Arsenic speciation in biomedical sciences: Recent advances and applications

Kaohsiung Journal of Medical Sciences (2011) 27, 382e389

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REVIEW ARTICLE

Arsenic speciation in biomedical sciences: Recent advances and applications Keng-Chang Hsu a, Chien-Che Sun b, Yeou-Lih Huang a,b,c,* a

Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan c Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan b

Received 6 September 2010; accepted 18 November 2010 Available online 8 July 2011

KEYWORDS Advance; Application; Arsenic speciation; Biomedical science

Abstract Speciation analysis of trace elements is an important issue in biomedical and toxicological sciences because different elemental species have different effects on health and the environment. For humans, arsenic (As) is a toxic element; the toxicity of As compounds is highly dependent on its chemical form. Although inorganic As compounds are human carcinogens, organic arsenicals are relatively less toxic. This article deals with recent advances and applications of methods for As speciation in biomedical sciences, with emphasis on the specimens commonly encountered in biomedical laboratories. Copyright ª 2011, Elsevier Taiwan LLC. All rights reserved.

Introduction Arsenic (As) is well known as a toxic element for human beings. The International Agency for Research on Cancer has classified As and its inorganic compounds as “carcinogenic to human” (Group 1); the organic arsenicals monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) are classified as “possibly carcinogenic to humans” (Group 2B); arsenobetaine (AsB) and other organic compounds that are not metabolized in humans are “not classifiable” (Group 3) [1,2]. Chronic As poisoning is a serious public * Corresponding author. Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, No. 100, Shih-Chuan 1st Road, Kaohsiung 807, Taiwan. E-mail address: [email protected] (Y.-L. Huang).

health problem worldwide; long-term exposure to inorganic As from drinking water is associated with skin lesions and malignant diseases [3e5]. As is a ubiquitous element that ranks 12th in abundance in the human body. Despite its notorious reputation as a poison, As has been used as a therapeutic agent for more than 2000 years [6]. Recently, As compounds have been examined extensively as potential anticancer agents toward some malignant diseases, especially for the treatment of acute promyelocytic leukemia (APL) [7,8], where low doses of arsenic trioxide was found to induce complete remission in individuals with APL who had a relapse, suggesting that induction of cell apoptosis might be one of the mechanisms of the therapeutic effect of arsenic trioxide. Because of the different toxic effects induced by different As species, speciation analysis is critical when evaluating

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the therapeutic efficiency of arsenic trioxide for treating individuals with APL. Measurement of essential or toxic elements has traditionally been performed using atomic spectrometry techniques, most notably flame atomic absorption spectrometry (AAS), electrothermal AAS, inductively coupled plasma atomic emission spectrometry, and inductively coupled plasma mass spectrometry (ICPeMS). ICPeMS is one of the most common analytical techniques for the determination of trace elements because of its multi-element analysis capability and its high sensitivity for most elements of interest [9]. For separation, high-performance liquid chromatography (HPLC) has the advantages of greater versatility and good reproducibility relative to other chromatographic separation methods (e.g. gas chromatography or capillary electrophoresis) [10]. The combination of HPLC with suitable detection systems for As speciation in biomedical sciences is the most attractive and most established approach. Ion-exchange chromatography is the most widely used separation technique, whereas ICPeMS is the most commonly used detection method. The low levels of As species in biomedical matrices make obtaining sensitive and reliable measurements a true challenge. ICPeMS provides a low detection limit and good sensitivity for the detection of trace element species; combined with the ease of coupling most HPLC systems with the ICPeMS, it is an excellent detection method for As speciation analysis. Table 1 lists the common As species found in biological materials. Because of the complexity of the sample matrix and the diversity of As species, the search for suitable analytical methods is a key issue for As speciation analysis in the field of biomedical sciences; this article focuses on some significant findings in the area, focusing on relevant reports published in the calendar years 2005e2009.

