反相HPLC法测定大鼠血浆中17-烯丙胺基-17-脱甲基格尔德霉素及其代谢产物17-氨基-17-脱甲基格尔德霉素

RP-HPLC Measurement of 17-Allylamino-17-Demethoxygeldanamycin and Its Active Metabolite 7-Amino-17-Demethoxygeldanamycin in Rat Plasma

目的:建立大鼠血浆中17-烯丙胺基-17-脱甲基格尔德雷素(17AAG)和17-氨基-17-脱甲基格尔德雷素(17AG)的反相高效液相色谱分析方法,用于17AAG 及其主要代谢产物17AG 的临床前药代动力学研究。方法:以α-萘酚为内标,用乙酸乙酯将17AAG 和17AG 从大鼠血浆中萃取分离吹干后,流动相复溶,HPLC 分析。色谱柱为日产迪玛 Inertsil-ODS 3色谱柱(250 mm×4.6 mm,5 μm),流动相为乙腈-25 mmol·L^(-1)磷酸盐缓冲液(含三乙胺10 mmol·L^(-1))(55:45,pH=3.0),流速1 mL·min^(-1),紫外检测波长313 nm。结果:17AAG、17AG 及内标α-萘酚与内源性干扰分离良好,17AAG 和17AG 在10.0-16000.0μg·L^(-1)浓度范围内呈良好线性关系,相关系数分别为0.9992和0.9987,平均萃取回收率均大于80%,内标回收率为85%。2种组分的最低定量限为10μg·L^(-1)。17AAG 日内 RSD 为2.9%～11.3%,日间 RSD 为6.5%～18.5%;17AG 日内RSD 为3.0%～3.8%,日间 RSD 为5.9%～16.4%。大鼠舌下静脉推注17AAG 15 mg·kg^(-1)后,其主要药动学参数为:CL=21.14 mL·min^(-1)·kg^(-1),AUC=206.5μg·min·mL^(-1),t_(1/2α)=24.82 min,t_(1/2β)=116.20 min,V\-c=1046.3 mL·kg^(-1)。结论:本法可满足17AAG 临床前药代动力学研究的要求。

Objective： To establish a sensitive RP- HPLC method for determining the concentration of 17 -allyl-amino - 17 - demethoxygeldanamycin （17AAG） and 17 - amino - 17 - demethoxygeldanamycin （17AG） in rat plasma. Methods： The plasma samples were extracted with ethyl acetate. The separation was performed on inertsil - ODS 3 column （4. 6 mm × 250 mm ,5 μm）and detected at UV wavelength of 313 nm. A mobile phase of 45 % （v/ v） 25 mmol · L^-1 sodium phosphate （pH 3.00）with 10 mmol · L^-1 triethylamine and 55% acetonitrile was run at a flow rate of 1 mL · min^ -1, α - Naphthoflavone as internal standard. Results： The linear ranges of 17AAG and 17AG were each 10. 0 ～ 16000. 0 μg · L^-1. The correlation coefficients of 17AAG and 17AG were 0. 9992 and 0. 9987. The lower limit of quantification was 10 μg · L^-1. The intra -day RSD were both less than 12% ;the inter -day RSD were both less than 19% and the average recoveries of 17AAG and 17AG were more than 80%. After single dose of 15 mg · kg^-1 in rat, the main pharmacokinetic parameters were estimated to be as follows：CL were 21.14 mL · min^-1 · kg^-1 ;AUC were 206. 5 μg · min ·mL ^-1, t1/2α=24.82min,t1/2β=116.20min,Vc=1046.3mL·kg^-1 , respectively. Conclusion： The established method is accurate and sensitive,It can be applied in the study of preclinical pharmacokinetics.