Rapid identification and characterization of known and new natural products directly from plant and marine sources without the necessity of isolation and purification can be achieved by various modern hyphenated techniques. Techniques like HPLC coupled to NMR or electrospray ionization tandem mass spectrometry (ESI-MS-MS) have been proven to be extremely powerful tools in natural product analysis, as they permit the fast screening of crude natural product extracts or fractions for detailed information about metabolic profiles, with a minimum amount of material.
The combined application of various hyphenated techniques even allows the discovery of new natural product, including complete and conclusive structure elucidation, and relative configurations prior to time-consuming and costly isolation and purification process. Some examples of the application of hyphenated techniques in natural products analysis are discussed here.
1. Isolation and analysis of natural products
Crude natural product extracts, which represent extremely complex mixtures of numerous compounds, can be analyzed successfully by using appropriate hyphenated techniques. Among the various hyphenated techniques.
LC-PDA and LC-MS are the two most extensively used for natural product analysis.
LC-NMR, as well as different multiple hyphenated techniques like LC-PDA-NMR-MS have also become popular most recently.
LC-MS, if the ionization technique is chosen appropriately, can be an extremely powerful and informative tool for screening crude plant extracts. The currently available various types of LC-MS systems allow the analysis of small nonpolar compounds to large polar constituents like oligosaccharides, proteins, and tannins present in natural product extracts.
Alkaloids
Alkaloids are a large group of nitrogen-containing secondary metabolites of plant, microbial, or animal origin.
Various hyphenated techniques have been used in the analysis of several types of alkaloids to date. With the development and wider availability of bench-top systems, GC-MS has become the method of choice for the analysis of various pyrolizidine and quinolizidine types of alkaloids.
Quinolizidine alkaloids, the main class of alkaloids found in the family Leguminosae, have been analyzed by GC-MS recently. Most of these alkaloids are sufficiently volatile and thermostable under GC conditions to permit analysis without chemical modification. However, some hydroxylated pyrolizidine alkaloids need to be analyzed as their trimethylsilyl derivatives. Ephedrine-type alkaloids, in dietary supplements containing the Chinese herb ma huang, were analyzed by GC-MS and GC-FTIR.
A number of protoberberine metabolites, differing in the number and the placement of various oxygen functions on the aromatic rings, have been identified prior to isolation from the Corydalis cell cultures by LC-NMR and LC-MS. This study provided the preliminary evidence for biosynthetic pathways to the formation of these alkaloids, especially the metabolic pathway to 2, 3, 10, 11-oxygenated tetrahydroprotoberberines in cultured cells.
An APCI interface was used in the LC-MS system, and the mass spectra were obtained with selected ion monitoring (SIM) and total ion monitoring (TIM) in the positive ion mode. Molecular ion information was obtained on the basis of protonated molecular ion [M + H]+ or a cluster ion [M + HCF3]+. The LC-NMR analysis was carried out on a Varian UNITY-INOVA-500 NMR spectrometer equipped with a PFG indirect detection LC-NMR probe with a 60 mL flow cell, using stop-flow mode. The LC operation was performed on a Cosmosil 5 C18-AR (4.6 × 150 mm) reversed-phase column, using a mobile phase composed of solvent A ¼ 0.1 M NH4OAc (0.05% TAF) and solvent B ¼ ACN. A gradient elution protocol was adopted as follows: 20–30% B in 10 min, 30% B in 10 min, and 30–155% B in 10 min, flow rate 1 mL/min, detection at 280 nm.
