n the present work, hollow polymer nanospheres (HPSs) were prepared and applied as reusable adsorbent in solid-phase extraction (SPE) to extract and determine trans, trans- muconic acid (tt-MA) from urine samples as a potential biomarker of low-level exposure to benzene. HPSs with controllable size and functional shells were synthesized through weak acid-base assembly under hydrothermal conditions. Hollow nanospheres can act as a storage reservoir or a nanoreactor, the functional shell not only provides a significant surface area for reactions, but also easily chelates target compounds through the abundant functional groups present in it. The main factors affecting the extraction performance, including sample volume, sample flow rate, elution volume, sample pH, type of washing solvent, and type of elution solvent, were evaluated and optimized through coupling to a high-performance liquid chromatography (HPLC-UV) analyzer. The highest recovery rate of tt-MA from urine with HPSs adsorbent was obtained at pH 7, sample volume 2 ml, sample flow rate 1 ml/min, deionized water/methanol (9,1 v/v) as washing solvent, and ethanol/acetic acid 3 % (8,2 v/v) as elution solvent. The optimized method was validated using three concentrations of 0.1, 25, and 50 μg/ml, and its within-day and day-to-day reproducibility was confirmed (RSD < 7.7 %). A good linear range (r 2 > 0.99) and low limits of detection (0.05 μg/ml) were obtained as well. The recovery of tt-MA from spiked urine samples in the quality control (QC) concentrations was more than 96 %, demonstrating the HPS-SPE method's applicability in the analysis of urine samples of benzene-exposed people.
