Currier Jenna, Saunders R Jesse, Ding Lan, Bodnar Wanda, Cable Peter, Matoušek Tomáš, Creed John T, Stýblo Miroslav
Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7461, USA.
J Anal At Spectrom. 2013 Jun 1;28(6):843-852. doi: 10.1039/C3JA30380B.
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.
无机砷（iAs）代谢过程中甲基亚胂酸（MAs）和二甲基亚胂酸（DMAs）的形成在长期接触iAs产生的不良反应中起重要作用。高效液相色谱 - 电感耦合等离子体质谱法（HPLC - ICP - MS）和氢化物发生 - 低温捕集 - 原子吸收光谱法（HG - CT - AAS）经常用于分析生物样品中的MAs和DMAs。虽然HG - CT - AAS一直能检测到MAs和DMAs，但HPLC - ICP - MS分析得到的结果却不一致且相互矛盾。本研究比较了这两种方法在由重组人砷（+3氧化态）甲基转移酶（AS3MT）、作为甲基供体的S - 腺苷甲硫氨酸、非硫醇还原剂三（2 - 羧乙基）膦以及亚砷酸盐（iAs）或MAs作为底物组成的甲基化体系中检测和定量MAs和DMAs的能力。结果表明，反相HPLC - ICP - MS可以识别和定量生物相关砷标准品的水性混合物中的MAs和DMAs。然而，甲基化混合物的HPLC分离导致MAs大量损失，特别是DMAs，总砷回收率低于25％。进一步分析表明，MAs和DMAs与AS3MT结合或与甲基化混合物的其他成分相互作用，形成不从柱上洗脱的复合物。在HPLC分离之前用HO氧化混合物，将三价砷转化为其五价类似物，使总砷回收率提高到约95％。相比之下，HG - CT - AAS分析发现在与iAs或MAs孵育的混合物中有大量甲基化三价砷，并提供了高（> 72％）的砷回收率。这些数据表明，基于HPLC的生物样品分析可能会低估MAs和DMAs的浓度，并且控制砷物种回收率对于避免假象至关重要。
