LC-MS/MS法测定大鼠血浆中的吴茱萸碱、吴茱萸次碱和吴茱萸内酯及其生物利用度的研究

Simultaneous determination and absolute oral bioavailability of evodiamine,rutaecarpine and evodin concentration in the rat plasma by LC-MS /MS

目的采用LC—ESI—MS／MS法同时测定大鼠血浆中的吴茱萸碱、吴茱萸次碱和吴茱萸内酯，并研究这3种活性物质在大鼠体内的药动学特征。方法取100灿血浆样品，用甲基叔丁基醚-二氯甲烷（3：1）萃取，选择右啡烷为内标。采用BDSHypersil C18色谱柱（100mm×2．1mm，2．4μm），乙腈-2mmol·L^-1乙酸铵（含0．05％甲酸）（60：40）为流动相，采用AB Sciex 4000 Q TRAP型三重四级杆串联质谱，ESI源，多反应监测（MRM）正离子模式。经检测，吴茱萸碱、吴茱萸次碱、吴茱萸内酯和内标的检测离子对分别为：m／z 304．3—134．4、m／z 288．1—273．3、m／z 471．3—425．2、m／z 258．0—201．0。结果血浆中吴茱萸碱、吴茱萸次碱和吴茱萸内酯的线性范围均为0．5～1×10^3 ng·mL^-1（r〉0．9960），提取回收率均〉70％，日内、日间RSD均〈15％。大鼠口服吴茱萸提取物后，血浆中吴茱萸碱、吴茱萸次碱及吴茱萸内酯的Cmax分别为19．02±2．15、19．83±5．83、41．61±7．48ng·L^-1，Tmax分别为1．17±0．29、1．25±0．66、0．63±0．14h，T1／2分别为4．64±2．14、5．72±1．34、4．25±1．72h；绝对生物利用度分别为16．96％±2．80％、12．02％±3．17％、5．43％±1．99％。结论所用方法准确、快速、灵敏，并成功用于临床前的药动学研究。

OBJECTIVE To establish LC - ESI - MS/MS for the simultaneous determination of evodiamine, rutecarpine and evodin in the rat plasma, and study the absolute oral bioavailahility of these three substances in healthy rats. METHODS The analytes were extracted from 100 μL plasma samples by liquid -liquid extraction using a mixed methyl tert -butyl ether： methylene chloride（ 3 ： 1 ） and dextrorpban was selected to be the internal standard. Chromatographic separation of the analytes was performed by a BDS Hypersil C18 column （ 100 mm × 2.1 mm,2.4μm）. The mobile phase consisted of aeetonitrile and 2 mmol·L^-1 ammonium containing 0.05% formic acid（ 60 ： 40 ）. Detection was catried out by electrospray ionization AB Sciex 4000 Q TRAP triple quadrnpole mass spectrometry in multiple reaction monitoring （MRM） mode. The ion combination of m/z 304.3→134.4 , m/z 288.1→273.3, m/z 471.3→425.2, m/z 258.0→201.0 was used to qualify Evodiamine, Rutaecarpine, Evodin and the internal standard, respectively. RESULTS Linear calibration curves were obtained in the concentration range of 0.5 - 1000 ng·mL^-1 for the three tested compouds （evodiamine, rntaecarpine and evodin）. Extraction recovery was over 70% in plasma. The intra - day and inter - day RSD were lower than 15% and recoverywas within ＋ 10% at three QC levels for all. The main pharmacokinetic parameters of C T T1/2 were 19.02 ± 2. 15 ng·L - 1 , 1. 17± 0. 29 h ,4. 64 ± 2. 14 h for Evodiamine, and 19.83 ± 5.83 ng. L-1, 1.25 ± 0.66 h, 5.72 ± 1.34 h for Rutaecarpine, and 41.61 ± 7.48 ng·L^-1,0.63 ± 0.14 h,4.25 ± 1.72 h for Evodin, respectively. Compared with iv administration, the absolute bioavailability of Evodiamine, Rutaecarpine and evodin were 16.96% ± 2.80% , 12.02% ± 3. 17% and 5.43% ± 1.99% , respectively. CONCLUSION This method was shown to be accurate, rapid and sensitive for the simultaneous determination of evodiamine, rutaecarpine and evodin in the rat plasma concentrations and could he successfully used for preclinical pharmacokinetic studies.