Mass spectrometric identification of novel arsinothioyl-sugars in marine bivalves and algae

Abstract

Following recent reports on the detection of the new group of thioarsenosugars in marine molluscs we have developed two chromatographic methods in order to explore the presence of additional species of this group in biological samples. A series of thioarsenosugar standards, containing four different thioarsenosugars in total, was prepared and used for method optimization. First, conventional anion exchange chromatographic conditions were modified to achieve efficient elution of the thioarsenosugars. This required relatively high contents of methanol in the mobile phase (up to 40%). Online hyphenation with electrospray tandem mass spectrometry provided good quality product ion mass spectra for the thioarsenosugars. These allowed the generation of collision induced dissociation breakdown curves for each compound which finally resulted in the development of a very sensitive and specific selected reaction monitoring approach. However, the high amount of methanol used with this method was not optimum for direct hyphenation with inductively coupled plasma mass spectrometry (ICP-MS) without a specially adapted interface. Therefore, reversed phase anion pairing HPLC with a low methanol content (5%) in the mobile phase was developed and used in this case. The combined application of both analytical techniques revealed the presence of two novel thioarsenosugars in marine bivalves and marine algae. Moreover, this is the first time that the detection of thioarsenosugars in marine algae is reported.

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Introduction

Recent publications have reported the identification of a series of arsine sulfide containing compounds in marine bivalves and mammalian urine.^1–3 In particular, Schmeisser et al. identified two arsine sulfide containing ribofuranosides (thioarsenosugars) in extracts of a canned mussel product by using a combination of high-performance liquid chromatography (HPLC) on-line with inductively coupled plasma mass spectrometry (ICP-MS) and electrospray mass spectrometry (ES-MS).¹ The structures of these thioarsenosugars, i.e. DMThioAsSugarGlycol and DMThioAsSugarPhosphate, are shown in Fig. 1. Independently, Fricke et al., using similar analytical techniques, demonstrated the presence of DMThioAsSugarPhosphate as a significant constituent in selected species of clams and mussels.²

Fig. 1 Structures and abbreviations of arsinoyl-sugars and arsinothioyl-sugars. The abbreviated nomenclature proposed for these compounds⁹ consists of 4 parts thus giving the most essential structural information: (1) the number of methyl groups bound to the arsenic atom: DM for dimethyl; TM for trimethyl; (2) only for the new group of thioarsenosugars (sulfur atom bound to the arsenic atom): Thio; (3) the common core for all arsenoribofuranoside-derivatives: AsSugar; (4) the characteristic functional group contained in the side chain: e.g. Glycol, Phosphate, Sulfonate, Sulfate.

As a result of these findings it is now pertinent when attempting to make risk assessments with respect to arsenic in seafood to take this new group of compounds into account. In addition, it is also relevant to investigate their metabolic behaviour in humans, as well as examine their toxicological properties and/or those of their metabolites and degradation products. Overall, the presence of the arsine sulfide containing sugars adds further complexity to the existing knowledge of arsenic biochemistry in marine organisms. For example, speculation regarding the origin of the thioarsenosugars suggests in vivo formation in mussels and clams from ingested arsinoyl-sugar analogues (oxoarsenosugars).¹ Alternatively, they may originate from marine algae, the major nutrient source for mussels.¹ Also, from an analytical chemistry perspective the recent identification of the two previously mentioned thioarsenosugars clearly highlights the limitations of most existing chromatographic methods which have been used extensively for the separation of water soluble arsenic species originating from marine organisms. The problem in this case stems from the fact that the thioarsenosugars are strongly retained on HPLC columns under the mobile phase conditions most commonly used in arsenic speciation analyses.^1–3 More specifically, the Hamilton PRP-X100 anion exchange column, used in the majority of arsenic speciation studies, exhibits, in addition to its anion exchange properties, strong hydrophobic characteristics which may be responsible for the extensive retention of the thioarsenosugars. Such elution behaviour is a major reason for this group of compounds evading detection for so long. Low chromatographic recovery of arsenic species is a significant problem when making risk assessments, as it may prevent detection of a significant portion of the arsenic species present in a particular sample. Another possible reason for the late discovery of the thioarsenosugars is the fact that no precautions are usually taken during sample storage and preparation in order to prevent the potential oxidation of arsinothioyl species to their corresponding arsinoyl-analogues.

