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.Other groups dealt with naphthy-lisoquinoline alcaloids [15 17], sesquiterpene lactones [6,18,19], triterpenesaponins [20] taxanes [21], lignanes [22], phenylphenalenones [23], polyhydroxysteroids [24], fasciculol triterpenes [25], tocopherols and tocotrienols [26], car-otenoid isomers [27] and flavonoids [28,29], as well as hop bitter acids [30,31].Remarkably, most applications published so far deal with the characterisa-tion of plant-derived mixtures, while applications to secondary metabolites ofmicro-organisms [32,33] or marine natural products [2,34,35] are still rare.In natural products analysis, most frequently the stop-flow mode is chosen1to acquire H spectra of the compounds of interest, or if further structural1information is required to perform two-dimensional H NMR spectra, such asCOSY, TOCSY, NOESY or ROESY.In many cases an on-flow NMR chro-matogram (usually at flow rates between 0.3 and 1 ml min 1) is recordedbeforehand, either to screen for the presence of particular groups of compoundsor to gain a general overview on the sample composition.(Heteronuclear LCNMR experiments, such as HSQC and HMBC of a natural product, have beenreported in the literature once [9]; however, this was of a highly enrichedfraction.) More recently,  time-sliced stop-flow [14,16] and on-flow approachesat low flow rates [34,35] have been applied to natural product extracts in orderto combine the advantages of both on-flow (a ready overview on the entiresample) and stop-flow (sufficient acquisition time for minor compounds)modes.The sensitivity of the NMR experiment is concentration-dependent, and thusat a given amount of sample, molecules with a lower molecular weight (MW)give a more intensive signal.On the other hand, larger molecules usually possessshorter relaxation times which allow more spectra to be accumulated per timeincrement by using shorter relaxation delays.The work published so far hasfocused mainly on the characterisation and identification of small molecules upto an MW of ca.700.However, LC NMR has been successfully applied to114 LC NMR and Related Techniquesidentify saponins of an MW up to 1400 [34,35] and to characterise largerbiomolecules such as glycosphingolipids [36].The first application of LC NMR MS to natural products analysis waspresented in 1999 [37].The additional mass spectroscopic information allowedthe identification of a further ecdysteroid in an extract of Silene otides whichcould not be identified by LC NMR alone [24].Further applications of thisdouble hyphenation dealt with the identification of napthodianthrones [38] andflavone glycosides [38,39] in natural products extracts.Taking the hyphenatedtechnique one step further, the suitability of an integrated LC UV IR NMRMS system for natural products analysis has been assessed [40]  again usingecdysteroids as an example.Such systems still suffer from different require-ments of the individual detectors (mainly in terms of sensitivity).However, theresults obtained are promising.Another application of LC NMR in natural products chemistry concernsbiosynthetic studies employing feeding experiments with stable isotope-labelled1compounds.H NMR spectra allow the determination of the amount of iso-topic label incorporated into metabolites, e.g.by observing signals that arise13from J-couplings of protons to C-labelled nuclei [41,42].A comprehensive recent review paper on the application of LC NMR inphytochemical analysis is recommended for further reading [1].In the following sections, some examples of the advantageous use of LCNMR and LC NMR MS in natural products analysis are presented in order todemonstrate the possibilities and limitations of these hyphenated techniques.5.1.2 APPLICATION OF LC NMR MS TO GLYCOSIDIC NATURALPRODUCTS OF MARINE ORIGIN5.1.2.1 INTRODUCTION  NEED FOR LC NMRIn the search for biologically active natural products for a technical application,constituents of marine organisms were systematically investigated [34,35].Bio-logical tests on extracts of the Baltic starfish Asterias rubens led to a classof closely related glycosidic compounds, the so-called asterosaponins.These9(11)are * -3( , 6) -dioxygenated steroids with a sulphate group attached at C-3and an oligosaccharide chain containing five or six sugar units at C-6 [ Pobierz całość w formacie PDF ]