Enantioseparations by High-Performance Liquid Chromatography Based on Chiral Ligand Exchange.

Although the first application of chiral ligand-exchange chromatography (CLEC) in HPLC dates back to late 1960s, this enantioselective strategy still represents the elective choice for the direct analysis of compounds endowed with chelating moieties. As a specific feature of the CLEC mechanism, the interaction between the chiral selector and the enantiomer does not take place in direct contact. Indeed, it is mediated by a central metal ion that, acting as a Lewis acid, simultaneously coordinates the two species, selector and analyte, through the activation of dative bonds. As a consequence, two diastereomeric mixed ternary complexes are generated in the column, ultimately leading to the stereoisomeric discrimination. CLEC applications can be carried out both with the chiral selector included in the mobile phase (chiral mobile phase, CMP), or as a part of the stationary phase. In the latter case, the chiral selector can be either covalently immobilized onto a solid support (bonded CSP, B-CSP) or physically adsorbed onto a conventional packing material, coated chiral stationary phase (C-CSP).In this chapter, a selection of CLEC applications with CMP- and C-CSP-based chiral systems is presented.

Cyclodextrins as Chiral Selectors in Capillary Electrophoresis Enantioseparations.

Due to their structural variability and their commercial availability, cyclodextrins are the most frequently used chiral selectors in capillary electrophoresis. A variety of migration modes can be realized depending on the characteristics of the cyclodextrins and the analytes. The basic considerations regarding the development of a chiral CE method employing cyclodextrins as chiral selectors are briefly discussed. The presented examples illustrate the separation modes of an acidic and a basic analyte with native and charged cyclodextrin derivatives as a function of the pH of the background electrolyte and the concentration of the cyclodextrin.

Graphene quantum dots for enhancement of fluorimetric detection coupled to capillary electrophoresis for detection of ofloxacin.

A new CE method with fluorescence detection is reported on the determination of ofloxacin in milk samples using graphene quantum dots (GQDs) for sensitivity enhancement. Injection of GQDs prior the standards/sample is crucial to increase the antibiotic fluorescence response. Clean-up and preconcentration steps allowed for a good linear correlation in a concentration range between 50 and 1000 ng/mL for the ofloxacin, detection and quantification limits being 10.7 and 35.5 ng/mL, respectively. Optimal CE conditions for the seven-fluoroquinolone separation method were assessed in terms of buffer type, pH, and voltage. The selective interaction of GQDs with ofloxacin as model analyte was subsequently studied finding a significant sensitivity improvement; therefore, the analytes would be detected at low concentrations by means of a commercial CE device equipped with a multi-wavelength photoluminescence detector. Due to the different maximum emission wavelengths of the target compounds and the limitations shown by the single-wavelength photoluminescence detector coupled to the CE system, we demonstrated the usefulness of the GQD-assisted sensitivity-enhanced CE method to determine ofloxacin in milk samples. This work opens an interesting possibility of using GQDs in separation techniques combined with photoluminescence detectors for lowering sensitivity levels typically provided by the mere device.

[Behaviors and Mechanisms of CIP and OFL Adsorption by Magnetic Biochar].

A magnetic reed biochar (MBC) was fabricated through chemical coprecipitation first and subsequent pyrolysis of reed stalk powder precipitated with Fe2+/Fe3+ at 873.15 K. The structure and properties of the MBC were characterized using SEM, BET, FTIR, and VSM. Adsorption experiments were carried out to investigate the adsorption behaviors and mechanisms of ciprofloxacin (CIP) and ofloxacin (OFL) onto the MBC. The MBC possessed roughness and pores within its structure and contained a large amount of oxygen-containing functional groups on the surface. The BET surface area of the MBC was 254.6 m2·g-1, and the total pore volume was 0.257 cm3·g-1, thus the MBC exhibited a relatively high porosity. The adsorption process was found to be pH and temperature dependent. The relative contributions of adsorbate species (cations, zwitterions, and anions) to overall adsorption varied for different pH values. Thermodynamic parameters indicated that the CIP and OFL adsorption onto MBC was a spontaneous, endothermic, and entropy-increasing process. Kinetics and isotherm data of CIP and OFL adsorption onto MBC were well depicted by the pseudo-second-order model and the Langmuir model. The equilibrium adsorption capacities of CIP and OFL onto MBC were 27.84 mg·g-1 and 22.00 mg·g-1, respectively. Pore-filling effects, π-π interaction between electron donors and acceptors, hydrogen bonding formation, hydrophobic interaction, and electrostatic interaction may be important mechanisms for CIP and OFL adsorption onto the MBC.