The present study was initiated as a result of our expectation that in addition to the two already identified thioarsenosugars in marine bivalves^1,2 at least two more should be present, i.e. DMThioAsSugarSulfonate and DMThioAsSugarSulfate. It is known that thioarsenosugars can be derived in vitro directly from their oxoarsenosugar analogues, following treatment with H₂S,⁴ therefore it is expected that a similar conversion can also take place in vivo. Thus because DMAsSugarSulfonate and DMAsSugarSulfate are present in marine bivalves and other marine organisms it is probable that their thio-analogues DMThioAsSugarSulfonate and DMThioAsSugarSulfate, which have so far evaded detection, are also present in these samples. In general, DMAsSugarGlycol and DMAsSugarPhosphate have been found to be the major arsenosugars present in marine bivalves⁵ so their thio-analogues are present at relatively high concentrations and can therefore be detected relatively easily using suitable chromatographic conditions. However, DMAsSugarSulfonate and DMAsSugarSulfate are usually present in much lower concentrations and hence the detection of their thio-analogues requires more sensitive analytical techniques.

In this study we report on the presence of two novel arsine sulfide containing arsenosugars (Fig. 1: DMThioAsSugarSulfonate and DMThioAsSugarSulfate) in marine bivalve samples, and also report the presence of all four thioarsenosugars in marine algae which to the best of our knowledge is for the first time. To achieve these identifications it was necessary to develop improved analytical methods. Thus anion-pairing (AP) reversed phase (RP) HPLC on-line with ICP-MS was developed and used for the analysis of extracts of various marine organisms. In addition, anion exchange chromatography was further optimized for the efficient elution of the thioarsenosugars and used in combination with electrospray (ES) tandem mass spectrometry (MS/MS) for definitive identification of these species. Both sensitive and highly selective detection was achieved by employing selected reaction monitoring (SRM). The two analytical methodologies developed and used in this study provided complementary evidence for the presence of arsine sulfide containing arsenosugars in extracts of marine bivalves and algae.

Experimental

Chemicals

Ammonium bicarbonate (puriss.), hydrochloric acid (37%, puriss., p.a.) and iron(II) sulfide sticks were obtained from Riedel-de Haen, Seelze, Germany. Ammonium hydroxide solution (puriss., p.a.), tetrabutylammonium hydroxide (TBAH) 30-hydrate (>99.0%) and malonic acid (>99.0%) were purchased from Fluka, Buchs, Switzerland. Methanol (gradient grade for HPLC) was supplied by Merck, Darmstadt, Germany.

Samples

Three commercially available marine algal samples were used in this study: two Canadian kelp powders, referred to as “old” and “new” Canadian kelp powder were purchased from Galloway’s, Richmond, BC, Canada, in 1999 (batch no. 231-0390-13) and 2005 (batch no. 231-0390-15), respectively, and one Icelandic kelp powder (Laminaria digitata, originating from Iceland) was purchased from Mountain Rose Herbs, Eugene, OR, USA. Fresh clams (Venus verrucosa) were purchased at a local market and kept frozen until they were dissected and extracted.

Extraction of kelp powder and marine bivalve samples

Kelp powder was suspended in deionised water (extractant–solid ratio typically 20 mL g⁻¹), shaken for 10 min and centrifuged for 15 min (2000 g; mini centrifuge, Labnet International, Inc., Woodbridge, NJ, USA). Clam samples were dissected and the digestive tissue was manually homogenised (glass Teflon® homogenizer) with deionised water (2 mL g⁻¹; water was degassed by sonication before use) and sonicated for 10 min. After centrifugation (15 min, 100% speed, Universal II, Hettich, Germany) the supernatant was filtered (HPLC syringe filter, 0.45 μm cellulose acetate, Alltech, IL, USA). The extracts were analyzed directly after preparation in order to avoid possible degradation during storage.

Total arsenic contents (mean ± SD, n = 3) were determined for the “old” Canadian kelp (18.9 ± 0.8 μg As g⁻¹) and the “new” Canadian kelp (39.6 ± 2.4 μg As g⁻¹) using microwave assisted acid digestion and quantification by ICP-MS (external calibration with indium as the internal standard). Extraction efficiencies for these samples were calculated as percentage ratio of the extracted arsenic content and the total arsenic content (n = 3): 86 ± 4% for the “old” Canadian kelp and 92 ± 6% for the “new” Canadian kelp.

Preparation of thioarsenosugar standards

Standard A. An aqueous extract of the “old” Canadian kelp powder, known to contain the 4 common oxoarsenosugars and dimethylarsinic acid (DMA),^6–8 was treated for approximately 10 min with H₂S (produced from reacting Fe(II) sulfide sticks with 10% HCl). Low amounts of a formed precipitate were removed by centrifugation prior to injection onto the HPLC. The resulting supernatant contained DMThioAsSugars with glycol-, phosphate-, sulfonate- and sulfate- aglycone (electrospray MS/MS data presented in results and discussion section) in an excess of H₂S. These compounds were stable for several days when stored frozen; no artefacts due to the unreacted H₂S were observed.

