Indirect Mercury Speciation in Biological Tissues by Closed‐Vessel Microwave‐Assisted Digestion and Flow‐Injection Cold‐Vapor Atomic Fluorescence Detection

Abstract

A simple and rapid method based on closed vessel microwave‐assisted extraction was developed to determine total, inorganic mercury and organomercury in biological tissues. Total mercury was extracted using HNO₃:H₂O₂ (4:1) mixture. In a separate subsample, extraction of mercury species was carried out with tetramethylammonium hydroxide (TMAH). The total and inorganic mercury analyses were carried out by flow‐injection cold‐vapor atomic fluorescence spectrometry (FI‐CV‐AFS). The organomercury concentration was calculated by difference. Considering a sample amount of 0.2 g, the detection limits were 4 and 26 ng/g for total and inorganic mercury, respectively. The accuracy of the procedures was checked by analyzing certified reference materials and recovery studies of spiked fish tissues.     

A rapid ultrasound-assisted thiourea extraction method for the determination of inorganic and methyl mercury in biological and environmental samples by CVAAS

A rapid ultrasound-assisted extraction procedure for the determination of total mercury, inorganic and methyl mercury (MM) in various environmental matrices (animal tissues, samples of plant origin and coal fly ash) has been developed. The mercury contents were estimated by cold vapour atomic absorption spectrometry (CVAAS). Inorganic mercury (IM) was determined using SnCl₂ as reducing agent whereas total mercury was determined after oxidation of methyl mercury through UV irradiation. Operational parameters such as extractant composition (HNO₃ and thiourea), sonication time and sonication amplitude found to be different for different matrices and were optimized using IAEA-350 (Fish homogenate), IM and MM loaded moss and NIST-1633b (Coal fly ash) to get quantitative extraction of total mercury. The method was further validated through the analysis of additional certified reference materials (RM): NRCC-DORM2 (Dogfish muscle), NRCC-DOLT1 (Dogfish liver) and IAEA-336 (Lichen). Quantitative recovery of total Hg was achieved using mixtures of 5% HNO₃ and 0.02% thiourea, 10% HNO₃ and 0.02% thiourea, 20% HNO₃ and 0.2% thiourea for fish tissues, plant matrices and coal fly ash samples, respectively. The results obtained were in close agreement with certified values with an overall precision in the range of 5-15%. The proposed ultrasound-assisted extraction procedure significantly reduces the time required for sample treatment for the extraction of Hg species. The extracted mercury species are very stable even after 24 h of sonication. Closed microwave digestion was also used for comparison purposes. The proposed method was applied for the determination of Hg in field samples of lichens, mosses, coal fly ash and coal samples     

Fish Oil Recovery from Viscera of Indian Mackerel (Rastrelliger kanagurta) by Supercritical Fluid: An Optimization Approach

Fish viscera, a waste of fish processing industry, can be exploited as a source of PUFA‐rich fish oil for use in food or pharmaceutical industry and at the same time the environment can be protected from pollution by fish wastes. Fish oil was recovered from viscera of Indian Mackerel (Rastrelliger kanagurta), by different supercritical fluid extraction (SFE) e.g. continuous, co‐solvent, soaking, and pressure swing techniques and the yields were compared with that of the solvent extraction method. The SFE parameters such as pressure, temperature, and CO₂ flow rate were optimised by employing Response Surface Methodology (RSM) with a view to maximize the oil yield and minimize CO₂ consumption. The central composite rotatable design (CCRD) consisting of three variables provided 20 experimental settings. Multiple regressions determined the coefficients of the second‐order polynomial equation. The optimum parameters for all 4 techniques applied were found to be 35 MPa, 60 °C, and 2 mL min^?1 with an Oil recovery ranging from 93 to 99%. The study demonstrated a lower CO₂ consumption by the soaking and pressure swing techniques at the optimized conditions. Thus, the soaking and pressure swing techniques were most effective for extracting oil from fish viscera.     

