Mercury speciation by coupling cold vapour atomic absorption spectrometry with flow injection on-line preconcentration and liquid chromatographic separation

A fully automated system for the direct determination of methylmercury (MeHg), ethylmercury (EtHg), phenylmercury (PhHg), and inorganic mercury (Hg(II)) at the ng/L level is described. It is based on solid phase extraction preconcentration incorporated in a flow injection (FI) system, high performance liquid chromatography (HPLC) separation, reduction combined with thermolysis and determination by cold vapour atomic absorption spectrometry (CVAAS). For preconcentration a microcolumn of bonded silica with octadecyl functional groups (C18 reversed phase material) was used as a sorbent for the mercury complexes formed on-line with ammonium pyrrolidine dithiocarbamate. Retained mercury species are eluted with a methanol-acetonitrile-water mixture and subjected to separation on an octadecylsilane (ODS) column before determination by CVAAS. The sensitivity of organo-mercury determination could be improved by using NaBH₄ as a reductant combined with a thermolysis step. In order to perform on-line measurements the preconcentration microcolumn was mounted in a pressure-tight casing. Limits of detection for MeHg, EtHg, PhHg and Hg(II) employing a sample volume of 58.5 mL were 9, 6, 10 and 5 ng/L, respectively. The relative standard deviation (RSD) calculated from 9 repeated measurements was found to be 3.6%, 5.5%, 10.4% and 7.6% for MeHg, EtHg, PhHg and Hg(II), respectively. Finally, the application of this method for speciation of mercury in fish and human urine is described. Received: 10 March 1997 / Revised: 29 January 1998 / Accepted: 5 February 1998     

Speciation of organomercury compounds by capillary electrophoresis with pre-column derivatization and on-line stacking

A simple capillary electrophoresis method was developed to separate and quantify methylmercury, ethylmercury, and phenylmercury with the enhancement of pre-column derivatization and on-line stacking.     

Determination of mercurial species in fish by inductively coupled plasma mass spectrometry with anion exchange chromatographic separation

This work demonstrated the feasibility of mercury speciation analysis by anion exchange chromatographic separation with inductively coupled plasma mass spectrometry detection. For the first time, by complexing with the mobile phase containing 3-mercapto-1-propanesulfonate into negatively charged complexes, fast separation of inorganic mercury (Hg²⁺), monomethylmercury (MeHg), ethylmercury (EtHg) and phenylmercury (PhHg) was achieved within 5 min on a 12.5-mm strong anion exchange column. The detection limits for Hg²⁺, MeHg, EtHg and PhHg were 0.008, 0.024, 0.029 and 0.034 μg L⁻¹, respectively. The relative standard deviations of peak height and peak area (5.0 μg L⁻¹ for each Hg species) were all below 3%. The determined contents of Hg²⁺, MeHg and total Hg in a certified reference material of fish tissue by the proposed method were in good accordance with the certified values with satisfactory recoveries. The relative errors for determining MeHg and total mercury were −2.4% and −1.2%, respectively, with an acceptable range for spike recoveries of 94–101%. Mercury speciation in 11 fish samples were then analyzed after the pretreated procedure. The mercury contents in all fish samples analyzed were found compliant with the criteria of the National Standards of China.     

Mercury speciation by coupling cold vapour atomic absorption spectrometry with flow injection on-line preconcentration and liquid chromatographic separation

A fully automated system for the direct determination of methylmercury (MeHg), ethylmercury (EtHg), phenylmercury (PhHg), and inorganic mercury (Hg(II)) at the ng/L level is described. It is based on solid phase extraction preconcentration incorporated in a flow injection (FI) system, high performance liquid chromatography (HPLC) separation, reduction combined with thermolysis and determination by cold vapour atomic absorption spectrometry (CVAAS). For preconcentration a microcolumn of bonded silica with octadecyl functional groups (C18 reversed phase material) was used as a sorbent for the mercury complexes formed on-line with ammonium pyrrolidine dithiocarbamate. Retained mercury species are eluted with a methanol-acetonitrile-water mixture and subjected to separation on an octadecylsilane (ODS) column before determination by CVAAS. The sensitivity of organo-mercury determination could be improved by using NaBH₄ as a reductant combined with a thermolysis step. In order to perform on-line measurements the preconcentration microcolumn was mounted in a pressure-tight casing. Limits of detection for MeHg, EtHg, PhHg and Hg(II) employing a sample volume of 58.5 mL were 9, 6, 10 and 5 ng/L, respectively. The relative standard deviation (RSD) calculated from 9 repeated measurements was found to be 3.6%, 5.5%, 10.4% and 7.6% for MeHg, EtHg, PhHg and Hg(II), respectively. Finally, the application of this method for speciation of mercury in fish and human urine is described.     

