Studies for monitoring the bioavailability of dietary flavonoid compounds generate great interest. Among them, low-molecular-weight phenolic acids, secondary metabolites present in colonic catabolism and urinary excretion, have been proposed as biomarkers of polyphenol intake. Using 4-hydroxyphenylacetic acid as a template, a molecularly imprinted polymer (MIP) was synthesized for selective extraction of these hydroxylated metabolites from human urine samples and posterior analysis in an HPLC-DAD-MS system. Polymers were characterized by Scanning electron microscopy (SEM), Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), Brunauer-Emmett-Teller (BET) method, and binding experiments. MIP presents specific recognition ability for template and analogues molecules. This capacity of recognition and the pH dependence of the binding strength was also studied. The method was validated over a concentration range of 0.25–40 mg/L, r2 > 0.995. In the optimized conditions, the recovery value was 94% with RSD 1.2%. The Limit of Detection (LOD) and Limit of Quantification (LOQ) were 1.22 and 3.69 mg/L, respectively. In our knowledge, it is the first time that this methodology is applied to analyze urinary catabolites of the polyphenol compound and to provide a specific method and simple analysis alternative. The selective extraction of these metabolites improves the application and results obtained by other less sensitive analysis methods than the validation method. It also facilitates the development of new screening methods.

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