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Analysis of nucleosides and monophosphate nucleotides from mushrooms with reversed‐phase HPLC
Author(s) -
Ranogajec Ana,
Beluhan Sunčica,
Šmit Zdenko
Publication year - 2010
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200900516
Subject(s) - chromatography , chemistry , high performance liquid chromatography , nucleotide , ionic strength , reversed phase chromatography , reagent , phosphate , quantitative analysis (chemistry) , phase (matter) , methanol , biochemistry , organic chemistry , aqueous solution , gene
The retention characteristics of five stationary phases were tested by using a selection of 5′‐mononucleotides and nucleosides with the aim to develop a simple, rapid and sensitive reversed‐phase liquid chromatography method without ion‐pair reagent usage. The method was optimized by changes in temperature, pH and ionic strength on a column showing a superior performance. The mobile phase consisted of a mixture of 0.05 M phosphate buffer and methanol, delivered at a flow rate of 0.4 mL/min and based on a gradient program. UV detection was used at a 254 nm wavelength. The method was validated for a quantitative analysis of 5′‐mononucleotides and nucleosides in wild edible mushrooms. For all nucleosides and nucleotides, the LOD and LOQ were less than 0.02 and 0.07 μg/mL, respectively. Validation parameters yielded recovery rates between 68.6 and 98.2%, with a precision expressed as a relative standard deviation of 7.6–15.3%. The content of 5′‐mononucleotides and nucleosides was determined for 10 samples of wild edible mushrooms found in Croatia and, accordingly, the equivalent umami concentrations were calculated.