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 (R ² > 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.

Short abstract

An innovative SPME strategy was developed for the efficient extraction of Tetracycline using polyaniline/phosphotungstic acid/Fe₃O₄ coated on stainless‐steel substrate.

Abbreviations

AMR

antimicrobial resistance

CV

cyclic voltammetry

EDS

energy‐dispersive spectroscopy

FE‐SEM

field‐emission scanning electron microscopy

FTIR

Fourier‐transform infrared spectroscopy

HPLC–DAD

high‐performance liquid chromatography with a diode array detector

LLE

liquid–liquid extraction

PANI

polyaniline

PTA

phosphotungstic acid

RR

relative recovery

RSD

relative standard deviation

SPE

solid‐phase extraction

SPME

solid‐phase micro extraction

TC

tetracycline

XRD

x‐ray diffraction

1. Introduction

Tetracyclines (TCs) are broad‐spectrum antibiotics extensively administered in both human and veterinary medicine. Their widespread use has led to frequent detection of TC residues in biological and environmental matrices, including human urine. These residues not only pose direct health concerns but also exacerbate the global threat of antimicrobial resistance (AMR), a crisis linked to nearly 5 million deaths in 2019 and expected to escalate in the future [1, 2].

Additionally, the adverse effects of antibiotic residues on immunocompromised individuals and emerging evidence of the role of bacterial infections in tumour progression highlight the pressing need for sensitive and reliable monitoring of TC residues in biological systems [3]. Structurally, TC consists of a linear, fused tetracyclic framework, decorated with multiple functional groups that determine its physicochemical properties and affinity for sorbent materials. Key features include a protonatable dimethylamino group at C‐4, hydroxyl substituents at positions C‐6, C‐10 and C‐12 that serve as hydrogen‐bond donors, and a β‐diketone moiety spanning C‐11 and C‐12a, which functions as both a strong hydrogen‐bond acceptor and a metal‐chelating site (Figure S1). These functional groups are critical in extraction processes, as they enable electrostatic interactions, hydrogen bonding and π–π stacking with functionalized sorbents [4, 5].

Liquid–liquid extraction (LLE) is a conventional sample preparation method in which analytes are distributed between two immiscible liquid phases. However, it typically requires large amounts of hazardous organic solvents and involves labour‐intensive handling steps. Solid‐phase extraction (SPE) offers a significant advancement by using a solid sorbent to selectively retain analytes, thereby improving selectivity, lowering solvent use and facilitating automation [6, 7]. Solid‐phase microextraction (SPME) represents a further miniaturization, combining sampling, extraction and preconcentration into a single, solvent‐free step. Compared with LLE and SPE, SPME is faster, more environmentally friendly and highly effective for trace analysis in complex matrices. However, its performance depends strongly on the properties of the sorbent coating. SPME has emerged as a robust sample preparation approach for trace‐level analysis due to its simplicity, minimal solvent consumption and compatibility with chromatographic techniques. The efficiency of SPME nanocomposites largely depends on the properties of the sorbent coating, which must ensure high selectivity, stability and adsorption capacity [8]. Conducting polymers, such as polyaniline (PANI), are particularly attractive as sorbent materials owing to their facile synthesis, environmental stability, low cost and diverse chemical functionalities. The conjugated backbone of PANI supports multiple interactions, including π–π stacking, hydrogen bonding and electrostatic forces, making it a suitable candidate for the enrichment of antibiotics and other polar pharmaceuticals [9].

To further improve the performance of PANI‐based sorbents, functional nanomaterials have been incorporated into the polymer matrix. Phosphotungstic acid (PTA), a heteropolyacid with a Keggin‐type structure, offers abundant negatively charged oxygen sites that enhance electrostatic interactions and hydrogen bonding with TC molecules [10, 11]. Magnetic iron oxide nanoparticles (Fe₃O₄ NPs) contribute additional surface area, stability and tunable surface chemistry, but when used alone, they suffer from aggregation and limited selectivity. Embedding Fe₃O₄ NPs and PTA within a PANI framework creates a synergistic system that overcomes these drawbacks and provides a robust sorbent with improved adsorption performance [12, 13, 14].

