Abstract

Size-exclusion high-performance liquid chromatography (SE-HPLC) was coupled on-line with axial inductively coupled plasma time-of-flight mass spectrometry (ICP-TOFMS). This hybrid approach was assessed for characterization of metallothioneins (MT) as well as for speciation of ultra-trace metals associated to the MT fraction in cytosol samples of the common carp. The separation of commercial gel-filtration standards with molecular weights that ranged from 1 355 to 540 000 Da was performed in a SUPELCO TSK gel G 3000 PWxL gel-filtration column using 30 mmol l⁻¹ Tris-HCl (pH 7.4, flow 0.8 ml min⁻¹) as a mobile phase. The proteins were first detected on-line spectrophotometrically at 254 and 280 nm, then specific detection of metals was performed using ICP-TOFMS. The potential of the ICP-TOFMS instrument as a fast transient detector for the quasi-simultaneous multi-elemental speciation of 16 isotopes bound to biomolecules of different size, after separation, was assessed using minute volumes of sample. The detection limits (3σ) for Cd, Zn and Cu bound to rabbit liver MT varied from 19.9 ± 0.5 pg (for ¹¹⁴Cd) to 158 ± 7 pg (for ⁶⁶Zn). Application of this hyphenated method to investigate the distribution of ultra-trace heavy metals among different size fractions of liver and kidney cytosols of carp and their relation with the induction of MT after exposure to Cd in water was addressed. The induction of Cd–MT in kidney was found to be a “biomarker” for exposure to ppb levels of Cd in water. Complementary anion-exchange chromatography was used in combination with ICP-TOFMS detection for multi-elemental speciation of MT fractions, previously isolated by SE-HPLC, in kidney and liver of stressed carp. Metal speciation of MT in vertebrate fish seemed to be similar to that observed for mammalian MT. A clear difference in terms of metal–protein associations was found between the MT fractions in carp kidney and liver tissues.