LC–SPE–NMR–MS: a total analysis system for bioanalysis (original) (raw)
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Liquid chromatography (LC)–nuclear magnetic resonance (NMR) combines the advantage of the outstanding separation power of liquid chromatography (LC) and the superior structural elucidating capability of nuclear magnetic resonance (NMR). NMR has proved that it is a standout detector for LC by providing maximum structural information about plant originated extracts, particularly on the isolating ability of isomeric (same molecular formula) and/or isobaric (same molecular weight) compounds as compared to other detectors. The present review provides an overview of the developmental trends and application of LC–NMR in natural product analysis. The different LC–NMR operational modes are described, and how technical improvements assist in establishing this powerful technique as an important analytical tool in the analysis of complex plant-derived compounds is also highlighted. On-flow, stop-flow and loop-storage modes, as well as the new offline mode LC–solid phase extraction (SPE)–NMR and...
In many metabolomics studies, metabolite identification by mass spectrometry (MS) is often hampered by the lack of good reference compounds; and hence, nuclear magnetic resonance spectroscopy (NMR) information is essential for structural elucidation, especially for the very large group of (plant) secondary metabolites. The classical approach for compound identification is to perform time-consuming and laborious high pressure liquid chromatography (HPLC) fractionations and purifications, before (re)dissolving the molecules in deuterated solvents for NMR measurements. Hence, a more direct and easy purification protocol would save time and efforts. Here, we propose an automated MS-guided HPLC-MSsolid phase extraction (SPE)-NMR approach, which was used to fully characterize flavonoid structures present in crude tomato plant extracts. NMR spectra of plant metabolites, automatically trapped and purified from LC-MS traces, were successfully obtained, leading to the structural elucidation of the metabolites. The MS-based trapping enabled a direct link between the mass signals and NMR peaks derived from the selected LC-MS peaks, thereby decreasing the time needed for elucidation of the metabolite structures. In addition, automated 1 H-NMR spectrum fitting further speeded up the candidate rejection process. Our approach facilitates the more rapid unraveling of yet unknown metabolite structures and can therefore make untargeted metabolomics approaches more powerful. structural elucidation, tomato background line of Moneymaker cultivar) 14, 15 was grown in a greenhouse in Wageningen, The Netherlands, harvested in June 2003, and pooled per plant, after which small pieces of tomato fruit or the frozen powder were stored at -80 ˚C.
Assessment of Compatibility between Extraction Methods for NMR- and LC/MS-Based Metabolomics
Analytical Chemistry, 2012
Because of the wide range of chemically and structurally diverse metabolites, efforts to survey the complete metabolome rely on the implementation of multiplatform approaches based on nuclear magnetic resonance (NMR) and mass spectrometry (MS). Sample preparation disparities between NMR and MS, however, may limit the analysis of the same samples by both platforms. Specifically, deuterated solvents used in NMR strategies can complicate LC/MS analysis as a result of potential mass shifts, whereas acidic solutions typically used in LC/MS methods to enhance ionization of metabolites can severely affect reproducibility of NMR measurements. These intrinsically different sample preparation requirements result in the application of different procedures for metabolite extraction, which involve additional sample and unwanted variability. To address this issue, we investigated 12 extraction protocols in liver tissue involving different aqueous/organic solvents and temperatures that may satisfy the requirements for both NMR and LC/MS simultaneously. We found that deuterium exchange did not affect LC/MS results, enabling the measurement of metabolites by NMR and, subsequently, the direct analysis of the same samples by using LC/MS with no need for solvent exchange. Moreover, our results show that the choice of solvents rather than the temperature determined the extraction efficiencies of metabolites, a combination of methanol/chloroform/water and methanol/water being the extraction methods that best complement NMR and LC/MS analysis for metabolomic studies.
Nuclear magnetic resonance spectroscopy (NMR) is arguably the most versatile analytical platform for complex mixture analysis. Specifically, interfacing liquid chromatography with parallel NMR and mass spectrometry (LC-NMR-MS) gives comprehensive structural data on metabolites of novel drugs in development. Applications in natural product, combinatorial chemistry and drug metabolism studies are reviewed.
Metabolomics, 2015
Liquid chromatography-mass spectrometry (LC-MS) methods using either aqueous normal phase (ANP) or reversed phase (RP) columns are routinely used in small molecule or metabolomic analyses. These stationary phases enable chromatographic fractionation of polar and non-polar compounds, respectively. The application of a single chromatographic stationary phase to a complex biological extract results in a significant proportion of compounds which elute in the non-retained fraction, where they are poorly detected because of a combination of ion suppression and the co-elution of isomeric compounds. Thus coverage of both polar and non-polar components of the metabolome generally involves multiple analyses of the same sample, increasing the analysis time and complexity. In this study we describe a novel tandem in-line LC-MS method, in which compounds from one injection are sequentially separated in a single run on both ANP and RP LC-columns. This method is simple, robust, and enables the use of independent gradients customized for both RP and ANP columns. The MS signal is acquired in a single chromatogram which reduces instrument time and operator and data analysis errors. This method has been used to analyze a range of biological extracts, from plant and animal tissues, human serum and urine, microbial cell and culture supernatants. Optimized sample preparation protocols are described for this method as well as a library containing the retention times and accurate masses of 127 compounds.
