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Clinical Chemistry 54: 163-171, 2008. First published November 2, 2007; 10.1373/clinchem.2007.092189
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Right arrow Drug Monitoring and Toxicology
(Clinical Chemistry. 2008;54:163-171.)
© 2008 American Association for Clinical Chemistry, Inc.

Drug Monitoring and Toxicology

Arsenic Speciation Analysis in Human Saliva

Chungang Yuan1,2, Xiufen Lu1, Nicole Oro1, Zhongwen Wang1, Yajuan Xia3, Timothy J. Wade4, Judy Mumford4 and X. Chris Le1,a

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.

aAddress 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{at}ualberta.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Determination of arsenic species in saliva 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 liquid chromatography (LC) and quantified them by inductively 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 determination of arsenite (AsIII), arsenate (AsV), and their methylation metabolites, monomethylarsonic acid (MMAV), and dimethylarsinic acid (DMAV), in >300 saliva samples collected from people who were exposed to varying concentrations of arsenic.

Results: The mean (range) concentrations (µg/L) in the saliva samples from 32 volunteers exposed to background levels of arsenic were AsIII 0.3 [not detectable (ND) to 0.7], AsV 0.3 (ND to 0.5), MMAV 0.1 (ND to 0.2), and DMAV 0.7 (ND to 2.6). Samples from 301 people exposed to increased concentrations of arsenic in drinking water showed detectable AsIII in 99%, AsV in 98%, MMAV in 80%, and DMAV in 68% of samples. The mean (range) concentrations of arsenic species in these saliva samples were (in µg/L) AsIII 2.8 (0.1–38), AsV 8.1 (0.3–120), MMAV 0.8 (0.1–6.0), and DMAV 0.4 (0.1–3.9). Saliva arsenic correlated with drinking water arsenic. Odds ratios for skin lesions increased with saliva arsenic concentrations. The association between saliva arsenic concentrations and the prevalence of skin lesions was statistically significant (P <0.001).

Conclusions: Speciation of AsV, AsIII, MMAV, and DMAV in human saliva is a useful method for monitoring arsenic exposure.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 metabolites are distributed in saliva by several mechanisms, including passive diffusion, active transport, and ultrafiltration (2). Previous studies on the use of saliva for biomonitoring 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 toxicity. Many studies on metabolism and toxicity of arsenic have relied on the analyses of biological fluids, including urine, blood, bile, and breast milk (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). No reports describe the detection of arsenic species in human saliva, however, although many chemical contaminants are metabolized and excreted to saliva (21).

A major challenge for detection of chemical contaminants 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 sensitivity 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 inductively coupled plasma mass spectrometry (ICP-MS)1 to detect arsenic species after separation by liquid chromatography (LC) and then applied electrospray tandem mass spectrometry (ESI-MS/MS) (16)(22) with multiple-reaction monitoring (MRM) to confirm the identification of arsenic species.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
instruments and reagents
The instruments used in the study included an MDS SCIEX Applied Biosystems 4000Q-Trap MS/MS analyzer equipped with an electrospray ion-source, an Agilent 7500cs octopole reaction system ICP-MS, operated with the helium mode, and an Agilent 1100 series liquid chromatograph, equipped with an autosampler and column temperature control. Chromatographic separation was achieved with a Phenomenex reversed-phase ODS-3 column (150 x 4.6 mm, 3-µm particle size) and a Hamilton PRP-X100 strong anion exchange column (150 x 4.1 mm) or a Hamilton PRP-X200 strong cation exchange column (150 x 4.1 mm).

We purchased arsenite (AsIII), arsenate (AsV), and dimethylarsinic acid (DMAV) from Aldrich and of monomethylarsonic acid (MMAV) from Chem Service and prepared stock solutions (1000 mg/L) from these materials. We prepared working solutions by appropriate dilutions of the above stock solutions with deionized water. MMAIII standard was prepared from the solid oxide (CH3AsO) and DMAIII from the iodide [(CH3)2AsI]. The precursors were prepared according to reported procedures (23) and were stored at 4 °C or –20 °C. Diluted solutions of the precursors were prepared fresh in deionized water. For quality control, we purchased the standard reference materials SRM1640 (Trace Elements in Natural Water) and SRM2670 (Trace Elements in Urine) from NIST. We used tetrabutylammonium hydroxide (Aldrich), malonic acid (Fisher), methanol (Fisher), and ammonium bicarbonate (Sigma) for preparation of the mobile-phase solutions, which were filtered through a 0.45-µm membrane before use for LC separation.

saliva collection
This study was conducted according to the recommendations of the World Medical Association Declaration of Helsinki (24) for international health research. All participants gave written informed consent.

