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.