Effect of temperature,collection mode,and modifier on the supercritical CO2 extraction of dicofol residues from fish samples

Toxicity evaluation of Dicofol to Astyanax bimaculatus schubarti, a characteristic fish species living in tropical rivers and lakes was carried out through LC₅₀ – 96 Hours. These experiments were performed under laboratory controlled conditions with atmospheric air flow and dilution water at 25°C in the static mode, supercritical fluid extraction (SFE) with pure CO₂ and CO₂ modified with hexane and methanol were used at 50, 70, 80, and 100°C and 300 atm. Several collection modes were studied to extract Dicofol from fish samples. The extraction efficiencies were directly comparedd with those obtained after 8 h of Soxhlet extraction using the same clean-up with Florisil and analysis by HRGC/ECD and HRGC/MS as a confirmatory analytical technique. The SFE recoveries at temperatures lower than 80°C were typically lower than soxhlet recoveries; however a temperature increase enhanced the efficiency of SFE. The results showed that under certain conditions, supercritical fluid gave higher extractio power (extracted 11 % more pesticide), shorter extraction time, and lower solvent consumption than Soxhlet, thus affording an excellent alternative to the conventional method for extracting Dicofol from fish sample.     

Robust microwave-assisted extraction protocol for determination of total mercury and methylmercury in fish tissues

A rapid and efficient closed vessel microwave-assisted extraction (MAE) method based on acidic leaching was developed and optimized for the extraction of total mercury (Hg), inorganic mercury (Hg²⁺) and methylmercury (CH₃Hg⁺) from fish tissues. The quantitative extraction of total Hg and mercury species from biological samples was achieved by using 5 mol L⁻¹ HCl and 0.25 mol L⁻¹ NaCl during 10 min at 60 °C. Total Hg content was determined using inductively coupled plasma mass spectrometry (ICP-MS). Mercury species were measured by liquid chromatography hyphenated with inductively coupled plasma mass spectrometry (LC-ICP-MS). The method was validated using biological certified reference materials ERM-CE464, DOLT-3, and NIST SRM-1946. The analytical results were in good agreement with the certified reference values of total Hg and CH₃Hg⁺ at a 95% confidence level. Further, accuracy validation using speciated isotope-dilution mass spectrometry (SIDMS, as described in the EPA Method 6800) was carried out. SIDMS was also applied to study and correct for unwanted species transformation reactions during and/or after sample preparation steps. For the studied reference materials, no statistically significant transformation between mercury species was observed during the extraction and determination procedures. The proposed method was successfully applied to fish tissues with good agreement between SIDMS results and external calibration (EC) results. Interspecies transformations in fish tissues were slightly higher than certified reference materials due to differences in matrix composition. Depending on the type of fish tissue, up to 10.24% of Hg²⁺ was methylated and up to 1.75% of CH₃Hg⁺ was demethylated to Hg²⁺.     

A new room temperature ionic liquid 1-butyl-3-trimethylsilylimidazolium hexafluorophosphate as a solvent for extraction and preconcentration of mercury with determination by cold vapor atomic absorption spectrometry

A new room temperature ionic liquid 1-butyl-3-trimethylsilylimidazolium hexafluorophosphate abbreviated as [C₄tmsim][PF₆] was synthesized and developed as a novel medium for liquid/liquid extraction of inorganic mercury in this work. Under optimal condition, o-carboxyphenyldiazoamino-p-azobenzene abbreviated as CDAA reacted with inorganic mercury to form a neutral Hg-CDAA complex, the complex was rapidly extracted into ionic liquid phase. After back-extracting into aqueous phase with sulfide sodium solution, the mercury concentration was detected by cold vapor atomic absorption spectrometry. The extraction and back-extraction efficiencies were 99.9 and 100.1% for 5.0 μg L⁻¹ standard mercury in 1000 mL of water solution, respectively. The detection limit, calculated using three times the standard error of estimate of the calibration graph, is 0.01 ng of mercury per milliliter water sample. The proposed method has been used to the determination of trace inorganic mercury in natural water with satisfactory results. Moreover, Zeta potential and surface tension of [C₄tmsim][PF₆] solution were measured and applied to explain the extraction mechanism of [C₄tmsim][PF₆] system.     

