PANI/PTA/Fe3O4 Nanocomposite Coated on Stainless‐Steel Towards Efficient Solid‐Phase Microextraction of Tetracycline From Human Urine Samples Prior to HPLC–DAD Analysis

ABSTRACT Tetracycline (TC) is a commonly prescribed broad‐spectrum antibiotic, and its widespread use together with its persistence in biological matrices, particularly urine, has raised serious concerns related to clinical safety, public health and the emergence of antimicrobial resistance, underscoring the need for its accurate and sensitive determination. In this work, an innovative solid‐phase microextraction (SPME) strategy was developed for the efficient extraction of TC from human urine samples using polyaniline/phosphotungstic acid/Fe3O4 (PANI/PTA/Fe3O4) nanocomposite coated on stainless‐steel substrate. Structural characterization of PANI/PTA/Fe3O4 using field‐emission scanning electron microscopy with energy‐dispersive, x‐ray diffraction (XRD) and Fourier‐transform infrared spectroscopy confirmed the successful incorporation of phosphotungstic acid (PTA) and Fe3O4 within the PANI framework. The resulting polymeric coating exhibited a rough and porous morphology, providing numerous active sites for efficient TC adsorption. Key extraction variables, including coating composition, film thickness, solution pH, ionic strength, extraction duration and desorption conditions, were systematically optimized. Under the optimized conditions, the PANI/PTA/Fe3O4 nanocomposite exhibited excellent analytical performance, with a limit of detection of 0.97 ng mL−1, a limit of quantification of 3.24 ng mL−1 and good linearity (R2 > 0.99) over a wide concentration range. The nanocomposite also exhibited strong reproducibility (intra‐day relative standard deviation [RSD] 2.6%–3.9%; inter‐day RSD 4.8%–5.0%) and high recoveries in spiked urine samples (99.5%–100.5%). The enhanced sorption performance was attributed to the combined effects of PANI (providing conductivity and π–π/π/hydrogen‐bonding interactions), PTA (contributing electrostatic and hydrogen‐bonding affinity) and Fe3O4 (imparting magnetic stability and surface activity). Overall, the PANI/PTA/Fe3O4 nanocomposite represents a robust and efficient SPME coating with strong potential for the determination of TC in complex biological matrices. The proposed SPME–high‐performance liquid chromatography (HPLC) methodology may be further adapted for the analysis of other structurally analogous antibiotics and pharmaceutical analytes in complex biological matrices by rational tailoring of the coating composition.