One of the greatest advances in natural product research over the last couple of decades has been the development of the online hyphenated techniques. These techniques allow for the prompt identification and characterisation of both known and unknown natural products, and can be applied to both plant and marine derived extracts (Bobzin S et al 2000). The prompt identification is a very important step in order to prevent the costly and time-consuming process of isolation and purification of known compounds (dereplication), which have little or no pharmacological significance.
The term 'hyphenation' was initially proposed by Hirschfeld T (1980), and refers to a technique in which the ability to elucidate a chemical structure is enhanced by coupling a powerful separation technique, which produces a highly purified eluent, which can then be subjected to analysis by a coupled detection system, which is connected in series or in the case of multiple hyphenated systems often in parallel with other detectors (LC-MS/NMR) (Patel K et al 2010). The detection systems can then process the relevant eluent simultaneously, producing information in the form of spectra, which can then be used to identify compounds using online databases.
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Figure 1: Illustrating set up for hyphenated systems, a multiple system can also be set up by the addition of multiple detection system, which can run in parallel LC-NMR/MS, (/) denoting a parallel detection system. (-) denoting a series system. The system would be connected to an online database, which identifies the spectra, if compounds are known.
The Role of Nuclear Magentic Resonance (NMR) in Natural Product Research
NMR has played an invaluable role in natural product structure elucidation, since 1957, in 1961 the first total assignment of a proton NMR was reported by Zavarin E and Anderson A (1961) on the structure of pygmaein, a tropolone from Cupressus pygmaea (Heartwood) (Marcias F et al 2007), since its inauguration NMR has improved immensely with the incorporation of high-field magnets (800-900MHz) (Patel K et al 2010), compared to the low-field magnets (60MHz) available during the period of its inauguration. Recent advances in hardware and software for the direct coupling of NMR with systems such as Capillary Electrochromatography (CE), and High Performance Liquid Chromatography (HPLC) have contributed further to the development of hyphenated techniques which incorporate NMR, a vital instrument in structure elucidation.
The development of 2D NMR in 1971, resulting in the formation of experiments such as TOCSY and NOESY further extended the potential of using NMR as part of a hyphenated technique in the investigation and structural elucidation of novel natural products.
HPLC/LC is a widely used technique for the separation of compounds from the complex matrices of natural products (Wolfender J 2009) but the detectors conventionally used in HPLC, provide no detailed structural information, the development of specialised interfaces has allowed HPLC to be coupled with analytical techniques that can provide structural information, at present one commonly used detector of this type in natural product research is MS, commonly found coupled to LC (LC-MS) and for volatile constituents coupled to GC is GC-MS, but a fast evolving and most promising technique for structure elucidation, rapidly emerging in natural product research currently is LC-NMR.
The first paper published describing the coupling of LC to NMR was published in 1978 by Watanabe N and Niki E (Exarchou V et al 2005) but there were several limitations in the inauguration of this hyphenated technique, as a consequence the technique could not be used as a robust analytical technique. Among one of the most prevalent issues was the lack of compatibility in solvents used in the systems, as NMR requires deuterated solvents, which are fairly expensive, with deuterium oxide (D2O) costing in the range of £250-£300 L-1 (Jaroszewski J et al 2005), another problem was the solvent signal was far greater than the analyte signal, due to low concentrations of analyte in eluent. This problem was resolved to some degree by introducing solvent suppression techniques, there are three main major methods, presaturation), (Simpson A and Brown S 2005), soft-pulse multiple irradiation, and WET (Smallcombe et al 1995)
Another issue of concern has been the lack of sensitivity of NMR itself. NMR although very useful in structure elucidation and characterisation, is probably the least sensitive technique and requires samples in the order of μg (10-6 g). Injecting samples of this magnitude would compromise the integrity of resolution in analytical HPLC columns, consequently resulting in peak broadening. According to Elipe M (2003) decreasing the flow rate to <1ml/min-1 would help prevent the degradation of resolution, providing the pump has a high degree of accuracy in this range. However, with the cryogenic probes which have recently become commercially available, the degree of sensitivity of LC-NMR can now be improved, by improving the signal to noise ratio, without compromising the resolution of HPLC, now even allowing samples in concentration ranges of ng (10-9 g) to be detected, which is also important as compounds of pharmacological interest often tend to be in very low concentrations in plants, the classic example being taxol, in the Taxus baccata trees for example the concentration of taxol was 0.055±0.008% (dry wt).
