Development of a derivatization method for the quantification of hydrogen sulfide and its application in vascular...

Hydrogen sulfide (H₂S) plays major functional and structural roles in diverse physiological functions and the pathogenesis of a variety of disorders in biological matrices. The significance of H₂S has prompted the development of sensitive and selective methods to determine its concentration in biological samples. The fluorescent reagent monobromobimane (MBB) has been widely used to measure various thiol-containing species through alkylation. MBB may prevent the oxidation of sulfide and the reaction of sulfide with several different species (such as superoxide radicals, hydrogen peroxide and peroxynitrite). An isomers of MBB, 3-(bromomethyl)-2, 6, 7-trimethyl-1H, 5H-pyrazolo [1,2-a] pyrazole-1, 5-dione (MMB), is cheaper than MBB and its use in the analysis of H₂S has not previously been reported. In the present study, we compared the derivatization reactions of hydrogen sulfide with MMB and MBB and developed a sensitive method to quantify H₂S in blood. In our method, H₂S was incubated in the dark with excess MMB in 0.1M Tris-HCl buffer (pH 10.1) at 50°C for 120min. 50μL aliquots of the derivatized product were analyzed using HPLC system with gradient elution of 0.1% (v/v) formic acid-acetonitrile. The limit of detection for the derivatized product was 0.03nmol/mL. The derivatization reaction was suitable for detecting low concentrations of H₂S. The derivate product is stable over time, permitting batch storage and analysis.