This research work presents the fabrication of a novel nanocomposite (PANI/PTA/Fe₃O₄) coated on stainless‐steel through electropolymerization technique. This method allows uniform coating formation, strong adhesion and precise control over film thickness and composition [15, 16]. The prepared nanocomposite was thoroughly characterized and used for direct‐immersion SPME to extract TC from human urine samples (IR.TBZMED.VCR.REC.1404.350), followed by high‐performance liquid chromatography with a diode array detector (HPLC–DAD) analysis (Figure 1). The combined functionalities of PANI, PTA and Fe₃O₄ significantly enhanced extraction efficiency, reproducibility and recovery. The results demonstrate a promising strategy for developing advanced nanocomposite coatings for SPME, contributing to more reliable monitoring of antibiotic residues in biological samples.

FIGURE 1.

A schematic overview of the electrochemical coating process, SPME and chromatographic analysis. PTA; phosphotungstic acid; SPME, solid‐phase micro extraction; TC, tetracycline.

2. Experimental Procedures

2.1. Materials

TC (96.5%), Fe₃O₄ nanoparticles, PTA (H₃[PW₁₂O₄₀]), aniline, nitric acid (65%), hydrochloric acid (37%), methanol, acetonitrile, sodium hydroxide, dimethylformamide, ammonium oxalate and dibasic ammonium phosphate were purchased from Merck (Germany). Stainless‐steel wires (0.25 mm in diameter, 4 cm in length) were used as substrates. All aqueous solutions were prepared with ultrapure water (18.2 MΩ cm, Milli‐Q, Millipore).

2.2. Instrumentation

HPLC was performed on an Agilent 1100 series system equipped with a DAD (G1315A). Separation was achieved using a ZORBAX C8 analytical column (250 × 4.6 mm², 5 µm) maintained at ambient temperature. The mobile phase consisted of dimethylformamide, 0.1 M ammonium oxalate and 0.2 M dibasic ammonium phosphate (27:68:5, v/v/v) at pH 7.6–7.7, delivered at a flow rate of 2.0 mL min⁻¹. The detection wavelength was set to 280 nm [17]. Fourier‐transform infrared (FTIR) spectra were obtained using a Bruker Tensor 27 spectrometer (Germany) to characterize functional groups of the synthesized sorbents. The surface morphology of the nanocomposites was examined by field‐emission scanning electron microscopy (FE‐SEM, TESCAN MIRA 3 LMU, Czech Republic), coupled with energy‐dispersive x‐ray spectroscopy (EDS) for elemental mapping. An ultrasonic bath (Elma Elmasonic P60H, Germany) was used to disperse the precursors.

2.3. Synthesis of the PANI/PTA/Fe₃O₄

The nanocomposite coating was fabricated via electropolymerization directly onto stainless‐steel wires. In brief, 25 mL of an electrolyte solution composed of 1.0 M HNO₃, 0.1 M aniline, 70 mg PTA and 100 mg of Fe₃O₄ NPs was sonicated for 5 min to achieve a homogeneous dispersion stainless‐steel wire (4 cm in length) as both anode and cathode. Anod wire pretreated sequentially with acetone, ethanol and ultrapure water was used as the electrodes, positioned 1 cm apart [18]. A direct current potential of 1.0 V was applied for 15 min, resulting in the deposition of a PANI/PTA/Fe₃O₄ composite film on the anode through aniline electropolymerization, during which Fe₃O₄ NPs and PTA were incorporated into the PANI network. The coating thickness was controlled by adjusting the electropolymerization time, whereas the film uniformity was improved by applying multiple deposition cycles [19, 20].

2.4. Standard Solutions

A stock solution of candidate drug (TC) (1000 mg L⁻¹) was prepared by dissolving 100 mg of TC in 100 mL of methanol under ultrasonic treatment. Working standard solutions were obtained by serial dilution with ultrapure water. Fresh calibration standards were prepared daily.

2.5. Sample Preparation

Human urine samples were sourced from Omega Pathobiology Laboratory (Tehran, Iran). All collections adhered to the ethical guidelines of the Ethics Committee of Amir Kabir University, with written informed consent obtained from each participant. Samples were anonymized, filtered through 0.22 µm membrane filters and stored at −20°C until analysis. Before extraction, the samples were diluted fourfold with ultrapure water [21].