Coumarins
The coumarins are the largest class of 1-benzopyran derivatives that are found mainly in higher plants. HPLC-PDA can be used successfully in the analysis of various phenolic compounds, including coumarins, because of the presence of significant amounts of chromophores in these molecules. The HPLC-PDA determination of coumarins, where absorption spectra are registered with a PDA detector, provides useful information about the identity of the molecule including oxidation pattern. The retention time together with the UV spectrum of individual peaks can be considered characteristic, and can easily be used to detect known coumarins in a crude extract. The coupling of MS to LC-PDA provides further structural information that is helpful for on-line identification of individual coumarins in any crude extract. Various coumarins together with other oxygen heterocyclic compounds, e.g., psoralens and polymethoxylated flavones, present in the nonvolatile residue of the citrus essential oils of mandarin, sweet orange, bitter orange, bergamot, and grapefruit, were analyzed by atmospheric pressure ionization (API) LC-MS system equipped with an APCI probe in positive ion mode. Recording MS spectra at different voltages provided information on molecular weight as well as fragment ions, and this allowed the identification of the main components in the extracts. In this study, cold-pressed citrus oils were analyzed by a Shimadzu LC system coupled with UV and MS detector with an APCI interface. The LC separation was carried out on a C18 Pinnacle column (250 × 4.6 mm, 5 mm), eluted isocratically or using a gradient at a flow rate of 1 mL/min with the solvent mixture: solvent A (THF:ACN:MeOH: water¼15:5:22:58) and solvent B (100% ACN). As coumarins are UV-absorbing compounds, they could be detected at 315 nm. The MS acquisition conditions were as follows: probe high voltage, 4 kV; APCI temperature, 400°C; nebulizing gas (N2) flow rate, 2.5 L/min; curved desolvation line (CDL) voltage, 25.5 V; CDL temperature, 230°C; deflector voltage, 25 and 60 V; and acquisition mode SCAN, 50–500 m/z.
Carotenoids
This group of natural products includes the hydrocarbons (carotenes) and their oxygenated derivatives (xanthophylls). LC-TLS has been applied successfully for the determination of carotenoids in four marine phytoplankton species, and a good degree of separation of diadinoxanthin, diatoxanthin, and other carotenoids has been achieved by isocratic HPLC elution with a greater sensitivity and selectivity than UV detection. This technique has allowed the monitoring of the interconversion of diadinoxanthin to diatoxanthin, and changes of other carotenoids under different light conditions. LC-TLS has also been found to be an ultrasensitive method for determination of b-carotene in fish oil-based supplementary drugs.
2. Essential oil and volatile components
GC-MS has been demonstrated to be a valuable analytical tool for the analysis of mainly nonpolar components and volatile natural products, e.g., mono- and sesquiterpenes. Chen et al. described a method using direct vaporization GC-MS to determine approx 130 volatile constituents in several Chinese medicinal herbs. They reported an efficient GC-MS method with EI for the separation and structure determination of the constituents in ether-extracted volatile oils of Chinese crude drugs, Jilin Ginseng, Radix aucklandiae, and Citrus tangerina peels. The components, predominantly monoterpenes, of the volatile oil of the oleoresin of Pestacia atlantica var. mutica have been analyzed recently by GC-MS together with the application of on-line databases.
Saponins
Saponins are steroidal or triterpenoidal glycosides that occur widely in plant species of nearly 100 families. As saponins are highly polar compounds and difficult to volatilize, the application of GC-MS is mainly restricted to the analysis of aglycones known as sapogenins or saponins. Apart from just a few, saponins do not have chromophores that are essential for UV detection. Owing to the lack of chromophores in saponins, it is not helpful to use a UV or PDA as the primary detection technique. An alternative primary detection technique, e.g., refractive index, could be used. Sometimes, precolumn derivatization of saponins can be used to attach a chromophore that facilitates UV detection at higher wavelengths. Among the hyphenated techniques, LC-MS, LC-NMR, and CE-MS could be useful for the rapid initial screening of crude extracts or fraction for the presence of saponins. While in LC-MS analysis TSP interface is used most extensively in phytochemical analysis, saponins having more than three sugars cannot be analyzed using this interface. For larger saponins (MW > 800), CF-FAB or ES is the method of choice. A combination of matrix solid-phase dispersion extraction and LC-NMR-MS was applied in the rapid on-line identification of asterosaponins of the starfish Asterias rubens. The LC-NMR-MS provided structural information in one single chromatographic run and was suitable for saponins in the molecular mass range 1200–1400 amu. This technique also allowed semiquantitative LC-NMR measurements through methyl signals (Me-18 and Me-19) of the steroidal skeleton.