Advances and applications of As speciation analysis in biomedical matrices Table 2 lists some selected recent (2005e2009) applications of As speciation in commonly used biomedical matrices. The analytical systems that have been used for As speciation in biomedical matrices, mainly from human beings, have typically combined chromatographic separation with Table 1

elemental or molecular mass spectrometric detection. Among these analytical systems, HPLC coupled with ICPeMS has been the most widely used method for determining As species in biomedical matrices. In the following sections, we describe the recent applications of As speciation analysis in urine, blood, hair and nail, and other biomedical matrices.

As speciation in urine Most of the method developments and applications of As speciation analysis in biomedical research have focused on the analysis of urine, which is generally considered a suitable sample for evaluating As metabolism. Notably, however, most medical laboratories measure only the total As concentration in urine, presumably because the practitioners are unaware that a high total-As concentration requires further speciation analysis, which might not be possible because of the lack of rapid and reliable methods. For high-throughput speciation in a medical laboratory, Heitland and Ko ¨ster [23] developed and validated a method using HPLC coupled with ICPeMS for the determination of five As species in human urine. They applied this method to compare urinary As speciation for various medical cases [22], suggesting that real concentration data for As species in urine are helpful for toxicologists and hygienists to understand the impact of As species in different situations. HPLCehydride generation (HG)eICPeMS, HPLCeHGe atomic fluorescence spectrometry (AFS), and HGeAAS are the three most commonly used analytical methods for determination of inorganic As and its metabolites in urine. From a comparison of the performance of these three methods, Lindberg et al. [18] suggested that, because of its considerably lower costs, HPLCeHGeAFS might be a good alternative in laboratories where the high cost of ICPeMS is not justified in relation to the intended use of the instrument. Occupational and environmental exposure to As compounds is associated with the increased prevalence of various kinds of diseases. Analytical methods for monitoring occupational or environmental exposure have focused on the determination of As metabolites in urine. Morton and Mason [54] developed an anion-exchange-based method coupled with ICPeMS that they subsequently used to

Arsenic species commonly found in biological materials

Name

Abbreviation

Chemical formula

Inorganic arsenic species Arsenite (arsenious acid) Arsenate (arsenic acid)

As(III) As(V)

As(OH)3 AsO(OH)3

Organic arsenic species Monomethylarsonous acid Monomethylarsonic acid Dimethylarsinous acid Dimethylarsinic acid Arsenobetaine Arsenocholine Trimethylarsine oxide Tetramethylarsonium ion

MMA(III) MMA(V) DMA(III) DMA(V) AsB AsC TMAO Me4Asþ

CH3As(OH)2 CH3AsO(OH)2 (CH3)2AsOH (CH3)2AsO(OH) (CH3)3AsþCH2COOe (CH3)3AsþCH2CH2OH (CH3)3AsO (CH3)4Asþ

