Spectrophotometric Determination of Cephalosporins in Pure Form and in Pharmaceutical Preparations

Abstract

A spectrophotometric determination is described for cephalosporins, offering adequate sensitivity and good precision. The procedure applies successfully to a wide variety of cephalosporins, also in pharmaceutical preparations: cephalothin, cefacetrile, cephapirin, cefotaxime, ceftizoxime, cephaloridine, cefazolin, cefamandole nafate, cephalexin, cefadroxil, cefoxitin and cefuroxime. The method employs a reaction with ammonium molybdate in sulphuric acid medium. The antibiotic is heated at 91.5°C for 15 min and the absorbance of the coloured product is measured at 670 nm against a reagent blank treated similarly. Beer's law is obeyed up to 125 to 150 μg of cephalosporin in the 5-ml final solution. The effect of reagent concentration and reaction conditions are discussed.     

Selective Densitometric Analysis of Cephalosporins Using Dragendorff’s Reagent

A simple, selective, precise, and stability-indicating thin-layer chromatographic method has been developed and validated for analysis of the cephalosporins cefpodoxime proxetil, ceftriaxone sodium, ceftazidime pentahydrate, cefotaxime sodium, cefoperazone sodium, cefazolin sodium, and cefixime in the bulk drug and in pharmaceutical formulations. TLC was performed on aluminium sheets precoated with silica gel G 60F₂₅₄ as stationary phase. The mobile phases chosen for development gave compact spots for all the drugs (R _F values 0.43–0.60). The separated compounds were visualized as orange spots by spraying with Dragendorff’s reagent. Linear regression analysis data for the calibration plots revealed good linear relationships between response and amounts of the drugs with correlation coefficients ranging from 0.9977 to 0.9998 and determination coefficients ranging from 0.9954 to 0.9996 over the concentration ranges 5–25 μg per spot for cefpodoxime proxetil, ceftriaxone sodium, and ceftazidime pentahydrate and 10–50 μg per spot for cefotaxime sodium, cefoperazone sodium, cefazolin sodium, and cefixime. The method was validated for precision, recovery, and robustness. Limits of detection and quantitation for the drugs ranged from 0.35 to 2.48 and from 1.07 to 7.50 μg per spot, respectively. The method was successfully applied to analysis of the drugs in their pharmaceutical dosage forms with good precision and accuracy. The method can also be used as a stability-indicating assay.     

Use of ammonium molybdate in the colorimetric assay of cephalosporins

A colorimetric method for the determination of five cephalosporins (cefoxitin sodium, cefotaxime sodium, cephapirin sodium, cephalothin sodium and cephaloridine), based on the blue colour formed by reaction of the cephalosporins with ammonium molybdate, is described. The effects of reagent concentration and reaction conditions are discussed. The proposed method has been applied to the analysis of cephalosporin injections, the results of which are in good agreement with those obtained by the official method of the British Pharmacopoeia.     

Synthesis and antibacterial activity of copper(II) complexes with sulphathiazole and cephalosporin ligands

Copper(II) reacts with cephalosporins plus sulphathiazole (Hstz) to form the following mixed complexes: [Cu(cefazol)(stz)(H₂O)], [Cu(cephalot)(stz)(H₂O)₂], [Cu(cefotax)(stz)], [Cu(ceftria)(Hstz)] and [Cu(cefepime)(stz)Cl] (Hcefazol = cefazolin, Hcephalot = cephalothin, Hcefotax = cefotaxime and H₂ceftria = ceftriaxone) which were characterized by physicochemical and spectroscopic methods. The spectra indicated that most of the cephalosporins are probably acting as monoanionic multidentate chelating agents, the exception being ceftriaxone which is dianionic. The complexes are insoluble in water and common organic solvents and probably have polymeric structures. They have been screened for activity against several bacteria, and the results are compared with the activity of cephalosporins.     

