High-resolution LC/MS for analysis of minor components in complex mixtures: negative ion ESI for identification of impurities and degradation products of a novel oligosaccharide antibiotic.

High-resolution mass spectrometry has been routinely used for structural confirmation and identification; however, it has mostly been applied to relatively pure samples. Exact mass measurement of minor components such as impurities, degradation products or metabolites in complex mixtures has been difficult without prior separation and isolation. Here we report the utilization of on-line liquid chromatography in combination with high-resolution mass spectrometry for the identification of impurities and base degradation products of Sch 27899, a member of the everninomicin class of antibiotics. Nine Sch 27899-related impurities and degradation products were detected by negative ion electrospray ionization using a magnetic sector mass spectrometer. Exact mass measurements were obtained at a resolution of 5000 using polyethylene glycol (PEG) sulfates as internal standards. Corresponding elemental compositions were determined within a 2 ppm error tolerance and structures were proposed for all components.

The role of mass spectrometry in the drug discovery process.

The structure characterization of biologically-active organic compounds, developed from synthetic and natural sources, is an integral part of the drug discovery effort to identify novel therapeutic agents. Mass spectrometric methods (electrospray ionization, matrix-assisted laser desorption/ionization, fast atom bombardment, electron ionization and chemical ionization) are uniquely qualified to solve a wide variety of structural identification problems with high speed and accuracy. This report provides an overview of the recent developments in mass spectrometry (MS) and discusses their contribution to several areas of pharmaceutical research: the automation of MS for high-throughput analysis to support new entity research, the use of liquid chromatography (LC)-MS for mixture analysis of degradation products and drug metabolites, the expanded role of highly sensitive MS for the structure elucidation of unknown organic compounds (especially natural products), the study of peptides and proteins, and the detection of non-covalent complexes.

Electrospray ionization mass spectrometry for the study of non-covalent complexes: an emerging technology.

The detection of non-covalent complexes in the mass range 19,000-34,000 Da, using electrospray ionization mass spectrometry (ESI-MS), is reviewed. The examples discussed include (1) a protein-ligand interaction (ras-GDP), (2) an inhibitor-protein-ligand interaction (SCH 54292/SCH 54341-ras-GDP), (3) a protein-protein interaction (gamma-IFN homodimer) and (4) a protein-metal complex [HCV (1-181)-Zn]. In each case, the ESI-MS method is capable of releasing the intact non-covalent complex from its native solution state into the gas phase in the form of multiply-charge ions. The molecular masses of these complexes were determined with a mass accuracy of better than 0.01%, which is far superior to the traditional methods of sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel permeation chromatography. The method provides the researcher with a quick, reliable and reproducible method for probing difficult biological problems. The key to success in the study of non-covalent complexes depends on careful understanding and manipulation of ESI source parameters and sample solution conditions; special care must be taken with the source orifice potential and the solution pH and organic co-solvents must be avoided. This paper also illustrates the usefulness of ESI-MS for addressing biological problems leading to the discovery of new therapeutics; the approach involves the rapid screening of potential drug candidates, such as weakly bound inhibitors.

Bacterial phospholipid analysis by fast atom bombardment mass spectrometry.

The structural analysis of naturally occurring bacterial phospholipids in mixtures by fast atom bombardment (FAB) mass spectrometry are reported. The bacterial strains examined included several genera of actinomycetes, two strains of Escherichia coli, and one strain each of Proteus mirabilis and Pseudomonas aeruginosa. FAB mass spectrometry proved to be a useful tool for the structural identification of phospholipids in mixtures and provided stable pseudo-molecular ions and characteristic fragment ions which permitted the identification of phosphatidylethanolamine and phosphatidyl choline. Information regarding the chain length of the fatty acids, their degree of unsaturation in the chains and the presence of hydroxyl groups was also obtained. The results obtained by FAB mass spectrometry were supported by high-resolution mass spectral data, tandem mass spectrometric studies and FAB mass spectrometry of components which had been separated and partially purified by thin-layer chromatography. Each organism displayed a highly characteristic phospholipid profile suggesting the possible use of FAB mass spectrometry as a method for rapid bacterial detection and identification.