Gentiopicroside is a natural secoiridoid glycoside that may require metabolic activation to exert pharmacological effects. In this study, two active metabolites of gentiopicroside (M1 and M2) were isolated from rat urines and identified with our previous method. Most importantly, a fast, sensitive and selective ultra high-performance liquid chromatography–tandem mass spectrometry method was developed to simultaneously determine gentiopicroside and its two metabolites in rat plasma. The analytes and internal standard (swertiamarin) were separated on an ACQUITY UPLC^® BEH C18 column (2.1 × 50 mm, 1.7 μm) using gradient elution by acetonitrile and 0.1% formic acid at a flow rate of 0.4 mL/min. The mass spectrometry detector was operated in the multiple reaction monitoring with positive ionization mode. The method had a good linearity over the concentration range of 0.2–10,000 ng/mL for gentiopicroside and 0.1–5000 ng/mL for the two metabolites. The validated method was successfully applied to the pharmacokinetic study of gentiopicroside and its metabolites after single oral administration of gentiopicroside (150 mg/kg) to rats (n = 8). The pharmacokinetic differences between gentiopicroside and its two metabolites were identified.Results provided the evidence for in vivo metabolism-based activation of gentiopicroside.

龙胆苦甙是天然的类蛇药苷，可能需要代谢激活才能发挥药理作用。在这项研究中，从大鼠尿液中分离了两种龙胆苦甙活性代谢物（M1和M2），并用我们以前的方法进行了鉴定。最重要的是，开发了一种快速，灵敏且选择性的超高效液相色谱-串联质谱法，用于同时测定大鼠血浆中的龙胆苦苷及其两种代谢物。分析物和内标物（獐牙菜苦）上分离的ACQUITY UPLC ^®BEH C18色谱柱（2.1×50 mm，1.7μm），使用乙腈和0.1％甲酸进行梯度洗脱，流速为0.4 mL / min。质谱检测器以正电离模式在多反应监测中运行。该方法在龙胆苦苷的浓度范围为0.2–10,000 ng / mL和两种代谢物的浓度范围为0.1–5000 ng / mL时具有良好的线性。经验证的方法已成功应用于龙胆苦甙和其代谢产物在大鼠中单次口服龙胆苦甙（150 mg / kg）的药代动力学研究（n = 8）。鉴定了龙胆苦甙和其两种代谢产物之间的药代动力学差异。结果为基于龙胆苦甙的体内代谢活化提供了证据。