الفهرس 
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Abstract
Aztreonam is totally synthetic antibacterial classified as a monobactam. It is active against a wide spectrum of gram-negative aerobic pathogens. This drug can effectively act as a complexing agent with metal ions. This study aimed to the development of a new analytical method of the drug, in bulk, dosage form, in presence of its degradation products as well as in human urine, by the separating ability of high performance liquid chromatographic technique. The proposed HPLC method depending on the principal of complexation chromatography. This study was initiated with the spectroscopic investigation of the chelation behavior of aztreonam with different metal cations which can be used for this study such as; Fe (NO3)3.9H2O, CrCI3.6H2O, Bi (NO3)3 , AI(NO3)3.9H2O,SbCI3, CuSO4.5H2O,NiSO4.6H2O,ZnSO4.7H2O,BaCI2.2H2O)CdSO4.8H2O, MnSO4.H2OICoSO4.7H2O,Mg(NO3)2.6H2Ol Pb(NO3)2) Hg(NO3)2.H2O and Hg2(NO3)2.H2O by reacting the titled drug with each metal cation separately in aqueous solution. A systematic selection of metal ions was completed, to find the most suitable candidate to be used as additive in the mobile phase of the proposed HPLC method. According to the results of each UV-absorption scans of each metal chelate they classified into three suggested categories; the first suggested category included Mg (II), Co (II), Cd(ll), Zn(ll), Mn(ll), and Ba(ll). With this category no significant change was observed in the original drug UV-absorption scan. The second suggested category included Bi(lll), Fe (III), CrIII), Al(lll), Pb(ll) and Sb(lll).this category showed distinguishable changes in the original drug UV-absorption scan but no red shift of maxima of the free drug. The third suggested category included Ni(ll), Cu (II), Hg(ll) and Hg(l). In this category there were significant changes in the original drug UV-absorption scan and showed a red shift of maxima of the free drug. The third category of metal ions was selected for further investigations, because, they showed a promising chelation liability with aztreonam. According to the study of the effect of standing time and metal salt concentration on the maxima of the corresponding chelate as a further selection of metal ions to be used as a metal ion additive in the mobile phase of a proposed HPLC method. Only the maxima of Hg(l) and Hg(li) complexes shifted to longer wavelength by increasing in standing time up to 25 min at room temperature and then it retained stable. While the maxima of Ni(ll) chelate shifted to longer wavelength by increasing in Ni(ll) salt in the reaction solution up to 36 x 10~5 M and then it retained stable. Cu(ll) -aztreonam chelate was the only complex which did not affected by the two last variables. Copper(ll)-aztreonam system was proved by UV and polarographically. The molar ratio was 1:1 and the most suitable pH for complexatiom was found to be 4. For the optimization of the HPLC method of analysis, the retention selectivity and elution order of aztreonam was studied as a function of methanol concentration, pH, heptanesulphonic acid sodium salt and copper salt concen-tration. Optimal separation of aztreonam was achieved by using a mobile phase consisted of 1.2x10”3 M cupric sulfate aqueous solution, pH 4 with sulfuric acid, 4.9x10”4 M heptanesulphonic acid sodium salt and 7% (v/v) methanol as organic modifier. The flow rate was 2 ml/min and the column effluent was monitored spectrophoto-metrically at 246 and 326 nm. Standard curves for aztreonam werawnstructed^y^plotti^g^e measured peak heights versus concentrations of the tested compound employed in nanograms of the final solutions. A linear relationship existed with a very good correlation coefficient and very small intercept. The ability of cupric ions to form a complex with aztreonam degradation products was proved UV, TLC and HPLC. The proposed HPLC method also was applied as stability indicating assay for aztreonam. It was found that, the degradation of the tested compound was stongly influenced by temperature (heating it in aqueous solution). A linear relationship between the logarithmic percent of remaining concentration and time existed from which the observed degradation rate constants (KObs) appeared to be pseudo first-order. Concerning the stability of aztreonam by