Thio arsenosugars identified as natural constituents of mussels by liquid chromatography-mass spectrometry

Article abstract of DOI:10.1039/b406917j

Two novel thio arsenosugars have been identified by liquid chromatography-mass spectrometry as significant arsenic constituents in samples of mussels.

Full text of DOI:10.1039/b406917j

Thio arsenosugars identified as natural constituents of mussels by liquid
chromatography-mass spectrometry
Ernst Schmeisser,a Reingard Raml,a Kevin A. Francesconi,*a Doris Kuehnelt,a Anna-Lena Lindberg,b
Csilla Sörösc and Walter Goesslera
a Karl-Franzens-University, Institute of Chemistry, Analytical Chemistry, Graz, Austria.
E-mail: kevin.francesconi@uni-graz.at; Fax: +43 316 3809845; Tel: +43 316 3805301
b Institute of Environmental Medicine, Karolinska Institute, Box 210 S-171 77, Stockholm, Sweden
c
Department of Applied Chemistry, BKÁE University, Villányi út 29-33, 1118 Budapest, Hungary
Received (in Cambridge, UK) 7th May 2004, Accepted 7th June 2004
First published as an Advance Article on the web 20th July 2004
Two novel thio arsenosugars have been identified by liquid
chromatography-mass spectrometry as significant arsenic con-
stituents in samples of mussels.
3 and 4 (Scheme 1). Although there have been no reports of such
species as natural products, a recent study8 showed the presence of
a thio arsenic species [(Me)2As(S)CH2COOH] in sheep urine. We
then synthesised9 analytical quantities of 3 and 4 to serve as
standards for HPLC-ICP MS and (later) HPLC-electrospray MS.
The chromatographic behaviour of the standard compounds 3 and
4 at pH 5.6 and pH 10.3 matched those for the two new arsenicals,
U1 and U2 respectively, present in the mussel extract. Spiking
experiments, whereby standard 3 or 4 was added to the mussel
extract, produced suitably enhanced undistorted peaks for U1 (pH
5.6, tR 16.8 min; pH 10.3, tR 4.3 min) or U2 (pH 10.3, tR 6.4 min),
respectively.
Further evidence for the proposed new arsenicals was provided
by HPLC-electrospray MS.10 Electrospray mass spectra11 for the
two standard thio arsenicals showed clear [M+H]+ protonated
molecular ions (for both 32S and 34S) at m/z 499/501 (compound 4)
and 345/347 (compound 3) in addition to characteristic product ions
at m/z 253/255 (loss of aglycone), and signals for 107 (AsS+), and
91 (AsO+, formed from traces of O2 in the N2 drying gas12); these
ions were selectively monitored in the HPLC-electrospray MS
analyses. Analysis of the mussel extract under the optimised
conditions produced the ions (m/z 501, 499, 253, 91) characteristic
for compound 4 at a retention time (tR = 13.3 min) exactly matching
The first report in 1977 of the innocuous arsenobetaine
[(CH3)3As+CH2COO2] as the major arsenical in lobster,1 and later
in many other marine animals,2 allayed toxicological concerns
about high arsenic concentrations in seafoods. Subsequent work,
however, revealed other naturally occurring organoarsenic com-
