Differential pulse polarographic study of the degradation of cephalexin: Determination of Hydrogen Sulphide and other Degradation Products

Differential pulse polarography (d.p.p.) is used to study the degradation of cephalexin. Hydrogen sulphide, evolved during the degradation of cephalexin solutions, was removed continuously in a stream of nitrogen and determined periodically. Other electroactive degradation products were observed by d.p.p. of the degraded sample solutions. The degradation mechanism is highly dependent on pH, the initial concentration of cephalexin, temperature, the particular buffer used, and the presence of dissolved oxygen. The formation and degradation of the diketopiperazine derivative formed by intramolecular aminolysis, particularly at neutral pH, can be followed by means of its polarographic peak at -0.9 V (pH 7.4). Approximately half the total sulphur originally present in cephalexin is liberated as hydrogen sulphide at pH 7.4 at 37°C. Increasing the degradation temperature to 80°C and sweeping out the hydrogen sulphide with nitrogen increases the yield of a major product which gives a peak at -1.26 V. At pH 8.5 (80°C. 100 μg cephalexin ml^-1) the percentage of the sulphur evolved as hydrogen sulphide increases with time, and a peak appears at -0.96 V (probably 2-hydroxy-3-phenyl-6-methylpyrazine) which increases as the peak at -1.26 V becomes smaller. Other products formed under different conditions (concentration, pH, temperature) are reported. At pH 3 (80°C) only 8% conversion via intramolecular aminolysis and 5% evolution of total sulphur is indicated after four hours.     

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.     

Electroanalytical studies of the pesticides menazon and its hydrolysis products

An electroanalytical study based on d.c. polarography, differential pulse polarography (d.p.p.) and coulometry is described for Menazon, S-[4, 6-diamino-1,3,5-triazin-2-yl)- methyl]-O,O-dimethylphosphorodithioate, and its alkaline hysdrolysis products in a (1 + 4) methanol/water medium. The responses of the different species are studied for analytical utility. Menazon itself produces two diffusion-controlled cathiodic waves or peaks at pH 4.78; the determination limit is 5.5 × 10^-7 M for the d.p. peak at about ?0.8 V vs. Ag/AgCl. The hydrolysis products obtained in 0.1 M sodium ydroxide give rise to four anodic waves. Acidification of the alkaline medium to pH 4.3 produces three well-defined waves; two cathodic and one anodic. The responses for the cathodic processes are linear with concentration over 2–2.5 orders of magnitude; the anodic wave is adsorption-controlled and provides linear response only at low concentrations. The detection limit for the hydrolysis products is 0.17 × 10^-6 M. Reaction mechanisms are proposed.     

Micellar Electrokinetic Chromatography of Cephalosporin Antibiotics

A simple micellar electrokinetic chromatographic (MEKC) method is described for the simultaneous separation of twelve cephalosporin antibiotics. The separation was performed in a phosphate buffer (0.1 M, pH 6.6) with sodium dodecyl sulfate (SDS) (0.1 M) as an anionic surfactant. The complete separation of twelve cephalosporins was attained in 36 min. Application of the method to the quantitative analysis of cefuroxime, cephalexin, cephapirin and cephradine in pharmaceutical preparations was demonstrated.     

Differential pulse polarographic determination of cephalexin based on the catalytic pre-wave of nickel(II)

A nickel-catalytic pre-peak at ?0.72 V vs. Ag/AgCl, is caused by cephalexin in sodium acetate (pH 8.3) medium. It is used to determine 2.4 × 10^?7–6 × 10^?6 M cephalexin; the relative standard deviation is 4% for 0.88 μg ml^?1 cephalexin. Other cephalosporins behave similarity; some amino acids can be tolerated in moderate amounts.     

