Arsenic-containing Fatty Acids Research Articles

Speciation analysis and toxicity evaluation of arsenolipids-an overview focusing on sea food.

Arsenic, which can be divided into inorganic and organic arsenic, is a toxic metalloid that has been identified as a human carcinogen. A common source of arsenic exposure in seafood is arsenolipid, which is a complex structure of lipid-soluble organic arsenic compounds. At present, the known arsenolipid species mainly include arsenic-containing fatty acids (AsFAs), arsenic-containing hydrocarbons (AsHCs), arsenic glycophospholipids (AsPLs), and cationic trimethyl fatty alcohols (TMAsFOHs). Furthermore, the toxicity between different species is unique. However, the mechanism underlying arsenolipid toxicity and anabolism remain unclear, as arsenolipids exhibit a complex structure, are present at low quantities, and are difficult to extract and detect. Therefore, the objective of this overview is to summarize the latest research progress on methods to evaluate the toxicity and analyze the main speciation of arsenolipids in seafood. In addition, novel insights are provided to further elucidate the speciation, toxicity, and anabolism of arsenolipids and assess the risks on human health.

Arsenolipids in raw and cooked seafood products in southwest China: A non-targeted analysis

Arsenolipids are the primary form of arsenic in the fat of marine organisms. Because seafood is a common source of arsenic exposure and some arsenolipids are toxic, studying the abundance and species of arsenolipids in seafood is crucial for health risk assessment. Current arsenolipid research is confined by analytical techniques and limited to raw seafood analysis, despite the fact that most seafood is ingested cooked. Therefore, the aim of this study is to evaluate which seafood contributes to arsenolipid dietary intake and investigate the changes in arsenolipids before and after cooking. In Chongqing, China, popular seafood such as clam, shrimp, oyster, abalone, hairtail, and yellow croaker were collected. The raw and cooked samples prepared from these seafood products were examined using a non-targeted screening approach established for arsenolipids, which coupled high-performance liquid chromatography with data-independent high-resolution quadrupole-time-of-flight electrospray ionization tandem mass spectrometry. Arsenic-containing hydrocarbons (AsHC330, AsHC332, and AsHC360), arsenic-containing fatty acids (AsFA362, AsFA390, AsFA404, AsFA418, and AsFA422), trimethylarsine oxide, and thiolated trimethylarsinic acid were detected. The species of arsenolipids in each type of seafood remained intact after heating in the microwave oven. In cooked samples, the concentrations of AsFA362 and AsFA390 were significantly lower than in raw samples, whereas the concentrations of other arsenolipids were unchanged. Microwave cooking did not result in the thiolation of the detected arsenolipids. The most detected species in raw and cooked samples were AsFA362, AsFA390, and AsFA418. Most arsenolipid species were found in the highest levels in hairtails and yellow croakers. It is the first time that arsenolipids have been found in the oyster, abalone, abalone liver, and yellow croaker. The present study contributes to a better understanding of arsenolipids exposure from seafood, which is useful for assessing the health risks of arsenic.

Elution with 1,2-Hexanediol Enables Coupling of ICPMS with Reversed-Pase Liquid Chromatography under Standard Conditions.

The inductively coupled plasma mass spectrometry (ICPMS) has been attracting increasing attention for many applications as an element-selective chromatographic detector. A major and fundamental limitation in coupling ICPMS with liquid chromatography is the limited compatibility with organic solvents, which has so far been addressed via a tedious approach, collectively referred to as the “organic ICPMS mode”, that can decrease detection sensitivity by up to 100-fold. Herein, we report 1,2-hexanediol as a new eluent in high-performance liquid chromatography–ICPMS which enables avoiding the current limitations. Unlike commonly used eluents, 1,2-hexanediol was remarkably compatible with ICPMS detection at high flow rates of 1.5 mL min–1 and concentrations of at least 30% v/v, respectively, under the standard conditions and instrumental setup normally used with 100% aqueous media. Sensitivity for all tested elements (P, S, Cl, Br, Se, and As) was enhanced with 10% v/v 1,2-hexanediol relative to that of 100% aqueous media by 1.5–7-fold depending on the element. Concentrations of 1,2-hexanediol at ≤30% v/v were superior in elution strength to concentrations at >90% v/v of the common organic phases, which greatly decreases the amount of carbon required to elute highly hydrophobic compounds such as lipids and steroids, enabling detection at ultra-trace levels. The proposed approach was applied to detect arsenic-containing fatty acids in spiked human urine, and detection limits of <0.01 μg As L–1 were achieved, which is >100-fold lower than those previously reported using the organic ICPMS mode. Nontargeted speciation analysis in Allium sativum revealed the presence of a large number of hydrophobic sulfur-containing metabolomic features at trace levels.

