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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 below 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 H 2 O 2 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 (&gt;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.

journal of analytical atomic spectrometry

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