Analytical speciation of mercury in fish tissues by reversed phase liquid chromatography-inductively coupled plasma mass spectrometry with Bi(3+) as internal standard.

In this work, the quantification of two mercury species (Hg(2+) and CH(3)Hg(+)) in fish tissues has been revisited. The originality of our approach relies on the use of Bi(3+) as internal standard (IS) and on the modification of typical extraction conditions. The IS (125 microl, 1000 microg l(-1) Bi(3+)) was added to the aliquot of fresh fish tissue (400-500 mg). A high-speed blender and ultrasound-assisted homogenization/extraction was carried out in the presence of perchloric acid (1.5 ml, 0.6 mol l(-1)), l-cysteine (500 microl, 0.75 mol l(-1)) and 500 microl toluene:methanol (1:1). Perchloric acid was used for protein denaturation and precipitation, toluene helped to destroy lipid structures potentially sequestering CH(3)Hg(+), L-cysteine was used to form water-soluble complexes with Bi(3+), Hg(2+) and CH(3)Hg(+). The excess of perchloric acid was eliminated by addition of potassium hydroxide (pH 5 with acetic acid). The obtained extract, was diluted with the mobile phase (1:1) and introduced (20 microl) to the reversed phase HPLC-ICP-MS system. The separation was achieved by isocratic elution (2.5 mmol l(-1) cysteine, 12.5 mmol l(-1) (NH(4))(2)HPO(4), 0.05% triethylamine, pH 7.0:methanol (96:4)) at a flow rate 0.6 ml min(-1). Column effluent was on-line introduced to ICP-MS for specific detection of (202)Hg, (200)Hg and (209)Bi. Analytical signal was defined as the ratio between (202)Hg/(209)Bi peak areas. The detection limits evaluated for Hg(2+) and CH(3)Hg(+) were 0.8 and 0.7 microg l(-1). Recovery of the procedure, calculated as the sum of species concentrations found in the sample with respect to total ICP-MS-determined Hg was 91.9% for king mackerel muscle and 89.5% for red snapper liver. In the standard addition experiments, the recovery results were 98.9% for Hg(2+) and 100.6% for CH(3)Hg(+). It should be stressed that the use of Bi(3+) as IS enabled to improve analytical performance by compensating for incomplete extraction and for imprecision of sample handling during relatively non-rigorous protocol.