摘要
目的:建立同时测定人尿液中新蛭素及其活性代谢产物水蛭素的超高效液相色谱-质谱联用(UPLC-MS/MS)方法,用于注射用重组新蛭素Ⅰ期临床试验健康受试者尿液的药动学研究。方法:采用新蛭素的类似肽MEH作为内标,50μL尿液样品加入等体积的50μL空白人血清经300μL甲醇沉淀蛋白后,通过Waters BEH300 C18色谱柱(300,2.1 mm×50 mm,1.7μm)分离,柱温为40℃,流动相为水(含0.1%甲酸)-乙腈(含0.1%甲酸),梯度洗脱,流速为0.3 mL·min-1,色谱运行时间为5 min,进样体积为5μL。采用电喷雾电离源正离子多反应监测检测。新蛭素,水蛭素和内标的监测反应质荷比(m/z)分别为1 042.4→1 280.4,1 152.1→1 352.8,1 063.3→1 332.1。对该方法的选择性与残留、标准曲线与定量下限、准确度与精密度、基质效应与回收率、稀释可靠性与稳定性进行了全面的验证。结果:新蛭素和水蛭素的线性范围均为10~2 000 ng·mL^-1,定量下限均为10 ng·mL^-1,新蛭素和水蛭素的批内和批间精密度为5.4%~13.9%和3.7%~12.2%,准确度为-6.6%^-0.2%;残留、基质效应与回收率、稀释效应与稳定性均在可接受的范围内。结论:该方法符合生物分析指导原则的要求,满足注射用重组新蛭素临床Ⅰ期药动学的尿液排泄研究。
Objective: To develop and validate a simple and practical ultra-performance liquid chromatography/tandem mass spectrometry(UPLC-MS/MS) method for simultaneous quantifying neorudin(EH)and its active metabolite hirudin variant 2-Lys47(HV2) in human urine,which will be applied to the clinical pharmacokinetic study of EH. Methods: MEH, an analog peptide of EH, was used as internal standard(IS). After the 50 μL urinary samples spiked with 50 μL human serum were extracted with 300 μL methanol,the analytes and internal standard were separated on a Waters BEH300 C18 column(300 ,2.1 mm×50 mm,1.7 μm)using a gradient elution procedure with mobile phase consisting of acetonitrile(containing 0. 1% formic acid) and water(containing 0. 1% formic acid) at a flow rate of 0. 3 mL·min-1. The injection volume was 5 μL and total run time was 5 min. Positive electrospray ionization was performed and multiple reaction monitoring(MRM) was conducted with transitions of m/z 1 042.4→1 280.4 for EH,m/z 1 152.1→1 352.8 for HV2,and m/z 1 063.3→1 332.1 for IS. This method was fully validated. Results: The within and between-run precision RSDs were in the range of 5. 4% ~ 13. 9% for EH and 3. 7% ~ 12. 2% for HV2,and the accuracy of both EH and HV2 was between-6.6% and -0. 2%. The extraction recoveries and matrix effect at three quality control(QC) levels for EH and HV2 were satisfactory. The stability of EH and HV2 during storage,preparation,and analysis was confirmed,and the carryover was also proved to be acceptable. Conclusion: This method was successfully applied to the urinary excretion study in Phase I clinical pharmacokinetic trials of EH following intravenous administration of 7. 6 mg·kg-1 to healthy volunteers.
作者
董晓娜
孟志云
顾若兰
朱晓霞
甘慧
靳继德
窦桂芳
DONG Xiao-na;MENG Zhi-yun;GU Ruo-lan;ZHU Xiao-xia;GAN Hui;JIN Ji-de;DOU Gui-fang(School of Basic Medical Sciences,Peking University,Beijing 100191,China;Institute of Radiation Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)
出处
《中国新药杂志》
CAS
CSCD
北大核心
2020年第4期427-436,共10页
Chinese Journal of New Drugs
基金
国家“重大新药创制”科技重大专项资助项目(2012ZX09102301-008).
