Instrumental Methods for Structure Elucidation of Drugs and Natural products

Introduction

Infrared spectroscopy provides information on the functional group of the constituents present as well as their interactions.
Hyphenated techniques combine chromatographic and spectral methods to produce selective information of fractions of chemical components in a mixture. Spectroscopy produces selective information for identification using standards or library spectra. This technique is simply the marriage of a separation technique and a spectroscopic detection technique. For the scope of this study, GC-MS, LC-MS AND LC-NMR will be covered.

INFRARED SPECTROSCOPY (IR)

Infrared spectroscopy is the analysis of infrared light interacting with a molecule. The main use of this technique in both organic and inorganic chemistry is to determine functional group in molecules. IR Spectroscopy measures atomic vibration, and based on this, the functional group is established.

Hyphenated Techniques

GC-MS

With MS as the preferred detection method, and single- and triplequadrupole, ion trap and time-of-flight (TOF) mass spectrometers as the instruments most frequently used, both LC-MS and GC-MS are the most popular hyphenated techniques in use today. GC-MS, which is a hyphenated technique developed from the coupling of GC and MS, was the first of its kind to become useful for research and development purposes. Mass spectra obtained by this hyphenated technique offer more structural information based on the interpretation of fragmentations. The fragment ions with different relative abundances can be compared with library spectra. Compounds that are adequately volatile, small, and stable in high temperature in GC conditions can be easily analyzed by GC-MS. Sometimes, polar compounds, especially those with a number of hydroxyl groups, need to be derivatized for GC-MS analysis. The most common derivatization technique is the conversion of the analyte to its trimethylsilyl derivative. In GC-MS, a sample is injected into the injection port of GC device, vaporized, separated in the GC column, analyzed by MS detector, and recorded [See the figure below]. The time elapsed between injection and elution is called “retention time” (Rt). The equipment used for GC-MS generally consists of an injection port at one end of a metal column (often packed with a sand-like material to promote maximum separation) and a detector (MS) at the other end of the column.
A carrier gas (argon, helium, nitrogen, hydrogen, to name a few) propels the sample down the column. The GC separates the components of a mixture in time and the MS detector provides information that aids in the structural identification of each component.
The GC-MS columns can be of two types: capillary columns and macro bore and packed columns. The following points need to be considered carefully regarding the GC-MS interface.

The interface transports efficiently the effluent from the GC to MS.
The analyte must not condense in the interface.
The analyte must not decompose before entering the MS ion source.
The gas load entering the ion source must be within the pumping capacity of the MS.

The most extensively used interfaces for a GC-MS are electron impact ionization (EI) and chemical ionization (CI) modes. However, in modern GC–MS systems, various other types can be used that allow identification of molecular ion. For example, an orthogonal TOF mass spectrometry coupled with GC is used for confirmation of purity and identity of the components by measuring exact mass and calculating elemental composition. Nowadays, a GC-MS is integrated with various on-line MS databases for several reference compounds with search capabilities that could be useful for spectra match for the identification of separated components.

Schematic diagram of a GC-MS system

LC-MS AND LC-MS/MS

This refers to the coupling of an LC with a mass spectrometer (MS).
The separated sample emerging from the column can be identified on the basis of its mass spectral data. A switching valve can help make a working combination of the two techniques. A typical automated LC-MS system consists of double three-way diverter in-line with an autosampler, an LC system, and the mass spectrometer. The diverter generally operates as an automatic switching valve to divert undesired portions of the eluate from the LC system to waste before the sample enters the MS.
An LC-MS combines the chemical separating power of LC with the ability of an MS to selectively detect and confirm molecular identity. MS is one of the most sensitive and highly selective methods of molecular analysis, and provides information on the molecular weight as well as the fragmentation pattern of the analyte molecule. The information obtained from MS is invaluable for confirming the identities of the analyte molecules.
This qualitative analysis makes it possible to reconstruct an unknown compound from MS data. The ionization techniques used in LC-MS are generally soft ionization techniques that mainly display the molecular ion species with only a few fragment ions. Hence, the information obtained from a single LC-MS run, on the structure of the compound, is rather poor. However, this problem has now been tackled by the introduction of tandem mass spectrometry (MS-MS), which provides fragments through collision-induced dissociation of the molecular ions produced. The use of LC-MS-MS is increasing rapidly. Hyphenated techniques such as HPLC coupled to UV and mass spectrometry (LC-UV-MS) have proved to be extremely useful in combination with biological screening for a rapid survey of natural products.

