Preparation and application of magnetic molecular imprinted polymers for extraction of cephalexin from pork and milk samples.

A highly selective and effective method was successfully developed using magnetic molecular imprinted polymers (MMIPs) as solid-phase extraction (SPE) coupled with high performance liquid chromatography-ultraviolet detector (HPLC-UV) to rapidly determine cephalexin (CFX) in complex animal-derived food. MMIPs were creatively synthesized via suspension polymerization using Fe3O4 magnetic nanoparticles as supporter, CFX as template, acrylamide (AM) as functional monomer, and ethylene glycol dimethacrylate (EGDMA) as cross-linker. The MMIPs were characterized using X-ray diffraction (XRD), Fourier transform infrared spectrometry (FT-IR), scanning electron microscopy (SEM), and vibrating sample magnetometry (VSM). The binding process fitted well with pseudo-second-order model with good selectivity. Scatchard plot analysis suggested that MMIPs have two types of binding sites with the Qmax of 24.18 mg g-1 and 40.25 mg g-1, respectively. And Langmuir model proved that the recognition sites were uniformly distributed in a monolayer on the surface of MMIPs. The methodological assessment showed good applicability of MMIPs with excellent recovery (85.5%-94.0%), precision (1.2%-2.4%), and stability (intra-day 1.3%-3.6%; inter-day 2.6%-4.3%) in determining CFX content. In addition, the linearity of the calibration curve was good in the range of 0.02-5.00 mg L-1, with a sensitive detection limit of 5.00 µg kg-1. The results above suggest that the obtained MMIPs exert good performance for separation of CFX in animal-derived food, and the proposed method is suitable for the reliable determination of CFX in complex samples.

Electrochemical Sensor Based on Molecularly Imprinted Polymer for the Detection of Cefalexin.

In this study, a new electrochemical sensor was developed for the detection of cefalexin (CFX), based on the use of a molecularly imprinted polymer (MIP) obtained by electro‒polymerization in an aqueous medium of indole-3-acetic acid (I3AA) on a glassy carbon electrode (GCE) and on boron-doped diamond electrode (BDDE). The two different electrodes were used in order to assess how their structural differences and the difference in the potential applied during electrogeneration of the MIP translate to the performances of the MIP sensor. The quantification of CFX was performed by using the electrochemical signal of a redox probe before and after the rebinding of the template. The modified electrode was characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The influence of different parameters on the fabrication of the sensor was tested, and the optimized method presented high selectivity and sensitivity. The MIP-based electrode presented a linear response for CFX concentration range of 10 to 1000 nM, and a limit of detection of 3.2 nM and 4.9 nM was obtained for the BDDE and the GCE, respectively. The activity of the sensor was successfully tested in the presence of some other cephalosporins and of other pharmaceutical compounds. The developed method was successfully applied to the detection of cefalexin from real environmental and pharmaceutical samples.

Comparison of Three Independent Methods of Cephalexin Determination by Means of Potassium Caroate / Comparación de tres métodos independientes para la detección de cefalexina usando el potasio de caroata

Aims.Three different assays for the quantity determination of Cephalexin in pure substance were developed and compared using potassium caroate as analytical reagent. Materials and Methods. Cephalexin pure substance was used and analytical reaction by means of KHSO5 as an oxidant was studied. Voltammetric, kinetic-spectrophotometric and iodometric methods were developed and validated. Results. The chemical reaction is finished with formation of the S-oxide of Cephalexin in the iodometric and voltammetric methods and the S-oxide hydrolysis product in the kinetic-spectrophotometric method. The reaction finishes in 1 min (observation time). The methods have been validated and good results including precision, accuracy and recovery were obtained. The recovery percent ranged from 98.7 to 101.4%, RSD from 1.42 to 3.0%. The methods proposed are linear in a wide range: voltammetry 2-45 μg mL-1, kinetic-spectrophotometry 1-16 μg mL-1, and iodometry 0.05-0.35 mg mL-1. LOD and LOQ were calculated. The kinetic-spectrophotometric method is the most sensitive (LOQ=1.0 μg mL-1). Conclusions. The methods proposed are rapid, simple and inexpensive and could be applied to pharmaceutical preparation

