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.