384 establish the levels of five As species in urine in unexposed and exposed persons. They found that urine samples from workers involved in timber treatment exhibited significantly higher levels of As(III), As(V), MMA, and DMA than did those from both unexposed groups and workers in the semiconductor industry. Using HPLCeICPeMS, Christian et al. [12] investigated the relationship between urinary As and Se excretion in pregnant women living in an As-contaminated region of Chile. They suggested that in populations exposed to As, Se intake might be correlated with urinary As excretion and may alter As methylation. Although chronic As exposure may not be rare in humans, subacute As poisoning is not common. Xu et al. [37] described the clinical manifestations and levels of As methylation after a rare subacute As-poisoning accident of workers who ingested high levels of As caused by a pipe leakage in a copper smelting factory. Studies of excreted urinary As species determined using HGeAAS with cold trapping reported increased urinary excretion of MMA and inorganic As compared with controls and concluded that biomethylation of As decreased in a dose-rate-dependent manner. Studies of dietary intake of As and its metabolism have gained much attention when using novel sample pretreatment methods coupled with ICPeMS. Many recent studies have examined As species in human urine in relation to dietary exposure. Using HPLC coupled with ICPeMS, Raml et al. [35] identified seven As metabolites in human urine after ingestion of an oxo-arsenosugar. In addition to previously identified metabolites, they reported three new As metabolites. By monitoring As metabolites using HPLCeICPeMS, Schmeisser et al. [39] investigated the As species excreted in urine after consumption of arsenolipids, significant As constituents of some seafood products, contained in commercially available canned cod liver products. Their study of two human volunteers provided data revealing that the arsenolipids present in cod liver are metabolized and excreted almost quantitatively in urine, chiefly as DMA and four As-containing fatty acids. Pearson et al. [55] described a method for the rapid separation of five As species [As(III), AsB, DMA, MMA, and As(V)] for subsequent quantitative determination using ICPeMS. The species were separated on a monolithic silica column using a mobile phase comprising tetrabutylammonium bromide, phosphate buffer, and methanol. They found a significant increase in DMA excretion in urine after rice consumption. Hata et al. [56] used HPLC coupled with ICPeMS for the quantitative determination of As(III), As(V), DMA, MMA, and AsB in urine samples from Japanese volunteers. They found that the mean and range of values for DMA and AsB were significantly higher than those reported for Western populations, presumably because of the high seafood consumption of the Japanese population. In contrast to most previous studies that have identified seafood as a significant contributor to the urinary excretion of As, Sirot et al. [25] found no correlation between dietary exposure and levels of urine biomarkers for As in a study of a French population with a high consumption of seafood. They suggested that biological monitoring results should be interpreted with caution. The main advantage of ion-pair HPLC is that it facilitates the separation of ionic species and uncharged molecular species. Table 2 reveals that, with the exception of ion-

K.-C. Hsu et al. exchange HPLC, ion-pair HPLC has been the most widely used separation technique for biomedical As speciation in recent years. Using ion-pair liquid chromatography (LC) coupled with ICPeMS, Pan et al. [32] developed a method for the separation and simultaneous determination of six As species [As(III), As(V), DMA, MMA, arsenocholine, and AsB] in human urine; this process featured a simple sample preparation method involving filtering through a 0.45-mm membrane and dilution with the mobile phase. Rabeih et al. [34] described a method for the determination of six As species [arsenite, arsenate, DMA(III), DMA(V), MMA(III), and MMA(V)] in human urine using ion-pair reversed-phase HPLC with isocratic elution coupled with ICPeMS. They used this technique to quantify As speciation in children’s urine samples obtained from an As-affected area in Brazil. To overcome the cost and time limitations, Afton et al. [31] developed a multi-element speciation method for the simultaneous speciation of As and Se. Based on ion-paired reversed phase chromatography followed by ICPeMS and electrospray ionization (ESI) ion-trap MS, they identified and quantified four As [As(III), As(V), MMA(V), and DMA(V)] and four Se species in various matrices, including urine samples, using tetrabutylammonium hydroxide as the ionpairing agent. The discrimination of As species in normal individuals and in diseased individuals is important for the evaluation of the therapeutic or toxicological effects of As compounds. To characterize inter- and intraindividual variabilities in the urine As metabolites of healthy individuals, Kile et al. [15] used HPLCeHGeAAS to determine several As species [As(III), As(V), DMA, and MMA] in urine samples collected from Bangladeshi individuals exposed to As in drinking water. They reported that concentrations of the urinary As metabolites were fairly reproducible within individuals but were poorly reproducible when expressed as ratios, suggesting that epidemiological studies should use both urinary As concentrations and the relative proportion of urinary As metabolites to minimize measurement errors and to facilitate the interpretation of the factors that influence As metabolism. Huang et al. [14] examined the association between urinary As speciation and the incidence of urothelial carcinoma in a cohort study. They used HPLCeHGeAAS method to measure As(III), As(V), MMA, and DMA in urine samples collected from a total of 1,078 residents of southwest Taiwan, finding that individuals diagnosed with urothelial cancer had greater percentages of MMA and lower percentages of DMA compared with the healthy individuals. A significant association existed between inefficient As methylation and the development of urothelial cancer in areas featuring high cumulative Asexposure strata in a region of southwest Taiwan endemic for arseniasis. Using the same method to measure the four As species of interest, Huang et al. [16] also investigated the association between As exposure and the risk of carotid atherosclerosis in a sample of the Taiwanese population. They concluded that individuals with greater exposure to As and a lower capacity to methylate inorganic As may be at a higher risk to develop carotid atherosclerosis. Several novel approaches developed for As speciation have been applied to urine samples. Zhu et al. [17] developed a novel hybrid atomizer based on atmospheric pressure dielectric barrier discharge plasma. The coupling of

