Backgroundβ-nicotinamide mononucleotide stands out as an essential breakthrough in “anti-aging” and consistently leads the list of top-selling nutritional supplements in terms of quantity. As the metabolites of β-nicotinamide mononucleotide, the detection of nicotinamide and N1-methylnicotinamide is of great significance for evaluating the nutritional effect of dietary supplements of β-nicotinamide mononucleotide. However, due to the extremely low concentration of nicotinamide and N1-methylnicotinamide in vivo and the serious matrix interference in biological samples, there is an increasing demand for materials and methods of pre-treatment. ResultsIn this study, Fe3O4@hydroxypropyl methyl cellulose@dodecylbenzenesulfonic acid magnetic fluid was synthesized for the first time, and it was used as pretreatment material to detect nicotinamide and N1- methylnicotinamide in urine samples by high performance liquid chromatography. Compared with other adsorption materials, Fe3O4@hydroxypropyl methyl cellulose@dodecylbenzenesulfonic acid nanoparticles are a kind of uniform magnetic fluid. Furthermore, they have more types and quantities of interaction sites (electrostatic interactions, hydrophobic interactions, hydrogen bonding interactions, and π-π interactions), so they own greater adsorption capacity. When the pH of the solution is 4, they can be adsorbed quickly within 10 s. The method successfully detected trace nicotinamide and N1-methylnicotinamide in urine samples in the linear range of 0.1–80 μg mL−1, the low detection limits were 0.30 ng mL−1 and 1.5 ng mL−1 respectively, and the quantification limits were 1.0 ng mL−1 and 5.0 ng mL−1, respectively. At the same time, the standard urine samples of nicotinamide and N1-methylnicotinamide showed satisfactory recovery rate 94.50–109.1 %. The results indicated that the established method can accurately and quantitatively determine trace nicotinamide and N1-methylnicotinamide in urine samples. SignificanceConsequently, low concentration of β-nicotinamide mononucleotide metabolites can be detected simultaneously, and the interference can be eliminated during the detection process, which provides an efficient and convenient pretreatment method and a rapid and sensitive detection method for the analysis of β-nicotinamide mononucleotide metabolites. What's more, it has a wide application prospect in the analysis of other similar metabolites in biological samples.