高效液相色谱荧光法测定人红细胞内甲氨蝶呤及其谷氨酸聚合物浓度

Determination concentration of methotrexate and its polyglutamates in human erythrocyte by high-performance liquid chromatographic fluorescence method

目的建立一种灵敏、准确、简单地测定人红细胞中甲氨蝶呤(MTX)及多聚谷氨酸甲氨蝶呤(MTXPG2和MTXPG3)浓度的方法。方法采用双三元液相色谱系统串联荧光检测器,使用C18-WP柱(20 mm×4 mm,5μm)作为在线固相萃取柱,Athena C_(18)-WP柱(150 mm×4.6 mm,3μm)作为分析柱。红细胞裂解液用硫酸锌-10%甲酸的甲醇(100∶90,v/v)沉淀蛋白,柱后添加H_(2)O_(2)光化学衍生MTXPGs,荧光检测激发波长为274 nm,发射波长为470 nm,柱温为40℃,进样体积为100μL。考察该方法的专属性、标准曲线与定量下限、精密度与回收率、稳定性。结果MTX、MTXPG2和MTXPG3在12.5~400.0 nmol·L^(-1)线性良好,MTX的标准曲线为y=763.8x-2961.1(R^(2)=0.9995),提取回收率为60.7%~66.1%;MTXPG2的标准曲线为y=1017.8x-239.8(R^(2)=0.9984),提取回收率为67.2%~67.3%;MTXPG3的标准曲线为y=1069.1x-819.6(R^(2)=0.9994),提取回收率为62.9%~70.1%;日内精密度RSD<8.8%,日间精密度RSD<10.8%。结论本方法准确、重现性好,采用在线固相萃取富集及分离目标化合物,简化了样品前处理步骤,提高分析效率,适用于检测类风湿性关节炎患者红细胞内MTX、MTXPG2和MTXPG3浓度。

Objective To establish a sensitive,accurate and simple method for the determination of methotrexate and methotrexate polyglutamates(MTXPG2 and MTXPG3)in human erythrocytes.Methods A dual three element gradient liquid chromatograph with a fluorescence detector was used,the C18-WP column(20 mm×4 mm,5μm)was used as the online SPE column,and the Athena C18-WP column(150 mm×4.6 mm,3μm)was used as the analytical column.Erythrocyte lysate was precipitated with zinc sulphate-10%formic acid methanol(100∶90,v/v),and postcolumn photo-oxidation of MTXPGs to fluorescent analytes using H2O2.The fluorescence excitation wavelength was 274 nm,the emission wavelength was 470 nm,the column temperature was 40℃,and the injection volume was 100μL.The specificity,standard curve,lower limit of quantitation,precision,recovery and stability of the method were investigated.Results MTX,MTXPG2 and MTXPG3 had good linearity in the range of 12.5-400.0 nm ol·L^(-1).The standard curve of MTX was y=763.8x-2961.1(R^(2)=0.9995),and the extraction recovery rate was 60.7%-66.1%;the standard curve of MTXPG2 was y=1017.8x-239.8(R^(2)=0.9984),and the extraction recovery rate was 67.2%-67.3%;the standard curve of MTXPG3 was y=1069.1x-819.6(R^(2)=0.9994),the extraction recovery rate was 62.9%-70.1%.Intra-day precision RSD<8.8%,inter-day precision RSD<10.8%.Conclusion This method is accurate and reproducibility,and the online solid-phose extraction enrichment and separation of target compounds simplify the sample pretreatment steps,improve the analysis efficiency,and is suitable for detecting the concentration of MTX,MTXPG2 and MTXPG3 in erythrocytes of patients with rheumatoid arthritis.