Structure elucidation of natural products usually relies on a combination of NMR spectroscopy with mass spectrometry whereby NMR trails MS in terms of the minimum sample amount required. In the present study, the usefulness of on-line solid-phase extraction (SPE) in LC-NMR for peak storage after the LC separation prior to NMR analysis is demonstrated. The SPE unit allows the use of normal protonated solvents for the LC separation and fully deuterated solvents for flushing the trapped compounds to the NMR probe. Thus, solvent suppression is no longer necessary. Multiple trapping of the same analyte from repeated LC injections was utilized to solve the problem of low concentration and to obtain 2D heteronuclear NMR spectra. In addition, a combination of the SPE unit with a recently developed cryoflow NMR probe and an MS was evaluated. This on-line LC-UV-SPE-NMR-MS system was used for the automated analysis of a Greek oregano extract. Combining the data provided by the UV, MS, and NMR spectra, the flavonoids taxifolin, aromadendrin, eriodictyol, naringenin, and apigenin, the phenolic acid rosmarinic acid, and the monoterpene carvacrol were identified. This automated technique is very useful for natural product analysis, and the large sensitivity improvement leads to significantly reduced NMR acquisition times.

Here we show, for the first time, evidence of the primary molecular structures in humic substances (HS), the most abundant naturally occurring organic molecules on Earth, and their associations as mixtures in terrestrial systems. Multi-dimensional nuclear magnetic resonance (NMR) experiments show us that the major molecular structural components in the mixtures operationally defined as HS are aliphatic acids, ethers, esters and alcohols; aromatic lignin derived fragments; polysaccharides and polypeptides. By means of diffusion ordered spectroscopy, distinct diffusion coefficients consistent with relatively low molecular weight molecules were observed for all the components in the mixtures, and saccharides were the largest single class of component present. Liquid chromatography NMR confirmed that HS components can be easily separated and nuclear Overhauser effect (NOE) enhancements support the finding that the components are of relatively low molecular weight

Bacteria are a major source of natural products that provide rich opportunities for both chemical and biological investigation. Although the vast majority of known bacterial metabolites derive from free-living organisms, increasing evidence supports the widespread existence of chemically prolific bacteria living in symbioses. A strategy based on bioinformatic prediction, symbiont cultivation, isotopic enrichment, and advanced analytics was used to characterize a unique polyketide, nosperin, from a lichen-associated Nostoc sp. cyanobacterium. The biosynthetic gene cluster and the structure of nosperin, determined from 30 μg of compound, are related to those of the pederin group previously known only from nonphotosynthetic bacteria associated with beetles and marine sponges. The presence of this natural product family in such highly dissimilar associations suggests that some bacterial metabolites may be specific to symbioses with eukaryotes and encourages exploration of other symbioses for drug discovery and better understanding of ecological interactions mediated by complex bacterial metabolites.biosynthesis | Peltigera membranacea | trans-acyltransferase polyketide synthase | 13 C nuclear magnetic resonance

LC-DAD-MS monitored fractionation of aHarpagophytum procumbens DC. (Pedaliaceae) root extract was combined with a hyphenated LC-DAD-MS/SPE-NMR technique, thus providing the spectral data needed for structure elucidation. This approach allowed the characterization of isobaric iridoid glycoside regioisomers present only as minor constituents. The analytes were identified as the (E/Z) pairs of 6′-O-(p-coumaroyl)harpagide (6′-PCHG) and 8-O-(p-coumaroyl)-harpagide (8-PCHG). The fact that 8-(Z)-PCHG constitutes a new natural product underlines the analytical power of this combined approach. Furthermore, derivatives 6′-(Z)-and 6′-(E)-PCHG are new constituents for H. procumbens.

Directly coupled HPLC-NMR-MS was used to identify and confirm the presence of quercetin O-glycosides and phloretin O-glycosides in an extract of apple peel. From the MS and MS/MS data, the molecular weights of the intact molecules as well as those of quercetin and phloretin and their sugar moieties were deduced. The NMR data provided information on the identity of the compounds as well as the alpha and beta conformations and the position of the glycosides on quercetin and phloretin. The following O-glycosides of quercetin could be identified: quercetin-3-alpha-L-rhamnosyl-(1-->6)-beta-D-glucoside (rutin), quercetin-3-beta-D-galactoside (hyperin), quercetin-3-beta-D-glucoside (isoquercitrin), quercetin-3-beta-D-xyloside (reynoutrin), quercetin-3-alpha-L-arabinofuranoside (avicularin), and quercetin-3-alpha-L-rhamnoside (quercitrin). Phloretin was present as phloretin-2'-beta-D-glucoside (phloridzin) and the 2'-beta-D-xylosyl-(1-->6)-beta-D-glucoside. Concentrations were between 0.2 and 5 mg/g of apple peel.

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When small B lymphocytes bind antigen in the context of suitable signals, a profound geno-proteomic metamorphosis is activated that generates antibody-secreting cells. To study the metabolic changes associated with this differentiation program, we compared the exometabolome of differentiating murine B lymphoma cells and primary B cells by monodimensional proton nuclear magnetic resonance spectroscopy and mass spectrometry coupled to liquid chromatography. Principal component analysis, a multivariate statistical analysis, highlighted metabolic hallmarks of the sequential differentiation phases discriminating between the proliferation and antibody secreting phases and revealing novel metabolic pathways. During proliferation, lactate production increased together with consumption of essential amino acids; massive Ig secretion was paralleled by alanine and glutamate production, glutamine being used as carbon and energy sources. Notably, ethanol and 5'-methylthioadenosine were produced during the last phase of protein secretion and the proliferative burst, respectively. Our metabolomics results are in agreement with previous genoproteomics studies. Thus, metabolic profiling of extracellular medium is a useful tool to characterize the functional state of differentiating B cells and to identify novel underlying metabolic pathways.

In this study, the hyphenation of LC-SPE-NMR-MS at 500 MHz was applied to the structural elucidation of a low concentrated paracetamol metabolite present in human urine. Single or multiple peak trapping of the mass detected metabolite on SPE cartridges was employed to increase the sensitivity and quality NMR measurement over the conventional LC-NMR method. After the elution of the metabolite from the SPE cartridge to the NMR flow probe using deuterated acetonitrile for initial NMR investigation, the fraction was revovered by flushing the sample out of the NMR probe head with nitrogen gas. On the recovered fraction, high resolution FT-ICR-MS measurements were conducted, giving exact mass information about the unknown metabolite. In addition, a cryogenic NMR micro probe head was used to enhance the sensitivity of the NMR measurement by a factor of 5 in order to run 2D experiments for structural elucidation of the unknown metabolite. The combination of both MS and NMR results, led unequivocally to the elucidation of the structure as the ether glucuronide of 3-methoxyparacetamol.