Chromatographic determination and in-situ cell imaging of thiol compounds based on a fluorigenic probe

This study combines a high-performance liquid chromatography-fluorescent detection method (HPLC-FLD) with in-situ cell imaging for the sensitive analysis of glutathione (GSH), cysteine (Cys) and homocysteine (Hcys), using BODIPY®507/545 IA as a labeling reagent. The analytical potential of BODIPY®507/545 IA in cell imaging was deeply explored, concerning fluorescent response, selectivity, cell-permeability, biotoxicity and so on. It is demonstrated that BODIPY®507/545 IA has good biocompatibility and the fluorescence intensity is enhanced remarkably after reacting with thiols. The best derivative condition was obtained in boric acid buffer (0.05 mmol/L, pH 9.5) at 45 °C for 15 min. For chromatographic method, two sensitive methods, HPLC-FLD and capillary electrophoresis-laser-induced fluorescence detection (CE-LIF) were both developed, validated, and compared. The detection limits for the thiols ranged from 5 to 10 nmol/L with HPLC-FLD and 0.5 nmol/L for the CE-LIF method. Finally, HPLC-FLD is adopted to quantify the thiols in HepG2 cell samples after cell imaging.