A saliva sample was collected from each of 32 volunteers (15 men and 17 women, age range 25 to 45 years) living in Edmonton, Canada. The concentration of arsenic in drinking water was <5 µg/L. Saliva was also collected from 301 people living in Ba Men, Inner Mongolia, China, where the concentrations of arsenic in drinking water (ground water) are as high as 826 µg/L. Demographic information on the participants has been reported for another biomarker study in the same population (25). The samples were collected at least 1 h after any food consumption; and before collection, participants rinsed their mouths at least 3 times to remove any food residue. The study participants provided saliva samples by spitting into 50-mL polyethylene centrifuge tubes. The samples were transported on dry ice via air courier and were stored at –20 °C until analysis.

pretreatment of saliva for arsenic speciation
The frozen saliva samples were thawed at room temperature and thoroughly vortex-mixed. We diluted the saliva 3-fold by pipetting 500 µL saliva and 1 mL deionized water into a 15-mL centrifuge tube. After vortex-mixing and ultrasonication, the dilute sample was filtered through a 0.45-µm filter immediately before LC analysis.

digestion of saliva for total arsenic
We pipetted 300 µL saliva and 1.0 mL concentrated nitric acid into a 5-mL glass vial. The mixture was then heated at 60 °C for 12 h. The acid in the sample was evaporated by heating at 80 °C until approximately 100 µL of the solution remained. The sample was finally diluted to 1.5 mL with deionized water and analyzed for total arsenic by ICP-MS.

quantification of arsenic species by LC-ICP-MS
We carried out ion pair separation by use of the Phenomenex ODS-3 reversed-phase column with a mobile phase containing 5 mmol/L tetrabutylammonium, 5% methanol, and 3 mmol/L malonic acid (pH 5.6), with a flow rate of 1.2 mL/min. The column temperature was controlled at 48 °C. AsIII, AsV, DMAV, MMAV, DMAIII, and MMAIII were separated in approximately 5 min. The eluent from the LC column was directly introduced to the inlet of the ICP nebulizer using polytetrafluoroethylene tubing and appropriate fittings (26). The ICP was operated at a radio frequency power of 1550 W. The flow rate of argon carrier gas was 0.8 L/min. Helium (3.6 mL/min) was used as the collision gas for the octopole reaction mode to reduce isobaric and polyatomic interference.

identification of arsenic species by both LC-ESI-MS/MS and LC-ICP-MS
Initially, we operated ESI-MS/MS in the enhanced MS scan mode, in 1:1 methanol/water solution, to obtain MS/MS spectra of the arsenic species. The optimization of operating conditions was carried out using 200 µg/L standard solutions of each arsenic species. The pH values of the MMAV and DMAV solutions were adjusted to 2.0–3.0 using formic acid, and MMAV and DMAV were monitored under the positive ionization mode. The optimal electrospray conditions were: curtain gas (N2) 1.1 L/min, ionspray voltage 5000 V, temperature 300 °C, declustering potential 30 V, collision energy 30 V, and cell exit potential 26 V. AsIII and AsV were monitored using the negative ionization mode, and the pH values of the AsIII and AsV standard solutions was adjusted to 8.0–10. The optimal ionspray parameter settings for AsIII and AsV were curtain gas (N2) 1.8 L/min, ionspray voltage –4500 V, temperature 300 °C, and collision energy –20 V. The declustering potential was –100 V for AsIII and –130 V for AsV.

