A simple HPLC method for selective determination of hydrogen sulfide and bound sulfane sulfur in human plasma

A simple and sensitive method using high-performance liquid chromatography (HPLC) with fluorometric detection has been developed for the determination of hydrogen sulfide ion (HS^-) and bound (sulfane) sulfur in biological materials. The bound sulfur is defined as divalent sulfur that is easily liberated as HS^- by reduction with dithiothreitol (DTT). HS^- and DTT-redactable sulfide were converted into a fluorescent derivative, thionine, through the reaction with p-phenylenediamine and FeCl₃. Thionine was then determined by reverse-phase HPLC with fluorometric detection. The calibration curve for HS^- and bound sulfur is linear in the range from 0.5 to 10.0 μM, and the sensitivity of this method allows detection of 10 nM bound sulfur or HS^- (0.5 pmol). Percent recoveries of bound sulfur for GSSSG spiked into human serum were 98.4 ± 11.5% and 96.3 ± 5.3% at 0.5 and 5.0 μM, respectively. The proposed method was applied to the determination of HS^-and bound sulfur in human plasma. The mean concentration in plasma from healthy subjects (n = 10) was 0.51 ± 0.06 μM for HS^- and 1.55 ± 0.35 μM for bound sulfur. Furthermore, using established methods, we conducted a detailed analysis of the plasma sulfide-binding capacity, which clearly indicates that plasma has a limited ability to sequester HS^- as bound sulfur. Overall, our results show that the present method is expected to be applied to determine the distribution and dynamics of HS^- and bound sulfur species across organs and organelles.