Comparative oxidation state specific analysis of arsenic species by high-performance liquid chromatography-inductively coupled plasma-mass spectrometry and hydride generation-cryotrapping-atomic absorption spectrometry

Abstract

The formation of methylarsonous acid (MAs^III) and dimethylarsinous acid (DMAs^III) in the course of inorganic arsenic (iAs) metabolism plays an important role in the adverse effects of chronic exposure to iAs. High-performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) and hydride generation-cryotrapping-atomic absorption spectrometry (HG-CT-AAS) have been frequently used for the analysis of MAs^III and DMAs^III in biological samples. While HG-CT-AAS has consistently detected MAs^III and DMAs^III, HPLC-ICP-MS analyses have provided inconsistent and contradictory results. This study compares the capacities of both methods to detect and quantify MAs^III and DMAs^III in an in vitro methylation system consisting of recombinant human arsenic (+3 oxidation state) methyltransferase (AS3MT), S-adenosylmethionine as a methyl donor, a non-thiol reductant tris(2-carboxyethyl)phosphine, and arsenite (iAs^III) or MAs^III as substrate. The results show that reversed-phase HPLC-ICP-MS can identify and quantify MAs^III and DMAs^III in aqueous mixtures of biologically relevant arsenical standards. However, HPLC separation of the in vitro methylation mixture resulted in significant losses of MAs^III, and particularly DMAs^III with total arsenic recoveries ≤25%. Further analyses showed that MAs^III and DMAs^III bind to AS3MT or interact with other components of the methylation mixture, forming complexes that do not elute from the column. Oxidation of the mixture with H₂O₂ which converted trivalent arsenicals to their pentavalent analogs prior to HPLC separation increased total arsenic recoveries to ∼95%. In contrast, HG-CT-AAS analysis found large quantities of methylated trivalent arsenicals in mixtures incubated with either iAs^III or MAs^III and provided high (≥72%) arsenic recoveries. These data suggest that an HPLC-based analysis of biological samples can underestimate MAs^III and DMAs^III concentrations and that controlling for arsenic species recovery is essential to avoid artifacts.