Cordyceps sinensis has been widely used as traditional Chinese medicines (TCM) in China.To date,nucleosides are believed to be the bioactive components in Cordyceps.Therefore,a specific and sensitive analysis method of these compounds is essential for the quality control of Cordyceps.Hydrophilic interaction liquid chromatography (HILIC) was proposed for the separation of highly hydrophilic compounds by Alpert in 1990,which was characterized by polar stationary phases and aqueous-organic mobile phases.Tandem mass spectrometry could provides low limits of detection and high selectivity capabilities,so the HILIC-ESI-MS/MS method would be more suitable for the quantitative analysis of high polar compound in complex matrix.In this study,a hydrophilic interaction chromatography (HILIC) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) method was developed for the identification and quantification of 15 nucleosides and nucleobases namely thymine,uracil,thymidine,2-deoxyuridine,cordycepin,uridine,hypoxanthine,adenine,adenosine,xanthine,inosine,cytosine,guanine,cytidine,guanosine in natural Cordyceps.The separation was achieved on a Waters Xbridge Amide column (250×4.6 mm I.D.,3.5 μm) and gradient elution using a solution of acetonitrile and buffer (0.20% acetic acid and 10 mmol/L ammonium acetate) as the mobile phase at a flow rate of 0.8 mL/min.Identification of the 16 target nucleosides and nucleobases was based on retention time,UV spectra,mass measurements of the protonated molecules ([M+H]+) and main fragment ions (ESI-MS/MS).Quantitative analysis of investigated compounds were performed using time programmed selective ion monitoring (SIM) or multiple reaction monitoring (MRM) in positive ion mode under optimized mass conditions.All calibration curves showed good linearity (r2 > 0.9951) within the test ranges.The overall intra- and inter-day RSD ranged from 2.45 to 5.23% and from 3.29 to 6.73%,respectively.The LOD were in the range of 0.15-10.00 ng/mL,respectively.The recoveries of the samples were in the range of 83.6–117.3% for Cordyceps.The developed method was applied successfully for the analysis of nucleosides and nucleobases in 12 batches of natural Cordyceps samples collected from different regions in China.The results showed that almost all of these Cordyceps samples were rich in the nucleosides and nucleobases.Total contents of all the 15 nucleosides and nucleobases ranged from 388.43 to 1622.84 μg/g.The developed HILIC-ESI-MS/MS method had advantages as a useful tool for the study of the highly polar bioactive components in Cordyceps as well as their quality control.