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There are several modes of operation described in literature, in the continuous flow mode the eluent is measured similarly to UV, since the eluent is measured without stopping the flow, in this mode a high volume of analyte is required, which would affect the resolution in the analytical column (Exarchou V et al 2005), one of the greatest draw backs of this method is that it has the least sensitivity because of the very short periods of time eluents dwell in the NMR flow cell, which according to Karagianis G and Waterman P (2006) at a flow rate of 1ml/min is 3.6sec. The other methods which are under static conditions, require lower volumes of analyte commonly known as the stop-flow methods, in these methods the chromatographic run is stopped and eluent is collected in the flow cell, which can then be used to carry out 2D experiments such as WET-COSY and WET-TOCSY (Elipe M 2003), other modes in static conditions include time-slice and Loop collection modes (Exarcho V et al 2005)
Spring O et al (1995) published one of the first papers using LC-NMR in natural product chemistry, in which they characterised individual sesquiterpene lactones from Zaluzania grayana, since then the frequency of natural product research papers utilising this technique has increased, including several publications in marine natural product chemistry. Iwassa K et al (2003) reported the whole metabolic pathway leading to the synthesis of 2,3,10,11- oxygenated tetrahydroprotoberberines in from the cultured cells of Corydalis spp by the application of LC-NMR and LC-MS, and the structure a number of isomeric metabolites in the pathway.
LC-MS and LC-NMR can be combined to form a very powerful multi-hyphenated system LC-MS/NMR, in theory the system can be connected in a series or parallel manner, but a parallel system is preferred, as it is often difficult to correlate a set of results with each system. In the parallel system the eluent can be split using a custom made splitter, which splits the eluent into a ratio of 1:100 or in some studies a ration of 5:100, thus delivering only 1-5% of eluent to the MS, whilst delivering 95-99% to the NMR, due to the variation in sensitivity of both experimental procedures, with NMR being least sensitive.
The use of deuterated solvents in LC-MS/NMR for NMR would naturally lead to exchange of 1H with 2H, resulting in equilibrium with solvent and active molecular hydrogens capable of disassociating from the compound (Elipe M 2003), thus resulting on closely related molecular peaks.
Solid Phase Extraction (SPE-NMR) /SPE Systems (LC-SPE-NMR)
SPE involves passing the solution sample through a conditioned sorbent bed, which could simply be the eluent with analyte eluting from LC column, provided a suitable sorbent bed is used the analyte of interest will be extracted on the sorbent bed (Lindon J et al 1995) this procedure is often found often to compose the interface between liquid chromatography and NMR (LC-SPE-NMR). The extracted eluent at this stage can be dissolved using deuterated solvents, which is more economical viable then to have deuterated solvents constituting mobile phases in LC.
Christophoridou S et al (2005) reflect the strength of LC-SPE-NMR in a study in which they not only identified and elucidated the structure of known phenolic constituents, in the polar part of olive oil, but also identified several new phenolic constituents.
Although the hyphenation of gas chromatography with mass spectrometry (GC-MS) is of great importance in natural product chemistry especially with volatile constituents, there is has been very little evidence of GC and nuclear magnetic resonance spectroscopy hyphenation in natural product research, although the first attempt was made by Brame et al (1965) (KÏ‹hnle M et al 2008) One of the greatest advantages of coupling GC and NMR as stated by Grynbaum M et al (2007) would be the lack of background solvent signal interference with the typical carrier gasses used in GC (He, Ar and N2) This technique has also been postulated to be able to resolve structure of those small organic compounds with low boiling points.
KÏ‹hnle M et al (2008) demonstrated the ability of this hyphenated technique in being able to isolate and identify volatile stereoisomers, with very small sample amounts.
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