Association between fluoroquinolone exposure and children's growth and development: A multisite biomonitoring-based study in northern China.

BACKGROUND: Although animal experiments found that antibiotic exposure during early life increased adiposity, limited human epidemiological evidence is available for the effects of veterinary antibiotic exposure on children's growth and development. OBJECTIVE: This study was conducted to examine the body burden of fluoroquinolones in northern Chinese children and assess its association with growth and development. METHODS: After recruiting 233 children aged 0-15 years from 12 different sites in northern China in 2020, we measured urinary concentrations of 5 respective fluoroquinolones (fleroxacin, ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin) by high performance liquid chromatography. Categories of children's growth and development were identified based on the Z score of body mass index. The health risks of individual and combined antibiotic exposure were estimated by the hazard quotient (HQ) and hazard index (HI), respectively. The association between children's growth and development with antibiotic concentrations was evaluated via multiple logistic regression analysis. RESULTS: In total, 4 antibiotics, fleroxacin, ofloxacin, ciprofloxacin, and enrofloxacin, were found in urine samples of northern Chinese children at an overall frequency of 57.08%. Due to diet and economic differences, antibiotic concentrations in urine samples differed by study area, and the highest concentrations were found in Tianjin, Henan, and Beijing. The percentage of the participants with HQ > 1 caused by ciprofloxacin exposure was 20.61%, and the HI values in 23.18% of samples exceeded 1, suggesting potential health risks. The odds ratio (95% confidence interval) of overweight or obesity risk of tertile 2 of enrofloxacin was 3.01 (1.12, 8.11), indicating an increase in overweight or obesity risk for children with middle-concentration enrofloxacin exposure. CONCLUSION: This is the first study to show a positive association of enrofloxacin internal exposure with overweight or obesity risk in children, demonstrating that more attention should be given to the usage and disposal of fluoroquinolones to safeguard children's health.

Establishment of an immunological detection method of fleroxacin by fluorescence-linked immunosorbent assay.

The direct and indirect competitive fluorescence-linked immunosorbent assay (FLISA and icFLISA) incorporating quantum dots (QDs) for the detection of fleroxacin (FLE) was established for the first time in this study. The monoclonal antibody specific for FLE was successfully conjugated with QDs after purification by the caprylic acid-ammonium sulphate method. The limits of detection of FLISA and icFLISA were 0.012 ng/mL and 0.006 ng/mL, respectively; IC50 were 0.32 ng/mL and 0.19 ng/mL; and the detection ranges were 0.012-24.490 ng/mL and 0.006-16.210 ng/mL. The recovery was 93.8%-112.4% and the coefficient of variation was below 11.75%. The fabricated FLISA and icFLISA are cost-effective, high sensitive and can be an alternative method in the detection of FLE residues.

Collection and Separation of Fleroxacin and Ciprofloxacin in Ultrasound-Assisted Ionic Liquid Salting-Out Microextraction System.

An ultrasound-assisted ionic liquid (IL) salting-out microextraction system was developed and applied for the extraction of quinolone antibiotics from urine. A precipitate was formed from the salt and IL, and it acted as the sorbent for the analytes. The precipitate containing the analyte was separated by filtration, redissolved, and the solution then was evaporated. The resulting extract was redissolved for high-performance liquid chromatographic analysis. Several parameters, including type and volume of IL, the type and amount of salts, sample pH, temperature and extraction time were optimized. Under the optimal experimental conditions, the limits of detection for fleroxacin and ciprofloxacin were 3.12 and 4.97 µg L-1, respectively. When the present method was applied to real urine sample analysis, the analyte recoveries ranged from 82.3 to 106.8%. This ultrasound-assisted IL salting-out microextraction system had the characteristics of high recoveries, shorter separation time and easy-to-perform collection procedure, which yielded the method to have potential for wide application.

Identification of Binding Epitopes of a Fluorinated Compound Bound to Proteins Using 1H and 19F NMR Spectroscopy.

