A tip-on-tip micro-solid-phase extraction HILIC-LC-MS/MS platform for the determination of urinary methylated nucleosides

Abstract

Methylated nucleosides (mNSs) excreted in urine are considered biomarkers associated with RNA turnover and disease states such as cancer and can be detected through non-invasive sampling. In this work, we report the development and validation of a sensitive hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-LC-MS/MS) method for the simultaneous quantification of fifteen mNSs and related nucleobases in human urine. Due to the complexity of the biological matrix and the need to quantify low-abundance biomarkers, a sensitive and selective micro-scale solid-phase extraction (µ-SPE) using a tip-on-tip format with ENV+ sorbent is proposed as the most convenient strategy for sample preparation. This approach offers the advantages of rapid extraction, minimal solvent consumption, and simplified sample handling. The method is simple, effective, affordable, reproducible, and easily scalable. Additionally, HILIC-based chromatographic separation enabled effective retention and high resolution. According to international guidelines on endogenous analytes, the proposed method demonstrated excellent linearity (R² > 0.99), low limits of detection (1–12 µg L⁻¹), and acceptable intra- and inter-day precision (CV < 15%). Recovery values ranged from 85% to 120% across all analytes. This novel platform enables reliable and high-throughput profiling of urinary mNSs and offers strong potential for clinical diagnostics applications and population-based biomonitoring of RNA epigenetic signatures.