Research in the area of new nanomaterials has been given high priority as having an enormous economic potential. Due to marked antimicrobial effect, silver nanoparticles (AgNPs) are one of the most commercialized and successfully exploited nanomaterials in a wide range of medical and consumer products.In biological and environmental compartments, AgNPs undergo different transformations including interaction with organic molecules, such as proteins, and dissolution. Hyphenated systems consisting of capillary electrophoresis (CE) coupled to sensitive element detection like ICP-MS can be considered as the promising methods for speciation analysis of AgNPs. Here, we investigated applicability of different CE methods hyphenated to ICP-MS for speciation of AgNPs in biological systems.The paper presents approach to analyze species formed in interaction of AgNPs with metallothionein (MT) as model protein. As AgNPs might be coated by MTs in bio-fiuids, we installed first a CE-speciation method for MT-1 and MT-2. Although this separation was successful, no reproducible and well separated peaks for AgNPs or Ag* were achieved. Therefore, we focused on developing methods for separating MT-1, MT-2, Ag* and AgNPs. Several buffer conditions were tested to improve their separation and to minimize Ag-sticking to capillary walls. All compounds of interest in this paper, i.e. MT-1, MT-2, Ag+ and AgNPs, were well separated from each other using tetramethyl-ammoniumhydroxide as electrolyte. In mixed samples, we observed Ag+ being completely associated with MT-1, while Ag*-association with MT-2 was less: The highest quantity of Ag* was associated with a compound having low Cd-concentration, while another relevant fraction was bound to MT-2. Free Ag* was also seen in minor amounts whereas another Ag-peak at 8.13 min remains unknown. Most AgNPs remained free. AgNPs were only little associated with MT-1, the latter being split into two peak signals, whereas association with MT-2 was high. Only 15% of AgNPs remained unbound.We demonstrated CE hyphenated to the ICP-MS as promising and elegant technique to study AgNPs in biological systems. (C) 2018 Elsevier B.V. All rights reserved.