The objective of this project is development of new mass spectral techniques in order to provide innovative and/or more rapid solutions to problems involving (1) chemical structure determination, (2) complex mixture analysis and (3) measurement of trace components in biological systems. Matrix-assisted laser desorption ionization (MALDI) mass spectrometry, electrospray ionization mass spectrometry (ESI/MS), tandem mass spectrometry (MS/MS), combined liquid chromatography-mass spectrometry (LC/MS), combined capillary electrophoresis-mass spectrometry (CE/MS) and accurate mass measurement are the techniques of current interest. On-line CE sample concentration techniques with subsequent peak collection are being investigated as a preparative-scale method for the off-line combination of CE with high resolution MALDI/MS and post-source decay fragmentation analysis. A collaborative mass spectrometric and molecular modeling study to investigate the factors controlling alkali metal ion cationization during ionization from the liquid phase (ESI/MS and FAB/MS) is ongoing. Under normal circumstances, alkali metal ion cationization is not desirable because it can decrease sensitivity and/or complicate spectral interpretation. However, for certain classes of compounds, such as oligosaccharides and sesquiterpenoids, selective and controlled cationization can confer an analytical advantage. Derivatives of the antimarial natural product artemesinin (qinghaosu), including a series of trioxane dimers with antitumor activity, are being used as model compounds since they require alkali metal ion cationization to obtain useful mass spectra. An initial investigation focusing on dihyroartemisinin shows that adduct formation is cation and ionization technique selective. For molecular modeling calculations to be predictive, a consideration of cation solvation in the liquid matrix is required rather than just gas phase cation binding energies. Thus it appears that cation adducts formed during ionization from a liquid phase represent at least partially the state of the analyte in the matrix. A similar cationization phenomenon is observed during MALDI/MS of small molecules where desorption and ionization occurs from the solid phase. Fast atom bombardment mass spectrometry (FAB/MS) is employed to support the LMC synthetic effort through structural characterization of new compounds and synthetic intermediates. A number of synthetic compounds built on a constrained glycerol scaffold (disubstituted DAG-lactone) have been identified as potent agonists of protein kinase C (PK-C). Depending on the structure of the substituents comprising R1 and R2, these DAG-lactones appear to have some degree of PK-C isozyme specificity. A solid-phase combinatorial approach is being applied in the LMC to investigate chemical diversity at R1 and R2 in order to produce more specific C1 domain ligands. It is important that these synthetic products be rapidly characterized and their structures established before biological evaluation. Strategies for the rapid mass spectral characterization of these combinatorial libraries have been developed, evaluated and implemented using FAB/MS. The positive ion FAB mass spectrum of a typical DAG-lactone derivative consists of the intact molecular ion (M+) or protonated molecule (MH+) as well as several fragment ions derived from both the acyl and alkylidene portions. This spectrum is predictable and can be used to identify the DAG-lactone derivatives present in simple mixtures. Initially, the members of one selected library column or row are examined individually to confirm that mass spectra are predictable and to evaluate the efficiency of the chemistry. This information is then used to assemble simple 4- to 6-component mixtures encompassing the entire chemical space; these mixtures are then analyzed and interpreted. Library components not detected during mixture analysis are subsequently analyzed individually. Mixture analysis correlates directly with individual analysis and substantially reduces the number of samples to be examined, resulting in enhanced analytical turn-around. A complete sequence of initial evaluation, sample preparation, mass spectral analysis, data interpretation and report generation can be completed in 2 days. Aliquots of each library component are also archived for possible future analysis should later biological evaluation warrant additional characterization. Flow-injection ESI/MS is not suitable for analysis of these DAG-lactones because of their high lipophilicity and limited basicity. However, flow injection-APCI/MS produces spectra that consist of MH+ as well as various fragment and solvent-adduct ions, and this approach appears to be complementary to FAB/MS in terms of spectral information. MALDI/MS is also being evaluated as a tool for the rapid characterization of these small molecule libraries. Our ultimate goal is the structural characterization of all 96 library components in one day. Studies to confirm the structural identity of compounds identified as HIV-1 integrase inhibitors through 3-dimensional database searching continue. A series of novel cytotoxic isoquinoline quinones and iminoquinones isolated from the marine bryozoan Caulibugula intermis have been characterized by accurate mass analysis as part of their structural elucidation.