VO2+ and Cu2+ Interactions with Ceftriaxone and Ceftizoxime. HPLC Kinetic Studies

[摘要] Proper antibiotic characteristics make Ceftriaxone or Ceftizoxime to be among the third generation cephalosporins used in medicine. Our previous work on VO2+ and Cu2+ chelation with penicillins has already been reported.1,2 Several methods have been proposed for the determination of cephalosporin, such as spectrophotometric, potentiometric or electrophoretic methods3-9 and, more recently, HPLC10 and capillary electrophoresis.11 In this work, Reversed Phase High Performance Liquid Chromatography (RP-HPLC), has been used to study Cu2+-ceftriaxone and VO2+-ceftriaxone and Cu2+-VO2+-ceftizoxime interactions, since this method permits the separation of Cu2+ and VO2+ cephalosporin chelates, the corresponding Cu2+ and VO2+ cephaloic acid chelates, free cephalosporin and degradated compounds of the cephalosporin molecule. The RP-HPLC method also provides evidence for the stoichiometry and the chelation mechanisms. Thus, the goal of the present work consists in evaluating the influence of metal chelation in ceftizoxime or ceftriaxone degradation. Apparently only the VO2+ and Cu2+ ions cause this processes to take place.  Experimental Chemicals and reagents Commercial sodium Ceftriaxone, with 99.80% purity, and commercial sodium Ceftizoxime, with 98.80% purity, tested by the standard methods of the USP12 at a concentration of 0.5 mg mL-1, and 7-ADCA (7-aminoceph-3-em-4-oic acid or 7-desacetoxy methyl aminocephalosporanic acid) were supplied by SKF Laboratories (Madrid, Spain). The copper (II) sulphate solution was prepared from Merck analytical grade CuSO4 pentahydrate. The vanadium (IV) sulphate solution was prepared from Merck analytical grade VOSO4 pentahydrate. All other chemicals were HPLC grade and supplied by Micron Analítica S.A. (Madrid, Spain).Apparatus and instrument conditionsRP-HPLC assays were performed with a liquid chromatographic system equipped with two Waters M6000A pumps (Waters, Milford, Massachusetts, USA), a variable-wavelength LDC SM 5000 diode array detector (supplied by Micron Analítica, Madrid, Spain) and a Kontron Auto Sampler 460 automatic injector (Kontron Instruments, Münich, Germany) connected to a water-bath thermostat, controlled from a computer, through a RS232C connection. A Spherisorb ODS-18 reversed-phase column (10 mm, 25 x 0.46 cm), supplied by Micron Analítica S.A., was employed. The automatic injector, pumps and detector were controlled by software (D450 v.2.0) from Kontron Instruments. The mobile phase consisted of a 20% (ceftriaxone) or 30% (ceftizoxime) solution (v/v) of methanol in 0.01 mol L-1 KH2PO4. The flow rate was 0.8 mL min-1. The effluent was monitored at 254 nm and the injection volume was 10 mL. IEX-HPLC assays were performed with a liquid chromatographic system equipped with a Waters M-510 pump, a Waters 430 conductivity detector and a Waters U6K 20 mL loop injection valve. A Kontron Data Station with D450 software was used to monitor the detector output. An Ion-210 (Waters, Milford, Massachusetts, USA) cation column was used. The mobile phase consisted of a solution containing 10 mmol L-1citric acid and 3.5 mmol L-1 ethylenediamine. The flow rate was 2 mL min-1. The copper sulphate pentahydrate standard solution used in the calibration plot was supplied by Merck (Darmstadt, Germany) and the concentration used was 0.002 mmol L-1 of metallic ion.Buffer SolutionsFor the general investigations we used the Sörensen buffer (Table 1). A constant ionic strength of 0.5 was maintained for each buffer by adding an appropriate amount of KCl. All solutions were freshly prepared and the pH values were taken by a Methrom-Herisau E 520 pHmeter.   Analytical procedureEffect of pHThe hydrolysis of the Cephalosporin-Metal complex was studied between pH 2 to pH 5 because, at pH values above 6, the hydroxo complexes formed precipitate and mask quantification by HPLC.Effect of temperatureThe effect of temperature was studied at 30º, 40º, 50º and 60ºC.Effect of ion concentrationThe influence of ion concentration was studied in the molar ratios of 0.5:1, 1:1, 2:1 and 3:1. The cephalosporin was kept constant at 1.26 mmol L-1.  Results and Discussion Chromatograms from Cu2+-ceftriaxone interactions show three peaks, at retention times of 2.8 min (peak I), 4.3 min (peak II) and 4.8 min (peak III), indicated in Figure 1, with absorption maxima at 201, 240 and 280 nm (peak I), 197, 238 and 279 nm (peak II) and 308 nm (peak III), respectively. Peak II was not seen in chromatograms run at pH 4 and pH 5. Besides, peaks II and III do not appear at the initial time. The chromatogram peaks were directly identified by their UV spectra from the Diode Array Detector and were compared with the spectra of free ceftriaxone and with the 7-ADCA degradation product.  