Applications of NMR Spectroscopy: Volume 9 E-Book
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- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Serie: Applications of NMR Spectroscopy
- Sprache: Englisch
Applications of NMR Spectroscopy is a book series devoted to publishing the latest advances in the applications of nuclear magnetic resonance (NMR) spectroscopy in various fields of organic chemistry, biochemistry, health and agriculture.
The ninth volume of the series features reviews that highlight NMR spectroscopic techniques in microbiology, food science, pharmaceutical analysis and cancer diagnosis. The reviews in this volume are:
- NMR spectroscopy for the characterization of photoprotective compounds in cyanobacteria
- Coffee assessment using 1H NMR spectroscopy and multivariate data analysis: a review
- Evaluation of structure-property relationship of coconut shell lignins by NMR spectroscopy: from biorefinery to high-added value products
- Application of NMR spectroscopy in chiral recognition of drugs
- NMR-based metabolomics: general aspects and applications in cancer diagnosis
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Seitenzahl: 338
Veröffentlichungsjahr: 2006
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PREFACE
Nuclear Magnetic Resonance (NMR) spectroscopy has emerged as one of the most powerful techniques for the identification of materials and the study of their dynamic properties. As a result, the technique has found tremendous uses in almost all fields of physical, natural, and health sciences.
Volume 9 of the book series entitled Applications of NMR Spectroscopy is comprised of 5 chapters. In chapter 1 of the book, Sinha et al. have discussed the usage of NMR spectroscopy in the identification of photoprotective compounds and its advantages and disadvantages for metabolomic studies. In chapter 2 of the book, Valderrama et al. present an overview of 1H NMR-based metabolomics analysis used in coffee quality control. Avelino and Lomonaco, in the next chapter of the book, discuss the development of the lignin field by reviewing the recent advances provided by NMR spectroscopy. In chapter 4 of the book, Vashistha et al. highlight the current developments in NMR spectroscopy for chiral recognition of pharmaceuticals reported during the past five years. da Silva Júnior et al., in the last chapter of the book, discuss NMR-based metabolomics, its general aspects and applications in cancer diagnosis.
We wish to thank all eminent scientists for their scholarly contributions. The editorial team of Bentham Science Publishers, particularly Ms. Asma Ahmed (Senior Manager Publications), Obaid Sadiq (in-charge books department), and team leader Mr. Mahmood Alam (Editorial Director), deserves our deepest appreciation for compiling an excellent volume in a time-efficient manner. We are confident that, like the previous volumes of this book series, the current treatise will also receive wide appreciation from both the readers and practitioners of NMR spectroscopy.
List of Contributors
NMR Spectroscopy For The Characterization of Photoprotective Compounds in Cyanobacteria
Abha Pandey1,Neha Kumari1,Sonal Mishra1,Jyoti Jaiswal1,Rajeshwar P. Sinha1,*Abstract
Cyanobacteria are ubiquitous in nature as they efficiently tolerate various extreme climatic conditions for survival, such as increasing effects of solar radiation, salinity, and temperature, etc. Cyanobacteria are important sources of secondary metabolites, which enable them to withstand these harsh environmental conditions. Small-molecular-weight secondary compounds are primarily implied in the defense mechanisms in the case of biotic and abiotic stresses. Various beneficiary secondary compounds are educed from cyanobacteria, such as UV-screening pigments (mycosporine-like amino acids, scytonemin, carotenoids, etc.), phytohormones, cyanotoxins, and antioxidants. Bioactivity-directed isolation techniques are used to identify these molecules from complicated matrices in pharmacognosy (discovery of biologically active compounds from natural sources). NMR spectroscopy has appeared as a specific major analytical technique applied in metabolomics. The easy sample preparation, the expertise to evaluate metabolite quantity, the notable investigational reliability, and the innately non-destructive quality of NMR spectroscopy have made it the first-line option for significant scientific metabolic analyses. Unlike some mass spectrometry methods, NMR is not discriminatory, depending on the metabolites' precursors or their ionization potential. Screening of metabolites needs maximum sensitivity, and it is a process with a broad scope. In this chapter, we have discussed the usage of NMR spectroscopy in the identification of photoprotective compounds and its advantages and disadvantages for metabolomic studies. We have also explored several new NMR techniques that have recently become available in order to fortify its advantages and overcome its inherent limitations in metabolomics applications.