2.6. Direct‐Immersion SPME Procedure

For extraction, 10 mL of sample solution was spiked with TC at concentrations of 50 and 100 µg L⁻¹ in SPME vials, followed by the addition of a 5 mm magnetic stirring bar. The pH was adjusted to 5.0 with a 1.0 M NaOH standard solution, and NaCl (20% w/v) was added to promote salting‐out. The prepared PANI/PTA/Fe₃O₄ nanocomposite, fixed in a custom needle‐type holder, was immersed in the stirred sample (800 rpm) for 25 min at ambient temperature. Following extraction, the nanocomposite was placed in an Eppendorf tube containing 2 mL of methanol/formic acid (0.1% v/v) and stirred for 10 min to achieve analyte desorption. All experiments were conducted in triplicate, and method precision was assessed as relative standard deviation (RSD%).

2.7. Method Performance

The relative recovery (RR%) was calculated using the following equation:

$$\mathrm{RR}\%=\frac{C_{\mathrm{found}}-C_{\mathrm{real}}}{C_{\mathrm{added}}}\times 100$$

where C _found is the measured concentration after spiking, C _real is the initial concentration in the sample, and C _added is the amount of TC spiked.

3. Results and Discussion

A key factor influencing SPME extraction efficiency is the type and composition of the sorbent coating. In this study, various factors, including the composition and thickness of the absorbent, extraction time, pH and stirring speed, were examined to optimize the extraction process. The developed method was then used to separate and quantify TC in real samples.

3.1. Optimization of Sorbent Composition

The extraction efficiency of SPME nanocomposites is primarily dictated by the chemical composition of the coating [22, 23, 24]. Nanocomposites coated solely with PANI showed poor recovery of TC, emphasizing the necessity for compositional modification [25]. As shown in Table 1, TCs recovery increased with rising PTA content up to 70 mg (S3), corresponding to the optimal PTA/Fe₃O₄ ratio. In this composition, PTA contributed abundant negatively charged oxygen atoms, which facilitated electrostatic attraction and hydrogen bonding with TC, whereas Fe₃O₄ nanoparticles provided additional active sites and enhanced film stability [26, 27]. When the PTA concentration exceeded this level (S4–S5), over‐protonation of the PANI backbone and reduced Fe₃O₄ incorporation likely occurred, leading to decreased porosity and restricted analyte diffusion. Conversely, at lower PTA amounts (S1–S2), the limited number of functional binding sites reduced adsorption efficiency. Overall, these findings highlight the importance of maintaining a balanced composition of PANI, PTA and Fe₃O₄ to maximize analyte‐sorbent interactions and ensure effective extraction.

TABLE 1.

Analytical figures of merit of the developed DI‐SPME/HPLC–DAD method for TC extraction.

Target analyte	Spiked level (µg L−1)	LOD (ng mL−1)	LOQ (ng mL−1)	R 2	Intra‐day RSD (%)	Inter‐day RSD (%)
TC	50	0.97	3.24	0.9994	3.4	5.0
TC	100	—	—	—	3.1	4.1

Abbreviations: HPLC–DAD, high‐performance liquid chromatography with a diode array detector; RSD, relative standard deviation; SPME, solid‐phase micro extraction; TC, tetracycline.

Different PTA/Fe₃O₄ ratios were evaluated while keeping the amount of aniline constant (235 mg). Among the tested formulations, the nanocomposite prepared with 70 mg PTA and 100 mg Fe₃O₄ (S3) showed the highest TC extraction recovery, whereas both lower and higher PTA loadings resulted in reduced efficiency (Table S1).

3.2. Applied Voltage Effect

The electro‐polymerization voltage was found to be a key factor in governing the formation and characteristics of the PANI/PTA/Fe₃O₄ composite coating. Voltages between 0.5 and 1.2 V were examined. At lower potentials (≤0.7 V), aniline polymerization remained incomplete, producing thin, uneven coatings with limited incorporation of Fe₃O₄ and PTA. In contrast, higher voltages (≥1.1 V) led to over‐oxidation, hindering nanoparticle incorporation, reducing porosity and limiting analyte accessibility. An intermediate potential of 1.0 V yielded the best outcome, resulting in a uniform, porous and stable film with balanced incorporation of Fe₃O₄ and PTA, thereby enhancing both structural integrity and sorption efficiency. These findings demonstrate that precise control of deposition voltage is essential for producing reproducible, high‐performance SPME coatings.

