Mobile Phase Aging and Its Impact on Electrospray Ionization of Oligonucleotides.

ABSTRACT

The implementation of fluoroalcohol/alkylamine mobile phase systems in oligonucleotide LC-MS provides a good balance between chromatographic separations and MS sensitivity. Since its introduction, several parameters including mobile phase composition, additive concentration, alkylamine hydrophobicity, and different fluoroalcohols have been carefully evaluated and optimized. While our understanding of this mobile phase system has increased over the years, there are challenges that continue to hinder method performance and remain poorly understood. One of these challenges is the constant loss of MS sensitivity over time, commonly termed mobile phase aging. This study investigates two aging mechanisms associated with loss of MS sensitivity alkylamine oxidation and aggregate formation. The relationship between pH, organic solvent, oxygen, and mobile phase aging is characterized, and mitigation strategies to extend mobile phase lifetime are discussed.