ARTICLE
pubs.acs.org/ac
Identification and Quantification of Arsenolipids Using
Reversed-Phase HPLC Coupled Simultaneously to High-Resolution
ICPMS and High-Resolution Electrospray MS without Species-Specific
Standards
Kenneth O. Amayo,† Asta Petursdottir,†,‡ Chris Newcombe,† Helga Gunnlaugsdottir,‡ Andrea Raab,†
Eva M. Krupp,†,§ and J€org Feldmann*,†
†
Trace Element Speciation Laboratory, Department of Chemistry, Meston Walk, University of Aberdeen, Aberdeen AB24 3UE,
Scotland, United Kingdom
‡
Matis, Icelandic Food and Biotech R&D, Vinlandsleid 12, 113 Reykjavik, Iceland
§
Aberdeen Centre of Environmental Sustainability, St. Machar Drive, Aberdeen, Aberdeen AB24 3UU, Scotland, United Kingdom
bS Supporting Information
ABSTRACT: Although it has been known for decades that
arsenic forms fat-soluble arsenic compounds, only recent attempts to identify the compounds have been successful by using
a combination of fractionation and elemental and molecular
mass spectrometry. Here we show that arsenolipids can directly
be identified and quantified in biological extracts using reversedphase high-performance liquid chromatography (RP-HPLC)
simultaneously online-coupled to high-resolution inductively
coupled plasma mass spectrometry (ICPMS) and high-resolution electrospray mass spectrometry (ES-MS) without having a
lipophilic arsenic standard available. Using a methanol gradient
for the separation made it necessary to use a gradient-dependent
arsenic response factor for the quantification of the fat-soluble
arsenic species in the extract. The response factor was obtained
by using the ICPMS signal of known concentration of arsenic.
The arsenic response was used to determine species-specific
response factors for the different arsenic species. The retention
time for the arsenic species was utilized to mine the ES-MS data for accurate mass and their tandem mass spectrometry (MS/MS)
fragmentation pattern to give information of molecular formula and structure information. The majority of arsenolipids, found in the
hexane phase of fish meal from capelin (Mallotus villosus) was in the form of three dimethylarsinoyl hydrocarbons (C23H38AsO,
C17H38AsO, C19H42AsO) with minor amounts of dimethylarsinoyl fatty acids (C17H36AsO3, C23H38AsO3, C24H38AsO3). One of
the dimethylarsinoyl fatty acids (C24H38AsO3), with an even number of carbon in the fatty acid chain, was identified for the first time
in this work. This molecular formula is unusual and in contrast to all previously identified arsenic-containing fatty acids with odd
numbers of carbon.
A
rsenic is enriched in marine biota and occurs mainly in watersoluble species such as arsenobetaine and arsenosugars.1,2
Even though it was already pointed out 40 years ago that arsenic
is also present in the fat or oil fraction of fish,3 only limited
attempts have so far been made to determine the molecular
structure of those fat-soluble arsenic species.46 Recently reversed-phase (RP) chromatography coupled to inductively
coupled plasma mass spectrometry (ICPMS) has been used to
identify different arsenic-containing compounds in cod liver oil,7
capelin oil,8 and tuna fish.9 The molecular structures were
determined either by fraction collection, cleanup steps, and offline identification by matrix-assisted laser desorption ionization
r 2011 American Chemical Society
time-of-flight mass spectrometry (MALDI-TOFMS),6 highresolution electrospray mass spectrometry (ES-MS),10 or indirectly by comparison with synthesized lipid-soluble arsenic
species which were fully characterized by molecular mass spectrometry. The analytical workup schemes used to determine their
molecular structures were, however, extensive, and the identification of potentially coeluting arsenic-containing compounds in
low concentrations was difficult. Since the origin and biological
Received: March 6, 2011
Accepted: March 30, 2011
Published: March 30, 2011
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activity of such compounds are unknown there is a need for
simpler methodologies which are capable to directly identify and
quantify all arsenic-containing compounds in a lipophilic extract.