Objetivo. Se han desarrollado tres métodos diferentes para la detección cuantitativa de Cefalexina en producto puro, usando el caroata de potasio como un reactivo analítico. Materiales y métodos. Fue estudiada la reacción analítica de cefalexina con KHSO5 como oxidante. Se han desarrollado y validado los métodos de voltamperimetría, de espectrofotometría cinética y de iodometría. Resultados. La reacción química se completa con la formación de s-óxido de Cefalexina dentro de los métodos de iodometría y de voltamperometría, y con la formación del producto de hidrólisis de s-óxido en el método de espectrofotometría cinética. La reacción se completa en 1 minuto (tiempo de observación). Los métodos han sido validados, y se han obtenido resultados buenos, incluida la exactitud, la fiabilidad y la reproducibilidad. El contenido de la sustancia básica oscila desde 98,7% hasta 101,4%. El RSD está entre 1,42 y 3,0%. Los métodos propuestos son lineales en un amplio espectro: la voltamperometría 2-45 μg ml-1, la espectrofotometría cinética 1-16 μg ml-1, y la iodometría 0,05-0,35 mg ml-1. Han sido calculados el límite de detección y el límite de cuantificación. El método de espectrofotometría cinética es el más sensible (el límite de cuantificación = 1,0 mcg ml-1). Conclusiones. Los métodos propuestos son rápidos, simples y baratos, y pueden ser utilizados en las preparaciones farmacéuticas

Molecularly imprinted polymers based stir bar sorptive extraction for determination of cefaclor and cefalexin in environmental water.

Although stir bar sportive extraction was thought to be a highly efficiency and simple pretreatment approach, its wide application was limited by low selectivity, short service life, and relatively high cost. In order to improve the performance of the stir bar, molecular imprinted polymers and magnetic carbon nanotubes were combined in the present study. In addition, two monomers were utilized to intensify the selectivity of molecularly imprinted polymers. Fourier transform infrared spectroscopy, scanning electron microscopy, and selectivity experiments showed that the molecularly imprinted polymeric stir bar was successfully prepared. Then micro-extraction based on the obtained stir bar was coupled with HPLC for determination of trace cefaclor and cefalexin in environmental water. This approach had the advantages of stir bar sportive extraction, high selectivity of molecular imprinted polymers, and high sorption efficiency of carbon nanotubes. To utilize this pretreatment approach, pH, extraction time, stirring speed, elution solvent, and elution time were optimized. The LOD and LOQ of cefaclor were found to be 3.5 ng · mL-1 and 12.0 ng · mL-1, respectively; the LOD and LOQ of cefalexin were found to be 3.0 ng · mL-1 and 10.0 ng · mL-1, respectively. The recoveries of cefaclor and cefalexin were 86.5 ~ 98.6%. The within-run precision and between-run precision were acceptable (relative standard deviation <7%). Even when utilized in more than 14 cycles, the performance of the stir bar did not decrease dramatically. This demonstrated that the molecularly imprinted polymeric stir bar based micro-extraction was a convenient, efficient, low-cost, and a specific method for enrichment of cefaclor and cefalexin in environmental samples.

Influence of process variables on extraction of Cefalexin in a novel biocompatible ionic liquid based-aqueous two phase system.

Despite the fact that ionic liquid-based aqueous two phase systems (ATPSs) have been widely studied for extraction purposes, the adequacy of biodegradable organic salts as salting out agents has been left unexploited. In this study, we investigated the ability of sodium-based organic salts in the formation of ATPS in the presence of a common ionic liquid, [C4mim]BF4. In the pioneering aspect of this work, Response Surface Methodology (RSM) based on three-variable central composite design (CCD) was employed for determination of the effect of pH and the initial concentration of phase components on the partition coefficient of Cefalexin. Consequently, regression model equations and contour plots were applied to evaluate the effect of system's parameters on biomolecule's extraction. The tie-line (TL) data were determined for each experimental run and their reliability was confirmed by Othmer-Tobias and Bancroft correlations. In order to investigate the salting-out ability the effective excluded volume (EEV) was determined from the binodal data. Furthermore, FTIR spectra confirmed no chemical interactions between Cefalexin and [C4mim]BF4 in the extraction process. The microscopic structure of the top phase was analyzed by DLS, conductivity and TEM in order to investigate the mechanism of extraction. Hydrophobic interaction, the salting-out effect and the aggregation phenomena played the dominant role in the study of the extraction process.

Removal of cephalexin from aqueous solutions by original and Cu(II)/Fe(III) impregnated activated carbons developed from lotus stalks Kinetics and equilibrium studies.

Lotus stalk activated carbon (AC) was produced by ultrasound digestion of lotus stalks in H(3)PO(4). Copper nitrate and iron nitrate were used to impregnate AC, producing Cu(II)-impregnated AC (AC-Cu) and Fe(III)-impregnated AC (AC-Fe). The modified ACs were characterized by N(2) adsorption-desorption isotherms, Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD). The adsorption kinetics and isotherms of cephalexin (CEX) in aqueous solution were studied for AC, AC-Cu and AC-Fe. The kinetics and equilibrium data agreed well with the pseudo-second-order kinetics model and Freundlich isotherm model for all three adsorbents. The results also showed that the adsorption capacities of AC-Cu and AC-Fe were larger than the capacity of AC and AC-Fe was found to be the most effective at the removal of CEX in solution. Furthermore, batch experiments were conducted to study the effects of pH (2.5-10.5), initial concentration of CEX (4-16 mg/L), ionic strength (10-1000 mM) on CEX removal.

