Chromatographic Separation and NMR An Integrated Approach in Pharmaceutical Development

Over the past 10 years, major improvements in the performance of LC-NMR have been realized. The addition of postcolumn SPE, advances in probe technology including cryogenic probes and microcoil probes, improved solvent suppression pulse sequences, and shielded magnets with better homogeneity have all contributed to rapid advancements in this technology. Application of LC-NMR to problems in pharmaceutical development has had a major impact on structure elucidation studies. LC-NMR has been successfully applied to determine the structures of degradation products, impurities, mixtures of compounds, and metabolites. Use of stop flow techniques with LC-NMR experiments has been a critical means of identifying unstable compounds and studying conformational kinetics. The integration of SPE as an intermediate step between the LC unit and the NMR spectrometer has vastly improved the power of the hyphenated technique in trace analysis applications. Online postcolumn enrichment of chromatographic peaks by SPE dramatically reduces the NMR acquisition times by allowing repeated injections to be trapped onto the same cartridge or different cartridges. Because protonated solvents can be easily removed with a drying procedure, solvents and buffers may be freely chosen for maximizing chromatographic separation without compromising NMR spectral quality. The compound of interest may then be eluted from an SPE cartridge using deuterated organic solvent, which helps to reduce dynamic range issues. When combined with cryogenically cooled microcapillary probes, the sensitivity of the NMR signal increases about 10-fold over conventional room temperature probes, enabling full structure characterization at the microgram level. Heteronuclear experiments with concentrations previously only possible in a limited number of cases have now become standard experiments. The availability of HSQC and HMBC experiments and microcoil/cryogenic technology opens the possibility of using LC-(SPE) NMR for the structural elucidation of complete unknowns on a microgram scale. To enable significant future downscaling beyond the current capabilities, improved performance of the LC-NMR interface and improved SPE cartridge retention need to be addressed. In addition, the active volume of the NMR flow cell or capillary tube will have to shrink along with the corresponding detection coils in order not to lose filling factor. As the size of the NMR probes become more efficient with respect to mass sensitivity, techniques such as CE or capillary electrochromatography (CEC) may be interfaced more successfully with NMR spectroscopy. Overall, the current state of the art in LC-NMR has demonstrated proven utility in a variety of applications. When combined with SPE and cryotechnology, LC-NMR has become an extremely valuable tool for mass limited samples, enabling structure elucidation without the need for laborious serial isolation and purification procedures.