Integrative strategy to determine residual proteins in cefaclor produced by immobilized penicillin G acylase.

There is a growing trend in the pharmaceutical industry towards substituting conventional chemical synthesis routes of semi-synthetic ß-lactam antibiotics (SSBAs) through environmentally sustainable enzymatic processes. These have advantages such as cost reduction in terms of solvent and waste treatment and time saving owing to fewer reaction steps. Penicillin G acylase (PGA) is an industrially important enzyme that is mainly used to catalyze the synthesis of SSBAs. In this study, we established an integrative strategy using three different analytical methods for determining the PGA-associated residual protein content, which is a critical quality issue in the end product. Cefaclor was taken as representative example of SSBAs. High-performance liquid chromatography coupled with fluorescence detection (HPLC-FD) allowed the routine analysis of PGA residual proteins and other low molecular weight (MW) impurities with high detection specificity and sensitivity, comparable to those of the Bradford assay and microfluidic protein chip electrophoresis. However, these latter two methods were superior for quantitative and qualitative analysis, respectively, and should be regarded as necessary adjuncts to the HPLC-FD method. By combining the three methods, trace levels of residual proteins were detected in four (out of 13) cefaclor bulk samples from two different manufacturers, with a major protein MW of ∼63 kDa. This suggests that the higher MW PGA subunit tends to persist in the end product. The integrative determination strategy described here can be used to evaluate SSBA bulk samples and monitor the process of SSBA manufacturing by enzymatic methods, especially in terms of inter-batch consistency and process stability.

Chromatographic studies of unusual on-column degradation of cefaclor observed in the impurity separation by HPLC.

An unpredictable ghost peak was intermittently observed during the impurity separation of cefaclor and formulation by high performance liquid chromatography (HPLC) with a content from below the reported threshold to approximately 0.3% in different laboratories. Through a series of investigations, the ghost peak was identified as an unusual on-column degradant of cefaclor formed under elevated column temperature but was not an actual sample impurity. The chemical structure of the degradant was determined by spectroscopic methods, including high resolution mass spectrometry (HRMS) and 1H-NMR. Consequently, the unknown peak was identified as a C-4 oxidative decarboxylation analog of cefaclor. The formation mechanism of the analog is proposed, and it is suggested that elevated column temperature during HPLC analysis has a profound effect on the degradation. Dissolved oxygen in the mobile phase may promote the formation of the ghost peak. The degradation can be suppressed by using a column temperature below 30 °C. Moreover, several other prevention measures are suggested based upon the results of the investigation.

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.

A comparative study of capillary zone electrophoresis and pH-potentiometry for determination of dissociation constants.

Acidity constants of six cephalosporin antibiotics, cefalexin, cefaclor, cefadroxil, cefotaxim, cefoperazon and cefoxitin are determined using capillary zone electrophoresis (CZE) and pH-potentiometric titrations. Since CZE is a separation method, it is not necessary for the samples to be of high purity and known concentration because only mobilities are measured. The effect on determination of dissociation constants of different matrices (serum, 0.9% NaCl, fermentation matrix) was examined. The advantages of CZE can be utilized in those fields where potentiometry has limitations (sample quantity, solubility, purity, simultaneous determinations), although pK(a) values that are close to each other can be determined by potentiometry with more accuracy.

Measurement of cefaclor and amoxicillin-clavulanic acid levels in middle-ear fluid in patients with acute otitis media.

Concentrations of cefaclor (CFC) or amoxicillin-clavulanic acid (AMX/CA) in middle-ear fluid collected preserving the stability and clearing the cell contents has been compared to those obtained using the traditional method. Sixty-seven children with effusive otitis media were treated orally with CFC (20 mg/kg of body weight) or AMX/CA (20 mg/kg) (4:1 ratio). The concentrations in cell-free fluid (C-) appear higher than those in the total fluid (C+) (as assayed traditionally).

Sensitive determination of a beta-lactam antibiotic, cefaclor by liquid chromatography with chemiluminescence detection.

We present a sensitive method for the determination of cefaclor (CCL), a beta-lactam antibiotic. It was based on the chemical derivatization of the drug with 4-(2'-cyanoisoindolyl)phenylisothiocynate (CIPIC) under the reaction conditions with heating at 80 degrees C for 7 min in the presence of pyridine. The CIPIC reagent could react with the primary amino group of the drug to form the CIPIC-conjugated CCL. The derivatives emitted not only fluorescence (FL) at maximum emission wavelength of 410 nm with irradiation at 310 nm, but also chemiluminescence (CL) in the presence of H(2)O(2), borate buffer (pH 9.6) and acetonitrile. After separation of the CIPIC derivatives of CCL and cephradine as internal standard in human serum by reversed-phase liquid chromatography, the derivatives could be monitored with both FL and CL detections. The detection limit (S/N=3) in the chromatograph was 1 pmol by the CL detection and 10 pmol by the FL detection. The proposed CL method permitted the most sensitive determination of CCL in the human serum after its oral administration.

