Dual labeling for simultaneous determination of nitric oxide, glutathione and cysteine in macrophage RAW264.7 cells by microchip electrophoresis with fluorescence detection

•Simultaneous measurement of NO and thiols was firstly realized in microchip format.•The MCE-FLD method was simple, rapid, sensitive and efficient.•The MCE-FLD method was verified to determine NO and biothiols in macrophage cells.

A simple, rapid and efficient method based on microchip electrophoresis coupled with fluorescence detection (MCE-FLD) was developed for simultaneous determination of nitric oxide (NO), glutathione (GSH) and cysteine (Cys) using dual labeling strategy. Two highly reactive fluorogenic probes, 1,3,5,7-tetramethyl-8-(3′,4′-diaminophenyl)-difluoroboradiaza-s-indacene (DAMBO) and 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene (TMPAB-o-M), were used for labeling NO and thiols, respectively, under physiological conditions. The rapid separation and sensitive detection of the derivatives were achieved on a glass microchip within 70 s in a running buffer of 20 mM H3Cit–Na2HPO4 solution (pH 7.4) containing 15% (v/v) acetonitrile at a separation voltage of 2400 V. The limits of detection (S/N = 3) for NO, GSH and Cys were 7.0, 3.0 and 2.0 nM, respectively. The proposed method was validated by measuring intracellular levels of NO and biothiols in macrophage RAW264.7 cells.