Simplified and miniaturized procedure based on ultrasound-assisted cytosol preparation for the determination of Cd and Cu bound to metallothioneins in mussel tissue by ICP-MS

Abstract

A simplified and miniaturized procedure for the determination of Cd and Cu bound to metallothioneins (MTs) by ICP-MS in mussel tissue has been developed. Cytosol preparation was based on the indirect sonication of slurries containing the lyophilized sample dispersed in 1 mL of extractant by means of a sonoreactor Cup-Horn. Rabbit liver MTs (Apo-MT-I, Apo-MT-II and Cd(7)-MT-II) and a conventional cytosol preparation procedure were used for validation purposes. The usual heating step and additional centrifugations of the conventional procedure for cytosol preparation can be omitted when using ultrasound treatment. The possible effect of denaturation on MTs and its effect on the metal bound to MTs were evaluated. Variables influencing the ultrasound-assisted cytosol preparation procedure were carefully optimized for simultaneous determination of both metals. Chromatographic conditions to separate the MT fraction from other proteins present in cytosols were also studied. Six samples can be processed within 3 min of sonication. An acid ultrasound-assisted extraction procedure with diluted acid was also proposed for determining total Cd and Cu. Finally, Cd and Cu bound to MTs as well as total Cd and Cu were determined in mussels from Pontevedra and Ares-Betanzos coastal inlets (Galicia, Spain).