pounds in marine samples, in particular a range of arsenic-
containing carbohydrates collectively termed arsenosugars which
occur in algae and organisms feeding on algae such as molluscs.2
The toxicology of arsenosugars is yet to be fully evaluated, but it is
likely to be complex because they are biotransformed in humans to
many as yet unidentified arsenic metabolites.3,4
As part of our studies on the human health consequences of
arsenic in foods, we have analysed food items with high
performance liquid chromatography using inductively coupled
plasma mass spectrometry as the arsenic selective detector (HPLC-
ICP MS).5 One sample, a canned mussel product,6 was of interest,
because analysis of an aqueous extract of the mussel showed the
presence of an unidentified arsenic compound (U1), in addition to
common arsenicals namely arsenosugars 1 and 2 (Scheme 1),
arsenobetaine, trimethylarsoniopropionate, and dimethylarsinate.
With time (several days at room temperature), however, peaks for
1 and 2 in the extract increased with a corresponding, although
smaller, decrease in the peak for U1, and on treatment of the sample
with H2O2 (note 7) the peak for U1 completely disappeared while
peaks for both 1 and 2 increased substantially (Fig. 1). Although it
first appeared as though U1 may be serving as a precursor to both
arsenosugars 1 and 2, mass balance calculations suggested that U1
was converting solely to arsenosugar 1, and the precursor of
arsenosugar 2 was a possible fourth arsenical which was being
retained on the HPLC column. Indeed, when the chromatography
was repeated under different conditions (pH 10.3, see note 5), an
additional peak (U2) was observed, and when the extract was
treated with H2O2 this peak disappeared with a concomitant
quantitative increase in the peak for arsenosugar 2 (Fig. 1; Table
1).
The observation that the unknowns U1 and U2 were readily
converted in air into arsenosugars 1 and 2 (both arsine oxides)
suggested that they may be the corresponding thio arsenic species
Fig. 1 HPLC-ICP MS of aqueous extract of canned mussel before and after
treatment with H2O2 at pH 5.6 (i), and pH 10.3 (ii) (see note 5).
Table 1 Concentrations of As species (mg As l21 a
) in a fresh extract of
canned mussel, and in the same extract after treatment with H2O2
c
As species
tRb/min
Extractc
Extract + H2O2
1
U1
2
3.5 (iii)
4.4 (ii)
6.0 (i)
7.9 (5.1%)
27.6 (3.8%)
7.5 (6.1%)
49.1 (5.0%)
34.5 (4.3%)
< 1.0
55.2 (4.2%)
< 1.0
U2
6.5 (ii)
a Values have been normalised to accommodate small differences in
volumes due to addition of H2O2. b Number in parenthesis represents one of
three HPLC conditions described in note 5. c Mean and relative standard
deviation of three HPLC measurements.
Scheme 1 Interconversion of novel thio arsenosugars with known
arsenosugars.
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C h e m . C o m m u n . , 2 0 0 4 , 1 8 2 4 – 1 8 2 5
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