1H/19F NMR-based screening methods were applied to a human serum albumin-fleroxacin complex. Fleroxacin contains three fluorine atoms in a molecule, which is suitable as a model fluorinated compound for NMR analysis with 1H and 19F detection. The 19F{1H} and 1H{1H} saturation transfer difference spectra were acquired and the 1H/19F spin-lattice relaxation rates were measured with and without any selective irradiation of protein resonance to identify the binding epitopes of fleroxacin. Because several 1H signals of fleroxacin resonated close to water, its precise signal intensities were unavailable. The 19F NMR-based screening methods successfully provide complementary information, indicating its importance in the analysis of fluorinated compounds.

Antibiotics and efflux: combined spectrofluorimetry and mass spectrometry to evaluate the involvement of concentration and efflux activity in antibiotic intracellular accumulation.

Background: In Gram-negative bacteria, passing through the double membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibiotic activity. Spectrofluorimetry has been developed to follow fluoroquinolone accumulation inside bacteria using intrinsic bacterial fluorescence as an internal standard. However, adaptation for non-fluorescent antibiotics is needed; quantitative methods based on MS offer the possibility of expanding the detection range obtained by spectrofluorimetry. Objectives: To validate, with spectrofluorimetry, the use of MS to measure antibiotic accumulation in cells and to determine the relationship between antibiotic concentrations and the amount of intrabacterial accumulation in different efflux backgrounds on the same batch of molecules. Methods: Spectrofluorimetry was performed in parallel with MS on the same samples to measure the ciprofloxacin and fleroxacin accumulation in cells expressing various efflux pump levels. A microplate protocol was set up to determine the antibiotic accumulation as a function of external antibiotic concentrations. Results: A correlation existed between the data obtained with spectrofluorimetry and MS, whatever the efflux pump or tested antibiotic. The results highlighted different dynamics of uptake between ciprofloxacin and fleroxacin as well as the relationship between the level of efflux activity and antibiotic accumulation. Conclusions: We have developed a microplate protocol and cross-validated two complementary methods: spectrofluorimetry, which contains a reliable internal standard; and MS, which allows detection of low antibiotic amounts. These assays allow study of the dose effect and the efflux impact on the intrabacterial accumulation of antibiotics.

Upconversion particle@Fe3O4@molecularly imprinted polymer with controllable shell thickness as high-performance fluorescent probe for sensing quinolones.

Localized photo-polymerization was ingeniously applied to prepare a multifunctional molecularly imprinted polymer (MIP) fluorescent probe using the "layer-by-layer" assembly of MIP and Fe3O4 nanoparticles on NaYF4: Yb3+, Er3+ upconversion particles (MUCPs@MIP). Enrofloxacin was used as the template and chosen as the target molecular during the investigation of the adsorption property. This ternary probe has magnetic and broad-spectrum molecular recognition capability, fast response, and upconversion fluorescence. The results of the fluorescence quenching analysis showed good linear ranges of 1.03nmol/L to 0.28µmol/L for enrofloxacin, 1.69nmol/L to 0.22µmol/L for fleroxacin, 6.92nmol/L to 0.28µmol/L for levofloxacin, 7.54nmol/L to 0.30µmol/L for ciprofloxacin, and 3.90nmol/L to 0.25µmol/L for enoxacin. This probe was further used to determine five quinolones in fish tissues and the recoveries ranging from 90.33% to 108.43% were obtained with relative standard deviation below 5.53%. This work offers a new and general strategy to synthesize a MUCPs@MIP upconversion fluorescence probe with magnetic and selective molecular recognition capability for rapid and accurate sensing of multiple chemical residues in the environment and agri-food products.

An automated magnetic dispersive micro-solid phase extraction in a fluidized reactor for the determination of fluoroquinolones in baby food samples.