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HPLC with HGeAAS and the optimized dielectric barrier discharge atomizer provided results for the speciation of As in NIST SRM-2670a (standard reference materials 2670a, human urine) urine that were in good agreement with those obtained using ICPeMS. Although photooxidation is a potential means for the conversion of organic As species,

Table 2

the reaction times in conventional photooxidation reactors is relatively long. Nakazato and Tao [38] developed a novel post-column photooxidation reactor that allows highefficiency photooxidation using a low-temperature mercury lamp. In the absence of any oxidizing agents, the oxidative decomposition of persistent organic arsenicals (e.g. AsB) to

Separation and detection systems used for arsenic speciation in biomedical matrices

Sample type

Arsenic species

Separation system

Detection system References

Urine As(III), As(III), As(III), As(III), As(III),

HGeICPeMS ICPeMS HGeAAS HGeDBD HGeAFS, HGeICPeMS As(III), As(V), MMA, DMA Anion- and cation-exchange HPLC ETAAS As(III), As(V), MMA, DMA Anion-exchange HPLC HGeAFS As(III), As(V), MMA, DMA, AsB Anion-exchange HPLC ICPeMS As(III), As(V), MMA(V), DMA(V), AsB Anion-exchange HPLC ICPeMS As(III), As(V), MMA, DMA, AsB, AsC, TMAO Anion-exchange HPLC ICPeMS As(III), As(V), MMA(V), DMA(V), MMA(III), Anion- and cation-exchange HPLC ICPeMS DMA(III), AsB As(III), MMA(V), DMA(V) Ion-pair HPLC ICPeMS As(III), As(V), MMA, DMA Ion-pair HPLC ICPeMS As(III), As(V), DMA, MMA, AsC, AsB Ion-pair HPLC ICPeMS PAA, PMAA, DPAA, PDMAO, DPMAO Ion-pair HPLC ICPeMS As(III), As(V), MMA(V), DMA(V), MMA(III), Ion-pair HPLC, cation-exchange ICPeMS DMA(III), AsB, AsC, Tetra (Me4Asþ), HPLC TMAsO DMA, oxo-DMAE, TMAO, oxo-DMAA, Anion-exchange HPLC ICPeMS thio-DMAA, thio-DMAE, thio-arsenosugar ICPeMS Thio-Gly, thio-SO4, TMAO, thio-PO4, thio- Reversed-phase HPLC DMAE, thio-SO3, thio-DMAA, thio-DMA iAs, DMA, MMA, TMA Cryogenic separation HGeAAS As(III), As(V), MMA(V), DMA(V), MMA(III), Anion- and cation-exchange HPLC ICPeMS DMA(III), AsB As(III), As(V), MMA, DMA, AsB, AsC, Cation-exchange LC HEPO/HGeICPeMS TMAO, MPhAs, DPhAs DMA, thio-DMAP, oxo-DMAB, thio-DMAB Anion- and cation-exchange HPLC ICPeMS Blood Serum Serum Plasma, blood cell whole blood

As(V), As(V), As(V), As(V), As(V),

MMA, MMA, MMA, MMA, MMA,

DMA DMA DMA DMA DMA

Anion-exchange Anion-exchange Anion-exchange Anion-exchange Anion-exchange

HPLC HPLC HPLC HPLC HPLC

[11] [12,13] [14e16] [17] [18] [19] [20] [21e24] [25e27] [28] [29] [30] [31] [32] [33] [34]