Discovery and Identification of Arsenolipids Using a Precursor-Finder Strategy and Data-Independent Mass Spectrometry.

Arsenolipids are a class of lipid-soluble arsenic species. They are present in seafoods and show high potentials of cytotoxicity and neurotoxicity. Hindered by traditional low-throughput analytical techniques, the characterization of arsenolipids is far from complete. Here, we report on a sensitive and high-throughput screening method for arsenolipids in krill oil, tuna fillets, hairtail heads, and kelp. We demonstrate the detection and identification of 23 arsenolipids, including novel arsenic-containing fatty acids (AsFAs), hydroxylated AsFAs, arsenic-containing hydrocarbons (AsHCs), hydroxylated AsHCs, thiolated trimethylarsinic acids, and arsenic-containing lysophosphatidylcholines not previously reported. The new method incorporated precursor ion scan (PIS) into data-independent acquisition. High-performance liquid chromatography (HPLC) electrospray ionization quadrupole time-of-flight mass spectrometry (ESI–qToF–MS) was used to perform the sequential window acquisition of all theoretical spectra (SWATH). Comprehensive HPLC–MS and MS/MS data were further processed using a fragment-guided chromatographic computational program Precursorfinder developed here. Precursorfinder achieved efficient peak-picking, retention time comparison, hierarchical clustering, and wavelet coherence calculations to assemble fragment features with their target precursors. The identification of arsenolipids was supported by coeluting the HPLC–MS peaks detected with the characteristic fragments of arsenolipids. Method validation using available arsenic standards and the successful identification of previously unknown arsenolipids in seafood samples demonstrated the applicability of the method for environmental research.

Variation in arsenolipid concentrations in seafood consumed in Japan

Variation in arsenolipid concentrations was assessed in 18 seafood samples including fish, shellfish, and crustaceans purchased in Japan. Analyses were performed by high performance liquid chromatography-inductively coupled plasma mass spectrometry/electrospray ionization tandem mass spectrometry. Stable isotope ratios for nitrogen and carbon were also measured in the samples for obtaining trophic level information of the species. Arsenic-containing hydrocarbons (AsHCs) and arsenic-containing fatty acids (AsFAs) were detected in the seafood samples; the toxic AsHCs were found in all of the seafood samples with large variation in the concentrations (83 ± 73 ng As/g fw, coefficient of variation = 88%). Our previous point estimate of health risk of AsHCs intake via seafood consumption in Japan, based on average AsHC concentration in seafood, suggested insignificant risk, and the present study supports our previous estimate. AsHC concentrations significantly correlated with lipid content of the seafood samples (r = 0.67, p < 0.01), a result expected because of the fat solubility of the compounds. The AsHCs concentrations, however, were not significantly correlated with nitrogen stable isotope ratios suggesting that AsHCs do not biomagnify. The source of the observed large variation in AsHC concentrations will be the subject of further investigation.

Estimation of daily intake of arsenolipids in Japan based on a market basket survey

