HPLC-MS/MS法测定白血病患者血浆甲氨蝶呤浓度

摘要

Objective:To establish a HPLC-MS/MS method to determine methotrexate in Leukemia patients plasma.Methods:The method employs a direct precipitation with methanol containing doxofylline as the internal standard and a reversed phase separation in an Ultimate XB-C18 column. Mobile phase was a mixture of water (containing 2mmol•L-1 ammonium acetate and 0.1% formic acid) and methanol (containing 0.1% formic acid). The flow rate was 0.75 mL•min-1 and the column temperature was 40℃. Mass spectrometric analysis was performed in positive ion mode and the multiple reaction monitoring transitions ofm/z 455.200→308.200 andm/z 267.100→181.100 were used to quantify methotrexate and doxofylline (internal standard), respectively.Results: The calibration curve of methotrexate was linear within range of 0.022-2.200 μmol•L-1,Y=4.56X+0.081,r=0.996 0.Conclusion: This method is convenient, accurate and rapid and it is suitable for detecting methotrexate in leukemia patients plasma.%目的：建立测定白血病患者使用大剂量甲氨蝶呤化疗后血浆中甲氨蝶呤浓度的HPLC-MS/MS法，并应用于临床治疗药物监测，为临床合理用药提供可靠依据。方法：采用多索茶碱作内标，血浆样品经含内标甲醇沉淀蛋白处理。色谱柱为Ultimate XB-C18，柱温40℃，流速0.75 mL·min-1，流动相为含0.1%甲酸和2 mmol·L-1乙酸铵的水溶液和含0.1%甲酸的甲醇溶液，梯度洗脱。质谱检测方式为电喷雾离子阱正离子模式，MRM扫描，监测甲氨蝶呤m/z 455.200→308.200，多索茶碱m/z 267.100→181.100。结果：甲氨蝶呤浓度在0.022~2.200μmol·L-1内线性关系良好，Y=4.56x +0.081(r＝0.9960)。结论：该方法简便、准确、快速，检测的特异性及灵敏度高，适用于甲氨蝶呤的血药浓度测定。