Comparison of methods with respect to efficiencies, recoveries, and quantitation of mercury species interconversions in food demonstrated using tuna fish

Eight different analytical extraction procedures commonly used to extract mercury species from biological samples were evaluated by analyzing Tuna Fish Tissue Certified Reference Material (ERM-CE464) certified for the content of total mercury and methylmercury. Speciated isotope dilution mass spectrometry (SIDMS; US Environmental Protection Agency’s method 6800) was utilized to evaluate and effectively compensate for potential errors during measurement and accurately quantify mercury species using all the extraction methods. SIDMS was used to accurately evaluate species transformations during sample pretreatment, preparation and analysis protocols. The extraction methods tested in this paper were based on alkaline extraction with KOH or tetramethylammonium hydroxide; acid leaching with HCl, HNO₃ or CH₃COOH; extraction with l-cysteine hydrochloride; and enzymatic digestion with protease XIV. Detection of total mercury and mercury species from all extraction methods was carried out by inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography–ICP-MS, respectively. Microwave-assisted extraction and ultrasound-assisted extraction were found to be the most efficient alkaline digestion protocols that caused the lowest levels of transformation of mercury species (6% or less). Extraction with 5 M HCl or enzymatic digestion with protease resulted in the second-highest extraction efficiency, with relatively lower transformation of methylmercury to inorganic mercury (3 and 1.4%, respectively). Despite frequent use of acid leaching for the extraction of mercury species from tuna fish samples, the lowest extraction efficiencies and the highest mercury species transformation were obtained when microwave-assisted extraction with 4 M HNO₃ or CH₃COOH was used. Transformations as high as 30% were found using some literature protocols; however, all the extractions tested produced accurate quantitation when corrected in accordance with the SIDMS method standardized in the US Environmental Protection Agency’s method 6800. Figure Determinative CRM Tuna Fish Tissue Methylmercury Calibration vs. Determinative Calculation.     

Determination of Carbon‐based Engineered Nanoparticles in Marketed Fish by Microwave‐assisted Extraction and Liquid Chromatography‐atmospheric Pressure Photoionization‐tandem Mass Spectrometry

An optimized method for the determination of two major carbon‐based engineered nanoparticles (C₆₀ and C₇₀) in marketed fish samples is described. The method involves the use of microwave‐assisted extraction (MAE) coupled with liquid chromatography ‐ tandem mass spectrometry with atmospheric pressure photoionization (LC‐APPI‐MS/MS). Factors affecting the extraction efficiency of the analytes from fish samples were optimized by a central composite design method. The optimal extraction temperature and time for MAE were found to be 233 °C for 22 min, and the extraction solution was composed of toluene and acetone in a ratio of 4.64:1. The limits of quantitation (LOQs) were 0.1 and 0.05 ng/g for C₆₀ and C₇₀, respectively. The precision for these analytes at two spiked levels, as indicated by relative standard deviations (RSDs), were less than 10% for both intra‐ and inter‐day analysis. Accuracy, expressed as the mean extraction recovery, was between 85 and 98%. The method was further validated based on EU Commission Decision 2002/657/EC, including a decision limit (CCα) and detection capability (CCβ) for marketed fish samples.     

Temperature‐ and pressure‐dependent study of 35Cl NQR frequency and spin lattice relaxation time in 2,3‐dichloroanisole

The temperature and pressure dependence of ³⁵Cl NQR frequency and spin lattice relaxation time (T₁) were investigated in 2,3‐dichloroanisole. Two NQR signals were observed throughout the temperature and pressure range studied. T₁ were measured in the temperature range from 77 to 300 K and from atmospheric pressure to 5 kbar. Relaxation was found to be due to the torsional motion of the molecule and also reorientation of motion of the CH₃ group. T₁ versus temperature data were analyzed on the basis of Woessner and Gutowsky model, and the activation energy for the reorientation of the CH₃ group was estimated. The temperature dependence of the average torsional lifetimes of the molecules and the transition probabilities were also obtained. NQR frequency shows a nonlinear behavior with pressure, indicating both dynamic and static effects of pressure. The pressure coefficients were observed to be positive for both the lines. A thermodynamic analysis of the data was carried out to determine the constant volume temperature coefficients of the NQR frequency. The variation of spin lattice time with pressure was very small, showing that the relaxation is mainly due to the torsional motions of the molecules. Copyright © 2010 John Wiley & Sons, Ltd.     