Determination of methylmercury and phenylmercury in water samples by liquid-liquid-liquid microextraction coupled with capillary electrophoresis

A novel method for determination of methylmercury (MeHg) and phenylmercury (PhHg) by liquid-liquid-liquid microextraction (LLLME) coupled with capillary electrophoresis (CE) with ultraviolet (UV) technique was developed. The method based on MeHg and PhHg was complexed with 1-(2-pyridylazo)-2-naphthol (PAN) to form hydrophobic complexes. When the sample solution was stirred, analytes were extracted into the organic layer (200 microL toluene) and back-extracted simultaneously into the 4.0 microL 0.1% (w/v) l-cysteine microdrop. The factors affecting on the LLLME of two mercury species, including sample pH, complex reagent concentration, extraction time, volume of organic solvent, stirring rate and phase volume ratio, were investigated. Under the optimized conditions, the detection limits (S/N=3) of MeHg and PhHg were 0.94 and 0.43 ngmL(-1) (as Hg), respectively. The precisions (RSDs, c=10 ngmL(-1), n=7) were in the range of 3.3-3.4% for migration time, 6.1-7.2% for peak area response, and 6.7-7.5% for peak height response for the two mercury species. The enrichment factors of 324 for MeHg and 210 for PhHg were obtained with 40 min LLLME. The developed method was successfully applied to the determination of trace amounts of MeHg and PhHg in water samples.     

Preconcentration speciation method for mercury compounds in water samples using solid phase extraction followed by reversed phase high performance liquid chromatography

A simple and rapid method for in situ preconcentration of inorganic and organic mercury compounds in water samples, based on solid phase extraction using dithizone immobilised on a reversed-phase C18 cartridge, has been developed. The adsorbed complexes were stable on the cartridge for at least 2 weeks. The speciation analysis of methylmercury (MeHg), phenylmercury (PhHg) and inorganic mercury (Hg (II)) were done by reversed-phase high performance liquid chromatography. The calibration graphs of MeHg, PhHg and Hg (II) were linear (r>0.999) from the detection limits (0.58, 0.66 and 0.54 ng) to 38, 25 and 26 ng of Hg, respectively. The average recoveries of MeHg, PhHg and Hg (II) from spiked samples (0.3-48.0 mug l(-1) Hg) were 98+/-3, 99+/-1 and 100+/-7%, respectively. By applying SPE procedure a 200-fold concentration of the sample was obtained.     

pH-mediated dual-cloud point extraction as a preconcentration and clean-up technique for capillary electrophoresis determination of phenol and m-nitrophenol

pH-mediated dual-cloud point extraction (dCPE) technique for capillary electrophoresis (CE) determination of phenol and m-nitrophenol is proposed in this paper. This technique for the preconcentration and clean-up of the two analytes includes two steps through simple pH-mediation. The analytes are transferred into surfactant-rich phase in the first step (under acidic condition) with Triton X-114 as the extractant because the two analytes become hydrophobic in acidic solution. They were back-extracted into alkaline aqueous phase in the second cloud point step. Because the concentration of Triton X-114 in the final aqueous solution after dCPE is only around critical micelle concentration, its adsorption on the inner wall of capillary and its possible influence on electrophoretic separation are eliminated. Baseline separation of phenol and m-nitrophenol is realized in a 60 cm x 75 microm i.d. capillary at 18 kV using 50 mM boric acid solution (pH 9.5). The preconcentration factors are 24.0 for phenol and 22.5 for m-nitrophenol. The detection limits of phenol and m-nitrophenol are 2.0 x 10(-6) mol L(-1) and 2.5 x 10(-6) mol L(-1), respectively. The proposed method was successfully applied to the determination of two analytes in spiked natural water samples.     

