PAMS 2019: Improving LC-MS Separations of Peptides with Difluoroacetic Acid Ion Pairing

Importance of the Topic

Peptide mapping by reversed‐phase liquid chromatography coupled to mass spectrometry (RPLC‐MS) is a cornerstone technique for the detailed characterization of biopharmaceutical proteins. Effective ion pairing agents are crucial to balance chromatographic resolution and MS sensitivity. Trifluoroacetic acid (TFA) provides excellent peak shape but suppresses MS signal, whereas formic acid (FA) enhances MS response at the cost of chromatographic performance. Difluoroacetic acid (DFA) emerges as a promising compromise, potentially improving both separation selectivity and mass spectral quality when purified to low metal content.

Objectives and Study Overview

• Assess chromatographic performance of 0.1% purified DFA versus FA and TFA in peptide mapping.
• Compare retention, peak capacity, and MS signal intensity on two sub-2 µm particle UPLC C18 chemistries.
• Evaluate impact of trace metal contamination on adduct formation and mass spectral clarity.

Methodology and Instrumentation Used

Peptide digests of NIST mAb were analyzed using ACQUITY UPLC systems with Peptide BEH C18 and Peptide CSH C18 columns (1.7 µm, 2.1×150 mm) at 80 °C. Mobile phases contained 0.1% FA, purified DFA or TFA in water (A) and acetonitrile (B) under identical binary gradients. UV detection wavelengths were optimized for each acid modifier. MS data were acquired on a Xevo G2‐XS QToF in full‐scan and fragmentation modes (100–2000 m/z). Purified DFA (IonHance DFA) certified at <100 ppb Na^+ and K^+ was compared with reagent‐grade DFA to assess metal‐related adduct formation. Instrumentation list:

• ACQUITY UPLC H-Class Bio
• ACQUITY UPLC TUV detector
• Xevo G2-XS QToF MS
• Peptide BEH C18 and Peptide CSH C18 columns
• IonHance Difluoroacetic Acid (low metals DFA)

Main Results and Discussion

• DFA‐modified separations yielded resolution and peak capacities approaching those of TFA, outperforming FA by 20–30% in capacity.
• MS sensitivity with DFA was slightly lower than FA but dramatically higher than TFA, delivering balanced performance.
• Purified DFA reduced sodium and potassium adducts compared to commercial DFA, improving spectral clarity for peptides like 2:T13 and 1:T37.
• Charge state distributions with DFA resembled FA more closely than TFA, suggesting similar ionization efficiencies for subunit and peptide analyses.

Benefits and Practical Applications

• Enhanced selectivity: DFA introduces distinct retention profiles useful for resolving challenging peptides.
• Improved MS quality: Low-metal DFA minimizes adducts and preserves signal intensity.
• Method flexibility: Single gradient conditions can be transferred across CSH and BEH chemistries with minimal adjustment.
• Biopharma analytics: Suitable for routine QC peptide mapping, impurity profiling, and lot‐to‐lot comparability studies.

Future Trends and Opportunities

• Expansion to intact protein and subunit analyses using DFA for top-down and middle-down workflows.
• Development of hybrid ion‐pairing modifiers combining DFA with other additives for enhanced selectivity.
• Integration with high‐throughput automation and microflow LC-MS for faster screening.
• Adoption of polymeric, trace-metal certified containers and fittings to further reduce metal leaching.

Conclusion

Purified difluoroacetic acid represents a compelling compromise between chromatographic resolution and MS sensitivity in peptide mapping. It delivers near-TFA peak capacities with FA-like signal intensity and mitigates metal adduct formation when certified low-metal grades and polymeric containers are employed. DFA thus enables high-quality, reproducible RPLC-MS workflows vital for biopharmaceutical characterization and QC.

References

1. Manuscript in review.
2. Kellett J., Birdsall R., Yu Y. Application of Difluoroacetic Acid to Improve Optical and MS Performance in Peptide LC-UV/MS. 2018. Waters Technical Brief (PN: 720006482EN).
3. Nguyen J. M., Liu X., Lauber M. A. Low Adduct Peptide LC-MS Obtained with IonHance DFA and Certified LDPE Containers. 2019. Waters Application Note (PN: 720006596EN).