Comparing ion-pairing reagents and sample dissolution solvents for ion-pairing reversed-phase liquid chromatography/electrospray ionization mass spectrometry analysis of oligonucleotides.

A sensitive and selective liquid chromatography/mass spectrometry (LC/MS) method is essential for quality control of synthetic oligonucleotides. However, researchers are still searching for improvements to ion-pairing reagents for ion-pairing reversed-phase LC/MS. This study performed a comprehensive comparison of six ion-pairing reagents to determine their performance as mobile phase modifiers for oligonucleotide LC/MS. The study was performed using a Waters ultra-performance liquid chromatography (UPLC®) system coupled to a Waters LCT premier XE ESI-TOF mass spectrometer by using a UPLC® OST column (2.1 mm × 100 mm, 1.7 µm). Buffer systems containing ion-pairing reagents (triethylamine, tripropylamine, hexylamine, N,N-dimethylbutylamine, dibutylamine, N,N-diisopropylethylamine) and hexafluoro-2-propanol were compared by measuring the adduct ion formation, chromatographic separation, and MS signal intensity of four oligonucleotides (10mer to 40mer). The effect of dissolution solvents on MS signal intensity and adduct ion formation was also investigated. Results showed that the type of dissolution solvent can have a signficiant impact on adduct ion formation with oligonucleotides. Results also showed that the maximum separation for small, medium and large oligonucleotides occured when using tripropylamine, N,N-dimethylbutylamine, and dibutylamine, respectively. However, on average 15 mM hexylamine and 50 mM hexafluoro-2-propanol provided the best chromtatographic performance (resolution values: 14.1 ± 0.34, 11.0 ± 0.17, and 6.4 ± 0.11 for the pairs of oligonucleotides T10 & T15, T15 & T25, and T25 & T40, respectively (3 replicates)). The impact of dissolution solvent on the MS signal of oligonucleotides depends on the type of ion-pairing reagent. Buffer combining 15 mM hexylamine and 50 mM hexafluoro-2-propanol produced the highest overall performance for oligonucleotides (10mer to 40mer) with respect to chromatographic resolution and mass detection.