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目的:建立同时测定大鼠血浆中水飞蓟宾和丹参素浓度的液相色谱-串联质谱法(LC-MS/MS),并用于水飞蓟宾-丹参素钠复方注射剂在大鼠体内的药动学研究。方法:血浆样品经乙酸乙酯液-液萃取后,以甲醇-0.2%甲酸水溶液(80∶20)为流动相,Zorbax Eclipse XDB-C18(150 mm×4.6 mm,3.5μm)色谱柱分离,采用电喷雾离子化源(ESI)三重四极杆串联质谱,以选择反应监测模式(SRM)进行检测。用于定量分析的离子反应m/z分别为481.1→300.9(水飞蓟宾)、196.9→135.1(丹参素)和153.0→109.1(内标,原儿茶酸)。结果:水飞蓟宾和丹参素分别在10~10 000 ng.mL-1和20~20 000 ng.mL-1浓度范围内呈良好的线性关系,最低定量下限(LLOQ)分别为10和20 ng.mL-1。方法的精密度、准确度和稳定性均符合要求。大鼠静脉注射低、中、高3个剂量的水飞蓟宾-丹参素钠复方注射剂后水飞蓟宾的主要药动学参数为:t1/2分别为(2.30±0.45)、(2.11±0.43)、(2.40±0.36)h,Ke分别为(0.31±0.06)、(0.34±0.07)、(0.30±0.04)h-1,AUC0~8 h分别为(3 264.0±789.4)、(7 706.1±1 750.0)、(21 171.1±5 018.7)ng.h.mL-1,AUC0~∞分别为(3 330.3±800.1)、(7 899.5±1 750.0)、(21 791.0±5 048.1)ng.h.mL-1;丹参素的主要药动学参数为:t1/2分别为(1.98±0.47)、(1.70±0.18)、(2.04±0.25)h,Ke分别为(0.37±0.10)、(0.41±0.04)、(0.34±0.04)h-1,AUC0~8 h分别为(3 758.9±1 066.1)、(7 065.8±921.5)、(17 228.5±3 705.2)ng.h.mL-1,AUC0~∞分别为(3 885.8±1152.1)、(7 250.7±923.1)、(17 791.3±3 720.2)ng.h.mL-1。结论:本方法具有专属性强、灵敏度高等优点,适用于复方制剂中水飞蓟宾和丹参素的临床前药动学研究。
OBJECTIVE: To establish a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method for the simultaneous determination of silybin and danshensu in plasma of rats and its application in silybin-sodium danshensu compound injection in rats Pharmacokinetic studies. Methods: The plasma samples were separated by liquid-liquid extraction with ethyl acetate and separated on a Zorbax Eclipse XDB-C18 column (150 mm × 4.6 mm, 3.5 μm) using methanol-0.2% formic acid in water Electrospray ionization source (ESI) triple quadrupole tandem mass spectrometry was performed with a selective reaction monitoring mode (SRM). The ion reaction m / z for quantitative analysis was 481.1 → 300.9 (silybin), 196.9 → 135.1 (danshensu) and 153.0 → 109.1 (internal standard, protocatechuic acid), respectively. Results: Silybin and Danshensu showed a good linearity in the range of 10 ~ 10 000 ng.mL-1 and 20 ~ 20 000 ng.mL-1, respectively. The lowest limit of quantification (LLOQ) was 10 and 20 ng.mL-1. The precision, accuracy and stability of the method are in line with the requirements. The main pharmacokinetic parameters of silybin after 3 doses of silybin-sodium danshensu compound injections were (2.30 ± 0.45) and (2.11 ± 0.43 ± 0.36, h (Ke) were 0.31 ± 0.06, 0.34 ± 0.07, 0.30 ± 0.04 h-1, respectively. The AUCs of 0-8 h were (3 264.0 ± 789.4), ± 1 750.0), (21 171.1 ± 5 018.7) ng.h.mL-1, and AUC0 ~ ∞ were (330.3 ± 800.1), (7 899.5 ± 1 750.0) and (21 791.0 ± 5 048.1) ng.h . The main pharmacokinetic parameters of Danshensu were (1.98 ± 0.47), (1.70 ± 0.18), (2.04 ± 0.25) h and (0.37 ± 0.10) and ± 0.84 ± 0.04 h-1, AUC0-8 h were (3 758.9 ± 1 066.1), (7 065.8 ± 921.5), (17 228.5 ± 3 705.2) ng.h.mL-1, respectively ~ ∞ were (3 885.8 ± 1152.1), (7 250.7 ± 923.1), (17 791.3 ± 3 720.2) ng.h.mL-1. Conclusion: The method has the advantages of strong specificity and high sensitivity, and is suitable for preclinical pharmacokinetic studies on silybin and danshensu in compound preparations.