INTRODUCTION:

Coupling Techniques combine chromatographic and spectral methods to increase the advantages of both that are chromatography and spectroscopy. Chromatography refers pure or nearly pure fractions of chemical components in a mixture. Spectroscopy produces selective information for identification of pure component using standards or library spectra. The coupling technique mainly used for analysis of natural products because of its complex structures. A natural product means herbs, herbal concoctions, dietary supplements, traditional Chinese medicine, or alternative medicine. Natural products are generally either of prebiotic origin or originate from microbes, plants, or animal sources. As chemicals, natural products include such classes of compounds as terpenoids, polypeptides, amino acids, peptides, proteins, carbohydrates, lipids, nucleic acid bases, ribonucleic acid (RNA), deoxyribonucleic acid (DNA). Natural products mainly present in crude form and having complex structure so coupling technology is useful tool for purification and identification of the structure ^[1].

Hirschfield defined the term hyphenated techniques as “the online combination of chromatographic techniques and one or more spectroscopic techniques”.

In recent years, hyphenated techniques have received a principle attention to solve complex analytical problems.

These techniques are carried out for qualitative and quantitative analysis of unidentified compound in complex natural products and complex synthetic mixtures.

Natural product research and development:

Modern hyphenated techniques are useful for Rapid identification and characterization of known and new natural products directly from plant and marine sources without the necessity of isolation and purification. 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. Many changes have been carried out in natural product research now days. With the various developments in the field of separation science, spectroscopic techniques, and micro plate-based ultrasensitive in vitro assays, natural product research mainly developed novel and interesting chemical scaffolds. The various available hyphenated techniques, e.g., GC-MS, LC-MS, LC-FTIR, LC-NMR, LC-NMR-MS, CE-MS, have been used for the pre-isolation analyses of crude extracts or fractions of it from different natural sources, separation and on-line revealing of natural products, chemotaxonomic studies, chemical finger printing, quality control of herbal products, dereplication of natural products, and metabolic studies. These developed changes have been carried out from extraction to resolve of the structures of purified products and their natural activity ^[2].

Analytical techniques used for natural products:

Mainly these techniques are used for purification, isolation, identification of complex compounds after synthesis or after extraction process.

A. Chromatography

B. Spectroscopy

C. Hyphenated Techniques (Coupling techniques)

Chromatography:

Chromatography shows that the greatest number of new developed methods were published for pharmaceutical analysis, including applications identification, purity testing, assay, stability testing, and content uniformity testing of drug products, intermediates, and raw materials, as well as analysis of drugs and their metabolites in biological samples. Chromatographic analysis plays a fundamental role in the drug development process. It is involved in release testing and stability study of identity and purity of active pharmaceutical ingredient (API) in both drug substance and drug product ^{[3, 4]}. In Column chromatography high-performance liquid chromatography (HPLC) is the most common technology employed for achiral Impurity Isolations and Chiral Impurity Isolations.

Spectroscopy:

Ultraviolet and visible absorption spectroscopy, Infrared spectroscopy, Fluorimetry and chemiluminescence, X-ray fluorescence spectrometry, Atomic absorption and flame emission spectroscopy, Atomic emission spectroscopy, Nuclear magnetic resonance spectroscopy, Mass spectrometry all these are the methods mainly used for the structural elucidation of natural products. When a mixture of compounds whose absorption spectra are known is analyzed, then the mixture’s composition can be determined. The method is based on absorption spectra of pure individual components and calibrating mixtures of well defined fraction components.

The Fourier transform infrared spectrometry offers numerous possibilities for the treatment of spectra and has applications for the analysis of structured micro samples (infrared microanalysis).

FTIR is used to study the dispersion of the active agent of a medication within its excipients, or to evaluate the impurities in a foodstuff for cattle. Statistical methods and chemo metric approaches as partial least square (PLS) or principal component analysis (PCA) are then very useful ^[5].

NMR has become one of the principal study techniques for inorganic crystals as well as molecular structures. Although NMR has for a long time been considered as not sensitive enough to be adaptable to environmental analysis.

Mass spectrometry (MS) is an analytical method of characterizing matter, based on the determination of atomic or molecular masses of individual species present in a sample. Miniaturization and the emergence of new ionization techniques allow to this method to be present in a variety of sectors: organic and inorganic chemistry, biochemistry, clinical and environmental chemistry, and geochemistry. This technique is even now becoming essential in the fields of genomics and proteomics ^[5].

