Phytochemistry in the microgram domain—a LC–NMR perspective

Abstract

Plants represent an extraordinary reservoir of novel molecules and there is currently a resurgence of interest in the vegetable kingdom as a possible source of new lead compounds for introduction into therapeutical screening programs. In order to discover potential new bioactive natural products, the dereplication of crude plant extracts performed prior to isolation work is of crucial importance for avoiding the isolation of a known constituent. In this respect, chemical screening strategies have been developed using hyphenated techniques (LC/UV-DAD, LC-MS and LC-NMR). In our laboratory, these techniques have been fully integrated into the isolation process and are used for the chemical screening of crude plant extracts in complement with on-line or at-line bioassays. LC-UV-MS is used as a first dereplication step in combination with UV and MS databases, while LC-NMR is performed in a second step for de novo on-line structure determination. This approach enables the partial or the complete on-line identification of natural products in complex matrices such as crude plant extracts. These methods also give a unique possibility to study unstable compounds, which rapidly degrade or which are not separable at a preparative level. In the multi-hyphenated approach used (hypernation), LC-NMR plays a key role since it provides the most detailed structural information. The relatively low sensitivity of this technique, however, requires that strategies for high loading of plant extracts are developed and compromises for solvent selection have to be made. For more demanding experiments, at-line strategies based on the microfractionation of the LC-peak of interest and recording of spectra in fully deuterated solvents in microflow probes represent a promising alternative.

Intra- and inter-metabolite correlation spectroscopy of tomato metabolomics data obtained by liquid chromatography-mass spectrometry and nuclear magnetic resonance

“…In plant metabolomics, these two technologies are commonly used as independent approaches for profiling plant systems (Le Gall et al 2003;Moco et al 2006a;Tikunov et al 2005;Ward et al 2003). The (online) combination of NMR and MS technologies (Jaroszewski 2005;Seger and Sturm 2007;Tatsis 2007;Wolfender et al 2005), in particular through LC-solid phase extraction (SPE)-NMR-MS (Exarchou et al 2003) has been used for the efficient detection, separation, isolation and unequivocal structural elucidation of metabolites from plant origin. This analytical approach seems therefore especially useful in the identification of biomarkers discovered in high-throughput LCMS and 1 H-NMR metabolomics studies.…”

HPLC microfractionation of flavones and antioxidant (radical scavenging) activity of Saccharum officinarum L.

“…In view of the potential role of sugarcane as a dietary source of flavonoids as well as its possible use as a functional food, this study involved an investigation into the antioxidant activity of Saccharum officinarum L. juice ("garapa") and the identification of active compounds from leaves and juice, using HPLC microfractionation 12 combined with TLC monitoring of the compounds with antioxidant activity ( -carotene test) and free radical scavenging activity (DPPH assays). 13 HPLC microfractionation consists of collecting the peak from an analytical-scale HPLC separation for more detailed examinations of the compounds on the microgram scale.…”

Emerging Applications of Metabolomics in Studying Chemopreventive Phytochemicals

“…Compared to MS, NMR is nondestructive in nature and capable of providing more structural information, but less sensitive for detecting lowabundance metabolites. Another advantage of NMR is that chromatographic separation is not required for NMR analysis even though LC-NMR technology has become a choice for conducting high-resolution analysis of complex chemical and metabolite mixture (38). In contrast, majority of MS-based metabolomics requires a separation process, such as GC, LC, and capillary electrophoresis (CE), prior to sample introduction and mass detection in MS system.…”