Development of mass spectrometric methods for detecting arsenic-glutathione complexes

It has been suggested recently that arsenic-glutathione (As-GSH) complexes play an important role in the methylation of arsenic. The present study describes the development of high-performance liquid chromatography (HPLC)-electrospray tandem mass spectrometry (ES-MS/MS), operated in the selected reaction monitoring (SRM) mode, and HPLC-inductively coupled plasma mass spectrometry (ICP-MS) methods suitable for the sensitive and selective identification of four As-GSH complexes. Method optimization was carried out using a series of synthetically prepared standards, i.e., three As-GSH species containing trivalent arsenic: tri(glutamyl-cysteinyl-glycinyl)trithio-arsenite (ATG), di(glutamyl-cysteinyl-glycinyl)methyl-dithio-arsonite (MADG), and (ã-glutamyl-cysteinyl-glycinyl) dimethyl-thio-arsinite (DMAG), as well as one As-GSH species containing pentavalent As: dimethylthioarsinic acid-glutathione (DMTA^V-GSH). The collision induced dissociation behavior of these compounds was investigated in detail to identify optimum SRM transitions for each complex. Both methods were based on reversed-phase chromatography using gradient elution with methanol, formic acid, and water as solvents. The amount of methanol that was used with this HPLC method (up to 12% vol/vol) was compatible with ICP-MS, without the need of a specially adapted interface. Subsequently, these analytical methods were applied to carry out a preliminary investigation about the role of As-GSH complexes in the methylation of arsenite by methylcobalamin (CH₃B₁₂) in the presence of glutathione (GSH). For the first time, the complexes ATG, MADG, and trace amounts of DMAG were detected as products of this reaction.