Because the mobile phase for the ion-pair chromatography was not compatible with ESI-MS/MS, a strong anion exchange column was used to separate arsenic species. The arsenic species were subsequently detected using both ICP-MS and ESI-MS/MS. Ammonium bicarbonate (20 mmol/L, pH 8.5) was used as the mobile phase at a flow rate of 0.8 mL/min. The LC effluent was split equally for ICP-MS and ESI-MS/MS detection. The flow to the ICP-MS was directly introduced to the nebulizer of ICP-MS. The flow to the ESI-MS/MS was mixed with a solution containing methanol and either formic acid or ammonium hydroxide. Formic acid was introduced to the flow (adjusting the pH to 2.0–3.0) for the ESI-MS/MS detection of MMAV and DMAV under the positive ionization mode. Ammonium hydroxide was added to the flow (adjusting the pH to 8.0–10) for the ESI-MS/MS analysis of AsIII and AsV under the negative ionization mode. ESI-MS/MS was operated under the MRM mode. The characteristic ion transitions 139/91 and 139/121 were used for detecting DMAV, 141/93 and 141/123 for MMAV, 107/91 and 125/107 for AsIII, and 141/123 for AsV.

determination of total arsenic by flow injection ICP-MS
We analyzed the acid-digested saliva samples for total arsenic using flow-injection ICP-MS. A 50–100 µL sample was injected into a carrier stream of water at a flow rate of 1 mL/min, and was introduced to the ICP-MS.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
separation and identification of arsenic species using LC with ICP-MS and ESI-MS/MS detection
Using ion-pair chromatography, we achieved the separation of 6 arsenic species, including AsIII, AsV, DMAV, MMAV, DMAIII, and MMAIII, in approximately 5 min. Speciation of saliva samples and samples enriched with arsenic species showed matching retention times for chromatographic peaks in saliva and the standards.

To confirm the identity of arsenic species in the samples, we further complemented the ICP-MS detection with ESI-MS/MS. Whereas ICP-MS provided detection of elemental arsenic (at m/z 75), ESI-MS/MS offered molecular information characteristic to each of the arsenic species. Therefore, we were able to achieve identification and quantification of arsenic species by using LC separation with both ICP-MS and ESI-MS/MS detection, as described below.

To obtain characteristic MS/MS information of the arsenic species, we initially carried out ESI-MS/MS analyses for each arsenic standard. MS/MS spectrum of MMAV showed the parent molecular ion [M+H]+ at m/z 141 and the characteristic product ions, including (CH3)As(O)OH+ at m/z 123, As(OH)2+ at m/z 109, HAsOH+ at m/z 93, (CH3)AsH+ at m/z 91, AsH2+ at m/z 77, and As+ at m/z 75. Similarly, the characteristic MS/MS spectrum of dimethylarsinic acid (DMAV) included the protonated molecular ion [M+H]+ at m/z 139 and its fragments at m/z 121, 109, 91, 89, 77, and 75. Mass spectra of AsIII and AsV were obtained using negative ionization mode. The m/z 141 molecular ion and m/z 123, 93, and 77 fragment ions were characteristic of AsV. The molecular and fragment ions for AsIII were m/z 125, 107, and 91, with m/z 107 being most abundant, corresponding to (O)AsO–.

Having characterized the MS/MS behavior of the arsenic species, we were able to select the characteristic MS/MS transitions for MRM detection of the particular arsenic species. Fig. 1 shows ion chromatography separation with ICP-MS monitoring of As at m/z 75 (Fig. 1a and 1b ), MRM for DMAV transitions 139/91 (Fig. 1c ) and 139/121 (Fig. 1d ), and MRM for MMAV transitions 141/93 (Fig. 1e ) and 141/123 (Fig. 1f ). The matching of chromatographic retention times between the saliva sample (Fig. 1b ) and arsenic standard solution (Fig. 1a ) suggests the presence of AsIII, DMAV, MMAV, and AsV in the saliva sample. MRM monitoring of DMAV transitions at m/z 139/91 (Fig. 1c ) and 139/121 (Fig. 1d ) supports that the 2nd peak (3.8 min) is indeed DMAV. Likewise, the specific MRM of MMAV transition at m/z 141/93 (Fig. 1e ) and 141/123 (Fig. 1f ) supports the identity of MMAV (15.0 min) in the saliva sample.


Figure 1
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  		Figure 1. Chromatograms from LC-ICP-MS and LC-ESI-MS/MS analysis of arsenic in a standard solution (a) and in a saliva sample (b–f).

