Optimization of a Monobromobimane (MBB) Derivatization and RP-HPLC-FLD Detection Method for Sulfur Species Measurement in Human Serum after Sulfur Inhalation Treatment.

(1) Background: Hydrogen sulfide (H 2 S) is a widely recognized gasotransmitter, with key roles in physiological and pathological processes. The accurate quantification of H 2 S and reactive sulfur species (RSS) may hold important implications for the diagnosis and prognosis of diseases. However, H 2 S species quantification in biological matrices is still a challenge. Among the sulfide detection methods, monobromobimane (MBB) derivatization coupled with reversed phase high-performance liquid chromatography (RP-HPLC) is one of the most reported. However, it is characterized by a complex preparation and time-consuming process, which may alter the actual H 2 S level; moreover, a quantitative validation has still not been described. (2) Methods: We developed and validated an improved analytical protocol for the MBB RP-HPLC method. MBB concentration, temperature and sample handling were optimized, and the calibration method was validated using leave-one-out cross-validation and tested in a clinical setting. (3) Results: The method shows high sensitivity and allows the quantification of H 2 S species, with a limit of detection of 0.5 µM. Finally, it can be successfully applied in measurements of H 2 S levels in the serum of patients subjected to inhalation with vapors rich in H 2 S. (4) Conclusions: These data demonstrate that the proposed method is precise and reliable for measuring H 2 S species in biological matrices and can be used to provide key insights into the etiopathogenesis of several diseases and sulfur-based treatments.