液相色谱-串联质谱法测定大鼠血浆中脱水葛根素浓度及其药代动力学研究
LC-MS/MS method for the determination of a new puerarin derivative and its application in pharmacokinetic studies in rats
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摘要: 目的:建立快速测定大鼠血浆中脱水葛根素浓度的液相色谱-串联质谱分析方法,并探讨脱水葛根素在大鼠体内的药代动力学。方法:血浆样品加入甲醇沉淀蛋白后,用LC-MS/MS进行测定分析。色谱柱为Zorbax SB-C18 column(4.6 mm×150 mm I.D.5.0 μm,Agilent,USA),流动相为甲醇(含10 mmol·L-1乙酸胺)-0.1%甲酸溶液(20:80,V/V),流速0.6 mL·min-1,温度40℃。检测应用三重串联四级杆质谱仪的多重反应监测模式(MRM),选择的检测离子如下:脱水葛根素399.1→281.0,染料木素(内标)271.0→215.0。结果:在大鼠血浆样品中,脱水葛根素浓度在1.5-5400 ng·mL-1之间线性关系良好,最低检测限为1.50 ng·mL-1。准确度在95.73%-103.18%之间,精密度RSD在4.33%-7.86%之间。结论:建立的LC-MS/MS联用方法简单准确,灵敏度高,适用于大鼠血浆中对脱水葛根素的药代动力学研究。Abstract: AIM:To establish a sensitive and rapid liquid chromatographic-tandem mass spectrometry(LC-MS/MS) method for the quantitative analysis of dehydrated puerarin in rat plasma,and its application for pharmacokinetic studies.METHODS:A plasma sample was pretreated by one-step protein precipitation by the addition of five volumes of methanol.The chromatographic separation was achieved on a Zorbax SB-C18 column(4.6 mm×150 mm I.D.5.0 μm,Agilent,USA) at 40℃ at a flow rate of 0.6 mL·min-1 by an isocratic elution consisting of 10 mmol·L-1 ammonium acetate in methanol and water containing 0.1% formic acid in a ratio of 20:80(V/V).Detection was performed on a triple quadrupole mass spectrometer in multiple-reaction monitoring(MRM) mode.An atmospheric pressure chemical ionization(APCI) interface in positive ionization mode was used by monitoring the transitions from m/z 399.1 →281.0(dehydrated puerarin) and m/z 271.0→215.0(internal standard,IS).RESULTS:Calibration curves were linear in the concentration range from 1.50 to 5400 ng·mL-1,and the lower limit of quantification(LLOQ) was 1.50 ng·mL-1 in rat plasma.The accuracy and precision values,which were calculated from three different sets of quality control samples analyzed in sextuplicate on three different days,ranged from 95.73% to 103.18%,and from 4.33% to 7.86%,respectively.CONCLUSION:The method was successfully applied to assess the pharmacokinetics of dehydrated puerarin after oral administration in rats.