Capillary electrophoretic determination of methotrexate, leucovorin and folic acid in human urine

A simple, rapid and sensitive procedure using capillary zone electrophoresis (CZE) to measure methotrexate, folinic acid and folic acid in human urine has been developed and validated. Optimum separation of methotrexate, folinic acid and folic acid was obtained on a 60 cm × 75 μm capillary using a 15 mM phosphate buffer solution (pH 12.0), temperature and voltage 20 °C and 25 kV, respectively and hydrodynamic injection. Under these conditions the analysis takes approximately 9.0 min. Good results were obtained for different aspects including stability of the solutions, linearity, accuracy and precision. Before CZE determination, the urine samples were purified and enriched by means of a solid phase extraction step with a preconditioned C18 cartridge and eluting the compound with a mixture 1:1 of methanol:water. A linear response over the urine concentration range 1.0-6.0 mg L−1 for MTX and 0.5-6.0 mg L−1 for folinic acid and folic acid was observed. Detection limits for the three compound in urine were 0.35 mg L−1. CZE was shown to be a good method with regard to simplicity, satisfactory precision, and sensitivity.