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

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. 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. 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