Speciation analysis of mercury in water samples by dispersive liquid–liquid microextraction coupled to capillary electrophoresis

In this study, a method of pretreatment and speciation analysis of mercury by dispersive liquid–liquid microextraction along with CE was developed. The method was based on the fact that mercury species including methylmercury (MeHg), ethylmercury (EtHg), phenylmercury (PhHg), and Hg(II) were complexed with 1‐(2‐pyridylazo)‐2‐naphthol to form hydrophobic chelates and l ‐cysteine could displace 1‐(2‐pyridylazo)‐2‐naphthol to form hydrophilic chelates with the four mercury species. Factors affecting complex formation and extraction efficiency, such as pH value, type, and volume of extractive solvent and disperser solvent, concentration of the chelating agent, ultrasonic time, and buffer solution were investigated. Under the optimal conditions, the enrichment factors were 102, 118, 547, and 46, and the LODs were 1.79, 1.62, 0.23, and 1.50 μg/L for MeHg, EtHg, PhHg, and Hg(II), respectively. Method precisions (RSD, n = 5) were in the range of 0.29–0.54% for migration time, and 3.08–7.80% for peak area. Satisfactory recoveries ranging from 82.38 to 98.76% were obtained with seawater, lake, and tap water samples spiked at three concentration levels, respectively, with RSD (n = 5) of 1.98–7.18%. This method was demonstrated to be simple, convenient, rapid, cost‐effective, and environmentally benign, and could be used as an ideal alternative to existing methods for analyzing trace residues of mercury species in water samples.     

Speciation analysis of mercury by dispersive solid‐phase extraction coupled with capillary electrophoresis

A pretreatment method of dispersive solid‐phase extraction (DSPE) along with back‐extraction followed by CE‐UV detector was developed for the determination of mercury species in water samples. Sulfhydryl‐functionalized SiO₂ microspheres (SiO₂−SH) were synthesized and used as DSPE adsorbents for selective extraction and enrichment of three organic mercury species namely ethylmercury (EtHg), methylmercury (MeHg), and phenylmercury (PhHg), along with L‐cysteine (L‐cys) containing hydrochloric acid as back‐extraction solvent. Several main extraction parameters were systematically investigated including sample pH, amount of adsorbent, extraction and back‐extraction time, volume of eluent, and concentration of hydrochloric acid. Under optimal conditions, good linearity was achieved with correlation coefficients over 0.9990, in the range of 4−200 μg/L for EtHg, and 2−200 μg/L for MeHg and PhHg. The LODs were obtained of 1.07, 0.34, and 0.24 μg/L for EtHg, MeHg, and PhHg, respectively, as well as the LOQs were 3.57, 1.13, and 0.79 μg/L, respectively, with enrichment factors ranging from 109 to 184. Recoveries were attained with tap and lake water samples in a range of 62.3−107.2%, with relative standard deviations of 3.5–10.1%. The results proved that the method of SiO₂−SH based DSPE coupled with CE‐UV was a simple, rapid, cost‐effective, and eco‐friendly alternative for the determination of mercury species in water samples.     

On-line hyphenation of capillary electrophoresis with flame-heated furnace atomic absorption spectrometry for trace mercury speciation

Capillary electrophoresis (CE) was directly interfaced to flame-heated furnace atomic absorption spectrometry (FHF-AAS) via a laboratory-made thermospray interface for nanoliter trace element speciation. The CE-FHF-AAS interface integrated the superiorities of stable CE separation, complete sample introduction, and continuous vaporization for AAS detection without the need of extra external heat sources and any post-column derivation steps. To demonstrate the usefulness of the developed hybrid technique for speciation analysis, three environmentally significant and toxic forms of methylmercury (MeHg), phenylmercury (PhHg), and inorganic mercury (Hg(II)) were taken as model analytes. Baseline separation of the three mercury species was achieved by CE in a 60 cm long x 75 microm inner diameter fused-silica capillary at 20 kV and using a mixture of 100 mM boric acid and 10% v/v methanol (pH 8.30) as running electrolyte. The precision (relative standard deviation, RSD, n = 7) of migration time, peak area and peak height for the mercury species at 500 microg x L(-1) (as Hg) level were in the range of 0.9-1.2%, 1.5-1.9%, and 1.4-2.0%, respectively. The detection limit (S/N = 3) of three mercury species was 3.0 +/- 0.15 pg (as Hg), corresponding to 50.8 +/- 2.4 microg x L(-1) (as Hg) for 60 nL sample injection, which was almost independent on specific mercury species. The developed hybrid technique was successfully applied to the speciation analysis of mercury in a certified reference material (DORM-2, dogfish muscle).     