Inhibitory effects of extracellular polymeric substances on ofloxacin sorption by natural biofilms.

Natural biofilms have strong affinities for organic contaminants, and their extracellular polymeric substances (EPS) have been thought to control the sorption process. However, the role of EPS in the sorption of antibiotics, an emerging concern, is poorly understood. Here, soluble (SEPS) and bound EPS (BEPS) were extracted from intact biofilms incubated at different lengths of time to obtain SEPS- and BEPS-free biofilms. Batch sorption experiments and infrared spectroscopy were used to investigate the role of EPS in the sorption of ofloxacin (OFL) by natural biofilms. The sorption capacities of OFL onto intact biofilms were lower than that those onto SEPS-free and BEPS-free biofilms. Partition and Langmuir adsorption contributed to the sorption of OFL onto these biofilms. SEPS and BEPS suppressed partitioning of OFL into biofilm organic matter. Meanwhile, the formation of hydrogen bonds could affect the Langmuir adsorption of OFL onto BEPS-free biofilms. These sorption mechanisms occurred simultaneously and enhanced the sorption capacities of biofilms after EPS removal. The information obtained in this study is beneficial for understanding the interaction mechanisms between antibiotics and natural biofilms.

Fabrication of magnetic Fe3O4@nSiO2@mSiO2-NH2 core-shell mesoporous nanocomposite and its application for highly efficient ultrasound assisted dispersive µSPE-spectrofluorimetric detection of ofloxacin in urine and plasma samples.

In this research, a sensitive, simple and rapid ultrasound assisted dispersive micro solid-phase extraction (USAD-µSPE) was developed using a synthesized core-shell magnetic mesoporous nanocomposite (Fe3O4@nSiO2@mSiO2-NH2) as an efficient adsorbent for the preconcentration and spectrofluorometric determination of ofloxacin (OFL) in biological samples. The synthesized adsorbent was characterized using FT-IR spectroscopy, transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), energy dispersive X-ray (EDX) spectroscopy, thermogravimetric analysis (TGA) and Brunauer-Emmett-Teller (BET) analysis. The application of this magnetic nanocomposite as a sensitive solid phase for removal, preconcentration and spectrofluorometric quantification of trace amount of OFL was developed. Influence of various variables including pH, sorbent dosage, desorption solvent properties and sonication time on present method response was studied and optimized. The results showed that using the proposed method OFL can be determined in the linear concentration range of 1.0-500.0µgL-1 with a limit of detection as low as 0.21µgL-1 and relative standard deviation less than 2.5 (%). The results of human urine and blood plasma analysis showed that the method is a good candidate for biological sample analysis purposes.

On-line Identification of chiral ofloxacin in milk with an extraction/ionization device coupled to Electrospray Mass Spectrometry.

The direct separation and analysis of chiral drugs in the complex matrix systems are meaningful and challenging. As the most common broad-spectrum antibiotic, levofloxacin has a strong antibacterial ability, but its enantiomer, dextrofloxacin can cause serious harm to human health. In this work, we reported a rapid on-line extraction/ionization device coupled with Electrospray Mass Spectrometry (ESI-MS) for chiral analysis of ofloxacin enantiomers in complex matrix of milk. Since ofloxacin is difficult to dissolve in water and most organic solvents, the procedure of separating ofloxacin in complex system is often complicated. Using the homemade apparatus, the sample pretreatment process was greatly simplified. Milk sample was directly injected and chiral ofloxacin in the sample was extracted at PTFE membrane for further ionization. It took less than 10s to finish all the procedures including sampling, extraction, reagents mixing, ionization and mass analysis. Utilizing reaction thermodynamics method, trimeric cluster ion [NiΙΙ(ref)2Ofloxacin-H]+ was formed and collisionally dissociated to get chiral resolution of levofloxacin and dextrofloxacin due to the different relative stabilities of the two diastereomeric clusters produced through the dissociation of NiΙΙ bound trimeric clusters. With the proposed method, qualitative and quantitative chiral analysis of ofloxacin in milk was successfully achieved in a simple and fast way.