that of the standard (Fig. 2). Retention time (tR = 5.1 min) matching
was also achieved for compound 3 in the mussel extract, but on this
occasion clear signals could be obtained only for [M+H]+ at m/z
345 and for m/z 91; the product ion m/z 253 was also detected but
the peak was not well resolved. Individual experiments in which the
mussel extract was spiked with synthesized 3 or 4 produced single
enhanced peaks for U1 and U2, respectively. On the basis of these
HPLC-electrospray MS data and the HPLC-ICP MS results, the
structures for the two arsenicals U1 and U2 in mussel were assigned
as the novel thio arsenosugars 3 and 4 respectively.
Because our sample was a canned mussel product, it seemed
possible that the new arsenic species present in the sample were
artefacts from the processing procedure. HPLC-ICP MS analysis of
an aqueous extract of fresh, unprocessed mussels (Perna canal-
iculus), however, similarly revealed the presence of the thio
compounds 3 and 4 indicating that they are natural products (we
cannot disregard the possibility of these changes being elicited
between time of sample collection and analysis). We have also
carried out preliminary investigations of other molluscs and a
crustacean sample, and have found 3 and 4 to be present in most
cases. These data suggest that thio arsenosugars are significant
seafood arsenicals, in molluscs at least, which raises the question as
to why they have so far eluded detection in the many previous
studies investigating arsenic species in marine samples. Possibly,
the unusual chromatographic behaviour of these thio arsenosugars
(they are strongly retarded on the anion exchange column PRP-
X100 at typically used pHs 5–6), together with their ready
conversion to the corresponding oxides have contributed to their
being overlooked until this study. The likelihood of other thio
arsenosugars occurring as natural products, and so far escaping
detection, must also be considered.
There appear to be two possible sources for the thio arsenosugars
in mussels. First, these compounds may be naturally present also in
algae, the major food item of the mussels examined here, and they
are then accumulated unchanged by the mussels. Alternatively, the
thio arsenic compounds may be formed in vivo by the mussels from
ingested arsenosugars 1 and 2. Although the question remains open,
available data suggest that the second explanation is more likely
because the thio compounds do not appear to be present in algae,
and, arsenosugars, which are major arsenicals in algae, are present
only as minor compounds in the mussels examined here.
The presence of thio arsenicals in marine animals including those
used as human foods, raises two important issues. First, the
metabolism in humans and possible toxicological properties of
these new compounds needs to be assessed. Second, it would be
interesting to investigate the role of these compounds in the
biotransformation of arsenic, especially their possible involvement
in the formation of arsenobetaine, the major form of arsenic in
marine animals.
Financial support from the Austrian Science Fund (FWF) under
project P16088-N03 and EC project No. QLK4-CT-2001-00264
(ASHRAM) is gratefully acknowledged.
Notes and references
1 J. S. Edmonds, K. A. Francesconi, J. R. Cannon, C. L. Raston, B. W.
Skelton and A. H. White, Tetrahedron Lett., 1977, 18, 1543–1546.
2 K. A. Francesconi and J. S. Edmonds, Adv. Inorg. Chem., 1997, 44,
147–189.
3 M. S. Ma and X. C. Le, Clin. Chem., 1998, 44, 539–550.
4 K. A. Francesconi, R. Tanggaard R, C. J. McKenzie and W. Goessler,
Clin. Chem., 2002, 48, 92–101.
5 HPLC-ICP MS was performed under three sets of chromatographic
conditions: (i) Anion-exchange pH 5.6 (Hamilton PRP-X100 column,
4.1 3 250 mm; 20 mM NH4H2PO4, pH 5.6; 40 °C); (ii) Anion-exchange
pH 10.3 (PRP-X100, 4.1 3 100 mm; 20 mM NH4HCO3, pH 10.3, 40
°C); (iii) cation-exchange (Zorbax 300-SCX, 4.6 3 150 mm; 10 mM
pyridinium formate pH 2.6, 30 °C). Flow rate of 1.5 ml min21 and 10 ml
or 20 ml injection volumes were used in all cases. Selective arsenic
detection was performed with ICP MS (Agilent 4500 or 7500c) at m/z
75.
6 The product comprised whole mussel tissue (species unknown) bathed
in an aqueous liquor. Analyses were mainly performed on an aqueous
extract of the tissue, prepared with 35% extraction efficiency by shaking
a portion of freeze-dried tissue (300 mg containing 11.2 mg As g21 dry
mass) with water (5.0 ml) overnight. The liquor (neat, untreated) was
also analysed and showed the same pattern of arsenic species.
7 H2O2 (20 ml, 30% solution) was added to 300 ml of the extract.
8 H. R. Hansen, R. Pickford, J. Thomas-Oates, M. Jaspars and J.
Feldmann, Angew. Chem., Int. Ed., 2004, 43, 337–340.
9 H2S was bubbled for several minutes through an aqueous solution
containing arsenosugar 1 (30 mg As in 10 ml) or arsenosugar 2 (1 mg As
in 1 ml), which resulted in essentially quantitative conversion to the
respective thio compounds (as determined by HPLC-ICP MS).
10 HPLC-electrospray MS was performed under anion-exchange condi-
tions (PRP-X100 column, 1 3 150 mm; 4 mM NH4HCO3, pH 10.3 and
methanol (95+5) at 40 °C). Flow rate was 0.1 ml min21 and injection
volume was 10 ml. Electrospray ionisation and mass detection was
performed in positive ion mode with an Agilent G1946D single
quadrupole MS.
11 Optimised fragmentor voltages for standard thio arsenicals 3 and 4 for
protonated molecular ions and product ions were: m/z 345 & 347, 499
& 501 (100 V); 253 & 255 (150 V); 107 (250 V) 91 (400 V).
12 D. Kuehnelt, W. Goessler and K. A. Francesconi, Rapid Commun. Mass
Spectrom., 2002, 17, 654–659.
Fig. 2 HPLC-electrospray MS chromatograms for standard 4 and aqueous
extract of canned mussel.
C h e m . C o m m u n . , 2 0 0 4 , 1 8 2 4 – 1 8 2 5
1825