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-Na(2)HPO(4) buffer (pH 7.40), the derivatization is completed in just 5 min under 37 degrees C. The linear range is 0.005-0.2 micromol 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%.