An automated magnetic dispersive micro-solid phase extraction procedure in a fluidized reactor was developed for the determination of fluoroquinolone antimicrobial drugs (fleroxacin, norfloxacin and ofloxacin) in meat-based baby food samples. A stepwise injection system was successfully combined with afluidized reactorand applied for the magnetic dispersive micro-solid phase extraction procedure automation. The developed automated procedure involved injection of the sample solution into the fluidized reactor followed by the on-line separation of the analytes from the sample matrix based on fluidized beds strategy using magnetic nanoparticles, elution and determination of the analytes using a high performance liquid chromatography system with fluorescence detection. The floating of the magnetic nanoparticles in a liquid sample phase was accomplished by air-bubbling. In the developed method Zr-Fe-C magnetic nanoparticles were used as an efficient sorbent for the determination of fleroxacin, norfloxacin and ofloxacin. Under the optimal conditions, the calibration graphs were linear over the concentration ranges of 10-1000 µg L-1 for fleroxacin (R2 = 0.996), 5-1000 µg L-1for norfloxacin (R2 = 0.998) and ofloxacin (R2 = 0.998). The limits of detection, calculated from the blank tests based on 3σ, were 3.0 µg L-1forfleroxacin, 1.5 µg L-1for norfloxacin and ofloxacin. The limits of quantification, calculated from the blank tests based on 10σ, were 10 µg L-1 forfleroxacin, 5 µg L-1for norfloxacin and ofloxacin. The method was applied for the determination of fluoroquinolonesin meat-based baby food samples and the results were compared with those obtained by the reference method. The recovery values for all analytes were within of 86-122% range.

Fluorescence spectroscopic analysis on interaction of fleroxacin with pepsin.

The interaction between fleroxacin (FLX) and pepsin was investigated by spectrofluorimetry. The effects of FLX on pepsin showed that the microenvironment of tryptophan residues and molecular conformation of pepsin were changed based on fluorescence quenching and synchronous fluorescence spectroscopy in combination with three-dimensional fluorescence spectroscopy. Static quenching was suggested and it was proved that the fluorescence quenching of pepsin by FLX was related to the formation of a new complex and a non-radiation energy transfer. The quenching constants KSV , binding constants K and binding sites n were calculated at different temperatures. The molecular interaction distance (r = 6.71) and energy transfer efficiency (E = 0.216) between pepsin and FLX were obtained according to the Forster mechanism of non-radiation energy transfer. Hydrophobic and electrostatic interaction played a major role in FLX-pepsin association. In addition, the hydrophobic interaction and binding free energy were further tested by molecular modeling study.

Mass spectrometic study of speciation in aluminium-fluoroquinolone solutions.

Fluoroquinolones (FQLs) are synthetic antibacterial agents containing a 4-oxo-1,4-dihydroquinoline skeleton. When concomintantly administered with other drugs which may contain metal ions, particularly Al(3+) (antacids, phosphate binders, vaccines etc) they may form metal-drug complexes. Pharmacokinetic studies showed that aluminium-quinolone interactions lead to reduced bio- availability and altered activity of the drug with possible development of the toxic effects of aluminum ion. Reliable speciation in Al(3+) - quinolone systems at micromolar concentration level is needed to better understand pharmaco- and toxicokinetics of the FQLs in the presence of Al. In this work, the speciation in solutions containing Al(3+) and FQL family members (fleroxacin, moxifloxacin and ciprofloxacin) was studied by electrospray mass spectrometry (ESI-MS), ESI-MS/MS, and laser desorption ionization (LDI) MS. The dominating species identified in all the three Al(3+)-FQL solutions, at ca 30-50 µmol L(-1) total Al concentration and 2:1 to 1:3 metal-to-ligand ratio in the pH range 3.0- 6.0, were the ions related to the complexes AlL(2+), AlL(2)(+) and AlL(3)(0) (L = ligand in the monodeprotonated form). Mixed protonated and hydroxo complexes were also formed at lower and higher pH values respectively and, as expected, dimeric and polymeric species were not observed in ESI spectra. LDI measurements confirmed the existence of the mononuclear complexes found by ESI, and indicated the formation of polymeric species. The ion [2Al(3+) +5(-)](+) was identified with all three FQLs. This ionic species most probably arises from Al(2)L(2) by clustering with free ligand anions. Comparison of literature potentiometric data with mass spectral data indicated good agreement between speciation schemes. The obtained results suggest the presence of strong interaction between FQLs and Al(3+) which may be important in affecting absorption of these drugs in the gastrointestinal tract.

