Volatile Analytes Formed from Arsenosugars: Determination by

Anal. Chem. 2004, 76, 418-423

Volatile Analytes Formed from Arsenosugars: Determination by HPLC-HG-ICPMS and Implications for Arsenic Speciation Analyses Ernst Schmeisser, Walter Goessler, Norbert Kienzl, and Kevin A. Francesconi*

Institute of Chemistry, Analytical Chemistry, Karl-Franzens-University, Universitaetsplatz 1, 8010 Graz, Austria

It is generally accepted that the use of the hydride generation method to produce volatile analytes from arsenic compounds is restricted to the two inorganic forms (As(III) and As(V)) and the three simple methylated species methylarsonate (MA), dimethylarsinate (DMA), and trimethylarsine oxide. We report here that arsenosugars, major arsenic compounds in marine organisms, produce volatile analytes by the hydride generation (HG) method without a prior mineralization/oxidation step and that they can be quantitatively determined using HPLCHG-ICPMS. The hydride generation efficiency depends on the type of hydride generation system and is influenced by the concentration of HCl and NaBH4. For the four arsenosugars investigated, the hydride generation efficiencies were ∼21-28% (or 4-6%, depending on the HG system) that obtained for As(III) under conditions optimized for As(III). This hydride efficiency was less than that shown by MA (∼68% relative to As(III)) and DMA (∼75%) but greater than that displayed by As(V) (∼18%). Analysis of two species of brown algae, Fucus serratus and Hizikia fusiforme, by HPLC-HG-ICPMS produced results comparable with those obtained from other techniques used in our laboratory (HPLC-ICPMS and LCESMS for F. serratus) and with results from other laboratories taking part in a round robin exercise (H. fusiforme). This study shows for the first time the quantitative determination of arsenosugars using the hydride generation method without a decomposition step and has considerable implications for analytical methods for determining inorganic arsenic based on the formation of volatile hydrides. The determination of arsenic species is an important research area because arsenicals are abundant and diverse in environmental and biological samples, and they display very different degrees of toxicity.1 Of major toxicological interest are the toxic inorganic forms of arsenic, namely, arsenite (As(III)) and arsenate (As(V)), and their products of human metabolism, predominantly methylarsonate (MA) and dimethylarsinate (DMA). About 25 additional organoarsenic species have been identified in organisms, and these are generally considered to be nontoxic. The most abundant * To whom correspondence should be addressed. Phone: +43 316 380 5301. Fax: +43 316 380 9845. Email: [email protected]. (1) Shibata, Y.; Morita, M.; Fuwa, K. Adv. Biophys. 1992, 28, 31-80.

418 Analytical Chemistry, Vol. 76, No. 2, January 15, 2004

Figure 1. Structures of the four major arsenosugars found in biological samples and investigated in this work.

naturally occurring organoarsenic compounds include arsenobetaine and a group of closely related riboside derivatives termed arsenosugars (Figure 1). Arsenosugars are the major arsenic compounds in marine algae, where they can occur at concentrations exceeding 100 mg As kg-1 dry mass, and they are also significant metabolites in marine animals that feed on algae.2 Recently, arsenosugars have also been reported in freshwater algae and vascular plants and in terrestrial organisms, including earthworms, fungi, and plants.2 Analytical methods are needed to determine the various chemical forms of arsenic, in particular, to distinguish between toxic and nontoxic species. Most methods employ a chromatographic system coupled to an arsenic-selective detector, with the most powerful combination in common use being high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICPMS).3 Detection by ICPMS offers many advantages, including high elemental selectivity, large linear range, and low limits of detection. The detection limits can be further improved by incorporating a hydride generation (HG) derivatization step.3 (2) Francesconi, K. A.; Kuehnelt, D. Environmental chemistry of arsenic; Frankenberger, W. T., Ed.; Marcel Dekker: New York, 2002, Chapter 3. (3) Goessler, W.; Kuehnelt, D. Environmental chemistry of arsenic; Frankenberger, W. T., Ed.; Marcel Dekker: New York, 2002, Chapter 2. 10.1021/ac034878v CCC: $27.50

