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目的:建立高效液相色谱-电化学法测定小鼠血浆中异丙肾上腺素浓度,为研究其在小鼠体内药代动力学过程提供检测手段。方法:采用SymmetryShield C18柱(4.6 mm×250 mm,5.0μm),流动相为含0.05 mol.L-1柠檬酸,0.05 mol.L-1无水乙酸钠,0.3 mmol.L-1EDTA-Na2,1.0 mmol.L-1IPR-B8离子对试剂,0.06 mmol.L-1二正丁胺,1.6%甲醇水溶液。流速:0.9 mL.min-1,柱温25℃。以3,4-二羟基苄胺为内标;用电化学检测器测定,检测电压:700 mV。测定小鼠皮下注射异丙肾上腺素(0.75 mg.kg-1)后不同时间的血浆药物浓度,用3P97软件拟合计算相关药动学参数。结果:IP在浓度范围为0.5~294.0 ng.mL-1内,线性关系良好(r=0.9998,n=7)。平均提取回收率为85.8%,平均回收率为100.9%,日内及日间RSD均小于3.9%(n=5)。最低检测限为0.08 ng.mL-1(S/N≥3)。相关药动学参数为AUC0-t=2424.76 min.ng.mL-1,Vc=2774 mL.kg-1,Cl=0.309 mL.g-1.min-1,Ka=(0.9817±0.174)min-1,Lag Time=(0.3076±0.0375)min。结论:本法准确、快速、稳定、灵敏度高,可用于生物样品中异丙肾上腺素浓度测定。
OBJECTIVE: To establish a method for the determination of isoprenaline concentration in mouse plasma by high performance liquid chromatography-electrochemistry, and to provide a test method for studying its pharmacokinetics in mice. Methods: A Symmetry Shielded C18 column (4.6 mm × 250 mm, 5.0 μm) was used. The mobile phase consisted of 0.05 mol·L -1 citric acid, 0.05 mol·L -1 anhydrous sodium acetate, 0.3 mmol·L -1 EDTA- 1.0 mmol.L-1 IPR-B8 ion pair reagent, 0.06 mmol.L-1 di-n-butylamine, 1.6% aqueous methanol. Flow rate: 0.9 mL.min-1, column temperature 25 ℃. 3,4-dihydroxybenzylamine as an internal standard; with electrochemical detector, detection voltage: 700 mV. The concentration of plasma drug was measured at different times after subcutaneous injection of isoproterenol (0.75 mg.kg-1) in mice, and the relevant pharmacokinetic parameters were calculated by 3P97 software fitting. Results: IP showed good linearity (r = 0.9998, n = 7) within the range of 0.5-294 ng · mL-1. The average extraction recovery was 85.8% with an average recovery of 100.9% with an intraday and intraday RSD of less than 3.9% (n = 5). The minimum detection limit was 0.08 ng.mL-1 (S / N≥3). The relevant pharmacokinetic parameters were AUC0-t = 2424.76 min.ng.mL-1, Vc = 2774 mL.kg-1, Cl = 0.309 mL.g-1.min-1 and Ka = 0.9817 ± 0.174 min- 1, Lag Time = (0.3076 ± 0.0375) min. Conclusion: This method is accurate, rapid, stable and sensitive. It can be used for the determination of isoproterenol in biological samples.