Escherichia coli adhesive coating as a chiral stationary phase for open tubular capillary electrochromatography enantioseparation.

Bacteria, the microorganism with intrinsic chirality, have numerous fascinating chiral phenomena such as various chirality-triggered biological processes and behaviors. Herein, bacteria were firstly explored as novel chiral stationary phases in open-tubular capillary electrochromatography (OT-CEC) for enantioseparation of fluoroquinolone enantiomers and simultaneous separation of six fluoroquinolone antibiotics. The model strain, i.e. non-pathogenic Escherichia coli (E. coli) DH5α, was adhered onto the inner surface of positively charged polyethyleneimine (PEI) modified capillaries based on the bacterial adhesion characteristics and strong electrostatic interaction. The morphology and thickness of the bacteria adhesive coatings in the capillary were characterized by field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM). Baseline separation of ofloxacin and partial separation of lomefloxacin enantiomers could be achieved by the E. coli coated columns. The preparation parameters including the coating time and concentration of bacteria that affecting the chiral resolution were intensively investigated. The electrophoretic parameters, including pH, buffer concentration and applied voltage, were also optimized. The developed method was validated (linearity, LOD, LOQ, intra-day, inter-day and column-to-column repeatability and recovery) and successfully utilized for the quantitative analysis of ofloxacin enantiomers in the ofloxacin tablets. Moreover, only a slight decrease in the separation efficiency was observed after 90 consecutive runs on the E. coli@capillary. These results demonstrated that bacteria are promising stationary phases for chiral separation in CEC.

Preparation of polysulfone materials on nickel foam for solid-phase microextraction of floxacin in water and biological samples.

Solid-phase microextraction with polysulfone and molecularly imprinted polymers as coating on nickel foam were used to adsorb and enrich floxacin drugs. The preparation method is simple and reproducible to obtain the materials with controlled thickness. After evaluation by scanning electron microscope and various adsorption experiments, the materials were used to adsorb analytes in water samples and biological samples. Coupling with chromatographic analysis, the method recoveries are satisfactory with 90.0-104.8% and 79.31-107.1% for water and biological samples. The method repeatability by intra- and interday experiments shows that the RSD values for water and biological samples were 1.0-9.9% and 1.7-10.3%, with the quantitative limits of three floxacin drugs as 3.0-6.2 µg L-1. Graphical Abstract Preparation diagram of polysulfone material.

Synergistic effect of aqueous removal of fluoroquinolones by a combined use of peroxymonosulfate and ferrate(VI).

This paper examines for the first time the combined use of ferrate(VI) (FeVIO42-, Fe(VI)) with peroxymonosulfate (PMS) to oxidize four fluoroquinolones (FQs) (flumequine (FLU), enrofloxacin (ENR), marbofloxacin (MAR), and ofloxacin (OFL)) at pH 7.0 and 25.0 °C. The results demonstrate that a combination of PMS and Fe(VI) synergistically enhance removal of FQs compared to the removal seen with either PMS or Fe(VI) alone. The oxidized products (OPs) of the reaction of FLU with the PMS-Fe(VI) system, identified by liquid chromatography high-resolution mass spectrometry (LC-HRMS) techniques, showed transformation products resulted from hydroxylation, ring cleavage, and de-fluorination processes. Thus, the combination of PMS and Fe(VI) can be developed as an innovative oxidative technology to remove contaminants from water.

Use of various ß-cyclodextrin derivatives as chiral selectors for the enantiomeric separation of ofloxacin and its five related substances by capillary electrophoresis.