Standard B. The Icelandic kelp powder (Laminaria digitata) contains a high amount of DMAsSugarSulfonate besides lower amounts of DMAsSugarPhosphate, DMAsSugarGlycol and DMAsSugarSulfate (data not shown). Solid phase extraction using a strong anion exchange cartridge was applied to prepare fractions enriched with DMAsSugarSulfonate, which was not available to us as a single standard. This was achieved by conditioning the cartridge with methanol, water and 2.5 mM ammonium acetate, applying the Icelandic kelp extract and eluting it with 2.5 mM ammonium acetate followed by 10 mM ammonium hydrogen carbonate at pH 10. Fractions of 1 mL were collected and analyzed using HPLC-ES-MS/MS to determine their arsenosugar content. The conversion of DMAsSugarSulfonate to DMThioAsSugarSulfonate was performed in the same way as for standard A; however centrifugation was not necessary in this case.

Standard C. Chromatographic fractions containing DMAsSugarSulfate were collected following the HPLC analysis (anion exchange column, PRP-X100, 10 mM NH₄HCO₃, pH 10, 1 mL min⁻¹) of an extract of the “old” Canadian kelp powder. The DMAsSugarSulfate containing fractions were pre-concentrated and combined. DMThioAsSugarSulfate was then prepared as described for standard A; however, centrifugation was not necessary in this case.

DMAsSugarGlycol and DMAsSugarPhosphate (structures shown in Fig. 1) were kindly donated from Prof. K. A. Francesconi (Karl-Franzens University, Graz, Austria). Their conversion to DMThioAsSugarGlycol and DMThioAsSugarPhosphate, respectively, was accomplished as described for standard A; centrifugation was not necessary.

Instrumentation and experimental conditions

Electrospray tandem mass spectrometry was performed in the selected reaction monitoring mode (SRM) using a TSQ Quantum (Thermo Finnigan, San Jose, CA, USA) in the positive ion mode with the following tune parameters: electrospray voltage 4.1 kV, sheath gas pressure 45 arbitrary units, auxiliary gas pressure 25 arbitrary units, capillary temperature 300 °C, source collision-induced dissociation (CID) 0 V. Anion exchange chromatography (PRP-X100, 250 × 4.1 mm with two PRP-X800 cation exchange pre-columns; Hamilton, Reno, NV, USA) was applied for online HPLC-ES-MS/MS hyphenation using a Surveyor HPLC-System with a quaternary gradient pump (including solvent degassing) and autosampler (20 μL injection volume). Gradient elution was performed with 20 mM NH₄HCO₃, pH 10 (eluent A) and 20 mM NH₄HCO₃, 40% methanol, pH 10 (eluent B) as previously optimised for the detection of 28 arsenic species, including 4 thioarsenosugars:⁹ 5 min at 25% B; in 1 min to 100% B; 24 min at 100% B; in 0.5 min to 25% B; for 4.5 min at 25% B (35 min total run time). The HPLC flow rate of 1 mL min⁻¹ was split postcolumn with 24% introduced into the ES source.

For HPLC-ICP-MS a reversed phase column (Discovery C18, 150 × 4.6 mm, 5 μm particle size, Supelco, Bellefonte, PA, USA) operated with a Marathon HPLC pump (Rigas Labs, Thessaloniki, Greece) at 1 mL min⁻¹ and manual injection (50 μL) was coupled online with an X-Series ICP-MS (Thermo Electron Elemental Analysis, Winsford, UK). A concentric nebulizer was used in combination with an impact bead spray chamber. A grounded torch shield was used in order to improve detection sensitivity. The ICP-MS was tuned daily for optimum intensity of ¹¹⁵In and ⁷⁵As. During the chromatographic analysis ⁷⁵As was monitored, in addition m/z 77 was used to detect possible chloride interferences, i.e. ArCl⁺. Based on a previously published rapid separation method for DMAsSugars,⁶ an anion-pairing reversed phase method was developed for the separation of DMThioAsSugars. The optimum mobile phase contained 5 mM tetrabutylammonium hydroxide (TBAH) in 5% aqueous methanol. Malonic acid was used to adjust the pH to 7.5. Calibration with arsenite and arsenate standard solutions was performed for the quantification of thioarsenosugars in the algal extracts.

