LC-MS/MS法测定大鼠脑组织中奥拉西坦的浓度

Concentration determination of oxiracetam in rat brain by LC-MS/MS

目的:建立一种简便、快速的LC-MS/MS法测定大鼠脑组织中奥拉西坦的浓度,并对SD大鼠静脉注射奥拉西坦后的脑组织分布进行研究。方法:SD大鼠脑组织样品用0.9%氯化钠溶液匀浆后,以吡拉西坦为内标,用乙腈沉淀蛋白,采用LC-MS/MS系统进行分离和测定。色谱柱为Agilent Poroshell 120 SB-C_(18)柱(100 mm×4.6 mm,2.7μm),流动相为30%甲醇-70%水(含10 mmol·L^(-1)甲酸铵,0.1%甲酸),流速为0.2 mL·min^(-1),柱温为30℃。质谱条件:电喷雾离子源(ESI),正离子模式,多反应离子监测(MRM),检测离子为奥拉西坦m/z 158.6→113.6,内标吡拉西坦m/z 142.6→125.4。SD大鼠分别单次静脉注射奥拉西坦500、1 000、2 000 mg·kg^(-1),于不同时间点分别采集脑组织,测定脑组织中奥拉西坦的浓度。结果:SD大鼠脑组织匀浆液质量浓度在1.0~100.0μg·mL^(-1)范围内线性关系良好,定量下限为1.0μg·mL^(-1),批内和批间精密度RSD均小于5.7%,准确度为96.3%~105.2%。大鼠分别单次静脉注射500、1 000、2 000 mg·kg^(-1) 3个剂量的奥拉西坦后,主要药动学参数t1/2分别为(3.56±0.27)h、(3.36±0.49)h、(3.60±0.79)h,C_(max)分别为(24.78±16.06)μg·g^(-1)、(50.79±25.92)μg·g^(-1)、(82.21±19.33)μg·g^(-1),MRT0-t分别为(2.54±0.02)h、(2.38±0.30)h、(2.57±0.08)h,AUC0-t分别为(48.59±10.92)μg·h·g^(-1)、(94.54±9.30)μg·h·g^(-1)、(201.35±18.87)μg·h·g^(-1)。结论:本测定方法可用于大鼠奥拉西坦药代动力学在脑组织中的分布研究;大鼠分别单次静脉注射3个剂量的奥拉西坦后,奥拉西坦进入脑组织的时间较快,浓度较高,持续时间较长,利于发挥活化和改善脑组织功能的作用。

Objective：To develop a simple and rapid HPLC-MS/MS method for the determination of oxiracetam concentration in rat brain tissue,and to study the distribution of oxiracetam in the brain tissues after being injected intravenously in SD rats.Methods：The tissue samples of SD rat brain were firstly homogenized with 0.9% sodiumchloride solution.Using piracetam as an internal standard（IS）and acetonitrile as a protein precipitant,the samples were separated and determined by LC-MS/MS system.Agilent Poroshell 120 SB-C_（18） analytical column（100 mm×4.6 mm 2.7 μm）was used in the system,and the mobile phase was prepared with methanol and water in a volume ratio of 30∶70,containing 10 mmol·L-1 ammonium acetate and 0.1% formic acid.The flow rate was 0.2 mL·min-1 and the column temperature was set at 30 ℃.The parameters for MS were as follows：electrospray ionization source（ESI）was operated in positive ion mode;the quantification was performed using multiple reaction monitoring（MRM）mode to monitor the precursor-to-product ion transitions of m/z 158.6 → 113.6 for oxiracetam and m/z 142.6 → 125.4 for the internal standard piracetam.SD rats were injected intravenously with single doses of 500,1 000 and 2 000 mg·kg-1 oxiracetam,respectively.The rat brain tissues were collected at different time intervals and the concentrations of oxiracetam in the brain tissues were determined.Results：The linear calibration curve of oxiracetam was obtained in the concentration range of 1.0-100.0μg·mL-1,and the lower limit of quantification（LLOQ）was 1.0 μg·mL-1. Both the intra-and inter-batch precisions of RSD were less than 5.7%.The accuracy was in the range of 96.3%-105.2%.The main pharmacokinetic parameters of oxiracetam in SD rats brain tissue after a single dose of 500,1 000,2 000 mg·kg-1 oxiracetam by intravenous route were as follows：t1/2=（3.56±0.27）h,（3.36±0.49）h,（3.60±0.79）h;Cmax=（24.78±16.06）μg·g-1,（50.79±25.92）μg·g-1,（82.21±19.33）μg·g-1;MRT0-t=（2.54±0.22）h,（2.38±0.30）h,（2.57±0.08）h;AUC0-t=（48.59±10.92）μg·h·g-1,（94.54±9.30）μg·h·g-1,（201.35±18.87）μg·h·g-1.Conclusion：The method can be used to investigate the pharmacokinetics of oxiracetam distributed in rat brain tissues.After being injected intravenously with a single dose of oxiracetam at 3 dosages,the uptake of oxiracetam into the brain tissue is very quick,maintained at a high concentration with a long duration.All the actions promote the activation and improvement of the brain functions.