Quantitative and comparative liquid chromatography-electrospray ionization-mass spectrometry analyses of hydrogen sulfide and thiol metabolites derivaitized with 2-iodoacetanilide isotopologues

Hydrogen sulfide (H2S), previously known as a toxic gas, is now recognized as a gasotransmitter along with nitric oxide and carbon monoxide. However, only few methods are available for quantitative determination of H2S in biological samples. 2-Iodoacetanilide (2-IAN), a thiol-reacting agent, has been used to tag the reduced cysteine residues of proteins for quantitative proteomics and for detection of cysteine oxidation modification. In this article, we proposed a new method for quantitative analyses of H2S and thiol metabolites using the procedure of pre-column 2-IAN derivatization coupled with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). 13C6-Labeled and label-free 2-IAN efficiently react with H2S and thiol compounds at pH 9.5 and 65 °C. The derivatives exhibit excellent stability at alkaline conditions, high resolution on reverse phase liquid chromatography and great sensitivity for ESI-MS detection. The measurement of H2S, l-cysteine, glutathione, and DL-homocysteine derivatives was validated using 13C6-labeled standard in LC-ESI-MS analyses and exhibited 10 nM-1 μM linear ranges for DL-homocysteine and glutathione and 1 nM-1 μM linear ranges for l-cysteine and H2S. In addition, the sequence of derivatization and extraction of metabolites is important in the quantification of thiol metabolites suggesting the presence of matrix effects. Most importantly, labeling with 2-IAN and 13C6-2-IAN isotopologues could achieve quantitative and matched sample comparative analyses with minimal bias using our extraction and labeling procedures before LC-MS analysis.