Organic,inorganic and total mercury determination in fish by chemical vapor generation with collection on a gold gauze and electrothermal atomic absorption spectrometry

A method for organic, inorganic and total mercury determination in fish tissue has been developed using chemical vapor generation and collection of mercury vapor on a gold gauze inside a graphite tube and further atomization by electrothermal atomic absorption spectrometry. After drying and cryogenic grinding, potassium bromide and hydrochloric acid solution (1 mol L^− 1 KBr in 6 mol L^− 1 HCl) was added to the samples. After centrifugation, total mercury was determined in the supernatant. Organomercury compounds were selectively extracted from KBr solution using chloroform and the resultant solution was back extracted with 1% m/v L-cysteine. This solution was used for organic Hg determination. Inorganic Hg remaining in KBr solution was directly determined by chemical vapor generation electrothermal atomic absorption spectrometry. Mercury vapor generation from extracts was performed using 1 mol L^− 1 HCl and 2.5% m/v NaBH₄ solutions and a batch chemical vapor generation system. Mercury vapor was collected on the gold gauze heated resistively at 80 °C and the atomization temperature was set at 650 °C. The selectivity of extraction was evaluated using liquid chromatography coupled to chemical vapor generation and determination by inductively coupled plasma mass spectrometry. The proposed method was applied for mercury analysis in shark, croaker and tuna fish tissues. Certified reference materials were used to check accuracy and the agreement was better than 95%. The characteristic mass was 60 pg and method limits of detection were 5, 1 and 1 ng g^− 1 for organic, inorganic and total mercury, respectively. With the proposed method it was possible to analyze up to 2, 2 and 6 samples per hour for organic, inorganic and total Hg determination, respectively.     

Alkylation of ionic mercury to methylmercury and dimethylmercury by methylcobalamin: Simultaneous determination by purge-and-trap GC in line with FTIR

A new approach was used to determine the reaction products of methylcobalamin and ionic mercury: purge-and-trap gas chromatography in line with Fourier transform infrared spectroscopy (PT GC/FTIR). This technique simultaneously and specifically determines the spectrum of dimethylmercury (DMeHg) and methylmercury produced by the reaction. No interference from other known organic mercury species could be detected. The method is different from others because it does not require solvent extraction of the organomercurials from aqueous solution, but relies on immediate volatilization from the reaction vessel by addition of 100 μl of 10 mM NaBH₄. The sample was purged with nitrogen for 10 min. The volatile species of mercury were trapped in a column at ?120°C, injected into the gas chromatograph and detected by FTIR. The efficiency of DMeHg and MeHg formation depended on different parameters: pH, temperature, reaction time, and the methylcobalamin/ionic mercury ratio. The initial reaction product was MeHg which was further transformed to DMeHg. The first methylation rate was two times faster than the second. MeHg formed first, reaching a maximum at higher temperatures (28°C and 37°C) and later decreasing as DMeHg formed. At lower temperatures (20°C) the rate of MeHg formation was slower, being similar to the formation rate of DMeHg. Different species of inorganic mercury such as HgSO₄, Hg(NO₃)₂, Hg(SCN)₂, HgCl₂ and Hgl₂ were used to study differences in methylation by methylcobalamin under standard conditions of acidity, temperature and cofactor Hg(II) ratio.     

Preconcentration of inorganic mercury and organic mercury with solvent extraction for neutron activation analysis

A preconcentration method combined with neutron activation technique for the analysis of organic and inorganic mercury in waste water samples at ppb levels is presented. The inorganic mercury is extracted in CCl₄ solution with lead diethyldithiocarbamate reagent and the organic mercury is extracted in C₆H₆ solution. Interfering activities of sodium and bromine are removed from the irradiated samples by this procedure. Two different solvent extraction procedures are also described in detail.     