Recent studies on arsenolipids have only focused on the polar
arsenolipids in fish oils and in tuna fish.810 Here we report the
use of reversed-phase chromatography which is coupled online
simultaneously to high-resolution ICPMS and high-resolution
ES-MS to gain quantitative information of the nonpolar arsenolipids in fish meal and molecular information from accurate mass
of the protonated molecular ion, its fragments, and tandem mass
spectrometry (MS/MS) pattern as well as the chromatographic
behavior recorded with ICPMS.
ARTICLE
Table 1. HPLCICPMS/ES-MS Parameters for Arsenolipid
Speciation Analysis
HPLC
column
Thermo Scientific
ACE C18; 4.6 mm 150 mm, 5 μm
column temperature
30 °C
injection volume
100 μL
buffer A
0.1% formic acid in water
buffer B
0.1% formic acid in methanol
splitter ratio
25:75 (ICPMS/ES-MS)
flow rate
gradient
1 mL/min
025 min: 4%/min, 10 min 100% B
ICPMS
’ EXPERIMENTAL SECTION
Chemicals and Standards. All chemicals used were of
analytical grade or better. Milli-Q water (18.2 MΩ cm) was used
for sample preparations. Formic acid and sodium arsenite were
supplied from Sigma-Aldrich (U.K.). Sodium dimethylarsinic
acid (98%, DMAV) and sodium arsenite (AsIII) used as calibration standards for quantification were obtained from ChemService (U.S.A.). Hexane, hydrogen peroxide (H2O2, 32%), and
methanol were obtained from Fisher Scientific; nitric acid
(HNO3, 65%) was from Fluka (U.K.).
Samples. The capelin meal samples were collected from
industrial producers in Iceland. The capelin fish (Mallotus
villosus) used for the meal was caught in Icelandic waters just
prior to meal production.
Digestion Method for Total Arsenic Concentration. Samples were digested with concentrated HNO3/H2O2 (1:2, V/V)
in the microwave (Mars-5, CEM instrument, U.K.) with the
following temperature program: 5 min at 50 °C (2 min temp
ramp), 5 min at 75 °C (2 min temp ramp), 25 min at 95 °C
(5 min temp ramp).
The total arsenic concentrations in the digests were determined by quadruple ICPMS (Agilent 7500c) with standard
ICPMS conditions, m/z 75 and m/z 77 were monitored for
possible interference by ArClþ and Ge (m/z 74) used as internal
standard. Quantification was carried out against standard solutions of sodium arsenite in the calibration range of 020 μg/L.
The accuracy of the results was evaluated by the measurement of
certified reference materials (CRM): seaweed 140 (International
Atomic Energy Authority) and fish muscle DORM-3 (National
Research Council Canada).
The measured values of the CRMs gave satisfactory results.
The DORM-3 gave a recovery of 100% with 6.9 ( 0.7 μg As/g (n
= 3), and the IAEA 140 measured at 44.18 ( 2.1 μg As/g (n = 3)
gave a recovery of 99.7%.
Extraction of Nonpolar and Polar Arsenolipids from Capelin Fish Meal. An amount of 50 g of fish meal was mixed with
400 mL of hexane to extract the nonpolar fraction of the
arsenolipids. The mixture was shaken overnight before separating the supernatant (hexane plus arsenolipids) from the residue.
After extraction the solvent was evaporated with a rota evaporator to give about 5 mL of the extract, which was further evaporated to dryness under the flow of nitrogen gas.
After the extraction of the nonpolar fraction with hexane, the
residue was further extracted following the same procedure with
400 mL of methanol/dichloromethane (MeOH/DCM, 1:2 v/v)
to recover the polar fraction of the arsenolipids. The solvent was
evaporated to dryness and the extracts stored in the fridge.