INTRODUCTION
UV-radiation levels on Earth's surface are increasing due to CFCs and reduced cloud cover [1]. Cyanobacteria are the principal origin for many metabolites, such as alkaloids, carbohydrates, flavonoids, pigments, phenols, saponins, steroids, tannins, terpenes and vitamins, which could be exploited in the biotechnology and industrial sectors [2]. Cyanobacteria are able to survive in high UV radiation through several photoprotective mechanisms. Quenching strategies are being used by distinct species, which frequently imply the DNA repair system, antioxidative enzymatic activities, and UV-screening compounds in combination.
UV-induced damaged DNA can be repaired by photoreactivation, excision and mismatch repair. Photoprotective compounds (PPCs), having UV-absorbing properties, such as mycosporine-like amino acids (MAAs) and scytonemin, are produced by cyanobacteria, which help in the protection against excessive UVR. Various environmental factors like the variations in the intensity and wavelength of UVR, nutrient deficiency curb, and a number of stresses affect the biosynthesis of these compounds [3].
In the past twenty years, quick and effective characterization of secondary metabolites has become a great challenge for the investigation of novel bioactive compounds and drug research. Many secondary metabolites have been identified by conventional techniques in cyanobacteria. Compounds were generally isolated from cyanobacterial extracts by semi-preparative liquid chromatography and identified using classical spectrometric techniques, namely ultraviolet-visible (UV-Vis) spectrophotometry, infrared (IR) spectroscopy, mass spectrometry (MS) or nuclear magnetic resonance (NMR). However, these processes are time-consuming. In order to expedite the isolation and purification of compounds from cyanobacterial extracts, various techniques are being used in metabolomics such as gas chromatography-mass spectroscopy (GC-MS), liquid chromatography-mass spectroscopy (LC-MS), and high-performance liquid chromatography (HPLC), often in combination with mass spectroscopy (HPLC-MS), or nuclear magnetic resonance (HPLC-NMR) [4-10]. Each technology has its own advantages and limitations. The selection of technology is focused on the inquiry and the nature of the samples and is also determined by the evaluation and its expertise availability. Recognition and evaluation of most of the metabolites are usually not determined by a single technology, and mostly, various technologies are being used for a complete study.
NMR is an extensively used technique for studying secondary metabolites from natural cyanobacterial extracts. It is the most effective technique for the structural identification of unknown compounds in a mixture. However, the isolation and at least partial purification of compounds have to be carried out prior to NMR spectroscopy. To reduce these time-consuming steps, physical coupling of liquid chromatography and NMR has been used in the last two decades. In practice, the routine applications of HPLC-NMR have been successfully applied only in the last ten years. NMR spectra of HPLC purified fractions from biological samples are now possible due to the introduction of flow-through probe heads.
Compared to other technologies, NMR has several ascendancies in being non-destructive, non-adherent, easily quantifiable; it does not require chromatographic separation, sample treatment, chemical derivatization, and the identification of novel compounds. NMR is fully automated and surprisingly reproducible, allowing high throughput [11], large-scale metabolic studies that can be done easily by NMR spectroscopy in comparison to LC-MS or GC-MS. NMR is also used for the characterization of sugars, organic acids, alcohols, and highly polar compounds. NMR analysis is not only used for biofluids or tissue extracts but is also applicable for whole tissues, organs, and other samples by using solid-state NMR (ssNMR) and magic-angle sample spinning (MAS) NMR [12-15]. Furthermore, NMR is exploited in the metabolite imaging of live samples via magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) [16-20]. Real-time metabolite characterization of living samples can be done by NMR spectroscopy [21, 22]. Molecules can be studied at the atomic level, not only based on 1H atoms but several other biologically reactive groups, including 13C, 15N and 31P [23-28]. Lack of sensitivity is a limitation of NMR as it only provides information on 50 to 200 recognized metabolites with concentrations greater than 1 μM. NMR spectroscopy requires large amounts of samples, as compared to HPLC, GC, MS, GC-MS and LC-MS. Nevertheless, this is typically not a major problem in microbiology. This chapter provides an overview of the utilization of NMR spectroscopy in the profiling and structural elucidation of photoprotective compounds in cyanobacteria.
