AI Article Synopsis
- Hexafluoroisopropanol (HFIP) has been commonly used in ion-pairing reversed-phase liquid chromatography/mass spectrometry (IP-RP-LC/MS) for oligonucleotide analysis, but researchers are exploring better alternatives.
- The study compared different ion-pairing reagents and fluoroalcohols, including HFIP and hexafluoro-2-methyl-2-propanol (HFTP), using an Agilent UHPLC system.
- Results indicate that while both HFIP and HFTP are effective, HFTP offers better desalting capabilities, and the choice of fluoroalcohols depends on the specific ion-pairing reagents used, with recommendations for different oligon
Article 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.
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