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.

The formation of methylarsonous acid (MAs) and dimethylarsinous acid (DMAs) 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 and DMAs in biological samples. While HG-CT-AAS has consistently detected MAs and DMAs, HPLC-ICP-MS analyses have provided inconsistent and contradictory results. This study compares the capacities of both methods to detect and quantify MAs and DMAs in an 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) or MAs as substrate. The results show that reversed-phase HPLC-ICP-MS can identify and quantify MAs and DMAs in aqueous mixtures of biologically relevant arsenical standards. However, HPLC separation of the methylation mixture resulted in significant losses of MAs, and particularly DMAs with total arsenic recoveries below 25%. Further analyses showed that MAs and DMAs 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 HO 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 or MAs and provided high (>72%) arsenic recoveries. These data suggest that an HPLC-based analysis of biological samples can underestimate MAs and DMAs concentrations and that controlling for arsenic species recovery is essential to avoid artifacts.