PRINCIPAL PHOTOPROTECTIVE COMPOUNDS IN CYANOBACTERIA
Scytonemin and MAAs are the chief UV-screening pigments biosynthesized by cyanobacteria mainly in response to harsh UV-irradiation. They are considered powerful UV-absorbing biomolecules.
Mycosporine-Like Amino Acids (MAAs)
MAAs are UV-absorbing, uncoloured, and water-soluble small molecules with molecular weights between 188 and 1050 Dalton. They are characterized by a cyclic-hexenone or cyclic-hexenimine chromophore combined with nitrogen or alternatively an amino acid or its imino alcohol, with absorption maxima between 309 and 362 nm [29-33]. The first MAA was reported by Shibata in 1969 isolated from a cyanobacterium from the Great Barrier Reef [34]. Thereafter, many MAAs such as mycosporine-glycine, shinorine, palythinol, palythene, porphyra-334, and asterina-330 have been characterized [35]. They are powerful UV-A/UV-B-absorbing compounds having high coefficients between 28,100 and 50,000 M cm-1 [36, 37]. They act as photoprotectants due to their characteristic UV absorption maxima, photostability, and resistance against various extreme physicochemical stressors such as high UV radiation or excessive temperature and pH.
MAAs help protect the cell from UV radiation by dissipating the energy as heat, thereby avoiding the production of reactive oxygen species (ROS) [38]. MAAs have become indispensable for the medicinal and cosmetic industries due to their photoprotective abilities. The chemical structures of some MAAs are shown in Fig. (1).
Fig. (1)) Chemical structures of several MAAs with their corresponding absorption maxima (λmax).Scytonemin
Cyanobacteria produce an extracellular sheath, which is made up of polysaccharides. Scytonemins, lipid-soluble yellow-brown dimeric pigments with indolic and phenolic subunits with a molecular weight of 544 Dalton are often embedded in the sheath. Scytonemins come in two forms; fuscorhodin is the reduced form (red colored), and fuscochlorin is the oxidized form (yellow colored) [39, 40]. The in vivo absorption maximum of scytonemin is at 370 nm, whereas the absorption maximum of isolated scytonemin is at 386 nm. In addition, the molecule significantly absorbs at 252, 278, and 300 nm. UV-A-induced inhibition of photosynthesis and photobleaching of chlorophyll a have been found to be reduced in the presence of scytonemin [41, 42]. The UV-protective capacity of scytonemin has also been established in the terrestrial cyanobacterium Chlorogloeopsis sp [43, 44]. The stability of scytonemin is maximal even in adverse or excessive conditions, such as harsh UV radiation, extreme temperatures, etc. Scytonemins are photoprotective even when cells are inactive physiologically, and other photoprotective strategies, such as repair of damaged machinery of cells, would be ineffective [45]. As MAAs, scytonemin may be exploited as a sunscreen in cosmetic industries due to its higher screening potential [46, 47]. The chemical structure of scytonemin and its derivatives are shown in Fig. (2). The applications of both scytonemin and MAAs are summarized in Table 1.
