HPLC-MS/MS法测定大鼠血浆中异绿原酸B浓度及其在药代动力学研究中的应用

摘要

建立大鼠血浆中异绿原酸B的LC-MS/MS测定方法,研究大鼠口服异绿原酸B后的血浆药代动力学特征.血浆样品中加入内标白藜芦醇后经甲醇沉淀蛋白.HPLC分离采用Zorbax SB-C18 (3.5 μm,2.1 mm×100 mm)柱,流动相为含0.1％甲酸的甲醇-水,流速为0.2 mL/min.质谱检测采用电喷雾离子源,负离子选择反应监测(SRM)模式检测m/z515.3→352.9(异绿原酸B)和m/z 227.1→143.1(白藜芦醇,内标).标准曲线在5-2500 ng/mL范围内线性良好(r2=0.9982),日内、日间精密度(RSD)小于12.46％,准确度在士5.80％范围内,血浆样品稳定性好.该方法已成功应用于大鼠血浆异绿原酸B的药代动力学研究.结果 表明,在5-20 mg/kg剂量范围内,异绿原酸B在大鼠体内呈非线性的药代动力学特征.%A sensitive LC-ESI-MS/MS method for determination of isochlorogenic acid B in rat plasma was developed and validated in the present study.Plasma samples were prepared by a simple protein precipitation with methanol containing resveratrol as intemal standard (IS).The chromatographic separation was performed on a Zorbax SB-C18 column (3.5 μm,2.1 mm× 100 mm,Agilent,USA) at a flow rate of 0.2 mL/min using methanol/water containing 0.1％ formic acid (v/v) as mobile phase.The detection was performed on a triple quadrupole tandem mass spectrometer equipped with Electronic Spray Ion by selected reaction monitoring (SRM) of the transitions at m/z 515.3→352.9 for isochlorogenic acid B and m/z 227.1→ 143.1 for IS,respectively.The calibration curve of the method was linear over the range of 5-2500 ng/mL (r2 =0.9982).The intra-and inter-day precisions (R.S.D.％) were less than 12.46％,and the accuracy (R.E.％) was within ±5.80％.Isochlorogenic acid B was sufficiently stable under all relevant analytical conditions.The validated method was successfully applied to the plasma pharmacokinetic studies of isochlorogenic acid B in rats.It was found that isochlorogenic acid B had non-linear pharmacokinetic characteristics in rats within the dosage ranges from 5 to 20 mg/kg.