Polarographic analysis of some cephalosporin antibiotics

Summary The cephalosporin derivatives moxalactam, cefazolin, cefuroxime, ceftriaxone and cefotaxime were studied by direct current, sampled direct current, differential pulse polarography and cyclic voltammetry. While moxalactam and cefazolin give rise to one reduction wave, cefuroxime, ceftriaxone and cefotaxime exhibit two reduction waves. The characteristics of corresponding electrode reactions and their analytical parameters are presented and discussed. Optimum pH-ranges for the determination of the five cephalosporins are given. Linear concentration ranges varied from 0.1 to 200 g/ml and limits of determination were of the order of 0.01 to 0.04 g/ml depending on the compound of interest. The precision of the proposed method is excellent with relative standard deviation around 1.3% at a concentration of 0.5 g/ml for all investigated cephalosporins.

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Polarographische Analyse einiger Cephalosporin-Antibiotica

     

Methodological approach to development of enzymatic technologies for semisynthetic betalactam antibiotic production

The algorithm for the research and design of optimized processes for synthesis of semisynthetic betalactam antibiotics with the use of peptidases with mechanism of action based on the acylenzyme interm ediate formation as a catalyst was formulated. The applicability of the proposed approach to the development of the processes for enzymatic synthesis of cefoxitin and cefazolin, semisynthetic cephalosporins, as an example, was demonstrated.     

Optimization of separation and migration behavior of cephalosporins in capillary zone electrophoresis

The influences of buffer pH, buffer concentration and buffer electrolyte on the migration behavior and separation of 12 cephalosporin antibiotics in capillary zone electrophoresis using three different types of buffer electrolyte, including phosphate, citrate, and 2-(N-morpholino)ethanesulfonate (MES), were investigated. The results indicate that, although buffer pH is a crucial parameter, buffer concentration also plays an important role in the separation of cephalosporins, particularly when cefuroxime and cefazolin, cephalexin and cefaclor, or cefotaxime and cephapirin are present as analytes at the same time. The electrophoretic mobility of cephalosporins and electroosmotic mobility measured in citrate and MES buffers are remarkably different from those measured in phosphate buffer. With citrate buffer, optimum buffer concentration is confined to a small range (35-40 mM), whereas buffer concentrations up to 300 mM can be used with MES buffer. Complete separations of 12 cephalosporins could be satisfactorily achieved with these three buffers under various optimum conditions. However, the separability of 12 cephalosporins with citrate or MES buffer is better than that with phosphate buffer. As a consequence of a greater electrophoretic mobility of cephalosporins than the electroosmotic mobility with citrate buffer at pH below about 5, some cephalosporins are not detectable. The cloudiness of the peak identification and of the magnitudes of the electrophoretic mobility of cefotaxime and cefuroxime reported previously are clarified. In addition, the pKa values of cephradine, cephalexin, cefaclor, and cephapirin attributed to the deprotonation of either an amino group or a pyridinium group are reported, and the migration behavior of these cephalosporins in the pH range studied is quantitatively described.     

An Isocratic HPLC Method for the Determination of Cephalosporins in Plasma

Abstract

An isocratic reversed-phase liquid chromatographic method for the determination of eight cephalosporins in human plasma using UV detection at 254 nm is described. Plasma proteins were precipitated using acetonitrile prior to injection of a 10 μl aliquot onto an octadecylsilane column. The mobile phases consisted of 6–11% acetonitrile in sodium dihydrogen phosphate (0.01M). The minimum detectable limit for each drug was less than 1 γg/ml of plasma. Possible interference from other drugs which might be administered concurrently is discussed. The reproducibility and precision of the method for cephalosporin assay are shown from the analysis of plasma containing 5–500 γg/ml of plasma. The chromatographic behavior of the eight cephalosporins was examined by varying mobile phase conditions.     

Spectrophotometric Assay of Certain Cephalosporins Based on Formation of Ethylene Blue

Abstract

The hydrolytic degradation of antibiotics is very often used as a preliminary step in the analytical procedures for their determination. Therefore, a procedure was developed for measuring small amounts of cefadroxil and cefotaxime in pure samples as well as in formulations.

