Evaluating IonHance Hexafluoroisopropanol (HFIP) for Enhanced LC-MS Oligonucleotide Analysis

Significance of the Topic

Liquid chromatography–mass spectrometry (LC-MS) is an essential tool for separating, identifying, and quantifying oligonucleotide therapeutics and research targets such as siRNA, antisense oligonucleotides, sgRNA, and mRNA. Ion-pair reversed-phase LC-MS relies on volatile buffers of alkyl amines and hexafluoroisopropanol (HFIP). Trace metal impurities in HFIP can bind analytes, leading to sodium and potassium adducts that obscure mass spectra and complicate data interpretation. High-purity HFIP is therefore critical for reliable spectral quality and reproducible analytical workflows.

Objectives and Study Overview

This application note investigates a highly purified form of HFIP (IonHance HFIP) produced via proprietary distillation to minimize Na+ and K+ levels (<100 ppb each). Comparative analyses assess the impact of IonHance HFIP versus undistilled and alternative vendor HFIP reagents on LC-MS performance for oligonucleotide standards ranging from 10- to 100-mers.

Methodology and Instrumentation

ICP-MS was used to measure sodium and potassium in multiple production batches of IonHance HFIP to confirm specifications. Oligonucleotide ladder (10–60 mer ssDNA) and a 100-mer standard were separated using an ACQUITY Premier Oligonucleotide BEH C18 300 Å, 1.7 µm, 2.1 × 50 mm column. Mobile phases contained 50–100 mM HFIP and DIPEA in water/acetonitrile gradients. System flushing protocols ensured comparison fairness between HFIP sources. Mass spectra were acquired under identical LC-MS conditions and normalized to base peaks for adduct analysis.

Main Results and Discussion

• ICP-MS confirmed Na and K levels below 100 ppb in IonHance HFIP.
• For the 10-mer ssDNA, distilled HFIP increased the intensity of the deprotonated base peak and halved sodium and potassium adduct formation at the [M-2H]2- charge state, relative to undistilled HFIP.
• Analysis of the 100-mer standard demonstrated cleaner raw and deconvoluted spectra with IonHance HFIP compared to a competing vendor reagent, with significantly reduced adduct peaks and improved signal clarity.

Benefits and Practical Applications

• Enhanced mass spectral clarity and sensitivity due to reduced metal adducts.
• Improved confidence in molecular weight assignment and purity assessment.
• Consistent reagent supply and reproducible performance supports high-throughput and QA/QC laboratories.

Future Trends and Potential Applications

Continued development of ultra-pure mobile phase additives will benefit emerging oligonucleotide modalities, including modified backbones and large mRNA constructs. Integration of such high-purity reagents into automated, high-throughput LC-MS platforms will further streamline peptide and nucleic acid analysis in pharmaceutical development and quality control. Additionally, tailored additive chemistries may optimize ionization for new biomolecule classes.

Conclusion

IonHance HFIP, through stringent purification, delivers exceptionally low sodium and potassium levels, leading to marked reductions in adduct formation and enhancement of LC-MS spectral quality for oligonucleotide analysis. Its consistent supply and performance make it a valuable reagent for analytical and QC laboratories focusing on nucleic acid therapeutics.

References

• Donegan M, Nguyen JM, Gilar M. Effect of Ion-Pairing Reagent Hydrophobicity on Liquid Chromatography and Mass Spectrometry Analysis of Oligonucleotides. J Chromatogr A. 2021;1666:46286.
• Guimarães GJ, Saad JG, Annavarapu V, Bartlett MG. Mobile Phase Aging and its Impact on Electrospray Ionization of Oligonucleotides. 2023.