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Magnetic nanomaterials find increasing application as separation agents to rapidly isolate target compounds from complex biological media (i.e., blood purification). The responsiveness of the used materials to external magnetic fields (i.e., their saturation magnetization) is one of the most critical parameters for a fast and thorough separation. In the present study, magnetite (Fe3O4) and non-oxidic cementite (Fe3C) based carbon-coated nanomagnets are characterized in detail and compared regarding their separation behavior from human whole blood. A quantification approach for iron-based nanomaterials in biological samples with strong matrix effects (here, salts in blood) based on platinum spiking is shown. Both materials are functionalized with polyethyleneglycol (PEG) to improve cytocompatibility (confirmed by cell toxicity tests) and dispersability. The separation performance is tested in two setups, namely under stationary and different flow-conditions using fresh human blood. The results reveal a superior separation behavior of the cementite based nanomagnets and strongly suggest the use of nanomaterials with high saturation magnetizations for magnetic retention under common blood flow conditions such as in veins. [ABSTRACT FROM AUTHOR]