Traditional analytical approaches including HPLC, GC, UV detection, etc., have become insufficient to effectively address the growing number of challenges in analyses of species- specificity and sensitivity. The hyphenated technique is developed from the combination of a separation technique that is chromatographic techniques and an on-line spectroscopic revealing technology. In recent years, hyphenated techniques have been used to solve complex mixtures, isolation, identification and purification by using analytical technologies that is spectroscopic techniques and chromatographic techniques has been carried out for both quantitative and qualitative analysis of unidentified compounds in complex natural product extracts or fractions^[7] .

Hyphenated techniques includes: LC-IR, LC-NMR, LC-MS, HPLC- NMR-MS, LC-ESI-MS and UPLC–MS, etc. Newly developed hyphenated analytical methods have significantly given their application in the analysis of biomaterial, especially natural products and apart from routine research, these techniques are also finding increasing application in commercial uses.

These hyphenated techniques offer

• Shorter analysis time

• Higher degree of automation

• Higher sample throughput

• Better reproducibility

• Reduction of contamination because it is a closed system

• Enhanced combined selectivity and therefore higher degree of information.

LC-IR:

This technique is the coupling of liquid chromatography and infrared spectroscopy or FTIR is known as LC-IR or HPLC-IR. IR or FTIR is a helpful spectroscopic technique as it shows spectrum of functional groups in mid IR region which helps in structural detection of compounds in a given sample. These hyphenated techniques 237 absorption bands of the mobile phase solvent are so huge in the mid-IR region that they often used the small signal generated by sample components. Mainly two approaches are used in these techniques that are flow cell approach and solvent elimination approach ^{[8, 9]}.

Fig: LC-IR^[10]

The flow cell approach is as like that of UV-visible or other detector used in HPLC. In this case absorption of mobile phase induces detection of sample component absorption bands. While in solvent elimination approach, after the mobile phase solvent has been eliminated IR detection is carried out in such a medium that have transference for IR region. Generally, KBR or KCL salts are used for the collection of sample components in the eluent, and heating up the medium before IR detection eliminates the volatile mobile phase solvents. There are two types of interfaces for the solvent elimination approach that is diffuse-reflectance infrared Fourier transform (DRIFT) approach and buffer–memory technique ^[11].

LC–NMR TECHNOLOGY:

This technique is the coupling of liquid chromatography and nuclear magnetic resonance. This is considered as powerful technique for structural elucidation of natural products because such extraction contains large number of closely related compounds which is difficult to separate.

LC–NMR coupling technology must deal with the interfacing of LC to NMR, the flow-through probe design and factors such as NMR sensitivity, solvent suppression, NMR- and LC-compatible solvents and the volume of the chromatographic peak versus the volume of the NMR flow cell. There are various modes of operation for LC–NMR, which can be distinguished by the status of the sample during the measurement. For example, in the on flow mode, the sample under study is flowing continuously through the NMR flow-cell during data acquisition.

Fig: A typical LC-NMR system ^[12]

In the Several applications of continuous-flow LC–NMR with environmental samples, biological fluids and natural products have shown that stopped-flow experiments must be carried out to achieve adequate data acquisition times for the structure elucidation of unknown compounds ^[13].

On-flow LC–NMR (continuous flow)

In this method the NMR the sample is measured without stopping the flow and the result is displayed as a two-dimensional (2D) time–frequency plot consisting of a set of one-dimensional spectra (frequency domain) versus retention time. The main problem of on-flow operation with short residence time is that 1H-NMR data acquisition and S/N for minor compounds is insufficient and the direct acquisition of 13C–NMR spectra even for the main constituents is not feasible. An additional complication occurs when solvent gradients are used for LC separation; the NMR chemical shifts of the solvent and analyte resonances depend on the solvent composition and vary continuously as spectra are acquired during the chromatographic run.

Several applications of continuous-flow LC–NMR given with environmental samples^{[14, 15]}. Biological fluids and natural products have shown that stopped-flow experiments must be carried out to achieve adequate data acquisition times for the structure elucidation of unknown compounds ^{[16, 17]}.

LC–NMR under static conditions

In this methods NMR measurements can be carried out under non flowing or static conditions: (i) use of a valve to stop the elution when the analyte reaches the flow-cell volume within the rf coil, the so-called stopped-flow mode or (ii) use of sample loops to store the individual analyte fractions obtained from the chromatographic separation. In both cases the analytes can be examined with more time intensive 1D and 2D NMR experiments ^{[18, 19]}.

