Chromatographic Performance Comparison in Ultrashort-Chain PFAS Analysis

Importance of the topic

Per- and polyfluoroalkyl substances (PFAS), including ultrashort-chain (USC, C1–C3) and short-chain (SC, C4–C7) species, are increasingly regulated contaminants in food due to their mobility, persistence, and potential health impacts. USC PFAS (e.g., trifluoroacetic acid) are highly polar, small, often volatile and water soluble, which complicates their extraction, chromatographic retention, and mass-spectrometric detection in complex food matrices. Robust chromatographic methods that increase retention and separate analytes from coextractives are therefore critical to improve detection sensitivity, reduce matrix effects, and support regulatory monitoring and food safety programs.

Objectives and overview of the study

This application study compared chromatographic performance for USC and SC PFAS in food matrix extracts using the new Agilent Altura Poroshell 120 PFAS mixed-mode column versus three other commercially available mixed-mode LC columns marketed for USC PFAS analysis. The comparison focused on retention, solvent‑effect tolerance (allowing direct injection of high‑organic food extracts), separation from matrix interferences, and mitigation of matrix effects. Four complex food matrices (baby food, whey protein powder, pet food powder, and shrimp) were extracted to produce EMR‑cleaned matrix blanks that were post‑spiked with USC/SC PFAS standards (spike levels 1 and 0.1 ng/mL) for column evaluation.

Methodology and sample preparation

Samples were extracted using QuEChERS (EN 15662) followed by EMR PFAS passthrough cleanup; EMR eluates were used directly for post‑spiking. Extraction solvent: acetonitrile (ACN) with 1% acetic acid. Because many solid food extracts contain very high organic content (90–100% ACN), the study evaluated whether columns could tolerate direct injection without offline solvent exchange (to avoid analyte loss for volatile USC compounds).

The study injected matrix blanks and fortified QC samples directly for the Altura column and commercial column 1. For commercial columns 2 and 3, extracts required 1:1 dilution with water (2× dilution) and use of a modified gradient to achieve acceptable chromatographic behavior.

Used instrumentation

• LC system: Agilent 1290 Infinity II high-speed pump combined with an Agilent 1290 Infinity III hybrid multisampler and 1290 Infinity II multicolumn thermostat; system modified with an InfinityLab PFAS analysis conversion kit and a Poroshell 120 PFAS delay column.
• Columns tested: Agilent Altura Poroshell 120 PFAS column (2.1 × 50 mm, 2.7 µm, P120 mixed‑mode C18 particles) and three other commercial mixed‑mode USC PFAS columns (all 2.1 × 50 mm).
• Mass spectrometer: Agilent 6495D triple quadrupole LC/MS with Jet Stream iFunnel ESI source; negative ion mode; fragmentor ~166 V; typical ESI gas settings summarized in the study.
• Consumables: Bond Elut QuEChERS EN kits, Captiva EMR PFAS Food cartridges (6 mL), polypropylene vials and tubes verified for PFAS cleanliness.

Analytical conditions

Mobile phases: A = 5 mM ammonium acetate + 0.05% acetic acid in water; B = 95:5 ACN:water. Two gradients were used: Gradient A (higher initial organic, used for Altura and column 1) and Gradient B (higher initial aqueous and hold time, used for columns 2 and 3). Injection volume 10 µL; column temperature 40 °C. A feed (online dilution) injection program on the 1290 Infinity III multisampler was also evaluated to mitigate solvent effects for columns with limited solvent tolerance.

Main results and discussion

Retention and separation

• The Altura Poroshell 120 PFAS column delivered substantially stronger retention for all evaluated USC and SC PFAS, with retention factors (k') > 3 for all analytes and a minimum k' of 9.4 for TFA (the earliest eluter). Analytes were well distributed across the run window, baseline-separated, and displayed good peak symmetry.
• Commercial column 1 produced performance broadly comparable to Altura but with slightly reduced retention and more pronounced tailing for some early analytes (TFA, PFPrA, PFBA). Resolution between PFPeA and PFBS on column 1 was poor (Re ≈ 0.48).
• Commercial columns 2 and 3 exhibited weak retention for early‑eluting USC analytes; even after applying a milder gradient (Gradient B), significant fronting/tailing and inadequate retention persisted. Those columns required 2× offline dilution of extracts (50% ACN) or use of feed injection online dilution to reach acceptable chromatography.

