LC-NMR-MS

There are examples in the literature for the application of LC-MS-NMR in the pharmaceutical industry. In the area of natural products, this technique has been applied as a rapid screening method of searching unknown marine natural products in chromatographic fractions [108] and for the separation and characterization of natural products from plant origin [109, 110]. Another application is in the area of combinatorial chemistry [111]. In the field of drug metabolism, LC-MS-NMR has been extensively applied for the identification of metabolites [112-120]. And finally, LC-MS-NMR has been used for areas such pharmaceutical research [35,121,122], drug discovery [123], degradation products [101], and food analysis [124,125]. 

Within the last decade hyphenated LC-MS, LC-NMR and LC-MS-NMR have become available analytical techniques. Since MS is a destructive tech- 

LC-MS-NMR cannot replace LC-MS, LC-NMR or even NMR techniques for the structural elucidation of compounds. There will always be cases where purihcation of the analyte(s) is required, when the structural problem is too complex or the separation of the chromatographic peak is not suitable. LC-MS-NMR. LC-MS, LC-NMR, and NMR have to be available to the analyst to choose the appropriate technique for each structural problem. The success rate of problems will depend on choosing the right technique, depending on the difficulty and nature of the problem. Each technique has its own advantages and limitations, and it is in the hands of the analyst to choose the one(s) that will help to solve the structural problems. 

The use of liquid chromatography-mass spectrometry for the identification of drug degradation products in pharmaceutical formulations, Biomed. Chromatogr. 14 (2000), 384-396. 

Clarke, D. Rindgen, W. A. Korfmacher, and K. A. Cox, Systematic LC/MS metabolite identification in drug discovery. Anal. Chem. 73 (2001), 430A 39A. 

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There has been significant advancement in the applications of NMR to the development of small-molecule pharmaceutical products. For example, advances in NMR automation (e.g., flow-injection analysis) and directly coupled methods (e.g., LC-MS-NMR analysis) have made analysis and characterization of small-molecule drugs much easier.23 25 These improvements have helped chemists to develop and characterize small-molecule combinatorial libraries and to screen for active compounds.4 6 It is likely some of these techniques can also be used in biopharmaceutical product development. 

LC/MS/NMR Liquid Chromatography/Mass Spectrometry/Nuclear Magnetic Resonance Spectroscopy... 

Keywords Citrus flavonoids health benefits phenolics determination sample preparation identification HPLC LC-MS NMR... 

During the last few years, more progress has been achieved by hyphenating LC-NMR to MS. The LC-NMR-MS or LC-NMR/MS (referred to as LC-MS-NMR in this chapter) has expanded the structure-solving capabilities by obtaining simultaneously MS and NMR data from the same chromatographic peak. There are some compromises that have to be taken into account because of the differences between MS and NMR, such as sensitivity, solvent compatibility, and destructive versus nondestructive technique, discussed below. LC-MS has been used for many years as a preferred analytical technique however, with the development of electrospray ionization techniques, LC-MS has been routinely used for the analysis of complex mixtures in the pharmaceutical industry. LC-MS-NMR is a combination of LC-MS with electrospray and LC-NMR presented below. 

Figure 20-1. Schematic setup for the LC-MS-NMR system. (Reprinted from reference 40, copyright 2003, with permission from Elsevier.)...

The capability of analyzing a complex mixture in a chromatographic run by the hyphenation of several techniques, such as NMR and MS, to HPLC is becoming more popular in the pharmaceutical industry. NMR and MS data on the same analyte are crucial for structural elucidation. When different isolates such as metabolites are analyzed by NMR and MS, one cannot always be certain that the NMR and the MS data apply to the same analyte, especially when the analytes have been isolated using analytical columns and prep columns for the MS and NMR analysis, respectively. HPLC conditions are not always reproducible when analytical and prep-HPLC columns are used to isolate different amounts of the analytes of interest. To avoid this ambiguity, LC-MS and LC-NMR are combined. MS data should be obtained initially because with NMR, data collection in the stop-flow mode can take hours or days, depending on the complexity of the structure and the amount of sample. This is why it is preferable to designate this operation as LC-MS-NMR rather than LC-NMR-MS or LC-NMR/MS. 

For the last 4-5 years, the LC-NMR-MS system has been commercially available only for the Bruker NMR instruments. For the Varian NMR instruments, the system has recently become available. The work presented here has been carried out by the author using a custom design of the LC-MS-NMR system on a Varian NMR instrument as explained above. 

As mentioned in the section of modes of operation for LC-NMR (Section 20.3.2), with the use of shielded cryomagnets, the location of the MS instrument will follow the same rule as for the HPLC. The most common modes of operation for LC-MS-NMR are on-flow and stop-flow. With stop-flow, the MS instrument can also be used to stop the flow on the chromatographic peak of interest that is to be analyzed by NMR. These two modes are presented here with an example. In the loop collection mode, the MS of the LC-MS-NMR system may also monitor the trapping of the chromatographic peak inside the loop. 

These experiments indicated that for sample mixtures, the on-flow mode of LC-MS-NMR is useful for obtaining structural information on the major components. If more detailed analysis is required, or the amount of sample is small and the compound(s) cannot be isolated because of instability or volatility, stop-flow is the mode of choice. LC-MS and LC-NMR chromatographic... 

M. V. Silva Elipe, LC-NMR and LC-MS-NMR Recent technological advancements, Encyclopedia of Chromatography, Marcel Dekker, New York, 2002, pp. 1-13. 

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