A rapid CE-potential gradient detection method for determination of quinolones.

A rapid CE coupled with potential gradient detection (PGD) for the separation and detection of four quinolones, namely, enoxacin, ofloxacin (OFL), fleroxacin, and pazufloxacin, was described. Separation was performed in a fused-silica capillary (75 microm x 8.5 cm) using a buffer of 30 mM Tris and 4 mM phosphoric acid at pH 8.9. Under the separation voltage of 3 kV, the quinolones were separated within 2.8 min with good linearity (r(2) >or= 0.985). The method was successfully applied in determining OFL in a pharmaceutical formulation. Also, a liquid-liquid extraction (LLE) method was developed and coupled to CE-PGD in determining quinolones that spiked in milk samples. With dichloromethane and hexane for enrichment and purification, the LLE recoveries of the four quinolones were in the range of 77-106%. The detection limits of the quinolones with LLE-CE-PGD were from 23 to 65 ng/mL. The proposed CE-PGD method was validated with an HPLC method, and the results indicated consistency between the two methods.

1H and 19F NMR relaxation studies of fleroxacin with Micrococcus luteus.

In order to investigate and characterize interaction processes between the fluoroquinolone fleroxacin and bacterial cells we used non-selective (all resonances are excited), selective (observed resonance is excited) spin-lattice relaxation rates and spin-spin relaxation measurements. The signals of three hydrogens at different moieties of the fleroxacin molecule were considered to get an insight in the complexation behavior. The enhancement of selective relaxation rates was observed with increasing fleroxacin concentrations and keeping the bacterial mass constant. The obtained relaxation rates of the affected hydrogens were analyzed via a Lineweaver-Burk-plot to determine the KD values. Furthermore, 19F NMR spectra were recorded and spin-spin relaxation rates (R2) were determined by a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence. Because of the dependency of the line width of NMR peaks on transversal relaxation time T2, we compared the line width at half-height at different fleroxacin concentrations in order to investigate the involvement of fluorine atoms in different positions in the complexation. All findings point to core quinolone moiety to be involved in the interaction with bacterial cells.

[Determination of fleroxacin by photochemical fluorescence of zinc-fleroxacin complex].

Based on the complex formed by Zn2+ which can strengthen the relative fluorescence intensity of fleroxacin evidently, a novel photochemical fluorescence method was developed. The effects of the acidity, the concentration ratio of Zn2+ to fleroxacin, and the time for illumination were studied. Under the optimum experiment conditions, the linear range of the determination was 5.00 x 10(-8) - 5.00 x 10(-6) mol x L(-1). The detection limit was 4.2 x 10(-8) mol x L(-1). The relative standard deviation of the determination of fleroxacin (5.0 x 10(-7) mol x L(-1)) was 1.7% (n = 20). The method was successfully applied to the determination of fleroxacin in specimens, and the recoveries were in the range of 95.0%-105%. The mechanism of this system is proposed.

[Ultraviolet spectral characteristics of charge-transfer reaction complex in micellar system and its application].

