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目的对比研究梓葛冻干粉针中葛根素在正常和脑缺血大鼠体内的药动学。方法建立测定大鼠血浆中葛根素的高效液相色谱-质谱联用法,以染料木素为内标,采用Diamonsil C18(4.6 mm×150 mm,5μm)色谱柱,以甲醇(含0.1%甲酸)-水(含10 mmol·L-1醋酸铵)(80∶20)为流动相,流速0.6 mL·min-1;采用电喷雾离子源负离子模式(ESI-),多反应监测(MRM)方式对葛根素(m/z 415.1→295.1)和内标物(m/z 269.2→133.1)进行测定。线栓法制备永久性脑缺血模型大鼠,正常和模型大鼠分别尾静脉注射给予梓葛冻干粉针(26.7 mg·kg-1),HPLC-MS/MS测定给药后不同时间点(3,5,10,30,60,120,180,300 min)血浆中葛根素浓度,数据经DAS 2.1软件计算药动学参数并用SPSS软件对数据进行统计学处理。结果血浆中葛根素浓度在0.02~100μg·mL-1内,呈良好线性(r=0.995 4);方法提取回收率大于86.3%,批内与批间精密度(RSD)均小于13.2%,准确度(RE)在-7.3%~10.5%内。正常组血浆中葛根素的实测达峰浓度(ρmax)为(56.5±3.4)μg·mL-1,经-时曲线下面积(AUC0-t)为(17.1±1.5)μg·h·mL-1,半衰期(t1/2)为(0.311±0.133)h,平均滞留时间(MRT0-t)为(0.269±0.044)h;在模型组,ρmax为(62.3±14.0)μg·mL-1,AUC0-t为(20.0±6.8)ng·h·mL-1,t1/2为(0.755±0.128)h,MRT0-t为(0.360±0.045)h。与正常组相比,梓葛冻干粉针中葛根素在模型组血浆中的t1/2、MRT0-t均显著延长(P<0.05)。结论建立的HPLC-MS/MS方法专属性强、灵敏度高、快速准确,可用于大鼠血浆中葛根素的测定和药动学研究。与正常大鼠相比,梓葛冻干粉针中葛根素在永久性脑缺血模型大鼠的生物利用度提高,消除减慢、滞留时间延长。
Objective To compare the pharmacokinetics of puerarin in Zige freeze-dried powder injection in normal and cerebral ischemia rats. Methods High performance liquid chromatography-mass spectrometry (HPLC-MS) was established for the determination of puerarin in plasma of rats. Genistein was used as internal standard. Diamonsil C18 column (4.6 mm × 150 mm, 5 μm) - water (containing 10 mmol·L-1 ammonium acetate) (80:20) as the mobile phase at a flow rate of 0.6 mL · min-1. The electrospray ionization negative ion mode (ESI-) and multiple reaction monitoring Puerarin (m / z 415.1 → 295.1) and internal standard (m / z 269.2 → 133.1) were measured. The rat model of permanent cerebral ischemia was established by thread occlusion method. Zi-Ge freeze-dried powder injection (26.7 mg · kg-1) was injected into the tail vein of normal and model rats, respectively, and determined by HPLC-MS / (3,5,10,30,60,120,180,300 min) plasma puerarin concentration, the data calculated by the DAS 2.1 software pharmacokinetic parameters and SPSS software for data processing. Results The concentration of puerarin in plasma ranged from 0.02 to 100 μg · mL-1, with a good linearity (r = 0.995 4). The extraction recovery was more than 86.3%. The intra-and inter-batch precision was less than 13.2% Degree (RE) within -7.3% ~ 10.5%. The peak plasma concentration of puerarin in the normal group was (56.5 ± 3.4) μg · mL-1, and the area under the curve of AUC0 was (17.1 ± 1.5) μg · h · mL-1 , The half life (t1 / 2) was (0.311 ± 0.133) h and the average retention time (MRT0-t) was 0.269 ± 0.044 h. In the model group, ρmax was (62.3 ± 14.0) μg · mL- t was (20.0 ± 6.8) ng · h · mL-1, t1 / 2 was (0.755 ± 0.128) h and MRT0-t was (0.360 ± 0.045) h. Compared with the normal group, the plasma levels of puerarin in the Gezi lyophilized powder injection in the model group, t1 / 2, MRT0-t were significantly longer (P <0.05). Conclusion The established HPLC-MS / MS method is specific, sensitive, rapid and accurate and can be used for the determination of puerarin in rat plasma and pharmacokinetic study. Compared with normal rats, puerarin freeze-dried puerarin in the permanent cerebral ischemia model rats increased bioavailability, elimination of slow, prolonged residence time.