Electrochemical reduction of 5,5′-dichlorohydurilic acid. mechanism and analytical applications

The electrochemical reduction of 5,5′-dichlorohydurilic acid has been studied at the dropping mercury electrode (DME) and the pyrolytic graphite electrode (PGE). At the DME the single polarographic reduction wave observed at pH 6–11 involves a direct 4e—2H⁺ reduction of the carbon-halogen bond to give hydurilic acid and chloride. The state of hydration or ionization of the 5,5′-dichlorohydurilic acid has no effect on the electrochemical reaction. At the PGE, 5,5′-dichlorohydurilic acid shows two voltammetric peaks. Peak I_c, observed between pH 5 and 7, arises from an overall 4e—2H⁺ reduction of 5,5′-dichlorohydurilic acid via a mechanism that involves initial electron attack at a carbonyl group alpha to a carbon-halogen bond with simultaneous elimination of chloride ion. The peak II_c process involves an initial 2e—1H⁺ reduction of a partially hydrated form of 5,5′-dichlorohydurilic acid with only one unhydrated halocarbonyl moiety available for reaction. Attack is again via the carbonyl group with simultaneous elimination of chloride and formation of 5-chlorohydurilic acid. A chemical dehydration step then occurs with a rate constant of ca. 0.24 s^?1 at pH 8.2, with formation of a further reducible halocarbonyl group. This is again reduced in an overall 2e—2H⁺ reaction to give hydurilic acid and chloride ion. The peak II_c process hence proceeds via an ECE mechanism. The different mechanisms observed for reduction of 5,5′-dichlorohydurilic acid at mercury and pyrolytic graphite electrodes are unusual. Analytical methods have been developed for the polarographic determination of 5,5′-dichlorohydurilic acid via its reduction wave at the DME, and for the voltammetric determination of hydurilic acid via its first oxidation peak at the PGE.     

Fluorescamine Post-Column Derivatization for the HPLC Determination of Cephalosporins in Plasma and Urine

Abstract

A post-column fluorescamine derivatization procedure is proposed for the determination of cephalosporins having an α-primary amino group in their side chain (cefaclor, cephalexin, cephradine, cefroxadine, cefaloglycine and cefadroxil). The linearity, repeatability and detection limits of fluorescence emission and UV absorption detection are compared under the same chromatographic conditions. Fluorescence detection is about two times more sensitive than UV absorption detection. Application to the determination of these cephalosporins in plasma and urine shows an improved selectivity by comparison with UV detection.     

Investigation of perchlorate, phosphate and ion-pairing eluent modifiers for the separation of cephalosporin epimers

The retention behavior of several pairs of 7 alpha- and 7 beta-cephalosporin epimers was investigated using perchlorate, phosphate and ion-pairing eluent modifiers. At pH 2.5, sodium perchlorate, sodium phosphate and sodium pentanesulfonate all provided separation of epimers with free 7-amino groups. When the 7-amino group was blocked, as in cephalexin and cefaclor, sodium perchlorate gave the best separation at pH 2.5. A tetrabutylammonium ion-pairing system at pH 7.0 provided separation of all epimer pairs containing a free carboxylic acid at the 3 position. Hydrophobic, residual silanol and ionic interactions were factors in the retention mechanism of the cephalosporins under the conditions investigated. An ionic interaction of perchlorate with the protonated amine of the cephalosporin was postulated as an explanation of the retention and selectivity effects observed with perchlorate as an eluent modifier.     

Polarographic study of uracil derivatives

Eighteen uracil derivatives were studied by d.c. polarography, differential-pulse (d.p.)polarography and d.p. cathodic stripping voltammetry. In a borax buffer at ca. pH 7.6, uracil, thymine and derivatives such as 5-halouracils, 5-trifluoromethyl-,5-aza-,5-acetyl-, 5-formyl- and 5-vinyl-uracil produced well-defined peaks at potentials between 0 V and ca. 160 mV vs. silver/ silver chloride (satd. KCl). The peaks are ascribed to the formation of sparingly soluble mercury salts. For the other derivatives tested (e.g., 5-nitro- and 5-ethynyl-uracil and 6-substituted uracils), the peaks were less well-defined and in some cases the polarographic curves were very complex. 2-Thiouracil produced a single peak at ca. ?400 mV, but only at pH 12.2. The shapes, heights and potentials of the peaks depended on the kind and position of the substituent on the pyrimidine ring. Rectilinear relationships of peak current vs. concentrations were found for most compounds (10^?5-10^?4 M) by d.p. polarography; d.c.polarography was not tested quantitatively. For 5-fluorouracil and 5-vinyluracil, linear calibrations were found for concentrations of 0.5–5 × 10^?7 M by d.p. cathodic stripping voltametry. Interference studies showed that small amounts of chloride and phosphate did not interfere but 5-fluorodeoxyuridine, which did not itself produce a peak, and proteins interfered seriously.     