Simultaneous determination of methylmercury and ethylmercury in rice by capillary gas chromatography coupled on-line with atomic fluorescence spectrometry

A comprehensive method for simultaneous determination of methylmercury (MeHg) and ethylmercury (EtHg) in rice by capillary gas chromatography (GC) coupled on-line with atomic fluorescence spectrometry was developed. The experimental conditions, including the pyrolyzer temperature and flow rates of the make-up gas and sheath gas, were optimized in detail. The absolute detection limits (3sigma) were 0.005 ng as Hg for both MeHg and EtHg. The relative standard deviation values (n=5) for 10 ng Hg/mL of MeHg and EtHg were 2.5 and 1.3%, respectively. The method was evaluated by analyzing 2 certified reference materials (DORM-2 and GBW08508), and the determined values of MeHg and total mercury concentrations were in good agreement with the certified values. In addition, the recoveries of MeHg and EtHg spiked into a rice sample collected from Jiangsu province in China were 86 and 77%, respectively. The proposed method was applied to analysis of MeHg and EtHg in 25 rice samples cultivated in 15 provinces of China. In all samples, MeHg was detectable and no EtHg was found. The MeHg contents in rice samples ranged from 1.9 to 10.5 ng/g, accounting for 7-44% of the total mercury measured.     

UV irradiation controlled cold vapor generation using SnCl2 as reductant for mercury speciation.

A simple and ultrasensitive method, which was based on cold vapor generation (CVG) coupled to atomic fluorescence spectrometry (AFS), was proposed for speciation analysis of inorganic mercury (Hg2+) and methylmercury (MeHg) in water samples. In the presence of UV irradiation, all the mercury (MeHg+Hg2+) in a sample solution can be reduced to Hg0 by SnCl2; without UV irradiation, only Hg2+ species can be determined. So the concentration of MeHg can be obtained from the difference of the total mercury and Hg2+ concentration; thus, speciation analysis of Hg2+ and MeHg was simply achieved without chromatographic separation. Under the optimized experimental conditions, the limits of detection were 0.01 ng mL-1 for both Hg2+ and MeHg. The sensitivity and limit of detection were not dependent on the mercury species, and a simple Hg2+ aqueous standard series can be used for the determination of both Hg2+ and MeHg.     

Dual-cloud point extraction and tertiary amine labeling for selective and sensitive capillary electrophoresis-electrochemiluminescent detection of auxins

The low concentrations of the auxins in samples of plant tissue necessitate the use of selective and sensitive techniques for their quantification. Herein a selective and sensitive method based on dual-cloud point extraction (dCPE) and tertiary amine labeling for the quantification of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) by capillary electrophoresis-electrochemiluminescence (CE-ECL) is proposed. The procedure for dCPE included two cloud point processes with Triton X-114 as the extractant. The two auxins became hydrophobic in an acidic solution and were extracted into surfactant-rich phase after the first cloud point procedure. They were then back-extracted into the alkaline aqueous phase during the second cloud point step. The extracted auxins were reacted with 2-(2-aminoethyl)-1-methylpyrrolidine (AEMP) in acetonitrile that contained N,N′-dicyclohexylcarbodiimide and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine to produce their AEMP-derivatives. The two auxin-AEMP-derivatives were subjected into CE and detected by Ru(bpy)₃²⁺-based ECL. The preconcentration factors for IAA and IBA with dCPE were 40.5 and 43.4, respectively. The on-capillary detection limits (S/N = 3) were 2.5 and 2.8 nM for IAA and IBA. This protocol presents a clear advantage in that it reduces the interference from the matrixes extensively and gives a high sensitivity for the detection of auxins. The proposed method was applied successfully to the detection of the two auxins in acacia tender leaves, buds, and bean sprout.     

Applicability of multisyringe chromatography coupled to cold-vapor atomic fluorescence spectrometry for mercury speciation analysis

In this paper, a novel automatic approach for the speciation of inorganic mercury (Hg(2+)), methylmercury (MeHg(+)) and ethylmercury (EtHg(+)) using multisyringe chromatography (MSC) coupled to cold-vapor atomic fluorescence spectrometry (CV/AFS) was developed. For the first time, the separation of mercury species was accomplished on a RP C18 monolithic column using a multi-isocratic elution program. The elution protocol involved the use of 0.005% 2-mercapthoethanol in 240 mM ammonium acetate (pH 6)-acetonitrile (99:1, v/v), followed by 0.005% 2-mercapthoethanol in 240 mM ammonium acetate (pH 6)-acetonitrile (90:10, v/v). The eluted mercury species were then oxidized under post-column UV radiation and reduced using tin(II) chloride in an acidic medium. Subsequently, the generated mercury metal were separated from the reaction mixture and further atomized in the flame atomizer and detected by AFS. Under the optimized experimental conditions, the limits of detection (3σ) were found to be 0.03, 0.11 and 0.09 μg L(-1) for MeHg(+), Hg(2+) and EtHg(+), respectively. The relative standard deviation (RSD, n=6) of the peak height for 3, 6 and 3 μg L(-1) of MeHg(+), Hg(2+) and EtHg(+) (as Hg) ranged from 2.4 to 4.0%. Compared with the conventional HPLC-CV/AFS hyphenated systems, the proposed MSC-CV/AFS system permitted a higher sampling frequency and low instrumental and operational costs. The developed method was validated by the determination of a certified reference material DORM-2 (dogfish muscle), and was further applied for the determination of mercury species environmental and biological samples.     

