Review for Analytical Methods for the Determination of Sodium Cephalothin.

Infections are the second leading cause of global morbidity and mortality, therefore it is highly important to study the antimicrobial agents such as cephalosporins. Cephalothin, an antimicrobial agent that belongs to the class of cephalosporins, has bactericidal activity and it is widely used in the Brazilian health system. In literature, some analytical methods are found for the identification and quantification of this drug, which are essential for its quality control, which ensures maintaining the product characteristics, therapeutic efficacy and patient's safety. The aim of this article is to review the available information on analytical methods for cephalothin. Thus, this study presents a literature review on cephalothin and the analytical methods developed for the analysis of this drug in official and scientific papers. It is essential to note that most of the developed methods used toxic and hazardous solvents, which makes necessary industries and researchers choose to develop environmental-friendly techniques, which will contribute to the harmonization of science, human, and environmental health.

Design of a sensitive fluorescent polarization immunoassay for rapid screening of milk for cephalexin.

In this paper we describe the development of a sensitive, fast, and easily performed fluorescence polarization immunoassay for determination of cephalexin in milk. The experimental work was performed to increase sensitivity and specificity. Therefore, the structures of the tracers were varied by synthesis of both cephalexin (CEX) and cephalotin (CET) conjugates with a variety of fluorescent labels. Two rabbit antisera containing antibodies against cephalexin and cephalotin were tested in homologous and heterologous combinations with the tracers. For every working antibody-tracer combination, the analytical conditions and cross-reactivity for structural analogues-cephalosporins and other antibiotics that could also be present in milk-were determined. It was found that the highest sensitivity was achieved by use of the homologous pair CET-EDF-anti-CET antibody (limit of detection (LOD) 0.4 µg kg(-1) for standard solutions prepared in buffer), but this combination was not appropriate because of high cross-reactivity with CET. For subsequent experiments, therefore, CEX- EDF-anti-CEX antibody were chosen (LOD 0.8 µg kg(-1) for standard solutions prepared in buffer). Part of this manuscript is devoted to the variation of precipitation agents for pretreatment of milk before analysis; milk is an extremely complicated matrix. The optimum protein precipitation agent was methanol. This technique for cephalexin determination was characterized by a limit of detection of 1 µg kg(-1). The method was validated by using naturally contaminated and spiked milk samples. The results obtained corresponded very well with those obtained by HPLC, which was used as confirmation method.

[Impurity profile study of cefalotin sodium by two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry].

A two-dimensional liquid chromatography-quadrupole time-of-flight mass spectrometry (2D-LC-QTOF MS) method to profile the impurities of cefalotin sodium was developed. A Symmetry C18 column (250 mm x 4.6 mm, 5 µm) was used in the first dimensional chromatography, with gradient elution using pH 2.5 phosphate buffer and acetonitrile as the mobile phases. The column temperature was maintained at 40 degrees C with an ultraviolet detection of 220 nm for analysis. An ACQUITY UPLC BEH C18 column (50 mm x 2.1 mm, 1.7 µm) was used in the second dimensional chromatography, with gradient elution using water containing 0.1% (v/v) formic acid and acetonitrile containing 0.1% (v/v) formic acid as the mobile phases. The column temperature was maintained at 40 degrees C. An HLB C18 column (30 mm x 2.1 mm, 20 µm) was used as the trap column. The data were collected in positive ion mode. The ion source temperature was set at 100 degrees C and the electrospray ionization (ESI) needle voltage was set at 1 000 V. The nebulizer gas temperature was set at 500 degrees C. The molecular formulas of the impurities were determined by their exact masses and isotope distributions. And the structures were determined by the protonated molecular ions and the manufacturing process of cefalotin sodium. Six impurities of cefalotin sodium were characterized and the origination of the impurities was deduced. Three of them were unknown impurities to the best of our knowledge. It was confirmed that the Chinese Pharmacopoeia 2010 has mistaken impurity A of cefalotin sodium. The results indicated that the 2D-LC-QTOF MS method could be used to investigate the impurity profile of cefalotin sodium, and it is simple and sensitive.

A prospective study on Aeromonas in outpatients with diarrhea in the central region of Rio Grande do Sul State

Aeromonas spp. were identified in five (2,7%) of 182 diarrheal stool cultures, A. caviae was predominant, resistant mainly to ampicillin and cephalotin. This is the first study showing the presence of Aeromonas spp. in diarrheal stools of outpatients in the central region of Rio Grande do Sul State, Brazil.

