龙胆苦苷在人尿中的含量测定及排泄研究

Determination of gentiopicroside and its excretion in human urine by LC/MS

目的：建立固相萃取液质联用方法测定人尿中龙胆苦苷浓度，研究人体静脉滴注注射用秦龙苦素的主要代谢途径。方法：尿样加入咖啡因内标，经固相萃取后LC—MS／MS分析。色谱备件为：Rescek C8柱（150mm×2．1mm，5μm），流动相为：甲醇-10mmol／L醋酸铵缓冲液-乙腈（50：40：10），流速为0．2mL/min，质谱检测采用多反应离子监测方式。12名健康受试者随机分组交叉静脉滴注80，240，400mg药物，用本法测定各时段尿样的浓度。结果：龙胆苦苷在30～9000ng／mL线性良好（r=0．9980），回收率为91．10％～96．21％，提取回收率为100．52％～103．83％，高、中、低浓度日内和日间变异系数均小于10％；24h内3个剂量下原形药物在尿中累积排泄率分别为（76．59±10．02）％、（71．95±12．12）％、（79．76±8．54）％，累积排泄量与给药剂量呈正比，排泄高峰为0～5．5h。结论：本方法适用于临床药物动力学研究，注射用秦龙苦素在人体内主要是以原形龙胆苦苷经肾脏排泄。

Aim： To develop a solid-phase extraction and LC/MS method for the determination of gentiopicroside excreted in human urine and assessment of its elimination route. Methods： Human urine samples, collected from 12 healthy volunteers exposed to 80 mg, 240 mg, and 400 mg of gentiopicroside by iv dosing in the randomized crossover design, added with caffeine as internal standard, handled by solid-phase extraction, then measured by LC-MS. Analyses were performed on Rescek C8 column（ 150 mm × 2.1 mm,5μm）with a mobile phase of methanol-10 mmol/L NH4OAC buffer-acetonitrile（50：40： 10）at a flow rate of 0.2 mL/min. Mass spectrometer with the mode of multiple reaction monitoring（MRM） was applied in the detection. Results： It indicated good linearity in the range of 30- 9000 ng/mL （r = 0.998 0） for assay of gentiopicroside in human urine. The extraction recovery and overall recovery were 100.52% ～ 103.83% and 91.10% ～ 96.21%, respectively. The intra-day and inter-day deviations at lower, middle and high levels were estimated to be less than 10%. The percentages of urinary excretive amount of the parent compound in 24 h following the administration of three dose sizes were estimated to be （76.59 ± 10.02） %, （71.95 ± 12.12）%, and （79.76 ± 8.54）%, respectively. The cumulative amount of the urinary excretion was shown to be directly proportional to the magnitude of dose. In addition, the maximal amount of the parent compound was excreted in urine in 0 - 5.5 h. Conclusion： This method proved to be sensitive, accurate, rapid, specific, and suitable for clinical pharmacokinetics. The results indicated that gentiopicroside, following iv dosing, is mainly eliminated via urinary route in the unchanged form.