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.

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.

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.

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.

Antibiotic levels in pericardial fluid.

An experimental model was designed to study the ability of antibiotics to enter the pericardial compartment. Noninfected and infected pericardial fluid and serum antibiotic activities were determined in adult mongrel dogs before and at intervals after antibiotic administration. After the administration of penicillin G, methicillin, cephaloridine, streptomycin, or gentamicin, clinically adequate antibiotic levels in the noninfected pericardial fluid were obtained within 1 h, and these levels approached or exceeded the serum levels within 2-4 h. Antibiotic levels obtained from infected dog pericardial fluids were higher than those from noninfected animals. Patients' serum and pericardial fluid antibiotic levels were measured after penicillin G, penicillin V, cephalothin, and gentamicin administration. We have found, both in the canine and human studies, that pericardial antibiotic levels taken at least 2 h after antibiotic administration are almost identical to those in the blood.

[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.

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.

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.

Rapid sensitive fluorometric analysis of cephalosporin antibiotics.

A rapid and sensitive fluorometric analysis for cephalosporins, which can also be applied to penicillins, is presented. The method involves reaction with 0.1 N sodium hydroxide at 100 degrees, producing stable fluorescent products. This method was applied to cephalexin and ampicillin with detection at concentrations as low as 0.01 mug/ml.

Quantitation of carbenicillin disodium, cefazolin sodium, cephalothin sodium, nafcillin sodium, and ticarcillin disodium by high-pressure liquid chromatography.

High-pressure liquid chromatographic (HPLC) methods for the quantitation of carbenicillin, cefazolin, cephalothin, nafcillin, and ticarcillin were developed. The stability of 2% solutions of the antibiotics in normal saline and in 5% dextrose in water were studied at 24 and 5 degrees. The assays were conducted using a previously reported colorimetric method, and some assays also were performed using HPLC. For discolored solutions of cephalothin, the colorimetric method was not stability indicating. The percent relative standard deviations by HPLC based on six injections were 1.69, 0.94, 1.30, 1.59, and 1.6 for carbenicillin, cefazolin, cephalothin, nafcillin, and ticarcillin, respectively. Both carbenicillin and ticarcillin apparently may be mixtures of two isomers at equilibrium with each other. The shelflives recommended by the manufacturers at 5 degrees may be too conservative.

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.

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.

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.

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.

Photometric extraction method for determination of cefalotin.

A selective, accurate and precise photometric method for the determination of cefalotin (1) is proposed. The method is based on a preliminary hydrolysis of the antibiotic in a strong sulphuric acid medium at 100 degrees C for 15 min. The 2-thienylacetic acid (2) formed as degradation product is determined photometrically with ninhydrin in strongly acidic solution. In these conditions 2 forms a yellow reaction product, which is extracted with chloroform and the absorbance measured at 423 nm. The reaction is practically specific as none of the initial substances used in the preparation of cefalotin or of its degradation products (except 2) reacts with ninhydrin under these conditions. The only other antibiotic which also gives a coloured product with ninhydrin in this strongly acidic medium is cefalexin. The latter however does not interfere at its coloured product absorbs at 520 nm. The method is simple, fast and with sufficient accuracy and precision (coefficient of variation 0,57--0,88% and relative error 0,41--0,64%).

[Photometric determination of cefalotin in biological fluids (author's transl)]. / Fotometrische bestimmung von cefalotin in biologischen flüssigkeiten.

The authors describe a simple chemical method for the determination of cefalotin in serum and urine. Cefalotin is submitted to acid hydrolysis; the hydrolysate thus obtained reacts with ninhydrin to form a condensation product, the absorption maximum of which lies at 440 nm.

Hydrolysis of cephalosporins in strongly acid medium.

The hydrolysis of cephalexin, cephalothin and cephaloridine in strongly acid medium (10--25% sulfuric acid) at high temperature (approximately 100 degrees C) was studied. The hydrolysis proceeds with a high rate (10--15 min are necessary for complete hydrolysis) and results in definite products with low mol.-wt. The hydrolysis under such hard conditions affects the side chain in 3-position, in some cases the C--N bond in the amido group in 7-position, and leads to a total cleavage of the beta-lactam ring, giving thiazine compounds. The reaction products were identified using IR and UV spectral data, thin-layer and gas chromatography, and some characteristic reactions of functional groups. The results of the investigation were used for development of photometric procedures for determination of cephalosporins.

Comparative biliary concentrations of cephazolin and cephalothin in patients with biliary tract disease.

The concentration of cephazolin in the serum, gall bladder bile, common duct bile, and gall bladder wall were consideredably higher than cephalothin especially with IV administration and indicate that cephazolin should be a useful antibiotic in the surgical treatment of acute cholecystitis.

[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.

[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.