Qualitative and quantitative analysis of nucleosides and nucleobases in Phylloporia ribis by UPLC-Q-TOF-MS and HPLC

Abstract

The dried fruit body of Phylloporia ribis(Hymenochaetaceae), which prefers to live on the stumps of Lonicera japonica(Caprifoliaceae), has a variety of activities, whereas its pharmacodynamic material basis is not completely clear and there are few reports on its quality control and evaluation. In this study, an UPLC-Q-TOF-MS method was used to analyze the nucleosides and nucleobases in P. ribis and a HPLC method was established for simultaneous determination of 10 nucleosides and nucleobases. MS and MS/MS data were acquired in positive ion mode. Based on the data comparison of the sample and the reference substance, the literature data and the compound databases of ChemSpider and PubChem, 18 nucleosides and nucleobases were identified qualitatively from the water extract of P. ribis for the first time. After optimization, the HPLC was performed using a Welch Ultimate AQ C_(18) column(4.6 mm×250 mm, 5 μm) by gradient elution with acetonitrile and water as mobile phase, the flow rate of 1.0 mL·min~(-1), the detection wavelength of 260 nm, and the column temperature of 30 ℃. Through the investigation of the extraction method, solvent and time, it was determined that the test solution should be obtained by cold water extraction for 18 h. At the present HPLC conditions, 10 components of uracil, cytidine, hypoxanthine, uridine, thymine, inosine, guanosine, 2'-deoxyinosine, 2'-deoxyguanosine and thymidine could be well separated(R > 1.5) and showed good linearity(r > 0.999 9) in the concentration ranges of 0.247-24.7, 0.283-28.3, 0.273-27.3, 0.256-25.6, 0.257-25.7, 0.318-31.8, 0.245-24.5, 0.267-26.7, 0.250-25.0 and 0.267-26.7 mg·L~(-1), respectively. The average reco-veries of 10 components were 95.78%-104.5%, and the RSDs were 2.2%-5.2%(n=6). The contents of 10 nucleosides and nucleobases in different samples of P. ribis varied greatly, which were 0.021-0.122, 0.004-0.029, 0.014-0.226, 0.009-0.442, 0.003-0.014, 0.002-0.146, 0.007-0.098, 0-0.054, 0.005-0.069, 0.004-0.081 and 0.072-1.28 mg·g~(-1) for uracil, cytidine, hypoxanthine, uridine, thymine, inosine, guanosine, 2'-deoxyinosine, 2'-deoxyguanosine, thymidine and total 10 components, respectively. These results demonstrated that the components had significant differences in the internal quality, and good quality control was needed to ensure the medical efficacy. This study provides a scientific basis for the discovery of pharmacodynamic ingredients, quality control and evaluation of P. ribis.