Established HPLC-ELSD Analytical Method For Glycosides Of Apostichopus Japonicus And Its Qualitative Identification

Sea cucumber containing multi-bioactive components is a traditional nourishuingfood in China. Sea cucumber saponins which have been reported to possess a widespectrum of pharmacological effects including anti-fungal, anti-bacterial, anti-tumor,hemolysis, cytotoxicity, and immunomodulatory, are the main secondary metabolitesof sea cucumber.This paper is aimed to study the glycosides of Apostichopus japonicus used asfood in northern China. Extraction methods of triterpene glycosides fromApostichopus japonicus have been primary optimized, and a high performance liquidchromatography-evaporative light scattering detector (HPLC-ELSD) method, whichhas been optimized on several relevant conditions, has been set up to analyze theglycosides extracts from Apostichopus japonicus. Furthermore, with the proposedHPLC condition, qualitative identification of glycosides of Apostichopus japonicus iscarried out by HPLC-MS/MS. A method, which is simple, accurate and rapid, hadbeen built to be able to detect quality of Apostichopus japonicus saponins and todistinguish the autheniticity of Apostichopus japonicus products. The results of thispaper provided theoretical foundation and scientific basis for quality discriminationand establishment quality assessment criterion of Apostichopus japonicus, and havethe great significance for Apostichopus japonicus industry developmentand regulating the consumer market of sea cucumber products.1. The four factors, including extraction solvent, ratio of sample to solution,extraction duration and extraction times, were investigated in the orthogonalexperiment to get the best extraction method for glycosides of Apostichopusjaponicus as follows:60%ethanol as extraction solvent, ratio of sample tosolution is1:800, extract5times with3hours each time. An optimized conditionwas obtained by regulating chromatographic column, mobile phase, flow rate,temperature and the parameters of ELSD. Ultimately, the HPLC-ELSD analysis was performed on a Waters Symmetry C18(150mm×4.6mm,5μm) column, at acolumn temperature of40℃and a ananlysis time of60minutes. The gradientelution system consisted of water (containing5mmol/L mammonium acetate) andacetonitrile. The flow-rate was set at0.7mL/min and the sample injection volumewas20μL. The parameters of ELSD: temperature of drift tube was60℃, thepressure of carrier gas was25psi, gain was100. After that the validation of themethod has been done.2. According to the established HPLC chromatographic conditions, the qualitativeidentification of glycosides extracts from Apostichopus japonicus has been gettenby high performance liquid chromatography-electrospray ionization-iontrap-tandem mass spectrometry (HPLC-ESI-IT-MS/MS) and the results showedthat the response signals were better in negative ion mode. The molecular weightis obtained from the [M-H]-/[M+NH4]+ions observed by ESI-IT-MS; Thecollision-induced dissociation data of the [M-H]－ions provided characteristicfragment ions information of triterpene glycosides, and compared with referenceliteratures we got the qualitative identification data of glycosides fromApostichopus japonicus. Its showed that:16chromatographic peaks was certifiedto be glycosides of Apostichopus japonicus in TIC chromatogram with retentiontime in14.0~30.5minutes, and the peak numbers and peak response value on theHPLC and TIC chromatogram are practically agreed. All above make thequalitative identification of glycosides of Apostichopus japonicas possible.