Element 2 (Thermo Scientific)
mode
HF
organic mode
1570 W
nebulizer
microconcentric
nebulizer gas
0.86 L/min
optional gas
20 mL/min O2
plasma gas
0.89 L/min
coolant gas
14.9 L/min
monitored masses
m/z 31 (P), m/z 32 (S),
(medium resolution)
ESI-MS
m/z 74 (Ge), m/z 75 (As), m/z 77
LTQ Orbitrap Discovery
(Thermo Scientific)
mode
spray voltage
positive
4.5 kV
normalized collision
35%
energy
capillary temperature
320 °C
capillary voltage
42 V
scan range
m/z 1002000
Fractionation of Hexane Extract by Vacuum Liquid Chromatography. The hexane extract was dissolved in 5 mL of
hexane and then fractionated by vacuum liquid chromatography
(VLC) with a column packed with silica gel 60 in order to isolate
the arsenic compounds from the complex matrix of hexane
fraction. Using gradient elution with varying compositions of
mixtures of the eluting solvents (hexane, ethyl acetate, and
methanol) in order of increasing polarity as shown in Supporting
Information Table S1, 12 fractions (F1F12) were separated
and each fraction was analyzed using HPLCICPMS.
Online Speciation Method (HPLCICPMS/ES-MS). The
arsenic species were separated using a reversed-phase column
(ACE-C18; 4.6 mm 150 mm) and a gradient of water and
methanol both with 0.1% formic acid see Table 1. After the
column the eluent was split into two with 25% to the highresolution (HR) ICPMS (Element 2, Thermo Scientific) and
75% to the ESI-MS (LTQ Orbitrap Discovery; Thermo
Scientific). HR-ICPMS was used in organic mode with platinum cones, 20% of oxygen, and a microconcentric nebulizer in
medium resolution. The signal was optimized to give a
maximum response for As intensities at m/z 75, and DMAV
was used as external standard for the quantification of the
arsenic species. In additional, m/z 74 was measured for Ge
which was used as internal standard. The instrument operating
parameters are summarized in Table 1.
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Figure 1. Reversed-phase HPLCICPMS chromatogram of F11 and F12 fractions from the precleaned hexane extract using normal phase VLC.
Each peak AF represents arsenic species (m/z 75).
Figure 2. Measurement of the relative intensities of postcolumn-added As and Ge by running the gradient elution program of the HPLC and injecting a
blank. The vertical areas indicate the elution of the identified arsenic species AF.
’ RESULTS AND DISCUSSION
Total Arsenic Concentrations in the Digests and Quality
Control. The results of the total arsenic analysis in the sample
digests showed that the fish meal contains 2.95 ( 0.11 μg As/g
fresh weight, the hexane extract contains 0.35 ( 0.01 μg As/g,
and MeOH/DCM extract was found to be 0.88 μg As/g. The
focus of this methodological paper is on the identification of fatsoluble compounds in the hexane phase, which makes up 12% of
the entire arsenic concentration.
HPLCICPMS of the Nonpolar Arsenolipids. The initial
extraction, followed by the VLC fractionation, resulted in 12
fractions with different lipophilicity. Total arsenic concentrations
of the fraction digests revealed that only fractions 11 and 12
contained significant amounts of arsenic, which were subsequently measured independently using RP-HPLCICPMS.
The less polar fractions (F1F10) did not show any significant arsenic peaks (eluting under RP conditions, Supporting
Table 2. Concentrations (μg As/g) of the Different Arsenic
Species, AF, of the Nonpolar Arsenolipids, Determined by
HPLCICPMSa
arsenolipids
Ab
B
Cc
D
E
F
sum total
concn (μg As/g) 0.003 0.0014 0.0077 0.061 0.175 0.081 0.33 0.35
% of species
0.8
0.4
2.2
17.4
50.0
23.1
94.3 100
% of species
0.1
<0.1
0.3
2.1
5.9
2.7
11.2 11.9
of total As
a
AsFA (B, C1, C2) and AsHC (D, E, F). b Probably introduced as
arsenate impurity from the glassware. c C1: 0.0030 and C2 0.0047 μg As/g
(see Figure 3).
