Recently efforts were undertaken to improve sensitivity of oligonucleotide LC/MS analysis while maintaining good resolution between different oligonucleotides. An HPLC column specifically designed for oligonucleotide separations, Clarity Oligo-RP, was used because this type of media will most likely show chromatographic changes during method optimization studies.
As MS sensitivity to oligonucleotides is believed to be most influenced by the MS suppression effects of ion-pairing reagents, different levels of ion-pairing reagents were evaluated for their effects on an oligonucleotide separation. A poly-dT 12–18 standard was used because the mixture is widely accepted in the industry as a good selectivity measure of a column’s performance. An overlay of the poly dT standard using different mobile phase conditions is shown in Figure 2.
Contrary to popular belief, MS sensitivity actually decreased with reduced ion-pairing reagent present in the mobile phase. Resolution also was seen to decrease with decreased ion-pairing reagent as retention of all analytes was greatly reduced. This reduction in analyte retention is actually the key factor in understanding MS sensitivity for oligonucleotides; the loss of ion pairing influenced retention results in oligonucleotides eluting in lower percentages of organic mobile phase.
It is well known that electrospray LC/MS efficiency is influenced by the percentage of organic present; increased retention of oligonucleotides caused by ion-pairing reagent makes up for any losses due to ion suppression, up to a point. The separation using 15 mM TEA/ 400 mM HFIP (a common concentration cited in the literature) shows a significant loss in MS signal due to ion-suppression effects. Thus it appears that there is an optimal balance between retention and ion suppression in maximizing LC/MS sensitivity for oligonucleotides.
For the Clarity Oligo-RP column that level appears to be around 2–6 mM TEA and 100–300 mM HFIP (these amounts are likely unique for each reversed-phase column).
In an attempt to further optimize both resolution and MS sensitivity, ratios between TEA and HFIP were investigated by fixing the amount of HFIP used (200 mm) for oligonucleotide separations and varying the levels of TEA used in the mixture. By varying the TEA and keeping HFIP constant, the pH of the mobile phase will change, giving some indication of the influence that pH may play on this separation.
Figure 3 is an overlay of the poly dT standard run at different HFIP/TEA ratios. Unlike the previous figure where dramatic changes in retention, resolution, and sensitivity are observed, varying the buffer ratios has a much less pronounced effect on sensitivity, though resolution did differ significantly between analyses.
Again there appears to be an optimal ratio between extremes, in this example using the Clarity Oligo-RP column the best condition evaluated was a mobile phase of 8 mM TEA with 200 mM HFIP (pH 8.0) in the aqueous mobile phase. These conditions provided both the best resolution and retention for the oligonucleotide mixture (which again will have an influence on sensitivity).
While these experiments only show an optimized separation for one oligonucleotide mixture (poly dT oligonucleotides), a common theme in both mobile-phase experiments is that there is an optimal balance of mobile-phase buffers that increases resolution and sensitivity for LC/MS applications. Such a methodology can be applied to other types of oligonucleotides (DNA, RNA, and phosphorothioates) to optimize a particular application. Of course, a key to success for any of these applications is proper instrumentation, column chemistry, and computational tools to generate, analyze, and interpret the oligonucleotide LC/MS data.