Fractionation platform for target identification using off-line directed two-dimensional chromatography, mass spectrometry and nuclear magnetic resonance.

The unambiguous identification of unknown compounds is of utmost importance in the field of metabolomics. However, current identification workflows often suffer from error-sensitive methodologies, which may lead to incorrect structure annotations of small molecules. Therefore, we have developed a comprehensive identification workflow including two highly complementary techniques, i.e. liquid chromatography (LC) combined with mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR), and used it to identify five taste-related retention time and m/z features in soy sauce. An off-line directed two-dimensional separation was performed in order to purify the features prior to the identification. Fractions collected during the first dimension separation (reversed phase low pH) were evaluated for the presence of remaining impurities next to the features of interest. Based on the separation between the feature and impurities, the most orthogonal second dimension chromatography (hydrophilic interaction chromatography or reversed phase high pH) was selected for further purification. Unknown compounds down to tens of micromolar concentrations were tentatively annotated by MS and structurally confirmed by MS and NMR. The mass (0.4-4.2 µg) and purity of the isolated compounds were sufficient for the acquisition of one and two-dimensional NMR spectra. The use of a directed two-dimensional chromatography allowed for a fractionation that was tailored to each feature and remaining impurities. This makes the fractionation more widely applicable to different sample matrices than one-dimensional or fixed two-dimensional chromatography. Five proline-based 2,5-diketopiperazines were successfully identified in soy sauce. These cyclic dipeptides might contribute to taste by giving a bitter flavour or indirectly enhancing umami flavour.

On-line coupling of high temperature GPC and 1H NMR for the analysis of polymers.

The on-line coupling of gel permeation chromatography (GPC) and 1H NMR operating at temperatures up to 130 degrees C is presented. A NMR flow probe with a cell volume of 120 microL and a stop-flow valve are developed for on-flow and stop-flow NMR measurements at high temperatures. To maintain high and constant temperatures through the whole probe, the flow probe contains two separate heating circuits. A modified stop-flow valve is developed as a control device for enabling on-flow and stop-flow experiments at high temperature conditions. Heated transfer lines connect the flow probe with the high temperature GPC system. Due to their semicrystalline nature, polyolefins can be studied by liquid chromatography only at temperatures above 100 degrees C. The novel high temperature GPC-NMR system is used for the separation of complex polyolefins regarding their molar mass and for the analysis of different chemical structures. Blends of polyethylene, poly(methyl methacrylate), and ethylene-methyl methacrylate copolymers are separated according to the molar masses of the components. The compositions of the components are directly studied by on-line NMR. Moreover, the chemical composition distribution of an ethylene-methyl methacrylate copolymer sample is analysed. Differences between results of on-flow and stop-flow measurements are discussed.

Application of high-performance liquid chromatography-nuclear magnetic resonance coupling to the identification of limonoids from mahogany tree (Switenia macrophylla, Meliaceae) by stopped-flow 1D and 2D NMR spectroscopy.

Separation and characterization of limonoids from Switenia macrophylla (Meliaceae) by HPLC-NMR technique has been described. Analyses were carried out using reversed-phase gradient HPLC elution coupled to NMR (600 MHz) spectrometer in stopped-flow mode. Separated peaks were collected into an interface unit prior to NMR measurements, which were performed with suppression of solvent signals by shaped pulses sequences. Structure elucidation of the limonoids was attained by data obtained from 1H NMR, TOCSY, gHSQC and gHMBC spectra without conventional isolation that is usually applied in natural products studies.

Characterization of a paracetamol metabolite using on-line LC-SPE-NMR-MS and a cryogenic NMR probe.

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.

Application of NMR spectroscopy and LC-NMR/MS to the identification of carbohydrates in beer.

The application of LC-NMR/MS for the direct identification of carbohydrates in beer has been studied. Carbohydrates are major beer components, and their structural characterization by NMR alone is seriously hindered by strong spectroscopic overlap. Direct analysis of beer by LC-NMR/MS enables the rapid (1-2 h) identification of dextrins with degree of polymerization (DP) of up to nine monomers, with degassing being the only sample treatment required. Although the presence of alpha(1-->6) branching points is easily indicated by NMR for each subfraction separated by LC, difficulties arise for the unambiguous assignment of linear or branched forms of high DP dextrins. The two beer samples investigated in this work were found to have significantly different oligosaccharide compositions, reflecting the different production conditions employed. The use of hyphenated NMR for the rapid characterization of the carbohydrate composition of beers may be the basis of a useful tool for the quality control of beer.

Identification of metabolites in human hepatic bile using 800 MHz 1H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS.

