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,2.2 µm）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 20 μL。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 10~4 000 pg·mL-1 和 25~8 000 pg·mL-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,2.2 μm）, 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 10-4 000 pg·mL-1 and 25-8 000 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.
[1] 中国后循环缺血专家共识组. 中国后循环缺血的专家共识[J].中华内科杂志, 2006, 45(9):786
China Posterior Circulation Ischemia Expert Consensus Group. Expert consensus on posterior circulation ischemia in China[J].Chin J Intern Med, 2006, 45(9):786
[2] 刘媛媛, 刘万敏.关于后循环缺血性眩晕的中西医研究进展[J].中西医结合心血管病电子杂志, 2020, 8(17):153
LIU YY, LIU WM.Progress of Chinese and western medicine research on posterior circulation ischemic vertigo[J].Cardiovasc Dis Ele J Integr Tradit Chin Western Med, 2020, 8(17):153
[3] WANG LY, WANG Y, ZHENG JQ, et al. Pharmacological profiles of an anticholinergic agent, phencynonate hydrochloride, and its optical isomers[J].Acta Pharmacol Sin, 2005, 26(5):527
[4] 刘传缋, 恽榴红.中枢解胆碱能抗晕动病新药盐酸苯环壬酯(英文)[J].中国药理学与毒理学杂志, 2005, 19(4):311
LIU CG, YUN LH.A new central anticholinergic anti-motion sickness drug phencynonate hydrochloride[J].Chin J Pharmacol Toxical, 2005, 19(4):311
[5] 刘丽群, 王丽韫.盐酸苯环壬酯对大鼠后循环缺血性眩晕和小鼠位置性眩晕的改善作用[J].中国药理学与毒理学杂志, 2017, 31(3):231
LIU LQ, WANG LY.Improvement of phencynonate hydrochloride on posterior circulation vertigo in rats and positive vertigo in mice[J].Chin J Pharmacol Toxicol, 2017, 31(3):231
[6] XU P, LIU Y, WANG L, et al.Phencynonate S-isomer as a eutomer is a novel central anticholinergic drug for anti-motion sickness[J].Sci Rep, 2019, 9(1):2000
[7] 李更, 王丽韫, 郑建全, 等.盐酸苯环壬酯左旋体及光学异构体抗动物晕动病药效学研究[J].中华航空航天医学杂志, 2006, 17(2):87
LI G, WANG LY, ZHENG JQ, et al.Comparative study on anti-motion sickness efficacy of R-phencynonate hydrochloride and its optical isomers[J].Chin J Aerospace Med, 2006, 17(2):87
[8] 李敬来. 盐酸苯环壬酯左旋体临床前药代动力学研究与系列手性合物的药代特性快速预测[D].北京:中国人民解放军军事医学科学院, 2007
LI JL.Preclinical Pharmacokinetic Study and Rapid Prediction of Pharmacokinetic Properties of Levophencynonate and A Series of Chiral Compounds[D].Beijing:Academy of Military Medical Science of PLA, 2007
[9] KOU Y, XU Y, XUE M, et al.Liquid chromatography-tandem mass spectrometry method for determination of phencynonate in rat blood and urine[J].J Chromatogr B Analyt Technol Biomed Life Sci, 2005, 828(1-2):75
[10] LI J, ZHANG Z, RUAN J, et al.Liquid chromatography-tandem electrospray mass spectrometry method for determination of serial chiral novel anticholinergic compounds of phencynonate in rat plasma[J].J Chromatogr B Analyt Technol Biomed Life Sci, 2007, 855(2):180
[11] KOU Y, LIU Y, XUE M, et al.Comparative pharmacokinetics and distribution kinetics in brain of phencynonate enantiomers in rats[J].Int J Pharm, 2008, 353(1-2):88
[12] 于博, 李敬来, 王晓英, 等.LC-MS/MS 法研究左旋去甲基苯环壬酯在比格犬体内的生物利用度[J].军事医学, 2012, 36(5): 365
YU B, LI JL, WANG XY, et al.Determination of levodesmethyl phencynonate hydrochloride by LC-MS/MS and its bioavailability in Beagle dog[J].Mil Med Sci, 2012, 36(5):365
[13] 曾媛, 郑露露, 熊磊, 等.HPLC-MS/MS 法测定犬体内盐酸苯环壬酯的血药浓度及其药动学研究[J].中国药房, 2015, 26(28): 3912
ZENG Y, ZHENG LL, XIONG L, et al.Plasma concentration of phencynonate hydrochloride in dogs by HPLC-MS/MS and its pharmacokinetic study[J].China Pharm, 2015, 26(28):3912
[14] LI J, LIAO S, WANG X, et al.A rapid and efficient analytical method for the quantification of a novel anticholinergic compound, R-phencynonate, by stable isotope-dilution LC-MS/MS and its application to bioavailability and dose proportionality studies[J].Biomed Chromatogr, 2017, 31(5):e3879
[15] LI B, QI WY, SHI AX, et al.Simultaneous determination of levophencynonate and its metabolite demethyl levophencynonate in human plasma by liquid chromatography tandem mass spectrometry[J].J Chromatogr B Analyt Technol Biomed Life Sci, 2016, 1027(1):221
[16] FDA Guidance.Guidance for Industry:Bioanalytical Method Validation[S].2018:70858
