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目的:建立超高效液相色谱-串联质谱法测定大鼠尿液和粪便中亚硫酸氢钠穿心莲内酯(ASB)含量,并用于研究大鼠静脉注射ASB在尿液和粪便中的排泄。方法:采用甲醇提取生物样品中的ASB,以脱水穿心莲内酯(DAG)为内标,在HypersilGold C18色谱柱(50 mm×2.1 mm,1.9μm)上以流动相甲醇-水梯度洗脱,流速0.2 mL·min-1,柱温35℃,分析时间6 min;在电喷雾离子化电离源上以选择反应监测方式进行负离子检测,用于定量分析的离子反应分别为m/z 413.2→287.2(ASB)和m/z 331.2→303.3(DAG)。大鼠自尾静脉注射80 mg.kg-1ASB,收集并测定给药后各时段的尿液和粪便中ASB浓度,计算各时段尿液和粪便中累计排泄率。结果:测定ASB的线性范围为50～5000 ng·mL-1,方法定量下限为50 ng·mL-1。日内和日间精密度(RSD)分别在11.2%和13.3%以内,准确度分别在85.8%～101.4%和87.9%～97.5%,提取回收率为96.1%～98.3%,基质效应为96.2%～98.1%。大鼠72 h尿液中累计排泄率为16.9%±3.9%,粪便中为34.1%±18.4%。结论:本方法速度快、专属性好、准确度高,适用于大鼠尿液和粪便中ASB含量测定,可应用于ASB大鼠排泄研究。
OBJECTIVE: To establish a method for determination of andrographolide (ASB) in rat urine and feces by ultra performance liquid chromatography-tandem mass spectrometry and to study the excretion of ASB in urine and feces of rats after intravenous injection. Methods: The ASB in biological samples was extracted with methanol, and the dehydroandrographolide (DAG) was used as internal standard. The mobile phase was eluted with methanol - water gradient on a HypersilGold C18 column (50 mm × 2.1 mm, 1.9 μm) 0.2 mL · min-1, the column temperature was 35 ℃ and the analysis time was 6 min. Negative ion detection was performed on the electrospray ionization source by selective reaction monitoring. The ion reactions for quantitative analysis were m / z 413.2 → 287.2 ASB) and m / z 331.2 → 303.3 (DAG). The rats were injected with 80 mg.kg-1 ASB from the tail vein to collect and measure the ASB concentration in the urine and feces of each time period after administration. The cumulative excretion rate of urine and feces in each time period was calculated. Results: The linear range of ASB was 50-5000 ng · mL-1, and the limit of quantification was 50 ng · mL-1. The intra-day and inter-day precision (RSD) were within 11.2% and 13.3%, respectively, with the accuracy of 85.8% -101.4% and 87.9% -97.5% respectively. The recovery rates were 96.1% -98.3% and the matrix effects were 96.2% 98.1%. The cumulative urinary excretion rate in rats at 72 h was 16.9% ± 3.9% and in feces 34.1% ± 18.4%. Conclusion: The method is rapid, specific and accurate. It is suitable for the determination of ASB in urine and feces of rats and can be applied to excretion of ASB rats.