3. Dereplication
The discrimination between previously tested or recovered natural product extracts and isolated single components found therein is essential to decrease the screening costs by reducing the large collections of isolates that are then subject to further detailed evaluation. Bioassay guided natural product isolation often leads to already known compounds of limited, or no chemical or pharmacological interest. Hence, appropriate methods that can distinguish at an early stage novel rather than known or already isolated natural compounds are essential for modern cost-effective natural product research.
Dereplication strategies employ a combination of separation science, spectroscopic detection technologies, and on-line database searching. Thus, the combination of HPLC with structurally informative spectroscopic detection techniques, e.g., PDA, MS, and NMR, could allow crude extracts or fractions to be screened not just for biological activity but also for structural classes. To perform an efficient screening of extracts, both biological assays and HPLC analysis with various detection methods are used.
Techniques such as HPLC coupled with UV photodiode array detection (LC-DAD-UV) and with mass spectrometry (LC-MS or LC-MS-MS) provide a large number of on-line analytical data of extract ingredients prior to isolation. The combination of HPLC coupled to NMR (LC-NMR) represents a powerful complement to LCUV-MS screening. These hyphenated techniques allow a rapid determination of known substances with only a small amount of source material.
LC-MS-MS spectra are generally reproducible. Therefore, the MS-MS databases of natural products can be used for dereplication purposes. For automated on-line dereplication purposes, Q-DIS/MARLINTM is one of the comprehensive and powerful analytical knowledge platforms available today, which is ideally suited for natural product dereplications. It permits quick identification of novel chemical classes based on LC-MS data. Q-DIS/MARLINTM is also perfect to automatically validate proposed chemical structures from combinatorial chemistry experiments of LC-MS data. Most of the dereplication protocols available to date for natural product analysis predominantly apply LC-PDA-MS. The LC-NMR, despite being able to provide more meaningful structural information, has achieved limited success owing to the lack of sensitivity, the dearth of general access to high-field NMR instruments, and the cost associated with the use of deuterated solvents.
3. Chemical fingerprinting and quality control of herbal medicine
The use of hyphenated techniques, e.g., LC-MS, CE-MS, LC-NMR, or LC-NMR-MS, in chemical fingerprinting analysis for quality control and standardization of medicinal herbs has attracted immense interest in recent years. Generally, in the context of drug analysis, fingerprinting method is used to highlight the profiles of the sample matrix, which is often sufficient to provide indications of the source and method of preparation. In herbal medicines, the profile depends not only on the preparation processes but also on the quality of the crude herb source material. The quality of the same herb can vary considerably depending on the geographic origins, sources, harvest times, and so on. The uniformity and stability of the chemical profiles thus represent the quality of the raw herbs. In both good agricultural practice (GAP) and good manufacturing practice (GMP), fingerprinting analysis is used to appraise the quality of the herbal material. In this process, the fundamental objective is to develop links between marker compound-based chromatographic or spectroscopic profiles and the efficacy of herbal products. Thin layer chromatography (TLC) has been the most widely used classical method for fingerprinting analysis in Chinese medicines. In the chemical fingerprinting method, wherever possible, the bioactive compounds or important chemical marker compounds are identified to allow consistent batch-to-batch fingerprinting analysis. For example, in the analysis of valerian (Valeriana officinalis) and feverfew (Tanacetum parthenium), the two marker compounds are valerenic acid and acetoxyvalerenic acid in the former case, and parthenolide and sesquiterpene lactones in the latter. GC-MS or LC-MS can be used to detect and confirm the identity of these trace marker compounds. ESI technique was used in the HPLCMS-based detailed chemical fingerprinting of danshen, sanqi, and ginkgo. In fingerprinting analysis, it is imperative to optimize all laboratory instrumentations and methodology to avoid any artifacts in the results. The relative intensity of the peaks is important, and chromatographic fingerprints must be specific for the substance being analyzed.
