高效液相色谱串联质谱法测定人尿液中左旋苯环壬酯及其代谢物去甲基左旋苯环壬酯的浓度

Determination of levophencynonate and its metabolite demethyl levophencynonate in human urine by HPLC-MS/MS

目的:建立高效液相色谱串联质谱法(HPLC-MS/MS法)同时测定人尿液中左旋苯环壬酯及其代谢物去甲基左旋苯环壬酯的浓度。方法:尿液样品利用自动化固相萃取仪进行处理,提取液直接进样,使用HPLC-MS/MS测定。采用SHIMADZU Shim-Pak XR C8(2.1 mm×50 mm,2.2μm)色谱柱,乙腈-0.5%甲酸水溶液为流动相以,梯度浇脱,流速0.4 mL·min-1,柱温40℃,进样量20μL。电喷雾离子化(ESI),正离子模式多重反应选择离子检测(MRM),左旋苯环壬酯、去甲基左旋苯环壬酯及内标d3-左旋苯环壬酯的检测离子对分别为:m/z 358.2→156.2、m/z 344.2→142.2和m/z 361.0→159.0。结果:尿液中左旋苯环壬酯和去甲基左旋苯环壬酯线性范围分别为10~4000 pg·m L^(-1)和25~8000 pg·m L^(-1),标准曲线的相关系数(r2)均大于0.99。批内、批间精密度及准确度的相对标准差和相对误差均小于15%,尿液基质对左旋苯环壬酯和去甲基左旋苯环壬酯测定无干扰。结论:本研究建立HPLC-MS/MS法同时定量测定人尿液中的左旋苯环壬酯和去甲基左旋苯环壬酯,灵敏度高,特异性强,耗时短,能够快速测定尿液样品,提供准确可靠的分析结果,已成功应用于临床样品的分析。临床研究结果显示,中国健康受试者单次服用1 mg和2 mg左旋盐酸苯环壬酯片后,尿液中左旋苯环壬酯的48 h累积排泄量分别为(0.185±0.150)μg和(0.598±0.352)μg。

Objective:To establish a method for simultaneous determination of levophencynonate and its metabolite demethyl levophencynonate in human urine by HPLC-MS/MS.Methods:The urine sample was extracted by using solid phase extraction.The extract was injected directly and was analyzed by the HPLC-MS/MS under the following conditions:Chromatographic column was SHIMADZU Shim-Pak XR C8(2.1 mm×50 mm,2.2μm),the mobile phase was acetonitrile-0.5%formic acid,with a flow rate of 0.4 mL·min-1 and column temperature at 40℃,and injected sample volume of 20μL.The results of levophencynonate,demethyl levophencynonate and their internal standard d3-levophencynonate by positive ion MRM detection were:m/z 358.4→156.4,344.5→142.2 and 361.0→159.0,respectively.Results:The linear ranges of levophencynonate and demethyl levophencynonate in urine were 10-4000 pg·mL^(-1)and 25-8000 pg·mL^(-1),respectively.The correlation coefficients(r2)of the standard curves were both greater than 0.99.The relative standard deviation and relative error of intra-batch and inter-batch precision and accuracy were all less than 15%.The urine matrix had no effect on the quantification of levophencynonate and demethyl-levophencynonate.Conclusion:The HPLC-MS/MS method established in this study is fully validated and proved to be rapid,sensitive and accurate for the simultaneous determination of levophencynonate and demethyl levophencynonate in human urine and applied to the analysis of clinical samples successfully.The results showed that the cumulative 48 h urinary excretion of levorphanolone in Chinese healthy subjects was(0.185±0.150)μg and(0.598±0.352)μg after a single dose of 1 mg and 2 mg levophencynonate hydrochloride tablets,respectively.