[35] [36] [37] [29] [38] [39]

As(III), MMA(V), DMA(V) Inorganic As, DMA, MMA As(III),As(V), MMA(V), DMA(V)

Ion-pair HPLC Anion, cation-exchange HPLC Anion-exchange HPLC

ICPeMS ICPeMS ICPeMS, HGAFS

[30] [13] [40]

As(III), As(V), MMA(V), DMA(V), AsB

Anion-exchange HPLC

ICPeMS

[27]

Hair and nail samples DPAA As(III), As(V) As(III), As(V), MMA, DMA As(III), As(V), MMA(V), DMA(V) As(III), As(V), MMA(V), DMA(V) As(III), As(V), MMA, DMA, AsB As(III), As(V), MMA, DMA, AsB As(III), As(V), MMA(V), MMA(III), DMA(V), DMA(III) Sulfur-containing arsenic metabolites

Gel-permeation HPLC ICPeMS Electrochemical HGeAAS Anion-exchange HPLC ICPeMS Anion-exchange HPLC ICPeMS Ion-pair HPLC HGeICPeMS Anion-exchange HPLC ICPeMS Anion- and cation-exchange HPLC ICPeMS Anion- and cation-exchange HPLC ICPeMS

[41] [42] [43,44] [45] [46] [24] [47] [48]

Size-exclusion HPLC

[49]

ICPeMS

(continued on next page)

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K.-C. Hsu et al.

Table 2 (continued) Sample type

Arsenic species

Other biomedical matrices Saliva As(III), As(V), MMA(V), DMA(V)

Separation system

Reversed-phase, anion, cationexchange HPLC Cryotrapping

Bladder Inorganic As, MMA, DMA epithelial cell Hepatocyte As(III), As(V), MMA(V), DMA(V), AsB, AsC Anion-exchange HPLC Fecal Me2AsSH, Me2AsSMe, Me2AsSSMe, Gas chromatography (Me2AsO2S, MeAs(SMe)(SEt)

Detection system References

ICPeMS

[50]

HGeCTeAAS

[51]

ICPeMS ICPeMS

[52] [53]

AAS Z Atomic absorption spectrometry; DBD Z Dielectric barrier discharge; DMAA Z Dimethylarsinoylacetate; DMAE Z Dimethylarsinoylethanol; DPAA Z Diphenylmethylarsinic acid; DPhAs Z Diphenylarsinic acid; DPMAO Z Phenyldimethylarsine oxide; ETAAS Z Electrothermal atomic absorption spectrometry; HEPO Z High-efficiency photooxidation; HGAFS Z Hydride generation atomic fluorescence spectrometry; MPhAs Z Monophenylarsonic acid; PAA Z Phenylarsonic acid; PDMAO Z Phenylmethylarsine oxide; PMAA Z Phenylmethylarsinic acid; Thio-Gly Z Thio-arsenosugarglycerol; TMAsO Z Trimethylarsine oxide.

arsenate occurs within an extremely short time (3.5 seconds). In addition, incorporating nanomaterials into the analytical system, Sun et al. [11] developed a novel digestion method for the determination of urinary As species using on-line nano-TiO2-catalyzed photooxidation coupled between microbore LC and ICPeMS. This method was applied for the determination of As(III), MMA, DMA, and AS(V) in urine samples.