A capillary electrophoretic method for the enantioseparation of ofloxacin and its five related substances (potential impurities, indicated as impurities B-F) was developed using ß-cyclodextrin derivatives as chiral selectors. To our knowledge, there are no previous studies about using capillary electrophoresis for the separation of impurities B-D. Six ß-cyclodextrin derivatives including cationic (piperidine- and cyclohexylamine-), neutral (dimethyl- and hydroxypropyl-), and anionic (carboxymethyl- and sulfated-) ß-cyclodextrin derivatives were tested and operational parameters such as buffer pH and concentration of ß-cyclodextrin derivatives were investigated. The best resolutions were all obtained with anionic ß-cyclodextrin derivatives: ofloxacin, impurities C-F could be best resolved with carboxymethyl-ß-cyclodextrin at satisfactory resolutions of 8.27, 9.98, 5.92, 8.49 and 6.78, respectively, while for impurity B, a particularly impressive resolution value, up to 21.38, was observed using sulfated-ß-cyclodextrin. The enhancement of enantioseparation observed for the tested analytes using anionic ß-cyclodextrin derivatives might be due to some favorable interaction between selectors and analytes. Given the fact that the selection of chiral selector depends on the structures of analytes, with the help of structural similarities and differences of the analytes, the structure-separation relationship was further discussed.

Synthesis and Characterization of Magnetic Molecularly Imprinted Polymer for the Enrichment of Ofloxacin Enantiomers in Fish Samples.

A new method for the isolation and enrichment of ofloxacin enantiomers from fish samples was developed using magnetic molecularly imprinted polymers (MMIPs). These polymers can be easily collected and rapidly separated using an external magnetic field, and also exhibit a high specific recognition for ofloxacin enantiomers. The preparation of amino-functionalized MMIPs was carried out via suspension polymerization and a ring-opening reaction using rac-ofloxacin as a template, ethylenediamine as an active group, glycidyl methacrylate and methyl methacrylate as functional monomers, divinylbenzene as a cross-linker, and Fe3O4 nanoparticles as magnetic cores. The characteristics of the MMIPs were assessed using transmission electron microscopy (TEM), X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FT-IR), and vibrating sample magnetometer (VSM) measurements. Furthermore, the adsorption properties were determined using Langmuir and Freundlich isotherm models. The conditions for use of these MMIPs as magnetic solid-phase extraction (MSPE) sorbents, including pH, adsorption time, desorption time, and eluent, were investigated in detail. An extraction method using MMIPs coupled with high performance liquid chromatography (HPLC) was developed for the determination of ofloxacin enantiomers in fish samples. The limits of quantitation (LOQ) for the developed method were 0.059 and 0.067 µg∙mL(-1) for levofloxacin and dextrofloxacin, respectively. The recovery of ofloxacin enantiomers ranged from 79.2% ± 5.6% to 84.4% ± 4.6% and ofloxacin enantiomers had good linear relationships within the concentration range of 0.25-5.0 µg∙mL(-1) (R² > 0.999). The obtained results demonstrate that MSPE-HPLC is a promising approach for preconcentration, purification, and simultaneous separation of ofloxacin enantiomers in biomatrix samples.

Quantitative analysis of antibiotics in aquifer sediments by liquid chromatography coupled to high resolution mass spectrometry.

A highly effective analytical method for multi-residue determination of antibiotics in aquifer sediments was first established in this study. Microwave-assisted solvent extraction (MASE) and solid-phase extraction were used for sample pre-concentration and purification, ultra-high performance liquid chromatography coupled to hybrid quadrupole-high resolution Orbitrap mass spectrometry (UHPLC-Q-Orbitrap) was applied for detection. For high resolution mass spectrometry (HRMS), the target compounds were tentatively identified by retention time and accurate mass which was measured with precursor ions in Target-SIM scan, and then confirmed by the monitoring of daughter ion fragments which were generated in dd-MS(2) scan. The results provided good mass accuracy with mass deviations below 2ppm (except norfloxacin with -2.3ppm) for quantitative analysis of the compounds by HRMS. Reasonable recoveries of all analytes were obtained more than 60% (except doxytetracycline) in fortification samples at concentrations higher than 10µgkg(-1). Relative standard deviations of repeatability and inter-day precision were below 21% and 11%. Limits of detection (LOD) ranged from 0.1 to 3.8µgkg(-1), whereas limits of quantification (LOQ) were established between 0.3-9.0µgkg(-1). The method was applied to analyze real aquifer sediment samples in different aquifer depth of 4.0, 7.5, 13.0 and 18.0m. Chlorotetracycline and ofloxacin were observed at relative high concentrations of 53 and 19µgkg(-1) respectively in 18.0m deepness. The exposure to low doses of these compounds in subsurface environment increases concerns on long-term ecological security of underground system.

