| Ziconotide is the synthetic version of o-conopeptide MVIIA, a 25-amino acid cone snail toxin from Conus magus. Its cysteine residues form 3 disulfide bridges, while its arginine, lysine residues and amidated C-terminal make it highly basic. It is a potent analgesic recently approved for the treatment of intense refractory pain in humans. The development of an analytical method for the qualitative and quantitative determination of this peptide in complex matrices is, therefore, of interest to many.;High performance liquid chromatography (HPLC) -- tandem mass spectrometric (MS/MS) analyses of intact and modified ziconotide were carried out in this study. The chromatographic performance of various columns for reversed-phase HPLC separation of ziconotide was investigated. The peak shape and retention of ziconotide on the columns used were compared. The effects of solvent composition on the charge state distribution and response of ziconotide were investigated. Optimization of various mass spectrometric parameters such as spray voltage, sheath gas and auxiliary gas flows was also conducted.;Analysis of intact ziconotide showed that the extensive cross-linking from multiple disulfide bridges reduces the structural information that can be derived from the MS/MS spectrum of ziconotide. Modification of the analyte by reducing the disulfide bonds and alkylating the resulting sulfhydryl groups eliminate cross-linking and increase the probability of fragmentation during collision-induced dissociation (CID). Fragment ions resulting from typical peptide bond cleavage (e.g., b and y ions) were observed after modification of ziconotide and the internal standard, o-conotoxin GVIA. The limit of quantitation obtained from the modified peptide experiments was 0.500 ng on column.;The HPLC-MS method developed for the analysis of intact ziconotide was successfully validated. The validated method was then used to evaluate the degradation of ziconotide in a test solution similar in composition to the commercially available drug formulation, PrialtRTM (Elan Pharmaceutical, Inc.). The plot of the natural logarithm of ziconotide concentration versus storage time at 50°C and half-life calculations indicated that the degradation of ziconotide could be a first- or pseudo first-order reaction. Results pertaining to the rate of degradation of a drug substance provide useful information for proper handling and storage of the ziconotide drug solution. (Full text of this dissertation may be available via the University of Florida Libraries web site. Please check http://www.uflib.ufl.edu/etd.html)... |
