微透析联合UPLC-MS/MS研究丹参酮ⅡA在大鼠 脑脊液中的药代动力学

Pharmacokinetics of Tanshinone ⅡA in rats cerebrospinal fluid by microdialysis combined with UPLC-MS / MS

关键词:  丹参酮ⅡA  微透析  超高效液相色谱-串联质谱法  大鼠脑脊液  药动学

Key words:  Tanshinone IIA  Microdialysis  Ultra-high performance liquid phase quadrupole time of flight mass spectrometry  cerebrospinal fluid of rats  Pharmacokinetics

摘要:

脑脊液中的药代动力学: 目的? 应用在体脑微透析联合UPLC-MS/MS探究丹参酮ⅡA(Tanshinone IIA,TSA)在正常大鼠及脑缺血/再灌注(cerebral ischemia reperfusion injury, CIRI)损伤模型大鼠脑脊液中的药代动力学特征。方法? 采用增量法和减量法考察灌流液流速和TSA的质量浓度对回收率和相对释放率的影响；减量法测定探针在大鼠体内相对释放率,考察其8 h内的稳定性。应用UPLC-MS/MS技术分析大鼠在体脑透析液样品中TSA浓度,绘制脑透析液药物浓度-时间曲线,计算药动学参数。探针体外回收率和相对释放率基本一致,且随灌流速度的增大而减小,与TSA的浓度无关；体内相对释放率在1.5 h～8.0 h内基本保持不变。UPLC-MS/MS方法学考察结果均符合生物样品要求。结果? 正常大鼠和大脑中动脉阻塞/再灌注损伤(Middle Cerebral Artery Occlusion/Reperfusion, MCAO/R)模型大鼠分别腹腔注射TSA后,脑脊液中药物浓度分别在60 min和80 min达到最高值,半衰期均为69.32 min。不同剂量给药后,脑透析液中TSA浓度差异显著(P<0.05),达峰浓度、药时曲线下面积随剂量而升高,平均驻留时间为102.90～170.48 min。结论? 本研究建立稳定可行的TSA在体脑微透析取样体系和UPLC-MS/MS定量方法,并用于治疗时间窗内TSA脑透析液药动学研究。TSA溶液在正常和模型大鼠脑脊液中分布迅速,病理状态使TSA脑内分布浓度增加。本研究为TSA早期预防和治疗CIRI用药合理性提供依据。

Abstract:

ABSTRACT: OBJECTIVE To investigate the pharmacokinetics of Tanshinone IIA(TSA) in vivo normal and cerebral ischemia/reperfusion(CIRI) injury rats cerebrospinal fluid by microdialysis(MD) combined with UPLC-MS/MS. METHODS The effects of different perfusion flow rates and TSA concentrations between the relative recovery rate and the relative loss rate were analyzed by incremental and reduction methods. The reduction method was also applied to investigate the stability of the probe recovery rate in 8 h. Cerebral dialysates were collected in normal and middle cerebral artery occlusion/reperfusion rats(MCAO/R) which were injected with TSA internationally. Then content of TSA in samples were quantified by UPLC-MS/MS that was established and verified. Meanwhile, drew the drug time curve and calculated the pharmacokinetic parameters. The relative recovery rate and relative loss rate of the MD probe in vitro were nearby. The increased of perfusion velocity, the decreased of the relative recovery rate as well as the relative loss rate. In addition, both of them were independent of TSA concentration. The relative loss rate in vivo was stable between 1.5 and 8.0 h. The methods of UPLC-MS/MS was excellent for the determination of TSA in rats cerebral dialysate. RESULTS TSA of cerebral dialysates in normal and MCAO/R rats was up to the highest at 60 min and 80 min respectively, but the half-life (T1/2) was the same of 69.32 min. Its concentration was significantly different (P<0.05) when the rats wereintervened by varied doses of TSA. The higher of TSA, the more peak concentration(Cmax) and area under curve(AUC). The mean residence times(MRT) in all groups were 102.90～170.48 min. CONCLUSION The established methods of TSA in MD and UPLC-MS/MS were applied to investigate the kinetic in vivo brain feasibly and accurately within treatment time window.The results indicated that TSA was able to cross the blood-brain barrier and enter the brain fast. The pathological state of CIRI would increase the distribution of TSA in the brain.This study provided a medical basis of TSA in the early prevention and treatment of CIRI.