Extending The Lower Limits Of Quantification Of A Therapeutic Oligonucleotide Through Microflow LC-MS/MS

By Daniel Warren, Sean McCarthy, Lei Xiong, SCIEX

The development of ionpairing reversed phase liquid chromatography (IP-RP LC) methodologies have paved the way for mass spectrometers to become a viable platform for oligonucleotide analysis by providing a strategy for sample introduction and orthogonal frontend separation that is compatible with electrospray ionization techniques common to modern mass spectrometers. IP-RP LCMS is often the preferred solution for both qualitative and quantitative oligonucleotide analysis; however, several fundamental challenges remain with this approach. The concentrations of ion pairing reagents needed to adequately retain and separate polar oligonucleotides on a reversed phase column, often an alkylamine paired with a fluorinated alcohol, contribute to charge competition resulting in electrospray ion suppression. This phenomenon compromises the mass spectrometer response and may prevent desired limits of detection and quantification from being achieved. The routine use of ion pairing reagents pose another challenge as their accumulation within the mass spectrometer contributes to contamination which accelerates front end cleaning and maintenance intervals.

Here a solution is presented to these widely recognized challenges through a novel  microflow LC-MS strategy. The sensitivity improvements that result from reduced flow rates entering the MS and the associated enhancement in ionization efficiency have been well documented and prompted microflow LC-MS assays to be deployed for myriad applications. In this instance the reduction of contaminants entering the mass spectrometer is proportional to the mobile phase flow offering an additional advantage of microflow LC-MS. This study was designed to characterize the improvement of quantification for a phosphorothioate antisense oligonucleotide assay scaled down to microflow LC-MS.