RP-HPLC同时测定William＇s E培养基中地塞米松、睾酮和6β羟基睾酮的含量

Simultaneous Determination of Dexamethasone,Testosterone and 6β-OH Testosterone in William's Medium E

目的建立同时测定William′sE培养基中地塞米松、睾酮和6β羟基睾酮含量的RP-HPLC检测方法,并用该方法研究地塞米松对大鼠原代肝细胞CYP3A的诱导作用。方法采用Agilent1100HPLC系统,Phenomenex色谱柱,以乙腈和磷酸溶液组成的流动相梯度洗脱分离,以酮康唑为内标,流速1.00mL.min-1,检测波长245nm,柱温20℃。结果地塞米松、睾酮和6β羟基睾酮分别在53～19291,161～29318和58～21091μg.L-1内呈线性关系;它们的日间和日内精密度均低于15%;地塞米松、睾酮和6β羟基睾酮的最小定量限分别为53.00,161.00和58.00μg.L-1。结论该方法简便、准确、可靠,可用于地塞米松作用于大鼠原代肝细胞体系后,地塞米松、CYP3A底物睾酮及其代谢产物6β羟基睾酮在William′sE培养基中的浓度测定。

OBJECTIVE To establish a RP-HPLC method with UV detection for the simultaneous determination of dexamethasone,testosterone and 6β-OH testosterone in William＇s Medium E,and to investigate in vitro induction of CYP3A by dexamethasone in primary rat hepatocyte culture. METHODS Aglient 1100 HPLC system was used in the drug analysis. The samples were separated on a reversed-phase HPLC column （phenomenex） with the mobile phase consisting of acetonitrile and 0. 01 mol·L^-1 phosphoric acid solution,using a linear gradient elution program. Ketoconazole was used as internal standard. The flow rate was set at 1.00 mL·min^-1. The detection wavelength was at 245 nm and the column temperature was 20℃. RESULTS Dexamethasone,testosterone and 6β-OH testosterone had good linear relationship over the range of 53～19 291,161～29 318 and 58～21 091 μg·L^-1,respectively. The intra-day and inter-day RSDs were less than 15%. The LOQ in William＇s Medium E of dexamethasone, testosterone and 6β-OH testosterone were 53.00,161.00 and 58. 00μg·L^-1, respectively. CONCLUSION The developed method is sample, accurate and reliable for determining dexamethasone ,testosterone and 6β-OH testosterone simultaneously in William＇s Medium E after induction of CYP3A by dexamethasone in primacy rat hepatocyte culture.