液相色谱-串联质谱法测定大鼠血浆中靛玉红的含量及其药动学

Pharmacokinetics of indirubin in rat plasma by liquid chromatography-tandem mass spectrometric method

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。

AIM To develop a sensitive and rapid liquid chromatography-tandem mass spectrometric method （LC-MS/MS） for the determination of indirubin in rat plasma, and pharmacokinetic study of indirubin. METHODS After a simple protein precipitation by acetonitrile, internal standard chrysophanol and indirubin in plasma were separated on an Agilent Cls column. The mobile phase consisted of acetonitrile-water （70 ： 30, V/V）, at a flow-rate of 0.30 mL .min-1 An API5000 tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the negative ion mode. Multiple reaction monitoring （MRM） using the precursor to product ion combinations of m/z 260.9-156.9 and m/z 253.0--225.0 was performed to quantify indirubin and the internal standard chrysophanol, respectively. Six rats were administrated with indirubin 20 mg.kg-1 by gavage. Rats＇ blood samples were collected before and at 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 12, 24, and 32 h after administration. Plasma drug concentrations were determined by LC-MS/ MS and drug concentration-time curve was drawn. The main pharmacokinetic parameters were calculated by statistical moment method. RESULTS The linear calibration curves of indirubin were obtained in the range of 0.1 - 10 ug.L-1 in rat plasma. The lower limit of quantification （LLOQ） was 0.1 ug.L-1. The intra-and interbatch precision （RSD） was below 8% calculated from quality control （QC） samples. The extraction recovery of low, medium, and high concentration QC samples and the internal standard were between 93.6% and 96.3%. The room temperature, freeze-thaw, and 30-day stability test results meet the requirements. After oral admini- stration of 20 mg.kg-1 dose of indirubin, the pharmacokinetic parameters of indirubin were （4.2 ± 1.0） h for tmax , （3.7 ± 0.7） ug.L-1 for pmax , （4.7 ± 2.5） h for t1/2 z, （22.8 ± 8.7） ug.h.L-1 for AUC0-t. CONCLUSION The method is simple, sensitive, as well as rapid, and is suitable for application in pharmacokinetic study of indirnbin.