using the inclusion complex forming ability of cyclodextrins. UV- spectroscopy, IR and DSC tested the formation of complexes between aztreonam with cyclodextrins. The effect of p-cyclodextrin concentration on the stability of aztreonam in water was also studied. The UV-spectra of aztreonam were performed in the absence and presence of a-, p- and y-cyclodextrin. It was found that, a-, y-cyclodextrin did not cause any significant change compared with the spectrum of the free aztreonam. This may indicate that a-cyclodextrin is too small to include aztreonam molecule and subsequently can not affect its chemical stability and y-cyclodextrin has the largest cavity. On the other hand, it was observed that, the UV spectrum of aztreonam with p-cyclodextrin was showed a significant difference than the spectrum of free molecule. The obtained IR spectra, of the physical mixtures of the drug and different types of cyclodextrins, showed changes in the positions of carbonyl groups of free aztreonam. This indicated the complex formation between the drug and the different types of cyclodextrins used. According to DSC data, aztreonam exhibited an exothermic melting curve with thaw and peak melting point at 204.4 and 225.5 °C, respectively. Its enthalpy was 286.5 Jg”1. The enthalpy of each physical mixture of aztreonam and a, b and g of 1:1 ratio was 36.14, 22.01 and 20.81 Jg”1. Obviously, no interaction occurred since the DSC thermograms of physical mixture of aztreonam with a-, p- and y- cyclodextrins, reflected the same characteristic feature of the drug alone. Some different appearances found in peak shape and height-to-width ratio were due to the possible geometric differences in the sample mixtures. The endothermic melting peaks of aztreonam seen in 1:1 drug-to-cyclodextrin ratio were reduced in intensity. The type of solubility curve of aztreonam-p-cyclodextrin complex was classified as type A phase diagram, indicating the formation of a 1:1 soluble complex, p-cyclodextrin exhibited AL type diagram with aztreonam, showing linear increase for aztreonam solubility as a function of p-cyclodextrin. The apparent stability constant, Kc, was 0.2 x 103 M”1. In the kinetic study, it was found that, among examined cyclodextrins, P-cyclodextrin had pronounced destabilizing effect on the degradation of aztreonam whereas on the other hand, a- and y-cyclodextrin did not exhibit any significant effect on the stability of tested compound. This may indicate that the labile moieties are not fit in p-cyclodextrin, which has larger cavity size than a-cyclodextrin and less than y-cyclodextrin. In comparing the results obtained under this investigation with those in the absence of cyciodextrins, its quite clear that exhibited accelerated destabilizing in aqueous solution with p-cyclodextrin. Conclusively the degradation of aztreonam in aqueous solution can be enhanced by inclusion complexation with cyciodextrins. Aztreonam is tightly bound with p-cyclodextrin while no significant effect was observed with a-cyclodextrin as well as with y-cyclodextrin. Thus, inclusion complexation of p-cyclodextrin may be a potentially means of decrease stability. In other words, the rate of degradation of aztreonam was significantly enhanced within the cyclodexthn complex than outside it in the solution. This effect of p-cyclodextrin on aztreonam will limit its usage in pharmaceutical preparations containing this drug. The proposed HPLC method was also applied for the analysis of aztreonam in biological fluids such as human urine. Aztreonam metabolite (unchanged aztreonam) has been identified by comparing its HPLC data, retention time and photodiode array UV-scan, with that of the pure drug. The parameters and correlation coefficient of the calibration plot indicated very good correlation coefficient and very small intercepts. The calibration curve was rectilinear over range of 3-20 ng/pl and the limit of quantification for aztreonam in urine is 3 ng/pl. The within-day coefficient of variation (CV%) ranged from 1 to 2.4%, and between-day (CV%) from 2.7 to 4.7% at three different concentrations. The absolute recoveries ranged from 97.5 to 97.8% and the relative recoveries from 99.7 to 100.5% at three different concentrations. The average excreted amount of aztreonam in human urine was about 66% of the IV administered dose.