The analysis system for 50-nucleotides was the same as that for
free amino acids determination. The assay was performed on a
Zorbax Eclipse XDB C18 column (250 4.6 mm, 5 lm, Agilent),
and the mobile phase was distilled water/methanol/acetic acid/
tetrabutylammonium hydroxide (894.5/100/5/0.5, v/v/v/v) with
injection volume of 20 lL at a flow rate of 0.7 mL/min, and the
50-nucleotides were detected by UV at 254 nm. Each 50-nucleotide
was identified using the authentic 50-nucleotide (Aladdin Reagent
(Shanghai) Co., Ltd, Shanghai, China) and quantified by the
calibration curve of the authentic compound relative to external
standards. The HPLC chromatograms of 50-nucleotide from fresh
button mushroom are shown in Fig. 1C.