Kinetics and mechanisms of hydrolysis and aminolysis of thioxocephalosporins

The effect of replacing the beta-lactam carbonyl oxygen in cephalosporins by sulfur on their reactivity has been investigated. The second-order rate constant for alkaline hydrolysis of the sulfur analogue is 2-fold less than that for the natural cephalosporin. The thioxo derivative of cephalexin, with an amino group in the C7 side chain, undergoes beta-lactam ring opening with intramolecular aminolysis by a reaction similar to that for cephalexin itself. However, the rate of intramolecular aminolysis for the S-analogue is 3 orders of magnitude greater than that for cephalexin. Furthermore, unlike cephalexin, intramolecular aminolysis in the S-analogue occurs up to pH 14 with no competitive hydrolysis. The rate of intermolecular aminolysis of natural cephalosporins is dominated by a second-order dependence on amine concentration, whereas that for thioxocephalosporins shows only a first-order term in amine. The Bronsted beta(nuc) for the aminolysis of thioxo-cephalosporin is +0.39, indicative of rate-limiting formation of the tetrahedral intermediate with an early transition state with relatively little C-N bond formation.     

Kinetic spectrofluorimetric determination of certain cephalosporins in human plasma

An accurate, reliable, specific and sensitive kinetic spectrofluorimetric method was developed for the determination of seven cephalosporin antibiotics namely cefotaxime sodium, cephapirin sodium, cephradine dihydrate, cephalexin monohydrate, cefazoline sodium, ceftriaxone sodium and cefuroxime sodium. The method is based on their degradation under an alkaline condition producing fluorescent products. The factors affecting the degradation and the determination were studied and optimized. The reaction is followed spectrofluorimetrically by measuring the rate of change of fluorescence intensity at specified emission wavelength. The initial rate and fixed time methods were used for the construction of calibration graphs to determine the concentration of the studied drugs. The calibration graphs are linear in the concentration ranges 0.2-1.2 μg mL⁻¹ and 0.2-2.2 μg mL⁻¹ using the initial rate and fixed time methods, respectively. The results were statistically validated and checked through recovery studies. The method has been successfully applied for the determination of the studied cephalosporins in commercial dosage forms. The high sensitivity of the proposed method allows the determination of investigated cephalosporins in human plasma. The statistical comparisons of the results with the reference methods show an excellent agreement and indicate no significant difference in accuracy and precision.     

Spectrophotometric assay of cephalosporins in pharmaceutical products,using chromotrope 2B and chromotrope 2R

A simple, rapid and sensitive Spectrophotometric method is proposed for the determination of cephadroxil (I), cephalexin (II) and cephradine (III). The method is based on ion-pair complex formation between these derivatives and Chromotrope 2B (C2B) or Chromotrope 2R (C2R), to give a highly coloured radical anion. The coloured products are quantified spectrophotometrically at 542 and 564 nm for C2B and C2R, respectively. The optimization of the experimental conditions is described. The method has been used for the determination of 0.4–15, 0.4–14 and 0.4–18 g/ml of drugs I, II and III, respectively. The accuracy of the method is indicated by the excellent recovery (100.0±1.7%) and the precision is supported by the low relative standard deviations 1.5%. The sensitivity of the method is discussed and the results are compared with the official method. The interference from common degradation products and excipients was also studied. The proposed method was applied successfully to the determination of the different cephalosporins in dosage forms, with good precision and accuracy. The results were compared with those given by the official B.P. 1993 method.     

Electrochemical determination of mesotrione and its degradation products on glassy carbon electrode

