LC-MS测定正常及非酒精性脂肪肝模型大鼠血浆中冬青素A的质量浓度

Quantitative determination of ilexgenin A in control and nonalcoholic fatty liver disease rats plasma by liquid chromatography coupled with mass spectrometry

目的建立正常及非酒精性脂肪肝模型大鼠血浆中冬青素A质量浓度的液质联用测定方法,同时比较其代谢差异。方法采集到的血浆样品以三七皂苷Rc为内标,经固相萃取柱处理后进行LC-MS分析。色谱条件:岛津Shim-pack CLC-ODS C18柱(150mm×6.0mm,5μm);流动相A为1mL.L-1甲酸水溶液,B为1mL.L-1甲酸乙腈溶液,梯度洗脱;质谱条件采用大气压电喷雾离子源(ESI源),负离子方式,选择离子检测(SIM),检测离子分别为m/z501.1(冬青素A)和m/z1 078.3(三七皂苷Rc)。结果方法学考察表明,该法定量限为2.5×10-3μg.mL-1,在2.5×10-3～5.0μg.mL-1范围内线性关系良好,日内和日间精密度(RSD)均小于10%,提取回收率在85%～115%范围内。结论该方法灵敏性高,专属性强,适用于正常及非酒精性脂肪肝模型大鼠血浆中冬青素A的药动学研究。

Objective To determine the concentration of ilexgenin A in control and nonalcoholic fatty liver disease(NAFLD) rats plasma by LC-MS,and compare the differences of those metabolism.Methods Using notoginsenoside Rc as the internal standard,the plasma samples were processed by solid-phase extraction and analyzed by LC-MS.Chromatographic separation was performed on a SHIMADZU Shim-pack CLC-ODS C18 column(150 mm ×6.0 mm,5 μm).The mobile phase consisted of A(1 mL·L-1 formic acid-water) and B(1 mL·L-1 formic acid-ACN),with gradient elution.Mass spectra were equipped with an electrospray ionization(ESI) source,operated in the negative ion detection mode.In selected ion monitoring(SIM),the ion combinations of m/z 501.1 and m/z 1 078.3 was used to qualify ilexgenin A and notoginsenoside Rc respectively.Result The proposed method was validated with a linear range of 2.5 × 10-3-5.0 μg·mL-1.The limit of quantification(LOQ) was 2.5 × 10-3 μg·mL-1.Intra-day and inter-day precision expressed as relative standard deviation(RSD) were less than 10%,and the average recovery was in the range of 85%-115%.Conclusion The method with high sensitivity and good specificity was used successfully for the pharmacokinetic study of ilexgenin A in control and NAFLD rats after oral administration of ilexgenin A in single dose.