An ion-pairing reversed-phase liquid chromatography-mass spectrometry (IP-RP-LC-MS) was developed for the determination of nucleotides, nucleosides and their transformation products in Cordyceps. Perfluorinated carboxylic acid, namely pentadecafluorooctanoic acid (PDFOA, 0.25. mM), was used as volatile ion-paring agent and a reversed-phase column (Agilent ZORBAX SB-Aq column) was used for the separation of three nucleotides namely uridine-5'-monophosphate (UMP, 0.638-10.200μg/mL), adenosine-5'-monophosphate (AMP, 0.24-7.80μg/mL) and guanosine-5'-monophosphate (GMP, 0.42-13.50μg/mL), seven nucleosides including adenosine (0.55-8.85μg/mL), guanosine (0.42-6.75μg/mL), uridine (0.33-10.50μg/mL), inosine (0.21-6.60μg/mL), cytidine (0.48-15.30μg/mL), thymidine (0.20-6.30μg/mL) and cordycepin (0.09-1.50μg/mL), as well as six nucleobases, adenine (0.22-6.90μg/mL), guanine (0.26-4.20μg/mL), uracil (0.38-12.15μg/mL), hypoxanthine (0.13-4.20μg/mL), cytosine (0.39-12.45μg/mL) and thymine (0.26-8.25μg/mL) with 5-chlorocytosine arabinoside as the internal standard. The overall LODs and LOQs were between 0.01-0.16μg/mL and 0.04-0.41μg/mL for the 16 analytes, respectively. The contents of 16 investigated compounds in natural and cultured Cordyceps were also determined and compared after validation of the developed IP-RP-LC-MS method. The transformations of nucleotides and nucleosides in Cordyceps were evaluated based on the quantification of the investigated compounds in three extracts, including boiling water extraction (BWE), 24. h ambient temperature water immersion (ATWE) and 56. h ATWE extracts. Two transformation pathways including UMP → uridine → uracil and GMP → guanosine → guanine were proposed in both natural Cordyceps sinensis and cultured Cordyceps militaris. The pathway of AMP → adenosine → inosine → hypoxanthine was proposed in natural C. sinensis, while AMP → adenosine → adenine in cultured C. militaris. However, the transformation of nucleotides and nucleosides was not found in commercial cultured C. sinensis. © 2010 Elsevier B.V.