The first application of high field NMR spectroscopy (800 MHz for (1)H observation) to human hepatic bile (as opposed to gall bladder bile) is reported. The bile sample used for detailed investigation was from a donor liver with mild fat infiltration, collected during organ retrieval prior to transplantation. In addition, to focus on the detection of bile acids in particular, a bile extract was analysed by 800 MHz (1)H NMR spectroscopy, HPLC-NMR/MS and UPLC-MS. In the whole bile sample, 40 compounds have been assigned with the aid of two-dimensional (1)H-(1)H TOCSY and (1)H-(13)C HSQC spectra. These include phosphatidylcholine, 14 amino acids, 10 organic acids, 4 carbohydrates and polyols (glucose, glucuronate, glycerol and myo-inositol), choline, phosphocholine, betaine, trimethylamine-N-oxide and other small molecules. An initial NMR-based assessment of the concentration range of some key metabolites has been made. Some observed chemical shifts differ from expected database values, probably due to a difference in bulk diamagnetic susceptibility. The NMR spectra of the whole extract gave identification of the major bile acids (cholic, deoxycholic and chenodeoxycholic), but the glycine and taurine conjugates of a given bile acid could not be distinguished. However, this was achieved by HPLC-NMR/MS, which enabled the separation and identification of ten conjugated bile acids with relative abundances varying from approximately 0.1% (taurolithocholic acid) to 34.0% (glycocholic acid), of which, only the five most abundant acids could be detected by NMR, including the isomers glycodeoxycholic acid and glycochenodeoxycholic acid, which are difficult to distinguish by conventional LC-MS analysis. In a separate experiment, the use of UPLC-MS allowed the detection and identification of 13 bile acids. This work has shown the complementary potential of NMR spectroscopy, MS and hyphenated NMR/MS for elucidating the complex metabolic profile of human hepatic bile. This will be useful baseline information in ongoing studies of liver excretory function and organ transplantation.

Virtual chromatographic resolution enhancement in cryoflow LC-NMR experiments via statistical total correlation spectroscopy.

A new approach to enhancing information recovery from cryogenic probe "on-flow" LC-NMR spectroscopic analyses of complex biological mixtures is demonstrated using a variation on the statistical total correlation spectroscopy (STOCSY) method. Cryoflow probe technology enables sensitive and efficient NMR detection of metabolites on-flow, and the rapid spectral scanning allows multiple spectra to be collected over chromatographic peaks containing several species with similar, but nonidentical, retention times. This enables 1H NMR signal connectivities between close-eluting metabolites to be identified resulting in a "virtual" chromatographic resolution enhancement visualized directly in the NMR spectral projection. We demonstrate the applicability of the approach for structure assignment of drug and endogenous metabolites in urine. This approach is of wide general applicability to any complex mixture analysis problem involving chromatographic peak overlap and with particular application in metabolomics and metabonomics.

Identification of isomeric tropane alkaloids from Schizanthus grahamii by HPLC-NMR with loop storage and HPLC-UV-MS/SPE-NMR using a cryogenic flow probe.

Two fully automated HPLC-NMR methods are reported and compared for the structure elucidation of four isomeric tropane alkaloids from the stem-bark of an endemic Chilean plant, Schizanthus grahamii Gill. (Solanaceae). The first approach interfaced a conventional HPLC column to NMR by means of a loop storage unit. After elution with a mobile phase consisting of deuterated water and standard protonated organic solvents, the separated analytes were momentarily stored in a loop cassette and then transferred one-at-a-time to the NMR flow probe for measurements. The second strategy combined HPLC with parallel ion-trap MS detection and NMR spectroscopy using an integrated solid-phase extraction (SPE) unit for post-column analyte trapping. The SPE cartridges were dried under a gentle stream of nitrogen and analytes were sequentially eluted and directed to a cryogenically cooled flow-probe with an NMR-friendly solvent. The structures of the four isomeric alkaloids, 3alpha-senecioyloxy-7beta-hydroxytropane, 3alpha-hydroxy-7beta-angeloyloxytropane, 3alpha-hydroxy-7beta-tigloyloxytropane and 3alpha-hydroxy-7beta-senecioyloxytropane, were unambiguously determined by combining NMR assignments with MS data.

The application of LC-NMR and LC-SPE-NMR to compositional studies of natural organic matter.

Non-living natural organic matter (NOM) is ubiquitous in the oceans, atmosphere, sediments, and soils, and represents the most abundant organic carbon reserves on earth. However, a large proportion is considered to be "molecularly uncharacterized" because the inherent complexity of NOM is problematic when applying conventional analytical techniques. This manuscript presents initial applications of LC-NMR (1H) and LC-SPE-NMR (1H) to the studies of NOM isolated from water and soil. LC-NMR is applied to dissolved natural organic matter (DNOM) collected from freshwater environments, and both LC-NMR and LC-SPE-NMR are applied to an alkaline soil extract. The polar and complex nature of the DNOM samples limits conventional reversed phase separation, which can be partially overcome with the use of an ion pair reagent, although such an approach further complicates the NMR detection. LC-SPE-NMR of the soil alkaline extract was encouraging, and specific components in the mixture could be assigned. This work demonstrates that it is both possible to separate and concentrate specific components in NOM such that NMR detection is possible. As NMR information will be critical in unraveling the novel and/or complex structures in NOM this represents a key analytical hurdle in this area.