Schematic diagram of an LC-MS system (electron spray ionization interface.
Nowadays, various types of LC-MS systems incorporating different types of interfaces are available commercially. The interfaces are designed in such a way that they offer adequate nebulization and vaporization of the liquid, ionization of the sample, removal of the excess solvent vapor, and extraction of the ions into the mass analyzer. The two most widely used interfaces, especially in relation to natural product analysis, are electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), due to their greater ionization stability and more sensitivity than others.
The latter is considered as “the chromatographer’s LC-MS interface” because of its high solvent flow rate capability, sensitivity, response linearity, and fields of applicability. With these interfaces, various types of analyzers, e.g., quadrupole, ion trap, or TOF, can be used. Each of these analyzers, however, offers varying degree of mass accuracy and resolution. In the LC-UV-MS mode, thermo spray (LC-TSP-MS) and continuous-flow FAB (LC-CF-FAB) interfaces can also be applied. For phytochemical analysis, the TSP has been found to be the most suitable interface as it allows introduction of aqueous phase into MS system at a flow rate (1–2 ml/min) compatible with that usually used in phytochemical analysis.
In LC operation for LC-MS, the preferred option is a reversed-phase system using a gradient or isocratic solvent mixture of water, ACN, or MeOH. Small amounts of acetic acid, formic acid, ammonium hydroxide/ammonia solution, or ammonium acetate can also be used in the mobile phase. In conjunction with these interfaces, different types of analyzers, e.g., quadrupole, ion trap, or TOF, can be used, and they offer various degrees of mass accuracy and MS-MS possibilities. LC-MS systems do not allow a complete and unambiguous on-line identification of a component, unless it is a well-known natural product, and complementary on-line spectroscopic information is available in databases. One of the main problems associated with LC-MS is that the quality of response strongly depends on various factors, e.g., nature of the compounds to be analyzed, the solvent and buffer used as the mobile phase, the flow rate and, of course, the type of interface used. For example, a crude natural product extract generally contains a number of various types of compounds that differ considerably in their physicochemical properties, solubility, molecular size and stability. It is therefore extremely difficult, if not impossible, to optimize the ionization conditions that can be suitable for all those different types of compounds. One way to get around this difficulty is to analyze the extract in different ionization modes.
Coupling LC-MS with MS helps in analysis of many metabolites as already mentioned. When simultaneously analyzing compounds belonging to structurally district groups, LC-MS is very efficient compared with GC-MS. LC-MS can determine even polar analytes without need for pior derivatization. With the combination of UV detection, confirmation of the identity of compounds in a sample with the help of standards and literature data is a possibility.
The fragments of the compounds could be obtained from the LC-MS data with reference to flavonoids, you can obtain information on the

Structure of the aglycone
Types of carbohydrate or substituent present
The sequence of glycan part
Interglycosyclic linkages
Attachment point of the substituents to the aglycone

Both positive ionization (PI) and negative ionization (NI) are applied to yield [M-H]+ and [M-H]– ions.
MALDI- Matrix assisted laser desorption ionization, MALDI-TOF- Matrix assisted laser desorption ionization (Time-of-Flight) and API – Atmospheric pressure photoionization are other ionization techniques for analysis of a wide range of biomolecules.
LC-UV-MS can be used to demostrate the existence of the same flavonoid glycosides in related taxa, and sufficient spectral data may be provided to assign the structures of common monoglycosides and di-glycosides by comparison of the standards. The complete structures of more complex glycosides can be obtained only by NMR; either by isolating the compound or possibly by LC-NMR.

