A HPLC-based methylene blue/methylene green method for accurate measurement of hydrogen sulfide in plasma.

Hydrogen sulfide (H2S) is an endogenously produced gasotransmitter with important physiological roles in cardiovascular, neurological, and metabolic health. Accurate quantification of circulating H2S remains technically challenging due to its volatility, reactivity, and rapid oxidation. Here, we present a high-performance liquid chromatography (HPLC)-based methylene blue method using methylene green as an internal reference (MBMG) for plasma H2S quantification with improved sensitivity, reproducibility, and compatibility. Using this approach, a linear standard curve was generated for NaHS concentrations between 0.05 and 10 μM. The method successfully detected significantly higher plasma H2S levels in wild-type (WT) mice (∼0.5 μM) compared to CSE-knockout (KO) mice (∼0.15 μM), with both groups showing expected increases following NaHS intraperitoneal injection. Specificity testing revealed minimal interference from biologically relevant sulfur species; only cysteine at supraphysiological concentrations generated a negligible signal. Plasma cysteine levels remained unchanged following NaHS injection. Post-reaction samples remained stable for at least six months at -80 °C, facilitating remote analysis and long-term storage. Collectively, the MBMG method provides a practical and sensitive platform for reliable plasma H2S measurement, with broad applicability in basic, translational, and clinical research settings.