Determination of cefaclor by selective sample enrichment/clean-up on silica gel bonded polyelectrolyte in ion-exchange column chromatography.

A silica gel-bound cationic polyelectrolyte, poly[N-chloranil N,N,N',N'- tetramethylethylene diammonium dichloride], modified as ion-exchanger capable of molecular recognition of beta-lactam antibiotic, was used in solid phase extraction through column chromatography for a sample clean-up and enrichment of analyte from a dilute solution. The optimum and selective sorption conditions for a model antibiotic, cefaclor, were established. The high selectivity of polymer at pH 9.5 and flow rate as high as 5 ml/min were observed for the quantitative sorption of cefaclor. The desorption by 0.1 N HCl at flow rate of 0.1 ml/min and subsequent heating at 80 degrees C for 2 h allowed the antibiotic to be detected as corresponding oxazolone form in UV-spectrophotometric and differential pulse adsorptive stripping voltammetric measurements. The potential of the suggested approach was illustrated by estimating cefaclor in urine and blood plasma samples.

Indirect polarographic and cathodic stripping voltammetric determination of cefaclor as an alkaline degradation product.

Cefaclor is not reducible at a mercury electrode, but it can be determined polarographically and by cathodic stripping voltammetry as its initial alkaline degradation product which is obtained in high yield by hydrolysis of cefaclor in Britton-Robinson (B-R) buffer pH 10 at 50 degrees C for 30 min (reduction peak at pH 10, -0.70 V). Differential pulse polarographic calibration graphs are linear up to at least 1 x 10(-4) mol/l(-1). Recoveries of 93% of the cefaclor (n = 3) were obtained from urine spiked with 38.6 microg/ml(-1) using this polarographic method with 1 ml urine made up to 10 ml with pH 10 buffer. Using cathodic stripping voltammetry and accumulating at a hanging mercury drop electrode at - 0.2 V for 30 s, linear calibration graphs were obtained from 0.35 to 40 microg/ml(-1) cefaclor in B-R buffer pH 10. A relative standard deviation of 4.2% (eta = 5) was obtained, and the limit of detection was calculated to be 2.9 ng/ml(-1). Direct determination of cefaclor in human urine (1 ml of urine was made up to 10 ml with pH 10 buffer) spiked to 0.39 microg/ml(-1) was made (recovery 98.6%).

Spectrofluorometric determination of alpha-aminocephalosporins in biological fluids and pharmaceutical preparations.

A selective and highly sensitive fluorometric method was developed for the determination of four alpha-aminocephalosporins, namely cefaclor, cefadroxil, cephalexin and cephradine. The method involves the reaction of the target compounds with fluorescamine at a specific pH, ranging from 7.8 to 8.4. The produced derivatives exhibit maximum fluorescence intensities at 472-478 nm after excitation at 370-372 nm. The method is highly specific because other alpha-aminocephalosporins whose alpha amino group was blocked do not react similarly and hence do not interfere. At the specific pH range of the reaction where no degradation may occur with that medium the proposed method can be utilised as a stability-indicating assay. The different experimental parameters affecting the derivatisation reaction were carefully studied and incorporated into the procedure. Under the described conditions, the proposed method is linear over the concentration range of 0.05(-1) microg/ml(-1) for both cefaclor and cephalexin, and 0.05-0.65 and 0.025-0.5 microg/ml(-1) for cefadroxil and cepharadine, respectively and the coefficients of determination were greater than 0.999 (n = 3). The recoveries of the title compounds from spiked serum ranged from 88.6 to 89.7% and from spiked urine from 92.2 to 93.3% with a limit of quantitation (LOQ) of 25-50 ng/ml(-1) and limit of detection (LOD) of 5 ng/ml(-1) (S/N = 2) for all drugs. The mechanism of the fluorometric reaction is proposed and the advantages of the proposed method are discussed.

[Studies on the simultaneous measurement of several cephalosporins by RP-HPLC (I)].