Solution cathode glow discharge induced vapor generation of mercury and its application to mercury speciation by high performance liquid chromatography-atomic fluorescence spectrometry

A novel solution cathode glow discharge (SCGD) induced vapor generation was developed as interface to on-line couple high-performance liquid chromatography (HPLC) with atomic fluorescence spectrometry (AFS) for the speciation of inorganic mercury (Hg(2+)), methyl-mercury (MeHg) and ethyl-mercury (EtHg). The decomposition of organic mercury species and the reduction of Hg(2+) could be completed in one step with this proposed SCGD induced vapor generation system. The vapor generation is extremely rapid and therefore is easy to couple with flow injection (FI) and HPLC. Compared with the conventional HPLC-CV-AFS hyphenated systems, the proposed HPLC-SCGD-AFS system is very simple in operation and eliminates auxiliary redox reagents. Parameters influencing mercury determination were optimized, such as concentration of formic acid, discharge current and argon flow rate. The method detection limits for HPLC-SCGD-AFS system were 0.67 μg L(-1) for Hg(2+), 0.55 μg L(-1) for MeHg and 1.19 μg L(-1) for EtHg, respectively. The developed method was validated by determination of certified reference material (GBW 10029, tuna fish) and was further applied for the determination of mercury in biological samples.     

Dielectric barrier discharge-plasma induced vaporization and its application to the determination of mercury by atomic fluorescence spectrometry

This paper describes a low-temperature dielectric barrier discharge (DBD)-plasma induced vaporization technique using mercury as a model analyte. The evaporation and atomization of dissolved mercury species in the sample solution can be achieved rapidly in one step, allowing mercury to be directly detected by atomic fluorescence spectrometry. The DBD plasma was generated concentrically in-between two quartz tube (outer tube: i.d. 5 mm and o.d. 6 mm, inner tube: i.d. 2 mm and o.d. 3 mm). A copper electrode was embedded inside the inner quartz tube and sample solution was applied onto the outer surface of the inner tube. The effects of operating parameters such as plasma power, plasma gas identity, plasma gas flow rate and interferences from concomitant elements have been investigated. The difference in the sensitivities of Hg(2+), methylmercury (MeHg) and ethylmercury (EtHg) was found to be negligible in the presence of formic acid (≥1% v/v). The analytical performance of the present technique was evaluated under optimized conditions. The limits of detection were calculated to be 0.02 ng mL(-1) for Hg(2+), MeHg and EtHg, and repeatability was 6.2%, 4.9% and 4.3% RSD (n = 11) for 1 ng mL(-1) of Hg(2+), MeHg and EtHg, respectively. This provides a simple mercury detection method for small-volume samples with an absolute limit of detection at femtogram level. The accuracy of the system was verified by the determination of mercury in reference materials including freeze-dried urine ZK020-2, simulated water matrix reference material GBW(E) 080392 and tuna fish GBW10029, and the concentration of mercury determined by the present method agreed well with the reference values.     

Development of a new hybrid technique for rapid speciation analysis by directly interfacing a microfluidic chip-based capillary electrophoresis system to atomic fluorescence spectrometry

