LC-MS/MS法同时测定大鼠血浆中L-色氨酸和L-犬尿氨酸浓度的研究

Simultaneous determination of L-tryptophan and L-kynurenine in rat plasma by LC-MS/MS

本研究运用液相色谱-二级质谱连用技术,以3-硝基酪氨酸（3-NT）为内标,建立了可同时、快速测定大鼠血浆中L-色氨酸（L-Try）和L-犬尿氨酸（L-Kyn）的LC-MS/MS法,通过比较肝癌成模过程中L-Kyn和L-Try的变化来为肝癌的诊断提供理论依据。色谱条件：RRHD Eclipse Plus C（18）色谱柱（3.0 mm×100 mm,1.8μm）,以水-乙腈（含0.1%甲酸）（90∶10）为流动相洗脱,流速0.25 mL/min,进样量5μL。质谱条件：采用MRM模式进行定量检测,化合物质荷比分别为205.12→146.10（L-Try）,209.09→146.10（L-Kyn）,227.09→181.10（3-NT）。结果显示：L-Try和L-Kyn的线性范围分别是9.670-9 670和9.973-9 973 ng/mL（r^2≥0.999 0）,L-Try和L-Kyn的定量限分别为9.670和9.973 ng/mL,日内、日间精密度均小于15%。整个分析物的回收率均大于81.17%,并没有表现出严重的基质效应,表明肝癌成模过程中大鼠血浆L-Kyn/L-Try比值呈现整体下降趋势。L-Kyn/L-Try比值的变化是肝肿瘤形成过程中一个非常明显的变化,可为药物代谢研究和肝癌机制研究发挥重要作用。该方法灵敏、快捷、准确,专一性强,可用于药物代谢的分析研究及药物对肝癌的作用机制研究。

To establish a simple and sensitive LC-MS/MS method for simultaneous determination of the concentration for L-tryptophan（L-Try）and L-kynurenine（L-Kyn）in rat plasma. The changesin the process of the liver tumors formation may provide a basis for the diagnosis of liver cancer. 3-Nitro-L-tyrosine（3-NT）were added as the internal standard for the determination of two active substances and the chromatographic analysis was performed on a RRHD Eclipse Plus C18 column（3. 0 mm×100 mm, 1. 8 μm）. The mobile phase was composed of water and acetonitrile（containing 0. 1%formic acid）（90 ∶10）at a flow rate of 0. 25 mL/min, and the injection volume is 5 μL. Detection and quantification were performed by mass spectrometry in multiple reaction monitoring mode with m/z 205. 12→146. 10（L-Try）, m/z 209. 09 →146. 10（L-Kyn）, m/z 227. 09→181. 10（3-NT）, respectively. The results show that thelinear ranges were 9. 670-9 670 ng/mL for L-Try, and 9. 973-9973 ng/mL for L-Kyn（r^2≥0. 9990）. The limit of quantitation were 9. 670 ng/mL for L-Try, and 9. 973 ng/mL for L-Kyn, respectively. The intra- and inter-day precisions were all less than15%; the recoveries ofthe two analytes were more than 81. 17% and severe matrix effect was not observed. The ratio of L-Try/L-Kyn determined by LC-MS/MS in rat plasma showed an overall downward trend, which could used effectively for the drug metabolism studies and researches on the action mechanism of medicine on liver cancer. A rapid, simple, sensitive and specific LC-MS/MS method has been successfully developed and could also be used effectively for the drug metabolism studies and researches on the action mechanism of medicine on liver cancer.