UPLC-MS/MS法同时检测大鼠体内7-乙基-14-氨基喜树碱及其代谢产物

Simultaneous Determination of 7-ethyl-14-Aminocamptothecin and Metabolite in Rat by UPLC-MS/MS Method

7-乙基-14-氨基喜树碱(EAC)是具有良好抗肿瘤活性的新型喜树碱衍生物,为测定静脉注射EAC后原形化合物及其酰胺型代谢产物(EAC-M)在体内的浓度,揭示体内动力学性质,现研究建立了同时定量测定SD大鼠血浆中EAC和EAC-M的UPLC-MS/MS方法。以7-乙基-14-硝基喜树碱(ENC)、吉咪替康(CMT)为内标,血浆样品加入内标溶液和乙腈沉淀蛋白后,以Phenyl-Hexyl柱(2.1 mm×50 mm,1.8μm)为分离柱,5 mmol/L醋酸铵水溶液(含0.1%甲酸)-乙腈为流动相梯度洗脱,流速0.5 mL/min。洗脱物经电喷雾离子源(ESI)正离子化,在三重四级杆质谱仪上以多反应监测模式(MRM)测定EAC(m/z392→291)和EAC-M(392→290)。方法学研究结果显示,内源性物质不干扰待测物和内标的测定。在0.500~250 ng/mL范围内,EAC和EAC-M的浓度与响应值的线性关系良好,定量下限均为0.500 ng/mL。方法的日内、日间精密度(RSD)≤7.6%,准确度(RE)为-4.2%~5.9%。提取回收率为84.4%~98.7%,基质效应为94.8%~104%。高于定量上限浓度的样品,用空白血浆稀释6倍至线性范围,测得的精密度(RSD)为1.8%,准确度(RE)为-10%^-3.0%。样品经设定的稳定性考察条件后,测得的准确度(RE)为-8.6%~11%,RSD<7.2%。药代动力学结果显示,静脉注射后EAC在大鼠体内的清除速率中等,组织分布高。给药后EAC-M在血浆中迅速检出,暴露量约相当于EAC的5%。连续给药5 d,EAC和EAC-M在大鼠体内无明显蓄积。所建立的方法简便、灵敏、选择性强,适用于SD大鼠血浆中EAC及代谢产物EAC-M的测定和药代动力学研究。

7-ethyl-14-Aminocamptothecin （EAC） is a novel derivate of camptothecin which exhibits favorable antitumor activity.To determine the concentrations of the parent drug and lactam form metabolite （EAC-M） in vivo after intravenous administration of EAC,an UPLC-MS/MS method was established to simultaneously quantify EAC and EAC-M in SD rat plasma.7-ethyl-14-Nitrocamp- tothecin （ENC） and Chimmitecan （CMT） were used as internal standards.After protein precipitation by adding solution of internal standards and acetonitrile,plasma samples were separated on a Phenyl-Hexyl colum （2. lmm-50 rmn, l.8 -tm） with the mobile phase consisting of 0.5 mmoL/L ammonium acetate solution （containing 0.1% formic acid）-acetonitrile.Gradient elution was employed at a flow rate of 0.5 mL/min.Eluted compounds were ionized by an electrospray ionization source （ESI） in positive mode.The parent to production transitions for both the parent drug （m/z 392→291） and the metabolite （m/z 392→290） were monitored on a triple quadrupole mass spectrometer,using the multiple reaction monitoring （MRM）.Based on the results from method validation,no interference was observed in blank plasma samples.The linear ranges for EAC and EAC-M were 0.500 - 250 ng/mL,the limits of quantitation were 0.500 ng/mL.The inter- and intraday precisions （RSD） were not more than 7.6%,the accuracies （RE） were -4.2% - 5.9%.The extraction recovery values were 84.4% - 98.7%,and the matrix effects were 94.8% - 104%.Samples beyond the upper limits of quantification were analyzed after a 6-fold-dilution with blank plasma,the precisions （RSD） were 1.8%,and the accuracies （RE） were -10% - -3.0%.In stability tests,the accuracies （RE） were -8.6% - 11% and the precisions （RSD） were blow 7.2%.Data from pharmacokinetic tests indicated that EAC is moderately cleaned and is widely distributed in tissues.EAC-M was rapidly detected in plasma after intravenous administration which showed a 5% exposure of EAC.No accumula- tion of EAC or EAC-M was observed after an administration for 5 consecutive days.In summary,the provided method is convenient,sensitive,se- lective,and suitable for investigation of EAC and EAC-M in pharmacokinetic research.