Determination of nucleotides, nucleosides and their transformation products in Cordyceps by ion-pairing reversed-phase liquid chromatography-mass spectrometry.

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, 0.25mM), 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, 0.638-10.200microg/mL), adenosine-5'-monophosphate (AMP, 0.24-7.80microg/mL) and guanosine-5'-monophosphate (GMP, 0.42-13.50microg/mL), seven nucleosides including adenosine (0.55-8.85microg/mL), guanosine (0.42-6.75microg/mL), uridine (0.33-10.50micro/mL), inosine (0.21-6.60microg/mL), cytidine (0.48-15.30microg/mL), thymidine (0.20-6.30microg/mL) and cordycepin (0.09-1.50microg/mL), as well as six nucleobases, adenine (0.22-6.90microg/mL), guanine (0.26-4.20microg/mL), uracil (0.38-12.15microg/mL), hypoxanthine (0.13-4.20microg/mL), cytosine (0.39-12.45microg/mL) and thymine (0.26-8.25microg/mL) with 5-chlorocytosine arabinoside as the internal standard. The overall LODs and LOQs were between 0.01-0.16microg/mL and 0.04-0.41microg/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), 24h ambient temperature water immersion (ATWE) and 56h 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. Copyright 2010 Elsevier B.V. All rights reserved.