A high-performance liquid chromatography with fluorescence detection for measurement of hydrogen sulfide in rat plasma

Hydrogen sulfide (H₂S), previously known as a toxic gas with a rotten egg odor, is now the third largest gasotransmitters after carbon monoxide (CO) and nitric oxide (NO). Precisely determining the levels of H₂S in organisms is crucial for elucidating the biological functions of H₂S and treating H₂S-related diseases. This study presents a novel and sensitive high-performance liquid chromatographic method coupled with fluorescence detection (HPLC-FL) for measuring the concentration of H₂S in plasma using (maleimide) ethyl 4-(pyren-1-yl) butanoate (MEPB) as a derivatization reagent. In particular, we employed the standard addition method to exclude potential interference from complex endogenous substances in plasma. This HPLC-FL approach was validated in accordance with ICH and FDA guidelines, confirming its specificity, linearity, accuracy, precision and stability. Compared with classical UV-Vis method, this HPLC-FL method provides higher sensitivity and specificity for more accurate detection of H₂S in plasma, attributable to the distinct sample pretreatment and derivatization conditions. HPLC-FL method was further utilized in assessing the release kinetics of H₂S from 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3thione (ADT-OH) in rat plasma. To our knowledge, this is the first application of MEPB for the determination of H₂S in plasma.