A simple, fast and sensitive analytical method was developed for the quantification of herbicide mesotrione (MES) and its degradation products: 4-(methylsulfonyl)-2-nitrobenzoic acid (MNBA) and 2-amino-4-(methylsulfonyl) benzoic acid (AMBA) using differential pulse voltammetry (DPV). Voltammetric measurements were performed using glassy carbon electrode (GCE). Potential range, pH of the electrolyte and the scan rate were optimised to achieve the lowest detection limits of analytes. The optimal conditions were obtained in a Britton–Robinson (BR) buffer at pH 4.0 for MNBA and at pH 6.0 for MES and AMBA, with the scan rate 0.08 V/s. The potential V for (1) nitro and carbonyl groups of MES, (2) nitro group of MNBA and (3) amino group of AMBA, obtained under optimised conditions, was plotted as a function of a peak current (I). The I(V) dependencies were measured for the following concentration ranges: 0.5–5.0 µM for the nitro group of MES and MNBA, 0.75–5.0 µM and 0.50–8.5 µM for the carbonyl groups of MES, and 0.25–8.5 µM for amino group of AMBA; whereas, the limit of detection was in range 0.07–0.23 µM (20–80 µg/L). The proposed method is the first one that allows the determination of both MES and its degradation products. The practical applicability of these newly developed voltammetric methods was verified by direct determination of MES and its degradation products in model samples of drinking and surface water.     

Synthesis and preliminary antimicrobial activities of new arylideneamino-1,3,4-thiadiazole-(thio/dithio)-acetamido cephalosporanic acids

New derivatives of 7-aminocephalosporanic acid 1-8 were synthesized by acylation of the 7-amino group of the cephem nucleus with various arylidinimino-1,3,4-thiadiazole-thio(or dithio)-acetic acid intermediates 3a-d and 5a-d, respectively, so the acyl side chains of these new cephalosporins contained a sulfide or disulfide bond. This unique combination of a Schiff base with the sulfide or disulfide bonds in the acyl side chain afforded new cephalosporins of reasonable potencies, some of which were found to possess moderate activities against the tested microorganisms. Their chemical structures were characterized by 1H-NMR, IR spectroscopy and elemental microanalysis. Preliminary in vitro antimicrobial activities of the prepared cephalosporins were investigated using a panel of selected microorganisms. Results indicated that the newly synthesized cephalosporins containing disulfide bonds (compounds 5-8) exhibited better activities against Staphylococcus aureus and Escherichia coli. The cephalosporins cross-linked by a sulfide bond (compounds 1-4) showed a slight change in antimicrobial activities when compared with that of the reference cephalosporin (cephalexin).     

Continuous flow molecular emission cavity analysis of cephalosporins by alkaline degradation to sulphide

A continuous flow method for the determination of some cephalosporins (cephradine, cephalexin, cephalosporin C, cefadroxil, cephapirin and cephalothin) in the general range 10.0-250.0 micrograms ml-1 is described. The sample is mixed with sodium hydroxide and remains for 20 min at 90 degrees C in the delay coil of an air-segmented system. The solution is then mixed with an excess of orthophosphoric acid, and the hydrogen sulphide evolved is continuously transferred into the cavity for generation of the S2 molecular emission. The analysis is automated, requires no sample pre-treatment and samples can be analysed at a rate of 30 per hour with a relative error of 1-2%. The method was evaluated by carrying out recovery experiments and by the analysis of commercial formulations. Results agreed well with those obtained by the standard methods.     

HPLC and 1H-NMR Studies of Alkaline Hydrolysis of Some 7-(Oxyiminoacyl)cephalosporins

Alkaline hydrolysis (pH 10.5) of the three 7-(oxyiminoacyl)cephalosporins 1a–c (cefuroxime, ceftazidime, and ceftriaxone) was studied at 37° using HPLC and ¹H-NMR techniques. The 7-epicephalosporin 2 , the 3-methylidene compound 3 , and the 6-epimer 4 of the 3-methylidene compound 3 were identified for each cephalosporin as the major degradation products under the conditions used; ceftazidime ( 1b ) yielded also the Δ²-isomer 5b (Scheme 1). A kinetic scheme was developed to account for the production of these compounds, and the different kinetic constants involved in the process were calculated. The experimental results show that the presence of a pyridinio group at position C–C(3) favours the appearance of the Δ²-isomer, which was detected mainly in cephalosporins bearing an ester function at C(4). The presence of an oxyimino group at C? CONH? C(7) facilitates epimerization at C(7) (→ 2 ), whereas that of an electron-withdrawing group at C? C(3) results in a increased formation constant for the 3-methylidene compound 3 . The 3-methylidene compounds 3a–c produced by the three cephalosporins on cleavage of the β-lactam ring all underwent epimerization at C(6) to yield the corresponding 6-epimer 4 .     

