The role of fluoroalcohols as counter anions for ion‐pairing reversed‐phase liquid chromatography/high‐resolution electrospray ionization mass spectrometry analysis of oligonucleotides

Abstract

Rationale Hexafluoroisopropanol (HFIP) has been widely used as a counter anion in the mobile phase for ion‐pairing reversed‐phase liquid chromatography/mass spectrometry (IP‐RP‐LC/MS) analysis of oligonucleotides. However, researchers are still searching for improvements to counter anions for LC/MS analysis of oligonucleotides. This study aimed to find alternatives to HFIP for analyzing oligonucleotides. Methods The study was performed using an Agilent 1290 ultra‐high‐performance liquid chromatography (UHPLC) system coupled to an Agilent 6540 mass spectrometer by using an oligonucleotide BEH C18 column (100 × 2.1 mm, 1.7 μm). Buffer systems containing ion‐pairing reagents (triethylamine, tripropylamine, hexylamine, dimethylbutylamine, diisopropylethylamine, N,N‐dimethylcyclohexylamine, and octylamine) and fluoroalcohols (HFIP and hexafluoro‐2‐methyl‐2‐propanol (HFTP)) were compared chromatographically and mass spectrometrically. Results Results showed that HFTP has better desalting ability than HFIP, but both HFIP and HFTP have comparable effects on the separation of oligonucleotides sized from 10mer to 40mer for most of ion‐pairing reagents, with the exception of triethylamine and N,N‐dimethylcyclohexylamine, where HFIP performed better than HFTP. Conclusions The choice of fluoroalcohols in IP‐RP‐LC/MS analysis of oligonucleotides depends on the type of ion‐pairing reagents used in the mobile phase. As a guideline, we would recommend to use either HA‐HFIP or HA‐HFTP for small oligonucleotides, but TPA‐HFTP for large oligonucleotides for IP‐RP‐LC/MS analysis of synthetic oligonucleotides.

HFIP in Organic Synthesis

“…For example, LC-MS/MS or MALDI-MS analysis of samples prepared with HFIP shows increased recovery of lipophilic molecules and proteins with enrichment for hydrophobic as well as membrane-associated proteins. Fluoroalcohols including HFIP serve as counterions in ion-pair reversed-phase LC-MS analysis of oligonucleotides. , HFIP has been shown to effect the retention of both acidic and basic analytes during LC-MS as well . In proteomics, HFIP is used to extract proteins from tissues , or as a component of cell lysis buffer for sample preparation. , …”

Recent developments in the characterization of nucleic acids by liquid chromatography, capillary electrophoresis, ion mobility, and mass spectrometry (2010–2020)

“…HFTP seemed to outperform HFIP regarding MS sensitivity when used together with more hydrophobic ion‐pairing reagents such as HA for small oligonucleotides and TFA for larger oligonucleotides [108]. Subsequently, the effects of different IP reagents together with different fluoroalcohols on the chromatographic separation and MS analysis of synthetic oligonucleotides in IP‐RP‐LC–MS were thoroughly investigated [109]. TEA, HA, dimethylbutylamine (DMBA), DIPEA, octylamine (OA), N , N ‐dimethylcyclohexylamine (DMCHA), and tripropylamine (TPA) were studied together with fluorinated modifiers HFTP and HFIP.…”

“…For the last two ion‐pairing reagents, HIFP performed better than HFTP. With this work, the authors showed that the choice of fluoroalcohols should be matched to the ion‐pair reagent used for IP‐RP‐LC and advised the use of HA‐HFIP or HA‐HFTP for small oligonucleotides and TPA‐HFTP for large nucleic acids [109]. IP‐RP‐MS was also successfully applied to the separation and determination of antisense oligonucleotides in biological samples [110].…”

Oligonucleotides: Current Trends and Innovative Applications in the Synthesis, Characterization, and Purification

“…The enhancement was significantly stronger than with any combination of HFIP and ion-pairing reagents. It is also important to point out that HFIP and HFMIP produce significantly different mass spectra, due to different charge-state distributionpatterns, for ONs [91]. In addition, HFMIP is characterized by more pronounced desalting ability with respect to HFIP, which usually allows to obtain mass spectra that are simpler to interpret.…”

“…From the analysis of different ON samples, by using different combinations of alkylamine and fluoroalcohols, Liu et al concluded that the use of hexylamine with either HFIP or HFMIP is more suitable for small ONs, while tripropylamine in combination with HFMIP has to be preferred for large synthetic ONs [91].…”