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

A new method using MCE with LIF detection was developed for the determination of hydrogen peroxide (H(2)O(2)). 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 H(2)O(2) 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 microM with an LOD (S/N=3) of 0.20 microM (19.1 amol). The proposed method was applied to determine H(2)O(2) in phorbol myristate acetate-stimulated RAW264.7 macrophages, the concentration of H(2)O(2) was found to be 1.86+/-0.05 microM; 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.