Information Figure S1), whereas both polar fractions F11 and
12 showed similar arsenic peaks with identical retention times
for the lipophilic species (Figure 1).
Additionally, sulfur and phosphorus were determined, in medium
resolution (m/z 32 and 31) using the HR-ICPMS, but no peaks
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Figure 3. Chromatogram of a capelin fish meal (hexane extract) using RP-HPLC and monitored online simultaneously for arsenic m/z 75 (ICPMS)
and the other masses (B = m/z 363.1875, C1 = m/z 437.2031, C2 = m/z 449.2031, D = m/z 405.2134, E = 333.2124, and F = 361.2448) by the ES
orbitrap MS. The m/z 75 signal for peak C indicates a broad signal illustrating the almost coelution of two molecular peaks (C1 and C2).
were recorded for those elements which coeluted with the arsenic
peaks (Supporting Information Figure S2). Although the detection
of sulfur and phosphorus is less sensitive compared to arsenic, a 1:1
molar ratio in the compounds should have shown a peak in the
ICPMS for both elements. Hence, the detected arsenic species are
neither thioarsenicals nor contain phosphorus. Thio-containing
organoarsenic compounds have been identified in many different
marine samples,9,11,12 and arsenic-containing phospholipids have
been tentatively identified in marine samples.4,6
Quantification of the Different Lipophilic Arsenic Species
by HPLCICPMS. The quantities of the unknown lipophilic
arsenic species, AF, Figure 1, were determined from the peak
areas with DMAV as a calibrant (Supporting Information Figure
S3) due to element-specific, rather than molecular-specific,
response in the ICPMS.
The arsenic response changes with increasing methanol concentration due to the use of a methanol/water gradient for the
separation.13,14 Although ICPMS produces an elemental arsenic
specific response, it is retention time dependent with increasing
methanol concentration in the mobile phase. This effect has been
observed by other elements with high ionization potential (IP)
such as sulfur13 and phosphorus.15,16 Thus, arsenic standards
eluting at the same retention times as the unknown arsenic
species would be needed for quantification. Currently there are
no fat-soluble arsenic standards available, and further, even if they
were available, calibrating for each individual unknown arsenic
species would be very time-consuming. A different calibration
concept is needed. The methanol concentration can be kept
constant, by adding postcolumn a compensatory additional
methanol gradient from another HPLC pump as described
before.15 This approach has, however, two disadvantages: an
extra HPLC pump is necessary, and the eluent is diluted. Here we
describe a combination of external classical calibration, using flow
injection with different concentrations of DMAV, while simultaneously running a gradient elution program of the mobile phase.
From the calibration graph an arsenic response factor (RAs) can
be determined and used for quantification (Supporting Information Figure S3). The benefit is a single chromatographic run, of
only approximately 45 min, for the calibration of all unidentified
arsenic species regardless of their retention time.
Table 3. Accurate Masses, Recorded by the Orbitrap, Which
Form Peaks at the Same Retention Times as Arsenic in the
ICPMS, Compared to the Calculated Masses of the Molecular
Formulas Given and the Associated Relative Error
peak
mol formula MHþ
calcd MHþ
exptl MHþ
Δm (ppm)
B
C1
C17H36AsO3
C23H38AsO3
363.1871
437.2027
363.1875
437.2031
þ1.10
þ0.91
C2
C24H38AsO3
449.2027
449.2031
þ0.89
D
C23H38AsO
405.2129
405.2134
þ1.23
E
C17H38AsO
333.2129
333.2124
1.50
F
C19H42AsO
361.2441
361.2448
1.94
In order to determine the correct response factor for every
arsenic compound (RtAs) eluting under the gradient program
a retention time specific arsenic response factor frt needs to
be determined. This was achieved by adding postcolumn a
solution containing Ge and As (approximately 100 μg As/L).
