离线紫外照射和高效液相荧光法测定他莫昔芬及代谢物在人血浆中的浓度

Determination of Tamoxifen and Two Metabolites in Human Plasma by Offline UV Irradiation and High Performance Liquid Chromatography with Fluorescence

目的建立高效液相荧光法测定人血浆中他莫昔芬及主要代谢物浓度。方法血浆样品经正己烷-正丁醇（98∶2）提取后,以甲醇-1%三乙胺水溶液（82∶18）为流动相,流速为1mL·min-1,色谱柱为Agilent Extend C18（4.6mm×150mm,5μm）,柱温为50℃,离线紫外照射（254nm）10.5min,使目标化合物转变成强荧光性的菲衍生物,荧光检测波长：激发波长（λex）260nm,发射波长（λem）375nm。结果血浆内源性杂质不干扰待测物测定,线性范围分别为：他莫昔芬为0.5~200μg·L-1;N-去甲他莫昔芬为0.5~300μg·L-1;4-羟基他莫昔芬为0.1~10μg·L-1。日内、日间精密度（RSD）均小于10%。样品4次冻融,以及在提取后,4℃下12h内稳定性良好。结论该法灵敏、快速、准确,操作简便、线性范围宽,可用于他莫昔芬的药动学研究及常规治疗药物监测。

OBJECTIVE To establish a high performance liquid chromatography with fluorescence detector for the determination of tamoxifen and two metabolites in human plasma.METHODS Plasma samples were extracted by n-hexane n-butanol（98 ： 2）. Offiine UV wavelength of 254 nm was used to convert the target compounds to highly fluorescence phenanthrene derivates for 10.5 min. Separation was carrid out on the Agilent Extend C18 column （4.6 mm×150 mm, 5 μm） at 50 ℃. The mobile phase consisted of methanol -1% triethylamine （82 ： 18） at a flow rate of 1 mL·min^-1. The fluorescence detector was operated at excitation wavelength of 260 nm and emission wavelength of 375 nm, respectively. RESULTS The intrinsic impurity of plasma did not interfere with the determination. The linear range of tamoxifen was 0.5-200 μg·L^-1. The linear range of N-desmethyltamoxifen was 0.5-300 μg·L^-1. The linear range of 4-hydroxytamoxifen was 0.1-10μg·L^-1. The within-day and between-day precisions were less than 10%. The stability of unprocessed and processed samples stored 12 h at 4 ℃ was good.After 4 cycles of freeze and thaw processes, the plasma samples were stable.CONCLUSION The established HPLC method is rapid, sensitive, accurate and can be applied to the clinical pharmacokinetic research and routine therapeutic drug monitoring.