As speciation in blood Most As speciation analyses of blood sources are commonly performed using ion chromatography coupled with ICPeMS to achieve the lowest detection levels from different matrix types. Arsenite, arsenate, MMA, DMA, AsB, arsenocholine, and other possible organic As species have all been determined this way. In recent studies of the speciation of As species in blood samples, determining the effects of As compounds as therapeutic drugs for leukemia has become a dominant issue. Fukai et al. [30] used HPLCeICPeMS to monitor the levels of serum and urine As [As(III), MMA, and DMA] in an individual with APL who was administered arsenic trioxide as a therapeutic agent. They concluded that nanomolar concentrations of As in serum were sufficient to produce a therapeutic effect against APL. Fujisawa et al. [57] also used HPLCeICPeMS to monitor plasma As and urinary As excretion after therapeutic administration of arsenic trioxide to individuals with APL. After intravenous administration, the methylated As species were rapidly excreted in the urine, with no measurable accumulation of arsenic trioxide in the blood. Sample preparation for As speciation is an important issue because potential artifacts may be presented after the preparation and storage of biological samples. To study analytical artifacts in the speciation of As in clinical samples, Slejkovec et al. [40] used HPLCeHGeAFS and HPLCeICPeMS to analyze blood samples from cancer individuals treated with high doses of arsenic trioxide. Because contemporary analytical protocols might not be completely immune to artifacts resulting from losses arising from the sampling to the detection stage, they recommended careful interpretation of the results, particularly regarding metabolic and pharmacokinetic effects, and independent

comparison of the sum of species with the total As concentration.

As speciation in hair and nail samples The convenient and noninvasive sampling of hair and nails is frequently applied to the biological monitoring of metal exposure. Analysis of As in human hair is commonly used for biomedical and epidemiological applications to assess exposure or therapeutic outcomes. Recently, speciation analysis of As in hair, fingernail, and toenail samples has become a new approach and powerful tool for analytical assessment in studies of As exposure [26,42e44,46,48]. Ya ´n ˜es et al. [46] developed a simple method for As speciation in human hair based on water extraction and HPLCeHGeICPeMS. From assessments of representative species in extracts from hair, they found that more than 98% of the total As in hair was inorganic As. Raab and Feldmann [48] described a new method for As speciation in hair by extracting with boiling water; they used it to investigate the stability of As species during extraction from hair and nail matrices. Under the extraction conditions used, they noted that inorganic and methylated As(V) species were stable, but methylated As(III) species were not. Furthermore, hair and nail samples from humans suffering from chronic As intoxication contained predominantly inorganic As, with small and strongly varying amounts of DMA(V) and MMA(V) present. Sanz et al. [44] used pressurized liquid extraction of As from hair and then performed As speciation analysis using HPLCeICPeMS. They validated these methods by analyzing a certified human hair reference material. The concentrations found in hair and nails coming from a heavily As-contaminated area were significantly higher (39-fold and 20-fold, respectively) than their background levels. A similar study, conducted by Button et al. [45], on the HPLCeICPeMS analyses of aqueous extracts from toenail samples revealed that inorganic arsenite was the dominant species extracted, with lesser amounts of inorganic arsenate and organic dimethylarsinate. Li et al. [42] developed a method for direct determination of trace levels of As(III) and As(V) through electrochemical HGeAAS without prereduction of As(V). Their method, based on electrochemical HGeAAS with a glassy carbon cathode, led to the simultaneous generation of AsH3 from As(III) and As(V) in the

Arsenic speciation in biomedical sciences presence of acid. Quantitation of these two As species in a certified human hair reference material was possible by controlling the electrolytic current. To investigate As metabolism through a comparative study of As levels in biological matrices, Brima et al. [24] determined the levels of total As in urine, fingernail, and hair samples and those of As metabolites in the urine of healthy volunteers from three different ethnic groups residing in the same area; they observed statistically significant differences, except for the As content in hair. Mandal et al. [49] used size-exclusion HPLCeICPeMS to identify sulfur-containing As metabolites in biological samples. For the identification of four thioarsenical compounds in human nail and urine samples, the proposed Size exclusion chromatographyeESIeMS technique provided better results when compared with those obtained through anion-exchange HPLCeICPeMS. A recent report has suggested that Napoleon Bonaparte’s death was brought on by chronic exposure to As and a medication error; Kintz et al. [47] used HPLCeICPeMS to perform As speciation in two samples of Napoleon’s hair. Their results identified massive amounts of inorganic As, which the authors considered to be consistent with chronic As intoxication.