Arsenolipid concentrations were measured in 17 food composites prepared from 152 food items purchased in Shizuoka city, Japan, to (1) determine the food contributing to daily intake of arsenolipids, and (2) estimate the daily intake of arsenolipids. Analysis of arsenolipids was performed by high performance liquid chromatography-inductively coupled plasma mass spectrometry/electrospray ionization tandem mass spectrometry (HPLC-ICP-MS/ESI-MS-MS). Arsenic containing hydrocarbons (AsHCs), arsenic containing fatty acids (AsFAs), and arsenosugar phospholipids (AsSugPLs) were detected only in “algae” and “fish and shellfish” of the 17 food composites in a concentration range of 4.4–233 ng As/g fresh weight (fw). Two cytotoxic arsenolipids, AsHC332 and AsHC360, were detected in “algae” and “fish and shellfish” in the concentrations range of 33–40 ng As/g fw. The estimated average daily intake of AsHC332 and AsHC360 was ca 3000 and 360 ng As/person/day, or 50 and 6.0 ng As/kg bw/day, respectively. The present study indicated that arsenolipids from “algae” and “fish and shellfish” consumption contributed to the daily intake of toxic AsHCs, though the margin of exposure for the AsHC332 and AsHC360 does not appear to pose a health risk for the general Japanese population.

Arsenic-containing hydrocarbons: effects on gene expression, epigenetics, and biotransformation in HepG2 cells.

Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids found in fish and algae, elicit substantial toxic effects in various human cell lines and have a considerable impact on cellular energy levels. The underlying mode of action, however, is still unknown. The present study analyzes the effects of two AsHCs (AsHC 332 and AsHC 360) on the expression of 44 genes covering DNA repair, stress response, cell death, autophagy, and epigenetics via RT-qPCR in human liver (HepG2) cells. Both AsHCs affected the gene expression, but to different extents. After treatment with AsHC 360, flap structure-specific endonuclease 1 (FEN1) as well as xeroderma pigmentosum group A complementing protein (XPA) and (cytosine-5)-methyltransferase 3A (DNMT3A) showed time- and concentration-dependent alterations in gene expression, thereby indicating an impact on genomic stability. In the subsequent analysis of epigenetic markers, within 72h, neither AsHC 332 nor AsHC 360 showed an impact on the global DNA methylation level, whereas incubation with AsHC 360 increased the global DNA hydroxymethylation level. Analysis of cell extracts and cell media by HPLC-mass spectrometry revealed that both AsHCs were considerably biotransformed. The identified metabolites include not only the respective thioxo-analogs of the two AsHCs, but also several arsenic-containing fatty acids and fatty alcohols, contributing to our knowledge of biotransformation mechanisms of arsenolipids.

Arsenic-containing hydrocarbons disrupt a model in vitro blood-cerebrospinal fluid barrier

Lipid-soluble arsenicals, so-called arsenolipids, have gained a lot of attention in the last few years because of their presence in many seafoods and reports showing substantial cytotoxicity emanating from arsenic-containing hydrocarbons (AsHCs), a prominent subgroup of the arsenolipids. More recent in vivo and in vitro studies indicate that some arsenolipids might have adverse effects on brain health.In the present study, we focused on the effects of selected arsenolipids and three representative metabolites on the blood-cerebrospinal fluid barrier (B-CSF-B), a brain-regulating interface. For this purpose, we incubated an in vitro model of the B-CSF-B composed of porcine choroid plexus epithelial cells (PCPECs) with three AsHCs, two arsenic-containing fatty acids (AsFAs) and three representative arsenolipid metabolites (dimethylarsinic acid, thio/oxo-dimethylpropanoic acid) to examine their cytotoxic potential and impact on barrier integrity. The toxic arsenic species arsenite was also tested in this way and served as a reference substance. While AsFAs and the metabolites showed no cytotoxic effects in the conducted assays, AsHCs showed a strong cytotoxicity, being up to 1.5-fold more cytotoxic than arsenite. Analysis of the in vitro B-CSF-B integrity showed a concentration-dependent disruption of the barrier within 72 h. The correlation with the decreased plasma membrane surface area (measured as capacitance) indicates cytotoxic effects. These findings suggest exposure to elevated levels of certain arsenolipids may have detrimental consequences for the central nervous system.

Effects of arsenolipids on in vitro blood-brain barrier model.

Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids (AsLs) occurring in fish and edible algae, possess a substantial neurotoxic potential in fully differentiated human brain cells. Previous in vivo studies indicating that AsHCs cross the blood-brain barrier of the fruit fly Drosophila melanogaster raised the question whether AsLs could also cross the vertebrate blood-brain barrier (BBB). In the present study, we investigated the impact of several representatives of AsLs (AsHC 332, AsHC 360, AsHC 444, and two arsenic-containing fatty acids, AsFA 362 and AsFA 388) as well as of their metabolites (thio/oxo-dimethylpropionic acid, dimethylarsinic acid) on porcine brain capillary endothelial cells (PBCECs, in vitro model for the blood-brain barrier). AsHCs exerted the strongest cytotoxic effects of all investigated arsenicals as they were up to fivefold more potent than the toxic reference species arsenite (iAsIII). In our in vitro BBB-model, we observed a slight transfer of AsHC 332 across the BBB after 6h at concentrations that do not affect the barrier integrity. Furthermore, incubation with AsHCs for 72h led to a disruption of the barrier at sub-cytotoxic concentrations. The subsequent immunocytochemical staining of three tight junction proteins revealed a significant impact on the cell membrane. Because AsHCs enhance the permeability of the in vitro blood-brain barrier, a similar behavior in an in vivo system cannot be excluded. Consequently, AsHCs might facilitate the transfer of accompanying foodborne toxicants into the brain.

Toxicity of two classes of arsenolipids and their water-soluble metabolites in human differentiated neurons.

Arsenolipids are lipid-soluble organoarsenic compounds, mainly occurring in marine organisms, with arsenic-containing hydrocarbons (AsHCs) and arsenic-containing fatty acids (AsFAs) representing two major subgroups. Recently, toxicity studies of several arsenolipids showed a high cytotoxic potential of those arsenolipids in human liver and bladder cells. Furthermore, feeding studies with Drosophila melanogaster indicated an accumulation of arsenolipids in the fruit fly's brain. In this study, the neurotoxic potential of three AsHCs, two AsFAs and three metabolites (dimethylarsinic acid, thio/oxo-dimethylarsenopropanoic acid) was investigated in comparison to the toxic reference arsenite (iAsIII) in fully differentiated human brain cells (LUHMES cells). Thereby, in the case of AsHCs both the cell number and cell viability were reduced in a low micromolar concentration range comparable to iAsIII, while AsFAs and the applied metabolites were less toxic. Mechanistic studies revealed that AsHCs reduced the mitochondrial membrane potential, whereas neither iAsIII nor AsFAs had an impact. Furthermore, neurotoxic mechanisms were investigated by examining the neuronal network. Here, AsHCs massively disturbed the neuronal network and induced apoptotic effects, while iAsIII and AsFAs showed comparatively lesser effects. Taking into account the substantial in vitro neurotoxic potential of the AsHCs and the fact that they could transfer across the physiological barriers of the brain, a neurotoxic potential in vivo for the AsHCs cannot be excluded and needs to be urgently characterized.

Determination of moderately polar arsenolipids and mercury speciation in freshwater fish of the River Elbe (Saxony, Germany)

Arsenic and mercury are frequent contaminants in the environment and care must be taken to limit their entrance into the food chain. The toxicity of both elements strongly depends upon their speciation. Total amounts of As and Hg as well as their species were analyzed in muscle and liver of 26 fishes of seven freshwater fish species caught in the River Elbe. The median concentrations of As were 162 μg kg−1 w.w. in liver and 92 μg kg−1 w.w. in muscle. The median concentrations of total Hg were 241 μg kg−1 w.w. in liver and 256 μg kg−1 w.w. in muscle. While this level of Hg contamination of the freshwater fish in the River Elbe is significantly lower than 20 years ago, it exceeds the recommended environmental quality standard of 20 μg Hg kg−1 w.w. by a factor of 5–50. However, the European maximum level of 500 μg Hg kg−1 for fish for human consumption is rarely exceeded. Arsenic-containing fatty acids and hydrocarbons were determined and partially identified in methanolic extracts of the fish by HPLC coupled in parallel to ICP-MS (element specific detection) and ESI-Q-TOF-MS (molecular structure detection). While arsenobetaine was the dominant As species in the fish, six arsenolipids were detected and identified in the extracts of liver tissue in common bream (Abramis brama), ide (Leuciscus idus), asp (Aspius aspius) and northern pike (Esox lucius). Four arsenic-containing fatty acids (AsFA) and two arsenic-containing hydrocarbons (AsHC) are reported in freshwater fish for the first time. With respect to mercury the more toxic MeHg+ was the major species in muscle tissue (>90% of total Hg) while in liver Hg2+ and MeHg+ were of equal importance. The results show the high relevance of element speciation in addition to the determination of total element concentrations to correctly assess the burden of these two elements in fish.

