[Current advances in the analysis of free RNA modified nucleosides by high performance liquid chromatography-tandem...

Post-transcriptional ribonucleic acid (RNA) modifications play crucial roles in regulating gene expression, with both eukaryotic and prokaryotic RNA exhibiting more than 170 distinct and ubiquitous modifications. RNA turnover generates numerous free nucleosides, including unmodified nucleosides and a variety of modified ones. Unlike unmodified nucleosides, modified nucleosides are not further degraded or used in the salvage-synthesis pathway owing to a lack of specific enzymes, which leads to the cytosolic accumulation or cellular efflux of modified nucleosides. These modified nucleosides can act as signaling molecules that regulate downstream pathways once transported to the extracellular space; alternatively, they are metabolized in the bloodstream and excreted in urine. Metabolized modified nucleosides are altered by cellular stress responses and mediate abnormal physiological states. Changes in the urinary and blood levels of modified nucleosides associated with cancer can serve as biomarkers for disease. Therefore, identifying and accurately quantifying nucleosides is vital for understanding RNA degradation and associated patterns of nucleoside metabolism. Such analyses are helpful when studying the biological functions and potential clinical applications of modified nucleosides. In this regard, high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) offers significant advantages in terms of sensitivity, selectivity, and efficiency, and has been widely used to analyze DNA and RNA nucleosides/nucleotides and their analogues. Multiple MS detection patterns and quantification methods have been established to detect nucleosides in biological samples, including cultured cells, urine, blood, and tissue samples. However, the development of an accurate HPLC-MS/MS method faces several challenges. Firstly, the presence of a complex biological matrix that contains macromolecules, small molecules, and salts can interfere with analysis. Salts and co-eluting substances in the extraction solution often affect mass-spectrometric responses for target analytes. Secondly, various nucleosides are present in vastly different abundances, with contents varying by up to four orders of magnitude; hence, accurately quantifying multiple nucleosides in a single assay is challenging. Thirdly, N-glycosidic bonds are favorably cleaved in most nucleosides during MS to produce the same characteristic fragment ions, which are often accompanied by nucleobases. This tendency poses challenges for distinguishing structural isomers and mass-analogs of modified nucleosides by MS. Post-transcriptional chemical modifications include methylation, hydroxylation, sulfur/oxygen substitution, and side-chain additions. Developing a unified method for simultaneously screening modified nucleosides is difficult owing to biochemical diversity; consequently, there is a need for advanced HPLC-MS/MS method capable of accurately quantifying such nucleosides. This review summarizes the development and applications of LC-MS technologies for analyzing endogenous nucleosides, covering sample preparation, chromatographic-separation and mass-spectrometric-detection conditions, and the development of quantification methods. Additionally, we discuss applications aimed at detecting and quantifying RNA-derived modified nucleosides in biological samples. The applications of HPLC-MS/MS technology are highlighted, the regulation and function of free modified nucleosides are discussed, and the potential functions of modified nucleosides as disease biomarkers for clinical applications are introduced.