Effect of loading rate and HRT on the removal of cephalosporin and their intermediates during the operation of a membrane bioreactor treating pharmaceutical wastewater.

The viability of treating high-concentration antibiotic wastewater by an anaerobic membrane bioreactor was studied using submerged flat sheet membrane. The objective of the study was to determine the effect of organic loading rate and hydraulic retention time on the removal of cephalosporin derivative, viz. cephalexin, and the intermediates 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and acyl group (phenyl acetic acid) in an anaerobic membrane bioreactor with enhanced biodegradation using the bioaugmentation technique. The pharmaceutical industry is looking for alternatives to either direct disposal of 7-amino-3-deacetoxycephalosporanic acid and phenyl acetic acid, or further degradation and disposal, which will essentially require additional costs and maintenance. The present regulatory standard, implemented at a global level, does not allow for such disposal alternatives and hence the present study was aimed at the complete removal of the intermediates 7-ADCA and phenyl acetic acid prior to discharge.

Synthesis of cephalexin in aqueous medium with carrier-bound and carrier-free penicillin acylase biocatalysts.

The use of very high substrate concentrations favors the kinetically controlled synthesis of cephalexin with penicillin acylase (PA) not only by Michaelian considerations, but also because water activity is depressed, so reducing the rates of the competing reactions of product and acyl donor hydrolysis. Commercial PGA-450, glyoxyl agarose immobilized (PAIGA) and carrier-free cross-linked enzyme aggregates of penicillin acylase (PACLEA) were tested in aqueous media at concentrations close to the solubility of nucleophile and at previously determined enzyme to nucleophile and acid donor to nucleophile ratios. The best temperature and pH were determined for each biocatalyst based on an objective function considering conversion yield, productivity, and enzyme stability as evaluation parameters. Stability was higher with PAIGA and specific productivity higher with PACLEA, but best results based on such objective function were obtained with PGA-450. Yields were stoichiometric and productivities higher than those previously reported in organic medium, which implies significant savings in terms of costs and environmental protection. At the optimum conditions for the selected biocatalyst, operational stability was determined in sequential batch reactor operation. The experimental information gathered is being used for a technical and economic evaluation of an industrial process for enzymatic production of cephalexin in aqueous medium.

A two-step, one-pot enzymatic synthesis of cephalexin from D-phenylglycine nitrile.

A cascade of two enzymatic transformations is employed in a one-pot synthesis of cephalexin. The nitrile hydratase (from R. rhodochrous MAWE)-catalyzed hydration of D-phenylglycine nitrile to the corresponding amide was combined with the penicillin G acylase (penicillin amidohydrolase, E.C. 3.5.1.11)-catalyzed acylation of 7-ADCA with the in situ-formed amide to afford a two-step, one-pot synthesis of cephalexin. D-Phenylglycine nitrile appeared to have a remarkable selective inhibitory effect on the penicillin G acylase, resulting in a threefold increase in the synthesis/hydrolysis (S/H) ratio. 1,5-Dihydroxynaphthalene, when added to the reaction mixture, cocrystallized with cephalexin. The resulting low cephalexin concentration prevented its chemical as well as enzymatic degradation; cephalexin was obtained at 79% yield with an S/H ratio of 7.7.

Micellar electrokinetic capillary chromatography for the separation of cefalexin and its related substances.

Micellar electrokinetic capillary chromatography (MEKC) was examined for analysis of cefalexin and its related substances. Good selectivity was obtained with two different buffer solutions: a sodium acetate buffer (50 mM, pH 5.25) containing sodium dodecyl sulfate (50 mM SDS) or sodium phosphate buffer (40 mM, pH 7.0) containing 100 mM SDS. Both methods permit cefalexin to be completely separated from its ten related substances within 20 min. The robustness of the method, using pH 5.25 acetate buffer, was examined by means of a full-fraction factorial design to test the influence of buffer pH, concentration of SDS and buffer concentration. The parameters for validation such as linearity, precision, limit of detection and limit of quantitation are also reported. The results show that method 1 is suitable for the analysis of cefalexin.

Use of aqueous two-phase systems for in situ extraction of water soluble antibiotics during their synthesis by enzymes immobilized on porous supports.

