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 (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.
Gong
Xiaocong
X
Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education, Key Laboratory of Molecular and Nano Probes, Shandong Normal University, Shandong, P. R. China.
Li
Qingling
Q
Xu
Kehua
K
Liu
Xin
X
Li
Hongmin
H
Chen
Zhenzhen
Z
Tong
Lili
L
Tang
Bo
B
Zhong
Hongbo
H
D000818:Animals; D002457:Cell Extracts / Q000737:chemistry; D002460:Cell Line; D046209:Electrophoresis, Microchip / Q000379:methods*; D004867:Equipment Design; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D016014:Linear Models; D008264:Macrophages / Q000737:chemistry* / Q000378:metabolism; D008353:Mannitol / Q000737:chemistry; D051379:Mice; D017382:Reactive Oxygen Species / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D002457:Cell Extracts; D005452:Fluoresceins; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; C543452:bis(p-methylbenzenesulfonyl)dichlorofluorescein; D008353:Mannitol; D006861:Hydrogen Peroxide