Assessment of sample preparation for single particle ICP-MS determination of mercury selenide nanoparticles in fish tissues within mercury speciation framework

Mercury selenide nanoparticles (HgSeNPs) play a crucial role in mercury (Hg) detoxification in aquatic organisms, and their inclusion in classical Hg speciation studies expands the understanding of Hg biotoxicological impact. In this context, single particle inductively coupled plasma mass spectrometry (spICP-MS) has emerged as a powerful tool for the detection and characterization of HgSeNPs. However, its application to solid samples, such as fish tissues, requires an efficient recovery without compromising the NP integrity. This work explores a unified extraction protocol for full Hg speciation using tetramethylammonium hydroxide (TMAH), a reagent commonly employed in classical Hg speciation, combined with different energy sources (microwave and ultrasound). Three microwave programs and key ultrasonic parameters (extraction time, power, and amplitude) were evaluated. Microwave-assisted extraction at 150 °C enabled reliable quantification of classical Hg species and provided size distribution information for HgSeNP. In contrast, ultrasound-assisted extraction under optimal conditions (1 minute, 28 W, 2 cycles at 10% amplitude) allowed quantitative recovery of HgSeNPs while preserving particle size, demonstrating superior performance for NP isolation. Due to the lack of certified reference materials (CRMs) for HgSeNPs, method optimization and performance assessment were carried out using a commercial HgSe material, supporting the feasibility of integrated speciation protocols. The optimized methods were successfully applied to tuna and swordfish samples. This study highlights the need for further validation and CRMs, including HgSeNPs, to ensure reliable interlaboratory comparison and comprehensive Hg speciation. • A unified sample preparation for chemical Hg speciation and HgSeNPs isolation was evaluated. • Microwave- and ultrasound-assisted alkaline extractions were optimized and compared. • Microwave extraction provided HgSeNP screening and size distribution information. • Ultrasound extraction enabled quantitative HgSeNP recovery with preserved size. • Use of CRMs for classical species as tools to validate nanoparticle analysis methods.