Platform of Reversed Phase_Strong Cation Exchange_Reversed Phase Two_Dimensional High Performance Liquid Chromatography for Determination of Methotrexat in Plasma / 分析化学

ABSTRACT

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, 5μm), 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, 5μm) for the second separation. The mobile phase used for the first dimension was 10 mmol/L ammonium acetate_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 μmol/L (r=0. 99998). The LOQ was 0. 005 μmol/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.