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2P-23 PDF
LC/MSを用いたオリゴヌクレオチドの分析条件の検討
In the quality control of synthetic oligonucleotides, HPLC is used for the confirmation of compounds and the analysis of impurities. Commonly, ion-pairing reversed phase chromatography is used for mass spectrometry detection. The solutions containing triethylamine (TEA) and hexafluoroisopropanol (HFIP) are used as additives in mobile phase for high sensitivity detection and the control of interaction between analytes and packing materials. However, since HFIP is expensive solvent, a method of using alkylammonium acetate as a substitute for TEA-HFIP solution has also been proposed, but there is a problem of sensitivity reduction due to ion suppression effect. In this study, we focused on trifluoroethanol (TFE) as an alternative to HFIP and mobile phase conditions by combination with various alkylamines were investigated for analysis of oligonucleotides.
Signal intensity by using solution containing TFE and alkyamine was equivalent to that containing HFIP and alkylamine. Difference of selectivity was observed between condition using TFE and HFIP. In particular, in the separation of oligonucleotides with same sequence but non- or full-phosphorothioated, better separation was obtained under condition using TFE. As a result of examining the optimum analytical conditions, it was suggested that dimethylpropylamine (DMPA) was suitable as additive with TFE in eluent.