脉络宁注射液中肉桂酸血药浓度的LC-MS-MS测定及人体药动学研究

Determination of plasma concentration of cinnamic acid by LC-MS-MS and study of pharmacokinetics in healthy volunteers after Mailuoning injection

目的:建立LC-MS-MS法测定人血浆中肉桂酸的浓度,研究单剂量和多剂量静脉滴注脉络宁注射液后肉桂酸在健康人体内的药动学。方法:血浆样品以酸化的乙酸乙酯-乙醚(1∶3)萃取;ZOBAX SB C18色谱柱(4.6 mm×150 mm,5μm)分离,柱温为30℃,流动相为甲醇-水(2 mmol.L-1醋酸铵)(45∶55),流速为500μL.min-1;电喷雾离子源,多反应监测。检测离子:肉桂酸,[M-H]-,m/z146.8/103.1;替硝唑(内标),[M-H]-,m/z245.6/126.0。10名受试者单、多次剂量静脉滴注脉络宁注射液后,采用LC-MS-MS法测定血浆中肉桂酸浓度,DASver1.0软件对药-时曲线进行拟合,并计算药动学参数。结果:肉桂酸在0.50～400μg.L-1呈现良好线性,最低定量限为0.50μg.L-1,日内、日间精密度均小于10%。受试者给药后,肉桂酸药时曲线符合二房室模型。单、多次剂量主要药动学参数分别为Cmax(μg.L-1):115.73±44.31,113.79±25.61;T1/2β(h):0.41±0.087,0.52±0.132;V(L.kg-1):0.519±0.134,0.651±0.322;CL(L.kg-1.h-1):0.899±0.295,0.830±0.222;AUC0～t(μg.L-1.h-1):158.64±56.019,166.49±46.788。结论:该法准确、灵敏、专属。单剂量和多剂量给药后肉桂酸主要药动学参数经统计学分析无显著差异;多剂量给药后,肉桂酸体内无蓄积现象,体内过程不受性别差异的影响;其在受试者体内的药动学存在明显个体差异。

Objective： To establish a LC-MSMS method for quantification of cinnamic acid in human plasma and study its pharmacokinetics after administration of Mailuoning injection at a single and the multiple doses to healthy volunteers.Method： Plasma samples were acidified with hydrochloric acid and extracted with ethyl acetate-ether.Cinnamic acid was determined by LC-MS-MS using a ZOBAX SB C18 column with a mobile phase of methanol-water（containing 2 mmol·L -1 ammonium acetic）（45∶55） at a flow rate of 0.5 mL·min-1 and detected using ESI with negative ionization mode.Ions monitored in the multiple reaction monitoring（MRM） mode were m/z 146.8 /103.1 -for cinnamic acid and m/z 245.6 /126.0 -for tinidazole（IS）.After administration of Mailuoning injection at a single and the multiple doses via intravenous guttae（ivgtt） to 10 healthy volunteers,the concentration of cinnamic acid in plasma was determined by LC-MS-MS method.The concentration-time curves were simulated by DASver 1.0 and the pharmacokinetic parameters were calculated.Result： The calibration curve was linear within the range of 0.5-400 μg·L-1.The LLOQ was 0.5 μg·L-1 and RSDs of intra and inter day were less than 10%.The concentration-time curves of cinnamic acid were consistent with the two-compartment model.The pharmacokinetic parameters after administration of Mailuoning Injection at a single and the multiple injections were as follows： Cmax（μg·L-1）,115.73±44.31 and 113.79±25.61;T1/2β（h）,0.41±0.087 and 0.52±0.132;V（L·kg-1）,0.519 ±0.134 and 0.651±0.322;CL（L·kg-1·h-1）,0.899±0.295 and 0.830±0.222;AUC0-tn（μg·L-1·h）,158.64±56.019 and 166.49±46.788.Conclusion： The developed LC-MS/MS method was sensitive and selective,and there was no interference from endogenous substances. After administration of Mailuoning injection via ivgtt to healthy volunteers,the pharmacokinetic parameters of cinnamic acid between a single and the multiple doses,and between the male and female were no significant difference.There was no accumulation with multiple injections for cinnamic acid,but there were significant individual differences in the pharmacokinetics of cinnamic acid in volunteers.