Peak I is assigned to the Cu2+-ceftriaxone complex, which has a similar UVspectrum to uncomplexed ceftriaxone. Peak III is due to a Cu2+-cephaloic complex, with typical absorption between 300-320 nm.1,2 Finally, peak II, with retention time and absorption spectra similar to 7-ADCA, a product of ceftriaxone degradation, can be ascribed to a Cu-7-ADCA chelate. Chromatograms of VO2+-ceftriaxone interaction show the same profile, but the retention times are different (2.3 min, peak I; 4.0 min, peak II, and 4.7 min, peak III). Peaks from free ceftriaxone (1.8 min)13 are not present at all in any chromatogram. The chromatogram from Cu2+-ceftizoxime interactions shows four peaks, at retention times of 6.8 min (peak I), 8.1 min (peak II), 4.3 min (peak III) and 5.8 min (peak IV), as indicated in Figure 2, with absorption maxima at 201, 240 and 280 nm (peaks I and II), 197, 238 and 279 nm (peak III) and 308 nm (peak IV), respectively. Peak III is not present in the chromatogram at pH=5. Peaks III and IV do not appear at the initial time. The chromatographic peaks were also identified by comparing the UV spectra from the Diode Array Detector with the spectra of free ceftizoxime and with that of the 7-ADCA degradation product.   Peaks I and II are attributed to two isomers of a Cu2+-ceftizoxime complex. Peak IV is due to a Cu2+-cephaloic complex, with an absorption maximum between 300-320 nm. Finally, peak III, with retention time and absorption spectra similar to 7-ADCA, a product of ceftizoxime degradation, can be ascribed to a Cu-7-ADCA chelate. The chromatogram of VO2+ ceftizoxime interaction shows the same profile, but the retention times of the peaks are different (5.8 min, peak I; 7.1 min, peak II, 3.9 min, peak III and 4.8 min, peak IV). The two peaks attributed to free ceftizoxime (1.4 min and 1.6 min)14 are not present in any of the chromatograms. Commercial ceftizoxime has two isomeric forms, Z-syn and E-anti, which are separated under our chromatographic conditions. It is reasonable to think, on the basis of the HPLC results from the reactions of ceftriaxone with Cu2+ and VO2+, that metal ions interact with the ceftriaxone through the formation of a five-member chelate (peak I). By this way, Cu2+ and VO2+ would accelerate the hydrolysis of ceftriaxone resulting in the formation of a cephaloic acid chelate (peak III). Additionally, Cu2+-7-ADCA and VO2+-7-ADCA complexes are formed (peak II) at pH between 2 and 3. The interaction mechanism for the Cu2+-ceftriaxone complex is shown in Figure 3. Similar effects happen with the ceftizoxime interaction.Chelate stoichiometryBy plotting the peak area of Cu2+-ceftriaxone and VO2+-ceftriaxone chelates (peak I) obtained by the RP-HPLC method versus the Cu2+/ceftriaxone and VO2+/ceftriaxone molar ratios (0.5:1 to 3:1), at pH 2 to pH 5, an inflexion point was observed at a 1:1 molar ratio (see Figure 4) in correlation with the stoichiometry of the complex. Plots obtained from ceftizoxime are similar.  Equilibrium reactionKinetic constantAccording to results observed using the RP-HPLC method, in order to determine the kinetic constants for the cupric and oxovanadium ceftriaxone and ceftizoxime chelate formations, we assume that the following equilibrium reaction may occur:With an equilibrium constant: Since the VO2+ ion was not detectable under our chromatographic conditions, we used VO2+ in excess, in order to consider, in the equilibrium constant, the unreacted VO2+ concentration as the initial analytical concentration. Values of log Ke at different values of pH are shown in Table 2.Thermodynamic constantsCalculations of DG°, DS° and DH° values are also shown in Table 2. The calculated enthalpy change for the complexation between Cu2+ and VO2+ and ceftriaxone was found to be between of 24-38 kJ mol-1 (37-46 kJ mol-1 for ceftizoxime). The change in free energy was around ¾3.5 kJ mol-1 and the entropy change in the range of 85 to 130 J mol-1 (127-147 J mol-1 for ceftizoxime). These DSº values are to be expected for chelate formation.Hydrolysis reactionNow, we assume that the next reaction of the Cu2+and VO2+ ceftriaxone or ceftizoxime complexes is a non-equilibrium hydrolysis reaction, as described in equation (3):We have evaluated the effects of pH, temperature and ion concentration on this hydrolysis reaction, through the observed kinetic constants and the Arrhenius heats of activation.Effect of pHThe logarithms of the observed kinetic constants versus pH, for the hydrolysis of the cupric-ceftriaxone chelate, show a minimum rate at pH 5 (Table 3). The series pH 2 > pH 3 > pH 4 > pH 5 indicates a better stability at pH 5. Similar processes were encountered for the VO2+-ceftriaxone complex. If we compare the observed constants for the free ceftriaxone hydrolysis reactions with those of the cupric-ceftriaxone complex, at a 1:1 molar ratio, a situation of lesser stability is seen in the ceftriaxone values at pH 2 (k3 of 0.584) and pH 3 (0.463) at 30 ºC,15 in relation to the Cu2+-ceftriaxone complex (0.082, pH 2 and 0.062, pH 3). These effects are less at pH 4 (0.162 vs. 0.065) and pH 5 (0.140 vs. 0.069). The same happens in the

[学科分类] 化学（综合）