Charge-transfer (CT) reaction of chloranil (TCBQ) as a pi-electron acceptor with fleroxacin (FLX) as an electron donor has been studied by ultraviolet spectrophotometry method. Experiment showed that FLX reacted with TCBQ in sodium dodecyl sulfate (SDS) micellar systems, and a stable complex was formed and the absorbency was remarkably enhanced. Therefore, a simple, rapid, accurate and sensitive method for the determination of FLX has been developed. Beer's law is obeyed in the range of 0.6-24 mg x L(-1) of FLX and r = 0.9993. The apparent molar absorptivity of CT complexes at 326 nm is 3.3 x 10(4) L x mol(-1) x cm(-1). The composition of CT complex was found to be 1:1 by Bent-French and curved intersection methods. The proposed method has been applied to the determination of ESL in tablets. The recoveries are 99.2%-99.7%. The relative standard deviation is 0.7%-2.1%. The proposed methods are suitable for the routine quality control of drug alone and in tablets or capsules without fear of interference caused by the excipients expected to be present in tablets or capsules.

Validation of an HPLC method for the determination of fleroxacin and its photo-degradation products in pharmaceutical forms.

HPLC determination of fleroxacin in dosage forms was carried out using either reversed-phase column YMC pack ODS-AQ or Supelco LC Hisep shielded hydrophobic phase column, with UV detection at 280 nm. The mobile phase for ODS column consisted of 50:50:0.5 v/v/v and for Hisep column 15:85:0.5 v/v/v acetonitrile-water-triethylamine. The pH of the mobile phase was adjusted to 6.30 for ODS column and to 6.85 for Hisep column, with H3PO4. Linear response was obtained in the concentration range of fleroxacin between 0.01 and 1.30 micrograms/mL. Detection limit was 4.8 ng/mL. Recovery test in the determination of fleroxacin in "Quinodis" tablets (Hoffmann La Roche, nominal mass 400 or 200 mg) was 98-101% for both columns. The effect of the composition and pH of the mobile phase on spectra, retention time and dissociation constants of fleroxacin was discussed. The proposed method could be also used for separation of the photo-degradation products of fleroxacin. Ten degradation products were separated on the ODS-AQ column, thus confirming the suitability of the proposed method for stability study of fleroxacin in pharmaceuticals.

[Fluorescence spectrometric determination of fleroxacin by charge-transfer reaction].

A simple and reliable fluorescence spectrometric method for the determination of fleroxacin (FLX) has been developed based on the charge-transfer reaction. The charge-transfer complex which formed between fleroxacin as the donor and 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ) as the acceptor has been studied by fluorimetry. Experiment shows that FLX is reacted with DDQ in a mixture of acetone and methanol at 30 degrees C for 30 min and a complex is formed. The composition of the charge-transfer complex is found to be 1:1 by Bent-French and curved intersection methods. The linear dynamic range is 0.4-8.4 mg.L-1, the detection limit is 0.1 mg.L-1 and the recovery of FLX is 96.6%-99.2%. At last, the charge-transfer reaction mechanism was explored. The method has been applied to the determination of FLX in tablets with satisfactory results.

Determination of fleroxacin in pure and tablet forms by liquid chromatography and derivative UV-spectrophotometry.

Derivative UV-spectrophotometric and liquid chromatographic (LC) methods for fleroxacin determination were validated. In the spectrophotometric assay, first-, second-, third-, and fourth-order measurements were applied with the use of peak-zero and peak-peak techniques. The linear correlation between amplitude of the peak and concentration of the examined drug ranged from 2.0 to 12.0 micro/mL. An isocratic LC analysis was performed on a Purospher ODS column with an acidic mobile phase containing tetrabutylammonium hydroxide. Measurements were made at a wavelength of 285 nm with 4-aminobenzoic acid (PABA) as internal standard. The calibration curve was linear (r = 0.9999) in the studied range of concentration (1.0-10.0 microg/mL). The accuracy (mean recovery, about 100%), precision (relative standard deviation < 1%), selectivity, and sensitivity of the elaborated methods were satisfactory.

Spectrophotometric and HPLC determination of fleroxacin in tablets.

The paper describes and compares spectrophotometric and HPLC determination of fleroxacin in commercial tablets. The optimum conditions for spectrophotometric assay were found to be at pH < 3.5 (0.1 M HCl) at a wave length of 286 nm. HPLC analysis was carried out on a Beckman ODS 5 microns column in a pH 3 phosphoric acid solution (detector wave length 254 nm).

