Chromatography Comparison of Ultra-Short-Chain PFAS in Complex Food Matrix Extracts

Summary: Chromatography Comparison of Ultra-Short-Chain PFAS in Complex Food Matrix Extracts

Significance of the topic

The increasing regulatory and public-health focus on ultra-short-chain per- and polyfluoroalkyl substances (USC PFAS) such as trifluoroacetic acid (TFA) demands reliable analytical workflows for food safety testing. USC PFAS are highly polar, low-molecular-weight compounds that are difficult to retain and separate by conventional reversed-phase LC, which leads to poor chromatographic performance, strong matrix effects, and potential false positives from isobaric interferences. Robust chromatography and injection strategies that allow direct analysis of high-organic food extracts with minimal matrix suppression are therefore essential to lower limits of quantification and provide confident identification in complex food matrices.

Study goals and overview

• Systematically compare chromatographic performance of several commercially available mixed-mode LC columns engineered for USC PFAS analysis using extracts from diverse food matrices (baby foods, whey protein powder, pet food, shrimp).
• Assess analyte retention, peak shape, mitigation of solvent effects from high-organic injections, and susceptibility to matrix effects and isobaric interferences.
• Evaluate the benefit of feed (direct) injection on chromatographic integrity versus classic flow-through (sandwich) injection.

Methodology

• Sample preparation: QuEChERS extraction followed by EMR mixed-mode passthrough cleanup to produce matrix-blank extracts and matrix-matched post-spike samples. Spiking levels of USC/SC standard were 1.0 ng/mL and 0.1 ng/mL for performance evaluation.
• Analytes: Eight USC PFAS including TFA, TFMS, PFPrA, PFEtS, PFBA, PFPrS, PFPeA and PFBS monitored by multiple reaction monitoring (MRM) transitions on a tandem quadrupole MS. Note PFBA and PFPeA shared single MRM transitions and thus lack a second qualifier ion.
• Chromatographic approaches compared: multiple mixed-mode USC PFAS columns and a typical C18 for reference; tests performed using both classic flow-through and feed (direct) injection modes.

Instrumentation used

• LC system: Agilent 1290 Infinity II (method details referenced) with Agilent 1290 Infinity III Hybrid Multisampler operating in feed or feed-adaptive modes.
• Columns evaluated: Altura Poroshell 120 PFAS HPLC Column (2.1 x 50 mm, 2.7 µm) with a Poroshell 120 PFAS Delay Column (4.6 x 30 mm) and three other mixed-mode USC PFAS columns (referred to as Column 1, Column 2, Column 3).
• Mobile phases: A = 5 mM ammonium acetate in water with 0.05% acetic acid; B = acetonitrile:water (95:5, v/v). Sample solvent commonly 90:10 ACN:water (some columns required more aqueous sample solvent).
• Chromatographic conditions: flow 0.5 mL/min, column temperature 40 °C, injection volume 10 µL, total run time ~12 min (post time variable per method).
• MS detection: Agilent 6495D triple quadrupole LC/TQ in negative polarity. Typical source settings included gas temperature 200 °C (nebulizer), sheath gas 300 °C, capillary voltage ~2500 V, with nozzle 0 V.
• Autosampler settings: feed injection mode with adaptive feed speed (10% of pump flow), specific inner/outer wash and seat-wash sequences to mitigate carryover and solvent effects.

