Stable isotope dilution mass spectrometry quantification of hydrogen sulfide and thiols in biological matrices

Hydrogen sulfide (H₂S), a gaseous signaling molecule that impacts multiple physiological processes including aging, is produced via select mammalian enzymes and enteric sulfur-reducing bacteria. H₂S research is limited by the lack of an accurate internal standard-containing assay for its quantitation in biological matrices.

After synthesizing [³⁴S]H₂S and developing sample preparation protocols that avoid sulfide contamination with the addition of thiol-containing standards or reducing reagents, we developed a stable isotope-dilution high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Total H₂S and other abundant thiols (cysteine, homocysteine, glutathione, glutamylcysteine, cysteinylglycine) in biological matrices, conducted a 20-day analytical validation/normal range study, and then both analyzed circulating Total H₂S and thiols in plasma from 400 subjects, and within 20 volunteers before and after antibiotic-induced suppression of gut microbiota.

Using the new assay, all analytes showed minimal interference, no carryover, and excellent intra- and inter-day reproducibility (≤7.6%, and ≤12.7%, respectively), linearity (r² > 0.997), recovery (90.9%-110%) and stability (90.0%-100.5%). Only circulating Total H₂S levels showed significant age-associated reductions in both males and females (p < 0.001), and a marked reduction following gut microbiota suppression (mean 33.8 ± 17.7%, p < 0.001), with large variations in gut microbiota contribution among subjects (range 6.0-66.7% reduction with antibiotics).

A stable-isotope-dilution LC-MS/MS method is presented for the simultaneous quantification of Total H₂S and multiple thiols in biological matrices. We then use this assay panel to show a striking age-related decline and gut microbiota contribution to circulating Total H₂S levels in humans.