Voltammetric analysis of certain 4-quinolones in pharmaceuticals and biological fluids.

The voltammetric behaviour of Enrofloxacin (I), Sparfloxacin (II) and Fleroxacin (III) was studied using direct current (DCt), differential pulse (DPP) and alternating current (ACt). All the drugs manifest cathodic waves in Britton Robinson buffer over the pH range of 4.0-11.98. The waves were characterized as being irreversible, diffusion-controlled with limited adsorption properties. The diffusion current concentration relationships were found to be rectilinear over the ranges 4 x 10(-5) x 10(-4) M, 1 x 10(-5)-2 x 10(-4) M, 1 x 10(-5)-4 x 10(-4) M using DCt mode for I, II and III, respectively and 1 x 10(-6)-4 x 10(-5) M, 1 x 10(-6)-1 x 10(-4) M, and 2 x 10(-6)-8 x 10(-5) M, using DPP mode for I, II and III respectively, with minimum detectability (S/N = 3) of 1 x 10(-7) M for I, II and 2 x 10(-7) M for III. The proposed method was successfully applied to the determination of the studied compounds either per se or in formulations and biological fluids. The results obtained were concordant to those given using reference methods.

Voltammetric methods for analytical determination of fleroxacin in quinodis tablets.

The direct current (dc) and differential pulse (dp) polarographic reduction of fleroxacin was done in a wide pH range from 2.48 to 13.00. The appropriate buffer choice was made for its dp polarographic determination in a range from 1.845 to 16.926 microg /ml, at pH 8.50. The adsorptive properties of fleroxacin were investigated in order to achieve an increase in sensitivity and a possibility of fleroxacin determination by applying the adsorptive stripping voltammetric method. The adsorptive processes at the hanging mercury drop electrode were investigated in Britton-Robinson and borate buffers. Adsorptive preconcentration followed by differential-pulse cathodic stripping showed one wave at approximately - 1.1 V being the most sensitive for analytical determination of fleroxacin. Two linear ranges were obtained, the first one from 18.465 to 258.51 ng/ml, and the second one from 3.693 to 18.465 ng/ml.

Formation of antigenic quinolone photoadducts on Langerhans cells initiates photoallergy to systemically administered quinolone in mice.

Quinolone antibacterial agents are well known to cause photoallergy as a side-effect. Murine photoallergy to fluoroquinolones is a T cell-mediated immune response, evoked either by systemic fluoroquinolone and subsequent exposure of skin to ultraviolet A light or by subcutaneous injection of fluoroquinolone-photomodified epidermal cells. In this photosensitivity, epidermal Langerhans cells may be photomodified initially with the drug and thus present photohaptenic moieties to sensitize and restimulate T cells. Although we have shown that Langerhans cells photocoupled in vitro with fluoroquinolones are capable of stimulating sensitized T cells, it remains unclear whether systemically given fluoroquinolone photomodifies Langerhans cells upon ultraviolet A irradiation of the skin and the Langerhans cells become photohapten-bearing, T cell-stimulatory cells. In a murine model of fleroxacin photoallergy induced by intraperitoneal injection of the drugs plus ultraviolet A irradiation of skin, we found that Langerhans cells as well as keratinocytes are photoderivatized with fleroxacin as demonstrated with a fluoroquinolone-specific monoclonal antibody. Langerhans-cell-enriched epidermal cells prepared from mice treated with fleroxacin and ultraviolet A induced proliferation of sensitized T cells, indicating that photomodified Langerhans cells are functional. There was an optimal range of ultraviolet A dose to quantitatively and qualitatively form fleroxacin-photomodified Langerhans cells, as excess ultraviolet A rather reduced the photoantigen-presenting capacity of Langerhans cells presumably because of drug phototoxicity. Our study suggests that Langerhans cells serve as photoantigen-presenting cells in drug photoallergy.