After injecting a blank sample the ICPMS arsenic response
was monitored while the gradient program was running. To
illustrate the change in sensitivity during the chromatographic
run the response of arsenic (m/z 75) at the start was set to
unity. The relative intensities (frt) of As and Ge are shown
in Figure 2. From the shape of the curve it is obvious that
the methanol-related response change is element-dependent.
Using the change of Ge, added routinely as a continuous
internal standard, would not give a satisfactory account of the
sensitivity change of the analyte As during a chromatographic
run. The reason is that Ge is probably completely ionized
due to its relatively low IP of 7.9 eV, whereas with As, with its
high IP of 9.8 eV, only a fraction is ionized, probably around
2030%. It has been suggested by Raber et al.14 that the response for arsenic does not change dramatically when methanol is added continuously at a high concentration so that the
overall response change of arsenic is minimized. If it does, like
in the illustrated example, a response factor for every arsenic
compound, eluting at different retention times, can be calculated using a simple relationship (eq 1). The factor frt can be
determined for every retention time by taking the intensity
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Figure 4. MS/MS, from the protonated molecular peaks F, E, and D, respectively, on panels a, b, and c with assigned fragments and the MS. Panel d
shows the MS at the retention time for peaks C1 (MHþ = 437.2031) and C2 (MHþ = 449.2031). In-source fragmentation is shown in the Supporting
Information.
data over the anticipated peak width of the unknown arsenic
compound.
R t As ðtÞ ¼ RAs frt
ð1Þ
It can be seen from Figure 2 that the response under the tuned
condition for arsenic increases by a factor of 4 with increasing
methanol content in the mobile phase. This is due to the fact that
the center core of the plasma is actually hotter because of
enhanced thermal conductivity of carbon and carbon with the
high IP of 11.3 eV transfers the charge to nonionized atoms with
a lower IP such as As.13,14,17 The response, however, decreases
again if the methanol concentration is so high that a high amount
of the plasma energy is consumed by methanol decomposition
and atomization. It should be mentioned the response change
with the methanol gradient can be changed when the ICPMS is
tuned, for example, by using arsenic with high amounts of
methanol. The increased response shown in Figure 2 has a
positive effect on the limit of detection (LOD) for the late-eluting
fat-soluble arsenic species, which, however, is counteracted by the
peak broadening of late-eluting peaks. The LOD, when the ICPMS
was set to medium resolution, was approximately 0.1 μg/L in the
extract for the late-eluting arsenic species (based on 3σ of the
baseline noise and the given RAs). Using low resolution the LOD
could even be reduced to 0.01 μg As/L for each species.
After the calibration, the As and Ge containing solution is
substituted by a continuous internal standard containing only
Ge, and the samples can be measured. The concentrations of the
unknown lipid-soluble arsenic species (AF) were determined
by using this calibration method (Table 2).
Figure 5. Proposed molecular structures of arsenic-containing fatty
acids (AsFA) and hydrocarbons (AsHC) extracted from capelin
fish meal.
To validate the calibration, the total arsenic concentrations of
the extract (0.35 μg As/g) were compared to the sums of all
species quantified in the extracts (0.33 μg As/g), giving a
chromatographic recovery of 94.3%. This is within the error of
consecutive analysis of approximately (5%.
No arsenic species were irreversibly adsorbed on the column;
this was checked by increasing the gradient to pure methanol and
increasing the running time. Thus, it should be safe to assume
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Figure 6. Modeled orbitrap data fitted to the arsenic peak C (m/z 75) in the ICPMS with relative response factors (rC1 = 0.8 for m/z 437 and rC2 = 1.2
for m/z 449) for the overlapping arsenic-containing compounds (C1 and C2). The pink signal is the modeled arsenic response from these two arseniccontaining organic species eluting at 23.0 min for C1 and 23.2 min for C2; the m/z 75 indicates traces of a possible third arsenic species.
that the arsenic species were correctly quantified using the
retention time specific response factor without having lipophilic
arsenic species available as standards.
