Mass β- Lactam Antibiotics And Their Cleavage Law Of Impurities And Toxicity Prediction And Evaluation

The drug quality and safety are not only the tremendous issues for drug regulation administrations and pharmaceutical companies, but also the main concerns for public. The drug quality is closely related to the adverse drug reactions found in the clinics, and the drug impurity analysis is the key process ensuring the drug quality. Therefore, the concept of impurity profiling is proposed, the definition of impurity profiling is "A description of the identified and unidentified impurities, present in a new drug substance", with the aim of detecting, identifying or elucidating the structure and quantitatively determining all the impurities in bulk drugs and pharmaceutical dosage forms. Impurity profiling is focused on the limit determination according to the biological activity of impurities. To achieve the profiling of drug impurities, three key technical issues should be considered:separation of the complex sample, structural elucidation of unknown impurities in trace amount, and toxicity evaluation of impurities. In this dissertation, the widely prescribed P-lactam antibiotics in clinics are chosen, the studies are mainly about the structural elucidation and toxicity evaluation.HPLC-MS is one of the most important tools in the drug quality control, the possible structures could be rapidly determined based on the knowledge of impurity source and fragmentation pathways in mass spectrometry. Thus, a deep understanding of the fragmentation behavior in mass spectrometry is desperately required for impurity profiling, the different fragmentation patterns in mass spectrometry, as well as the universal pathways are very useful information for structural elucidation. The differentiation of stereoisomeric impurities using mass spectrometry, infrared multiple photon dissociation (IRMPD) spectroscopy and computational chemistry is also highlighted. For toxicity prediction and evaluation, theoretical calculations were used to study the molecular conformations in aqueous solution and to confirm the concept of toxic functional groups of the C-3and C-7side chains. Molecular polarities were calculated to predict the passive absorption in Zebrafish embryo toxicity testing. The following four parts are included in this study:1. The universal fragmentation behaviors of the cleavage of P-lactam ring in Penems were studied by collision-induced dissociation mass spectrometry and computational chemistry in the positive ion mode. The protonation sites and structures of several Penams were explored in the gas-phase, four β-lactam ring cleavage mechanisms were proposed, the thermodynamic characteristics of these pathways were also investigated, the most favorable pathway for each compound was located. The study indicated that the protonation sites have crucial impacts on the cleavage.2. The differentiation of some cephalosporins and their A-3isomers were comprehensively investigated by mass spectrometry in both the positive and negative ion mode. The characteristic product ions enabled distinguish of these positional isomers. IRMPD spectroscopy and computational chemistry were applied for structural determination and fragmentation mechanism study.3. In the positive ion mode, cephalosporins with (Z)-imine configuration of the C-7side chain, together with their (E)-isomers were studied by electrospray tandem mass spectrometry and computational chemistry. The results suggested that the fragmentation behavior of (Z)-and (E)-isomers are significantly influence by the nature of their C-3side chain. The (Z)-and (E)-isomers could only be distinct when the cleavage of C-3side chain occurred, the (E)-isomers were more likely to lose the substituents of the imine moieties, leading to the characteristic ions for differentiation. If the C-3side chain kept intact under collision-induced dissociation, the (Z)-and (E)-isomers would have the same fragmentation pattern.4. The computational chemistry was applied to study the conformations and polarities of cephalosporins and their impurities in the aqueous solution, also, Zebrafish embryo toxicity testing was used to evaluate and validate the toxic effects. The results indicated that the basic structure of the cephalosporin was almost nontoxic, while the C-3and C-7side chains were the main toxic functional groups. The conformations of the side chain in the aqueous solution affected the potency of the toxic effect, moreover, they determined the polarity of the molecules. Consequently, the conformations would influence the ability of passive absorption through the embryo membrane. The decreased polarity would increase the membrane permeability and finally gave rise to more potent toxic activity.