Direct spectrofluorimetric determination of glutathione in biological samples using 5-maleimidyl-2-(m-methylphenyl) benzoxazole

A new thiol fluorescence probe, 5-maleimidyl-2-(m-methylphenyl)benzoxazole (MMPB) has been developed for the direct determination of reduced glutathione (GSH) in real samples. Compared to the reported N-substituted maleimide type of thiol reagents, the main advantage of MMPB is its rather high selectivity for GSH to cysteine (Cys), which often coexists with GSH in biological samples. Under mild conditions similar to the physiological environment, MMPB reacted with GSH to give a highly fluorescent derivative with the excitation and emission wavelengths of 299.2 and 355.8 nm, respectively. In the presence of 0.40-fold (molar ratio) of Cys, a linear relationship was found in the range of 0-1.62×10⁻⁷ mol l⁻¹ with the detection limit (3σ) of 3.23×10⁻¹⁰ mol l⁻¹ for GSH determination. Many other amino acids (100-fold) did not interfere with the determination. Since the molar ratio of Cys to GSH in mammalian tissues and blood does not exceed the value of 0.40:1, the proposed method has been used in the direct determination of GSH in these kinds of biological samples, such as human blood, pig’s liver and heart with the recoveries of 94.3-104.5%