Chromatographic recoveries for the AP-RP-HPLC method were determined offline by fraction collection. First, the sample was injected onto the HPLC system and the eluent was collected for the entirety of the chromatographic run (fraction I). Second, injection of the same sample was repeated, but without the HPLC column, and again the mobile phase was collected (fraction II). In both cases, blank fractions consisting of mobile phase collected before the extract injection and after the chromatographic runs resulting in fractions I and II, were used to correct for the arsenic background in fractions I and II. The arsenic response of all fractions was measured using ICP-MS with indium as the internal standard. The percentage chromatographic recovery for arsenic was calculated as: [(blank-corrected arsenic intensity of fraction I)/(blank-corrected arsenic intensity of fraction II)] × 100.

Results and discussion

Development of analytical methods for the identification of thioarsenosugars

Arsinothioyl-sugars (thioarsenosugars) were prepared by reacting their corresponding arsinoyl-sugar analogues with H₂S. A detailed description of the procedure and starting materials used for the preparation of the arsinothioyl-sugars is given in the experimental section of this paper. Anion exchange HPLC online with ES tandem MS was used to characterize the thioarsenosugar content of all the standards. For this purpose tandem mass spectra and collision induced dissociation (CID) breakdown curves (Fig. 2) were obtained for each of the protonated molecules of the thioarsenosugar compounds. From the recorded data it was possible to propose a fragmentation scheme for this group of compounds (Scheme 1). It should be noted that all four of the thioarsenosugars examined exhibited similar CID behaviour. A common feature was the formation of a product ion with m/z 253, believed to correspond to the dimethylarsinothioylpentose ion (Scheme 1a). This is in analogy to the already reported CID behaviour of the arsinoyl-sugars,^10,11 all of which afford a product ion at m/z 237, corresponding to their dimethylarsinoylpentose moiety (Scheme 1b). Another common CID feature for all 4 thioarsenosugars is the formation of a product ion with m/z 97. This ion is believed to correspond to [C₅H₅O₂]⁺, arising from the elimination of As(CH₃)₂SH and H₂O molecules from the dimethylarsinothioylpentose product ion (m/z 253). Once again this behaviour is similar to that observed for the arsinoyl-sugars during their analysis under both low and high energy MS/MS conditions.^9–11 Only DMThioAsSugarSulfate gave a significant additional product ion, observed at m/z 345, believed to form following the loss of SO₃. Therefore, in some cases in-source conversion of DMThioAsSugarSulfate ([M + H]⁺, m/z 425) to DMThioAsSugarGlycol ([M + H]⁺, m/z 345) may occur. Thus in order to avoid any possibility for misidentification, chromatographic separation of the two compounds is required prior to ES-MS detection. Even more critical is the fact that DMAsSugarSulfate and DMThioAsSugarSulfonate have molecular ions of the same m/z ratio 409. In the SRM mode both compounds exhibit the transition 409 → 97, however, the second transition 409 → 237 and 409 → 253, respectively, is unique.

Scheme 1 Proposed fragmentation scheme for both (a) arsinothioyl-sugars and (b) arsinoyl-sugars.

Fig. 2 Collision induced dissociation breakdown curves for the four thioarsenosugar compounds: (a) DMThioAsSugarGlycol, (b) DMThioAsSugarPhosphate, (c) DMThioAsSugarSulfonate, (d) DMThioAsSugarSulfate.

The breakdown curves presented in Fig. 2 were further used in order to find the most efficient conditions for generating the product ions to be used in the selected reaction monitoring (SRM) mode. The SRM approach is invaluable as it enables both sensitive and selective detection of the thioarsenosugars when appropriate conditions are used. The breakdown curves presented in Fig. 2 also constitute a valuable source of reference data for researchers to whom thioarsenosugar standards are not readily available.

For the identification of the thioarsenosugars at the low μg L⁻¹ level an HPLC-ES-MS/MS method, operated in the SRM mode was set up to monitor two transitions for each thioarsenosugar. The intensity ratios of the two transitions used for each thioarsenosugar were determined (Table 1) following the analysis of standard A (contains all four thioarsenosugars). Relative standard deviations of less than 10% (n = 2) for these ratios indicate good reproducibility of product ion formation under the experimental conditions applied.