An anion exchange column (Hamilton PRP-X100, 150 x 4.6 mm, 5-µm particle size) was used to separate the arsenic species. The mobile phase contained 20 mmol/L ammonium bicarbonate and 5% methanol at pH 8.5. The mobile phase flow rate was 0.8 mL/min. The LC effluent was split equally to ICP-MS and ESI-MS/MS. ICP-MS was used to monitor elemental arsenic (m/z 75) in a standard solution (a) and in a saliva sample (b). The LC effluent to ESI-MS/MS was mixed online with formic acid to adjust the pH to 2.0–3.0 before being introduced to the ESI-MS/MS. The parent/daughter transitions of m/z 139/91 (c) and 139/121 (d) originating from DMAV, and 141/93 (e) and 141/123 (f) originating from MMAV were monitored by ESI-MS/MS using the MRM mode.

Fig. 2 shows separation of arsenic in another saliva sample with ICP-MS detection of arsenic species (Fig. 2a ), MRM monitoring for AsIII transitions at m/z 107/91 (Fig. 2b ) and 125/107 (Fig. 2c ), and MRM monitoring for a specific AsV transition at m/z 141/123 (Fig. 2d ). Retention times of the peaks detected by both ICP-MS (Fig. 2a ) and ESI-MS/MS (Fig. 2b-d ) match those of standards AsIII and AsV (Fig. 1aUp ). Cation exchange separation was also performed on selected samples to confirm the presence of AsIII and AsV in the saliva sample.


Figure 2
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  		Figure 2. Chromatograms from LC-ICP-MS (a) and LC-ESI-MS/MS (b–d) analyses of a saliva sample.

The chromatographic conditions were the same as in Fig. 1Up . The LC effluent was split equally to ICP-MS and ESI-MS/MS. ICP-MS was used to monitor elemental arsenic (m/z 75) in the saliva sample (a). The LC effluent to ESI-MS/MS was mixed online with ammonium hydroxide to adjust the pH to 8.0–10 before being introduced to the ESI-MS/MS. The parent/daughter transitions of m/z 107/91 (b) and 125/107 (c) originating from AsIII, and 141/123 (d) originating from AsV were monitored by ESI-MS/MS using the MRM mode.

determination of saliva arsenic concentrations
Having identified AsIII, AsV, MMAV, and DMAV species in saliva, we further quantified the concentrations of these arsenic species in saliva samples from volunteers (Table 1 ) and from a group of people who were exposed to varying concentrations of arsenic in drinking water (Table 2 ). Speciation analyses of saliva samples from 32 volunteers showed that AsIII, AsV, and DMAV were the major arsenic species. MMAV was detected in 2 samples. Other arsenic species, if present, were below detection limits. Additional analyses of total arsenic, directly by ICP-MS without LC separation, showed consistent results with the sum of AsIII, AsV, MMAV, and DMAV concentrations obtained from the speciation analyses. The total arsenic concentration in the saliva samples ranged from 0.2 to 3.3 µg/L.


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  		Table 1. Summary of arsenic species concentration in saliva samples collected from volunteers in Edmonton, Canada.


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  		Table 2. Summary of arsenic species concentration in saliva samples collected from Inner Mongolia.

The saliva samples from Inner Mongolia showed higher concentrations of arsenic (Table 2Up ). The total arsenic concentrations ranged from 0.4 to 140 µg/L. The major arsenic species were inorganic AsIII and AsV, accounting for 85% of total arsenic. Most of the samples (98%–99%) contained detectable AsIII and AsV, 80% of samples had detectable MMAV, and 68% of samples had detectable DMAV. The mean percentages of individual arsenic species concentration over the mean total arsenic concentration were: AsIII 28%, AsV 57%, MMAV 7%, and DMAV 4%. In addition, an unknown arsenic species was present in 20% of the samples and accounted for approximately 13% of total arsenic concentration in the saliva samples. The retention time (5.3 min) of this species was longer than that of AsV and did not match that of any arsenic species available to us, including the 4 arsenic species shown in Fig. 1Up , plus MMAIII, DMAIII, arsenobetaine, trimethylarsine oxide (TMAO), and dimethylthiolarsenate (DMTA) produced by reacting DMAV with hydrogen sulfide (H2S) (27)(28)(29). We confirmed no retention time match between the unknown and the above arsenic species, using 3 chromatography systems: ion-pair chromatography (13), strong anion exchange (Figs. 1Up and 2Up ), and strong cation exchange (PRP-X200 column with 5 mmol/L pyridine and 5% methanol as mobile phase, pH 2.9). In addition, we used LC-ESI-MS/MS to analyze the saliva sample containing the unknown species. The ion transitions did not match those of our arsenic standards. The exact composition of the unknown arsenic species remains to be characterized.