Solvent-effect mitigation and sample injection

• The Altura column and commercial column 1 tolerated direct injection of high‑organic extracts (90–100% ACN) without compromising peak shape, reducing the need for solvent-exchange and thus lowering the risk of analyte loss (important for volatile USC compounds like TFA).
• Columns 2 and 3 lacked sufficient solvent‑effect mitigation; even with feed‑injection online dilution, their early‑eluting analytes showed broader peaks compared with Altura and column 1.

Matrix effects and sensitivity

• Matrix effect testing across five food extracts showed that the Altura column produced matrix effects within ~90–110% for all analytes except TFA, indicating minimal ion suppression/enhancement and a consistent response in complex matrices.
• Other mixed‑mode columns showed lower and more variable matrix‑effect values, indicating stronger and heterogeneous ion suppression likely caused by poorer separation of analytes from polar coextractives (e.g., sugars, salts).

Separation from isobaric interferences and confirmatory potential

• Altura’s stronger retention allowed chromatographic separation of PFBA from a common isobaric interference in plant‑based baby food extracts; on traditional RP‑C18 columns PFBA frequently coeluted with the interference, increasing false positives and LOQs. Altura’s orthogonal selectivity offers a chromatographic confirmatory approach that can reduce reliance on high‑resolution MS for PFBA/PFPeA confirmation.

Benefits and practical applications

• Higher analyte retention and improved separation reduce matrix-induced ion suppression and improve detection sensitivity and quantitation reliability for USC/SC PFAS in complex food extracts.
• Tolerance for direct injection of high‑organic extracts simplifies sample workflows (avoids drying/reconstitution), reduces potential analyte loss (especially important for volatile USC analytes), and increases throughput.
• Greater method flexibility: Altura allows use of stronger starting organic in gradients and standard injection strategies without complex online dilution hardware or extensive sample dilution.
• Chromatographic confirmation of analytes that lack qualifier ions (PFBA, PFPeA) may reduce dependence on more costly HRMS instruments for routine confirmation in some matrices.

Future trends and potential applications

• Broader method validation: expand performance verification across more food types and a wider panel of PFAS (including emerging analogs) to support regulatory compliance (e.g., AOAC SMPRs and EU limits).
• Chromatographic confirmation workflows: further develop and validate orthogonal LC separation strategies (like Altura) as cost‑effective alternatives or complements to HRMS for confirmatory analysis of analytes lacking qualifier ions.
• Integration with online dilution and automated sample handling: optimize feed‑injection or online dilution approaches for columns with limited solvent tolerance to improve robustness without extensive offline processing.
• Method harmonization and standardization: adoption of robust mixed‑mode columns in interlaboratory studies and standard methods to harmonize USC PFAS monitoring in foods.
• Coupling with HRMS for non‑target screening: utilize strong chromatographic retention to improve identification confidence and reduce matrix interferences in suspect and non‑target workflows.

Conclusion

The Agilent Altura Poroshell 120 PFAS mixed‑mode column demonstrated markedly improved chromatographic retention, solvent‑effect tolerance, and separation of USC and SC PFAS from matrix interferences compared with three other commercially available mixed‑mode columns. These attributes enabled direct injection of high‑organic food extracts, reduced matrix effects and ion suppression, improved sensitivity and quantitation reliability, and offered chromatographic separation that can aid confirmatory identification of analytes prone to isobaric interferences. Overall, Altura provides practical advantages for routine PFAS monitoring in complex food matrices and supports method simplification and robustness for laboratories addressing regulatory requirements.

References

1. European Commission. Commission Regulation (EU) 2023/915 of 25 April 2023 on Maximum Levels for Certain Contaminants in Food and Repealing Regulation (EC) No 1881/2006. EUR‑Lex 2023.
2. AOAC. 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.
3. Battisti I, Trentin AR, Franzolin E, Nicoletto C, Masi A, Renella G. Uptake and Distribution of Perfluoroalkyl Substances by Grafted Tomato Plants Cultivated in a Contaminated Site in Northern Italy. Sci Total Environ. 2024;915:170032.
4. Zhao L, Giardina M, Parry E. Determination of 30 PFAS in Baby Food. Agilent Technologies Application Note, publication 5994‑7367EN, 2025.
5. Zhao L, Parry E. Determination of 30 PFAS in Fish Oil, Coffee Powder, and Protein Powder. Agilent Technologies Application Note, publication 5994‑8610EN, 2025.
6. Zhao L. Determination of 43 PFAS in Beer and Wine. Agilent Technologies Application Note, publication 5994‑8813EN, 2025.