Abstract: AIMTo establish a simple pre-treated method and high performance liquid chromatographic method for separation and determination of ginkgolic acids in Ginkgo biloba leaves. METHODSThe ginkgolic acids in the German standard sample were identified by LC/DAD/ESI/MS. The methods for pre-treament and high performance liquid chromatography determination of ginkgolic acids were studied. Ginkgo biloba leaves were extracted with n-hexane in Soxhlet apparatus,then concentrated under vacuum. The ginkgolic acids can be determined directly by HPLC after one-pre-purified-step by silica gel column chromatography. The eluant was petroleum ether-diethyl ether-formic acid (89∶11∶1). The chromatographic column was Inertsil ODS-2; the mobile phase was methanol-3% acetic acid (92∶8); the flow rate was 1.0 mL·min^-1; the column temperature was 40℃; the detection wavelength was at 310 nm. RESULTS There were five kinds of ginkgolic acid (C13∶0, C15∶1, C17∶2, C15∶0 and C17∶1) in ginkgo biloba leaves. The relative percentage content of ginkgolic acids C15∶1 and C17∶1 was about 85%. Ginkgolic acid C17∶2 had not been reported in China. The HPLC indicates that there was nearly no impurities except ginkgolic acids after treated by column chromatography. The results showed that the content of ginkgolic acids in the leaves of Ginkgo biloba collected in April, May and June was 1.48%, 1.19% and 1.11% respectively. The average recovery of Ginkgo biloba leaves collected in June was 97.0%,RSD was 1.7% (N=6). CONCLUSIONThe method is accurate,simple and reliable,and can be used for determination of ginkgolic acids in Ginkgo biloba leaves.