Oxidized nucleosides are biochemical markers for tumors, aging, and neurodegenerative diseases. However, during the last decade, the analytical methods for nucleosides by gas chromatography/ mass spectrometry (GC/MS) and high-performance liquid chromatography (HPLC) with singleparameter detectors like electron-capture detection (ECD) have not been sufficiently rapid or reliable to detect nucleosides in urine and to analyze clinical samples. It has been reported (Dudley et al., Rapid Commun Mass Spectrom. 2000; 14: 1200) that liquid chromatography with electrospray mass spectrometry (LC/ESI-MS) is more specific and sensitive for analysis of nucleosides than HPLC with conventional detectors; however, this method required complex extraction steps. In the present work a direct LC/ESI-MS method for nucleosides without extraction of urine samples has been developed. Analysis of nucleosides using positive-ion mode with selected reaction monitoring effectively eliminated potential interferences from endogenous constituents of the urine. This highly selective and sensitive method made it possible to analyze urinary nucleosides with a lower limit of quantitation of 0.2 nmol/mL. The method has been validated, with both excellent linearity and reproducibility, in the calibration range from 0.2-400 nmol/mL. The correlation coefficients of the calibration curves were higher than 0.987. The coefficients of variation were in the range 0.03-14.92% (inter-day) and 0.54-14.39% (intra-day), respectively. Copyright # 2004 John Wiley & Sons, Ltd.Oxidized nucleosides represent excellent biomarkers for determining the extent of damage in genetic material, which has long been of interest in understanding the mechanism of aging, neurodegenerative diseases, and carcinogenesis. [1][2][3][4][5] Various methods have been developed for quantifying modified nucleosides, based on an enzyme-linked immunosorbent assay (ELISA), 6,7 32 P-post-labelling, 8 capillary electrophoresis, 9,10 HPLC with photodiode array detection 11,12 or ECD, 13 and GC/MS. 14-16 However, GC/MS analysis faced additional problems in the hydrolysis and derivatization steps. 17 HPLC/ECD analysis has limitations in that the requirement for electroactive components meant that the method was mainly applicable to the detection of only 8-oxodeoxyguanosine. 18 Due to their chemical characteristics in HPLC analysis, various kinds of nucleosides could not be analyzed using just one kind of detector.To overcome these problems, in the present work, we developed an analytical method for determining many kinds of nucleosides at one time using LC/MS with ESI (electrospray ionization) detection. We confirmed that mass spectrometric detection with ESI is more specific and sensitive than any other HPLC detection method. We also developed a direct analytical method for nucleosides without extracting urine samples. This approach is both more convenient and more efficient in conducting studies with large sample numbers, e.g., a clinical study, than analytical methods involving complex extrac...