Table 1 SRM ratios determined for thioarsenosugar standards and thioarsenosugars in kelp powder extracts using HPLC-ES-MS/MS detection

Analyte	Selected reactions monitored (collision energy/eV)		Intensity ratio of SRM-1/SRM-2 (peak areas)
	SRM-1	SRM-2	Standard A (n = 2)	“New” Canadian kelp powder day 1 (n = 3)	“New” Canadian kelp powder day 2 (n = 3)
DMThioAsSugarGlycol	345 → 97 (20)	345 → 253 (10)	1.10 ± 0.07	1.04 ± 0.10	1.09 ± 0.07
DMThioAsSugarPhosphate	499 → 253 (15)	499 → 97 (27)	2.22 ± 0.01	2.65 ± 0.23	2.50 ± 0.39
DMThioAsSugarSulfonate	409 → 97 (25)	409 → 253 (15)	1.70 ± 0.14	1.66 ± 0.34	1.56 ± 0.18
DMThioAsSugarSulfate	425 → 97 (30)	425 → 253 (15)	1.33 ± 0.02	1.41 ± 0.19	1.54 ± 0.11

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It is obvious that a necessary requirement for the successful identification of the thioarsenosugars is their efficient elution from the HPLC column. In most published arsenic speciation methodologies that have employed the Hamilton PRP-X100 column with ICP-MS detection the mobile phase has not been reported to contain significant amounts of organic solvent (e.g. methanol), mainly because all the water soluble arsenic species that have been reported until recently in the literature elute off this column within a reasonable retention time. However using typical conditions (only aqueous mobile phase, pH 10, 25 cm column, 1 mL min⁻¹) with this anion exchange column, very long retention times for the thioarsenosugars were observed. Fricke et al. even term the DMThioAsSugarPhosphate as “unchromatographable”.² In earlier experiments, we found that the addition of methanol to the mobile phase significantly shortens the retention times of the thioarsenosugars.⁹ The methanol content of the mobile phase was therefore optimized for the efficient elution of all four thioarsenosugars from the Hamilton PRP-X100 column. To achieve this it was convenient to use ES-MS detection which is easily compatible with elevated levels of methanol in the mobile phase. On the other hand, adding high amounts of methanol to the mobile phase with ICP-MS detection would require a specially adapted interface (e.g. cooled spray chamber, membrane desolvator or post-column dilution), which were either not available in our study or have negative effects on the analytical performance, e.g. loss of some species at the membrane or higher limits of detection. Another option would be the use of a microbore HPLC-column. In the present study the optimized chromatographic mobile phase which allowed for reasonable retention and sufficient separation of the four thioarsenosugars contained up to 40% methanol.

To overcome the requirement for high organic content in the mobile phase an alternative chromatographic separation mode was investigated for the development of a complementary HPLC-ICP-MS method suitable for the detection of the thioarsenosugars DMThioAsSugarSulfonate and DMThioAsSugarSulfate. More specifically, anion pairing (AP) reversed phase (RP) HPLC with a mobile phase containing 5 mM TBAH, pH 7.5 and 5% methanol was used in the isocratic mode at a flow rate of 1.0 mL min⁻¹. At pH 7.5 adequate retention, efficient separation and symmetrical chromatographic peaks were observed for the 4 thioarsenosugars present in standard A. The addition of 5% methanol to the mobile phase resulted in further improvement of peak symmetry and shorter retention times for the thioarsenosugars (Fig. 3a). The fact that the thioarsenosugars elute readily with a small amount of methanol in the mobile phase is important as it enhances the chances for detecting DMThioAsSugarSulfonate and DMThioAsSugarSulfate and possibly other more strongly retained species in marine organisms. The synthetic DMThioAsSugar standard solutions prepared from isolated/enriched DMAsSugar -Glycol, -Phosphate, -Sulfonate (standard B) and -Sulfate (standard C) were used to confirm the identity of the detected peaks. The peaks indicated with asterisks (*) elute at the same retention times as peaks obtained for an aqueous dimethylarsinic acid (DMA) solution after reaction with H₂S. In analogy to the previously described synthesis of the thioarsenosugars this reaction is expected to convert DMA to thio-dimethylarsinic acid species. The presence of two oxygen atoms bound to the arsenic in DMA may account for the presence of more than one reaction product. Fig. 3b shows the chromatographic behaviour of the arsinoyl-sugars present in aqueous extracts of the “old” Canadian kelp powder (not derivatized with H₂S). It is evident that these early eluting arsinoyl-sugars are not fully resolved, however, because the objective for the development of this chromatographic method was the efficient elution and separation of DMThioAsSugarSulfonate and DMThioAsSugarSulfate, this was not a problem. It should be mentioned, however, that under different mobile phase conditions all four of the arsinoyl-sugars can be resolved.⁶

Fig. 3 AP RP HPLC-ICP-MS chromatograms of a H₂S treated extract of the “old” Canadian kelp sample (standard A) containing 4 thioarsenosugars (a) and a non-derivatized extract of the same algal sample containing 4 oxoarsenosugars (b). In both cases m/z 75 (⁷⁵As⁺) was monitored. Arrows indicate the correlation between the peaks of the oxoarsenosugars and their corresponding thioarsenosugars produced upon reaction with H₂S. Asterisks (*) indicate thio dimethylarsinic acid species.