Main results and discussion

• Impact of feed injection: Using the Hybrid Multisampler in feed injection mode substantially improved peak shapes and retention for USC PFAS, most notably on columns that otherwise suffered from solvent-related injection effects (Columns 2 and 3). Feed injection reduced solvent mismatch artifacts arising from high-organic sample solvent.
• Altura Poroshell 120 PFAS column performance: This column delivered the strongest retention for USC PFAS, excellent peak symmetry, and robustness to injections of high-organic extracts. It allowed more flexible gradient settings and direct analysis of high-organic sample extracts without the severe solvent effects seen on other mixed-mode columns.
• Comparative column behavior: Column 1 demonstrated acceptable performance with somewhat shorter retention and some peak tailing for analytes such as TFA, PFPrA and PFBA. Columns 2 and 3 required samples in weak solvent/high aqueous to approach acceptable peak integrity and still showed poorer performance relative to Altura.
• Matrix effects: Matrix-matched experiments across five food matrices showed the Altura PFAS column produced matrix effects within approximately 90–110% for most analytes (indicating minimal suppression or enhancement) except for TFA which was more variable. Column 1 was the next best in mitigating matrix suppression. Columns 2 and 3 exhibited greater ion suppression and lower reproducibility in matrix extracts.
• Resolution of isobaric interferences: In plant-based baby food extracts a polar isobaric interference co-eluted with PFBA on a traditional C18 column, masking PFBA and jeopardizing confident identification (especially since PFBA has only one MRM). The Altura PFAS column achieved baseline separation of PFBA from the interfering component, reducing false positives and enabling a more reliable confirmatory workflow for PFBA and PFPeA.

Benefits and practical applications

• Direct analysis of high-organic food extracts without additional dilution or solvent-exchange steps when using a column with robust tolerance (e.g., Altura PFAS), which saves time and reduces sample handling that can introduce variability.
• Improved analyte retention and peak shape reduce ion suppression in the source and lower method LOQs, enhancing detection confidence for USC PFAS at sub-ng/mL levels in complex matrices.
• Better chromatographic separation of isobaric interferences reduces false positives and supports reliable confirmation when limited MRM transitions are available (critical for short-chain PFAS with single transitions).
• Feed injection capability on modern multisamplers is an effective operational approach to mitigate solvent-mismatch injection artifacts across a range of LC columns.

Future trends and applications

• Method standardization: Expect growing harmonization of LC/MS methods and acceptance criteria for USC PFAS in food matrices, driven by regulatory guidance and SMPRs.
• Column chemistry development: Further refinement of stationary phases tailored to extreme polar analytes will increase retention and selectivity while maintaining robustness to diverse sample solvents.
• Enhanced sample handling: Continued interest in simplified workflows (e.g., direct injection with improved columns, automated cleanup cartridges such as EMR PFAS) to reduce matrix effects and increase throughput.
• Analytical sensitivity and confirmation: Greater use of high-sensitivity tandem MS, improved internal standards (isotope-labelled USC PFAS), and orthogonal confirmation strategies to address single-transition analytes.
• Application breadth: Expansion of validated USC PFAS methods across a wider range of foodstuffs, beverages, and environmental samples to support surveillance and regulatory compliance.

Conclusions

• Feed injection on a modern hybrid multisampler effectively mitigates solvent-related injection artifacts and improves chromatographic performance for USC PFAS, particularly on columns sensitive to sample solvent strength.
• The Altura Poroshell 120 PFAS column outperformed other evaluated mixed-mode USC PFAS columns by providing stronger retention, better peak symmetry, tolerance to high-organic injections, superior matrix-effect mitigation, and effective separation of isobaric matrix interferences (e.g., PFBA in plant-based baby food).
• Combining appropriate sample cleanup (QuEChERS + EMR passthrough), feed injection, and a PFAS-optimized column offers a practical workflow to improve accuracy, lower LOQs, and reduce false positives in complex food matrices.

References

1. Agilent Technologies. InfinityLab and Altura Poroshell 120 HPLC columns. Agilent publication details, 2026.
2. Agilent Technologies. Captiva EMR PFAS Cartridges product information, 2026.
3. Agilent Technologies. Comparative Study of Chromatographic Performance for Ultra Short Chain PFAS in Food Matrices, publication 5994-9182EN, 2026.
4. Agilent Technologies. Reliable Ultra Short Chain PFAS Analysis in Water and Landfill Groundwater, publication 5994-9019EN, 2026.
5. Kamuf M. More Sensitive Quantification of PFAS by LC/MS with the Agilent 1260 Infinity II Hybrid Multisampler. Agilent application note 5994-6994EN.
6. AOAC International. Standard Method Performance Requirements (SMPRs) for Per- and Polyfluoroalkyl Substances (PFAS) in Produce, Beverages, Dairy Products, Eggs, Seafood, Meat Products, and Feed (AOAC SMPR 2023.003), 2023.