Label-free microfluidic free-flow isoelectric focusing, pH gradient sensing and near real-time isoelectric point determination of biomolecules and blood plasma fractions.

We demonstrate the fabrication, characterization and application of microfluidic chips capable of continuous electrophoretic separation via free flow isoelectric focussing (FFIEF). By integration of a near-infrared (NIR) fluorescent pH sensor layer under the whole separation bed, on-line observation of the pH gradient and determination of biomolecular isoelectric points (pI) was achieved within a few seconds. Using an optical setup for imaging of the intrinsic fluorescence of biomolecules at 266 nm excitation, labelling steps could be avoided and the native biomolecules could be separated, collected and analysed for their pI. The fabricated microchip was successfully used for the monitoring of the separation and simultaneous observation of the pH gradient during the isoelectric focussing of the proteins α-lactalbumin and ß-lactoglobulin, blood plasma proteins and the antibiotics ampicillin and ofloxacin. The obtained pIs are in good agreement with literature data, demonstrating the applicability of the system. Mass spectra from the separated antibiotics taken after 15 minutes of continuous separation from different fractions at the end of the microchip validated the separation via microfluidic isoelectric focussing and indicate the possibility of further on- or off-chip processing steps.

Separation of Ofloxacin and Its Six Related Substances Enantiomers by Chiral Ligand-Exchange Chromatography.

A chiral ligand-exchange high-performance liquid chromatography method was developed for the enantioseparation of ofloxacin and its six related substances termed impurities A, B, C, D, E, and F. The separation was performed on a conventional C18 column. Different organic modifiers, copper salts, amino acids, the ratio of Cu(2+) to amino acid, pH of aqueous phase, and column temperature were optimized. The optimal mobile phase conditions were methanol-water systems consisting of 5 mmol/L copper sulfate and 10 mmol/L L-isoleucine (L-Ile). Under such conditions, good enantioseparation of ofloxacin and impurities A, C, E, and F could be observed with resolutions (RS ) of 3.54, 1.97, 3.21, 3.50, and 2.12, respectively. On the relationship between the thermodynamic parameters and structures of analytes, the mechanism of chiral recognition was investigated. It was concluded that ofloxacin and impurities A, C, E, and F were all enthalpically driven enantioseparation and that low column temperature was beneficial to enantioseparation. Furthermore, the structure-separation relationship of these analytes is also discussed.

In-tube magnetic solid phase microextraction of some fluoroquinolones based on the use of sodium dodecyl sulfate coated Fe3O4 nanoparticles packed tube.

In-tube magnetic solid phase microextraction (in-tube MSPME) of fluoroquinolones from water and urine samples based on the use of sodium dodecyl sulfate (SDS) coated Fe3O4 nanoparticles packed tube has been reported. After the preparation of Fe3O4 nanoparticles (NPs) by a batch synthesis, these NPs were introduced into a stainless steel tube by a syringe and then a strong magnet was placed around the tube, so that the Fe3O4 NPs were remained in the tube and the tube was used in the in-tube SPME-HPLC/UV for the analysis of fluoroquinolones in water and urine samples. Plackett-Burman design was employed for screening the variables significantly affecting the extraction efficiency. Then, the significant factors were more investigated by Box-Behnken design. Calibration curves were linear (R(2)>0.990) in the range of 0.1-1000µgL(-1) for ciprofloxacin (CIP) and 0.5-500µgL(-1) for enrofloxacin (ENR) and ofloxacin (OFL), respectively. LODs for all studied fluoroquinolones ranged from 0.01 to 0.05µgL(-1). The main advantages of this method were rapid and easy automation and analysis, short extraction time, high sensitivity, possibility of fully sorbent collection after analysis, wide linear range and no need to organic solvents in extraction.

Selective solid-phase extraction of artificial chemicals from milk samples using multiple-template surface molecularly imprinted polymers.