Identification of thioarsenosugars in marine organisms

The two analytical methodologies developed in this study, AP RP HPLC-ICP-MS and HPLC-ES-MS/MS, were used to investigate the presence of naturally occurring thioarsenosugars in extracts of marine bivalve samples and a commercially available kelp powder (“new” Canadian kelp powder). A mild extraction procedure was applied using only deionised water. Sonication was only applied for a 10 min period for the marine bivalve extractions. At the beginning of the study we used extracts of the “old” Canadian kelp powder as starting material for the preparation of standard A. Later we obtained a new batch of kelp powder from the same supplier. In contrast to the “old” kelp powder, the “new” powder already contained thioarsenosugars in significant amounts. Therefore, the extracts of this algal sample were characterised in detail. Initially, HPLC-ES-MS/MS was used in the SRM mode to examine the presence of both arsinoyl-sugars and arsinothioyl-sugars in extracts of “new” Canadian kelp powder. The resulting chromatogram (Fig. 4) revealed the presence of 8 arsenosugars (structures shown in Fig. 1). Most importantly, these findings demonstrate the identification of the thioarsenosugars DMThioAsSugarSulfonate and DMThioAsSugarSulfate in marine organisms and for the first time the presence of thioarsenosugars in marine algae. The SRM intensity ratios of the 2 transitions monitored, shown in Table 1, agree with those obtained for the standards and thus support the reported identification of the four thioarsenosugars. Even more conclusive evidence was obtained from the excellent quality tandem mass spectra obtained for each of the thioarsenosugars present in the extract of commercial “new” Canadian kelp powder (Fig. 5). These spectra are in good agreement with those obtained for the synthetic standards. Quantification of these compounds using electrospray MS was not attempted as this would require the use of more rigorously purified and quantified thioarsenosugar standards, which is an ongoing objective of our research.

Fig. 4 HPLC-ES-MS/MS chromatograms obtained for arsenosugars in an extract of the “new” Canadian kelp powder. Two SRM transitions were monitored for each arsenosugar. The traces correspond to the following arsenosugars: (a) DMAsSugarGlycol, (b) DMAsSugarPhosphate, (c) DMAsSugarSulfonate, (d) DMAsSugarSulfate, (e) DMThioAsSugarGlycol, (f) DMThioAsSugarPhosphate, (g) DMThioAsSugarSulfonate, (h) DMThioAsSugarSulfate.

Fig. 5 Tandem mass spectra of the protonated thioarsenosugars present in the “new” Canadian kelp powder extract. Precursor ions are indicated with an asterisk (*). The collision energy used was 10 eV. The tandem mass spectra correspond to: (a) DMThioAsSugarGlycol, (b) DMThioAsSugarPhosphate, (c) DMThioAsSugarSulfonate, and (d) DMThioAsSugarSulfate.

The presence of DMThioAsSugarSulfonate and DMThioAsSugarSulfate in extracts of “new” Canadian kelp powder was further confirmed using AP RP HPLC-ICP-MS. More specifically peaks 6 and 7 in the chromatogram shown in Fig. 6 were found to correspond to the novel thioarsenosugars DMThioAsSugarSulfonate and DMThioAsSugarSulfate, respectively. Identification in this case was achieved by matching their retention times with those obtained from standards B and C, respectively. In addition, a series of other arsenic species labelled as peaks 1, 3, 4, 5, and zone 2 (consisting of several overlapping arsenic species) were detected. Only tentative identification of these peaks was carried out as they were not the focus of the present study. Thus, based on retention time data, we propose peak 1 arising from DMAsSugarGlycol, peak 3 from DMAsSugarSulfate, peak 4 from DMThioAsSugarPhosphate, peak 5 possibly from a thio derivative of dimethylarsinic acid, and finally zone 2 from DMAsSugarPhosphate, DMAsSugarSulfonate and DMThioAsSugarGlycol. The presence of all four arsinoyl-sugars in this particular sample was clearly demonstrated using HPLC-ES-MS/MS (Fig. 4). In addition the retention times for the arsinoyl-sugars under these specific chromatographic conditions are known from the analysis of “old” Canadian kelp powder extracts (Fig. 3b). Peak 5 exhibits the same retention time as one of the species produced following the reaction of dimethylarsinic acid with hydrogen sulfide. Peak 4 has the same retention time as DMThioAsSugarPhosphate present in standard A. Finally the identity of the overlapping arsenic species giving rise to zone 2 was suggested based on the species identified in this sample by using HPLC-ES-MS/MS (Fig. 4). Quantification was done for the well resolved arsenic species in extracts of the “new” kelp sample (n = 2): DMThioAsSugarPhosphate 0.25 ± 0.06 μg As g⁻¹, DMThioAsSugarSulfonate 2.6 ± 0.5 μg As g⁻¹ and DMThioAsSugarSulfate 3.3 ± 0.5 μg As g⁻¹. According to this the two novel thioarsenosugars constitute about 8% of the total eluted arsenic of the “new” Canadian kelp powder. Column recovery for the arsenic species present in the extract of this sample was determined to be (77 ± 7)% (n = 2).