Five samples had a detectable peak retention time identical to that of MMAIII, and 3 samples had a peak retention time matching that of DMAIII. Because of the low concentration and the insufficient sample volume, however, we were not able to use MRM to confirm their identity in the saliva samples.

standard reference materials
In the absence of standard reference material (SRM) for saliva arsenic, we analyzed Natural Water 1640 and 2 urine SRMs (SRM 2670 and CRM no. 18) (11)(13). Eleven repeat speciation analyses of Natural Water 1640 using LC-ICP-MS showed that the mean (SD) concentrations of AsIII and AsV were 0.76 (0.05) µg/L and 25.62 (0.61) µg/L, respectively. Another 11 replicate analyses of this SRM by ICP-MS determined the mean (SD) total arsenic concentration to be 26.56 (0.36) µg/L. These results are in excellent agreement with the certified value of total arsenic [26.67 (0.41) µg/L, 99.6% accuracy]. Results from the analyses of urine SRM also showed good agreements with the certified values.

limits of detection
With the LC-ICP-MS method, the limits of detection, defined as the concentration of arsenic that produced a chromatographic peak having intensity equal to 3 times the SD of the baseline noise (30), were 0.03 g/L for AsIII, MMAIII, and DMAV; 0.05 g/L for MMAV; 0.08 g/L for AsV; and 0.1 g/L for DMAIII. Limits of quantification ranged from 0.1 g/L for AsIII and DMAV to 0.2 g/L for MMAV, and 0.3 g/L for AsV.

recovery and comparison between the sum of arsenic species and total arsenic concentrations
To evaluate the possible effect of the saliva sample matrix on the separation and quantification of arsenicals, we measured recovery using the saliva samples from the volunteers separately enriched with each arsenic species. The enriched concentrations were 0.5–50 µg/L for AsIII and AsV and 0.5–5 µg/L for the methylated arsenic species. The recoveries of AsIII, AsV, DMAV, and MMAV were 98%–100%. The recoveries of MMAIII and DMAIII were 82%–97%. The saliva sample matrix did not affect the separation of arsenic species, as shown by the identical retention times between arsenic standards and saliva samples (Figs. 1Up and 2Up ).

Each saliva sample was separately analyzed for individual arsenic species and for total arsenic concentrations. Results in Tables 1Up and 2Up show that the sum of arsenic species concentrations (obtained from LC-ICP-MS and LC-ESI-MS/MS) was in good agreement with the total arsenic concentrations (obtained from ICP-MS analyses without LC separation).

correlation between saliva arsenic species and skin lesions
To evaluate the relationship between the presence of skin lesions and saliva arsenic concentrations, saliva arsenic measures were log (base 10) transformed and categorized into quartiles, and the adjusted odds ratios were calculated using logistic regression models. Table 3 summarizes the adjusted odds ratios for skin lesions by quartiles of arsenic concentrations. For all 4 arsenic species, with the increase of their concentrations in saliva, the number of people with skin lesions increases and the odds ratio for skin lesions also increases. The highest odds ratio (8.545) for skin lesions is associated with the highest quartile of AsIII concentration in saliva. Odds ratios for skin lesions associated with the highest quartiles of AsV, MMAV, and DMAV concentrations were 3.246, 3.396, and 5.595, respectively. These associations were statistically significant (P <0.001).


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  		Table 3. Adjusted odds ratios for skin lesions by quartile of salvia arsenic species.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Before this study, no data had been reported on arsenic concentration or speciation in human saliva. This report is the first to describe the detection of arsenic species in saliva and show the evidence of methyl arsenical metabolites, MMAV and DMAV, as well as inorganic AsIII and AsV in saliva.

The detection and identification of trace arsenic species in saliva are possible because of the coupling of highly sensitive and selective analytical techniques (LC-ICP-MS and LC-ESI-MS/MS). LC-ICP-MS enables selective quantification of arsenic species, and LC-ESI-MS/MS provides confirmation of the species identities. The mass spectra of arsenic species obtained in our experiment are in agreement with those previously obtained using a single quadrupole mass spectrometer (22).