Determination of Organic and Inorganic Mercury Species as Emetine Dithiocarbamate Complexes by High-Performance Liquid Chromatography with Electrogenerated Tris(2,2′-bipyridine)ruthenium(III) Chemiluminescence Detection

A sensitive method for simultaneous determination of organic and inorganic mercury species has been developed and is presented in this study. The method is based on complex formation of mercury species with the emetine dithiocarbamate (emetine-CS₂) ligand, HPLC separation, and tris(2,2′-bipyridine)ruthenium(III) chemiluminescence detection. The complexation reactions of the mercury species and emetine-CS₂ ligand occurred instantaneously upon the addition of emetine-CS₂ solution to the solution containing the mercury species. The complete separation of these complexes was achieved using an ODS column with 20 mM NaH₂PO₄-acetonitrile (52:48, v/v) containing 30 mM NaClO₄ as an ion-pair reagent. The calibration graphs of these complexes were linear in the range from 1–100 µg/L. The detection limits were 0.27 µg/L, 0.33 µg/L, 0.39 µg/L, and 0.17 µg/L for methylmercury, ethylmercury, phenylmercury, and the mercury ion, respectively, at a signal-to-noise ratio of 3. The developed technique was validated by analyzing certified reference materials, CRM7402-a (cod fish, NMIJ) and CE464 (tuna fish, ERM), in combination with sonication-assisted acid leaching and liquid-liquid extraction. The emetine-CS₂ ligand has been used for extraction, separation, and detection of mercury species. The results determined using the proposed method were in good agreement with the values of the certified reference materials. The MeHg⁺ and EtHg⁺ recoveries for the spiked samples were found to be almost 100%.     

Solvent extraction of copper(II) from sulfate medium with N -(2-hydroxybenzylidene)aniline

Solvent extraction of copper(II) from sulfate medium with N-(2-hydroxybenzylidene)aniline is studied with the following parameters: pH, concentration of the extractant, nature of diluent, and temperature. The extraction of copper(II) proceeds by a cation exchange mechanism and the extracted species are CuL₂ in cyclohexane and toluene and CuL₂ with some CuL₂HL in chloroform. The equilibrium constants have been calculated as well as thermodynamic parameters ΔH°, ΔS°, and ΔG°. The temperature effect on the solvent extraction of copper(II) with N-(2-hydroxybenzylidene)aniline in cyclohexane is discussed.     

Sulfhydryl-functionalised magnetic nanoparticles as sorbent in dispersive solid-phase extraction for the rapid enrichment of mercury species from natural water samples

The sulfhydryl-functionalised core-shell Fe₃O₄@SiO₂ magnetic nanoparticles (Fe₃O₄@SiO₂–RSH MNPs)-based dispersive solid-phase extraction method was developed. The goal of this method is the extraction of mercury species from natural water samples. An interesting aspect of the method is that, thanks to the spontaneously aggregate, the MNPs with a sub-30-nm-size range could be fast and efficiently extracted by 0.45 μm pore size mixed cellulose esters membrane filter. Thus, the elution step can be conducted by passing small amounts eluent through the MNPs on the membrane. It is also found that addition of Ag⁺ to water sample could improve the elution efficiency, and furthermore, minimises the matrix effects during the extraction of mercury species from natural water samples. The feasibility of the method was studied, and extraction efficiency was evaluated. The results showed that, calculated at 5 ng/L spiked concentration levels, absolute recoveries were 89.4%, 91.9% and 64.2%, and enrichment factors (EFs) were 596, 613 and 428, for inorganic mercury, methylmercury and ethylmercury, respectively. The high EFs were achieved in 5 min of overall extraction time. The method was applied to groundwater and river water samples. The results showed that its suitability for use in fast extracting trace levels of mercury species from natural water samples.     

