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目的建立风湿免疫病患者服用甲氨蝶呤的液相色谱-质谱联用血药浓度测定方法。方法采用甲醇沉淀蛋白,以氨基蝶呤作为内标,用Epic C18色谱柱(4.6 mm×50 mm,3μm),甲醇(含0.1%甲酸)-水(含0.1%甲酸)为流动相,梯度洗脱,流速0.6 m L·min~(-1),柱温40℃。用电喷雾离子化源,正离子模式,多反应监测(MRM)扫描方式进行监测。考察方法的专属性、标准曲线、定量下限、精密度、准确度、回收率、基质效应和稳定性。结果甲氨蝶呤在0.5~200.0 ng·m L~(-1)范围内线性良好(r=0.999 2),定量下限为0.5ng·m L~(-1),日内、日间精密度及准确度RSD<15%,回收率为76.80%~85.49%。结论该检测方法快速、灵敏,满足对服用甲氨蝶呤72 h内的风湿免疫病患者血药浓度的准确测定。
Objective To establish a method for the determination of methotrexate in patients with rheumatoid disease by liquid chromatography-mass spectrometry. Methods The protein was precipitated with methanol and aminopterin was used as an internal standard. The mobile phase consisted of Epic C18 column (4.6 mm × 50 mm, 3 μm), methanol (containing 0.1% formic acid) and water Off, flow rate 0.6 m L · min -1, column temperature 40 ℃. Electrospray ionization, positive ion mode, multiple reaction monitoring (MRM) scanning were used. Investigate method specificity, standard curves, lower quantitation limits, precision, accuracy, recovery, matrix effects and stability. Results Methotrexate showed a good linearity (r = 0.999 2) within 0.5-200.0 ng · m L -1 and a lower limit of quantitation of 0.5 ng · m L -1. The intra- and inter-day precision and Accuracy RSD <15%, the recovery rate was 76.80% ~ 85.49%. Conclusion The detection method is rapid and sensitive, and can meet the requirement of accurate determination of plasma concentration of rheumatoid immunity in patients taking methotrexate within 72 h.