3.3. Stirring Speed Effect

By inducing fluid motion, stirring enhances mass transport of ions and charged molecules to the electrode surface, preventing concentration gradients and ensuring a uniform deposition process. Optimal stirring speed is essential for achieving the desired coating thickness, microstructure and adhesion. Insufficient stirring can lead to non‐uniform coatings, whereas excessive agitation may cause turbulence and undesirable effects. Precise control of stirring speed is therefore crucial for achieving consistent and high‐quality coating materials [28]. The study examined stirring rates between 500 and 1400 rpm and ultimately determined that 800 rpm optimized reaction kinetics and mass transfer. Higher stirring speeds led to instability as the magnetic stirrer collided with the vial, creating excessive turbulence and bubbles. Consequently, 800 rpm was maintained throughout the experiment.

3.4. Characterization of the PANI/PTA/Fe₃O₄ SPME Sorbent

3.4.1. Morphology and Thickness

FE‐SEM analysis (Figure 2a–e) demonstrated apparent morphological differences between the prepared coatings. Nanocomposites coated with pure PANI exhibited relatively smooth surfaces with low porosity, whereas the incorporation of PTA and Fe₃O₄ resulted in markedly rougher, more porous textures. This structural modification introduced numerous active sites and substantially increased the effective surface area for analyte adsorption. Elemental mapping further confirmed the homogeneous distribution of Fe, C, N, O, W and P throughout the coating, verifying the successful integration of PTA and Fe₃O₄ within the PANI matrix.

FIGURE 2.

FE‐SEM images of (a) PANI, (b) PANI/PTA, (c) PANI/PTA/Fe₃O₄, (d) SEM‐EDS elemental analysis of PANI/PTA/Fe₃O₄, (e) elemental EDX‐mapping of PANI/PTA/Fe₃O₄ and (f) cross‐sectional FE‐SEM image of PANI/PTA/Fe₃O₄ nanocomposite.

Cross‐sectional SEM images (Figure 2f) revealed an average film thickness of approximately 94 µm. Although thicker coatings generally provide more binding sites, they may also slow equilibration and limit mass transfer. The optimized thickness achieved in this work offered a suitable balance between adsorption capacity and extraction kinetics, thereby ensuring both high efficiency and reproducibility.

3.4.2. FTIR Spectra

FTIR spectra (Figure S2) confirmed the successful fabrication of the nanocomposite. Distinctive PANI bands were identified at 3272 cm⁻¹ (N–H stretching), 2927 cm^{− 1} (C–H stretching), 1457 cm⁻¹ (C═C stretching) and 1505 cm⁻¹ (C═N stretching) [29, 30, 31]. The absorption band at 1290 cm⁻¹ is indicative of the protonated conducting state of PANI and is attributed to the C–N⁺ stretching vibration associated with the polaronic structure [32, 33, 34, 35]. Additional absorption features in the 900–1100 cm⁻¹ range, attributed to P═O and W═O vibrations, further validated the incorporation of PTA into the polymer framework [36, 37]. In the ternary PANI/PTA/Fe₃O₄ composite, new bands appeared at 450–580 cm⁻¹, corresponding to Fe–O stretching, confirming the successful integration of Fe₃O₄ nanoparticles [38].

3.4.3. X‐Ray Diffraction (XRD) Studies

The crystalline properties of PANI, PTA, Fe₃O₄ and their composites were investigated by XRD. Pristine PANI displayed a broad diffraction halo centred at 2θ = 20°–30°, typical of its amorphous nature [39]. Pure PTA, in contrast, exhibited sharp reflections at 2θ ≈ 10.5°, 20.0°, 25.4°, 29.3°, 34.7°, 42.0°, 50.6° and 55.1°, corresponding to its highly crystalline Keggin‐type structure [40]. Distinct peaks for Fe₃O₄ nanoparticles appeared at 2θ ≈ 30.2° (2 2 0), 35.6° (3 1 1), 43.3° (4 0 0), 53.5° (4 2 2), 57.0° (5 1 1) and 62.6° (4 4 0), in agreement with the cubic spinel structure of magnetite [41, 42].

