Simultaneous determination of superoxide and hydrogen peroxide in macrophage RAW 264.7 cell extracts by microchip electrophoresis with laser-induced fluorescence detection.
A method for the first time to simultaneously determine superoxide and hydrogen peroxide in macrophage RAW 264.7 cell extracts by microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF) was developed. 2-Chloro-1,3-dibenzothiazolinecyclohexene (DBZTC) and bis(p-methylbenzenesulfonyl) dichlorofluorescein (FS), two probes that can be specifically derivatized by superoxide and hydrogen peroxide, respectively, were synthesized and used. Parameters influencing the derivatization and on-chip separation were optimized. With the use of a HEPES (20 mM, pH 7.4) running buffer, a 50 mm long separation channel, and a separation voltage of 1800 V, baseline separation was achieved within 48 s for the two derivatization products, DBZTC-oxide (DBO) and 2,7-dichlorofluorescein (DCF). The linearity ranges of the method were 0.08-5.0 and 0.02-5.0 microM with detection limits (signal-to-noise ratio = 3) of 10 nM (1.36 amol) and 5.6 nM (0.76 amol) for superoxide and hydrogen peroxide, respectively. The relative standard deviations (RSDs) of migration time and peak area were less than 2.0% and 5.0%, respectively. The recoveries of the cell extract samples spiked with 1.0 microM standard solutions were 96.1% and 93.0% for superoxide and hydrogen peroxide, respectively. With the use of this method, superoxide and hydrogen peroxide in phorbol myristate acetate (PMA)-stimulated macrophage RAW 264.7 cell extracts were found to be 0.78 and 1.14 microM, respectively. The method has paved a way for simultaneously determining two or more reactive oxygen species (ROS) in a biological system with high resolution.
Li
Hongmin
H
College of Chemistry, Chemical Engineering and Materials Science, Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan 250014, People's Republic of China.
Li
Qingling
Q
Wang
Xu
X
Xu
Kehua
K
Chen
Zhenzhen
Z
Gong
Xiaocong
X
Liu
Xin
X
Tong
Lili
L
Tang
Bo
B
D000818:Animals; D045744:Cell Line, Tumor; D046209:Electrophoresis, Microchip / Q000379:methods*; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000032:analysis*; D007834:Lasers; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000032:analysis*
D005452:Fluoresceins; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; C543452:bis(p-methylbenzenesulfonyl)dichlorofluorescein; D013481:Superoxides; D006861:Hydrogen Peroxide