Fig. (2)) Chemical structures of scytonemin and its derivatives.NMR SPECTROSCOPY
One-Dimensional (1D) NMR Spectroscopy
The principles involved in NMR spectroscopy are as follows:
Each metabolite is composed of atoms that constitute nuclei. Each nucleus is comprised of positive charges that are responsible for the spin of the nuclei.If a magnetic field is applied externally, the energy is absorbed by the atoms, and the atoms undergo a transition into a higher excited state. The energy transfer occurs at a particular wavelength that corresponds to the frequency.When the external magnetic field is removed, the atoms return to their original state, i.e., a lower energy level. The energy is emitted at a specific frequency during this process.The NMR spectrum of that specific nucleus is characterized by these energy transfers and frequencies [48].1H NMR Spectroscopy
1H NMR spectroscopy is widely used in the studies of metabolites that are based on the NMR technique due to the presence of 1H atoms in most of the organic compounds and, hence, in nearly every known metabolite. 1D 1H NMR is greatly automatable, authentic, and rapid, so one-dimensional 1H NMR spectra are mainly used for the study of metabolites. The chemical details present in a 1D 1H NMR spectrum of biological samples extracted from the tissues are sufficient for the characterization and the subsequent quantification of multiple metabolites at a time [49, 50] based on a library of many references. 1H NMR spectra of identified metabolites are found in several public databases, which analyse 1D 1H NMR spectra within a second [51-54]. Also, metabolites can be identified and quantified by using this technique [55, 56]. 1D 1H NMR spectra are mainly used for the quantification of metabolites since they can be performed easily and quickly without complicated sample preparation, and no polarisation transfer methods are used. There is an important role of solvent suppression in metabolomics studies because 1D 1H NMR spectra are conducted in water. On the basis of the types of metabolites to be investigated, various methods can be utilized. NMR-based metabolomic studies involve the collection of dozens to hundreds of spectra, which is critical for excluding solvent effects due to parameter changes. NMR samples can be analysed, stored, and repeatedly reanalysed to check previous findings on the same sample due to the extended storage of biological samples at -80 0C; it shows minute effects on the observed results of NMR [57, 58]. Some limitations of this technique show that there are chances of overlapping peaks leading to vagueness in the characterisation and quantification of compounds. Peak overlapping can be avoided by using higher magnetic fields during NMR experiments, and due to this, spectral dispersion is increased. When compared to 1D 1H NMR spectroscopy, two-dimensional 1H NMR spectroscopy can resolve overlapping peaks because it can determine and identify new metabolites.
13C NMR Spectroscopy
13C NMR spectroscopy is characterised by narrow line widths with broad chemical shift dispersion and thus allows better resolution as compared to 1H NMR. The availability of 13C is low in natural conditions because it has a low sensitivity of the 13C nucleus, hindering its application in metabolomics. Distortionless enhancement by polarisation transfer (DEPT) [59] can be utilized to enhance the 13C signal intensity. The 13C signal can also be enhanced by using glucose having 13C; this technique has been used for a long time for marking metabolites in the research of microbial metabolites and mammalian cell lines [60, 61]. Shanaiah et al. [62] used to mark the unmarked metabolites with 13C [63] as 13C enrichment does not work for mammalian or human studies. Cryoprobe technology in which the NMR probes are frozen to absolute zero to mitigate the electronic noise can be used to enhance the signal. Hyperpolarisation techniques can also be used for the enhancement of 13C NMR signals. The 13C NMR can be used for isotope tracing experiments [64, 65], and can help in the direct carbon determination in biosynthetic pathways and their chemistry.
15N NMR Spectroscopy
15N NMR spectroscopy has characteristics of a broad chemical shift and relatively narrow line widths. Due to its poor sensitivity, direct detection is not possible. Naturally, it is found in low abundance, and the gyromagnetic ratio is also less; therefore, the 15N nucleus is less sensitive than the 1H nucleus. To improve its effect, indirect ion detection is done by the enhancement of isotopes along with 1H. 15N NMR spectroscopy is involved indirectly in the structural analysis of proteins, RNA and DNA, but it cannot be used for studying metabolites. The Raftery group has developed a technique for the detection of NMR-based metabolomics with the 15N isotope [27]. It involves selective tagging of metabolites that provide an individual peak for each marked metabolite and conquers the cues from non-tagged molecules, increasing the sensitivity and peak dispersion. Hundred quantifiable metabolites can be detected from a single class of molecules using a two-dimensional 1H-15N HSQC approach. 15N-cholamine has dual characteristics, which help in the efficient determination of marked metabolites and also in chemical shift [66].
