HPLC-ELSD在肝微粒体中银杏萜内酯的测定及其代谢研究中的应用

 目的为研究银杏萜内酯的代谢作用机制和进一步开发利用,建立其体外代谢的HPLC-ELSD测定法。方法以Dia-monsil^TMODS-C₁₈为色谱柱;甲醇-水(37:63)为流动相,流速1.0mL·min^-1;蒸发光散射检测器(ELSD)检测:漂移管温度60℃,雾化气(N₂)压力0.25MPa。鼠肝微粒体孵育液中银杏萜内酯用乙醚提取,空气流下挥干乙醚,残渣用适量流动相溶解,取40μL进样,测定剩余银杏内酯A,B,C和白果内酯的含量。对鼠肝微粒体孵育液中银杏萜内酯的HPLC-ELSD进行方法学研究,并应用于银杏萜内酯的体外代谢研究。结果白果内酯、银杏内酯A、银杏内酯B和银杏内酯C分别在3.30～133.60,6.8～136.0,5.40～109.0和3.6～72.0mg·L^-1内呈现良好的线性关系,相关系数r分别为0.9936,0.9964,0.9959和0.9930(n=5)。测得定量限分别为3.30,6.80,5.40和3.60mg·L^-1(RSD<15%,n=3);检测限分别为12,32,20和16ng(S/N=3);方法回收率分别为85.9%～102.9%,85.9%～98.3%,88.6%～98.9%和85.4%～101.8%(n=5),日内、日间精密度小于10.5%。孵育液中其他成分不干扰银杏萜内酯的测定。结论本法简便、准确,可用于银杏萜内酯的体外代谢研究。

Studies on determination and in vitro metabolism of ginkgolides and bilobalide in rat liver microsomes by using HPLC-ELSD method

OBJECTIVE To estabolish a HPLC-ELSD method for determining of ginkgolides and bilobalide in rat hepatic micro-somes. METHODS Ginkgolides and bilobalide in rat hepatic microsomal incubates were extracted with diethylether. The determination was performed on a Diamonsil^TM ODS -C₁₈ column (4.6 mm×250 mm, 5μm) with the mobile phase of methalnol-water( 37:63) at a flow rate of 1.0 mL·min^-1 . ELSD was used with the drift tube temperature of 60℃ and the pressure of nebulizing gas (nitrogen)of 0.25 Mpa.RESULTS The calibration curve was in good linearity over the range of 6.80～136.0 mg·L^-1 of ginkgolide A, 5.40～109.0 mg·L^-1 of ginkgolide B, 3.60～72.0 mg·L^-1 of ginkgolide C,3.30- 133.60 mg·L^-1 of bilobalide . The limits of detection were as follows:bilobalide 12 ng, ginkgolide A 32 ng, ginkgolide B 20 ng, ginkgolide C 16 ng(S/N=3) .The limits of quantification were as follows:bilobalide 3.30 mg·L^-1, ginkgolide A 6.80 mg·L^-1 ,ginkgolide B 5.40 mg·L^-1 ,ginkgolide C 3.60 mg·L^-1(RSD<10% ,n = 3 ). The average recoveries and RSDs were in the range of 85.4%～102.9% and 1.2%～10.5%.The method was used to study the in vitro metabolism of ginkgolides in rat liver microsome. CONCLUSION The method is simple, accurate and can be used to study the metabolism of ginkgolides and bilobalide in the rat hepatic microsomes.