Study On The Chromatographic Analysis Of Active Ingredients From Ginkgo Biloba

Ginkgo biloba extract(EGB761)had been widely used in clinical practice.In this paper,the chromatographic analysis methods of the active substances such as ginkgolic acid,lactone and biflavonoid were studied.(1)Ai ming at the problem that the sensitivity of the pharmacopoeia detection method of ginkgolic acid was low and the interference of the matrix was obvious.The dispersed liquid phase microextraction-high performance liquid chromatography method was established for detection of ginkgolic acid from ginkgo biloba extract.The low concentration of ginkgolic acid in the sample could be quickly and efficiently enriched in the organic phase by dispersing the liquid phase microextraction,and the coexisting strong polar substances were allowed to enter the aqueous phase,thus reducing the matrix interference.The results showed that a 1.00 mL sample solution was mixed with 200 ?L of chloroform and dispersed in 10.0 mL of water,the extraction equilibrium was achieved within 30 s,and ginkgolic acid could be enriched more than 7 times,while the concentration of matrix impurities significantly less than before extraction.The chromatographic conditions were as follows: the C-18 column was eluted with isocratic solution containing 0.01 mol/L trifluoroacetic acid in methanol-water(92: 8)solution,and the ginger apricot was separated at a column temperature of 40 °C.The 240 nm wavelength detection had the highest signal to noise ratio.Through methodological validation,this method had a good linear relationship in the concentration range of 0.1~5 mg/L,the detection limit was 0.51~1.08 mg/kg and the repeatability of 3.4%~8.1%,spiked recovery rate was carried out with 90%~103%.After compared with the method of European Pharmacopoeia EP8.0,the results were consistent,and the accuracy was higher.(2)Ai ming at the problem that the use of tetrahydrofuran-water-n-propanol mobile phase was easy to produce peroxides by pharmacopoeia method,a method of methanol-water system as mobile phase was proposed.The results showed that from the 20% methanol aqueous solution to 60% methanol aqueous solution for gradient elution,the five lactone components and matrix impurities in the sample could be effectively separated.And the column efficiency was better than tetrahydrofuran-water-n-propanol mobile phase system,the system adaptability was better.Verified by methodology,this method had the advantages of good linearity in the range of 50 ~ 3000 mg/L,and the detection limit was 0.507~1.003 g/kg.It could meet the requirements of pharmacopoeia detection,reproducibility between 2.1% to 9.8%,plus the standard recovery rate of 96% to 106% between.After compared experiment with the method of Chinese Pharmacopoeia 2015 version,the results were consistent.(3)In view of the lack of corresponding standard samples in the research process of ginkgo biloba biflavonoids,the biflavonoids in the crude extract of Ginkgo biloba were isolated,enriched and separated by preparative chromatography.The chromatographic conditions were as follows: the C-18 column was gradiently eluted with methanol-water solution containing 0.01 mol/L acetic acid,the wavelength was 330 nm.Three biflavonoid components were obtained.It was identified by infrared spectroscopy,UV spectroscopy,hydrogen nuclear magnetic resonance and high resolution mass spectrometry.The biflavonoids corresponding to the three components were bilobetin,ginkgelin(isomeric mixture)and sciadopitysin.The molecular weights were 552,566 and 580 respectively.The purity of those three kinds of biflavone were 90.5%,95.7%,93.9%.