This paper reported the research on the simultaneous separation and determination of six cephalosporins by RP-HPLC. Six cephalosporins are cefalcor, cefalexin, cefradine, cefadroxil, cefominox and cefoxitin. The analytical conditions for this method were as follows: a Hypersil ODS C18(200 mm x 4.6 mm i.d., 5 microns), detection wavelength: 254 nm; a mobile phase solution of 50 mmol/L monopotassium phosphate (pH 3.4)-acetonitrile (87.5:12.5) and DAD detector. The flow rate was 1.0 mL/min. The calibration curves of the six compounds were linear, the correlation coefficients were 0.9951 for cefominox, 0.9999 for the others, the range were 164 ng-16.4 micrograms for cefominox, 99 ng-9.934 micrograms for cefadroxil, 104 ng-10.358 micrograms for cefalcor, 122 ng-12.224 micrograms for cefalexin, 107 ng-10.702 micrograms for cefradine and 115 ng-11.506 micrograms for cefoxitin. The recovery rates were 103.5% for cefominox, 99.3% for cefadroxil, 101.4% for cefalcor, 101.5% for cefalexin, 98.7% for cefradine and 97.6% for cefoxitin. Six cephalosporins were all stable in 50 mmol/L monopotassium phosphate (pH 3.4-4.6). When preparations of these cephalosporins were determined, it is indicated there were no difference between the results by using this method and the pharmacopoeia methods. The total separation time of these cephalosporins was within fifteen minutes. This method is simple, sensitive, rapid and accurate.

HPTLC assay of cephalexin and cefaclor in pharmaceuticals.

A simple and reliable HPTLC method for the simultaneous determination of cephalexin and cefaclor is developed and validated. The methanol-ethyl acetate-acetone-water (5:2.5:2.5:1.5 v/v/v/v) solvent system is used for the quantitative evaluation of chromatograms. The chromatographic zones, corresponding to the spots of cephalexin and cefaclor on the silica gel plates, are scanned in the reflectance/absorbance mode at 265 nm. The method is found to be reproducible and convenient for the quantitative analysis of cephalexin and cefaclor in its dosage forms.

Analytical investigation of beta-lactam antibiotics in pharmaceutical preparations. IX. Colorimetric determination of six cephalosporins of second and third generation in the range of micromolar concentrations.

A sensitive, accurate, precise and the same time simple and rapid method for the colorimetric determination of some cephalosporins of the second and third generations, such as: cefoxitin sodium (CFXT), cefaclor (CFCL), cefamandole nafate (CFMD), ceforanide l-lysine (CFRN), cefotaxime sodium (CFTX), and cefurozime sodium (CFRX) was described. The new method proposed is based: a) On the reduction of Fe(III) to Fe(II) by the drug analysed and b) On complexation of Fe(II) formed with o-Phenanthroline (O-Phen) consistently the formation of the well known highly stable orange-red coloured chelate complex [Fe(II)-(o-Phen)3]2+ which exhibits an absorption maximum at lambda = 510 nm (pH 4.50 +/- 0.2). Beer's law is obeyed for: 1.0 - 37.5 microgram mL-1 for CFX, 1.0 - 25.0 microgram mL-1 for CFMD, CFRN, and CFTX and 2.0 - 37.5 microgram mL-1 for CFTX and CFCL, while the apparent molar absorptivity ( epsilon in L mol-1cm-1) and the Sandell's sensitivity in (ngcm-2) both referred to the drug analyzed, are 1.29 x 10(4); 34.7 (CFXT), 7.61 x 10(3); 50.7 (CFCL), 3.33 x 10(4); 15.4 (CFMD), 2.60 x 10(4); 17.6 (CFRN) respectively. The regression line equation for each one of the above studied cephalosporins were calculated with a correlation coefficient 0.9997 < r < 1.0000; the accuracy and the precision of the method was considered as very satisfactory, while the results of a statistical analysis by means of the Student's t-test and the variance ratio F-test prove that no significant difference was observed between the results of the proposed method and those of official one.

Pharmaceutical properties of loracarbef: the remarkable solution stability of an oral 1-carba-1-dethiacephalosporin antibiotic.

Loracarbef is an oral 1-carba-1-dethiacephalosporin antibiotic structurally related to cefaclor. Like many beta-lactam antibiotics, loracarbef exists in several hydrated crystalline forms. The pH-solubility profile curve for loracarbef monohydrate is U-shaped, resembling those of other zwitterionic cephalosporins. Loracarbef was found to be much more stable in solution than cefaclor. For example, in pH 7.4 phosphate buffer, loracarbef was unexpectedly found to be 130-150 times more stable than cefaclor and 10-12 times more stable than cephalexin, depending on the phosphate concentration. The pH-stability profile is U-shaped, similar to that of other zwitterionic cephalosporins, and shows maximum stability at the isoelectric point. At any given pH, loracarbef is more stable in solution than any other therapeutically useful cephalosporin. Acetate, borate, citrate, and especially phosphate buffers have catalytic effects on the rate of loracarbef hydrolysis.