Determination of inorganic and total mercury by vapor generation atomic absorption spectrometry using different temperatures of the measurement cell

A simple and inexpensive laboratory-built flow injection vapor generation system coupled to atomic absorption spectrometry (FI-VG AAS) for inorganic and total mercury determination has been developed. It is based on the vapor generation of total mercury and a selective detection of Hg^2 + or total mercury by varying the temperature of the measurement cell. Only the inorganic mercury is measured when the quartz cell is at room temperature, and when the cell is heated to 650 °C or higher the total Hg concentration is measured. The organic Hg concentration in the sample is calculated from the difference between the total Hg and Hg^2 + concentrations. Parameters such as the type of acid (HCl or HNO₃) and its concentration, reductant (NaBH₄) concentration, carrier solution (HCl) flow rate, carrier gas flow rate, sample volume and quartz cell temperature, which influence FI-VG AAS system performance, were systematically investigated. The optimized conditions for Hg^2 + and total Hg determinations were: 1.0 mol l^− 1 HCl as carrier solution, carrier flow rate of 3.5 ml min^− 1, 0.1% (m/v) NaBH₄, reductant flow rate of 1.0 ml min^− 1 and carrier gas flow rate of 200 ml min^− 1. The relative standard deviation (RSD) is lower than 5.0% for a 1.0 μg l^− 1 Hg solution and the limit of quantification (LOQ, 10 s) is 55 ng g^− 1. Certified samples of dogfish muscle (DORM-1 and DORM-2) and non-certified fish samples were analyzed, using a 6.0 mol l^− 1 HCl solution for analyte extraction. The Hg^2 + and CH₃Hg⁺ concentrations found were in agreement with certified ones.     

The fate of mercury species injected into coelomic fluid of starfish Leptasterias polaris

Mature starfish Leptasterias polaris, collected in the St Lawrence Estuary (eastern Canada), were exposed to two mercury species (HgCI₂ and CH₃HgCI) via injections into the coelomic fluid. In vivo effects of some complexing agents (glutathione, mercaptoethanol and EDTA) on the distribution of ²⁰³Hg-labelled species in starfish organs and tissues and their possible role in mercury transport through membranes were studied over a 24 h period. The excretion of ammonia and mercury was also measured. When injected alone, inorganic mercury and methylmercury [CH₃Hg(II)] were distributed in all organs, with a preferential adsorption in gonads, pyloric caeca and stomach. Mercury excretion was very low under all conditions studied. Mercaptoethanol, a small thiol ligand, was very efficient in reducing both mercury species in the coelomic fluid and seems to have promoted translocation towards most organs of the starfish. Its action is attributed to the formation of small and neutral complexes, HgL₂ and CH₃HgL, which can diffuse through membranes preserving their integrity. Glutathione increased the translocation of CH₃Hg(II) towards surrounding organs, but had no apparent effect on inorganic mercury. EDTA promoted the transport of inorganic mercury only. These results highlight (1) the particular interest of starfish to workers studying in vivo chemical complexation of mercury species, and (2) the potential role of complexing molecules in the biotransport of mercury species through living membranes.     

Thermodynamic consideration on the interface formation of water and ethylene glycol isobutyl ether mixture

The interfacial tension of the binary two-phase mixture of water and ethylene glycol isobutyl ether (EIB) was measured as a function of temperature in the vicinity of the lower critical solution temperatureT _c under atmospheric pressure. The interfacial tension decreased with decreasing temperature and approached zero atT _c . The thermodynamic quantities of interface formation were evaluated by applying equations developed previously to the interfacial tension vs temperature curves. The results were compared with those of the water and diethylene glycol diethyl ether system examined previously, and the effect of the molecular structure of the ether molecule on its interfacial behavior was discussed. It was suggested that the hydration of the ethylene oxide groups of ether molecules was an important factor in the interface formation as well as in the mixing of component molecules of the systems investigated here.     

Supercritical fluid extraction of mercury species

Supercritical fluid extraction was used to recover organic and inorganic mercury species. Variations in pressure, water, methanol, and chelator create methods that allowed separation of inorganic from organic mercury species. When extracted using a compromised set of extraction conditions, the order of extraction was methyl, phenyl and inorganic mercury. For the individually optimized conditions, quantitative recoveries were observed. Level as low as 20 ppb were extracted and then determined using ICP.