Simultaneous determination of methotrexate and its eight metabolites in human whole blood by capillary zone electrophoresis

A simple and selective capillary electrophoretic method was established for the simultaneous determination of methotrexate (MTX), 7-hydroxymethotrexate (7-OHMTX), 2,4-diamino-N10-methylpteroic acid (DAMPA), and polyglutamate derivatives [MTX–(Glu)n, n=2–7] in whole blood. After extraction, those analytes were separated by fused-silica capillary and a running buffer containing glycine (1.2 M, pH 9.3). The quantitative ranges were 10–50 μM for each analyte. The intra- and inter-day RSD and RE values were all less than 6 and 11%, respectively. The limits of detection (S/N=3, injection 5 s) were found to be 1 μM for MTX, 7-OHMTX, MTX–(Glu)2, MTX–(Glu)3, and MTX–(Glu)4; 3 μM for MTX–(Glu)5 and MTX–(Glu)6; 5 μM for MTX–(Glu)7, and 8 μM for DAMPA. All recoveries were greater than 94%. This method was applied to blood MTX monitoring in a patient with acute lymphoblastic leukemia.