Simultaneous analysis of reduced and oxidized forms of multiple biological aminothiols in cells and tissues by double derivatization combined with liquid chromatography‒mass spectrometry.

Biological aminothiols (BATs) typically exist in both reduced and oxidized forms, each exhibiting diverse biological activities. Monitoring the levels and ratios of the two forms is crucial for clinical diagnosis and understanding their roles in biological systems. In this study, we developed a method for simultaneous analysis of both reduced and oxidized BATs using a double derivatization approach combined with liquid chromatography-mass spectrometry (LC-MS). The method employed a sequential derivatization strategy: initially, 2‑bromo-N,N-dimethylacetamide (Br-DMA) reacted with the thiol groups of reduced BATs, followed by derivatization of both reduced and oxidized BATs with stable isotope labeling reagents, [d0]-/[d3]-6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate ([d0]-/[d3]-DMMIC). The methodology validation showed excellent linearity (R2 > 0.99), accuracy (85.07-119.94 %), precision (intraday: 5.26-18.78 %; interday: 6.52-19.01 %), recovery (70.09-119.27 %), and matrix effect (92.69-126.79 %). Finally, the method was successfully applied to nontargeted BAT screening in lung A549 cells, assessing changes in BAT levels in A549 cells upon treatment with the anticancer compounds triptolide and bufalin, and comparing differences in BAT levels between lung adenocarcinoma and paracarcinoma tissues. The results indicated that the developed method could be a comprehensive practical protocol and serve as a platform for profiling reduced and oxidized BATs in biological samples while meeting various analysis demands.