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目的:建立HPLC-MS/MS法测定大鼠血浆中金丝桃素的浓度。方法:血浆样本加入适量内标,经乙腈直接沉淀蛋白后采用HPLC-MS/MS进行分析。色谱柱采用Ultimate C18柱(150 mm×2.1 mm,5.0μm);流动相由乙腈-5 mmol·L-1醋酸铵(含0.1%甲酸)(90∶10)组成,柱温35℃;流速0.5 ml·min-1;采用电喷雾离子源(ESI),以多反应监测方式(MRM)进行定量分析。金丝桃素和内标吡格列酮在负离子模式下定量分析离子对分别为m/z 503.2→m/z 405.1和m/z 355.0→m/z 41.9。结果:金丝桃素在0.1~13.2 ng·ml-1线性关系良好(r>0.99),最低定量限为0.1 ng·ml-1,提取回收率为84.19%~98.71%,日内、日间精密度均不高于18.47%。结论:该方法分析速度快、灵敏、准确,为临床进一步研究金丝桃素提供了基础。
OBJECTIVE: To establish a HPLC-MS / MS method for the determination of hypericin in rat plasma. Methods: The plasma samples were spiked with appropriate amount of internal standard, and the proteins were directly precipitated by acetonitrile and analyzed by HPLC-MS / MS. The column was equipped with an Ultimate C18 column (150 mm × 2.1 mm, 5.0 μm). The mobile phase consisted of acetonitrile-5 mmol·L-1 ammonium acetate (0.1% formic acid) (90:10) ml · min-1. Quantitative analysis was carried out by multiple reaction monitoring (MRM) using electrospray ionization (ESI). Hypericin and the internal standard pioglitazone quantified ion pairs in negative ion mode were m / z 503.2 → m / z 405.1 and m / z 355.0 → m / z 41.9, respectively. RESULTS: Hypericin showed a good linearity (r> 0.99) at a linear range of 0.1-13.2 ng · ml-1 with a minimum limit of quantification of 0.1 ng · ml-1. The recoveries ranged from 84.19% to 98.71%. The intra- and inter- Degrees are not higher than 18.47%. Conclusion: The method is rapid, sensitive and accurate and provides the basis for clinical further study of hypericin.