OBJECTIVE:To develop HPLC method for determination of the concentration of methotrexatate ( MTX) in human plasma.METHODS: The one point method was compared to the standard curve method in determining the concentration of methotrexatate ( MTX ) in human plasma.RESULTS: In HPLC determination of the concentration of methotrexatate (MTX) in human plasma, the linear range of MTX was 0.05-5.00 g/ml by the standard curve method with the recovery and RSD up to the standards for the determination and the linear range of MTX was 0.36-0.65 g/ml by one point method with the recovery and RSD up to the standards for the determination.The two methods showed similar results in determination of the concentration of methotrexatate (MTX) in human plasma at 44 hours after oral administration of MTX, and there was no statistically significant difference (P>0.05).CONCLUSIONS:The one point method was simple, accurate, precise and reproducible and it is suitable fore clinical determination of plasma concentration of MTX.%目的：建立甲氨蝶呤（ MTX）血浆浓度的高效液相色谱测定方法。方法：利用外标一点法检测甲氨蝶呤血浆浓度，并与标准曲线法进行对比。结果：使用高效液相色谱法测定甲氨蝶呤血浆浓度时，标准曲线法在0.05～5.00μg／ml范围内药物浓度与峰面积呈现良好的线性关系，回收率、相对标准偏差符合测定要求；外标一点法在0.36～0.65μg／ml范围内药物浓度与峰面积呈现良好的线性关系，回收率、相对标准偏差符合要求。2种方法对患者服药44 h后甲氨蝶呤血浆浓度测定结果一致，差异无统计学意义（ P＞0.05）。结论：外标一点法简单、快速，精密度和重复性较好，适用于临床甲氨蝶呤血浆浓度测定。
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