微透析-液相色谱-化学发光法测定大鼠脑组织中丹参酚类化合物浓度及其药代动力学研究

目的基于丹参素(DSS)、原儿茶醛(PAL)和丹酚酸B(SalB)对HAuCl4-luminol-H2O2化学发光的增强作用,建立测定大鼠脑微透析液中3种水溶性酚类化合物浓度的液相色谱-化学发光法(HPLC-CL),探讨该分析法在大鼠脑组织药代动力学研究中的应用。方法采用微透析体内连续采样技术,6只SD大鼠腹腔注射水合氯醛麻醉后,将微透析脑探针植入左侧皮质,用林格液2.0μl·min-1的流速灌流。尾静脉注射丹参滴注液(DSS2.5mg·kg-1、PAL0.4mg·kg-1、SalB0.4mg·kg-1)并收集脑透析液样品,脑透析液收集时间间隔为15min。用HPLC-CL法测定给药后大鼠脑透析液中的药物浓度,利用DAS软件拟合并计算其药代动力学参数。结果丹参素、原儿茶醛和丹酚酸B的脑透析液浓度分别在1.64～430.40、5.30～528.00和8.00～800.00μg·L-1范围内线性关系良好,其检测限分别为0.33、0.22和2.56μg·L-1。以质控样品计算,在各浓度水平下,日间和日内精密度小于5.2%,符合生物样品分析要求。大鼠单剂量静注丹参滴注液后,丹参素在脑组织中的主要药代动力学参数T12、AUC0-∞、MRT0-∞和k分别为(0.64±0.20)h、(369.39±114.77)ng·h·ml-1、(0.64±0.18)h和1.36±0.27。结论建立的HPLC-CL法具有高选择性和高灵敏度等优点,结合微透析采样技术能够实现脑组织中游离丹参素浓度的动态监测,可用于丹参素的生物样品定量分析及药代动力学研究。其中丹参素的脑组织药代动力学参数为国内外首次报道。

Determination of phenolic compounds of Salvia miltiorrhiza in rat brain by liquid chromatography with chemiluminescence detection using microdialysis sampling and application to pharmacokinetic study

Aim To develop a novel method using high-performance liquid chromatography coupled with chemiluminescence detection for the simultaneous determination of danshensu(DSS),protocatechuic aldehyde(PAL),and salvianolic acid B(SalB) of Salvia miltiorrhiza based on the chemiluminenscent enhancement by three phenolic compounds of the HAuCl4-luminol-H2O2 system,and to apply the proposed method to the pharmacokinetic study in rats'brain.Methods Microdialysis probes were inserted into SD rats' brain cerebral cortex under anesthesia by chloral hydrate and perfused with Ringer's solution at 2.0 μL· min-1.Danshen injection was administered(DSS 2.5 mg·kg-1,PAL 0.4 mg·kg-1,SalB 0.4 mg·kg-1) via ranine vein after a 2 h post-surgical stabilization period,and brain microdialysis was collected every 15 min.The analytes in brain microdialysates were quantified by the proposed HPLC-CL method.The main pharmacokinetic parameters of danshensu in brain were estimated by DAS 2.0 software.Results Danshensu,protocatechuic aldehyde,and salvianolic acid B showed good linearities over the ranges of 1.64 ～ 430.40,5.30 ～ 528.00,and 8.00 ～ 800.00 mg·L-1,with the detection limits of 0.33,0.22,and 2.56 μg·L-1,respectively.The inter-and intra-day precisions of the quality control samples were well within the acceptance criteria with the RSD below 5.2%.After single i.v.administration of danshen injection to SD rats,the pharmacokinetic parameters of T1 2,AUC0-∞,MRT0-∞,and K of danshensu in rat brain were(0.64 ± 0.20) h,(369.39 ±114.77) μg·h·L-1,(0.64 ± 0.18) h,and 1.36 ± 0.27,respectively.Conclusions The proposed method of high selectivity and sensitivity can achieve dynamic monitoring of unbound danshensu in rat brain and allow for the high-throughput quantitation of biological samples for the pharmacokinetic studies.The pharmacokinetic parameters of danshensu in rat brain were reported for the first time.