Studies on orally active cephalosporin esters. II. Chemical stability of pivaloyloxymethyl esters in phosphate buffer solution

The degradation kinetics of pivaloyloxymethyl (POM) esters of cephalosporins in phosphate buffer solution (pH 6-8) were investigated. The degradation of the starting delta 3 cephalosporin ester proceeded mainly via isomerization to the delta 2 ester and subsequent hydrolysis to the delta 2 acid. Hydrolysis to the delta 3 acid (the parent acid) was very slow. Analysis of the rate constants indicated that the isomerization rate k12 was approximately equal to the apparent degradation rate of the delta 3 ester kdeg, and slower than the hydrolysis rate of the delta 2 ester k24. The isomerization process to the delta 2 ester was found to be the rate-determining step in the degradation of cephalosporin esters. The substituent at the C-3 position of the cephalosporins affected the degradation kinetics. The degradation was accelerated by increase of pH, buffer concentration and added protein.     

Applications of Spectrometric Full Spectrum Quantitation (FSQ) Software for Multicomponent Analysis and Stability Studies of Pharmaceuticals

ABSTRACT

A rapid, simple, accurate and specific spectrophotometric procedure for analysis of multi-component mixtures and for assessment of drug stability in pharmaceutical products has been described. The proposed method relies on the application of the spectrophotometric full spectrum quantification (FSQ) software to overcome severe spectral overlap and to permit measurement of the individual components in mixtures with satisfactory degree of accuracy and precision. The potential of FSQ software for multicomponent analysis was elucidated by analyzing complex mixtures of highly overlapping spectra (aspirin/caffeine/salicylic acid) and mixtures of structurally similar compounds (cephalexin/ cephradine) and (ceftriaxone/ ceftazidime /cefotaxime). Furthermore, the robustness of the FSQ for stability studies was assessed by analyzing chloramphenicol in presence of its alkali-induced degradation products and famotidine in presence of its acid-induced degradation products. The validity of FSQ software for multicomponent analysis and stability studies was evaluated by comparing the results with those of HPLC methods. Statistical analysis indicated that the FSQ and HPLC data were consistent, however the FSQ method is faster, easier and more reliable.     

Simultaneous Determination of Cephradine,L‐Arginine,and Cephalexin in Cephradine for Injection by Capillary Zone Electrophoresis

Abstract

Applying capillary zone electrophoresis (CZE) to separate the components of Cephradine for Injection: cephradine, and L‐arginine, as well as cephalexin, which is the degradation product of cephradine was studied. The best results were achieved with background electrolyte consisting of 50 mM disodium hydrogen phosphate buffer at pH 6.5 and an applied voltage of 20 kV in a bare fused‐silica capillary. The samples were injected at 50 mbar for 4 s. The capillary temperature was 25°C and the UV detection was performed at a wavelength of 195 nm. Histidine was used as internal standard (IS) to ensure acceptable precision data. The linear ranges of cephradine, L‐arginine, and cephalexin were 93.8–6255.6 µg/mL, 47.9–3195.2 µg/mL, and 6.1–405.4 µg/mL, respectively. Quantitative parameters such as accuracy, precision, limit of detection (LOD), and limit of quantitation(LOQ) were all established in CZE mode.     

901 — Electrode process of glutamate dehydrogenase in differential pulse polarography

The anodic and cathodic responses of sulfhydryl groups in glutamate dehydrogenase (GDH) were studied using the d.p.p. method. The current peaks were detected at the potentials U_p,anod and U_p,cath of approx. −1.45 V and −1.50 V, respectively, versus s.c.e. in phosphate buffer, pH 7. Both the cathodic and the anodic responses of GDH showed autoinhibiting diffusion-controlled adsorption effects. Both peak potentials U_p,anod and U_p,cath are shifted positively with increasing pH up to pH 8.5 (U_p,anod by 40 mV/pH, U_p,cath by 60 mV/pH) and then become pH-independent. The preceding chemical reaction (field-induced homolysis) with pH-dependent rate yields the electroactive form, the thiyl radical, through abstraction of the hydrogen atom from the -SH group. The radical could be reduced, yielding the thiol anion after the uptake of one electron. The two-electron anodic oxidation in d.p.p. involves the product of this reduction process and another thiol anion in an adjacent position. The dissociation rate constant of the thiol group near that undergoing homolysis was estimated from the d.p.p. data (log k_d = 2.9 ± 0.2).