Selective spectrofluorimetric determination of glutathione in clinical and biological samples using 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene

This work reports the development of a selective, sensitive and rapid spectrofluorimetric method for the determination of reduced glutathione (GSH) in the presence of relatively high levels of cysteine (Cys) in clinical and biological samples using 1,3,5,7-tetramethyl-8-phenyl-(2-maleimide)-difluoroboradiaza-s-indacene (TMPAB-o-M). The fluorescence from TMPAB-o-M is strongly quenched by its maleimide moiety, but after reaction with thiol, the fluorescence is restored with a 350-fold intensity increase (fluorescence quantum yield from 0.002 to 0.73). In H(3)Cit-Na2HPO4 buffer (pH 7.40), the derivatization is completed in just 5 min under 37 degrees C. The linear range is 0.005-0.2 mu mol L-1. with detection limit of 1.1 x 10(-10) mol L-1 (signal-to-noise ratio=3). Almost all amino acids, including Cys, impose no interference even if present at relatively high concentrations (amino acids:GSH = 100: 1. Cys:GSH = 1:1, molar ratio, C-GSH = 3 x 10(-7) mol L-1). The sample can be used directly without further treatment after the protein is removed. The developed method is precise with a relative standard deviation (R.S.D.) lower than 5.0% (n = 6) and has been applied to the determination of GSH in human blood and pig's liver with recoveries between 94.4 and 105.6%. (C) 2008 Published by Elsevier B.V.