Comprehensive analysis of arsenic metabolites by pH-specific hydride generation atomic absorption spectrometry

Abstract

In a variety of biological systems, inorganic arsenic (iAs) is metabolized to yield methylated arsenicals that contain arsenic in +5 or +3 oxidation states. Atomic absorption spectrometry (AAS) coupled with a pH-specific generation of arsines has been used for selective analysis of trivalent and pentavalent inorganic, mono-, and dimethylated arsenicals in biological matrices. We have optimized this method to permit simultaneous detection and quantification of all relevant metabolites of iAs, including trimethylarsine oxide (TMAs^VO). The optimization includes increasing the density of the chromatographic adsorbent used for cold-trapping of generated arsines and modification of the temperature gradient for release of arsines from the cold trap. These modifications improve the boiling-point separation of arsine, methylarsine, dimethylarsine, and trimethylarsine before the detection by AAS. Arsines from trivalent arsenicals and from TMAs^VO are selectively generated at pH 6. At pH 1, arsines are generated from both tri- and pentavalent arsenicals. Thus, the optimized technique permits analysis of arsenite (iAs^III), arsenate (iAs^V), monomethylarsonic acid (MAs^V), monomethylarsonous acid (MAs^III), dimethylarsinic acid (DMAs^V), dimethylarsinous acid (DMAs^III), and TMAs^VO. The detection limits range from 0.14 ng As (for TMAs^VO) to 0.40 ng As (for iAs^V). Calibration curves are linear over the concentration range of 0.5–100 ng As. Recoveries vary between 85 and 124%. The precision of the method in various biological matrices ranges from 1.0 to 14.5%. Using the optimized technique, both trivalent and pentavalent methylated and dimethylated arsenicals, but not TMAs^VO, have been detected in cultured primary human hepatocytes exposed to iAs^III. In contrast, TMAs^VO was detected as the final product of in vitro methylation of iAs^III by rat As^III-methyltransferase, cyt19. TMAs^VO was also detected in the urine of mice treated with MAs^V or DMAs^V. Thus, the optimized method improves the efficiency of arsenic speciation analysis in biological matrices, providing a more comprehensive picture of the role of metabolism in the disposition and action of iAs.