Arsenic-containing hydrocarbons and arsenic-containing fatty acids: Transfer across and presystemic metabolism in the Caco-2 intestinal barrier model.

Arsenic-containing hydrocarbons (AsHCs) and arsenic-containing fatty acids (AsFAs) represent two classes of arsenolipids occurring naturally in marine food. Toxicological data are yet scarce and an assessment regarding the risk to human health has not been possible. Here, we investigated the transfer and presystemic metabolism of five arsenolipids in an intestinal barrier model. Three AsHCs and two AsFAs were applied to the Caco-2 intestinal barrier model. Thereby, the short-chain AsHCs reached up to 50% permeability. Transport is likely to occur via passive diffusion. The AsFAs showed lower intestinal bioavailability, but respective permeabilities were still two to five times higher as compared to arsenobetaine or arsenosugars. Interestingly, AsFAs were effectively biotransformed while passing the in vitro intestinal barrier, whereas AsHCs were transported to the blood-facing compartment essentially unchanged. AsFAs can be presystemically metabolised and the amount of transferred arsenic is lower than that for AsHCs. In contrast, AsHCs are likely to be highly intestinally bioavailable to humans. Since AsHCs exert strong toxicity in vitro and in vivo, toxicity studies with experimental animals as well as a human exposure assessment are needed to assess the risk to human health related to the presence of AsHCs in seafood.

Synthesis of two new arsenolipids and their identification in fish

Arsenolipids (AsL) of the type arsenic‐containing fatty acids (AsFA) and hydrocarbons (AsHC) are natural organoarsenic compounds present in high abundance in seafood. Since little is known about their toxicity, there is a significant concern about their health risk. Here we report the synthesis of two new AsL and their identification in fish by liquid chromatography (LC) coupled simultaneously to inductively coupled plasma mass spectrometry (ICP‐MS) and to high resolution mass spectrometry (HR‐MS). The molecular structure was elucidated on the basis of their exact mass and their product ion mass spectra. One of the synthesized AsL has been already identified in fish samples, meanwhile, the other arsenolipid is reported here as a natural product in fish.Practical applications: The synthesized AsL can be used as analytical standards to confirm the identification of AsL in different biological samples and to optimize analytical methods for their determination by liquid chromatography coupled simultaneously to ICP‐MS and HR‐MS. The material can be further used for toxicity testing of AsL and to study their uptake, bioavailability, and biotransformation.We report the synthesis of two new arsenolipids and their identification in fish by liquid chromatography (LC) coupled simultaneously to inductively coupled plasma mass spectrometry (ICP‐MS) and to high resolution mass spectrometry (HR‐MS). One of the synthesized arsenolipid has been already identified in fish samples, meanwhile, the other arsenolipid is reported for the first time here as a natural product in fish.

A study of lipid- and water-soluble arsenic species in liver of Northeast Arctic cod (Gadus morhua) containing high levels of total arsenic

In the present study liver samples (n=26) of Northeast Arctic cod (Gadus morhua), ranging in total arsenic concentrations from 2.1 to 240mg/kg liver wet weight (ww), were analysed for their content of total arsenic and arsenic species in the lipid-soluble and water-soluble fractions. The arsenic concentrations in the lipid fractions ranged from 1.8 to 16.4mg As/kg oil of liver, and a linear correlation (r2=0.80, p<0.001) was observed between the total arsenic concentrations in liver and the total arsenic concentrations in the respective lipid fractions of the same livers. The relative proportion of arsenolipids was considerably lower in liver samples with high total arsenic levels (33–240mg/kg ww), which contained from 3 to 7% of the total arsenic in the lipid-soluble fraction. In contrast liver samples with low arsenic concentrations (2.1–33mg/kg ww) contained up to 50% of the total arsenic as lipid-soluble species. Arsenic speciation analysis of the lipid-soluble fractions of the livers, using reversed-phase high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC–ICP-MS), revealed the presence of several arsenolipids. Three major arsenic-containing hydrocarbons (C17H39AsO, C19H41AsO and C23H37AsO) and five arsenic-containing fatty acids (C17H35AsO3, C19H39AO3, C19H37AsO3, C23H37AsO3 and C24H37AsO3) were identified using HPLC coupled to quadrupole time-of-flight mass spectrometry (qTOF-MS). Arsenobetaine was the major arsenic species in the water-soluble fraction of the livers, while dimethylarsinate, arsenocholine and inorganic arsenic were minor constituents. Inorganic arsenic accounted for less than 0.1% of the total arsenic in the liver samples.