© 2004 American Chemical Society Published on Web 12/06/2003

Hydride generation is a general term applied to techniques for analyzing metals and metalloids and their species following their chemical conversion to volatile analytes (usually hydrides).4 The application of the hydride generation method for determining arsenic species, however, is restricted: although there are many naturally occurring arsenicals, only five of them (As(III), As(V), MA, DMA, and trimethylarsine oxide) are reported to be hydrideactive (i.e. can be converted to volatile analytes).5 For this reason, the hydride generation technique for determining arsenicals is used mainly for samples that contain predominantly inorganic As, MA, and DMA, namely, natural waters, drinking water, and human urine.6 The range of arsenic species amenable to analysis by the hydride generation method can be extended by incorporating a post-HPLC column decomposition step whereby non-hydrideactive arsenicals are converted first to As(V) and then to AsH3.7,8 By running the system with and without a decomposition step, the method is claimed to provide further evidence for the identification of various compounds on the basis of their being hydride-active or not.9-14 This distinction between hydride-active and nonactive arsenic species also has broader implications: future techniques for determining toxicologically relevant arsenic species in food items may be based on the assumption that they are hydride-active, whereas other arsenicals in food, mainly arsenobetaine and arsenosugars, are not.15,16 A potential pitfall with this approach was revealed recently in the study of Sanchez et al.,17 who reported small responses for arsenosugars when analyzed by the hydride generation method without a decomposition step. This observation has considerable implications for arsenic speciation analyses based on hydrideactive compounds and the possible future application of those methods to distinguish toxic inorganic arsenicals from nontoxic organoarsenicals.15 We report here a systematic investigation describing the quantitative analysis of arsenosugars using HPLCHG-ICPMS. The technique was optimized for four major arsenosugars and applied to the determination of these compounds in aqueous extracts of two species of brown algae. EXPERIMENTAL SECTION Standards and Reagents. Standard solutions of the following arsenic species were prepared in Milli-Q water (18.2 MΩ cm): As(III) from NaAsO2 and As(V) from Na2HAsO4‚7H2O purchased from Merck (Darmstadt, Germany); dimethylarsinate (DMA) from sodium dimethylarsinate trihydrate purchased from Fluka (Buchs, (4) Campbell, A. D. Pure Appl. Chem. 1992, 64, 227-244. (5) Wei, X.; Brockhoff-Schwegel, C. A.; Creed, J. T. J. Anal. At. Spectrom. 2001, 16, 12-19. (6) Anonymous. In Arsenic in Drinking Water; National Academy Press: Washington, DC, 1999, Chapter 3. (7) Dagnac, T.; Padro´, A.; Rubio, R.; Rauret, G. Talanta 1999, 48, 763-772. (8) Gomez-Ariza, J. L.; Sanchez-Rodas, D.; Giraldez, I.; Morales, E. Talanta 2000, 51, 257-268. (9) Le, X. C.; Ma, M.; Wong, N. A. Anal. Chem. 1996, 68, 4501-4506. (10) Howard, A. G. J. Anal. At. Spectrom. 1997, 12, 267-272. (11) Ma, M.; Le, C. X. Clin. Chem. 1998, 44, 539-550. (12) Gallagher, P. A.; Wei, X.; Shoemaker, J. A.; Brockhoff, A.; Creed, J. T. J. Anal. At. Spectrom. 1999, 14, 1829-1834. (13) Slejkovec, Z.; Van Elteren, J. T.; Byrne, A. R. Talanta 1999, 49, 619-627. (14) Go´mez-Ariza, J. L.; Sa´nches-Rodas, D.; Gira´ldez, I.; Morales, E. Analyst 2000, 125, 401-407. (15) Capelo, J. L.; Lavilla, L.; Bendicho, C. Anal. Chem. 2001, 73, 3732-3736. (16) Andersons, S. L.; Pergantis, S. A. Talanta 2003, 60, 821-830. (17) Sa´nches-Rodas, D.; Geiszinger, A.; Go´mez-Ariza, J. L.; Francesconi, K. A. Analyst 2002, 127, 60-65.

