LC-NMR-MS systems

The first attempts to include a mass spectrometer in the LC-NMR set-up were made several years ago. Nowadays, LC-NMR/MS systems are commercially available, and the usage of this kind of set-up will be briefly evaluated below [1-7]. 

The use of a mass spectrometer in an LC-MS set-up is now a standard technique. However, if a mass spectrometer is brought into an LC-NMR/MS system, some further problems will occur which are mainly due to the relatively high sample loadings used in NMR spectroscopy. In the case of an ion-trap mass spectrometer, some of the already established routines are as follows ... 

As preferably one user should be able to operate the whole system, it becomes clear that a highly integrated system is needed. Even if the peak selection is carried out by the user, the positioning of the sample, followed by the start of the NMR and MS experiments, must take place automatically. An integrated LC-NMR/MS system therefore has to contain the following components ... 

The use of LC-NMR as an analytical tool in combinatorial chemistry remains limited. Its major application within the pharmaceutical industry is in the identification of drug metabolites.38 For this purpose LC-NMR has been further coupled to a mass spectrometer to generate an LC-NMR-MS system.43 The combination of the three techniques provides a powerful tool for structural characterization. 

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. 

Fig. 10 Schematic of an LC/NMR/MS system. In this configuration, NMR is used to provide structural, stereochemical, or ligand binding information while MS is used to provide molecular weight information. (Courtesy of Bruker Daltonics, Billerica, MA.)...

Figure 3.46 An LC-NMR-MS system showing the LC, the MS, the NMR-MS interface and the NMR itself (The magnet is not shown. Image courtesy of Bruker BioSpin Limited).

Fig. 11.14 LC chromatogram of pipeline and partially purified metabolites at 300 nm obtained using LC/NMR/MS system. Separation was performed on ODS-AQ coliunn [19]...

Multiple hyphenation ( hypemation ) provides comprehensive spectroscopic information from a single separation. The first doubly hyphenated HPLC-NMR-MS appeared in 1995 [661], and its value is now accepted meanwhile fully integrated on-line LC-NMR-MS and MSn systems (QMS, QTTMS) are commercially available. On-line LC-NMR-MS coupling is by no means trivial. For example, the sensitivity of NMR is limited, while MS is incompatible with non-volatile buffers. The... 

Fig. 19.23. Schematic showing an LC-SPE-NMR/MS system. The dotted arrows represent electronic controls and the solid lines represent the flow path of the eluent.

The coupling of LC (liquid chromatography) with NMR (nuclear magnetic resonance) spectroscopy can be considered now to be a standard analytical technique. Today, even more complex systems, which also include mass spectrometry (MS), are used. The question arises as to how such systems are handled efficiently with an increasing cost and a decreasing availability of skilled personal. LC-NMR and LC-NMR/MS combine the well-established techniques of LC, NMR and MS. For each of those techniques, various automation procedures and software packages are available and used in analytical laboratories. However, due to the necessary interfacing of such techniques, completely new demands occur and additional problems have to overcome. 

The analysis of a sample in a complex system such as LC-NMR or even LC-NMR/MS can be split into several steps, as follows ... 

The first application of LC-NMR-MS to natural products analysis was presented in 1999 [37]. The additional mass spectroscopic information allowed the identification of a further ecdysteroid in an extract of Silene otides which could not be identified by LC-NMR alone [24], Further applications of this double hyphenation dealt with the identification of napthodianthrones [38] and flavone glycosides [38,39] in natural products extracts. Taking the hyphenated technique one step further, the suitability of an integrated LC-UV-IR-NMR-MS system for natural products analysis has been assessed [40] - again using ecdysteroids as an example. Such systems still suffer from different requirements of the individual detectors (mainly in terms of sensitivity). However, the results obtained are promising. 

It is generally accepted that an analysis by LC/UV/MS coupled with a H NMR is sufficient to characteri.se a compound and provide an estimation of purity. The massive increase in sample numbers through combinatorial approaches has led to an urgent need for the above techniques to be available to the chemist on demand. This has cataly.sed the development of high-throughput walk-up open-access LC/UV/MS systems. 

D. Bao, V. Thanabal, W.F. Pool, Determination of tacrine metabolites in microsomal incubate by LC-NMR-MS with a column trapping system, J. Pharm. Biomed. Anal., 28 (2002)23. 

LC/NMR is often used within multi-hyphenated systems in different fields of application, especially LC/NMR/MS. In this case, MS is used as an efficient and selective detection method to trigger NMR experiments only on the peaks of interest, which render the targeted measurement extremely valuable. 

Because of the use of microfluidic systems, it might be possible in the future to use online miniaturized chip separation with miniaturized MS for rapid on-site analysis. Additionally, coupling of LC-NMR/MS will be a invaluable system that will allow the obvious identification of metabolites. 

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