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