On-line stacking capillary electrophoresis for analysis of methotrexate and its eight metabolites in whole blood.

Therapeutic drug monitoring of methotrexate (MTX) and its metabolites is significant for the evaluation of treatment response of acute lymphoblastic leukemia and the prevention of adverse effects. Those eight metabolites including 7-hydroxymethotrexate, MTX-polyglutamate derivatives (MTX-(Glu) n , n = 2-7), and 2,4-diamino-N 10 -methylpteroic acid, especially the MTX-(Glu) n , only exist in blood cells and also present antifolate effects. Therefore, whole blood analysis has importance in clinical therapy. This study combined solid-phase extraction and on-line stacking capillary electrophoresis to examine the levels of MTX and its eight metabolites in whole blood. A higher conductivity buffer (HCB) was used to accumulate large amount of samples into a narrow zone, which resulted in effective stacking and sharp peaks. A fused-silica capillary was filled with phosphate buffer (80 mM, pH 2.5) containing 15% v/v tetrahydrofuran and 100 mM sodium dodecyl sulfate. Then HCB (100 mM phosphate, pH 2.5; 1 psi, 60 s) was injected for accumulating large volume of analytes (1 psi, 200 s). Owing to the pH difference between sample zone and HCB, dynamic pH junction was generated for focusing. Meanwhile, sweeping made further stacking by using sodium dodecyl sulfate in phosphate buffer. The limits of detection (S/N=3) were 0.1 μM for MTX, 0.2 μM for 7-hydroxymethotrexate and 2,4-diamino-N 10 -methylpteroic acid, 0.3 μM for MIX-(Glu) n = 2-5 , and 0.5 μM for MTX-(Glu) n = 6-7 . During validation, this method showed good linearity (r> 0.9914) and reproducibility (relative standard deviation and relative error, both less than 13%). The applications were performed to monitor blood MTX and its metabolites in acute lymphoblastic leukemia patients. The blood concentrations could provide some information related to therapeutic response and adverse effects.