Universal quantitative NMR analysis of complex natural samples
Nuclear Magnetic Resonance (NMR) is a universal and quantitative analytical technique. Being a unique structural tool, NMR also competes with metrological techniques for purity determination and reference material analysis. In pharmaceutical research, applications of quantitative NMR (qNMR) cover mostly the identification and quantification of drug and biological metabolites. Offering an unbiased view of the sample composition, and the possibility to simultaneously quantify multiple compounds, qNMR has become the method of choice for metabolomic studies and quality control of complex natural samples such as foods, plants or herbal remedies, and biofluids. In this regard, NMR-based metabolomic studies, dedicated to both the characterization of herbal remedies and clinical diagnosis, have increased considerably.
LC-NMR coupling technology: recent advancements and applications in natural products analysis
Magnetic Resonance in Chemistry, 2005
An overview of recent advances in nuclear magnetic resonance (NMR) coupled with separation technologies and their application in natural product analysis is given and discussed. The different modes of LC-NMR operation are described, as well as how technical improvements assist in establishing LC-NMR as an important tool in the analysis of plant-derived compounds. On-flow, stopped-flow and loop-storage procedures are mentioned, together with the new LC-SPE-NMR configuration. The implementation of mass spectrometry in LC-NMR is also useful on account of the molecular weight and fragmentation information that it provides, especially when new plant species are studied. Cryogenic technology and capillary LC-NMR are the other important recent developments. Since the plant kingdom is endless in producing potential drug candidates, development and optimization of LC-NMR techniques convert the study of natural products to a less-time-consuming task, speeding up identification.
F1000 - Post-publication peer review of the biomedical literature
A novel strategy is introduced that combines high-resolution mass spectrometry (MS) with NMR for the identification of unknown components in complex metabolite mixtures encountered in metabolomics. The approach first identifies the chemical formulas of the mixture components from accurate masses by MS and then generates all feasible structures (structural manifold) that are consistent with these chemical formulas. Next, NMR spectra of each member of the structural manifold are predicted and compared with the experimental NMR spectra in order to identify the molecular structures that match the information obtained from both the MS and NMR techniques. This combined MS/NMR approach was applied to E. coli extract where the approach correctly identified a wide range of different types of metabolites, including amino acids, nucleic acids, polyamines, nucleosides and carbohydrate conjugates. This makes this approach, which is termed SUMMIT MS/NMR, well suited for high-throughput applications for the discovery of new metabolites in biological and biomedical mixtures overcoming the need of experimental MS and NMR metabolite databases.
Current approaches and challenges for the metabolite profiling of complex natural extracts
Journal of Chromatography A, 2014
Metabolite profiling is critical in many aspects of the life sciences, particularly natural product research. Obtaining precise information on the chemical composition of complex natural extracts (metabolomes) that are primarily obtained from plants or microorganisms is a challenging task that requires sophisticated, advanced analytical methods. In this respect, significant advances in hyphenated chromatographic techniques (LC-MS, GC-MS and LC-NMR in particular), as well as data mining and processing methods, have occurred over the last decade. Together, these tools, in combination with bioassay profiling methods, serve an important role in metabolomics for the purposes of both peak annotation and dereplication in natural product research. In this review, a survey of the techniques that are used for generic and comprehensive profiling of secondary metabolites in natural extracts is provided. The various approaches (chromatographic methods: LC-MS, GC-MS, and LC-NMR and direct spectroscopic methods: NMR and DIMS) are discussed with respect to their resolution and sensitivity for extract profiling. In addition the structural information that can be generated through these techniques or in combination, is compared in relation to the identification of metabolites in complex mixtures. Analytical strategies with applications to natural extracts and novel methods that have strong potential, regardless of how often they are used, are discussed with respect to their potential applications and future trends.
Journal of Separation Science, 2002
Isolation of circulating metabolites in drug discovery using high-performance liquid chromatography, and their identification by liquid chromatography coupled with tandem mass spectrometry and nuclear magnetic resonance spectroscopy One of the major components of modern drug discovery is the structural determination and the assessment of biological activity of plasma metabolites. LC-MS/MS has played a prominent role in the identification of metabolites; however, fragmentation patterns alone may not be sufficient for identification. Consequently, it may be necessary to isolate the metabolites for NMR or LC-NMR analysis. This report describes the isolation and identification of the major plasma metabolites of two lead compounds (SCH X and SCH Y). The major metabolite of SCH X in monkey plasma constituted 65% of total compound-derived materials. Incubation of rat liver microsomes with SCH X gave the mono-hydroxylated metabolite found in monkey plasma; however, the yield was low. Incubations with microsomes from rats pre-treated with various cytochrome P450 inducers showed that the highest yield was obtained from pregnenolone 16a-carbonitrile (PCN)-induced animals. For SCH Y, two metabolites were found in bile and plasma of both rats and monkeys. Various in vitro systems did not produce amounts sufficient for isolation. Therefore, the metabolites of SCH X and SCH Y were isolated from PCN-induced rat liver microsomal incubation and rat bile, respectively. The chemical structures of the metabolites were unambiguously determined using LC-NMR analyses.