In vitro toxicological characterisation of arsenic-containing fatty acids and three of their metabolites.

Arsenic-containing fatty acids are a group of fat-soluble arsenic species (arsenolipids) which are present in marine fish and other seafood. Recently, it has been shown that arsenic-containing hydrocarbons, another group of arsenolipids, exert toxicity in similar concentrations comparable to arsenite although the toxic modes of action differ. Hence, a risk assessment of arsenolipids is urgently needed. In this study the cellular toxicity of a saturated (AsFA 362) and an unsaturated (AsFA 388) arsenic-containing fatty acid and three of their proposed metabolites (DMAV, DMAPr and thio-DMAPr) were investigated in human liver cells (HepG2). Even though both arsenic-containing fatty acids were less toxic as compared to arsenic-containing hydrocarbons and arsenite, significant effects were observable at μM concentrations. DMAV causes effects in a similar concentration range and it could be seen that it is metabolised to its highly toxic thio analogue thio-DMAV in HepG2 cells. Nevertheless, DMAPr and thio-DMAPr did not exert any cytotoxicity. In summary, our data indicate that risks to human health related to the presence of arsenic-containing fatty acids in marine food cannot be excluded. This stresses the need for a full in vitro and in vivo toxicological characterisation of these arsenolipids.

Arsenolipids in commercial canned cod liver: An occurrence and distribution study

Arsenolipids (AsL) of the type arsenic‐containing fatty acids (AsFA) and hydrocarbons (AsHC) are known to occur in seafood and their identification is today a challenge due to the need of data for toxicological assessment. The aim of this investigation was to enhance the data concerning AsL in commercial canned cod liver. Therefore, the present study focuses on the quantification and identification of AsL by LC–ICP‐MS combined with ESI‐MS. The molecular structure of 17 AsL was elucidated on the basis of their exact mass and their product ion spectra and two new AsFA tentatively identified. The total arsenic concentration in four different canned cod liver analyzed by ICP‐MS ranged from 2.6 to 5.5 mg As/kg with an extraction recovery of 98%. The AsL detected by RP‐IPC–MS comprise 58–95% of the total arsenic content, indicating that AsL are a major class of arsenic in canned cod liver. Much of the AsL detected in the canned cod liver extracts are, however, not yet identified.Practical applications: The results obtained in the present research show the possibility to apply the current extraction and detection method in different biological samples for the determination of AsL. Additionally, this information can be used for further studies to assess the risk of AsL for consumer health. On the other hand, the MS/MS results could be used for the future identification of AsL without the ICP‐MS detection and thus, low the cost of the analysis.Numerous arsenolipids (AsL) were detected and identified by HPLC hyphenated online with ICP‐MS (element specific detection) and ESI‐Q‐TOF‐MS (molecular structure detection) in commercial canned cod liver. ESI‐MS and ESI‐MS/MS spectra confirmed the proposed molecular structure of two new AsL of the type arsenic‐containing fatty acid identified for the first time in this study.

