目的 建立HPLC-荧光检测法来同时测定大鼠血浆中伊伐布雷定及其活性代谢物N-去甲基伊伐布雷定的浓度，研究单剂量口服伊伐布雷定在大鼠体内的药动学。方法 以盐酸曲马多为内标，采用C₂柱固相萃取法处理血浆。色谱条件：色谱柱： Kromasil-C₁₈色谱柱（4.6 mm×250 mm，5 μm）；流动相：乙腈-10 mmol·L-1的磷酸二氢钾溶液（含0.1%的1.2 mol·L-1盐酸）（22∶78）；流速：1.8 mL· min-1；柱温：25 ℃；荧光检测波长：激发波长λ_ex=283 nm，发射波长λ_em=328 nm。大鼠灌胃给予伊伐布雷定水溶液1.5 mg·kg-1后，按规定时间点取血并测定血浆中的伊伐布雷定及代谢物N-去甲基伊伐布雷定的浓度，绘制药-时曲线，采用DAS 2.0软件计算药动学参数。结果 伊伐布雷定及其活性代谢物N-去甲基伊伐布雷定的血药浓度线性范围分别为0.5～80，0.8～100 μg·L-1；相关系数（r）均大于0.999，最低定量限分别为0.5和0.8 μg·L-1；方法回收率分别为96.67%～103.47%，90.5%～101.25%；批内精密度分别为9.34%～13.73%，9.14%～12.35%；批间精密度分别为5.9%～10.42%，4.18%～11.39%。伊伐布雷定及其活性代谢物N-去甲基伊伐布雷定在大鼠体内的血药浓度-时间曲线符合二室模型，其主要药动学参数：t_1/2分别为（2.84±1.43）和（5.732±2.89） h，ρ_max分别为（217.83±86.04）和（9.76±2.79）μg·L-1，t_max分别为（0.57±0.16）和（0.54±0.13）h，AUC_0-t分别为（534.46±179.20）和（25.93±4.34）μg·h·L-1。结论 本实验所建立的同时测定血浆中伊伐布雷定及其活性代谢物N-去甲基伊伐布雷定的HPLC-固相萃取-荧光测定的方法简便、快速、灵敏、准确、可靠，能够满足血药浓度和药动学研究的需要。

OBJECTIVE To develop a HPLC method with fluorescence detection for the simultaneous determination of ivabradine and its active metabolite，N-desmethylivabradine，in rat plasma， and to investigate their pharmacokinetic behaviors after single oral dose in rat. METHODS Tramadol hydrochloride was used as internal standard, and the drugs were extracted from plasma by SPE method with C₂ column. Separation was achieved on a C₁₈ reversed-phase analytical column（4.6 mm×250 mm，5 μm）at 25 ℃.The mobile phase consisted of acetonitrile-10 mmol·L-1 KH₂PO₄ （containing 0.1% 1.2 mol·L-1 hydrochloride） （22∶78），and delivered at 1.8 mL· min-1. The excitation and emission wavelengths were 283 and 328 nm，respectively. The rats were given ivabradine solution at 1.5 mg·kg-1 via ig. The plasma concentrations of ivabradine and its metabolite were determined， and their pharmacokinetic parameters were processed by DAS 2.0 pharmacokinetic software. RESULTS A good linearity was obtained over the range of 0.5-80 μg·L-1 for ivabradine and 0.8-100 μg·L-1 for its metabolite N-desmethylivabradine with both the correlation coefficient （r）﹥0.999. The lower limits of quantitation of ivabradine and its metabolite were 0.5 and 0.8 μg·L-1，respectively. The recoveries of method were 96.67%-103.47% for ivabradine and 90.5%-101.25% for N-desmethylivabradine. The precisions of intra-batch were 9.34%-13.73% and 9.14%-12.35%，respectively. The precisions of inter-batch were 5.9%-10.42% and 4.18%-11.39%，respectively. The RESULTS indicated that the in vivo kinetic processes of ivabradine and N-desmethylivabradine in rats were all fitted to a two-compartment model. The main pharmacokinetic parameters of ivabradine and N-desmethylivabradine were as follows： t_1/2（2.84±1.43）and（5.732±2.89）h，ρ_max（217.83±86.04）and（9.76±2.79）μg·L-1，t_max（0.57±0.16）and（0.54±0.13）h ，AUC_0-t（534.46±179.20）and（25.93±4.34）μg·h·L-1， respectively. CONCLUSION The established method is proved to be simple，sensitive，exclusive and reliable，which meets the requirements of the plasma concentration determination and pharmacokinetic study.