In the field of natural products, the combination of on-flow mode and stopped-flow has been used extensively ^[20,
21].

Cryogenic technology in LC–NMR

This technology carried out to increase NMR sensitivity. It has been realized that cooling of NMR rf coil and preamplifier electronics to cryogenic temperature, which eliminates the thermal electronic noise related with initial stages of signal detection and increases the coil quality factor. This leads to improvement in the S/N ratio by a factor of 3-4^[22,
23].

CapLC–NMR or micro flow NMR

This is the most promising development in NMR probe design, apart from the cryogenic probes, which gives improvement in mass sensitivity, is the introduction of miniaturized solenoid micro coils. The first approach to fabricate such micro coils involved the miniaturization of the classical Helmholtz or Saddle type coil used in conventional probes. Solenoid micro coils with an active detection volume of 1.5 microlitre allow detection limits in the low nanogram range for low molecular weight metabolites .the small detection volume is suited for the eluting peak volumes of capillary separation .therefore; microprobe with solenoid rf coil is mainly used in miniautherised hyphenated systems e.g. Capillary high-performance liquid chromatography NMR (capLC–NMR), capillary electrophoresis NMR (CE–NMR), capillary electro chromatography NMR (CEC NMR), and capillary isotachophoresis NMR (cITP–NMR).

The on-going development of LC–NMR holds much promise for advances in the fields of plant metabolomics and natural-products analysis. Detailed LC–NMR investigations of medicinal plants could contribute to new leads to drug development ^{[24, 25, 26]}.

LC–SPE–NMR

This technique is used to increase the sensitivity of the concentrate ample within a small volume and along with small volume NMR rf coils. Solid phase extraction useful technique for reproducible rapid and selective sample preparation .The use of guard column after the LC separation, the concentrated elute compounds are carried out for NMR analysis, as well as use of SPE cartridge connected to an NMR flow probe have been described to enhance the sensitivity of LC-NMR.LC-SPE-NMR technology recently used to identify in a commercial rosemary extract and extract of Rhaponticum Carthamoides ^{[27, 28]}.

LC-MS:

LC-MS or HPLC-MS is the coupling of liquid chromatography with a mass spectrometer; here identification of sample depends upon the mass spectral data which should be separated from the column. This is a powerful technique used for various applications which has very high sensitivity and selectivity.

LC-MS combines separating power of LC along with MS which is selectively detect and confirm molecular identity. In this system various types of interfaces are commercially available, such as nebulization and vaporization of liquid, ionization of the sample, removal of the excess solvent vapor and extraction of the ion into the mass analyser.Specially in natural product analysis electron spray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are used, which gives high solvent flow rate capability, sensitivity, response linearity and fields of applicability.

In LC reverse-phase system using gradient or isocratic solvent mixture of water, ACN or MeOH and small amount of acetic acid, ammonia solution used as mobile phase and in mass spectrometer different types of analyzers like quadrapole, ion trap or TOF can be used and results of this technique mainly depends on various factors that is nature of compounds to be analysed, solvent and buffer used as mobile phase, the flow rate and the type of interface used ^[29].

HPLC-NMR-MS:

This technique is the coupling of high performance liquid chromatography, nuclear magnetic resonance and mass spectrometry. Natural products extracts are a logical choice for characterization by hyphenated HPLC-NMR-MS. In HPLC-NMR effluent from a chromatographic column is directly analyzed in the NMR spectrometer without the need for peak isolation and sample preparation. These technologies Used to increase the impact of natural products in HTS based drug discovery ^{[30, 31]}.

Early applications of LCNMR used either 1) on-flow detection, where the spectrum is measured as the column effluent passes through the NMR flow-probe, or 2) stopped-flow detection, where the chromatographic flow is switched off in a timed fashion to ‘park’ the peak of interest in the flow probe for measurement. The drawback to these early methods is that in on flow measurements, the limited measuring time and sample flow reduce the quality of the spectrum.

Advances in the HPLC-NMR technique were offered by the development of peak capture and peak management methods that effectively eliminated the need to perform the NMR measurement during the chromatographic run ^{[32, 33]}.

LC-ESI-MS:

This technique is the coupling of liquid chromatography-electro spray ionization-mass spectrometry (LC-ESI-MS).By using this technique we detected several minor alkaloids such as dehydrogenated forms of α-tomatine, demissine and commersonine. Total GA content, expressed as the sum of the four main alkaloids α-solanine, α-chaconine, solamargine and solasonine differed from species to species.