The method depends on forming a vis-absorbing compound with N,N-diethyl-p-phenylenediamine sulphate (N,N-DPPD) (ethylene blue dye), after the hydrolysis of cefadroxil and cefotaxime in sodium hydroxide solution to give hydrogen sulphide. The method is selective for cephalosporins, since other β-lactam compounds such as penicillins do not give hydrogen sulphide under alkaline hydrolysis. Variables such as pH, temperature, reagent concentrations and stability of the colour produced have been evaluated to permit selection of the most advantageous technique. Beer's law obeyed over the concentration range 0.5–10 μg/ml and 0.5–7 μg/ml for cefadroxil and cefotaxime, respectively. The detection limit being 0.1 μg/ml and 0.05 μg/ml (defined as the amount of the drug that gave a signal of twice the background noise) for cefadroxil and cefotaxime, respectively. The method has been successfully applied to the analysis of some pharmaceutical formulations.

The results have been statistically compared with those obtained by the official method. The relative standard deviation (for 10 replicates) was 1.12 (9 μg/ml) and 0.49% (5 μg/ml) for cefadroxil and cefotaxime, respectively. Procedural details and data for the effect of operating parameters are presented.     

Microcalorimetric studies on the antimicrobial actions of different cephalosporins

Microcalorimetry was applied to study the effect of cephalosporins (cefazolin sodium and cefonicid sodium) on the E. coli growth. The microbial activity was recorded as power-time curves through an ampoule method with a TAM Air Isothermal Microcalorimeter at 37°C. The parameters such as the growth rate constant (k), inhibitory ratio (I), the maximum power output (P _m) and the time corresponding to the maximum power output (t _m) were calculated. The change tendencies of k, with the increasing of concentration (C) of the two cephalosporins, are similar which show that cefazolin sodium and cefonicid sodium have the same inhibitory mechanism. The experimental results reveal that cefonicid sodium has a stronger antibacterial activity towards E. coli than that of cefazolin sodium and this was coincide with the clinical manifestations.     

Chromatographic studies of some cephalosporins on thin layers of silica gel G–zinc ferrocyanide

A simple, selective and precise thin‐layer chromatographic method has been developed for the analysis of eight cephalosporin antibiotics, namely cephadroxil, cephalexin, cefixime, cefaclor, cefpodoxime proxetil, cefuroxime axetil, cefotaxime sodium and ceftriaxone sodium. The hR_F values of these cephalosporins were investigated on silica gel G–zinc ferrocyanide layers. Mixing of zinc ferrocyanide with silica gel G resulted in a decrease in hR_F values, removal of tailing and better resolutions. The influence of silica gel G–zinc ferrocyanide ratio and mobile phases on the chromatographic behavior of cephalosporins on thin layers was investigated. Cephalosporins were selectively separated in their binary and ternary synthetic mixtures and pharmaceutical formulations. Quantitative separations of cephalosporins from their synthetic mixtures were also achieved with good recoveries (97.8–100.3%). Copyright © 2010 John Wiley & Sons, Ltd.     

Serum and Pelvic Tissue Concentrations of Ceftriaxone and Cefazolin at Hysterectomy

Abstract

Ceftriaxone and cefazolin concentrations were assayed by high-pressure liquid chromatography in serum and pelvic tissue. Specimens were obtained at uterine removal subsequent to a 1-g intramuscular preoperative dose given to 117 women scheduled for elective vaginal or abdominal hysterectomy. The mean serum concentration of cefazolin was 43.2 ± 13.1 and 39.8 ± 15.4 μg/ml after vaginal and abdominal hysterectomy, respectively. For ceftriaxone they were 59.2 ± 16.8 and 56.1 ± 18.3 μg/ml for vaginal and abdominal hysterectomy, respectively. Mean tissue concentrations of ceftriaxone were 22.5 ± 10.4, 17.4 ± 6.9, 27.9 ± 10.7, and 16.4 ± 6.3 μg/g for vagina, myometrium, fallopian tube, and ovary, respectively, and respective mean tissue concentrations for cefazolin were 15.8 ± 7.6, 14.4 ± 8.5, 15.6 ± 8.0, and 12.4 ± 5.8 pg/g. Pelvic tissue concentrations of cefazolin were similar, but concentrations of ceftriaxone in fallopian tube and vagina were higher than those in ovary and myometrium. Tissue to serum ratios of ceftriaxone remained constant throughout the time intervals studied, whereas cefazolin ratios increased with time.     

