Additional studies were performed to determine if different ratios between TEA and HFIP provide differences in resolution
and MS sensitivity. The previous experiment suggests that obtaining maximum resolution and MS sensitivity revolves around
maintaining good retention of the ion-paired oligonucleotide. Different ratios were investigated based upon using a fixed
amount of HFIP (200 mM) and TEA concentrations ranging from 1 mM to 14 mM. Results are shown in Figure 4. The pH of the resulting
mobile phase was tested and is shown in the figure. Findings suggest that the ratio between HFIP and TEA is critical in obtaining
the maximum retention, MS sensitivity, and selectivity. Similar to what was observed in Figure 3, results suggest that the
optimal mobile phase for maximum MS sensitivity and oligonucleotide separation is a balance between obtaining maximum retention
(via ion-pairing reagent concentration) and minimizing MS suppression. It appears that mobile phase conditions using 200 mM
HFIP and 8 mM TEA (pH 8.0) provided the best results for LC–MS analysis for this mixture. Separating a different sample or
using a different HPLC column likely will require different optimal conditions.
Unlike other techniques for oligonucleotide characterization, reversed-phase LC–MS offers the capability to positively identify
the analytes in an oligonucleotide mixture. Reducing extracolumn volume by removing the UV detector from the flow path as
well as optimizing MS conditions can improve oligonucleotide LC–MS resolution significantly. Improved resolution can decrease
the complexity of the MS spectra, making the results generated from deconvolution software more useful.
Decreasing the amount of ion-pairing reagent can improve MS signal for oligonucleotides, but only to a point. Loss of retention
under conditions with low ion-pairing reagent concentration actually can reduce MS signal (due to earlier elution in lower
organic mobile phase). Reduced ion-pairing reagent also reduces resolution of closely eluted oligonucleotides. For this study,
the optimal mobile phase conditions for obtaining good resolution and MS signal were found to be 200 mM HFIP–8 mM TEA. While
not tested here, different types of oligonucleotides (RNA or phosphorothioate DNA) likely will have different optimum conditions
based upon their different hydrophobicities.
Greg Scott and Michael McGinley are with Phenomenex, Inc., Torrance, California. Matthew Champion is with Applied Biosystems, Foster City, California.
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