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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Methylated nucleosides (mNSs) in urine are key biomarkers for RNA turnover and diseases like cancer.
• Non-invasive detection of these biomarkers is crucial for early disease diagnosis.

Purpose of the Study:

• To develop and validate a sensitive HILIC-LC-MS/MS method for simultaneous quantification of fifteen mNSs and related nucleobases in human urine.
• To establish a reliable platform for clinical diagnostics and population-based biomonitoring of RNA epigenetic signatures.

Main Methods:

• Development of a sensitive hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry (HILIC-LC-MS/MS) method.
• Implementation of a micro-scale solid-phase extraction (µ-SPE) using ENV+ sorbent for efficient sample preparation.
• Validation of the method according to international guidelines for endogenous analytes.

Main Results:

• Simultaneous quantification of fifteen mNSs and related nucleobases achieved.
• Excellent linearity (R² > 0.99), low limits of detection (1-12 µg L⁻¹), and acceptable precision (CV < 15%) demonstrated.
• Recovery values ranged from 85% to 120%, indicating method reliability.

Conclusions:

• The developed HILIC-LC-MS/MS method with µ-SPE is sensitive, selective, and suitable for high-throughput urinary mNSs profiling.
• This novel platform supports clinical diagnostics and population-based biomonitoring of RNA epigenetic signatures.
• The method is simple, effective, affordable, reproducible, and scalable for broader applications.