
Stephan Buckenmaier, PhD, R&D Principal Scientist, Agilent Technologies
Stephan has worked in the field of separation sciences for well over 20 years. He joined Agilent in 2006 and is currently a principal scientist in R&D at Waldbronn, Germany, for their liquid phase separation division.
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