LC-NMR

Among the spectroscopic techniques available to date, NMR is probably the least sensitive, and yet it provides the most useful structural information toward the structure elucidation of natural products. Technological developments have allowed the direct parallel coupling of HPLC systems to NMR, giving rise to the new practical technique HPLC-NMR or LC-NMR, which has been widely known for more than last 15 years. The first on-line HPLC-NMR experiment using superconducting magnets was reported in the early 1980s. However, the use of this hyphenated technique in the analytical laboratories started in the latter part of the 1990s only. LC-NMR promises to be of great value in the analysis of complex mixtures of all types, particularly the analysis of natural products and drug-related metabolites in biofluids.
LC-NMR experiments can be performed in both continuous-flow and stop-flow modes. A wide range of bioanalytical problems can be addressed using 500, 600, and 800 MHz systems with 1H, 13C, 2H, 19F, and 31P probes. The main prerequisites for on-line LC-NMR, in addition to the NMR and HPLC instrumentation, are the continuous-flow probe and a valve installed before the probe for recording either continuous-flow or stopped-flow NMR spectra.
A UV–vis detector is also used as a primary detector for LC operation. Magnetic field strengths higher than 9.4 T are recommended, i.e., 1H resonance frequency of 400 MHz for a standard HPLC-NMR coupling. The analytical flow cell was initially constructed for continuous-flow NMR acquisition. However, the need for full structural assignment of unknown compounds, especially novel natural products, has led to the application in the stopped-flow mode.
In fact, the benefits of the closed-loop separation–identification circuit, together with the prospect of using all presently available 2D and 3D NMR techniques in a fully automated way, have prompted the development of stopped-flow modes, e.g., time-slice mode. A typical experimental arrangement of LC-NMR is shown in the figure below. Generally, in LC-NMR system, the LC unit comprises auto sampler, LC pump, column, and a non-NMR detector (e.g., UV, DAD, EC, refractive index, or radioactivity). From this detector, the flow is guided into the LC-NMR interface, which can be equipped with additional loops for the intermediate storage of selected LC peaks. The flow from the LC-NMR interface is then guided either to the flow-cell NMR probe-head or to the waste receptacle. Following passage through the probe-head, the flow is routed to a fraction collector for recovery and further investigation of the various fractions analyzed by NMR. An MS can also be attached to the system via a splitter at the output of the LC-NMR interface. In most of the LC-NMR operations, reversed-phase columns are used, employing a binary or tertiary solvent mixture with isocratic or gradient elution. The protons of the solvents of the mobile phase cause severe problems for obtaining an adequate NMR spectrum. The receiver of the NMR spectrometer is not quite able to handle the intense solvent signals and the weak substance signals at the same time. To overcome this problem, solvent signal suppression can be achieved by one of the three major methods: presaturation, soft-pulse multiple irradiation or water suppression enhancement through T1 effects (WET) presaturation employing a z-gradient. This problem can also be minimized by considering the following guidelines:

Using eluents that have as few 1H NMR resonances as possible, e.g., H2O, ACN, or MeOH.
Using at least one deuterated solvent, e.g., D2O (approx $290/L), ACN-d3 (approx $1600/L), or MeOD (approx $3000/L).
Using buffers that have as few 1H NMR resonances as possible, e.g., TFA or ammonium acetate.
Using ion pair reagents that have as few 1H NMR resonances as possible, e.g., ion pairs with t-butyl groups create an additional resonance.