This paper represents the first study on direct interfacing of microfluidic chip-based capillary electrophoresis (chip-CE) to a sensitive and selective detector, atomic fluorescence spectrometry (AFS) for rapid speciation analysis. A volatile species generation technique was employed to convert the analytes from the chip-CE effluent into their respective volatile species. To facilitate the chip-CE effluent delivery and to provide the necessary medium for subsequent volatile species generation, diluted HCl solution was introduced on the chip as the makeup solution. The chip-CE-AFS interface was constructed on the basis of a concentric "tube-in-tube" design for introducing a KBH4 solution around the chip effluent as sheath flow and reductant for volatile species generation as well. The generated volatile species resulting from the reaction of the chip-CE effluent and the sheath flow were separated from the reaction mixture in a gas-liquid separator and swept into the AFS atomizer by an argon flow for AFS determination. Inorganic mercury (Hg(II)) and methylmercury (MeHg(I)) were chosen as the targets to demonstrate the performance of the present technique. Both mercury species were separated as their cysteine complexes within 64 s. The precision (relative standard deviation, RSD, n = 5) of migration time, peak area, and peak height for 2 mg.L(-1) Hg(II) and 4 mg.L(-1) MeHg(I) (as Hg) ranged from 0.7 to 0.9%, 2.1 to 2.9%, and 1.5 to 1.8%, respectively. The detection limit was 53 and 161 microg.L(-1) (as Hg) for Hg(II) and MeHg(I), respectively. The recoveries of the spikes of mercury species in four locally collected water samples ranged from 92 to 108%.     

On-line coupling of flow injection displacement sorption preconcentration to high-performance liquid chromatography for speciation analysis of mercury in seafood

A simple and cost-effective method for speciation analysis of trace mercury in seafood was developed by on-line coupling flow injection microcolumn displacement sorption preconcentration to high-performance liquid chromatography (HPLC) with UV detection. The methodology involved the presorption of the Cu-PDC (pyrrolidine dithiocarbamate) chelate onto a microcolumn packed with a cigarette filter sorbent, simultaneous preconcentration of Hg(II), methylmercury (MeHg), ethylmercury (EtHg), and phenylmercury (PhHg) onto the microcolumn through a displacement reaction with the presorbed Cu-PDC, and their subsequent elution from the microcolumn for on-line HPLC separation. Interferences from heavy metal ions with lower stability of their PDC chelates relative to Cu-PDC were minimized without the need of any masking agents. With the consumption of 4.0 ml of sample solution, the enrichment factors were about 80. The detection limits were 10-25 ng g(-1) (as Hg) in fresh tissue. Precision (R.S.D. (%), n = 5) ranged from 2 to 3% at the 500 microg l(-1) (as Hg) level. The developed technique was validated by analyzing a certified reference material (DORM-2, dogfish-muscle), and was shown to be useful for mercury speciation in real seafood samples.     

汞的形态分析研究 Ⅱ.固相萃取预富集-液相色谱分离测定不同形态的有机汞

本文报道了固相萃取预富集处理样品继以液相色谱分离测定不同形态痕量有机汞的方法.二乙基二硫代氨基甲酸钠(DDTC)作络合剂及甲醇作洗脱液的预富集系统能在线富集甲基汞(MeHg)、乙基汞(EtHg)和苯基汞(PhHg).用于测定加标海水中MeHg、EtHg和PhHg,回收率分别为96.9％、102.4％和98.0％;相对标准偏差分别为3.5％、5.0％和5.0％;检测下限分别为 1.0μg/L、1.2 μg/L和 1.2 μg/L.     

A methodological study of mercury speciation using dogfish liver CRM (DOLT-2)

The purpose of the study was to optimise analytical methods for determination of the chemical speciation of mercury in studies of protective mechanisms of selenium. Optimisation of the methods was performed using CRM DOLT-2 (Dogfish liver), both in its original form and after separation of various fractions. The sample was homogenised with 10 mM Tris-HCl buffer (pH 7.6) and ultracentrifuged. The soluble phase obtained was applied to a size exclusion chromatography column (Sephadex G-75 column) for separation of various protein fractions. Total mercury (total Hg), monomethyl mercury (MeHg) and selenium (Se) were determined in whole dogfish liver tissue and its soluble and insoluble phases (pellet). Different approaches for determination of total Hg and MeHg were compared. Simultaneous determination of MeHg and inorganic mercury (Hg2+) was based on alkaline dissolution and/or acid leaching, followed by ethylation, room temperature precollection, isothermal gas chromatography (GC), pyrolysis and detection with cold vapour atomic fluorescence spectrometry (CVAFS). The sum of MeHg and Hg2+ was compared to total Hg results obtained by acid digestion and CVAAS detection. The accuracy of MeHg determination was checked by its determination using acid leaching at room temperature, solvent extraction, back extraction into Milli-Q water, ethylation, GC and CVAFS detection. For the insoluble phase it is recommended to use solvent extraction for MeHg and acid digestion CVAAS for total Hg. For determination of MeHg and Hg2+ in the lyophilised sample and water soluble fractions containing low concentrations of mercury species, the simultaneous measurement of MeHg and Hg2+ after alkaline dissolution is the most appropriate method.