In the binary PANI/PTA composite, characteristic PTA reflections were still present but reduced in intensity, suggesting partial incorporation of PTA into the PANI framework [40]. The ternary PANI/PTA/Fe₃O₄ material exhibited peaks corresponding to both PTA and Fe₃O₄, although with attenuated intensities compared to the pure components. This weakening and broadening of signals indicates uniform dispersion of PTA and Fe₃O₄ within the amorphous PANI matrix rather than phase segregation [38]. The coexistence of Fe₃O₄ reflections (3 1 1, 4 0 0, 5 1 1), PTA peaks and the PANI halo provides clear evidence of the successful integration of all three constituents into a single composite.

Overall, the XRD results confirm that the PANI/PTA/Fe₃O₄ nanocomposite preserved the crystalline signatures of PTA and Fe₃O₄ while embedding them within the amorphous PANI phase, yielding a structurally robust hybrid sorbent well suited for SPME applications (Figure S3).

3.4.4. Cyclic Voltammetry (CV) Examination

CV was used to assess the electrochemical behaviour of the synthesized coatings with potassium ferricyanide/potassium chloride (1:20) as the redox probe. As shown in Figure S4, the bare PANI electrode (Curve c) displayed the expected oxidation and reduction peaks corresponding to the leucoemeraldine emeraldine transition, though with relatively low current density. Incorporation of PTA into the polymer matrix (Curve b) enhanced the redox response, attributed to the acidic sites and proton‐donating properties of the Keggin‐type heteropolyacid, which facilitated more efficient electron transfer [43]. The ternary PANI/PTA/Fe₃O₄ composite (Curve a) exhibited the highest current density and the most pronounced redox peaks, highlighting the synergistic contribution of Fe₃O₄ nanoparticles in providing electroactive sites, improving conductivity and accelerating charge transfer across the polymer–electrolyte interface [44].

The marked increase in both anodic and cathodic peak currents for the PANI/PTA/Fe₃O₄ electrode confirms that the combined incorporation of Fe₃O₄ and PTA within the PANI backbone not only enhances charge mobility but also stabilizes the redox transitions of the polymer. These results are consistent with previous studies on PANI‐based composites, where the integration of metal oxides or heteropolyacids improved electrochemical performance and durability [45]. Overall, the superior electrochemical activity of the PANI/PTA/Fe₃O₄ nanocomposite underscores its potential as a stable and efficient sorbent coating, directly contributing to improved porosity, robustness and extraction efficiency of the fabricated SPME nanocomposite (Figure S4).

3.4.5. Composite Formation and Adsorption Mechanism

The PANI/PTA/Fe₃O₄ nanocomposite was formed through the electropolymerization of aniline in the presence of PTA and Fe₃O₄ nanoparticles. PTA functions both as a protonating dopant and as a polyoxometalate species, establishing strong ion–pair interactions with the imine group of PANI. This situation not only stabilizes the conductive emeraldine salt form but also introduces numerous negatively charged oxygen sites [46, 47]. Fe₃O₄ nanoparticles, uniformly embedded within the PANI network, reinforce the polymer structure, prevent collapse and expand the available surface area [48]. The resulting architecture provides a synergistic platform: PANI contributes a conjugated backbone for π–π stacking and hydrogen bonding, PTA supplies electrostatic and hydrogen‐bonding affinity, and Fe₃O₄ enhances mechanical stability and adsorption capacity [49].

Several complementary interactions drive TC adsorption onto the nanocomposite. At pH 5, TC exists primarily in its zwitterionic state, allowing the protonated dimethylamino group to interact electrostatically with the negatively charged oxygen atoms of PTA. In contrast, phenolic and β‐diketone groups engage in hydrogen bonding with functional sites on PANI and PTA. Simultaneously, the aromatic rings of TC undergo π–π stacking with the conjugated PANI backbone. The β‐diketone moiety (C‐11/C‐12a) also serves as a strong hydrogen‐bond acceptor for the –NH groups of PANI, further stabilizing the analyte sorbent complex [46, 47, 48]. Collectively, these cooperative mechanisms account for the excellent extraction efficiency, reproducibility and recovery achieved by the developed nanocomposite.

Considering the structural characteristics of TC together with the functional groups in the PANI/PTA/Fe₃O₄ coating, the adsorption mechanism can be explained by a combination of interactions, as depicted in Figure 3.

FIGURE 3.

Illustration of TC adsorption onto PANI/PTA/Fe₃O₄ at pH 5, showing π–π stacking, hydrogen bonding and electrostatic interactions, with Fe₃O₄ enhancing surface area and stability.

