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

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 (V) O). The optimization includes increasing the density of the chromatographic adsorbent used for cold- trapping of generated arsines and modi. cation 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 (V) O 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 (V) O. The detection limits range from 0.14 ng As ( for TMAs O-V) 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 (V) O, have been detected in cultured primary human hepatocytes exposed to iAs (III). In contrast, TMAs (V) O was detected as the final product of in vitro methylation of iAs (III) by rat As (III)- methyltransferase, cyt19. TMAs (V) O 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.