31P NMR Spectroscopy
Inspite the abundance of 31P and its wide spectral dispersion, the sensitivity is 660 times lower than 1HM, therefore, it has limited use in metabolomics studies; due to its comparatively broad spectral dispersion and susceptibility and the absence of phosphorus atoms in most of the metabolites. It is used to study several phosphorus-containing compounds like metabolites containing phospholipids and nucleoside (ATP, GTP, NADP, etc.) that play an essential role in energy metabolism [67]. In this isotope, tagging is used for the detection of several hydrophobic compounds. For the tagging of lipid metabolites having hydroxyl, aldehyde, and carboxyl groups, the 31P reagent 2-chloro-4,4,5,5-tetramethyldioxaphospholane (CTMDP) is used. One-dimensional 31P NMR having high resolution is used for the detection of tagged metabolites.
2D-NMR Spectroscopy
This spectroscopy is used in the identification of molecules, illustration of structures, and study of kinetics [68-70]. It may resolve the problem of overlaying resonances by proliferating the peaks, which depend on different “orthogonal” physical characteristics of the atoms of interest. With 2D NMR having an additional resolution, more metabolites can be identified as compared to 1D NMR. Many homonuclear 2D 1H-1H-NMR [71] and nuclear overhauser effect (NOESY) demonstrations with heteronuclear 1H, 13C coherence have been used to study metabolomics for many years [72-74]. Diffusion ordered spectroscopy (DOSY) [75, 76] and two-dimensional J-resolved NMR spectroscopy (J-Res) [77] are the forms of 2D NMR investigations that have also been used in various metabolomics studies. Here, we will discuss different types of 2D NMR spectroscopy.
Correlation Spectroscopy (COSY)
This is the easiest 2D NMR spectroscopy, and it is used to identify homonuclear correlations between paired nuclei and the molecular structure [78-81]. The COSY sequence consists of a 900 radio frequency (RF) pulse [82] followed by an evolution time (t1). Thereafter, a second 90° pulse (the length of a pulse, usually in microseconds), followed by a measurement period time (t2), is applied. It is helpful in metabolomics study due to its specific characteristics such as it is simple, fast, can be easily performed, and interpreted [83-87]. Unknown metabolites can be identified by COSY cross-peaks represented via bond coupling between coupled nuclei. This spectroscopic technique is being used for the investigation of several unknown and known metabolites, while 1D NMR is restricted only to the known metabolites.
Total Correlation Spectroscopy (TOCSY)
This is also known as homonuclear Hartmann-Hahn (HOHAHA) spectroscopy, in which the chemical shifts of two nuclei are related to each other in the total spin system of a specified compound. Not only cross-peaks of short-range (e.g., 3JHH) combined with protons can be seen, but peaks for protons that are joined by a series of scalar couplings are shown. More time is required for the collection of 2D, while 1D TOCSY requires less time and gives a simple 1D NMR spectrum that can be easily analysed. Selective excitation TOCSY spectroscopy is another version of the TOCSY useful for resolving spectral overlapping problems and the identification of metabolites in a given sample [88-90].
2D J-Resolved Spectroscopy (J-Res)
2D J-Resolved Spectroscopy was initiated by Ernst et al. and is one of the ancient 2D NMR spectroscopy techniques, which analyze both the type of chemical shifts, i.e., J-couplings and chemical shifts [91]. It increases the dispersion of peaks as compared to 1D NMR spectroscopy and helps in spectral assignments. 2D J-Res NMR spectroscopy is used for a broad range of NMR-based metabolomics analyses due to its speed [92]. The major disadvantage of 2D NMR spectroscopy is longer time required for a large number of samples. Frydman et al. (2002) [93] optimised and developed a single-scan 2D NMR method, which is known as planar imaging [94], coupled with the 2D J-Res experiment. This helps in the reduction of the J-Res accession time below one minute [95]. It has low sensitivity, and a high concentration of metabolites is required for metabolomic studies.
