Abstract: A new high performance liquid chromatography/mass spectrometric method for the analysis of 5’-nucleotide in lentinus edodes was reported. 5’-nucleotide could be separated on a ODS column by using a mobile phase containing 0.05% phosphoric acid water-methanol (90/10,V/V). The 5’-nucleotide in lentinus edodes were identified as 5’-inosine monophosphate, 5’-guanosine monophosphate, 5’-uridine monophosphate and 5’-adenosine monophosphate by using HPLC and LC/MS. The content of four 5’-nucleotides in lentinus edodes was determined by external standard method. Their recoveries were respectively 100.4%, 99.0%, 97.8%, 97.2%, their relative standard deviation were respectively 2.07%, 2.18%, 1.91%, 2.76% and their detection were respectively 2.5, 2.1, 3.2, 3.5μg/ml. The method was accurate, simple and easy.

Key words: high performance liquid chromatography/mass spectrometry, 5’-nucleotide, lentinus edodes