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目的:建立测定人血清中甲氨喋呤及其代谢产物7-羟基甲氨喋呤的浓度的LC-MS/MS法,并应用于临床治疗药物监测。方法:采用D3-甲氨蝶呤同位素内标,血清样品经含内标乙腈沉淀蛋白处理。HPLC色谱柱为Sepax GP-pHenyl,柱温35℃,流速0.3 ML·min~(-1),流动相为0.1%甲酸和乙腈,梯度洗脱。质谱检测方式为ESI正离子模式,MRM扫描,监测甲氨蝶呤m/z 455～308,7羟基甲氨蝶呤m/z 471～324,D3甲氨蝶呤(内标)m/z 458～311。结果:甲氨蝶呤浓度在5～4 000 ng·ml~(-1)范围内与峰面积线性关系良好(r=0.999 9),7-羟基甲氨蝶呤浓度在1～800 ng·ml~(-1)范围内与峰面积线性关系良好(r=0.999 7)。甲氨蝶呤及7-羟基甲氨蝶呤日内及日间RSD<9.0%。甲氨蝶呤平均回收率为94.34%～115.08%,7-羟基甲氨蝶呤平均回收率为96.41%～103.04%。结论:本方法简便、准确、快速,适用于甲氨喋呤及其代谢产物7-羟基甲氨喋呤的血药浓度测定。
OBJECTIVE: To establish a LC-MS / MS method for the determination of methotrexate and its metabolite 7-hydroxy methotrexate in human serum and its application in the monitoring of clinical therapeutic drugs. METHODS: D3-methotrexate isotope internal standard was used and serum samples were treated with acetonitrile-precipitated protein containing internal standard. The HPLC column was Sepax GP-pHenyl. The column temperature was 35 ℃ and the flow rate was 0.3 mL · min -1. The mobile phase consisted of 0.1% formic acid and acetonitrile with gradient elution. Mass spectrometry detection mode was ESI positive mode, MRM scanning, monitoring methotrexate m / z 455 ~ 308,7 hydroxy methotrexate m / z 471 ~ 324, D3 methotrexate (internal standard) m / z 458 ~ 311. Results: The concentration of methotrexate in the range of 5 ~ 4 000 ng · ml -1 had a good linear relationship with the peak area (r = 0.999 9), and the concentration of methotrexate in the range of 1 ~ 800 ng · ml ~ (-1) and the peak area of a good linear relationship (r = 0.999 7). Methotrexate and 7-hydroxy methotrexate day and day RSD <9.0%. The average recoveries of methotrexate ranged from 94.34% to 115.08%, and the average recoveries of 7-hydroxymethothotathimines ranged from 96.41% to 103.04%. Conclusion: The method is simple, accurate and rapid and suitable for the determination of methotrexate and its metabolite 7-hydroxy methotrexate.