3.5. Extraction of TC

Optimization of extraction parameters was carried out to enhance the performance of the PANI/PTA/Fe₃O₄ nanocomposite. Key factors, including pH, extraction time, sorbent loading, temperature and ionic strength, were systematically evaluated to achieve consistent and efficient TC recovery.

3.5.1. Effect of pH

Systematic investigation of pH from 4.0 to 8.0 revealed that extraction efficiency was maximized at pH 5.0. This behaviour is consistent with the acid–base speciation of TC, which exhibits multiple dissociation constants (pK _a ≈ 3.3, 7.7 and 9.7) that determine its ionic distribution in aqueous solution [1]. At this pH, TC is mainly present in its zwitterionic state, retaining a protonated dimethylamino group, whereas the phenolic/β‐diketone moieties are deprotonated. Under such conditions, the PANI/PTA/Fe₃O₄ coating supports complementary binding mechanisms: electrostatic attraction between cationic TC sites and negatively charged PTA clusters [43]; hydrogen bonding between TC functional groups (–OH/–NH) and oxygen atoms of PTA or nitrogen atoms of PANI [50]; and π–π stacking with the conjugated PANI backbone [50]. At lower pH values (≤4.0), overprotonation of both TC and the PANI matrix increases competition with H⁺ ions and weakens π–π interactions, thereby lowering extraction efficiency. At higher pH (≥6.0), the reduced cationic nature of TC diminishes electrostatic interactions and may promote partial degradation or epimerization [51], thereby decreasing uptake. Therefore, pH 5.0 (10–20 mM acetate buffer with 20% w/v NaCl) was selected as the optimum condition, providing the best balance of sorbent–analyte interactions, analyte stability and signal reproducibility.

3.5.2. Extraction Time

Extraction time plays a pivotal role in establishing equilibrium between analyte and sorbent. Although extending the duration can improve analyte uptake, it also prolongs analysis and may lead to desorption or matrix interferences. Thus, careful optimization is necessary to balance sensitivity, reproducibility and analytical efficiency [52]. Experiments conducted over 5–45 min showed a steady increase in TC adsorption up to 15 min, after which uptake plateaued, indicating equilibrium had been established. Insufficient extraction was observed at shorter times (<10 min), whereas extending the duration beyond 20 min did not improve recovery and could even promote nanocomposite fouling or analyte back‐diffusion. Accordingly, 15 min was chosen as the optimal extraction time, providing efficient mass transfer and reproducible adsorption while avoiding unnecessary prolongation of the analytical procedure.

3.5.3. Temperature

Temperature is an important parameter affecting both the kinetics and thermodynamics of extraction. Although elevated temperatures can enhance analyte diffusion toward the sorbent, they may also lower distribution coefficients or promote analyte degradation. For TC analysis, tests conducted at sub‐ambient, ambient, and moderately elevated conditions revealed that room temperature (25°C) provided the most favourable outcome. At temperatures above 35°C, partial degradation occurred, resulting in reduced recoveries, whereas cooling decreased diffusion rates and lowered extraction efficiency. Therefore, ambient temperature was selected for subsequent experiments, as it ensured analyte stability along with efficient adsorption kinetics.

3.5.4. Salting out Effect

The salting‐out effect is a crucial factor that can significantly enhance SPME efficiency [53, 54, 55]. In SPME, adding salt to the sample matrix reduces the solubility of target analytes. A PANI/PTA/Fe₃O₄ nanocomposite was exposed to solutions containing different levels of salt (NaCl) and TC. The study found that adding 20% salt to the solution significantly increased the amount of TC absorbed by the nanocomposite. As a result, subsequent experiments used a 20% salt concentration.

3.5.5. Desorption Time

The effect of desorption time was evaluated under magnetic stirring using the selected solvent. The peak area increased sharply within the first few minutes, then levelled off, indicating complete release of the analyte from the nanocomposite. Extending desorption beyond this equilibrium point provided no additional benefit and only lengthened the workflow. This trend aligns with desorption kinetics, in which elution becomes diffusion‐limited once the readily accessible analyte fraction has been removed, rendering longer times ineffective for improving signal [56]. Therefore, a desorption period of 10 min was selected as the optimal condition, ensuring quantitative recovery of TC from the PANI/PTA/Fe₃O₄ coating with minimal carry‐over in subsequent blanks.

