TiO₂-Based Solid Phase Extraction Strategy for Highly Effective Elimination of Normal Ribonucleosides before Detection of 2′-Deoxynucleosides/Low-Abundance 2′-O-Modified Ribonucleosides

A novel TiO2-based SPE strategy was developed for eliminating normal ribonucleosides before mass spectrometry (MS) analysis of 2'-deoxynucleosides and 2'-O-modified ribonucleosides. The chromatographic research for the retention behavior of ribonucleosides and 2'-deoxynucleosides on TiO2 materials was investigated using TiO2 separation column. The results indicated a specific affinity interaction mechanism between TiO2 and cis-diol-containing ribonucleosides, and the interaction was proved effective even under a wide range of pH conditions and salt concentrations. Benefiting from these features, a TiO2-based solid phase extraction (SPE) method was developed for highly efficient elimination of RNA contamination from genomic DNA. Compared with the widely used enzymatic digestion method, the proposed TiO2-based SPE method showed much more efficiency for the removal of RNA as well as provided high recoveries for the 2'-deoxynucleosides. In addition, the sample processing time is dramatically shortened using the TiO2-based SPE method (~5 min) compared to the traditional enzymatic digestion method (~12 h). Finally, the purification of 2'-O-methylated ribonucleosides from RNA was successfully achieved in HeLa cells by the TiO2-based SPE method, which provided a proof-of-concept for the purification of relevant modified ribonucleosides from bulky normal ribonucleosides. Taken together, this strategy developed in the current study offers a promising option to purify 2'-deoxynucleosides/2'-O-modified ribonucleosides for their sensitive and accurate determination by eliminating normal ribonucleosides in biological samples.