Techniques for increasing the throughput of flow injection mass spectrometry.

Improvements to the design and operation of a Gilson 215 multiprobe liquid-handling system have resulted in a significant increase in the throughput for flow injection molecular weight characterization of combinatorial chemistry libraries. The rapid injection sequence, and subsequent increased sample throughput, is effected by directing the entire mobile-phase flow through each of the injection loops sequentially while isolating or "dead-ending" the remaining nonactive loops. This mode of operation was accomplished by incorporating column-switching valves prior to and following the set of eight parallel injectors. Analysis rates are achieved without sacrificing the integrity of the flow injection peak profile as baseline resolution is maintained for all samples. Using this system, the total analysis time for a 96-well microtiter plate has been reduced to approximately 5 min.

Advances in high-throughput mass spectrometry.

The evolution of high-throughput drug discovery is readily apparent as the pharmaceutical industry continues to stress the rapid progression of new chemical entities and biological agents through drug discovery and development pipelines. Mass spectrometry and high performance liquid chromatography-mass spectrometry have played an instrumental role in the support and advancement of all facets of high-throughput drug discovery. The introduction of new instrumentation has extended the breadth of mass spectrometric-based capabilities from the characterization of high-throughput organic synthesis products to early adsorption, distribution, metabolism and excretion profiling. Additionally, advances in the capacity and throughput of mass spectrometry systems have concurrently led to the introduction of data management tools to address automated data reduction, archival and mining, as well as analytical data integration to chemical and biological databases.

Application of packed capillary liquid chromatography/mass spectrometry with electrospray ionization to the study of the human biotransformation of the anti-emetic drug dolasetron.

Packed capillary liquid chromatography/mass spectrometry (LC/MS) using electrospray ionization (ESI) was used to study the human biotransformation of the anti-emetic drug dolasetron. Urine from subjects given a single 100 mg intravenous dose, containing 14C-labeled dolasetron (50 microCi), was de-salted and concentrated for LC/MS with minimal loss of radioactivity (97% recovery). Aliquots of the de-salted material were injected directly onto a C8 packed capillary column (25 cm x 0.32 mm i.d.) and eluted with an acetonitrile-water gradient, buffered with 1% acetic acid, at a flow rate of 2 microliters min-1. Five metabolites were detected by LC ESI-MS which, yielded molecular mass information but no fragmentation. The identity of each metabolite was confirmed in a subsequent analysis using product ion scans in conjunction with collisionally induced dissociation. Precursor ion scanning was also employed and did not reveal any new biotransformation products. In addition to defining the major routes of biotransformation, the data obtained were compared with a 14C radioprofile prepared in a separate experiment. Qualitative agreement in the two chromatographic profiles enabled the major clusters of radioactivity to be assigned to specific metabolites of dolasetron. An important observation in this comparison was that the signal obtained by ESI did not provide an accurate assessment of the quantity of each metabolite. This was especially true for acidic conjugates (i.e. glucuronides, sulfates), which in the case of dolasetron can exist as zwitterions (no net charge). The results demonstrate the power of packed capillary LC ESI-MS for use in drug biotransformation studies and suggest that caution should be exercised when interpreting relative metabolite abundances from ESI data in the absence of actual reference standards.

Rapid analysis of antibiotic-containing mixtures from fermentation broths by using liquid chromatography-electrospray ionization-mass spectrometry and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry.

A crucial step in the isolation of antibiotic substances is establishing whether or not the isolated material represents a new chemical entity. Because of the importance of molecular weight to this process-known as dereplication-mass spectrometry has traditionally played an active role. In this communication a strategy for utilizing liquid chromatography-mass spectrometry (LC/MS) for novelty assessment is described. Crude extracts (20-50 µg) are chromatographed by conventional bore high-performance liquid chromatography (1 mL/min) after which a postcolumn split to divert roughly one-tenth of the sample to the mass spectrometer for molecular weight determination by electrospray ionization (ESI) mass spectrometry. The majority of the effluent is sent to a UV detector and ultimately collected as 1-min fractions for biological testing. As a secondary confirmation of molecular weight, an aliquot of each fraction (< 5%) is taken for analysis by matrix-assisted laser desorption ionization (MALDI). The improved efficiency of this approach over more traditional schemes utilizing off-line fraction collection and conventional ionization methods can be explained by several factors. First, the superior sensitivity of ESI and MALDI means that less material is required for successful analysis. Second, on-line LC/MS optimizes the efficiency of sample transfer and saves both time and labor. Furthermore, the concentration dependence of ESI allows a majority of the material injected for LC/MS to be recovered for biological testing without compromising the signal available for molecular weight determination. As a validation of the above method, crude extracts containing two well-characterized antibiotics-teicoplanin and phenelfamycin-were examined. Results from these analyses are presented along with data from the analysis of a potent unknown antifungal sample.