A new route for simple and rapid determination of hydrogen peroxide in RAW264.7 macrophages by microchip electrophoresis

A new method using MCE with LIF detection was developed for the determination of hydrogen peroxide (H2O2). Bis(p-methylbenzenesulfonyl)dichlorofluorescein, a new fluorogenic reagent synthesized by our laboratory was employed as a labeling reagent, the derivatization reaction was performed in 0.10 M HEPES buffer (pH 7.4) for 30 min at 37 degrees C. The detection of H2O2 was accomplished in 55 s, using a 40 mM HEPES buffer, 20% mannitol, pH 7.4, on a glass microchip. The RSDs of migration time and peak area were 1.8 and 3.7%, respectively. Method validation showed the linear response ranging from 0.50 to 50 mu M with an LOD (S/N = 3) of 0.20 mu M (19.1 amol). The proposed method was applied to determine H2O2 in phorbol myristate acetate-stimulated RAW264.7 macrophages, the concentration of H2O2 was found to be 1.86 +/- 0.05 mu M; recoveries for macrophage samples were from 96.7 to 97.8%, within-days and between-days accuracies were 4.5% (n = 5) and 6.8% (n = 5), respectively.