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Abstract
The number of small oligonucleotides being used to improve human health is increasing rapidly. There are currently four FDA-approved therapeutic oligonucleotides and small endogenous RNA molecules have come to the forefront of both basic and translational research. For example, there have been enormous efforts made to investigate correlations between disease state and changes in the expression of various microRNAs. Improved methods to interrogate oligonucleotides will enhance our ability to use these powerful molecules in therapeutics and diagnostics. To date, such advances have been impeded by significant deficiencies in our ability to track and accurately quantify these macromolecules.During the past decades, ion-pair reversed phase liquid chromatography combined with negative ion ESI-MS has become the method of choice for direct oligonucleotide analysis. LC-MS of various oligonucleotides has been accomplished using a mobile phase consisting of triethylamine (TEA) and hexafluoroisopropanol (HFIP). However, combinations of alternative alkylamines with HFIP were never carefully examined until studies from our laboratory showed that a mobile phase consisting of diisopropylethylamine (DIEA) and HFIP significantly increased the MS signal intensity for phosphorothioate DNA oligonucleotides when compared to the standard TEA/HFIP mobile phase. Early studies revealed that the choice of optimal ion-pairing agents should be made on a case-by-case basis as it is strongly influenced by the physicochemical properties of the oligonucleotides and the mobile phase. This represented a major hurdle for researchers as they needed to rigorously investigate and optimize the mobile-phase composition prior to each study. To solve this issue, we developed a computational algorithm that can automate the process of ion-pair selection by introducing ion-pair and oligonucleotide parameters into a statistical regression model which will predict the MS signal intensity based on these inputs. Moreover, we demonstrated that HFIP can be substituted with other fluorinated alcohols and they would successfully act as the counter ions for the alkylamine ion pairing agents. Hexafluoromethylisopropanol (HFMIP), particularly showed superior performance compared to HFIP when utilized with more hydrophobic ion-pairs. All such improvements in the sensitivity of LC-MS methods, finally allowed for the detection and quantitation of miR-451 in plasma.