Purification And Preparation Of Ginkgo Acid Monomers And The Study Of Its Analytical Method

Because ginkgolic acid(GKA) has allergenicity, embryo toxicity, immune toxicity and cell toxicity, so it has been considered as the main side effects constituents in EGb and its preparation. Its concentration has been restricted to 5 ppm and has become a major control target of the quality of Ginkgo. The aim of this subject is to establish purification and preparation methods of high purity GKA monomers and analysis methods of GKA.In this paper, crude GKA extract which was extracted from sarcotestas of Ginkgo biloba was purified by middle pressure silica gel column and the total GKA was obtained. Further the total GKA was separated by preparative high performance liquid chromatography to obtain GKA monomers and their purity and structure had been analysed. Two analysis methods of GKA by UPLC and LC/MS/MS had been discussed.The effect of factors such as solvent, eluent, flow rate and sample amount had been studied to obtain the optimal conditions:using petroleum ether (60～90℃) soak extraction at room temperature and petroleum ether (60～90℃)-ether-formic acid (89:11:1) as eluent, the flow rate was 15.0 mL·min-1 and the sample amount was 10.0 g. Under the conditions the yield after purified by silica gel column was 87.46%.The conditions of preparing GKA monomers were as follows: chromatography column, Waters XBridgeTM C18(19×150 mm,5μm); mobile phase, methanol-1%aqueous acetic acid; flow rate,25 mL-min-1; sample amount,75～85 mg; detection wavelength,310 nm. Under the above conditions, GKA can be separated successfully and the separation time was also reasonable. Six monomers were obtained with cis- and trans- isomers of 6-(8-seventeen carbon alkenyl) salicylic acid separated for the first time and the purity of them were C13:099.65%; C15:1 99.84%; C17:299.78%; C15:0 99.76%; cis-C17:1 99.65%; trans-C17:1 99.75%. Moreover, the structures of the six monomers were identified by UV, IR, MS and NMR to in sure that they were all ginkgo acids.We had preliminarily studied the two analysis methods of GKA by UPLC and LC/MS/MS. The linear and precision of both were good enough for determination of GKA.