HPLC-ECD法测定小鼠血浆中异丙肾上腺素浓度

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,5.0 μm),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^-1 EDTA-Na₂,1.0 mmol·L^-1 IPR-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AUC_0-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 develop an HPLC-ECD method for determination of isoproterenol(IP) concentration in plasma. Methods: IP can be separated on SymmetryShield C₁₈(4.6 mm×250 mm,5.0 μm)column,the mobile phase was consisted of 0.05 mol·L^-1citric acid monohydrate,0.05 mol·L^-1sodium acetate anhydrous,0.3 mmol·L^-1EDTA-Na₂,1.0 mmol·L^-1IPR-B₈,0.06 mmol·L^-1 dibutylamine and 1.6% Methanol,Flow rate was 0.9 mL·min^-1,column temperature was 25 ℃.The electrochemical detector was set at 700 mV,and dihydroxybenzy-lamine(DHBA)was used as internal standard.IP concentration in mice plasma were determined after sc.administration(0.75 mg·kg^-1),the data were processed with 3P97 pharmacokinetic program. Results: The isoproterenol was linear over the range 0.5-294.0 ng·mL^-1,the correlation coefficient was 0.9998(n=7).The average extraction recovery was 85.8%,the average recovery was 100.9%,RSD of intra-day and inter-day were less than 3.9%(n=5).The detection limit was 0.08 ng·mL^-1(S/N≥3).The main pharmacokinetic parameters were estimated to be as follows:AUC_0-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. Conclusion: The method is accurate,rapid and high sensitivity,it was suitable for the determination of isoproterenol in biological sample.