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目的建立及优化准确、简便的用于测定人血清中甲氨蝶呤浓度的HPLC方法。方法色谱柱:ACE 5C18-AR柱,流动相:甲醇-3%冰醋酸溶液(pH 3.5)(20∶80),检测波长306nm,柱温40℃。血清样品用高氯酸沉淀蛋白,取上清液,再用氢氧化钠中和后,取40μL进样分析。结果色谱峰分离良好,无干扰。甲氨蝶呤低浓度(0.05~3.40μmol·L-1)和高浓度(3.40~220.0μmol·L-1)的标准曲线线性均良好,r均=0.999 7。日内及日间RSD均<5.0%。结论本优化方法操作简便、灵敏准确,且重复性好,适用于甲氨蝶呤血药浓度检测。
Objective To establish and optimize an accurate and simple HPLC method for the determination of methotrexate in human serum. Methods Column: ACE 5C18-AR column, mobile phase: methanol-3% glacial acetic acid (pH 3.5) (20:80), detection wavelength 306nm, column temperature 40 ℃. Serum samples were precipitated with perchloric acid, the supernatant, and then neutralized with sodium hydroxide, 40μL injection analysis. Result peak separation, no interference. The standard curves of methotrexate with low concentration (0.05-3.40μmol·L-1) and high concentration (3.40-220.0μmol·L-1) showed good linearity with r = 0.999 7. RSDs were both <5.0% during and after the day. Conclusion The optimized method is simple, sensitive and accurate, and reproducible. It is suitable for the detection of methotrexate blood concentration.