Arsenic Speciation Analysis in Human Saliva Chungang Yuan, 1,2 Xiufen Lu, 1 Nicole Oro, 1 Zhongwen Wang, 1 Yajuan Xia, 3 Timothy J. Wade, 4 Judy Mumford, 4 and X. Chris Le 1* BACKGROUND: Determination of arsenic species in sa- liva is potentially useful for biomonitoring of human exposure and studying arsenic metabolism. Arsenic speciation in saliva has not been reported previously. METHODS: We separated arsenic species in saliva using liq- uid chromatography (LC) and quantified them by induc- tively coupled plasma mass spectrometry. We further confirmed the identities of arsenic species by LC coupled with electrospray ionization tandem mass spectrometry. These methods were successfully applied to the determi- nation of arsenite (As III ), arsenate (As V ), and their meth- ylation metabolites, monomethylarsonic acid (MMA V ), and dimethylarsinic acid (DMA V ), in 300 saliva sam- ples collected from people who were exposed to varying concentrations of arsenic. RESULTS: The mean (range) concentrations (g/L) in the saliva samples from 32 volunteers exposed to background levels of arsenic were As III 0.3 [not detectable (ND) to 0.7], As V 0.3 (ND to 0.5), MMA V 0.1 (ND to 0.2), and DMA V 0.7 (ND to 2.6). Samples from 301 people exposed to increased concentrations of arsenic in drinking water showed detectable As III in 99%, As V in 98%, MMA V in 80%, and DMA V in 68% of samples. The mean (range) concentrations of arsenic species in these saliva samples were (in g/L) As III 2.8 (0.1–38), As V 8.1 (0.3–120), MMA V 0.8 (0.1– 6.0), and DMA V 0.4 (0.1–3.9). Saliva ar- senic correlated with drinking water arsenic. Odds ratios for skin lesions increased with saliva arsenic concentra- tions. The association between saliva arsenic concentra- tions and the prevalence of skin lesions was statistically significant (P 0.001). CONCLUSIONS: Speciation of As V , As III , MMA V , and DMA V in human saliva is a useful method for monitoring arsenic exposure. © 2007 American Association for Clinical Chemistry Biomonitoring of chemicals and their metabolites in the human body is an important area of investigation (1 ). Arsenic in saliva, which may indicate the concentrations of arsenic in the body, may serve as a new biomarker for studying arsenic exposure and metabolism. Salivary glands have high blood flow, and chemicals and their me- tabolites are distributed in saliva by several mechanisms, including passive diffusion, active transport, and ultrafil- tration (2 ). Previous studies on the use of saliva for bio- monitoring have focused on herbicide (3 ), insecticide (4, 5), lead (6, 7), cadmium (7 ), phthalate (8 ), and drug (9 ) concentrations in humans or animal models. The concentrations of chemical contaminants in saliva have been shown to reflect their concentrations in plasma (4 ). Saliva sampling is noninvasive and has advantages over urine collection, particularly from young children still in diapers (7, 9). Studying arsenic species in saliva may increase our understanding of arsenic exposure, metabolism, and tox- icity. Many studies on metabolism and toxicity of arsenic have relied on the analyses of biological fluids, including urine, blood, bile, and breast milk (10 –20 ). No reports describe the detection of arsenic species in human saliva, however, although many chemical contaminants are me- tabolized and excreted to saliva (21 ). A major challenge for detection of chemical contam- inants in saliva is that the concentrations of the chemical contaminants are usually very low, often 1 to 2 orders of magnitude lower than in blood (4, 5). To achieve the sen- sitivity necessary for the detection of arsenic in saliva and to identify the arsenic species at ultratrace concentrations, we developed 2 highly specific techniques. We used in- ductively coupled plasma mass spectrometry (ICP-MS) 5 to detect arsenic species after separation by liquid chro- matography (LC) and then applied electrospray tandem mass spectrometry (ESI-MS/MS) (16, 22 ) with multiple- reaction monitoring (MRM) to confirm the identifica- tion of arsenic species. 1 Analytical and Environmental Toxicology, Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, T6G 2G3, Canada; 2 School of Environmental Sciences and Engineering, North China Electric Power University, Baoding 071003, Hebei Province, P. R. China; 3 Inner Mongolia Center for Endemic Disease Control and Research, Huhhot 010020, Inner Mongolia, P. R. China; 4 National Health and Environmental Effects Research Laboratory, Human Studies Division, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, U.S.A. * Address correspondence to this author at: Department of Laboratory Medicine and Pathology, Faculty of Medicine and Dentistry, University of Alberta, 10-102 Clinical Sciences Building, Edmonton, Alberta, Canada T6G 2G3. Fax +1-780- 492-7800; e-mail xc.le@ualberta.ca. Received May 17, 2007; accepted October 11, 2007. Previously published online at DOI: 10.1373/clinchem.2007.092189 5 Nonstandard abbreviations: ICP-MS, inductively coupled plasma mass spec- trometry; LC, liquid chromatography; ESI-MS/MS, electrospray ionization tan- dem mass spectrometry; MRM, multiple reaction monitoring; As III , arsenite; As V , arsenate; MMA V , monomethylarsonic acid; MMA III , monomethylarsonous acid; DMA III , dimethylarsinous acid; SRM, standard reference material. 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