Tissue accumulation of cephalothin in burns: a comparative study by microdialysis of subcutaneous interstitial fluid cephalothin concentrations in burn patients and healthy volunteers.

Burn tissue sites are a potential source of bacteremia during debridement surgery. Burn injury is likely to affect the distribution of antibiotics to tissues, but direct evidence of this is lacking. The aim of this study was to directly evaluate the influence of burn trauma on the distribution of cephalothin to peripheral tissues. We used subcutaneous microdialysis techniques to monitor interstitial fluid concentrations of cephalothin in the burnt and nonburnt tissues of adult patients with severe burns following parenteral administration of 1 g cephalothin for surgical prophylaxis. Analogous simultaneous studies conducted with healthy adult volunteers provided reference tissue concentration data. Equivalent tissue exposures were seen for burn and nonburn sites, giving overall median interstitial cephalothin concentrations (from 0 to 240 min) of 2.84 mg/liter and 3.06 mg/liter, respectively. A lower overall median interstitial cephalothin concentration of 0.54 mg/liter was observed for healthy individuals, and the patient nonburnt tissue and volunteer control tissue cephalothin concentrations exhibited significantly different data distributions (P < 0.001; Kolmogorov-Smirnov nonparametric test). The duration of tissue residence for cephalothin was longer for burn patients than for healthy volunteers. The results demonstrate the potential fallibility of using healthy population models to extrapolate tissue pharmacodynamic predictions from plasma data for burn patients.

Determination of cephalosporins in solid binary mixtures by polarized IR- and Raman spectroscopy.

Quantitative IR- and Raman spectroscopic determinations of four cephalosporin antibiotics in six solid binary mixtures have been conducted. This is a new approach for spectroscopic determination of these antibiotics, since the corresponding quantitative analysis in solution only has been reported so far. The correlation coefficient r2 was found to be in the confidence intervals within 99.32-99.88% and 99.90-95.54% for the systems under study by using the absorption ratios of the characteristic bands at 800 cm(-1) and 721 cm(-1) present in the IR- and Raman spectra of the antibiotic compounds cephalexin, cephalotin, cephaloglycin and cephamandole, respectively. Solid-state linear dichroic infrared (IR-LD) spectral analysis of the solid mixtures was carried out in order to obtain experimental IR-spectroscopic assignment of the compounds studied. Independent high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis was performed for the validation of the vibrational spectroscopic data. The application of this instrumental analytical tool for the analysis of 10 tablets of the commercial products Cefamandole and Cefalotin (Actavis) was also studied.

Crystallization of cephalothin sodium during lyophilization from tert-butyl alcohol-water cosolvent system.

PURPOSE: Because cephalothin sodium (I) does not crystallize readily when freeze-dried from aqueous solutions, organic cosolvents were used to increase the crystallinity of lyophilized I. METHODS: Compound I was lyophilized from water-organic cosolvent (5% w/w) systems of each ethanol, ispropanol, and tert-butyl alcohol (TBA). RESULTS: When frozen solutions of I (10% w/w) in each of these cosolvent systems was characterized by DSC, the presence of cosolvent in the freeze-concentrate was evident. Moreover, the presence of the cosolvent accelerated the solute crystallization. This observation was based on the XRD of these systems during the various stages of freeze-drying. High initial solute concentration and annealing of frozen solutions facilitated the formation of a highly crystalline lyophile. The accelerated crystallization is attributed to supersaturation in cosolvent systems, facilitating nucleation during freezing with subsequent growth during annealing. Lyophiles obtained from water-isopropanol and water-ethanol systems collapsed, while the use of TBA as a cosolvent yielded a friable and pharmaceutically elegant cake, containing fine needle-shaped crystals of I. Gas chromatography revealed a residual TBA concentration of approximately 0.001% w/w in the crystalline lyophiles. In general, residual cosolvent levels were higher in lyophiles with lower crystallinity. CONCLUSIONS: TBA-water was found to be a suitable freeze-drying medium to promote crystallization of I and yielded a lyophile with desirable product characteristics.

Electrochemical determination of Cephalothin antibiotic by adsorptive stripping voltammetric technique.