3.5.6. Desorption Solvent

Efficient desorption requires a solvent system capable of disrupting the π–π, hydrogen‐bonding and electrostatic interactions between TC and the PANI/PTA/Fe₃O₄ coating. Various eluents were tested, including protic and aprotic solvents (methanol and acetonitrile), aqueous mixtures and acidified formulations. Acidified methanol consistently produced the highest peak areas and cleanest baselines, whereas acetonitrile and aqueous mixtures gave weaker signals and broader peaks. Methanol with a small amount of formic acid (≈0.5%–1% v/v) was therefore chosen for routine use. In this medium, protonation of TC's dimethylamino group and of PANI's basic sites reduces electrostatic binding with PTA. It disrupts hydrogen bonding, enabling rapid analyte release while maintaining compatibility with HPLC–DAD analysis. Increasing the acid concentration above 2% v/v did not further improve recovery and risked coating leaching or structural changes; thus, methanol + 1% (v/v) formic acid was selected as the optimal desorption solvent.

The effect of different experimental parameters on extraction efficiency of TC is summarized in Figure 4a–g.

FIGURE 4.

Optimization of extraction and desorption parameters for TC using PANI/PTA/Fe₃O₄ nanocomposites, including sorbent type (a), pH (b), ionic strength (c), extraction time (d), stirring speed (e), desorption time (f) and solvent choice (g). PANI, polyaniline; PTA, phosphotungstic acid.

3.6. Method Validation

3.6.1. Chromatographic Analysis

Figure S5 shows representative HPLC–DAD chromatograms obtained under optimized extraction conditions. A sharp, symmetric TC peak was consistently observed at ∼8.0 min in both standard solutions (50 and 100 µg L⁻¹) and spiked urine samples, confirming the stability and reproducibility of the separation. Blank urine samples showed no interfering peaks, demonstrating the method's high selectivity for TC in complex biological matrices. The stable baseline and well‐resolved peaks further highlight the robustness of the chromatographic conditions. Calibration curves were constructed using standard solutions at concentrations of 10, 50, 100, 500 and 1000 µg L⁻¹.

According to obtained results, a sharp, well‐resolved TC peak was observed in both the standard and the spiked sample. At the same time, the blank urine showed no endogenous interference, confirming the method's selectivity and suitability for TC determination in biological matrices.

The chromatographic profiles obtained under optimized DI‐SPME/HPLC–DAD conditions are shown in Figure S5. In the blank urine sample (Figure S5a), no signals appeared at the TC retention time, confirming the absence of endogenous compounds that could affect selectivity. The standard TC solution (50 µg L⁻¹) (Figure S5b) produced a sharp, symmetric peak at ∼8.0 min, establishing the reference retention time. Spiked urine samples at 50 and 100 µg L⁻¹ (Figure S5c,d) yielded well‐defined peaks at the same retention time, with peak intensities proportional to concentration. These findings confirm the method's accuracy, reproducibility and linearity in complex biological matrices. Overall, the chromatograms demonstrate that the PANI/PTA/Fe₃O₄ nanocomposite enables selective and sensitive extraction of TC from a real urine sample, with minimal matrix interference.

The analytical performance characteristics of the developed DI‐SPME/HPLC–DAD method, including sensitivity, precision, linearity and recovery, are summarized in Table 1. LOD and LOQ were calculated on the basis of the signal‐to‐noise ratios of 3 and 10, respectively.

To further evaluate the applicability of the developed method, the DI‐SPME/HPLC–DAD procedure was applied to real urine samples spiked with TC at two concentration levels. The results, presented in Table 2, confirm the method's accuracy and reproducibility in complex biological matrices.

TABLE 2.

Determination of TC in real urine samples using the developed DI‐SPME/HPLC–DAD method.

Sample	Target analyte	Added concentration (µg L−1)	Found concentration (µg L−1)	RSD% (n = 3)	Recovery (%)
Urine	TC	50	49.7	2.6	99.5
Urine	TC	100	99.5	3.0	100.5

Abbreviations: HPLC–DAD, high‐performance liquid chromatography with a diode array detector; RSD, relative standard deviation; SPME, solid‐phase micro extraction; TC, tetracycline.

