| With the improvement of people’s living standards and quality, food safety and environmental pollution problem were getting people’s attention. In recent years, the incidents of food safety and environmental pollution occured frequently because of drug residues, which have become the focus of attention in food and the environment. Fluoroquinolones, as a class of synthetic broad-spectrum antimicrobial drugs, were widely used for the prevention and treatment of bacterial infections in poultry and aquatic animals. Owing to abuse, irrational use, and non-compliance with the withdrawal period, the drug entered into the human body or the environment through the food chain, and thus caused harm and threats to human health and survival. Therefore, it was very necessary for the analysis of drug residues in food and the environment.Analysis of drug residues consisted of two parts, sample pretreatment and determination, sample pretreatment was a more critical step in drug residue analysis. So far, analysis of fluoroquinolone residues was confined to developing new detection techniques, and less focused on study of derivatization and sample pretreatment techniques. In view of trace fluoroquinolones in food and the environment, it was very important for development of derivatization and pretreatment techniques in these samples. In this thesis, fluoroquinolones were selected as the research objects. The application of derivatization including charge transfer reaction, complexation reaction, and inclusion was to sensitize fluorescence of analytes, sample pretreatment techniques (aqueous two-phase extraction, cloud point extration, and supramolecular solvent extraction) were applied for separation and enrichment of the analytes. Focusing on solving core technology of derivatization and sample pretreatment, coupled with analytical instruments, rapid, sensitive, eco-friendly and economical methods were established for analysis of trace fluoroquinolones in samples.The main research contents of this thesis were as follows:(1) Derivatization of four typical fluoroquinolones (FQs) namely norfloxacin (NOR), ofloxacin (OFL), ciprofloxacin (CIP), and gatifloxacin (GAT) with1,4-dihydroxyanthraquinone (1,4-DHAQ) was studied by fluorescence spectroscopy. Through fluorescence quenching of1,4-DHAQ and quantitative analysis by external standard method, FQs were analyzed by fluorescence spectrometer in water samples. Under the optimum conditions, there was a good linear relationship between the fluorescence intensities of four FQs and the concentration in the range of0.02～2.35μg/mL, the detection limits varied from0.015to0.020μg/mL. The average recoveries were95.9-103.5%with the relative standard deviations from0.7to2.5%by adding standards of known concentration in pharmaceutical preparations and environmental water samples for recovery experiment. The mechanism of fluorescence quenching was discussed, binding constants, binding sites, and free energy changes were determined for intermolecular derivatization, it was figured out that the main binding force was Van der Waals force between molecules.(2) Derivatization of four FQs (NOR, OFL, CIP, GAT) with2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) and fluorescence intensity of derivatives were studied by fluorescence spectroscopy. β-cyclodextrin (β-CD) was used for inclusion of derivatives, spectral property and fluorescence intensity of inclusion complex were investigated. A new method based on inclusion of FQs derivatives by β-CD coupled with fluorescence spectrometry for analysis of FQs in honey was established. Under the optimized conditions of derivatization and inclusion, a good linear relationship existed between the fluorescence intensities of four FQs and the concentration in the range of42.5-1340μg/kg, the limits of detection ranged from11.6to15.4μg/kg. By adding standards of known concentration in honey, the recoveries of four FQs between80.9%and92.8%were obtained, with the relative standard deviations in the range of1.6～4.0%. Derivatization mechanism of FQs with DDQ was studied, and inclusion ratio and inclusion constants were also determined.(3) Complexation reaction of FQs (NOR, OFL, CIP, GAT) with Al3+was studied by fluorescence spectroscopy, and salting-out assisted liquid-liquid ultrasonic extraction (SALLUE) was applied for extraction of FQs-Al3+complexes. The results showed that FQs and Al3+could form stable complexes with strong fluorescence in aqueous solution at pH4～5. Complexes were extracted into the organic phase through acetonitrile-ammonium sulfate aqueous two-phase system, and analyzed by spectrofluorometer. A new method based on Al+derivatization coupled with SALLUE, followed by fluorescence spectroscopy was established for analysis of FQs in milk. Under the optimized conditions of derivatization and extraction, there was a good linear relationship between the fluorescence intensities of four FQs and the concentration in the range of0.015～2.25μg/mL, the detection limits were in the range of0.009～0.016μg/mL. The average recoveries of four FQs between80.4%and96.8%were obtainded by adding standards of known concentration in milk, the relative standard deviations ranged from0.5to2.5%.(4) Vortex assisted acid-induced cloud point extraction (VAACPE) was studied for extraction of four FQs including NOR, CIP, SAR, and GAT in environmental water samples, the extracts were analyzed by spectrofluorometer. A new method based on VAACPE coupled with fluorescence spectroscopy was established for the determination of FQs in water samples. Under the optimum conditions, a good linear relationship was obtained between the fluorescence intensities of four FQs and the concentration in the range of0.045～0.90μg/mL, the limits of detection were in the range of0.007～0.013μg/mL. The average recoveries of four FQs ranged from83.0%to96.7%by adding standards of known concentration in water samples, and the relative standard deviations were in the ranged of0.9-3.7%.(5) Derivatization of four FQs (NOR, CIP, SAR, GAT) with4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD-C1) was studied, FQs derivatives were formed through pre-column derivatization, and extracted by ultrasound-assisted cloud point extraction (UACPE). A new method based on NBD-C1derivatization coupled with UACPE and HPLC analysis was established for the determination of FQs in eggs. The conditions of derivatization and extraction were optimized, under the optimum conditions, four FQs were completely separated through Agilent TC-C18(4.6×250mm,5μm) column whitin30min. A good linear relationship presented between the peak areas of four FQs and the concentration in the range of1.2-73.0μg/kg, the limits of detection ranged from0.2to0.5μg/kg. The recoveries of four FQs varied from86.2%to103.5%, with the relative standard deviations of0.6～4.3%by adding standards of known concentration in egg samples.(6) Supramolecular solvent extraction (SMSE) of four FQs namely pefloxacin (PEF), difloxacin (DIF), enrofloxacin (ENR), and danofloxacin (DAN) was studied, a new method was established based on nonanoic acid (NoA)-tetrabutylammonium hydroxide (TBAH) supramolecular solvent extraction followed by HPLC-FLD analysis. The SMSE conditions were optimized, under the optimum conditions, there was a good linear relationship between the peak areas of four FQs and the concentration in the ranged of10～1200μ/kg, the limits of detection were1.7～4.5μg/kg. The recoveries of four FQs, investigated by adding standards of known concentration in infant formula, were in the range of85.5～104.3%, with the relative standard deviations of0.9-3.9%. The mechanism of SMSE for FQs was investigated by1H NMR, the main driving force of extraction was intermolecular hydrogen bonding and hydrophobic interaction between NoA and FQs. |
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