HPLC法测定血清甲氨蝶呤浓度的方法建立及其与荧光偏振法的比较

An HPLC assay for the determination of methotrexate concentration in serum and its comparison to fluorescence polarization immunoassay

目的建立测定血清甲氨蝶呤（MTX）浓度的高效液相色谱（HPLC）法,并与荧光偏振法（FPIA）作比较,评价其临床实用性。方法血清样本经20%高氯酸沉淀蛋白质,吸取上清液进行HPLC分析。以MTX标准品外标法建立标准曲线,检测62例用药后患者血清MTX浓度,同时以FPIA测定。结果 HPLC法在0.049~25 ng/L内线性关系良好,日内及日间相对标准偏差（RSD）均〈6%,最低检测浓度为0.025 ng/L。HPLC法和FPIA具有良好的相关性（r=0.973,P〈0.01）,2种方法测定值的差异无统计学意义（P=0.705）。结论 HPLC法具有便捷、准确、成本低等特点,与FPIA相关性良好,适用于临床MTX的血药浓度监测。

Objective To establish a high performance liquid chromatography（HPLC） assay for the determination of methotrexate（MTX） concentration in serum and evaluate its application value by the comparison of fluorescence polarization immunoassay（FPIA）.Methods The serum after deproteinization with 20% perchloric acid was determined by HPLC.The standard curve was established with outer standard assay with standardization of MTX.The concentrations of MTX in 62 patients were determined by FPIA.Results By HPLC assay,the linear range was 0.049-25 ng/L.The relative standard deviations（RSD） of intra-day and inter-day were both below 6%.The limit of determination quantitation was 0.025 ng/L.The result of HPLC assay was relative to that of FPIA（r=0.973,P0.01）.There was no significant difference between the results of two methods（P=0.705）.Conclusions The method of HPLC for determination of serum MTX concentration are convenient,accurate,high sensitive and low-cost.The results of determination are relative to those of FPIA,thus this method is suitable to monitor the blood MTX concentration clinically.