Statistical analysis clearly showed that the prevalence of skin lesions increased with increasing concentrations of saliva arsenic species (Table 3Up ). For example, among those with the lowest quartile of AsIII concentrations, the prevalence of skin lesions was 9.8%, but this increased to 39.2% among those in the highest quartile (adjusted odds ratio = 8.545, P = 0). Similarly, the odds ratios for skin lesions increase with the concentrations of AsV, MMAV, and DMAV (Table 3Up ). For all saliva measures the trend with the prevalence of skin lesions was significant (P = 0.0001). Skin lesions are an important clinical symptom of chronic arsenic exposure and poisoning. The good correlation between the saliva arsenic concentration and the prevalence of skin lesions suggests that saliva arsenic can be a useful biomarker. Furthermore, correlation coefficients between arsenic species in saliva and total arsenic in drinking water were AsIII 0.470, AsV 0.589, MMAV 0.663, and DMAV 0.485 (P <0.001 for all).

Arsenic in drinking water is mostly in inorganic forms, AsV and AsIII (31)(32)(33)(34)(35). Various organic arsenic species are present in food (34)(35). The presence of MMAV and DMAV in saliva samples suggests direct absorption of these arsenic species or methylation of inorganic arsenic (34)(35)(36)(37). During this biomethylation process, monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII) can be formed as intermediates (13)(14)(36)(37). These trivalent methylarsenic species are more toxic than the pentavalent arsenic species (36)(37)(38)(39)(40). A few saliva samples showed detectable peaks matching those of MMAIII and DMAIII standards by LC-ICP-MS analysis; however, we were not able to provide confirmation by ESI-MS/MS analysis because of the limited sample volume and very low concentrations of these species.

In conclusion, inorganic arsenicals and their methylation metabolites, MMAV and DMAV, were quantified in saliva samples collected from people who were exposed to various concentrations of arsenic. The ability to quantify the individual arsenic species and the noninvasive collection of saliva samples make arsenic speciation in saliva a potentially useful biomonitoring approach for assessing human exposure to arsenic.


   Acknowledgments
 
Grant/funding Support: This study was supported by the Canadian Water Network, the Metals in the Human Environment Research Network, the National Cancer Institute of Canada, Alberta Health and Wellness, and the International Consortium of Persistent Toxic Substances (Chinese Academy of Sciences). Dr. C. Yuan acknowledges the research funding support from North China Electric Power University.

Financial Disclosure: None declared.

Acknowledgments: This research has been reviewed by US EPA and approved for publication. Approval does not signify that the contents reflect views of the Agency or endorse the trade names mentioned. We thank Dr. W. R. Cullen (University of British Columbia, Canada) for providing the MMAIII and DMAIII standards.


