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 3589
dx.doi.org/10.1021/ac2005873 | Anal. Chem. 2011, 83, 3589–3595
Analytical Chemistry 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. 3590
dx.doi.org/10.1021/ac2005873 |Anal. Chem. 2011, 83, 3589–3595
Analytical Chemistry
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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