Hence, it is necessary to check fingerprints obtained from related botanical products and known adulterants to ensure that the method developed can distinguish true from false identifications. Several analytical protocols based on LC-MS fingerprinting have been developed and integrated into a high-throughput analytical program incorporating standard methods, template structure determination, and structural libraries. For example, LC-MS was used to characterize mixtures of taxanes from Taxus brevifolia extracts and to develop a taxane database. The sensitivity of the currently available hyphenated techniques permits minimum sample preparation, thus saving analysis time and reducing unnecessary degradation of the components.
Medicinal properties of herbs used in traditional systems of medicine, e.g., traditional Chinese medicine or Ayurveda, are attributed to the presence of various types of biologically active molecules. Any variation, either qualitative or quantitative, in the chemical profile of the herb can lead to the total loss of medicinal properties, decreased potency, or even increased toxicity. Therefore, it is essential, for quality control purposes, to ascertain the presence of certain molecules in the herbal preparation or extract, and also to determine the quantity of each of the active principles by applying a suitable method, which allows on-line detection of molecules present in the herbal extract. TLC or paper chromatography used to be the method of choice for the quality control of most of the ancient herbal medicines. Nowadays, with the advent of modern hyphenated techniques, it is possible to obtain comprehensive chemical profiles of herbal medicine preparations or extracts. GC-MS and LC-MS are now being used quite extensively for direct on-line analysis of components present in the herbal preparations and for ensuring the quality of the herb. These techniques have been used in the traditional Chinese medicine.
The mass spectra of various components present in the extracts of Chinese medicine have been obtained on-line from the LC-MS run and matched with known standards for structural confirmation. Integrated MS databases have also been useful for identification of these compounds. In this way, GC-MS, LC-MS, and MS-MS fingerprinting profiles of the active ingredients of various Chinese herbal extracts have been obtained, and information has been stored in the form of an electronic database, which can be used for routine comparison of chemical profiles of individual herb extracts for quality control purposes. The GC or LC retention time and mass spectral data are reproducible, provided the chromatographic and spectroscopic conditions are kept constant. LC-NMR and LC-NMR MS have also been used to this purpose. A simple protocol for the chemical fingerprinting of Ephedra using HPLC-PDA has been recently described. Ephedra sinica (family: Ephedraceae), known as “ma huang,” is one of the oldest medicinal herbs used in traditional Chinese medicine. In the West, dietary supplements containing E. sinica have emerged as one of the top selling weight loss and endurance enhancing products used by over a million consumers.
4. Chemotaxonomy
Chemical taxonomy or chemotaxonomy is based on the principle that the presence of certain secondary metabolites is dictated by various enzymes involved in the biosynthesis of these compounds. These enzymes are strictly related to the genetic make-up of the organism. Hence, chemical profiling of these secondary metabolites, either by complete isolation and identification, or by separation and on-line identification using modern hyphenated techniques, could provide useful information with regard to the taxonomic or even phylogenetic relationships among various species. Introduction of hyphenated techniques in chemotaxonomic work can reduce the time and cost considerably by allowing on-line detection and identification of secondary metabolites present in extracts. Kite et al. described the application of GC-MS in the chemotaxonomic studies based on quinolizidine alkaloid profile in legumes. Using GCMS, it was possible to obtain data on the quinolizidine alkaloids of less readily available taxa by analyzing crude extracts made from small fragments of herbarium specimens, and thus compile a well-founded knowledge base on the distribution of such compounds in various species of legumes. On the basis of this distribution pattern of quinolizidine alkaloids, various chemotaxonomic inferences could be made. For example, from the GC-MS data analysis, it was observed that Poecilanthe contained quinolizidine alkaloids, and the particular combination of structures present suggested a similarity with members of Brongniartieae.
