A new route for simple and rapid determination of hydrogen peroxide in RAW264.7 macrophages by microchip electrophoresis
✍ Scribed by Xiaocong Gong; Qingling Li; Kehua Xu; Xin Liu; Hongmin Li; Zhenzhen Chen; Lili Tong; Bo Tang; Hongbo Zhong
- Publisher
- John Wiley and Sons
- Year
- 2009
- Tongue
- English
- Weight
- 170 KB
- Volume
- 30
- Category
- Article
- ISSN
- 0173-0835
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✦ Synopsis
Abstract
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°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 μM with an LOD (S/N=3) of 0.20 μM (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 μM; 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.