Solvent extraction, sonication, and microwave-assisted extractions in the presence of extraction agents (thioacetic acid, citric acid, cysteine, 2-mercaptoethanol, HCl + NaCl, etc.) were tested for the isolation of mercury species. A mixture of 6 M HCl and 0.1 M NaCl was selected as the most suitable extraction agent. The extraction efficiency was about 10% higher and the RSD below 3.3% when microwave-assisted extraction was applied instead of sonication. The liquid chromatography-cold vapour atomic fluorescence spectrometry (LC/CV-AFS) method was optimised and used for separation and determination of inorganic mercury cations and alkylated and arylated mercury species. Isocratic elution at a flow rate of 0.15 mL/min (with a mobile phase containing 0.05% 2-mercaptoethanol (pH = 5) and 7% methanol and with a stepwise increase of methanol content up to 100% MeOH in the 15th min) was used for separation of mercury species on a Hypersil BDS C18 RP column. The limits of detection of the LC/CV-AFS system were estimated as 0.2 microg/L (3%) for MeHg+, 0.07 microg/L (5.3%) for inorganic Hg, 0.06 microg/L (3.4%) for PhHg+, and 0.12 microg/L (4.4%) for EtHg with the corresponding RSDs at 5 microg/L (n = 10) given in parentheses. The concentrations (2-10 mg/kg fresh weight) of total mercury and methylmercury (90-99% of the total mercury) in selected fish obtained by HPLC/CV-AFS were in good agreement (absolute deviations 0.05 mg/kg) but more precise (RSDs <5.4% at 5 mg/L, n = 10) than those determined by GC coupled to an electron capture detector. The RSDs (3.1-8.2% and 4.1-9.0%) of the overall analytical procedure for the determination of total mercury (AMA 254) and methylmercury (HPLC/CV-AFS) were determined for intra-day and inter-day assays, respectively.
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