Switzerland); methylarsonate (MA) synthesized from As2O3 with CH3I in NaOH (Meyer reaction); arsenosugar 1 (Figure 1) synthesized according to McAdam et al.;18 and arsenosugars 2, 3, and 4 (Figure 1) previously isolated from natural sources.19 Ammonium dihydrogen phosphate (p.a.), aqueous ammonia solution (25%, Suprapur), sodium borohydride (p.a.), sodium hydroxide (p.a.), hydrochloric acid (p.a. 32%), and nitric acid (p.a) were all obtained from Merck, and phosphoric acid and sulfuric acid were purchased from Fluka. Aqueous sodium borohydride solutions at concentrations of 1.0, 0.7, 0.5, and 0.3% NaBH4 in 0.1 mol dm-3 NaOH were prepared fresh. A solution of 3 mol dm-3 HCl was prepared and diluted to prepare solutions of 1.0, 0.5, 0.1, 0.05, and 0.025 mol dm-3 HCl. Extracts of Algae. Fucus serratus samples were from a homogeneous batch of extracts previously prepared from the brown alga F. serratus. The arsenic species (mainly arsenosugars 1-4) in these extracts have been previously characterized and quantified by Madsen et al.20 using HPLC-ICPMS and HPLCESMS. A freeze-dried sample of the brown alga Hizikia fusiforme was prepared by GDCh (Gesellschaft Deutscher Chemiker) for use in a “round robin” study21 evaluating methods of arsenic speciation. A portion (100 mg weighed to 0.1 mg) of this material was extracted with 5.0 cm3 Milli-Q water and shaken overnight. The mixture was centrifuged, and the supernatant was filtered (0.22 µm) prior to analysis by HPLC-ICPMS and HPLC-HGICPMS. Chromatographic Separation of the Arsenic Compounds. The high performance liquid chromatography (HPLC) system consisted of a Hewlett-Packard 1100 series system (HewlettPackard, Waldbronn, Germany) equipped with a quaternary pump, a vacuum degasser, column oven, and an autosampler with a variable 100-mm3 injection loop. The separation of As(III), As(V), MA, DMA, and the four arsenosugars was performed on a Hamilton PRP-X100 (Reno, NV) anion-exchange column (100 × 4.1 mm i.d.) with aqueous 10 mmol dm-3 NH4H2PO4 at pH 6.0 (adjusted with 25% aqueous NH3) as the mobile phase. The column was operated at 40 °C, and the flow rate was 1.5 cm3 min-1. The injection volume was 20 mm3. For investigations without the hydride generation system, the outlet of the HPLC column was connected to the ICPMS (equipped with a Babington-type nebulizer) with PEEK (polyetheretherketone) capillary tubing (0.125mm i.d.). Hydride Generation of the Arsenic Compounds. Hydride Generation Accessory (Agilent Technologies). The HPLC system was connected directly to a continuous flow hydride generation ICPMS with ISIS (integrated sample introduction system) (Agilent, Waldbronn, Germany). The hydride generation system (Figure 2a) was equipped with a concentric nebulizer, a modified cyclonic spray chamber, and a membrane filter. Hydride generation was performed with NaBH4 (flow rate 0.5 cm3 min-1, optimized for As(III)) as reducing agent and HCl (flow rate 0.3 cm3 min-1, (18) McAdam, D. P.; Perera, A. M. A.; Francesconi, K. A. Aust. J. Chem. 1987, 40, 1901-1908. (19) Francesconi, K. A.; Edmonds, J. S.; Stick, R. V. J. Chem. Soc., Perkin Trans. 1 1992, 1349-1357. (20) Madsen, A. D.; Goessler, W.; Pedersen, S. N.; Francesconi, K. A. J. Anal. At. Spectrom. 2000, 15, 657-662. (21) Fecher, P.; Schuffenhauer, C. Bestimmung von anorganischem Arsen in Algen mit Hydrid-Atomabsorption; Poster presentation at Jahrestagung Chemie 2003, Munich, 2003.

Analytical Chemistry, Vol. 76, No. 2, January 15, 2004

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Table 1. Operating Conditions for the Agilent 4500 ICPMS (Agilent Technologies, Waldbronn, Germany) instrumental settings

without HG system

HG (Agilent Technologies)

HG (Cetac Technologies)

rf power, W plasma gas, dm3 min-1 carrier gas, dm3 min-1 blend gas, dm3 min-1 nebulizer spray chamber sampling cone skimmer cone sampling depth acquisition parameters: detector mode monitored masses (m/z) integration time/mass

1350 15.0 0.98 0.00 Babington type Peltier cooled 2 °C nickel, 1.0-mm orifice nickel, 0.4-mm orifice 6.5 mm

1300 15.0 0.33 0.85 none none nickel, 1.0-mm orifice nickel, 0.4-mm orifice 7 mm

1300 15.0 1.16 0.00 none none nickel, 1.0-mm orifice nickel, 0.4-mm orifice 7 mm

pulse 75 and 77 0.1 s

pulse 75 and 77 0.1 s

pulse 75 and 77 0.1 s

Figure 2. (a) Hydride generation accessory (Agilent Technologies), (b) HGX-100 (Cetac Technologies).

