Development of mass spectrometric methods for detecting arsenic-glutathione complexes

Abstract

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 HPLCinductively 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)methy1-dithio-arsonite (MAOC), and (a-glutemyl-cysteinyl-glycinyl) dimethyl-thio-arsinite (DMAG), as well as one As-GSH species containing pentavalent As: dimethylthioarsinic acid-glutathione (DMTAv-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 methy1cobalamin (CH 3Bu) 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. . The predominant forms of arsenic in drinking water are arsenite and arsenate (iAs III and iAs", respectively), the inorganic species which are regarded as human carcinogens [2,3]. Inorganic arsenic compounds are transformed in the body by repetitive reduction and oxidative methylation reactions, leading to the formation of organoarsenic species, the major of which are monomethylarsonic acid (MMAv) and dimethylarsinic acid (DMAV) [3,4].The precise pathway through which inorganic arsenic is metabolized in the organism is not yet known in sufficient detail. The generally accepted methylation mechanism involves the initial conversion of pentavalent arsenic species iAsv to trivalent species iAs IlI through a two-electron reduction, while oxidative addition of a methyl group follows [4,5]. Glutathione (GSH) can act as a reducing agent and 5-adenosylmethionine (SAM) as a methyl donor. The major site for the methylation seems to be the liver. However, Hayakawa et al. recently proposed Address reprint requests to Dr. S. A. Pergantis, Environmental Chemical Processes Laboratory, Department of Chemistry, University of Crete, Voutes, Heraklion 71003, Crete, Greece. E-mail: spergantis@chemistry. uoc.gr an altemative mechanism for the methylation of inorganic arsenic in biological systems [6]. According to this methylation mechanism, complexes of arsenic with gluta...

Enhanced determination of As–phytochelatin complexes in Chlorella vulgaris using focused sonication for extraction of water-soluble species

“…Fig S4-S16 and Table S1). The fragments found and the fragmentation pathways agree with previously published data 4, 5,8,19 . Focused sonication was compared with extraction by grinding using liquid nitrogen.…”

“…†) The fragments found and the fragmentation pathways agree with previously published data. 4,5,8,19 Focused sonication was compared with extraction by grinding using liquid nitrogen. The results for quantication of arsenic are shown in Fig.…”

“…5 The most problematic steps in the analysis of these complexes are their extraction from the biological material and the maintenance of their stability during HPLC separation. 6 Although conditions for stability of the complexes in a mobile phase and HPLC column have been achieved, 5,7,8 relatively little progress has been made in ensuring the integrity and representativeness of As-GS/PC complexes when extracting from solid samples or cells such as the green microalga Chlorella vulgaris. Murray et al 9 studied the biotransformations of arsenate in C. vulgaris, and found that when extracting with 1 : 1 (v/v) methanol-water followed by 10 min in a sonication bath, the extraction efficiency was between 11% and 27%, a surprisingly low gure compared to other algae.…”

“…The detailed elution prole was as follows: 0-20 min linear increase 0-20% B, 20-30 min 20% B, 30-32 min 20-0% B and 32-40 min 0% B. The ow rate was 0.75 mL min À1 8. The injection volume was 75 mL.…”

Proposal for novel metabolic pathway of highly toxic dimethylated arsenics accompanied by enzymatic sulfuration, desulfuration and oxidation

“…RF power; 1400 W, coaxial-type nebulizer, copper sampling cone, nickel skimmer cone, monitoring mass; m/z 75( 75 As + ), 35( 35 Cl + ), 72( 72 Ge + ). DMA III -SG was identified based on the method described by Kanaki and Pergantis [32].…”