A novel multiple-template surface molecularly imprinted polymer (MTMIP) was synthesized using ofloxacin and 17ß-estradiol as templates and modified monodispersed poly(glycidylmethacrylate-co-ethylene dimethacrylate) (PGMA/EDMA ) beads as the support material. Static adsorption, solid-phase extraction and high-performance liquid chromatography were performed to investigate the adsorption properties and selective recognition characteristics of the polymer templates and their structural analogs. The maximum binding capacities of ofloxacin and 17ß-estradiol on the MTMIP were 9.0 and 6.6 mg/g, respectively. Compared with the corresponding nonimprinted polymer, the MTMIP exhibited a much higher adsorption performance and selectivity toward three quinolones and three estrogens, which are common drug residues in food. The MTMIP served as a simple and effective pretreatment method and could be successfully applied to the simultaneous analysis of multiple target components in complex samples. Furthermore, the MTMIP may find useful applications as a solid-phase absorbent in the simultaneous determination of trace quinolones and estrogens in milk samples, as the recoveries were in the range 77.6-98.0%.

Preparation of temperature sensitive molecularly imprinted polymer for solid-phase microextraction coatings on stainless steel fiber to measure ofloxacin.

A kind of new temperature sensitive molecularly imprinted polymer (MIP) with ofloxacin (OFL) as template was prepared for the coating of solid phase microextraction (SPME). Dopamine was self-polymerized on stainless steel fiber (SSF) as the SPME support followed by silanization. Then MIP was synthesized as SPME coating on the modified SSF in a capillary, with N-isopropyl acrylamide as temperature sensitive monomer and methacrylic acid as functional monomer. The synthesis could be well repeated with multiple capillaries putting in the same reaction solution. The obtained MIP fiber was evaluated in detail with different techniques and various adsorption experiments. At last the MIP fiber was used to extract the OFL in milk. Satisfied recoveries between 89.7 and 103.4% were obtained with the limit of quantification (LOQLC) of 0.04 µg mL(-1) by the method of SPME coupled with high performance of liquid chromatography (HPLC).

Adsorption of zwitterionic fluoroquinolone antibacterials to goethite: a charge distribution-multisite complexation model.

Fluoroquinolone (FQ) antibacterials are aquatic contaminants of emerging concern (CEC), and adsorption to mineral surfaces is expected to play an important role in the fate, transport, and treatment of FQs. This study characterizes and models the adsorption of a zwitterionic FQ, ofloxacin (OFX), to goethite (α-FeOOH) over a wide range of pH (3-11), OFX concentration (20-500 µM), and electrolyte compositions (0.001-0.1M NaCl and NaClO4). Comparing OFX adsorption to structural analogues demonstrates that the carboxylate group is essential for binding to goethite. ATR-FTIR measurements indicate that FQs complex to goethite surfaces through carboxylate and carbonyl oxygen atoms, and that ClO4(-) co-adsorbs with OFX. Adsorption of the zwitterionic OFX increases with increasing ionic strength and is enhanced in NaClO4 relative to NaCl electrolyte, whereas adsorption of a non-zwitterionic analogue is insensitive to ionic strength. A CD-MUSIC (charge distribution-multisite complexation) model, incorporating multiple modes of surface complexation constrained by spectroscopic measurements and the crystallographic distribution of goethite surface sites, yields accurate predictions over wide-ranging solution conditions. According to the model, OFX adsorbs predominantly by inner-sphere complexation on terminal surfaces of the rod-shaped goethite crystals in NaCl electrolyte, and OFX-ClO4(-) ion pairing in NaClO4 induces formation of additional inner- and outer-sphere surface complexes on multiple crystal faces of goethite.

Chiral discrimination of ofloxacin enantiomers using DNA double helix regulated by metal ions.

DNA-based chiral selectors are constructed to discriminate ofloxacin enantiomers through metal-ion anchoring on a special DNA double helix that contains successive GC pairs. The effects of metal ions involving Mg(2+), Ni(2+), Cu(2+), Ag(+), and Pt(2+) were studied on the regulation of DNA chiral discrimination towards ofloxacin enantiomers. It is shown that DNA-Cu(II) complexes exhibit the highest enantioselectivities at the [Cu(2+)]/base ratio of 0.1. The enantiomeric excess can reach 59% in R-enantiomer after being adsorbed by the RET-Cu(II) complex. Stereoselective recognition of ofloxacin enantiomers on the double helix is tunable via external stimulus, providing a programmable desorption process to regenerate DNA. This DNA-based chiral selector exhibits excellent reusability without apparent loss of enantioselectivity after three cycles of adsorption and desorption.