   Footnotes
 
1 Nonstandard abbreviations: ICP-MS, inductively coupled plasma mass spectrometry; LC, liquid chromatography; ESI-MS/MS, electrospray ionization tandem mass spectrometry; MRM, multiple reaction monitoring; AsIII, arsenite; AsV, arsenate; MMAV, monomethylarsonic acid; MMAIII, monomethylarsonous acid; DMAIII, dimethylarsinous acid; SRM, standard reference material.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Barr DB, Wang RY, Needham LL. Biologic monitoring of exposure to environmental chemicals throughout the life stages: requirements and issues for consideration for the national children’s study. Environ Health Perspect 2005;113:1083-1091.[Web of Science][Medline] [Order article via Infotrieve]
  2. Hold KM, de Boer D, Zuidema J, Maes RAA. Saliva as an analytical tool in toxicology. Int J Drug Test 1995;1:1-36.
  3. Denovan LA, Lu C, Hines CJ, Fenske RA. Saliva biomonitoring of atrazine exposure among herbicide applicators. Int Arch Occup Environ Health 2000;73:457-462.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  4. Lu C, Irish RM, Fenske RA. Biological monitoring of diazinon exposure using saliva in an animal model. J Toxicol Environ Health Part A 2003;66:2315-2325.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  5. Timchalk C, Poet TS, Kousba AA, Campbell JA, Lin Y. Noninvasive biomonitoring approaches to determine dosimetry and risk following acute chemical exposure: Analysis of lead or organophosphate insecticide in saliva. J Toxicol Environ Health Part A 2004;67:635-650.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  6. Barbosa F, Tanus-Santos JE, Gerlach RF, Parsons PJ. A critical review of biomarkers used for monitoring human exposure to lead: advantages, limitations, and future needs. Environ Health Perspect 2005;113:1669-1674.[Web of Science][Medline] [Order article via Infotrieve]
  7. Gonzalez M, Banderas JA, Baez A, Belmont R. Salivary lead and cadmium in a young population residing in Mexico City. Toxicol Lett 1997;93:55-64.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  8. Silva MJ, Reidy JA, Samandar E, Herbert AR, Needham LL, Calafat AM. Detection of phthalate metabolites in human saliva. Arch Toxicol 2005;79:647-652.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  9. Gorodischer R, Koren G. Salivary excretion of drugs in children: Theoretical and practical issues in therapeutic drug monitoring. Dev Pharmacol Ther 1992;19:161-177.[Web of Science][Medline] [Order article via Infotrieve]
  10. Mandal BK, Suzuki KT. Arsenic round the world: a review. Talanta 2002;58:201-235.
  11. Ma M, Le XC. Effect of arsenosugar ingestion on urinary arsenic speciation. Clin Chem 1998;44:539-550.[Abstract/Free Full Text]
  12. Wang Z, Zhou J, Lu X, Gong Z, Le XC. Arsenic speciation in urine from acute promyelocytic leukemia patients undergoing arsenic trioxide treatment. Chem Res Toxicol 2004;17:95-103.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  13. Le XC, Lu X, Ma M, Cullen WR, Aposhian HV, Zheng B. Speciation of key arsenic metabolic intermediates in human urine. Anal Chem 2000;72:5172-5177.[Medline] [Order article via Infotrieve]
  14. Del Razo LM, Styblo M, Cullen WR, Thomas DJ. Determination of trivalent methylated arsenicals in biological matrices. Toxicol Appl Pharmacol 2001;174:282-293.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  15. Yoshida K, Inoue Y, Kuroda K, Chen H, Wanibuchi H, Fukushima S, Endo G. Urinary excretion of arsenic metabolites after long-term oral administration of various arsenic compounds to rats. J Toxicol Environ Health Part A 1998;54:179-192.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  16. Inoue Y, Date Y, Sakai T, Shimizu N, Yoshida K, Chen H, Kuroda K, Endo G. Identification and quantification by LC-MS and LC-ICP-MS of arsenic species in urine of rats chronically exposed to dimethylarsinic acid (DMAA). Appl Organomet Chem 1999;13:81-88.[CrossRef]
  17. Gregus Z, Gyurasics Á, Csanaky I. Biliary and urinary excretion of inorganic arsenic: monomethylarsonous acid as major biliary metabolite in rats. Toxicol Sci 2000;56:18-25.[Abstract/Free Full Text]
  18. Concha G, Vogler G, Nermell B, Vahter M. Low-level arsenic excretion in breast milk of native Andean women exposed to high levels of arsenic in the drinking water. Int Arch Occup Environ Health 1998;71:42-46.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  19. Zhang X, Cornelis R, De J, Mees L, Vanderbiesen V, Cubber AD, Vanholder R. Accumulation of arsenic species in serum of patients with chronic renal disease. Clin Chem 1996;42:1231-1237.[Abstract/Free Full Text]
