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                <term xml:lang="en">gadolinium-based contrast agents</term>
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              <p>Background: Gadolinium-based contrast agents (GBCA) are widely used in magnetic resonance imaging (MRI) to enhance image contrast. However, their interactions with biological macromolecules remain an important area of investigation due to potential safety concerns. Developing robust analytical methods to study these interactions is essential for improving safety assessments and guiding the design of next-generation contrast agents.Results: In this study, we employed Frontal Analysis Continuous Capillary Electrophoresis (FACCE) to investigate the binding interactions between lysozyme, a model protein, and several GBCA, including Gd-DTPA (Gadopentetate, Magnevist®), Gd-BOPTA (Gadobenate, Multihance®), Gd-DOTA (Gadoterate, Dotarem®), Gd-PCTA D2 (Gadopiclenol, Elucirem™) and Gd-HP-DO3A (Gadoteridol, Prohance®). Our results demonstrate that GBCA exhibit varying degrees of cooperative binding to lysozyme, with stoichiometry (n) of 4 for Gd-DTPA, Gd-BOPTA, and Gd-DOTA. The strength of interactions followed the trend: Gd-BOPTA (Multihance®) &amp;gt; Gd-DTPA (Magnevist®) &amp;gt; Gd-DOTA (Dotarem®), while Gd-PCTA D2 (Elucirem™) showed no significant binding. However, FACCE was unable to resolve interactions for Gd-HP-DO3A (Prohance®) due to interference with lysozyme.Significance: FACCE is a well-adapted method to investigate GBCA-protein interaction. It requires low sample volumes and allows determining the stoichiometry, binding constant, and cooperativity of the interaction. This work emphasizes the cooperative nature of the GBCA-lysozyme interactions and indicates that the Hill model is suitable for analysing the interactions within the tested concentration range of GBCAs.</p>
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