Identification. Identification of arsenic species using ICPMS,
by comparing retention times of available standards, is taken with
caution. Hence, the simultaneous hyphenation to the ES orbitrap
gives valuable information of the accurate masses of species that
elute at the same time as the arsenic peaks obtained by the
ICPMS. Figure 3 shows masses that coincide with peaks detected
with the ICPMS on m/z 75.
The three large peaks, D, E, and F, make up more than 90% of
the arsenic in the extract. They correspond to three compounds,
detected with the orbitrap, with the same peak shape and
retention times as the arsenic signal. The accurate masses from
the protonated molecular masses, recorded by the orbitrap,
suggest that all three compounds contain one arsenic atom per
molecule. Table 3 lists the recorded accurate masses and
compares to the most likely formulas containing one oxygen
atom in addition to one arsenic atom. The error between
experimental and calculated accurate mass was generally lower
than (2 ppm. The intensities were high enough to allow MS/MS
experiments of the [M þ H]þ peak. The accurate mass fragments
confirm that the compounds have a dimethylarsinoyl moiety and
one large hydrocarbon chain (Figure 4). This is not unexpected,
since previously identified fat-soluble arsenic species in capelin
oil were also arsenic-containing hydrocarbons (AsHC).6 While
AsHCs, E and F, are saturated, AsHC, D, is unsaturated as
illustrated in Figure 5. Additionally to these AsHCs, the other
arsenic peaks (B, C1, C2) contain presumably arsenic-containing
fatty acids (AsFA), since the accurate masses indicate three
oxygen atoms per arsenic in the molecule. The arsenic is bound
again as a dimethylarsinoyl moiety to a fatty acid (FA). Whereas
B is presumably a saturated AsFA, and well-separated from any
other arsenic species, the molecular formulas of C1 and C2 point
to unsaturated AsFAs. The location of the double bonds can only
be assumed by comparing the AsFA with the structures of the
known unsaturated fatty acids found in fish oil.
The arsenic peaks B and C in ICPMS (Figure 3) aligned with
AsFAs in the orbitrap. Peak A in the ICPMS showed no direct
match with arsenic-containing molecular peaks. Due to the
elution in the void it is likely that this compound is introduced
during sample preparation as arsenate from glass impurities;
hence, there are no ES-MS peaks.
The two almost coeluting arsenic AsFAs (C1 and C2) gave rise
to the unusually broad arsenic signal C, in the ICPMS (Figure 3).
Assuming similar ionization of the molecules, the added molecular signals of both masses should give the same peak shape and
retention time as the broad arsenic peak (C) in ICPMS. This
means even when the chromatographic resolution of the HPLC
method is not good enough for separating all arsenic-containing
species, the orbitrap signals can be used to convolute the arsenic
signal and quantify the overlapping arsenic compounds independently. If, however, the ICPMS peak is not matched by assuming
the same response factor for both arsenic compounds, a speciesspecific response factor could be modeled until the peak shapes
match. Practically all arsenic-containing molecules detected by
orbitrap, which overlap with the arsenic peak in ICPMS, can be
used to model the arsenic response by optimizing species-specific
responses. Here the intensities of peak C1 (m/z 437) and C2
(m/z 449) detected in the orbitrap, IC1, IC2, were multiplied with
the species-specific responses (rC1, rC2), and IAs can be compared
with As (m/z 75) as shown in Supporting Information Figure S4.
It, however, should be mentioned here that these relative
response factors are not only influenced by the compounds but
also by the coeluting matrix.
IAs ¼ rC1 IC1 þ rC2 IC2
ð2Þ
When the same sensitivity is assumed, the resulting arsenic peak
has not the same peak form as in ICPMS. Compound C1 shows a
higher sensitivity than C2 when compared to the arsenic signal;
hence, the rC1 of 0.8 and rC2 of 1.2 gave the best match (Figure 6).