A sensitive and reliable stripping voltammetric method was developed to determine Cephalothin antibiotic drug. This method is based on the adsorptive accumulation of the drug at a hanging mercury drop electrode and then a negative sweep was initiated, which yield a well defined cathodic peak at -625 mV versus Ag/AgCl reference electrode. To achieve high sensitivity, various experimental and instrumental variables were investigated such as supporting electrolyte, pH, accumulation time and potential, drug concentration, scan rate, convection rate and working electrode area. The monitored adsorptive current was directly proportional to the concentration of Cephalothin and it shows a linear response in the range from 4 x 10(-7) to 1.2 x 10(-6) mol l(-1) (correlation coefficient=0.9995) and the detection limit (S/N=3) is 3.3 x 10(-9) mol l(-1) at an accumulation time of 3 min. The developed AdSV procedure shows a good reproducibility, the relative standard deviation R.S.D.% (n=10) at a concentration level of 5 x 10(-7) mol l(-1) was 0.94%. Possible interferences by other pharmaceutical drugs and surfactants have been also evaluated. The applicability of this approach was illustrated by the determination of Cephalothin in pharmaceutical preparation and biological fluids such as serum and urine.

pKa calculations for class A beta-lactamases: influence of substrate binding.

Beta-Lactamases are responsible for bacterial resistance to beta-lactams and are thus of major clinical importance. However, the identity of the general base involved in their mechanism of action is still unclear. Two candidate residues, Glu166 and Lys73, have been proposed to fulfill this role. Previous studies support the proposal that Glu166 acts during the deacylation, but there is no consensus on the possible role of this residue in the acylation step. Recent experimental data and theoretical considerations indicate that Lys73 is protonated in the free beta-lactamases, showing that this residue is unlikely to act as a proton abstractor. On the other hand, it has been proposed that the pKa of Lys73 would be dramatically reduced upon substrate binding and would thus be able to act as a base. To check this hypothesis, we performed continuum electrostatic calculations for five wild-type and three beta-lactamase mutants to estimate the pKa of Lys73 in the presence of substrates, both in the Henri-Michaelis complex and in the tetrahedral intermediate. In all cases, the pKa of Lys73 was computed to be above 10, showing that it is unlikely to act as a proton abstractor, even when a beta-lactam substrate is bound in the enzyme active site. The pKa of Lys234 is also raised in the tetrahedral intermediate, thus confirming a probable role of this residue in the stabilization of the tetrahedral intermediate. The influence of the beta-lactam carboxylate on the pKa values of the active-site lysines is also discussed.

In situ degradation: a new concept for system suitability tests in monographs of the European Pharmacopoeia.

Monographs of the European Pharmacopoeia describe in the LC-test for related substances usually a system suitability test in order to ensure the adequate separation of impurities. Since the reference substances required are often not available a recent approach to avoid this problem is the generation of the required impurity by 'in situ degradation' of the active principle. This paper describes some typical applications of this technique as well as recent examples, such as the controlled degradation of cefalotin sodium, imipenem and spiramycin.

Spectrofluorimetric study of the degradation of alpha-amino beta-lactam antibiotics catalysed by metal ions in methanol.

The alpha-aminopenicillins ampicillin and amoxicillin and a cephalosporin, cephalothin, give rise to a fluorescent product when their methanolic solutions are incubated for prolonged time periods. The process also occurs in the presence of the metal ions Cd2+, Co2+ and Zn2+. The effects of the different ions on the emission and excitation wavelengths and the appearance rate of the fluorophore were studied. The appearance of the fluorescent product was zero order for ampicillin and amoxicillin in metal ion-free solution and solutions with Cd2+ and Zn2+, whereas in the presence of Co2+ ion it was first order under the experimental conditions used; for cephalothin it was first order in all cases. Apparent fluorescent compound formation rates were calculated in the zero-order reactions and rate constants in the first-order reactions. The activation energy of the formation reaction of the fluorescent products of amoxicillin and ampicillin was calculated from a study of the reactions at four temperatures; all the values recorded were between 34 and 118 kJ mol-1. As a possible mechanism for the formation of these products, cyclization of the penamaldic derivative of the antibiotic, which is formed in the first stage of the methanolytic reaction, is proposed.

Analysis of binary mixtures of cephalothin and cefoxitin by using first-derivative spectrophotometry.

A method for the analysis of two-component mixtures of cephalothin and cefoxitin using zero-crossing first-derivative spectrophotometry is described. This technique permits the quantification of these drugs with closely overlapping spectral bands without any separation step. Linear calibration graphs of first-derivative values at 235.00 and 236.75 nm for cephalothin and cefoxitin, respectively, with negligible intercepts were obtained versus concentration in the range 4.0-32.0 micrograms ml-1 for both antibiotics. This paper presents a systematic examination of the experimental data by applying an exhaustive statistical analysis to demonstrate the validity of the method. The results of the determination of these antibiotics in mixtures of injectable dosage forms are also presented, together with their determinations in physiological serum and glucosed physiological serum.

