超高效液相色谱-串联质谱法测定去氢骆驼蓬碱衍生物DH-330血药浓度及其在大鼠体内的药动学评价Δ

目的:建立大鼠血浆中去氢骆驼蓬碱衍生物DH-330的测定方法,并对大鼠灌胃DH-330后的药动学行为进行评价。方法:以替硝唑为内标,血浆样品以乙腈沉淀蛋白处理后,采用超高效液相色谱-串联质谱法测定血药浓度。色谱分析采用色谱柱为Waters ACQUITY BEH C1(850mm×2.1mm,1.7μm),流动相为乙腈-甲醇-0.5%甲酸水溶液(15∶55∶30,V/V/V),流速为0.4mL/min,柱温为30℃,进样量为5μL;质谱分析采用电喷雾电离源,正离子扫描,离子源温度为124℃,DH-330检测质荷比(m/z)为335.8→334.8,内标m/z为247.0→81.0。取6只Wistar大鼠,灌胃DH-330混悬液(50mg/kg),分别于给药前(0 h)及给药后0.25、0.5、1、2、4、6、8、12、24 h时于大鼠眼底静脉丛采血,测定DH-330血药浓度并绘制血药浓度-时间曲线,并采用Kinetica 5.0软件计算其药动学参数。结果:DH-330血药浓度的线性范围为25.05~2 004ng/mL(r=0.999 8),定量下限为25.05ng/mL;日内、日间精密度RSD均小于10%;准确度相对误差(RE)为-9.76%~4.55%,提取回收率大于85%(RSD<5%);稳定性RE为-2.53%~2.29%;不受基质效应或进样残留效应的影响。大鼠灌胃DH-330后达峰浓度为(1 162.43±241.72)ng/mL,药-时曲线下面积为(3 242.93±652.31)ng·h/mL,半衰期为(1.93±0.61)h,平均滞留时间为(3.23±0.30)h,清除率为(16.80±5.30)L/(h·kg),稳态表观分布容积为(54.78±19.64)L/kg。结论:本研究建立的方法具有操作简便,专属性强,灵敏度、精密度及回收率高等优点,可用于大鼠血浆中DH-330血药浓度的测定。大鼠灌胃给药后,DH-330的半衰期短,吸收迅速,表观分布容积大,表明其具有高的亲脂性,可能主要集中分布于组织中。

Determination of Plasma Concentration of Harmine Derivative DH-330 by UPLC-MS and Its Pharmacokinetics Evaluation in Rats

OBJECTIVE:To establish a method for the determination of harmine derivative DH-330 in rat plasma and to use it for pharmacokinetic behavior evaluation of DH-330 in rats after intragastric administration.METHODS:Using tinidazole as internal standard,after pre-treatment of acetonitrile precipitated protein,UPLC-MS method was adopted to determine the plasma concentration of DH-330.UPLC analysis was performed on Waters ACQUITY BEH C18 column(50 mm×2.1 mm,1.7μm)with mobile phase consisted of acetonitrile-methanol-0.5%formic acid aqueous solution(15∶55∶30,V/V/V)at flow rate of 0.4 mL/min,while the column temperature was 30℃,and sample size was 5μL.MS analysis was conducted by electrospray ionization source,positive ion scanning,ion source temperature at 124℃,DH-330 detection of mass to charge ratio(m/z)of 335.8→334.8,and internal standard m/z of 247.0→81.0.Six Wistar rats were given DH-330 suspension(50 mg/kg)intragastrically.Blood samples were collected from fundus venous plexus capillary before administration(0 h)and 0.25,0.5,1,2,4,6,8,12,24 h after administration.Plasma concentration of DH-330 was determined and plasma concentration-time curves were drawn.Pharmacokinetic parameters were calculated by using Kinetica 5.0 software.RESULTS:The linear ranges of DH-330 were 25.05-2 004 ng/mL(r=0.999 8),and the limits of quantitation was 25.05 ng/mL.RSDs of intra-day and inter-day were all less than 10%.The accuracy RE was-9.76%to 4.55%.The extraction recovery was higher than 85%(RSD<5%).Stability RE was-2.53%to 2.29%.They were not affected by matrix effect or residual effect of injection.The pharmacokinetic parameters of DH-330 in rats after intragastric administration included that cmax was(1 162.43±241.72)ng/mL,AUC0-∞was(3 242.93±652.31)ng·h/mL,t1/2 was(1.93±0.61)h,MRT was(3.23±0.30)h,CL was(16.80±5.30)L/h·kg,Vss was(54.78±19.64)L/kg.CONCLUSIONS:The established method is simple,specific,sensitive,precise and recovery,which can be used for the plasma concentration determination of DH-330 in rats.DH-330 has short half-life,rapid absorption and large apparent distribution volume after intragastric administration in rats,which indicates that it has high lipophilicity and may be mainly distributed in tissues.