As speciation in other body fluids and biological matrices Although the value of salivary trace element measurements for the biomonitoring of exposure has been questioned by researchers for many years, saliva has continued to attract attention as a noninvasive matrix. As speciation analysis in saliva is potentially useful for evaluating human exposure and exploring As metabolism. Yuan et al. [50] separated As species in saliva using LC and quantified them through ICPeMS. They determined several As species [As(III), As(V), DMA, and MMA] in saliva using LCeICPeMS and then confirmed the identity of the species using ESI MS/MS. They found a statistically significant relationship between the salivary As concentration and the presence of As-induced skin lesions; the salivary As concentrations also correlated with the drinking-water As concentration. The authors concluded that speciation of As(III), As(V), DMA, and MMA in human saliva is a useful method for monitoring As exposure. The cell culture model for demonstrating the possible effects of As intoxication has gained much attention in recent years; speciation analysis has, therefore, become necessary for studying the metabolism of As compounds. Herna ´ndez-Zavala et al. [51] investigated the speciation of As in exfoliated bladder epithelial cells from individuals exposed to As in drinking water. Using HGecryotrappinge AAS to determine the concentrations of inorganic As, MMA, and DMA, they observed detectable concentrations of all three species in both urine and epithelial cells. The authors suggested that urinary levels of inorganic metabolites do not necessarily reflect the levels of these metabolites in the bladder epithelium because no correlation existed between the concentrations of the species in the two sample types. Thus, As speciation analysis in bladder epithelial cells may be an effective tool for risk assessment of bladder cancer and other urothelial diseases associated

387 with exposure to inorganic As. Cao et al. [52] used HPLC coupled with ICPeMS to determine the levels of total As and As metabolites in human liver hepatocytes. Relative to direct sampling, they compared the effects of three digestion methods in the determination of As in liver hepatocytes after ultrasonication. Speciation of the lysate using HPLCeICPeMS revealed the presence of As(III), As(V), DMA, and a DMA intermediate metabolite. The application of microorganisms in trace element speciation is an interesting and alternative technology. The ability of cecal microflora to methylate inorganic As species has been described; Harrington et al. [58] studied the anaerobic and aerobic degradation of AsB by microorganisms associated with the human large intestine. They used HPLCeICPeMS to determine the levels of total As and As species in samples obtained from the 30-day incubation of human fecal matter with the microorganisms. Analysis of the aerobic system revealed significant degradation of AsB to DMA, dimethylarsinoylacetate, and trimethylarsine oxide after 7 days; in contrast, the anaerobic system exhibited no AsB degradation. The authors correlated these results with the biocatalytic ability of AsB degradation within the human GI tracts. Diaz-Bone et al. [53] analyzed the volatile As compounds formed from intestinal microorganisms, using simultaneous electron-impacte MS and ICPeMS detection after gas chromatographic separation to rapidly identify volatile As species in the headspace of fecal incubations. They identified three As speciesdmethylmethylthioethylthioarsine, dimethylmethylselenoarsine, and thiobis(dimethylarsine)dthat had not been reported previously from either human or environmental matrices, concluding that the intestinal metabolism of As is a process that should be considered during the risk assessment of ingested As.

Conclusion and future trends The importance of As speciation analysis in biomedical fields will continue to grow because of potential exposure to or contamination from As compounds and because of the potential applications of certain As compounds in cancer therapy. Although chromatographic separation coupled with ICPeMS detection has been used predominantly for As speciation analysis in biomedical research, some efforts have been undertaken to develop nonchromatographic speciation approaches. Methodological developments for As speciation have resulted in new hyphenation techniques and new detection systems. Overcoming the obstacles posed by low As levels, complicated matrices, and high diversity of As species in biomedical specimens remains a great challenge for As speciation and is facilitating the development of novel hyphenated methods. In addition, investigating As metabolism through speciation analysis of bodily fluids (e.g. saliva, sweat, or cerebrospinal fluid) appears to be a new trend awaiting further investigation.

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