In real urine analysis, no TC was detected in the unspiked samples, confirming the absence of endogenous TC residues. Spiked urine samples at 50 and 100 µg L⁻¹ produced well‐defined peaks at ∼8.0 min, identical to those of the standard solution, with no evidence of matrix interference. The relative recoveries (99.5%–100.5%) confirmed the reliability, accuracy and practical applicability of the DI‐SPME/HPLC–DAD method for TC determination in complex biological matrices.

3.6.2. Comparison With Prior Methods

Table S2 presents representative SPME/MSPE‐based methods developed for TC determination in various matrices [57, 58, 59, 60, 61, 62, 63, 64]. Many reported strategies employ molecularly imprinted polymers (MIPs), ionic liquids or advanced nanostructured sorbents, such as zeolites, MOFs and graphene oxide, to enhance selectivity and sensitivity. Although some approaches (e.g., MIP–SPE–HPLC–DAD and MSPE–LC–MS) achieved remarkable detection limits as low as 0.01 ng mL⁻¹, they are often hindered by labour‐intensive preparation, high costs and limited reusability. Simpler sorbents, such as conventional MIPs or hydrophilic–lipophilic balanced (HLB) coatings, have shown moderate recoveries (70%–90%) but generally exhibit poorer reproducibility.

In comparison, the DI‐SPME/HPLC–DAD method developed with PANI/PTA/Fe₃O₄ nanocomposite delivered competitive sensitivity (LOD 0.97 ng mL⁻¹), excellent recoveries (99.5%–100.5%) and robust reproducibility (RSD ≤ 5.0%). These results equal or surpass those of most advanced sorbent systems, whereas the electro‐polymerization process provides a straightforward, reproducible and controllable approach for nanocomposite fabrication. Unlike MOF‐ or IL‐based strategies that require complex synthetic steps, this method combines high analytical performance with operational simplicity, making it attractive for routine applications.

The enhanced performance is attributed to the synergistic roles of PANI (conductivity and π–π and hydrogen‐bonding interactions), PTA (electrostatic and hydrogen‐bonding affinity) and Fe₃O₄ (surface area and structural stability). Collectively, these components produce a durable, efficient sorbent that reliably extracts TC from complex biological matrices. Overall, the PANI/PTA/Fe₃O₄ nanocomposite emerges as a practical, reproducible and high‐performance alternative for the monitoring of antibiotic residues in real biological samples.

4. Conclusion

In summary, a novel PANI/PTA/Fe₃O₄ nanocomposite coating was fabricated on stainless‐steel nanocomposites via electro‐polymerization and applied for direct‐immersion SPME of TC in human urine. Structural and compositional analyses (SEM/EDS, FTIR, XRD and CV) confirmed the successful incorporation of PTA and Fe₃O₄ into the PANI framework, yielding a stable and porous coating. The optimized nanocomposite demonstrated strong analytical performance, with limits of detection of 0.97 ng mL⁻¹, limits of quantitation of 1.60–3.24 ng mL⁻¹, recoveries of 99.5%–100.5% and RSDs of <5.0%. These results highlight superior sensitivity, accuracy and reliability compared with many existing SPME coatings reported for TC determination. The enhanced efficiency of the PANI/PTA/Fe₃O₄ nanocomposite is attributed to the complementary functions of its components: PANI provided π–π and hydrogen‐bonding interactions, PTA contributed abundant electrostatic and hydrogen‐bonding sites, and Fe₃O₄ nanoparticles improved surface area and mechanical stability. This synergy enabled rapid equilibration, strong affinity toward TC and excellent reproducibility. Taken together, the developed nanocomposite offers a simple yet effective alternative to more complex sorbent systems, particularly for bioanalytical applications where matrix interferences are challenging. The straightforward electro‐polymerization strategy also provides a versatile platform that can be adapted for other pharmaceuticals and environmental pollutants. Future studies may explore its use for multi‐analyte monitoring and integration with advanced chromatographic or mass spectrometric techniques to expand its applicability in pharmaceutical and clinical analysis.

Author Contributions

Hossein Salar Amoli: writing – review and editing, supervision, project administration, conceptualization. Sanaz Naghinejad Orang: writing – original draft, methodology, validation. Mohammad Hasanzadeh: writing – review and editing, visualization, supervision, methodology.

Ethics Statement

This study is ethically approved by Tabriz University of Medical science.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File: ansa70086‐sup‐0001‐SuppMat.docx.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.