超高效液相色谱-串联质谱法测定人血浆中非索非那定的浓度及其应用

目的：建立超高效液相色谱-串联质谱法（UHPLC-MS/MS）测定人血浆中非索非那定的浓度，并用于灯盏花素对人P-糖蛋白体内活性影响的研究。方法：200 μL血浆经500 μL乙腈沉淀蛋白后，采用Waters ACQUITY UPLC^® BEH C₁₈柱（50 mm×2.1 mm，1.7 μm）为色谱柱，以乙腈（A）-0.1%甲酸水（B）为流动相梯度洗脱，流速为0.3 mL·min^-1；质谱采用电喷雾正离子模式离子化（ESI⁺）和多反应监测模式（MRM）扫描，非索非那定和苯海拉明（内标）的定量离子对分别为m/z 502.2→466.5和m/z 256.1→167.1。结果：非索非那定和苯海拉明的保留时间分别为2.19 min和1.89 min。人血浆中非索非那定的提取回收率、方法过程效率和基质效应均值分别为84.3%~89.6%、86.3%~90.8%和101.2%~104.2%；线性范围为1.00~1 000 ng·mL^-1（r=0.999 5）；日内、日间精密度RSD ≤ 10.9%，准确度RE%为-7.6%~5.0%；稳定性RSD和RE均在±15%内。18名志愿者给予灯盏花素或安慰剂后，非索非那定的达峰浓度C_max为（699±321）vs（710±331）ng·mL^-1、0到24 h的血浆浓度-时间曲线下面积AUC_0-24为（2 687.3±1 188.8）vs（3 015.0±1 550.2）ng·h·mL^-1、表观口服清除率CL/F为（51.3±23.8）vs（49.6±26.3）L·h^-1。结论：建立的基于UHPLC-MS/MS测定人血浆中P-糖蛋白底物非索非那定浓度的分析方法灵敏、简单，并成功应用于灯盏花素对人P-糖蛋白体内活性影响的研究。

Determination of fexofenadine in human plasma by UHPLC-MS/MS and its application

OBJECTIVE To develop a simple, sensitive and specific UHPLC-MS/MS method for the determination of the P-glycoprotein substrate fexofenadine in human plasma, and to study the effect of breviscapine on the activity of human P-glycoprotein.METHODS Sample processing was accomplished through a simple protein precipitation with 500 μL of acetonitrile to 200 μL plasma sample. The analytes were separated on a Waters ACQUITY UPLC^® BEH C18 column (50 mm×2.1 mm, 1.7 μm) using a mobile phase consisting of acetonitrile (A)-0.1% formic acid in water(B) by gradient elution. The flow rate was 0.3 mL·min^-1. The ionization was performed using electrospray ionization in positive ion (ESI+) by multiple reaction monitoring (MRM) mode for fexofenadine and diphenhydramine. The quantitative ion pairs of fexofenadine and diphenhydramine (internal standard) were m/z 502.2→466.5 and m/z 256.1→167.1, respectively.RESULTS The retention times of fexofenadine and diphenhydramine were 2.19 min and 1.89 min, respectively. The mean extraction recovery, mean process efficiency and mean matrix effect of fexofenadine were 84.3%-89.6%, 86.3%-90.8% and 101.2%-104.2%, respectively. The calibration curve was linear in the range of 1.0-1 000 ng·mL^-1(r=0.999 5). Intra-day and inter-day RSD% of precision were ≤ 10.9%, RE% of accuracy were in the range of -7.6%-5.0%, RSD% and RE% of stability were within 15%. After 18 volunteers were administrated with breviscapine or placebo, the peak concentration (Cmax), area under the plasma concentration time curve from 0 to 24 h (AUC_0-24) and apparent oral clearance (CL/F) of fexofenadine were (699±321) vs. (710±331) ng·mL^-1, (2 687.3±1 188.8) vs. (3 015.0±1 550.2) ng h·mL^-1 and (51.3±23.8) vs. (49.6±26.3) L·h^-1, respectively.CONCLUSION The method based on UHPLC-MS/MS for the determination of fexofenadine in human plasma is sensitive and simple, and is successfully applied to the study of the effect of breviscapine on the activity of human P-glycoprotein.