UPLC-MS/MS法同时检测人尿液中布格呋喃代谢物M1和M2

Simultaneous determination of metabolites M1 and M2 of buagafuran in human urine by UPLC-MS/MS

目的:建立UPLC-MS/MS法同时测定人尿液中布格呋喃2个代谢物M1和M2的浓度。方法:尿液样本用葡萄糖醛酸酶进行水解并稀释。采用Acquity BEH C_(18)(2.1 mm×50 mm,1.7μm)色谱柱,以甲醇-0.1%甲酸水溶液(75∶25)为流动相,流速0.4 m L·min^(-1),柱温35℃,进样量为10μL;采用串联四极杆质谱在ESI正离子电离模式下,应用多反应监测(MRM)扫描模式测定M1(m/z 279.1→243.1)、M2(m/z 277.2→151.2)和内标AF67(m/z 279.1→243.1)。样品分析时间为5 min。结果:M1和M2的质量浓度在10~2 000 ng·m L^(-1)范围内线性关系良好(r>0.99),批内、批间精密度和准确度以及稳定性考察项目的结果均符合要求。在单次和多次口服布格呋喃后,尿液中M1的累积排泄量分别为0.83%和1.18%,M2的累积排泄量分别为0.44%和0.72%。结论:本文建立了同时测定人尿液中布格呋喃2个代谢物(M1和M2)的UPLC-MS/MS方法,灵敏度高,特异性好,各项方法学考核的结果表明,本法可用于布格呋喃人体药代动力学的临床研究。考察了布格呋喃在中国健康人体的物料平衡和安全性等信息,结果显示尿液中M1和M2的累积排泄量很低,需要进一步研究才能获得符合要求的物料平衡数据。另外,多次口服布格呋喃后,代谢物在体内有一定量的蓄积。

Objective：To establish a UPLC-MS/MS method for simultaneous determination of two metabolites of buagafuran in human urine. Method：The urine samples were hydrolyzed and diluted. The UPLC separation was performed on an Acquity BEH C_（18） column（2.1 mm×50 mm,1.7 μm）thermostatted at 35 ℃ using methanol-0.1% formic water solution（75∶25）as mobile phase,and the isocratic flow rate was 0.4 m L·min^（-1）. Detection was performed on a tandem quadrapole mass spectrometer using positive electrospray ionization,and the multiple reactions monitoring（MRM）scan mode was adopted to detect mass transitions of M1（m/z 279.1 → 243.1）,M2（m/z 277.2 → 151.2）and AF67（m/z 279.1 → 243.1）,respectively. Each analytical run was 5 min. Results：The calibration curves were linear over the mass concentration range of 10-2 000 ng·m L^（-1）（r0.99）. The intra and inter batch precision（RSD）and accuracy（RE）and stability results met the acceptance criteria. The cumulative excretions in human urine were 0.83% and 1.18% for M1,0.44% and 0.72% for M2 after single and multiple oral doses of buagafuran,respectively. Conclusion：A sensitive and specific UPLC-MS/MS method was developed for the determination of two metabolites of buagafuran in human urine. The method was validated with respect to specificity,accuracy,precision and stability. This method was successfully applied to pharmacokinetic study of buagafuran to investigate the mass balance and safety of buagafuran after multiple oral doses to healthy Chinese volunteers. The data demonstrated that the cumulative excretions of M1 and M2 were very low,and further study was needed to obtain the satisfactory mass balance data. Furthermore,a certain degree of accumulation of metabolites was detected after multiple doses of buagafuran.