Fig. 6 AP RP HPLC-ICP-MS chromatogram of an extract of the “new” Canadian kelp powder obtained from monitoring m/z 75 (⁷⁵As⁺). The insert shows a magnification of the late eluting peaks, of which 6 and 7 are assigned as DMThioAsSugarSulfonate and DMThioAsSugarSulfate.

However, it must be considered that using arsenate as the calibrant for the quantification of thioarsenosugars with HPLC-ICP-MS requires that the arsenic response for the calibrant and all analytes is the same and more critically that the chromatographic recovery is the same for each involved arsenic species. The latter is usually not proven which leaves some doubt about the accuracy of the results. In future it will be necessary to prepare reproducible and stable standard solutions of the thioarsenosugars, e.g. on the basis of extracts from the “new” Canadian kelp powder, to investigate these aspects in detail.

Our results provide a conclusive identification of thioarsenosugars in the analysed “new” Canadian kelp powder. The analysis of fresh marine algae will be necessary to confirm that the thioarsenosugars are naturally occurring in the algae. However, already the presence of thioarsenosugars in the kelp powder supports the hypothesis that thioarsenosugars originate from marine algae and are subsequently taken-up by marine bivalves. It should be stressed, however, that this does not exclude the possibility for their in vivo formation in marine bivalves. It should also be mentioned that the kelp from which this commercial powder was produced has undergone several processing stages, however, it is unlikely that any of these can result in the formation of thioarsenosugars from their oxo-analogues. It is also important to note that these relatively high amounts of DMThioAsSugarSulfonate and DMThioAsSugarSulfate in the “new” Canadian kelp powder should allow for its use as a reference standard in future studies. Also, the commercial availability of significant amounts of the “new” Canadian kelp powder opens up the possibility for the production of a reference material containing all 8 arsenosugars.

Moreover, the presence of DMThioAsSugarSulfonate and DMThioAsSugarSulfate was detected in clam extracts by using AP RP HPLC-ICP-MS. Due to the very low amounts of these compounds present and the large amount of sample matrix HPLC-ES-MS/MS was not sensitive enough to confirm their presence. During the AP RP HPLC-ICP-MS analysis the “new” Canadian kelp powder extract was used as a source for these two compounds (Fig. 6) in order to compare their retention times with those of the late eluting peaks originating from the clam extract. Even though a satisfactory match was achieved, it was also necessary to conduct spiking experiments in which case a small amount of the kelp powder extract was spiked into the clam extract. Spike-to-extract ratios were chosen so that the amount of spiked compound approximately equalled the amount of the corresponding compound in the clam extract. The results obtained from this spiking experiment, i.e. single non-distorted peaks (Fig. 7 insert), further support the presence of DMThioAsSugarSulfonate and confirm the presence of DMThioAsSugarSulfate in the clam extract. Column recovery for the clam extract with the AP RP HPLC method was estimated to be 91% (n = 1). It should be noted that a preliminary report on the identification of DMThioAsSugarSulfonate and DMThioAsSugarSulfate in molluscs was presented simultaneously by us¹² and Kahn et al.¹³

Fig. 7 AP RP HPLC-ICP-MS chromatograms obtained monitoring m/z 75 (⁷⁵As⁺) following the injection of: (a) clam extract, (b) clam extract spiked with “new” Canadian kelp powder extract 5 ∶ 1, and (c) “new” Canadian kelp powder extract; peaks labelled 6 and 7 correspond to DMThioAsSugarSulfonate and DMThioAsSugarSulfate, respectively. Arsenic peaks eluting in zone I were not identified as this would require additional chromatographic separation which was not the objective of the present study. Offsets for chromatograms (b) and (c) are 500 cps and 1000 cps, respectively.