High-performance liquid chromatographic determination of cephalosporin antibiotics using 0.3 mm I.D. columns.

Four cephalosporins, cefazolin, ceftizoxime, cefaloridine and cefaclor, were determined using a novel microbore high-performance liquid chromatographic system designed to be entirely compatible with direct liquid interfacing (DLI) for mass spectrometric analysis. The chromatographic support was a 5-micron C18 column of 0.3 mm I.D., compared with the usual microbore column diameters of 1-2 mm. The mobile phase contained no buffers or salts which may have caused column blocking or mobile phase crystallization, and the use of a concentration column allowed the injection of large volumes of analyte (up to 500 microliters). The assay was reproducible, the relative standard deviations being less than 20% within-day and between-day for all the drugs. The detection limit for cefaloridine and cefazolin was 1 ng and for cefaclor and ceftizoxime 5 ng.

Solid-state chemistry and crystal structure of cefaclor dihydrate.

Cefaclor [7-(D-2-amino-2-phenylacetamido)-3-chloro-3-cepham-4-carboxy lic acid] crystallizes as the dihydrate. Crystals belong to space group P21, with a = 10.626(3), b = 7.1288(9), c = 14.124(3), and beta = 121.6(2). The structure was solved using direct methods and refined to an R of 0.0535. The bond lengths, angles, and conformation determined are as expected for cephalosporins. The two water molecules are held in the crystal differently. The 13C solid-state NMR spectrum of cefaclor dihydrate is also reported and is consistent with its crystal structure.

Micellar modification of drug stability: analysis of the effect of hexadecyltrimethylammonium halides on the rate of degradation of cephaclor.

The intra- and intermolecular rates of degradation of cephaclor were determined with and without hexadecyltrimethylammonium bromide (CTABr). Micellar-derived spectral shifts were used to measure the association of the ionic forms as well as to determine the effect of CTABr on the apparent acid dissociation constant of the antibiotic. The rate of degradation of cephaclor increased with detergent and was salt sensitive. Micellar effects were analyzed quantitatively within the framework of the pseudophase ion exchange model. All experimental data were fitted to this model which was used to predict the combined effects of pH and detergent concentration. Micelles increased the rate of OH- attack on cephaclor; most of the effect was due to the concentration of reagents in the micellar pseudophase. The intramolecular degradation was catalyzed 25-fold by micelles, and a working hypothesis to rationalize this effect is proposed. The results demonstrate that quantitative analysis can be utilized to assess and predict effects of detergents on drug stability.

Concentrations of antimicrobial agents in middle ear fluid, saliva and tears.

Antibiotics concentrations in middle ear fluid (MEF), saliva and tears were measured in children with persistent middle ear effusions undergoing tympanostomy tube placement. In 31 children given cefaclor, specimens of serum, saliva and MEF were collected at 0.5, 1, 2, 3 or 5 h after a dose. Another group of 37 children were randomized to receive a single dose of penicillin V, amoxicillin, ampicillin, erythromycin estolate, erythromycin ethylsuccinate, trimethoprim-sulfamethoxazole or cefaclor. Concentrations of antibiotics in saliva and tears bore no consistent relationship to those in MEF. Mean concentrations of all drugs in MEF were several-fold greater than the usual minimal inhibitory concentrations (MIC) of pneumococci, but only with trimethoprim and cefaclor were they greater than in usual MIC's for Haemophilus influenzae. Concentrations of antibiotics in MEF in persistent effusions were comparable to those previously reported in acute purulent effusions.

Concentration of cefaclor in human prostatic tissue.

Concentration of cefaclor, a new oral cephalosporin, was measured in prostatic tissue of ten patients undergoing suprapubic prostatectomy. The average prostatic tissue concentrations were 0.51 +/- 0.22 microgram and 0.74 +/- 0.67 microgram per gram of tissue following the oral administration of 250-milligram and 500-milligram doses, respectively. The prostate/plasma ratios of cefaclor were approximately 0.7 indicating no evidence of accumulation of the drug in prostatic tissue. Levels of cefaclor achieved in human prostatic disease are equal to or less than the minimum inhibitory concentration of strains of known facultative bacterial pathogens associated with prostatitis.