Heteronuclear Single Quantum Correlation Spectroscopy (HSQC)
Correlation spectroscopies such as COSY and TOCSY-like spectroscopy are related to the measurement of both homonuclear and heteronuclear correlations. Heteronuclear correlations NMR can be employed for the enhancement of the signal by transferring the nuclear spin polarisation via J-coupling from the I nucleus (usually the proton) to the S nucleus (usually the heteroatom). The magnetisation is first transferred from the high susceptible nucleus 1H towards the low susceptible nucleus 13C or 15N and then goes back to 1H for direct measurement. The 1H, 15N-HSQC spectrum provides the chemical shifts of proton and nitrogen atoms that are bonded directly, presenting individual cross-peaks for each H–N coupled pair. It is used to resolve and assign overlapping proton signals for the metabolites from complex mixtures. Fast metabolite quantification (FMQ) is used for the characterization, and subsequent quantification of the most abundant metabolites, with accuracy, which requires less time for the spectra collection. Heteronuclear multiple-quantum correlation spectroscopy (HMQC) is another correlation similar to the HSQC experiment but differs in the use of the approach of 2D for the transfer of magnetisation from 1H to the heteronuclear. HMQC also produces significantly broader peaks than HSQCs, therefore, it is less used for metabolomics.
Heteronuclear Multiple Bond Correlation (HMBC) Spectroscopy
HMB is also a 2D heteronuclear spectroscopy related to the chemical shifting between two different nuclei. This analysis is being used to understand the relations between nuclei, which are separated by chemical bonds. A low-pass filter is used for the elimination of the single bond correlation where only more minor J-couplings are optimised for detection. HMBC is performed to eliminate a single C-H bond, which is used to assign signals from quaternary and carbonyl carbons that cannot be detected by HSQC or HMQC experiments. HMBC combined with HSQC or HMQC is used for the identification of molecules and chemical structure elucidation.
Some More NMR Techniques Used in Metabolite Identification
High-Resolution Magic-Angle Spinning NMR Spectroscopy (HRMAS)
Due to the robust dipolar coupling and chemical shift anisotropy, broad and unresolved resonances are obtained, mainly magnetic susceptibility gradients responsible for broad spectra [96, 97]. Line widening caused by dipolar interaction and chemical shift anisotropy can be averaged by rotating samples at high speed with respect to a persistent magnetic field. For examining tissues, sample extraction or preparation steps are not needed [98, 99]. HRMAS is very useful and links the metabolic depiction for the biofluids and the histology analysis. After NMR analysis, the microstructure of the tissue specimens remains intact. The tissues do not disintegrate at the time of NMR analysis because the samples are examined at low temperatures in a very short period of time [100]. Other analyses such as histopathology can also be done by using the same tissue specimens for future confirmation due to the non-destructive, and non-disruptive nature of HRMAS.
Hyperpolarisation Methods
Dynamic Nuclear Polarization (DNP)
To enhance the intensity of an NMR signal in weak magnetic fields, DNP is used. DNP is based on the concept of conveying the Boltzmann polarisation by saturating the electron resonances to convey the polarisation of the electron spins to the nuclear spins. A temporary hyperpolarisation is induced in the spin-active nuclei via shifting of electrons to the nuclei of interest. The sample is rapidly melted and relocated to the NMR spectrometer to record the increased NMR signals [101]. The signal increment is analysed by comparing the signals of NMR intensities prior and after the application of DNP.
Parahydrogen-Induced Polarisation (PHIP) and Signal Amplification by Reversible Exchange (SABRE)
The hyperpolarisation approach, which relies on parahydrogen approaches like SABRE and PHIP, has been developed. This technique is successfully used to determine chemical process kinetics, investigate catalytic mechanisms, and study short-lived reaction intermediates [102]. PHIP has been used for the analysis of short-lived reaction intermediates, catalytic mechanisms and kinetics involved in chemistry [103]. PHIP technique produces a constant hyperpolarised gas flow leading to the polarisation, which is being conveyed to the nuclei of a compound. The nontoxic propane gas is used for this and could be generated in a hyperpolarised state through the propene hydrogenation with parahydrogen.