To date, three main types of data acquisition modes have been introduced: continuous-flow acquisition, stopped-flow acquisition, and time-sliced acquisition. Whatever may be the acquisition mode, an optimized HPLC separation is crucial to any LC-NMR analysis. As the sensitivity of LC-NMR is much less than other hyphenated techniques, e.g., LC-MS, or LC-PDA, it is imperative to develop a suitable LC separation where the quantity of the available separated compound is concentrated in the smallest available elution volume. LC-NMR represents a potentially interesting complementary technique to LC-UV-MS for detailed on-line structural analysis. Indeed, recent progress in NMR technology has given a new impulse to LC-NMR, which is now emerging as a powerful analytical tool. The development of efficient solvent suppression techniques enables the measurement of high-quality LC-1H-NMR spectra, both on-flow and stop-flow, with reversed-phase HPLC conditions. Non deuterated solvents such as MeOH or MeCN can be used, while water is replaced by D2O.
Recent advances in both hardware and software for the direct coupling of LC and NMR have given a new life to this hyphenated technique. These developments include new coil and flow-cell design for high sensitivity, new RF system for multiple solvent suppression and improved dynamic range gradient elution capability, and automatic peak-picking/storing capabilities. As a result, this method is a powerful tool used in many areas such as natural products, organic molecules, bio molecules, drug impurities, by-products, reaction mixtures, and drug degradation products. The potential of HPLC-NMR for the investigation and structural elucidation of novel natural products has been enormously extended by the advent of powerful solvent suppression schemes, and their combination with a series of homo- and heteronuclear 2D NMR experiments such as 2D total correlation spectroscopy (TOCSY) or 2D nuclear Overhauser enhancement spectroscopy (NOESY). LC-NMR, despite being known for about last two decades, has not quite become a widely accepted technique, mainly because of its lower level of sensitivity and higher cost compared to other available hyphenated techniques. However, the recent advances in technology, especially in relation to the developments in pulse field gradients and solvent suppressions methods, the improvement in probe technology, and the introduction of high-field magnets (800–900 MHz) have offered new impetus to this technique.
Recently, on-line or stopped-flow LC-NMR technique has become very useful to natural product research. Knowledge of the differentiation of isomers and substitution pattern are some of the advantages of LC-NMR; high cost of instrument, low sensitivity and long run times are already identified disadvantages.
For flavonoids, NMR helps to differentiate isomers, sugar configurations and substitution patterns on the aromatic ring system.MS techniques gives information on molecular mass and functional groups. Stopped flow mode was used in most recent times to aid very long scan times or very low flow rates to record NMR spectra. However, for a complete structural elucidation of a novel natural product. Preparative isolation is often very necessary.

LC-NMR SCHEMATIC DIAGRAM:

LC-IR

The hyphenated technique developed from the coupling of an LC and the detection method infrared spectrometry (IR) or FTIR is known as LC-IR or HPLC-IR. While HPLC is one of the most powerful separation techniques available today, the IR or FTIR is a useful spectroscopic technique for the identification of organic compounds, because in the mid-IR region the structures of organic compounds have many absorption bands that are characteristic of particular functionalities, e.g., –OH, –COOH, and so on. However, combination of HPLC and IR is difficult and the progress in this hyphenated technique is extremely slow because the hyphenated technique’s 237 absorption bands of the mobile phase solvent are so huge in the mid-IR region that they often obscure the small signal generated by the sample components.
In addition, as a detection technique, IR is much less sensitive compared to various other detection techniques, e.g., UV and MS. The recent developments in HPLC-IR technology have incorporated two basic approaches based on interfaces applied in HPLC-IR or HPLC-FTIR. One is a flow-cell approach and the other is a solvent-elimination approach. The approach used with the flow cell in LC-IR is similar to that used in UV–vis and other typical HPLC detectors. In this case, absorption of the mobile phase induces the interference of the detection of sample component absorption bands, but some transparent region of the mid-IR range produces detection possibility. For example, if one uses a mobile phase of a deuterated solvent such as heavy water or perdeuterated methanol, IR can monitor many organic compounds that have C–H structures in the molecules. The solvent-elimination approach is the preferred option in most of the LC-IR operations. After the mobile phase solvent is eliminated, IR detection is carried out in some medium that has a transparency for IR light.
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: diffuse-reflectance infrared Fourier transform (DRIFT) approach and buffer-memory technique. A unified interface for GC, HPLC, and SFC hyphenation to FTIR applying IR microscopic technique is also available today.
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