Direct Kinetic Determination of Bromhexine Hydrochloride in Pharmaceutical Formulations

Abstract

A very simple, automatic, fast method for the photometric determination of bromhexine hydrochloride based on the application of kinetic methodology and the stopped-flow mixing technique to the coupling reaction between the diazotized bromhexine derivative and N-(1-naphthyl)ethylenediamine is proposed. The high initial rate of this reaction allows analytical measurements to be made within only 0.5–1 s, which makes the method applicable to automatic routine analyses. The calibration graph is linear over the range 1.5–65.0 μg mL^?1 and the precision (as %RSD) is less than 2%. The presence of antibiotics such as penicillins and cephalosporins in a 100-fold excess has no effect on the analyte determination. The proposed method was satisfactorily used for direct analysis of pharmaceutical formulations containing these antibiotics.     

Determination of cefazolin by oscillographic polarography as its S,S′-dioxide

A simple rapid method is proposed to determine cefazolin in neat substances and powders for preparing injection solutions based on the preliminary oxidation of the preparation in weak acidic media by potassium peroxomonosulfate to respective S,S′-dioxide followed by its determination by oscillographic polarography. The concentration of the title material in the cefazolin substance was found to be 96.7%, RSD = 3%, δ= 2.4% and in the powder, 97.7%, RSD = 3%, δ = 2.3%.     

Vanadophosphoric Acid as a Modified Reagent for the Spectrophotometric Determination of Certain Cephalosporins and their Dosage Forms

Summary.  Vanadophosphoric acid in acidic medium is proposed as a modified reagent for the spectrophotometric determination of cephalexin, cephaprine sodium, cefazolin sodium, and cefotaxime in pure samples and in pharmaceutical preparations. The method is based on acid hydrolysis of cephalosporins and subsequent oxidation with vanadophosphoric acid. The resulting solution exhibits maximum absorption at about 516 nm. The effects of reaction conditions were investigated. Lambert-Beer’s law was obeyed over a concentration range of about 0.4–45 μg · cm⁻³. For more accurate results, Ringbom optimum concentration ranges were obtained, and the molar absorptivities and Sandell sensitivities were derived. The proposed method was applied to the determination of the drugs in pharmaceutical formulations; the results demonstrated that the proposed method is as accurate, pecise, and reproducible as the official methods. Received August 13, 1999. Accepted (revised) December 7, 1999     

Spectrophotometric Determination of Trace Amounts of Ascorbic Acid

Abstract

Colourless silver-gelatin complex is quantitatively reduced by ascorbic acid to yellow silver sol in water within the pH range 7.5–10.0 at room temperature. The determination of 1–10μg/ml of ascorbic acid is possible at 415 nm in the presence of glycine, alanine, fructose, sucrose, citric, tartaric, oxalic, malic, succinic acids and also in the presence of various reducing agents. The molar absorptivity of ascorbic acid at the δ_max is found to be 21500 lit mol^?1 cm^?1 and the Sandell sensitivity of the sol is 8.18x10^?3 μg ascorbic acid cm^?2 for 0.001 absorbance. The relative standard deviation is ±0.22% and the confidence limit (20 determinations, 95%) being 8.806±0.0093%.     

