[Determination of tetracycline and fluoroquinolone residues in fish by polydopamine nanofiber mat based solid phase extraction combined with ultra performance liquid chromatography-tandem mass spectrometry]

Abstract

Tetracyclines and fluoroquinolones are common antibacterial drugs used in aquaculture, and their residues may pose a risk to human health. The low concentration of drug residues and complex matrixes such as fats and proteins in aquatic products necessitate the urgent development of efficient sample pretreatment methods. Solid phase extraction (SPE) is the most common sample pretreatment method, in which the core is an adsorbent. Compared with traditional SPE adsorbents, nanofiber mat (NFsM) has more interaction sites because of their large specific surface area. Furthermore, NFsMs modified with specific functional groups can significantly improve the extraction efficiency of tetracyclines and fluoroquinolones. Polydopamine (PDA) is spontaneously synthesized by the oxidative self-polymerization of dopamine-hydrochloride in alkaline solutions (pH>7.5). Because of its rich amino and catechol groups, PDA can form π-π stacking, electrostatic attraction, hydrophobic interaction, and hydrogen bonding interactions with target molecules. By exploiting the above advantages, polystyrene (PS) NFsM, as a template, was prepared by the electrostatic spinning method, and PDA-PS NFsM was obtained by functional modification of PDA through self-polymerization. Fourier transform infrared spectroscopy (FT-IR) and field-emission scanning electron microscopy (FESEM) were used to characterize the synthesized PS NFsM and PDA-PS NFsM. It was proved that PDA was successfully modified on the PS NFsM, with the SEM images revealing a rough outer core shell structure and an inner honeycomb structure. Subsequently, the handmade SPE column with PDA-PS NFsM was completed. A novel and efficient screening analytical method based on PDA-PS NFsM for the simultaneous determination of three tetracyclines (tetracycline (TET), chlortetracycline (CTC), and oxytetracycline (OTC)) and three fluoroquinolones (enrofloxacin (ENR), ciprofloxacin (CIP), and norfloxacin (NOR)) in fish by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established. The SPE procedure was optimized to develop an efficient method for sample preparation. Evaluate parameters including the amount of NFsM usage, ionic strength, flow rate of the sample solution, composition of eluent, and breakthrough volume were investigated. Only (20±0.1) mg of PDA-PS NFsM was sufficient to completely adsorb the targets, and the analytes retained on NFsM could be eluted by 1 mL of formic acid-ethyl acetate (containing 20% methanol) (1∶99, v/v). The residues were redissolved in 0.1 mL 10% methanol aqueous solution containing 0.2% formic acid. In addition, no adjustment of the pH and ionic strength of the sample solutions was required, and the breakthrough volume was 50 mL. The limits of detection (LODs) and limits of quantification (LOQs) of the six target compounds were measured at 3 times and 10 times the signal-to-noise ratio (S/N), respectively. The LODs and LOQs were 0.3-1.5 μg/kg and 1.0-5.0 μg/kg, respectively. The linear ranges of the six target compounds were LOQ-1000 μg/kg, and the coefficient of determination (R²) was greater than 0.999. To evaluate the accuracy and precision, blank spiked samples at three levels (low, medium, and high) were prepared for the recovery experiments, and each level with six parallel samples (n=6). The recoveries ranged from 94.37% to 102.82%, with intra-day and inter-day relative standard deviations of 2.38% to 8.06% and 4.10% to 9.10%, respectively. To evaluate the purification capacity of PDA-PS NFsM, the matrix effects before and after SPE were calculated and compared. Matrix effects before SPE were -12.98% to -38.68%. After the completion of SPEbased on PDA-PS NFsM, the matrix effect of each target analyte was significantly reduced to -2.15% to -7.36%, which proved the significant matrix removal capacity of PDA-PS NFsM. Finally, the practicality of this method was evaluated by using it to analyze real samples. This SPE method based on PDA-PS NFsM is efficient, practical, and environmentally friendly, and it has great potential for use in the routine monitoring of drug residues in fish.

Keywords: drug residues; fish; nanofiber mat (NFsM); polyaniline (PDA); solid phase extraction (SPE); ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).

图 1. 自制SPE小柱示意图

图 2. PS NFsM和PDA-PS NFsM的傅里叶红外光谱图

图 3. (a)PS NFsM和(b)PDA-PS NFsM的场发射扫描电镜图

图 4. (a)吸附剂的使用量、(b)离子强度、(c)样品溶液的流速和(d)洗脱液对6种目标化合物萃取效率的影响(n=4)

图 5. 突破体积对6种目标化合物萃取效率的影响(n=6)

图 6. 6种目标物的提取离子流色谱图