Rapid and selective simultaneous quantitative analysis of modified nucleosides using multi-column liquid chromatography-tandem mass spectrometry

Background: The profiles of modified nucleosides could act as useful biomarkers for cancer and cellular stress-induced diseases. However, there are no reports of high throughput and simultaneous quantitative methods for using biomarker evaluation and discovery at the bedside. Methods: Modified nucleosides were separated on two CAPCELL PAK ADME S3 (100 mm × 2.1 mm i.d.; 3-μm particle size) analytical columns coupled with a CAPCELL PAK ADME cartridge (10 mm × 2 mm i.d.; 3-μm particle size) guard column. Both columns were used in tandem during multi-column LC analysis to reduce analysis time. Two mobile phases were used, including 20 mM ammonium acetate adjusted to pH 5.3 using acetic acid and 1.0 M ammonium acetate/acetonitrile/water/acetic acid (1/95/5/0.03, v/v/v/v), with the post-column addition of methanol to enhance ionization efficiency. Tandem mass spectrometry detection was performed using a triple quadrupole mass spectrometer equipped with a heated electrospray ionization source in selected reaction monitoring mode. Results: Four major nucleosides and 11 modified nucleosides, including guanosine, adenosine, uridine (U), cytidine, inosine, 1-methyladenosine, 5-methylcytidine, 2′-O-methylcytidine, 3-methylcytidine, 7-methylguanosine (m⁷G), 5-methyluridine (m⁵U), pseudouridine, 2-thiocytidine, N²-methylguanosine (m²G), N²,N²-dimethylguanosine, 2-fluoro-2′-deoxyadenosine as an internal standard, and its isotopic isomers were separated within 7 min and analyzed within 10 min. This resulted in limits of quantitation of 0.50–5.00 ng mL⁻¹, except for m²G (10.0 ng mL⁻¹), m⁷G (12.5 ng mL⁻¹), U (12.5 ng mL⁻¹), and m⁵U (50.0 ng mL⁻¹). This method provides a wide range of linearity, with correlation coefficients greater than 0.99 for all nucleosides. Both the accuracy and precision of this method satisfied criteria of <15% for higher concentrations and <20% for the lowest concentrations. Conclusions: In this study, we describe a rapid and selective method that uses multi-column liquid chromatography with tandem mass spectrometry (LC-MS/MS) to simultaneously quantify modified nucleosides. This global analysis will be useful for evaluating modifications in RNA.