Hydrophilic additives enhancing electrospray ionization of oligonucleotides in hexafluoro-2-propanol-free reversed-phase ion-pair liquid chromatography/mass spectrometry

The characterization of oligonucleotide therapeutics is commonly performed by liquid chromatography/mass spectrometry (LC/MS) using reversed-phase ion-pair (RP-IP) chromatography with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and triethylamine (TEA) due to its efficient electrospray ionization (ESI) performance. However, HFIP poses several challenges, including environmental concerns as a per- and polyfluoroalkyl substance (PFAS), limited mobile phase stability, and high reagent costs. Traditional HFIP-free alternatives, such as acetic acid (AA) and TEA, generally result in lower MS sensitivity. This study aimed to develop an HFIP-free analytical method for oligonucleotides and explored the use of AA/TEA-based mobile phase additives to improve MS sensitivity. An AA/glycine/TEA-based mobile phase prepared by adding glycine to an AA/TEA-based mobile phase, exhibited significantly enhanced MS sensitivity compared to the original AA/TEA system, reaching levels comparable to those achieved with HFIP/TEA. Comprehensive screening of amino acids revealed that aliphatic amino acids, particularly those with high hydrophilicity, contributed to enhanced ionization. These findings suggest that hydrophilic additives may promote the localization of oligonucleotides at the droplet surface and facilitate ion release, thereby enhancing ionization efficiency. Moreover, the AA/glycine/TEA system maintained MS sensitivity for a longer duration than the HFIP/TEA system. Although glycine is typically avoided in LC/MS due to its non-volatile nature, its use at sufficiently low concentrations did not lead to contamination or performance degradation. These findings support the potential of hydrophilic additives like glycine to enhance ionization efficiency in HFIP-free RP-IP-LC/MS for oligonucleotide analysis.