反向模式-强阳离子交换-反向模式二维色谱法测定人血浆中甲氨蝶呤浓度

Platform of Reversed Phase-Strong Cation Exchange-Reversed Phase Two-Dimensional High Performance Liquid Chromatography for Determination of Methotrexat in Plasma

建立了反向模式-强阳离子交换-反向模式（Reversed phase-strong cation exchange-reversed phase）二维色谱平台测定人血浆中甲氨蝶呤浓度的方法。样品经三氯醋酸沉淀蛋白后,在ASTON C8一级柱（100 mm×4.6 mm,5μm）上完成初分离,通过六通阀切换,经ASTON SCX中间级（20 mm×4.6 mm,5μm）二次分离和储存,在SAC C8二级柱（100 mm×4.6 mm,5μm）上完成最后分离,并测定。一级柱流动相为10 mmol/L醋酸铵-乙腈（90∶10,V/V,以醋酸调至pH 3.8）,流速为1.0 mL/min;中间级流动相为10 mmol/L H3PO4溶液（pH 3.0）;二级柱流动相为50 mmol/L醋酸铵-乙腈（87∶13,V/V,以醋酸调至pH 5.2）,流速为1.2 mL/min,检测波长306 nm。单次分析时间4 min,线性范围0.09～5.1μmol/L,检出限为0.005μmol/L,日内RSD小于1.8%,日间RSD小于3.5%,相对回收率99.1%～101.25%,绝对回收率85.7%～86.4%。本方法简便、准确,适合日常血药浓度监测和药代动力学研究。

A two-dimensional HPLC method was developed for the determination of methotrexat （MTX） in human plasma. The samples were treated with trichloroacetate for sedimentation and high speed centrifugation, and the obtained supernatant was taken for analysis. The analytes in sample were separated on the first dimension column （ASTON C8 100 mm × 4.6 mm, 5 μm） , and trapped on the middle column （ASTON SCX 20 mm × 4.6 mm, 5 μm） using valve-switching technique for purification and storage. Finally, the trapped analytes were transferred to the second-dimension column （SAC C8 100 mm ×4.6 mm, 5 μm） for the second separation. The mobile phase used for the first dimension was 10 mmol/L ammonium aeetate-acetonitrile（9： 1, V/V, pH = 3.8 ） with a flow rate of 1 mL/min and the mobile phase used for middle column was 10 mmoL/L phosphoric acid （pH = 3.0）. The mobile phase used in second-dimension was a mixed solution of 50 mmol/L ammonium acetate and acetonitrile （87：10, V/V, pH = 5.2）. UV detection was carried out at 306 nm and completed in 4 min. The calibration curve showed a linearity range from 0. 0879 to 5. 154 p, mol/L （r=0. 99998）. The LOQ was 0. 005 p, mol/L. The intra-and inter-day precisions were lower than 1.5% and 1. 8%, respectively. The relative recovery and the absolute recovery were 99. 1% -101. 2% and 85.67%-86.35%, respectively. The assay is simple, accurate, reproducible, and suitable for the therapeutic drug monitoring of MTX in the hospital and the study on the pharmacokinetics of MTX.