Yields of kinetically controlled synthesis of antibiotics catalyzed by penicillin G acylase from Escherichia coli (PGA) have been greatly increased by continuous extraction of water soluble products (cephalexin) away from the surroundings of the enzyme. In this way its very rapid enzymatic hydrolysis has been avoided. Enzymes covalently immobilized inside porous supports acting in aqueous two-phase systems have been used to achieve such improvements of synthetic yields. Before the reaction is started, the porous structure of the biocatalyst can be washed and filled with one selected phase. In this way, when the pre-equilibrated biocatalyst is mixed with the second phase (where the reaction product will be extracted), the immobilized enzyme remains in the first selected phase in spite of its possibly different natural trend. Partition coefficients (K) of cephalexin in very different aqueous two-phase systems were firstly evaluated. High K values were obtained under drastic conditions. The best K value for cephalexin (23) was found in 100% PEG 600-3 M ammonium sulfate where cephalexin was extracted to the PEG phase. Pre-incubation of immobilized PGA derivatives in ammonium sulfate and further suspension with 100% PEG 600 allowed us to obtain a 90% synthetic yield of cephalexin from 150 mM phenylglycine methyl ester and 100 mM 7-amino desacetoxicephalosporanic acid (7-ADCA). In this reaction system, the immobilized enzyme remains in the ammonium sulfate phase and hydrolysis of the antibiotic becomes suppressed because of its continuous extraction to the PEG phase. On the contrary, synthetic yields of a similar process carried out in monophasic systems were much lower (55%) because of a rapid enzymatic hydrolysis of cephalexin.

[Separation of drugs cephalexin, ampicillin, and their biosynthetic precursors L-Phe-L-Cys-D-Val and D-Phe-L-Cys-D-Val by capillary zone electrophoresis].

The separation of drugs cephalexin, ampicillin and their biosynthetic precursors L-Phe-L-Cys-D-Val and D-Phe-L-Cys-D-Val was investigated by using capillary zone electrophoresis in acidic Tris-H3PO4 buffer at pH 2.3. With 50 mmol/L Tris-H3PO4 (pH 2.3) buffer, L-Phe-L-Cys-D-Val and D-Phe-L-Cys-D-Val were only partially separated whereas near baseline separation was achieved between cephalexin and ampicillin. The experiments showed that the addition of beta-type cyclodextrins (beta-CD) to the running buffer can influence the separation selectivity. When beta-CD was used as buffer additives at 12 mmol/L, the separation between L-Phe-L-Cys-D-Val and D-Phe-L-Cys-D-Val was much improved, however, the separation between cephalexin and ampicillin was impaired. When using 50 mmol/L Tris-H3PO4 containing 12 mmol/L DM-beta-CD (pH 2.3) buffer system, all compounds investigated were completely separated. This separation method is simple, fast and easy to operate. It involves only the conventional reagents with a much lower run cost than using HPLC. It is expected to be used in the synthetic process monitoring.

Isolation and structural elucidation of an impurity of cefradine.

An impurity of unknown identity was isolated from commercial cefradine by liquid chromatography on poly (styrene-divinylbenzene) with HOAc (0.01 M)-CH3CN (94:6, v/v) as the mobile phase. The structure was elucidated as 4',5'-dihydrocefradine using nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). The structure was confirmed by comparison with the chromatographic retention characteristics and photodiode-array detected ultraviolet spectrum of the synthetic compound and with its infrared, NMR and MS spectra. The presence of 4',5'-dihydrocefradine in cefradine has not been described previously.

Reversed-phase high-performance liquid chromatography of amphoteric beta-lactam antibiotics: effects of columns, ion-pairing reagents and mobile phase pH on their retention times.

The separation of five amino beta-lactam antibiotics by reversed-phase high-performance liquid chromatography was studied as an insight into their retention behaviour. These five amphoteric compounds are cephradine, cephalexine, cefaclor, ampicillin and amoxicillin. Both octadecylsilane-bonded silica (C18) columns and phenyl-bonded silica (phenyl) columns were used, with mobile phase pH values between 2.5 and 7.4. In the absence of ion-pairing reagents the retention times for all the five compounds were the shortest at pH 4-6. The phenyl column was found to improve the separation between cephradine and ampicillin at pH values lower than 3, when these two compounds appeared as fused peaks on the C18 on C18 columns, with mobile phases both with and without ion-pairing reagents, were compared. The addition of 0.005 or 0.02 M tetraethylammonium acetate to the mobile phase did not result in significant ion-pair formation, except at pH values higher than 5.5. A strong ion-pairing effect was obtained at pH values higher than 6 with 0.005 or 0.02 M tetrabutylammonium phosphate, and the retention was decreased at pH values lower than 4. On the other hand, 0.005 M heptanesulphonic acid exhibited an ion-pair retention effect at pH values lower than 5. The molecular structures and pK(a) values were used to account for the retention behaviour of these antibiotics in the various mobile phases.

De-esterification of cephalosporin para-nitrobenzyl esters by microbial enzymes.

Bacteria and actinomycetes were screened for esterase enzymes capable of removing the para-nitrobenzyl ester from cephalosporins. An esterase preparation from Bacillus subtilis was used to prepare cephalexin and 7-ADCA from the corresponding para-nitrobenzyl esters.