优化的HPLC法测定人血清中甲氨蝶呤的浓度及临床应用

目的:建立及优化测定人血清中甲氨蝶呤(MTX)浓度的方法。方法:血样采用15%高氯酸溶液处理后采用高效液相色谱法(HPLC)进样测定。色谱柱为大连依利特ODS C18,流动相为甲醇-10 mmol/L磷酸二氢钾(20∶80,用氢氧化钠调pH 6.5),流速为1.0 ml/min,柱温为35℃,紫外检测波长为303 nm。结果:MTX血药浓度在0.1⁶4μmol/L范围内线性关系良好(r=0.999 5)。MTX低、中、高3种浓度的日内RSD分别为2.70%、1.08%、0.94%,日间RSD分别为4.36%、2.71%、1.95%(n=5),提取回收率分别为70.9%、78.3%、75.4%,最低检测限为0.03μmol/L。结论:优化后的方法准确可靠、灵敏度高、重现性好,适用于大剂量MTX血药浓度监测。

Concentration Determination of Methotrexate in Human Serum by Optimized HPLC and Clinical Application

OBJECTIVE： To establish and optimize HPLC method for concentration determination of methotrecate （MTX） in human serum. METHODS： Blood samples were treated with 15% Perchloric acid solution. The determination was performed on ODS C18 column with mobile phase consisted of methanol-10 mmol/L potassium dihydrogen phosphate （20：80, pH adjusted to 6.0 using sodium hydroxide） at the flow rate of 1.0 ml/min. The column temperature was set at 35℃ , and UV detection wavelength was set at 303 nm. RESULTS： The linear range of MTX was 0.1-64 μmol/L （r：0.999 5）. Intra-day RSDs of low, medium and high concentrations of MTX were 2.70%, 1.08% and 0.94%, and inter-day RSDs of them were 4.36%, 2.71% and 1.95%, re- spectively （n=5）. The extraction recoveries were 70.9%, 78.3% and 75.4%, respectively. The lowest detection limit was 0.03μmol/L. CONCLUSIONS： The optimized method is accurate, reliable, sensitive and reproducible, and can be used for the serum concentration monitoring of large-dose MTX in the clinic.