Arsenic-containing fatty acids and hydrocarbons in marine oils – determination using reversed-phase HPLC–ICP-MS and HPLC–qTOF-MS

Arsenolipids are the major arsenic species present in marine oils. Several structures of arsenolipids have been elucidated the last 5 years, demonstrating the chemical complexity of this trace element in the marine environment. Several commercial fish oils and marine oils, ranging in total arsenic concentrations from 1.6 to 12.5 mg kg(-1) oil, were analyzed for arsenolipids using reversed-phase high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The arsenolipids were quantified using three different arsenic-containing calibration standards; dimethylarsinate (DMA), triphenylarsinoxide (Ph₃AsO) and a synthesized arsenic-containing hydrocarbon (AsHC) (dimethylarsinoyl nonadecane; C₂₁H₄₃AsO). The observed variation in signal intensity for arsenic during the gradient elution profile in reversed-phase HPLC was compensated for by determining the time-resolved response factors for the arsenolipids. Isotopes of germanium ((74)Ge) and indium ((115)In) were suited as internal standards for arsenic, and were used for verification of the arsenic signal response factors during the gradient elution. Dimethylarsinate was the most suitable calibration standard for the quantification of arsenolipids, with recoveries between 91% and 104% compared to total arsenic measurements in the same extracts. A range of marine oils was investigated, including oils of several fish species, cod liver and seal, as well as three commercial fish oils. The AsHCs - C₁₇H₃₈AsO, C₁₉H₄₂AsO and C₂₃H₃₈AsO - were identified as the major arsenolipids in the extracts of all oils by HPLC coupled with quadrupole time-of-flight mass spectrometry (qTOF-MS). Minor amounts of two arsenic-containing fatty acids (AsFAs) (C₂₃H₃₈AsO₃ and C₂₄H₃₈AsO₃) were also detected in the oils. The sum of the AsHCs and the AsFAs determined in the present study accounted for 17-42% of the total arsenic in the oils.

Determination and identification of hydrophilic and hydrophobic arsenic species in methanol extract of fresh cod liver by RP-HPLC with simultaneous ICP-MS and ESI-Q-TOF-MS detection

The present study was focused on the determination and identification of arsenic species in methanolic extracts of cod liver. Arsenic species were fractionated and the fractions analysed by RP-HPLC-ICP-MS coupled with ESI-Q-TOF-MS. The total concentration of arsenic in the fresh cod liver was analysed by ICP-MS to be 1.53±0.02mgAskg−1w.w. and the extraction recovery was ca. 100% and the column recovery >93%. Besides polar inorganic and methylated arsenic species (>70%) more hydrophobic arsenic-containing fatty acids and hydrocarbons occurred. Based on the mass spectrometric data proposals for molecular structures were elaborated for 20 of the organic As species included 10 arsenic-containing fatty acids (AsFA) and an arsenic-containing hydrocarbon (AsHC) mentioned for the first time in fresh cod liver. Arsenobetaine was found as main water-soluble arsenic compound in cod liver followed by higher molecular mass arsenic-containing fatty acids and hydrocarbons.

A method for screening arsenolipids in fish oils by HPLC-ICPMS

We present a method for screening lipid-soluble arsenic compounds (arsenolipids) in fish oils by reversed-phase HPLC-ICPMS using a gradient elution with ethanol and acetate buffer at pH 6. Two different approaches were tested to reduce changes in arsenic response due to the carbon effect: addition of a supplementary methanol solution directly to the spray chamber or addition of methanol post-column through a T-piece. The latter method proved to be the best option for maintaining constant response for several arsenolipids covering a wide range of polarities. With the optimized method it is possible to perform a screening of at least three groups of arsenolipids with different polarities in 90 min with detection limits ranging from 5 to 11 μg As L−1, depending on the analyzed compound. The method was applied to the screening of arsenolipids in fractions obtained from cod liver oil and capelin oil, which include arsenic-containing fatty acids, arsenic-containing hydrocarbons and another group of lower polarity and unknown character.

Identification and Quantification of Arsenolipids Using Reversed-Phase HPLC Coupled Simultaneously to High-Resolution ICPMS and High-Resolution Electrospray MS without Species-Specific Standards

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 reversed-phase 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 (C(23)H(38)AsO, C(17)H(38)AsO, C(19)H(42)AsO) with minor amounts of dimethylarsinoyl fatty acids (C(17)H(36)AsO(3), C(23)H(38)AsO(3), C(24)H(38)AsO(3)). One of the dimethylarsinoyl fatty acids (C(24)H(38)AsO(3)), 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.