Acceptable HPLC separation using a reversed-phase C18 column eluting a gradient of ACN–water or MeOH–water mixture could be achieved for flavonoids and other phenolic compounds. LC-MS was also found to be useful in chemotaxonomic studies based on flavonoid profiles in legumes. Both ES and APCI sources could ionize flavonoids in these mobile phases, and acceptable ionization could be achieved in both positive and negative modes to yield [M + H] and [M – H]- ions, respectively. This technique allowed the analysis of various crude aqueous methanolic extracts of leaves or seeds of several legume species (without further 100 mL/500 mg of dry cells) and filtered, and the solvent was removed under vacuum to yield a dry extract. The residue was dissolved in acetone (100 mL) prior to injection into an HP1090M HPLC-PDA-MS system.
The separation of isoprenoid quinones in the extracts was carried out using an Intersil ODS-2 (1.5 ± 250 mm) column eluted isocratically with MeOH-isopropyl alcohol (7:3 v/v) at a flow rate of 0.11 mL/min. For postcolumn mixing of matrix and splitting, a solution of 6% m-nitrobenzyl alcohol in MeOH was used as a matrix and mixed with HPLC eluent at 1 mL/hour. The solution was split at a ratio of 25:1. On-line MS analysis was performed on a JEOL JMS-SX 102 mass spectrometer equipped with a Frit-FAB ion source and a FAB gun. Xe_ was employed as a primary beam at 10 mA of emission current.
5. Metabolomics (metabolomes analysis)
The term “metabolome” refers to the entire complement of low molecular weight metabolites inside a biological cell, and is also used to describe the observable chemical profile or fingerprint of the metabolites in whole tissue. The metabolomes represent the life history of an individual organism, including age and environmental factors such as soil type, moisture content, temperature, and stress factors. The study involving the detailed analysis of these metabolomes is referred to as “metabolomics,” which is a newly emerging area in natural product research in the postgenome era.
The aim of metabolomics, for example, plant metabolomics, is to provide a better understanding of metabolic or other physiological phenotypes through global genome-related technology. In metabolomics, nontargeted metabolite profiling and linkage of these profiles to genotypes or transcript profiles are regarded as the key issues. The metabolite profiling, without the isolation of individual metabolites, requires sophisticated high-throughput analytical techniques, e.g, various types of hyphenated techniques. A typical example of metabolomic study can be demonstrated from the metabolite profiling, which is the first trial of a metabolomic approach of an anthocyanin chemotype, in red and green forms of Perilla fructescens using LC-PDA-MS, CE for anion analysis and LC for amino acid analysis. In addition, studies on cell-specific anthocyanin accumulation and localization of anthocyanidin synthase, and gene expression using mRNA differential display of two chemo-varietal forms of P. fructescens, were also carried out.
CONCLUSION:
The technique developed from the coupling of a separation technique and an on-line spectroscopic detection technology is known as hyphenated technique. The remarkable improvements in hyphenated analytical methods over the last two decades have significantly broadened their applications in the analysis of biomaterials, especially natural products. In this article, recent advances in the applications of various hyphenated techniques, e.g., GC-MS, LC-MS, LC-FTIR, LC-NMR, CE-MS, etc. in the context of preisolation analyses of crude extracts or fraction from various natural sources, isolation and on-line detection of natural products, chemotaxonomic studies, chemical fingerprinting, quality control of herbal products, dereplication of natural products, and metabolomic studies are discussed with appropriate examples. Particular emphasis is given on the hyphenated techniques that involve LC as the separation tool.
References
- Patel, Kalpesh, et al. “Introduction to hyphenated techniques and their applications in pharmacy.” Pharmaceutical Methods, vol. 1, no. 1, 2010, p. 2. Gale OneFile: Health and Medicine. Accessed 19 Aug. 2021.
- Sarker SD, Nahar L. Hyphenated techniques and their applications in natural products analysis. Methods Mol Biol. 2012;864:301-340. doi:10.1007/978-1-61779-624-1_12