HPLC-MS/MS同时测定广金钱草中8种成分在大鼠胆汁中的浓度及其排泄动力学研究

Simultaneous determination of 8 constituents of Desmodii Styracifolii Herba in rat bile with HPLC-MS/MS and their excretion pharmacokinetics

建立同时测定灌胃给予大鼠广金钱草提取物后胆汁中7种黄酮和1种酚酸类化合物浓度的HPLC-MS/MS,并用于胆汁排泄研究。大鼠灌胃给予广金钱草提取物后,分别在0~1,1~2,2~4,4~6,6~8,8~12,12~24 h时间段收集胆汁。色谱柱采用Diamonsil C18column(4.6 mm×150 mm,5μm),柱温40℃。以甲醇-0.01%乙酸水溶液为流动相,梯度洗脱,流速0.8 m L·min^(-1)。质谱采用电喷雾离子源(ESI),负离子模式下检测,多反应离子监测(MRM)模式进行定量。结果表明8种成分在测定浓度范围内线性关系良好(r≥0.991 5),日内、日间精密度RSD<15%,准确度RE-15%~15%,稳定性良好。提取回收率>63.2%,内标归一化的基质因子RSD均未超过15%,符合要求。大鼠胆汁排泄研究结果显示,8种成分在胆汁中的原型排泄率均比较小,并且不同个体之间差别较大。该研究方法灵敏度高,选择性好,样品处理简单,可用于灌胃给予大鼠广金钱草提取物后胆汁中8种成分的排泄动力学研究。

To establish a HPLC-MS/MS quantitative method for analysis of 8 constituents in rat bile. The method was applied in the biliary excretion study after oral administration of Desmodii Styracifolii Herba extract. The rat bile samples were collected during 0-1,1-2,2-4,4-6,6-8,8-12,12-24 h. Diamonsil C_（18） column（ 4. 6 mm × 150 mm,5 μm） was adopted and eluted with methanol and0. 01% acetic acid in a gradient mode. The flow rate was 0. 8 m L·min^（-1）,and the column temperature was set at 40 ℃. The detection was carried out by a triple quadrupole linearion trap mass spectrometer in the negative ion mode with an electrospray source. Multiple reactions monitoring（ MRM） mode was employed. The calibration curves showed a good linearity,with correlation coefficients（ r≥0. 991 5） for all of the analytes within the concentration range. The intra-day and inter-day precisions（ RSD） were both less than15%,and the accuracies（ RE） ranged from-15% to 15%. The method had a good stability,and was suitable for the content determination of 8 constituents in rat bile. The extraction recoveries of the 8 analytes were more than 63. 2%,and the RSD of IS-normalized matrix factors were no more than 15%,which met the requirements for analysis. According to the biliary excretion study,the bile excretion rates of the 8 analytes were relatively low,with great difference among the individuals. The results showed that the established method was simple,selective and specific,thus could be applied in the biliary excretion study of the 8 analytes in rat bile.