人参皂苷CK在正常大鼠和大脑中动脉阻断模型大鼠体内药物代谢动力学比较研究

Development of an LC-MS/MS-based method for comparative study of pharmacokinetics of ginsenoside CK in normal rats and middle cerebral artery occlusion model rats

目的建立一种高灵敏度的液相色谱-质谱/质谱联用(LC-MS/MS)法测定人参皂苷CK在大脑中动脉阻断(MCAO)模型大鼠体内的含量,研究口服给药后大鼠体内的药物代谢动力学参数变化,为人参皂苷CK对脑神经保护以及抗炎作用奠定理论基础。方法正常大鼠及MCAO模型大鼠100 mg/kg人参皂苷CK单次灌胃给药后于不同时间取血及脑组织。大鼠血浆及脑组织匀浆后均采用蛋白沉淀法进行样品前处理,并构建LC-MS/MS法对人参皂苷CK进行检测。采用DAS 2.0软件的非房室模型计算大鼠给药后的药物代谢动力学参数以及MCAO模型大鼠给药后不同时间点血浆及脑组织的药物浓度。结果人参皂苷CK在1～500 ng/ml范围内线性关系良好,最低定量下限为1 ng/ml,日内、日间精密度小于13.6%,准确度偏差范围为-5%～13.4%。与正常大鼠相比较,MCAO模型大鼠血浆t1/2更长[(2.5±1.1) h对(11.0±10.9) h],AUC值更大[(14 944±2 170) ng/ml·h对(28 882±11 119) ng/ml·h];MCAO模型大鼠达峰浓度(Cmax)比正常大鼠略低[(2 834±768) ng/ml对(2 634±638) ng/ml]。在同一时间点,人参皂苷CK在MCAO模型大鼠血浆中比脑组织中分布更多;人参皂苷CK在正常大鼠体内吸收更快,血药浓度更高。结论本研究建立的LC-MS/MS方法灵敏度高、重现性好、样品处理过程方便快速,适用于人参皂苷CK生物样品分析。模型大鼠人参皂苷CK脑血含量比很低,脑内分布较少,提示人参皂苷CK可能并非通过增加脑内物质基础的途径而产生神经保护作用。

Objective To establish a high sensitive liquid chromatography coupled with tandem mass spectrometry(LCMS/MS) method for the determination of ginsenoside CK in the rats with middle cerebral artery occlusion(MCAO),and to study the pharmacokinetics parameters of ginsenoside CK in rats.Methods The plasma and brain tissue of rats were taken at different time points after single intragastric administration.Sample preparation was achieved by protein precipitation with methanol.Ginsenoside CK was detected by LC-MS/MS.The non-compartment model of DAS 2.0 software was used to calculate the pharmacokinetics parameters and the drug concentration in plasma and brain tissue in MCAO rats at different time points after administration.Results The method was linear over the range of 1~500 ng/ml with the lower limit of quantitation 1 ng/ml.The accuracy and precision of intra-and inter-day,recovery and stability of the method were all within the acceptable limits.Compared with the normal rats,the plasma t1/2 of the MCAO model rats was longer [(2.5+1.1) h vs(11.0±10.9) h],and the AUC value was higher [(14 944+2 170) ng/ml?h vs(28 882+11 11 9) ng/ml?h];the Cmax of MCAO model rats was slightly lower [(2 834+768)ng/ml vs(2 634+638) ng/ml].At the same time,the distribution of ginsenoside CK in the plasma of MCAO model rats was more than that in the brain tissue;the absorption of ginsenoside CK in normal rats was faster,and the blood concentration of ginsenoside CK was higher.Conclusion LC-MS/MS used for ginsenoside CK determination is a reliable approach.The low cerebral blood content of ginsenoside CK in MCAO model rats indicates that ginsenoside CK cannot penetrate the brain to effect.