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目的建立灵敏、快速的液相色谱-串联质谱(LC-MS/MS)法测定大鼠血浆中靛玉红的浓度,并研究其在大鼠体内的药动学特点。方法血浆经乙腈沉淀蛋白后取上清进样分析,采用Agilent C_(18)柱,以乙腈:水(70:30,V/V)为流动相,ESI源,负离子检测,检测离子对为m/z 260.9→156.9(靛玉红)和m/z 253.0→225.0(大黄酚,内标)。大鼠禁食过夜后按20 mg·kg~(-1)剂量靛玉红混悬液灌胃,给药前及给药后0.25、0.5、0.75、1、2、3、4、6、8、10、12、24和32 h采血。LC-MS/MS法测定血浆药物浓度,绘制血药浓度-时间曲线图,统计矩法计算主要药动学参数。结果靛玉红线性范围为0.1～10μg·L~(-1),定量下限为0.1μg·L-1。低、中、高浓度质控样品的批内和批间精密度均小于8%,低、中、高浓度质控样品和内标提取回收率在93.6%～96.3%,室温稳定性、冻融稳定性及30 d稳定性考察结果均符合要求。药动学参数为t_(max)(4.2±1.0)h,p)(max)(3.7±0.7)μg·L~(-1),t_(1/2z)(4.7±2.5)h,AUC_(0-t)(22.8±8.7)μg·h·L~(-1)。结论所建立的LC-MS/MS法简便快捷、重复性好、灵敏度高,适合于靛玉红的体内药动学研究。
Objective To establish a sensitive and rapid liquid chromatography-tandem mass spectrometry (LC-MS / MS) method for the determination of indirubin in rat plasma and to study its pharmacokinetics in rats. Methods The plasma was precipitated with acetonitrile and the supernatant was injected into the sample. The Agilent C_ (18) column was used with mobile phase of acetonitrile and water (70:30, V / V) / z 260.9 → 156.9 (indirubin) and m / z 253.0 → 225.0 (chrysophanol, internal standard). Rats were fasted overnight by 20 mg · kg ~ (-1) dose of indirubin suspension intragastrically before administration and after administration 0.25,0.5,0.75,1,2,3,4,6,8 , 10,12,24 and 32 h blood sampling. The concentration of plasma drug was determined by LC-MS / MS, the plasma concentration-time curve was drawn, and the main pharmacokinetic parameters were calculated by statistical moment method. Results The linear range of indirubin was 0.1-10μg · L -1 and the lower limit of quantitation was 0.1μg · L -1. The intra- and inter-batch precision of low, middle and high quality control samples were less than 8%, and the recovery rates of low, medium and high quality control samples and internal standard samples were between 93.6% and 96.3% at room temperature, Stability and 30 d stability of the inspection results are in line with the requirements. The pharmacokinetic parameters were as follows: t max 4.2 ± 1.0 h, P max 3.7 ± 0.7 μg · L -1, t 1/2 / 4.7 ± 2.5 h, AUC_ ( 0-t) (22.8 ± 8.7) μg · h · L -1. Conclusion The established LC-MS / MS method is simple, rapid, reproducible and sensitive and suitable for the in vivo pharmacokinetics of indirubin.