DMMIC derivatization-assisted liquid chromatography-mass spectrometry method for metabolite profiling of the glutathione anabolic pathway in esophageal cancer tissues and cells

In this work, a new pyrylium derivatization-assisted liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolite profiling of the glutathione anabolic pathway (GAP) in cancer tissues and cells. The pyrylium salt of 6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate (DMMIC) was used to label the amino group of metabolites, and a reductant of dithiothreitol (DTT) was employed to stabilize the thiol group. By combining DMMIC derivatization with LC-MS, it was feasible to quantify the 13 main metabolites on the GAP in complex biological samples, which had good linearity (R² = 0.9981−0.9999), precision (interday precision of 1.6%–19.0% and intraday precision of 1.4%–19.8%) and accuracy (83.4%–115.7%). Moreover, the recovery assessments in tissues (82.5%–107.3%) and in cells (98.1%–118.9%) with GSH-¹³C2, ¹⁵N, and Cys-¹⁵N demonstrated the reliability of the method in detecting tissues and cells. Following a methodological evaluation, the method was applied successfully to investigate difference in the GAP between the carcinoma and para-carcinoma tissues of esophageal squamous cell carcinoma (ESCC) and the effect of p-hydroxycinnamaldehyde (CMSP) on the GAP in KYSE-150 esophageal cancer cells. The results demonstrate that the developed method provides a promising new tool to elucidate the roles of GAP in physiological and pathological processes, which can contribute to research on drugs and diseases.