optimized for As(III)). The volatile analytes were transported via a 0.5-m sample transfer line (4-mm i.d.) directly to the torch of the ICPMS. HGX-100 (Cetac Technologies). The HPLC system was connected directly to a continuous flow hydride generation system (HGX-100, Cetac Technologies, Omaha, NE). The hydride generation system (Figure 2b) was equipped with a peristaltic pump and a spray chamber as a gas liquid separator, but was not equipped with a concentric nebulizer. Hydride generation was performed with NaBH4 (flow rate 1.5 cm3 min-1, optimized for As(III)) as reducing agent and HCl (flow rate 1.2 cm3 min-1, optimized for As(III)). The volatile analytes were transported via a 0.5-m sample transfer line (4-mm i.d.) directly to the torch of the ICPMS. For safety reasons, the wastes from the hydride generation reactions were collected in 2 mol dm-3 NaOH. Inductively Coupled Plasma Mass Spectrometry for ArsenicSpecific Detection. An Agilent 4500 inductively coupled plasma mass spectrometer (Agilent, Waldbronn, Germany) served as the arsenic-selective detector. The instrumental and acquisition parameters for HPLC-ICPMS and HPLC-HG-ICPMS are summarized in Table 1. For data evaluation, the chromatographic software G1824C version C.01.00 was used. RESULTS AND DISCUSSION HPLC-HG-ICPMS of the Arsenosugars. Analysis of the four standard arsenosugars 1-4 by HPLC-HG-ICPMS gave signals indicating that a volatile analyte was being formed directly from these compounds (i.e., without a prior decomposition step). Having established that arsenosugars were indeed hydride-active, we then investigated quantitative aspects of the method. Calibra420 Analytical Chemistry, Vol. 76, No. 2, January 15, 2004

Figure 3. HPLC-HG-ICPMS chromatograms of standard arsenosugars 1-4, each at concentrations of 1.00, 5.00, 10.0, 20.0, 50.0, 100, and 200 ng As cm-3. Conditions: Hamilton PRP-X100 anion exchange column; 10 mmol dm-3 aqueous NH4H2PO4 at pH 6.0; flow rate 1.5 cm3 min-1; column temperature 40 °C; injection volume 20 mm3; 3 mol dm-3 HCl (flow rate 0.3 cm3 min-1); 0.7% NaBH4 in 0.1 mol dm-3 NaOH (flow rate 0.5 cm3 min-1), hydride generation accessory (Agilent Technologies).

tion curves prepared with standard arsenosugars at concentrations ranging from 1 to 200 ng As cm-3 showed excellent linearity, with R2 > 0.999 for all compounds, calculated from chromatograms shown in Figure 3. Comparison of Different Hydride Generation Systems. Both hydride generation systems produced signals, but the response from the Agilent system was about 5-fold as intense as that produced by the Cetac system. The data are presented in Table 2, expressed as percent response relative to As(III), the most hydride-active of the arsenicals, and demonstrate the strong hydride-activity of the arsenosugars. Their response is ∼40% that obtained for MA and DMA and is up to 50% higher than the signal obtained for As(V). In view of this observed hydride-activity, it is surprising that arsenosugars have not previously been reported in hydride generation analyses. Possibly, their activity is strongly dependent on the type of hydride generation system, the conditions employed, or both. Certainly, the responses we obtained appear to be much greater than those reported by Sanchez et al.,17 although on that occasion, no attempt was made to optimize or to quantify the signals. Optimization of the Hydride Generation System for the Determination of Arsenosugars. Because the Agilent system

Table 2. Relative Hydride Generation Efficiency for As(III), DMA, MA, As(V) and Arsenosugars 1-4 for the Two Hydride Systems Investigateda

arsenic species

hydride generation accessory (Agilent Technologies)

HGX-100 (Cetac Technologies)

As(III) DMA MA As(V) arsenosugar 1 arsenosugar 2 arsenosugar 3 arsenosugar 4

100 74.8 ( 2.7 67.8 ( 1.7 18.4 ( 1.1 28.3 ( 3.2 24.0 ( 2.5 21.4 ( 2.6 20.6 ( 2.4

100 66.7 ( 1.4 65.5 ( 1.7 38.3 ( 1.1 6.5 ( 0.2 5.6 ( 0.6 4.5 ( 0.6 3.9 ( 0.7

a Values in % ( SD, (n ) 3) are recorded relative to As(III) taken as 100%.

was much more sensitive than the Cetac system, further optimization of the technique in terms of the hydride generation reagents was carried out with the Agilent system. Figure 4 shows the influence of the concentrations of NaBH4 and HCl on the hydride activity of the arsenosugars. The concentration of HCl had a marked effect: maximal response was obtained at 1 mol dm-3 HCl, and the response dropped dramatically at HCl concentration of