Capillary Electrophoresis Technology In The Rapid Screening Of Marine Medicinal Organisms Common Methods Of Nucleoside Compounds

Nucleoside compounds include nucleobases, nucleosides, nucleic acids and theirramifications or analogue, which are the foundations of DNA and RNA. Besides, there arelots of free nucleoside compounds in organisms. These compounds exist in terrestrials andin halobios. At present, the obstacles of studying biological active ingredients usingtraditional method of natural products chemistry faced with free nucleoside compoundswere extraction in duplicate, separation, purification and characterization. As a result,there are a lot of same compounds found in different marine medicinal organisms whichconsumes a lot of resources and reduces the efficiency of finding new compounds.Therefore, rapid exclusion of common nucleoside compounds during the process ofscreening active ingredients is imperative. In this study, we developed some new methodsfor rapid separation and screening15common nucleoside compounds, including cytidine,cytosine, adenosine, cordycepin, thymidine, uridine, guanosine,2’-deoxyguanosine,inosine, thymine, adenine, guanine, uracil, hypoxanthine and xanthine, using capillaryzone electrophoresis (CZE), micelle electrokinetic capillary (MEKC) and capillaryelectrophoresis-electrospray ionization time of flight mass spectrometry(CE-ESI/TOFMS), which provided a reference to the activity detection and duplicatesexclusion of this kind of compounds. The content and results of this study are as follows:1. For attaining the best buffer system, the different adaptivities of15nucleosidecompounds were investigated with diode array detector by using volatile salts (sodiumacetate, sodium bicarbonate, ammonium acetate and1,2-diamino-ethane (DEA)) andnonvolatile salts (sodium tetraborate decahydrate and disodium hydrogen phosphate) in anuncoated capillary tube. The results showed that, sodium tetraborate and disodiumhydrogen phosphate buffer system could not separate15nucleoside compoundssimultaneously in CZE mode. The two kinds of nonvolatile salts were more suitable forthe analysis of samples containing a small number of nucleoside compounds. Among thevolatile salts, DEA buffer could separate15nucleoside compounds completely after aseries of optimization. However, the DEA buffer was not stable and had to be replacedafter using several times. In MEKC mode, all15compounds got a better separation when the sodium lauryl sulfate (SDS) was added in the buffer except DEA.2. Instability of the volatile buffer separation system brings out accurate qualitativeand quantitative analysis problems in actual smaples. To solve the problems, disodiumhydrogen phosphate was chosen as buffer to develop a new MEKC method for screening15common nucleoside compounds in marine medicinal organisms. Factors such asmicelle concentration, pH, concentration of buffer, etc. were investigated in order toobtain the optimum conditions for the separation. The optimized separation procedureemployed25mmol/L disodium hydrogen phosphate buffer containing70mmol/L sodiumdodecyl sulfate (SDS) at pH8.7(without adjusted) as background electrolyte,25℃ofcapillary temperature and20kV of separation voltage. UV detection was at260nm andinjection was hydrodynamic at50mbar for3sec. The result showed that all peaks havethe best shape, symmetry and a rapid separation speed. The method validation wasimplemented. Precisions expressed by the R.S.D. values range from1.4%-7.6%. TheR.S.D. values of intra-day were all better than8.2%and inter-day were in the range of1.2%-10.8%. The recovery of15nucleoside compounds was between80.2%and112.9%.The sample was stable in24h.12nucleoside compounds were identified from a kind ofmarine medicinal organism-Anthopleura lanthogrammica Berkly by compared withmixed standard. The method was shown to be stable and reliable, which suiltable for rapidsimultaneous analysis15common nucleoside compounds in complex matrices of marinemedicinal organisms.3. A new capillary electrophoresis-electrospray ionization time of flight massspectrometry (CE-ESI-TOF/MS) method was developed for screening multi-nucleosidecompounds in marine drugs. The separation conditions of capillary electrophoresis anddetection conditions of mass spectrum were optimized and the compounds were separatedin an uncoated silica capillary tube.30mmol/L ammonium acetate containing5%methanol (pH=9.9) was used as background electrolyte. A50%methanol aqueoussolution containing0.25%formic acid was used as sheath liquid with the flow rate of3μL/min. Mass spectrometry analysis was performed in positive mode. At the optimizedcondition, the deviation of accurate mass detection was less than four millionths, which proved that it is reliable to use the accurate molecular weight to screen the nucleosidecompounds. The method validation indicated that all calibration curves showed goodlinearity (R2>0.9933). Meanwhile, the inter-day reproducibility of15nucleosidecompounds was between9.38%and24.34%, which could satisfy the sample analysis inpractice. The developed approach had been successfully applied to the analysis ofnucleoside compounds in Hippocampus and syngnathus. The results showed that thismethod was an effective way to screen multi-nucleoside compound rapidly andsimultaneously in complex matrix of marine drugs.