Structure, function, and inhibition along the reaction coordinate of CTX-M beta-lactamases

CTX-M enzymes are an emerging group of extended spectrum beta-lactamases (ESBLs) that hydrolyze not only the penicillins but also the first-, second-, and third-generation cephalosporins. Although they have become the most frequently observed ESBLs in certain areas, there are few effective inhibitors and relatively little is known about their detailed mechanism. Here we describe the X-ray crystal structures of CTX-M enzymes in complex with different transition-state analogues and beta-lactam inhibitors, representing the enzyme as it progresses from its acylation transition state to its acyl enzyme complex to the deacylation transition state. As the enzyme moves along this reaction coordinate, two key catalytic residues, Lys73 and Glu166, change conformations, tracking the state of the reaction. Unexpectedly, the acyl enzyme complex with the beta-lactam inhibitor cefoxitin still has the catalytic water bound; this water had been predicted to be displaced by the unusual 7alpha-methoxy of the inhibitor. Instead, the 7alpha-group appears to inhibit by preventing the formation of the deacylation transition state through steric hindrance. From an inhibitor design standpoint, we note that the best of the reversible inhibitors, a ceftazidime-like boronic acid compound, binds to CTX-M-16 with a K(i) value of 4 nM. When used together in cell culture, this inhibitor reversed cefotaxime resistance in CTX-M-producing bacteria. The structure of its complex with CTX-M enzyme and the structural view of the reaction coordinate described here provide templates for inhibitor design and intervention to combat this family of antibiotic resistance enzymes.     

反相高效液相色谱法同时测定多种头孢菌素的研究(I)

 建立了同时分离测定６种头孢菌素（头孢米诺、头孢羟氨苄、头孢克罗、头孢氨苄、头孢拉定和头孢西丁）的高效液相色谱法。流动相为Ｖ（５０ｍｍｏｌ／Ｌ的磷酸二氢钾缓冲液,ｐＨ３．４）ｖＶ（乙腈）＝８７．５ｖ１２．５的混合液,色谱柱为ＨｙｐｅｒｓｉｌＯＤＳＣ１８５μｍ,２００ｍｍ×４．６ｍｍｉ．ｄ．,紫外检测波长为２５４ｎｍ,流速为１．０ｍＬ／ｍｉｎ。各个组分的线性范围：头孢米诺为１６４ｎｇ～１６．４μｇ,头孢羟氨苄为９９ｎｇ～９．９３４μｇ,头孢克罗为１０４ｎｇ～１０．３５８μｇ,头孢氨苄为１２２ｎｇ～１２．２２４。     

The Kinetics and Mechanism of the Hydrolysis of Creatinine in Urine

Abstract

Creatinine in urine concentrations are routinely measured at Aldermaston by an autoanalyser, using the Jaffe reaction, as an index of urinary excretion rates. These values are used in calculations to estimate the body content of radionuclides from their urinary excretion rates.

Unfortunately, creatinine in urine concentrations gradually decrease with sample age due to pseudo first order hydrolysis of creatinine to give creatine in the presence of ammonia. This reaction may be arrested or reversed by mineral acid.

After storage at ambient temperatures for several weeks the creatinine in urine concentration falls by around 20%, so it is good practice to analyse samples soon after provision.

The activation energy for the hydrolysis of creatinine in urine is around 60 KJ/mol over the range 20–70 °C. Hence, raising the temperature by 10 [ddot]C approximately doubles the reaction rate.     

Microbial sensor for new-generation cephalosporins based in a protein-engineered β- lactamase

A protein-engineered β-lactamase, constructed by site-directed mutagenesis inEscherichia coli (E104M/G238S), and having broadened specificity, was able to degrade cephalosporins of first, second, and third generations. Manipulations of culture conditions allowed an increase in β-lactamase specific activity by up to twofold. The resultant bacteria were used to construct an immersable whole-cell biosensor for the detection of new-generation cephalosporins. Cells were immobilized on agar membranes, which in turn were attached to the surface of a flat pH electrode, thus constituting a biosensor based on the detection of pH changes. The sensor was able to detect second- and third-generation cephalosporins: cefamandole (0.4-4 mM), cefotaxime (0.4-3.5 mM), and cefoperazone (0.3-1.85 mM). Response times were between 3.5 and 11 min, depending on the kind of cephalosporin tested. The biosensor was stable for at least 7 d, time during which up to 100 tests were performed.