Additionally, when comparing the peak broadening of the
ICPMS signal to the modeled peak it indicates the elution of
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a third arsenic species, which could not be identified by the
orbitrap due to its trace amount.
Using this model and the species-specific responses the
concentration of the two major individual AsFA can be calculated
assuming only the two coeluting arsenic-containing compounds
make up the arsenic peak in the ICPMS. C1 would have a
concentration of 0.0030 μg As/g, whereas C2 is in slightly higher
concentration in the fish meal 0.0047 μg As/g. Additionally,
without having the standard compounds available relative response factors for every lipophilic arsenic compound for orbitrap
detection can be calculated and then used solely for further
routine analysis of lipophilic arsenic compounds.
Although the concentrations of the AsFA were too low for an
MS/MS experiment, the transient signals of some fragments
generated by in-source fragmentation (such as m/z 122 for the
dimethylarsinoyl moiety) confirm the identity of those compounds as AsFA with a dimethylarsinoyl moiety (Supporting
Information Figure S5). Although a class of compounds of
arsenic-containing long-chain fatty acids had been found in other
seafood previously,57 the C2 species (C24H38AsO3) is novel.
This compound might have been overlooked before, due to the
similar chromatographic behavior, since previous studies used
methods which relied on the identification of arsenicals in
fractions collected after chromatographic separation. Additional
confirmation of the assigned molecular structures has been
drawn from their chromatographic behavior. Fatty acids are
more polar and elute earlier than the hydrocarbons. Shorter
carbon chains increase polarity, whereas saturation of the carbon
chain decreases polarity. Hence, this would explain that the
longer, but unsaturated, fatty acid, C24H38AsO3, elutes almost at
the same time as C23H38AsO3 and the fact that the unsaturated
C23H38AsO elutes before the saturated C17H38AsO. The locations of the double bonds, of the AsFAs and AsHC, have not been
determined with certainty but were assumed to be similarly
formed biologically as the non-arsenic-containing ones. Rumpler
et al.7 claim that the dimethylarsinoyl moiety replaces the
terminal CH3 of fatty acids. That is, instead of an even
number of C’s in the fatty acids, normally found in fish oil, the
arsenic-containing fatty acids would have one less C and,
therefore, chains with odd numbers. The significance of the
novel C2 compound is that this As species seems to be in a
similar concentration as the other arsenic fatty acids but has an
even number of carbons in the chain. This is not directly
negating the hypothesis that it is nature’s infidelity during the
biosynthetic pathway which creates these AsFA.7 However,
the even-numbered carbon chain in the arsenic-containing
fatty acid must have a different starting product than the oddnumbered fatty acids.
’ CONCLUSIONS
The combination and online hyphenation of HPLC to highresolution ICPMS and ES-MS enables the direct identification of
arsenic compounds, even when no arsenic compounds with
similar chromatographic behavior are available. Furthermore,
with the ICPMS signals, relative response factors could be
determined for every arsenic-containing compound detected
by the orbitrap, even when they are not fully separated by
chromatography. This methodology could be used for any
class of compounds with an element which is amenable to
ICPMS detection such as bromine, phosphorus, sulfur, or any
other metal and metalloid.
ARTICLE
’ ASSOCIATED CONTENT
bS
Supporting Information. Additional information as noted
in text. This material is available free of charge via the Internet at
http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected].
’ ACKNOWLEDGMENT
K. O. Amayo thanks the Ambrose Alli University in collaboration with the Education Trust Fund, Nigeria for his scholarship;
C. Newcombe thanks the College of Physical Science and the
TESLA research fund. A. Petursdotttir and H. Gunnlaugsdottir
thank the AVS R&D Fund of Ministry of Fisheries in Iceland
for its financial support. The Síldarvinnslan hf (SVN) and
Vinnslust€odin hf (VSV) in Iceland are further acknowledged for
the samples provided.
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