[New method for selectivity optimization in thin layer chromatography and its application in pharmaceutical analysis].

A new experimental design method named the step climbing method (SCM), as well as a new global criterion (NRC), are proposed for the selectivity optimization of thin layer chromatography. The method offers chromatographers a convenient means to decide the optimum developer composition for thin layer chromatography. The principle of the method is described, and examples of applications in pharmaceutical analysis are given to certify the feasibility of the method. Clearly, it may also be used for the selectivity optimization in high performance liquid chromatography.

[Non-crystallinity determination by thermal analysis of freeze-dried cephalothin sodium].

Cephalothin sodium (CET-Na) prepared according to the conventional freeze-drying methods is known to easily develop color during storage. Since amorphous CET-Na has been reported to be markedly unstable, the color development is thought to be due to the presence of traces of CET-Na in an amorphous state observed with scanning electron microscopy. Quantitation by use of the powder X-ray diffractometry of such traces of amorphous CET-Na has proved to be of little use. Thermogravimetry (TG) and differential scanning calorimetry (DSC) have demonstrated that the freeze-dried CET-Na by the conventional methods contains three types of CET-Na: amorphous (unstable phase), quasi-crystalline (metastable phase) and crystalline (stable phase). Pyrolysis initiation temperatures of these three types of CET-Na have been demonstrated to become higher in this order. A new parameter for the evaluation of non-crystallinity of CET-Na has been introduced, in which the ratio is calculated from the data of the total weight loss observed through the pyrolysis of both amorphous and quasi-crystalline CET-Na against the total weight loss of all components during the pyrolysis of sample specimen. The ratio thus calculated is defined as "non-crystallinity". This new parameter has successfully been introduced to establish a good correlation to the degree of increasing color intensity with aging of freeze-dried CET-Na.

Potentiometric determination of cephalothin.

The composition of a pseudo-liquid potential-determining phase for the cephalothin-selective electrode has been determined and the following basic electrode parameters were examined: measurement range, slope, limit of detection, selectivity and lifetime. This paper discusses the effect of side-chain substituents on the electrode properties, the electrode having been used for cephalothin determination in the range 43-436.5 mg l-1 (standard deviation, 0.64-4.5 mg l-1).

Zero-crossing derivative spectrophotometry for the determination of mixtures of cephaloridine and cephalothin in pure and dosage forms.

First- and second-derivative spectrophotometry, with a zero-crossing technique of measurement, has been used for the quantitation of two-component mixtures of cephaloridine and cephalothin Na, which are cephalosporins with closely overlapping spectral bands. Beer's Law is followed for up to 28 and 36 micrograms/mL of cephaloridine in the first- and second-derivative modes, respectively, and up to 36 micrograms/mL of cephalothin Na in both modes. Detection limits at the 0.05 level of significance were calculated to be 0.13 and 0.37 micrograms/mL of cephaloridine and cephalothin Na, respectively, in the first-derivative mode, and 0.25 and 0.29 micrograms/mL, respectively, in the second-derivative mode. The recovery of these antibiotics in mixtures of injectable dosage forms is also reported.

Cephalothin: hydrolysis of the C-3'-acetoxy moiety of a 7-aminocephalosporanic acid; observation of both acyl-oxygen bond cleavage and reversible alkyl-oxygen bond cleavage.

The aqueous solution chemistry of the C-3'-acetoxy moiety of cephalothin sodium (1b) was examined with the use of isotopically labeled H2(18)O and [2-13C]acetate anion. The 18O incorporation studies indicate that the hydrolysis (at pH 4.7 +/- 0.1) of 1b to the deacetyl derivative of cephalothin (2b) proceeds via two pathways: alkyl-oxygen bond cleavage (55-63%) and acyl-oxygen bond cleavage accounting for the remainder. The incorporation of [2-13C]acetate into 1b suggests that the alkyl-oxygen cleavage pathway is a reversible reaction.

How certain drugs interfere negatively with the Jaffé reaction for creatinine.

Phenacemide and cephalothin may interfere either positively or negatively with the determination of creatinine by the Jaffé reaction, depending on the analytical system. The drug-picrate chromophores maximally absorb within the first minute of reaction (21 s for phenacemide, 45 s for cephalothin), after which the absorbances decrease. Thus, these drugs negatively interfere in analytical systems involving kinetic measurements when the time interval of measurement includes the period of decreasing absorbance.