Fast NMR Methods
Fast NMR methods include Nonlinear sampling (NLS), which decreases the value of indirectly determined enhancements and, at the same time, maintains a high resolution [95, 104], and Hadamard spectroscopy [105, 106], in which the structure of the particular metabolites in samples and their chemical shifts can be determined. Ultrafast 2D NMR spectroscopy is faster than rapid 2D NMR spectroscopy, which depends on the indirect increments. A significant drawback of this method is its low sensitivity; to overcome this, another technique called multiscan hybrid ultrafast 2D NMR has been employed that increases the susceptibility of this method.
Pure-Shift NMR
Indirect spin-spin couplings (J) and chemical shifts are two major features for defining the NMR spectra of molecules, and these are important for the characterisation of undetermined metabolites. Pure-shift NMR spectroscopy has better decoupling and, even more significance than spectral simplification. A complex spectrum of biological mixtures is seen due to peak multiplicity resulting from J couplings, but due to its resolution power and sensitivity, it is used to analyse complex mixtures [107, 108]. Recently, a real-time pure shift HSQC-SI sequence, having sensitivity enhancement over ordinary pure-shift NMR experiments, has been optimised for metabolomics studies [108].
LC-NMR and Other Hybrid NMR Approaches
NMR spectroscopy combined with LC-MS allows chromatographic techniques to elucidate the complexity of mixtures. LC-NMR may be performed by the use of fraction collectors and NMR tubes or flow probes; the NMR tube is much faster and simpler. LC-NMR equipped with mass spectrometry allows the identification of the structure of new compounds [109]. The characteristics of NMR spectroscopy are summarised in Table 2.
Applications of NMR in Metabolites
The applications of NMR in various fields are discussed in this section (Fig. 3).
Fig. (3)) Applications of NMR in various fields.Identification and Detection of Metabolite Structures
NMR has been used for a long time for the determination of the chemical structures and conformity of molecules [110]. It is the ultimate method for the complete study of chiral molecules, apart from the single-crystal X-ray diffraction method. NMR provides complete information about the purified small molecular structure. NMR is used to identify the complexity of cells, tissues or biofluids extracts, consisting of several molecules having a very broad range of concentrations. The main objective is the determination of many metabolites as possible representing several routes within the wide metabolic chains. Identification of all of the intermediates is not essential but desirable for the mapping of known pathways. Some part of spectral attributes in metabolite studies cannot be identified. NMR can be used to identify relative changes of unknown metabolites. For the identification of metabolites, several NMR variables, like shifting of chemicals and their reliance on pH, spin heterogeneity, homonuclear and heteronuclear covalent affinity, are involved and several database search tools are available for the identification of metabolites. The sample spectra should be matched by using the standard spectra present in the databases because NMR variables can be susceptible to pH, temperature, concentration, and ionic conditions [111, 112]. This is an important step for NMR data-based metabolite allocation since extraction procedures vary between laboratories. There is a degeneration of chemical shifts at some pH for some metabolites like lactate and threonine at neutral pH. So, 1D databases depending firstly on the shifting of chemicals, could not be authentic, and only a few characteristics are identified due to multiple spectra. To study homogeneous samples, assignments can be verified with the help of multidimensional NMR techniques, which depict the carbon skeleton, along with TOCSY, HSQC, HMBC, and/or involves both HSQC-TOCSY. Multidimensional NMR, including heteronuclear NMR methods, which involve nuclei other than 13C, like 15N and 31P, are used for the identification of unknown spectral features [111, 113]. Compounds can be identified by combining heteronuclear scalar coupling with chemical shift information present in databases and by comparing chemical shifting occurring in compounds with various functional groups. For functional group determination, derivable reagents targeting specific functional groups can be made. Isotope-editing characteristic of NMR can be used for the detection of metabolites having targeted functional groups like 15N ethanolamine and 15N cholamine, and it is used to determine metabolites having a carboxylate group using a 2D 1H (15N)-HSQC technique with more resolving power.
Stable Isotope Tracer Studies
Isotopically utilized improved trackers are crucial to interpret metabolic routes and pathway dynamics. Because it is very sensitive, it can easily detect radioactive metabolites, and methods involving tracers being radiolabeled have been broadly used in life sciences for resolving the routes involved in metabolic reactions. This technology was developed in the 1950s [114, 115