  20. Francesconi KA, Tanggaard R, McKenzie CJ, Goessler W. Arsenic metabolites in human urine after ingestion of an arsenosugar. Clin Chem 2002;48:92-101.[Abstract/Free Full Text]
  21. Polkowska Z, Kozlowska K, Namiesnik J. Biological fluids as a source of information on the exposure of man to environmental chemical agents. Crit Rev Anal Chem 2004;34:105-119.[CrossRef]
  22. Florêncio MH, Duarte MF, Bettencourt AMM, Gomes ML, Vilas Boas LF. Electrospray mass spectra of arsenic compounds. Rapid Commun Mass Spectrom 1997;11:469-473.[CrossRef][Web of Science]
  23. Cullen WR, McBride BC, Manji H, Pickett AW, Reglinski J. The metabolism of methylarsine oxide and sulfide. Appl Organometal Chem 1989;3:71-78.[CrossRef]
  24. Mo J, Xia Y, Wade TJ, Schmitt M, Le XC, Dang R, Mumford JL. Chronic arsenic exposure and oxidative stress: OGG1 expression and arsenic exposure, nail selenium, and skin hyperkeratosis in Inner Mongolia. Environ Health Perspect 2006;114:835-841.[Web of Science][Medline] [Order article via Infotrieve]
  25. . World Medical Association. World Medical Association Declaration of Helsinki 1989 Hong Kong. .
  26. Le XC, Lu X, Li X-F. Arsenic speciation. Anal Chem 2004;76:27A-33A.
  27. Hansen HR, Raab A, Jaspars M, Milne BF, Feldmann J. Sulfur-containing arsenical mistaken for dimethylarinous acid [DMA(III)] and identified as a natural metabolite in urine: Major implications for studies on arsenic metabolism and toxicity. Chem Res Toxicol 2004;17:1086-1091.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  28. Raml R, Goessler W, Traar P, Ochi T, Francesconi KA. Novel thioarsenic metabolites in human urine after ingestion of an arsenosugar, 2',3'-dihydroxypropyl 5-deoxy-5-dimethylarsinoyl-β-d-riboside. Chem Res Toxicol 2005;18:1444-1450.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  29. Fricke MW, Zeller M, Sun H, Lai VWM, Cullen WR, Shoemaker JA, Witkowski MR, Creed JT. Chromatographic separation and identification of products from the reaction of dimethylarsinic acid with hydrogen sulfide. Chem Res Toxicol 2005;18:1821-1829.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  30. . ACS Committee on Environmental Improvement. Guidelines for data acquisition and data quality evaluation in environmental chemistry. Anal Chem 1980;52:2242-2249.
  31. Terlecka E. Arsenic speciation analysis in water samples: a review of the hyphenated techniques. Environ Monit Assess 2005;107:259-284.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  32. Watt C, Le XC. Arsenic speciation in natural waters. Cai Y. eds. Biogeochemistry of Trace Elements 2002:11-32 American Chemical Society Symposium Series Washington, DC. .
  33. Gong Z, Lu X, Watt C, Wen B, He B, Mumford J, Ning Z, Xia Y, Le XC. Speciation analysis of arsenic in groundwater from Inner Mongolia with an emphasis on acid-leachable particulate arsenic. Anal Chim Acta 2006;555:181-187.[CrossRef][Web of Science]
  34. Cullen WR, Reimer KJ. Arsenic speciation in the environment. Chem Rev 1989;89:713-764.[CrossRef][Web of Science]
  35. . National Research Council. Arsenic in Drinking Water 1999 National Academy Press Washington, DC. .
  36. Styblo M, Drobna Z, Jaspers I, Lin S, Thomas DJ. The role of biomethylation in toxicity and carcinogenicity of arsenic: a research update. Environ Health Perspect 2002;110(Suppl.):767-777.[Web of Science][Medline] [Order article via Infotrieve]
  37. Aposhian HV, Aposhian MM. Arsenic toxicology: Five questions. Chem Res Toxicol 2006;19:1-15.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  38. Styblo M, Serves SV, Cullen WR, Thomas DJ. Comparative inhibition of yeast glutathione reductase by arsenicals and arsenothiols. Chem Res Toxicol 1997;10:27-33.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  39. Styblo M, Del Razo LM, Vega L, Germolec DR, LeCluyse EL, Hamilton GA, Reed W, Wang C, Cullen WR, Thomas DJ. Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells. Arch Toxicol 2000;74:289-299.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  40. Petrick JS, Ayala-Fierro F, Cullen WR, Carter DE, Aposhian HV. Monomethylarsonous acid (MMAIII) is more toxic than arsenite in Chang human hepatocytes. Toxicol Appl Pharmacol 2000;163:203-207.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]



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S. Suzuki, L. L. Arnold, D. Muirhead, X. Lu, X. C. Le, J. A. Bjork, K. B. Wallace, T. Ohnishi, S. Kakiuchi-Kiyota, K. L. Pennington, et al.
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