With the discovery of hydrogen sulfide as a signaling molecule and a potential therapeutic, measurement of free sulfide in blood – as hydrogen sulfide or hydrosulfide anion – has taken on importance. Here, we demonstrate and validate a method of free sulfide measurement whereby the free sulfide in whole blood is derivatized with excess monobromobimane. The resulting sulfide‐dibimane is subsequently extracted into ethyl acetate, followed by quantitation of sulfide‐dibimane via reverse‐phase HPLC with fluorescence detection. Reaction conditions are validated through 1) characterization of rate of conversion from sulfide to sulfide‐dibimane, 2) analysis of reaction in the presence of potential interferants, and 3) recovery of standard samples from a whole‐blood matrix. We found that reaction conditions of a mixture of acetonitrile and HEPES buffer (50 mM pH 8) gave rapid, clean conversion of sulfide to sulfide‐dibimane in the presence of excess monobromobimane. For whole blood, a 1:1:1 reaction mixture of 200 μl each acetonitrile:HEPES:blood proved optimal. Using this protocol, standard samples were consistently recovered in approximately 76% yield over the range of the assay. Baseline levels of free sulfide in rat blood were found to be about 0.3 – 0.5 μM. Subsequent work has proved the method effective in generating whole‐blood sulfide PK data in multiple species.

Hydrogen sulfide is best known as an environmental pollutant and human health hazard. Recently, sulfide has gained recognition as an endogenous biological mediator and is being examined for use as a therapeutic agent. As sulfide may be administered by both inhaled and parenteral routes, the question arises of how exposure to sulfide compares between the two modes of administration. Sprague Dawley rats were exposed for up to two hours of hydrogen sulfide gas up to 400 ppm or received up to 20 mg/kg sodium sulfide by continuous intravenous infusion. Sulfide concentrations in venous blood were quantified as sulfide dibimane derivative. Both modes of administration lead to a dose‐dependent elevation in blood sulfide concentrations over baseline concentrations and reached a new steady‐state concentration within two hours. Steady state blood sulfide concentrations show a simple linear relationship to hydrogen sulfide gas concentration or sodium sulfide dose. Identical blood sulfide concentrations can be achieved by either route of administration suggesting bioequivalency of hydrogen sulfide inhalation and sodium sulfide intravenous infusion in modulating blood sulfide concentrations. This suggests that both modes of administration may exert similar therapeutic effects.