Recently published papers for the identification of DMThioAsSugarGlycol and DMThioAsSugarPhosphate have reported the use of synthetically prepared arsinothioyl-sugar standards as spike solutions.^1,2 Compared to this approach the use of the “new” Canadian kelp extract as a spike solution has the important advantage that it contains no excess of H₂S. In a recently published study the reaction mixture was purged with argon to remove excess H₂S; however the efficiency of this procedure needs to be proven.¹³ Spiking experiments with a solution that contains unreacted H₂S may lead to the conversion of DMAsSugars present in the sample to their corresponding DMThioAsSugars. As a result peak areas in the chromatogram of the spiked extract may not equal the sum of the corresponding peak areas in the chromatograms of the nonspiked extract and the spike solution after consideration of the dilution factor. Moreover, it is even possible that additional peaks of arsinothioyl species may occur in the chromatogram of the spiked extract.

In all cases, the addition of H₂O₂ to the extracts resulted in the disappearance of the DMThioAsSugarSulfonate and DMThioAsSugarSulfate peaks from the chromatogram.

The possibility of conversion of oxoarsenosugars to thioarsenosugars during sample storage was also considered. The “old” kelp powder used in this study had been stored for several years, but still no thioarsenosugars were detected in this material. This supports the hypothesis that thioarsenosugars are not formed in the dried kelp powder during prolonged storage of the material. In our opinion the critical time for such a conversion to occur would be between harvesting and drying of the kelp. To our knowledge the kelp is cleaned after harvesting and put on racks for drying. Since the product is sold for human consumption measures are taken to prevent fouling that could lead to the formation of H₂S. The clam samples were obtained fresh i.e. alive from a local market. Storage in the freezer was limited to a maximum of a few days.

Conclusions

In this study we have demonstrated the presence of two novel thioarsenosugars in marine bivalves and marine algae. Moreover, this is the first report of any thioarsenosugars in marine algae. To achieve these identifications it was necessary to develop advanced analytical methods suitable for the detection of thioarsenosugars at the low μg L⁻¹ level in crude extracts containing high levels of matrix. Two different HPLC separation modes were optimized and applied along with element specific and molecular mass spectrometric detectors. We expect that these findings will promote several new directions in arsenic speciation research as questions relating to the biochemical, toxicological and environmental properties of this group of compounds need to be addressed.

It should also be stressed that future research in the development of improved extraction procedures is expected to enhance thioarsenosugar recoveries from various sample matrices. In addition, further work aimed towards investigating the stability of these compounds is still necessary.

Acknowledgements

The authors thank the European Commission for the funding of a Marie Curie Excellence Grant (Contract No. MEXT-CT-2003-002788).

References

1. E. Schmeisser, R. Raml, K. A. Francesconi, D. Kuehnelt, A. Lindberg, C. Sörös and W. Goessler, Chem. Commun., 2004, 1824–1825.
2. M. W. Fricke, P. A. Creed, A. N. Parks, J. A. Shoemaker, C. A. Schwegel and J. T. Creed, J. Anal. At. Spectrom., 2004, 19, 1454–1459.
3. H. R. Hansen, R. Pickford, J. Thomas-Oates, M. Jaspars and J. Feldmann, Angew. Chem., Int. Ed., 2004, 116, 337–340.
4. H. R. Hansen, M. Jaspars and J. Feldmann, Analyst, 2004, 129, 1058–1064.
5. Z. Slejkovek, J. T. Van Elteren and A. R. Byrne, Talanta, 1999, 49, 619–627.
6. S. Wangkarn and S. A. Pergantis, J. Anal. At. Spectrom., 2000, 15, 627–633.
7. M. Miguens-Rodriguez, R. Pickford, J. E. Thomas-Oates and S. A. Pergantis, Rapid Commun. Mass Spectrom., 2002, 16, 323–331.
8. R. Pickford, M. Miguens-Rodriguez, S. Afzaal, P. Speir, S. A. Pergantis and J. E. Thomas-Oates, J. Anal. At. Spectrom., 2002, 17, 173–176.
9. V. Nischwitz and S. A. Pergantis, Anal. Chem., 2005, 77, 5551–5563.
10. S. McSheehy, M. Marcinek, H. Chassaigne and J. Szpunar, Anal. Chim. Acta, 2000, 410, 71–84.
11. S. A. Pergantis, K. A. Francesconi, W. Goessler and J. E. Thomas-Oates, Anal. Chem., 1997, 69, 4931–4937.
12. K. Kanaki, V. Nischwitz and S. A. Pergantis, European Winter Conference on Plasma Spectrochemistry, Budapest, Hungary, 2005, poster T43.
13. (a) M. Kahn, R. Raml, W. Goessler, E. Schmeisser and K. A. Francesconi, European Winter Conference on Plasma Spectrochemistry, Budapest, Hungary, 2005, poster T41; (b) Now published M. Kahn, R. Raml, E. Schmeisser, B. Vallant, K. A. Francesconi and W. Goessler, Environ. Chem., 2005, 2, 171–176.

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