ABSTRACT
This work analyzed the thermal denaturation process of defatted bovine serum albumin (BSA). DSC measurements were performed on changing the pH, the ionic strength and the sodium dodecyl sulfate (SDS) concentration. These data have been compared with those previously obtained by us and other authors. The purpose of these measurements was to study the correlation between the three-dimensional organization of BSA native protein structure and its thermodynamic stability and to clarify the non-covalent interactions between the globular proteins and amphipathic molecules. These measurements have shown that the thermal denaturation is always irreversible regardless of pH, ionic strength and SDS concentration. The nature of the irreversible process superimposed on the protein unfolding is discussed. The strong stabilizing effect of NaCl on the BSA native structure has been found for the range 0-1.0 M. It is worth noting that the calorimetric curves, confined to the pH region studied, could not be represented by a two-state transition model; they were deconvoluted as the sum of two independent two-state transitions. These transitions were correlated to the domain structure of BSA. Sodium dodecyl sulfate has a net stabilizing effect up to a molar ratio of 10:1 (ligand to protein). In this range of concentrations the presence of SDS cause a biphasic profile of excess heat capacity. A simple thermodynamic model was developed in attempt to reproduce the experimental DSC profiles and collect information regarding the binding equilibrium of SDS.
Subject(s)
Calorimetry, Differential Scanning/methods , Serum Albumin, Bovine/chemistry , Sodium Dodecyl Sulfate/pharmacology , Binding Sites , Dose-Response Relationship, Drug , Hydrogen-Ion Concentration , Models, Chemical , Osmolar Concentration , Protein Denaturation/drug effects , Protein Folding , Serum Albumin, Bovine/drug effects , Serum Albumin, Bovine/metabolism , Sodium Dodecyl Sulfate/chemistry , Surface-Active Agents/chemistry , Surface-Active Agents/pharmacology , ThermodynamicsABSTRACT
A new amoxicillin sodium impurity was detected by reversed-phase HPLC in commercial injectable preparations only when examined very soon after the drug was dissolved in the solvent vial (within about 10 min). The stability of this impurity was investigated by the degradation kinetic of its aqueous solutions. Ionspray mass spectrometry with flow-injection analysis and HPLC-MS methods were used to establish its nature. Some hypotheses concerning its chemical structure were formulated. The most likely assumption referred to the (5S,6R) amoxicillin piperazinedione diasteroisomer. The presence of the amoxicilloic acid methyl ester, an intermediate of the amoxicillin degradation process, was also hypothesized.
Subject(s)
Amoxicillin/analysis , Drug Contamination , Chromatography, High Pressure Liquid/methods , Flow Injection Analysis , Mass SpectrometryABSTRACT
The level of particulate contamination in water small volume injections available in Italy has been determined. The influence of the ampoule size, breaking system, manual method of ampoule opening and the type of instrument used for the determination have been evaluated. The distribution of the particles inside the batch of the sample was also studied.