Fig: Schematic of an LC-MS (electro spray ionization interface) system ^[34]

The identification of individual GAs was carried out with a LC-ESI-MS system consisting of a solvent system from Latek Laboranalysen (Heidelberg, Germany) coupled with a Micromass VG Quattro II mass spectrometer (Waters, Manchester, UK). ESI-MS was performed using Mass Lynx version 4.0 (Waters, Manchester, UK). Nitrogen was used as a nebulizing and drying gas and was generated by a Parker nitrogen generator (Parker, Etten-Leur, and The Netherlands). An electro spray interface was used for ionization. LC separation was carried out using a LiChrospher 100 RP-18 (250 mm × 4 mm, 5 μm) analytical column from Merck (Darmstadt, Germany). Separation of the GAs at ambient temperature was achieved using a binary gradient system consisting of water (solvent A) and acetonitrile (solvent B), both containing 0.1% formic acid. The pump programme ranged from 10 to 60% solvent B within 20 min, followed by 100% solvent B in another 10 min with a constant flow rate of 1 ml min−1. Sample volumes of 20 μl were injected by a Rheodyne injection valve.

Post flow splitting was set to 1:5.Mass-spectrometric detection of positively charged ions was performed in the positive ion mode over the range m/z 50–1,200 and the instrument was set to the following tuning parameters: nebulizing gas pressure 13 l h−1, drying gas pressure 350 l h−1, capillary voltage 3.5 kV, high-voltage lens 0.5 kV, cone voltage 90 V, temperature of the heated transfer capillary 120 °C. Chromatograms were processed using Mass Lynx version 4.0.

In LC-ESI-MS spectra, dominating peaks were from protonated ions. Because interglycosidic bonds have very low binding energies, partly in-source fragmentation took place. This offered additional structural information. Alkaloids of S. tarijense, S. alandiae, S. pascoense, S. raphanifolium, S. maglia and S. chomatophilum were analysed in detail by this techniques ^[35].

The identification of individual glycosides was carried out with a LC-ESI-MS system Consisting of a solvent system from Latek Laboranalysen coupled with a Micromass VG Quattro II mass spectrometer’s-MS was performed using Mass Lynx version.

In LC-ESI-MS spectra, dominating peaks were from protonated ions. Because interglycosidic bonds have very low binding energies, partly in-source fragmentation took place. This offered additional structural information. Stobiecki et al. (2003) and Cataldi et al. (2005) already showed that cleavage at the interglycosidic bonds is the dominant process in the fragmentation of α-tomatine or the solanidine and solasodine type alkaloids, respectively. Chen et al. (1994) explained the cleavage by protonation of the interglycosidic oxygen followed by hydrogen transfer and cleavage of the interglycosidic bond distal to the aglycone ^[36].

UPLC–MS:

This technique is the combination of ultra performance liquid chromatography-mass spectrometry using three different cone voltage, and both positive and negative ionization modes.

This system consist of Acquity UPLC chromatograph coupled in series to a tunable dual-wavelength ultra/visible detector and a micomass Quattro macro API mass spectrometer was used for analysis and electro spray probe was used as the ionization source.

Mobile phase used in HPLC like methanol, ethanol and acetic acid, Milli-Q water: Milli-Q water as stimulant a, 3% (W/V) acetic acid in water as stimulant C, and 95 %( v/v) ethanol in water as stimulant D. The standard compounds like diethylene glycol, terphthalate (PET).

UPLC-MS technique is used for screening of nonvolatile contaminants in recycled polyethylene terphthalate (PET).Mainly in developed countries the increasing use of plastics and the environmental pressure associated with them have prompted authorities to establish regulations to reduce the amounts of plastics that are deposited in waste dump.Consiquently there is increasing pressure to recycle plastics such recycled material is cheaper than the virgin material.

PET can be successfully mechanically recycled, and a number of studies have investigated the scope for using recycled PET in various applications, especially applications in which it would come into contact with food ^{[37, 38]}.

The most problem linked with the use of recycled material such as PET in food containers is the likely migration of chemicals from the plastic container. Specific migration tests are carried out to ensure the materials can be used safely. From the study it is clear that standard protocol for screening of nonvolatile compounds is needed. Many steps are carried out to check the chemicals are present in samples that are not present in background or blank sample: (a) direct perfusion of the sample and ion monitoring for screening purposes; (b) UPLC–MS, monitoring the main ions; (c) a chemical and bibliographic study to identify the compounds that are likely to be present and the ions they are likely to generate; d) the roles of the compounds found and their origins. This study was concerned with the nonvolatile compounds in recycled PET, which was carried out by UPLC–MS ^[39].

