Mass spectrometry analysis of new chemical entities for pharmaceutical discovery.

In this Section, we review the applications of mass spectrometry for the analysis and purification of new chemical entities (NCEs) for pharmaceutical discovery. Since the speed of synthesis of NCEs has dramatically increased over the last few years, new high throughput analytical techniques have been developed to keep pace with the synthetic developments. In this Section, we review both novel, as well as modifications of commonly used mass spectrometry techniques that have helped increase the speed of the analytical process. Part of the review is devoted to the purification of NCEs, which has undergone significant development in recent years, and the close integral association between characterization and purification to drive high throughput operations. At the end of the Section, we review potential future directions based on promising and exciting new developments.

Development of a mass-directed preparative supercritical fluid chromatography purification system.

In this paper, we report the development of a mass-directed supercritical fluid chromatography (SFC) purification system. We have addressed issues on software compatibility, the interface between the preparative SFC and the mass spectrometer, and fraction collection. Good peak shape and signal were achieved in the mass spectrometry (MS) trace, allowing accurate peak detection and reliable fraction collection. Simple modifications on a commercially available fraction collector enabled fractionation at atmospheric pressure with high recovery. The SFC/MS purification system has been used in support of high-throughput library purification and has been proven to be a valuable tool in complementing our reversed-phase high-performance liquid chromatograph (RP-HPLC/MS)-based technology platform.

Buffer system for the separation of neutral and charged small molecules using micellar electrokinetic chromatography with mass spectrometric detection.

An organic buffer system will be discussed that is suitable for the separation of neutral as well as charged molecules be means of micellar electrokinetic chromatography (MEKC). The buffers are based on the combination of a long chain alkyl acid, such as lauric acid with ammonium hydroxide or an organic base such as tris-hydroxymethylaminomethane (Tris). The resulting buffer system is able to separate neutral compounds based on its micellar properties. These buffers exhibit much reduced conductivity compared to traditional MEKC buffers, such as sodium dodecylsulfate (SDS), which contain inorganic salts. They also have inherent buffer capacity at high pH resulting from the basic buffer component, which in our studies had pK values from about 8-11. The separations that were observed showed high efficiency with plate counts in many cases above 500,000 plates per meter. The reduced conductivity allowed for the application of much higher electric fields, resulting in very fast analysis times. Alternatively, an increase in detection sensitivity could be achieved, as the reduced conductivity allowed for the use of capillaries with lager internal diameters. Combinations of different alkyl acids and organic bases provided for significant flexibility in selectivity tuning. Finally, the fact that the organic micellar buffer systems discussed here do not contain inorganic ions, allows for coupling with mass spectrometric (MS) detection. The possibility of MS detection combined with the high speed in analysis that can be obtained using these organic buffer systems, could make this approach an interesting option for high throughput analysis of combinatorial libraries.

High-throughput microcoil NMR of compound libraries using zero-dispersion segmented flow analysis.

An automated system for loading samples into a microcoil NMR probe has been developed using segmented flow analysis. This approach enhanced 2-fold the throughput of the published direct injection and flow injection methods, improved sample utilization 3-fold, and was applicable to high-field NMR facilities with long transfer lines between the sample handler and NMR magnet. Sample volumes of 2 microL (10-30 mM, approximately 10 microg) were drawn from a 96-well microtiter plate by a sample handler, then pumped to a 0.5-microL microcoil NMR probe as a queue of closely spaced "plugs" separated by an immiscible fluorocarbon fluid. Individual sample plugs were detected by their NMR signal and automatically positioned for stopped-flow data acquisition. The sample in the NMR coil could be changed within 35 s by advancing the queue. The fluorocarbon liquid wetted the wall of the Teflon transfer line, preventing the DMSO samples from contacting the capillary wall and thus reducing sample losses to below 5% after passage through the 3-m transfer line. With a wash plug of solvent between samples, sample-to-sample carryover was <1%. Significantly, the samples did not disperse into the carrier liquid during loading or during acquisitions of several days for trace analysis. For automated high-throughput analysis using a 16-second acquisition time, spectra were recorded at a rate of 1.5 min/sample and total deuterated solvent consumption was <0.5 mL (1 US dollar) per 96-well plate.

One-minute full-gradient HPLC/UV/ELSD/MS analysis to support high-throughput parallel synthesis.

High-throughput parallel synthesis of library compounds for early drug discovery requires high-throughput analytical methods to confirm synthesis, identify reaction products, and determine purity. An ultrafast 1.0-min HPLC/UV/ELSD/MS method was developed and compared to our standard 2.5- and 5.0-min methods in order to determine if the faster method was appropriate to evaluate compound synthesis and determine purity. In addition to using standard test mixtures, a 400-member library produced by high-throughput parallel synthesis was used for comparing the various methods. Mass spectrometric detection was used for compound identification, while UV and ELSD data offered purity assessment. Compared to our longer separations, chromatographic separation achieved using the 1.0-min method was sufficient for compound evaluation and purity assessment. This ultrafast 1.0-min HPLC/UV/ELSD/MS method is expected to increase analytical throughput tremendously, provide important information faster, and reduce the overall cycle time from synthesis to screening.

A straightforward means of coupling preparative high-performance liquid chromatography and mass spectrometry.

Flow splitting to a mass spectrometer is a common way of coupling a highly specific detector to preparative or semi-preparative high-performance liquid chromatography (HPLC) purification of combinatorial libraries, drug metabolites, and characterizable impurities. The sensitive mass spectrometer consumes only a small fraction of the analyte while providing online structure-specific detection, and its output can thus be used to trigger collection of the desired fraction. Coupling mass spectrometry to preparative HPLC is difficult due to the susceptibility of the detector to fouling under conditions of high analyte concentration or solute amount, or to changes in solvent composition. We report here on a device, the mass rate attenuator (MRA), which automatically produces split ratios over a range of 100:1 to 100 000:1 under programmable user control. The MRA is a flow-control device that periodically gates a small aliquot from one liquid stream into another. The design allows the user to set the frequency of the gating without interruption of the HPLC flow stream. The MRA also allows control of the volume of the aliquot that is transferred between the flow streams. This additional control, compared to passive splitting devices, facilitates optimization of the tubing connecting the separation, detection and collection events. We demonstrate that such optimization can reduce the volume of the collected fraction without compromising recovery, thus reducing the time spent in evaporating solvents to reclaim purified products.