目的 采用液相色谱-质谱联用法(LC-MS/MS)测定人血浆中乌苯美司的药物浓度,并应用于健康受试者体内药动学研究。方法 以文拉法辛为内标,血浆样品经甲醇沉淀蛋白后,以甲醇-醋酸铵(2 mmol·L^-1,含 0.2% 甲酸)水溶液(50:50)为流动相,Waters 公司 XTerra^® MS C₁₈(150 mm×4.6 mm,5 μm)色谱柱分离,体积流量为 900 μL·min^-1,每个样品的分析时间为 4.20 min。样品经电喷雾离子源正离子化后,通过三重四极杆串联质谱仪,在多反应监测模式下测定乌苯美司(m/z 309.3→120.2)和内标文拉法辛(m/z 278.2→215.1)的浓度。20 名受试者空腹口服乌苯美司胶囊 20 mg,250 mL温开水送服,于用药前和用药后10、20、30、45 min及1.0、1.5、2.0、3.0、4.0、6.0、8.0、10.0 h由肘静脉取血3 mL,制备血浆,按建立的LC-MS/MS法测定血浆中乌苯美司浓度,计算药动学参数。结果 乌苯美司的血浆质量浓度在1.0~2 500.0 ng·L^-1范围内线性关系良好,定量下限为 1.000 ng·mL^-1,批内、批间精密度(RSD)均在 2.2%~5.1%,相对偏差(RE)在±15% 范围内。乌苯美司血浆样品室温放置 24 h,反复冻融(-20℃)3 次及冰冻(-20℃)保存 50 d 的情况下均稳定。健康受试者空腹口服乌苯美司胶囊20mg后,血浆中乌苯美司的达峰浓度(C_max)为(1 375±298) ng·mL^-1, 药时曲线下面积(AUC_{0~10 h})为(2 106±296) h·ng·mL^-1,AUC_0~∞为(2 116±299) h·ng·mL^-1,半衰期(t_1/2)为(1.38±0.20)h,达峰时间(t_max)为(0.71±0.23) h。结论 建立的LC-MS/MS分析方法简便、选择性高、灵敏度高,可用于受试者空腹口服 20 mg 乌苯美司胶囊后血浆样品中乌苯美司的药动学研究,乌苯美司胶囊口服吸收迅速,0.71 h 后血药浓度可达峰值。

Objective To establish an LC-MS/MS method for the determination of ubenimex, which was used subsequently to investigate the pharmacokinetics of ubenimex in healthy Chinese volunteers. Methods Venlafaxine was used as internal standard. Following a deproteinization procedure, ubenimex and the internal standard venlafaxine were eluted isocratically using a mobile phase containing of methanol-2 mmol·L^-1 ammonium acetate and 0.2% formic acid aqueous solution (50: 50) at a flow rate of 900 μL·min^-1 within 4.20 min. The analysis was carried out by using Waters XTerra^® MS C₁₈ column (150 mm × 4.6 mm, 5 μm). Ubenimex and the internal standard were measured by a triple-quadrupole mass spectrometer in positive electron electronic spray ion (ESI) mode using multiple reaction monitoring (MRM). The extracted ions monitored following MRM transitions were m/z 309.3→ 120.2 for ubenimex and m/z 278.2→215.1 for the internal standard venlafaxine. Twenty subjects took orally 20 mg of Ubenmex Capsule on an fasted state, 250 mL of warm boiled water, and took 3 mL of blood from the elbow vein before administration and 10, 20, 30, 45 minutes and 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 10.0 h after administration, to prepare plasma. The concentration of ubenmex in plasma was determined by the established LC-MS/MS method, and the pharmacokinetic parameters were calculated. Results The calibration curve of ubenimex in human plasma was linear over the concentration rang of (1.0-2 500.0) ng·mL^-1. The lower limit of quantitation was 1.000 ng·mL^-1. The intra-and inter-run precisions at three quality control levels were within 2.2%-5.1%, the relative deviation of the assay was within -15.0%-15.0%. The plasma samples were stable at room temperature for 24 h, at -20℃ for 50 days and during three freeze-thaw cycles. After the healthy subjects took Ubenimex Capsule 20 mg on an fasted state, the main pharmacokinetic parameters of ubenimex were as follows: C_max was (1 375 ± 298) ng·mL^-1, AUC_0-10h was (2 106 ± 296) h·ng·mL^-1, AUC_0-∞ was (2 116 ± 299) ng·h·mL^-1, t_1/2 was (1.38 ± 0.20) h, t_max was (0.71 ± 0.23) h. Conclusion The method was proved to be convenient, accurate and sensitive. The method was proved to be suitable for the pharmacokinetics of ubenimex in healthy Chinese volunteers after a single oral dose of 20 mg Ubenimex Capsule. Ubenimex Capsule were rapidly absorbed by oral administration, and the blood drug concentration reached the peak after 0.71 hours.

R285.61

苏州市药学会-江苏恒瑞临床药学科研基金项目(Syhky201807)