Applications:

Hyphenated techniques mainly used for identification and characterization of natural products from plant material. In which HPLC along with NMR or electro spray ionization tandem mass spectrometry have been proven to be extremely useful tool in natural product analysis.

Isolation and analysis of natural products:

The analysis of crude drugs from the plant extracts is a very difficult task. The above discussed hyphenated techniques are well capable of separating the drugs from natural product sources. They allow analysis of small nonpolar compounds to large polar compounds like oligosaccharides, proteins and tannins. Various alkaloids, coumarins, tannins, resins and essential volatile oil components can be separated by these techniques.

Alkaloids:

Alkaloids are the large group of nitrogen-containing secondary metabolites of plant, microbial, or animal origin. For the isolation of alkaloids various hyphenated techniques mainly used. Mainly GC-MS used for analysis of various pyrolizidine and quinolizidine type of alkaloids found in the family Leguminosae. However some hydroxylated pyrolizidine alkaloids analysed as their trimethylsily derivatives and Ephedrine alkaloids were also analysed by GC-MS and GC-FTIR in dietary supplement containing the Chinese herb ma huang ^[40].

Many number of protoberberine metabolites, differing in the number and position of oxygen atom in aromatic rings have been checked after isolation from the corydalis cell cultures by LC-NMR and LC-MS ^[41].

Coumarins:

The coumarins are the largest class of 1-benzopyran derivatives that are found mainly in higher plants. HPLC-PDA mainly used successfully in the analysis of phenolic compounds, including coumarins, because of the presence of chromophores in these molecules. In which absorption spectra are registered with a PDA detector gives information about the identity of the molecule involves in oxidation. The retention time along with UV spectrum gives individual peaks which is characteristic one and easily used to detect coumarins in a crud extract.

The coupling of MS to LC-PDA provides the structural information useful for the identification of individual coumarins in any crude extract. Various coumarins along with oxygen heterocyclic compounds, e.g., psoralens and polymethoxylated flavones, present in the non-volatile 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 ^[42].

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 ^[43].

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 ^{[44, 45]}.

Saponins:
Saponins are steroidal or triterpenoidal glycosides that occur widely in plant species of nearly 100 families. Saponins are highly polar compounds hence difficult to volatilize for which the use of LC-MS is essential for analysis of aglycones known as sapogenins or Saponins.

LC-MS, LC-NMR, CE-MS, LC-NMR-MS useful for detection of Saponins. 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 chromophores that facilitate UV detection at higher wavelengths ^[46,
47].

Dereplication:

The discrimination between previously tested or recovered natural product extracts and isolated single components found is essential to decrease the screening costs by reducing the large collections of isolates that are then subject to further detailed evaluation. 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 and with mass spectrometry 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 LC-UV-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 reproducible. Therefore, the MS-MS databases of natural products can be used for dereplication purposes. For automated on-line dereplication purposes, most of the dereplication protocols available for natural product analysis. The LC-NMR, being able to provide more meaningful structural information, has achieved limited success due to the lack of sensitivity, lack of general access to high-field NMR instruments, and the cost associated with the use of deuterated solvents^[48].

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 sesquiterpenes lactones in the latter. GC-MS or LC-MS can be used to detect and confirm the identity of these trace marker compound ^{[49, 50]}.

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.

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. 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 flavonoids 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 ^[51].

Metabolomics:
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. LC-MS is also used in the study of proteomics where again components of a complex mixture must be detected and identified in some manner. The bottom-up proteomics LC-MS approach to proteomics generally involves protease digestion and denaturation (usually trypsin as a protease, urea to denature tertiary structure and iodoacetamide to cap cysteine residues) followed by LC-MS with peptide mass fingerprinting or LC-MS-MS (tandem MS) to derive sequence of individual peptides. LC-MS-MS is most commonly used for proteomic analysis of complex samples where peptide masses may overlap even with a high-resolution mass spectrometer. Samples of complex biological fluids like human serum may be run in a modern LC-MS-MS system and result in over 1000 proteins being identified ^[52].

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.

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Received on 27.07.2012 Modified on 13.08.2012

Research J. Pharm. and Tech. 5(9): September 2012; Page 1145-1153