Chiral analysis

Chiral analysis refers to the quantification of component enantiomers of racemic drug substances or pharmaceutical compounds. Other synonyms commonly used include enantiomer analysis, enantiomeric analysis, and enantioselective analysis. Chiral analysis includes all analytical procedures focused on the characterization of the properties of chiral drugs. Chiral analysis is usually performed with chiral separation methods where the enantiomers are separated on an analytical scale and simultaneously assayed for each enantiomer.
Many compounds of biological and pharmacological interest are chiral. Pharmacodynamic, pharmacokinetic, and toxicological properties of the enantiomers of racemic chiral drugs has expanded significantly and become a key issue for both the pharmaceutical industry and regulatory agencies. Typically one of the enantiomers is more active pharmacologically. In several cases, unwanted side effects or even toxic effects may occur with the inactive enantiomer. Even if the side effects are not that serious, the inactive enantiomer has to be metabolized, this puts an unnecessary burden on the already stressed out system of the patient. Large differences in activity between enantiomers reveal the need to accurate assessment of enantiomeric purity of pharmaceutical, agrochemicals, and other chemical entities like fragrances and flavors become very important. Moreover, the moment a racemic therapeutic is placed in a biological system, a chiral environment, it is no more 50:50 due enantioselective absorption, distribution, metabolism, and elimination process. Hence to track the individual enantiomeric profile there is a need for chiral analysis tool.
Chiral technology is an active subject matter related to asymmetric synthesis and enantioselective analysis, particularly in the area of chiral chromatography. As a consequence of the advances in chiral technology, a number of pharmaceuticals currently marketed as racemic drugs are undergoing re-assessment as chiral specific products or chiral switches. Despite the choice to foster either a single enantiomer or racemic drug, in the current regulatory environment, there will be a need for enantioselective investigations. This poses a big challenge to pharmaceutical analysts and chromatographers involved in drug development process. In pharmaceutical research and development stereochemical analytical methodology may be required to comprehend enantioselective drug action and disposition, chiral purity assessment, study stereochemical stability during formulation and production, assess dosage forms, enantiospecific bioavailability and bioequivalence investigations of chiral drugs. Besides pharmaceutical applications chiral analysis plays a major role in the study of biological and environmental samples and also in the forensic field. Chiral analysis methods and applications between the period 2010 and 2020 are exhaustively reviewed recently. There are number of articles, columns, and interviews in relating to emerging trends in chiral analysis and its application in drug discovery and development process.
For chiral examination there is a need to have the right chiral environment. This could be provided as a plane polarized light, an additional chiral compound or by exploiting the inborn chirality of nature. The chiral analytical strategies incorporate physical, biological, and separation science techniques. Recently an optical-based absolute chiral analysis has been reported. The most frequently employed technique in enantioselective analysis involve the separation science techniques, in particular chiral chromatographic methods or chiral chromatography. Today wide range of CSPs are available commercially based on various chiral selectors including polysaccharides, cyclodextrins, glycopeptide antibiotics, proteins, Pirkle, crown ethers, etc. to achieve analysis of chiral molecules.

Chiral chromatography

This term has become very popular and commonly used in practice. But the appropriate expression is "enantioselective chromatography". Chiral chromatography has advanced to turn into the most preferred technique for the determination of enantiomeric purity as well as separation of pure enantiomers both on analytical and preparative scale. Chiral chromatographic assay is the first step in any study pertaining to enantioselective synthesis or separation. This includes the use of techniques viz. gas chromatography, high performance liquid chromatography, chiral supercritical fluid chromatography, capillary electrophoresis and thin-layer chromatography. The result of a literature survey done identifies HPLC-based chiral assays as the most dominating technology in use. An overview of various analytical methods engaged for chiral separation and analysis are listed in the table.

Method	Brief narrative of principle and application
Chromatographic
Chiral HPLC	Chiral HPLC is used to separate enantiomers either by direct or indirect separation mode. Widely employed to check enantiomeric purity, provided the reference standards of the racemate or the two enantiomers are available. Capable of distinguishing between enantiomers and from the racemate; from and
Chiral GC	Majority of chiral separations using GC are done with cyclodextrin derivatives as chiral selector. This method can be used to distinguish between enantiomers and from the racemate; from and
Supercritical fluid chromatography	Principle is very similar to that of HPLC. But SFC typically uses carbon dioxide as the mobile phase. Hence there is a need to pressurize the entire chromatographic flow path. SFC can differentiate enantiomers and enantiomer from the racemate; from and
Chiral capillary electrophoresis	Chiral CE is based largely on separation of enantiomers by complex formation with cyclodextrins which is used as the chiral selector. Able to differentiate between enantiomers and from the racemate; from and
Spectroscopic
Polarimetry	Polarimetry uses the innate property of chiral molecules to rotate the plane-polarized light in equal and opposite direction. This method can be used to distinguish between enantiomers and from the racemate; from and
Optical rotatory dispersion	ORD is a curve obtained by plotting the measured optical activity of a chiral compound as a function of the wavelength of the light used. Distinguish between enantiomers and from the racemate; from and
Circular dichroism	CD measures the differential absorption of left and right circularly polarized light by a chiral compound. These chiroptical techniques can be employed to identify and/or determine enantiomers
Nuclear magnetic resonanc	NMR spectroscopy done using chiral shift reagents or chiral solvating reagents. Capable of discriminating enantiomers as well as the racemate
Infrared	Differentiate the racemate and its enantiomers but not between the enantiomeric pair; or from
Calorimetry
Differential scanning calorimetry	The underlying principle is to measure the energy absorbed or evolved by a sample as a function of temperature. Data can distinguish between an enantiomer and the racemate, but not one enantiomer from its mirror-mage

Principle - separation of enantiomers

In an isotopic/achiral environment, enantiomers exhibit identical physicochemical properties, and therefore are indistinguishable under these conditions. For the separation of chiral molecules the challenge is to construct the right chiral environment. In a chromatographic system there are three variables namely, the chiral analyte, mobile phase and stationary phase, that can be manipulated to provide the crucial chiral environment. The strategy is to make these variables to interact with a chiral auxiliary whereby it forms a diastereomeric complex which has different physicochemical properties and makes it possible to separate the enantiomers. Based on the nature of the diastereomeric complex formed between the CS-CA species, enantiomer separation mythologies are categorized as indirect and direct enantiomer separation mode

Indirect separation of enantiomer

Indirect enantiomer separation involves the interaction between the chiral analyte of interest and the suitable reactive CS leading to the formation of a covalent diastereomeric complex that can be separated with an achiral chromatographic technique. Therapeutic agents often contain reactive functional groups in their structures. They are converted into covalently bonded diastereomeric derivatives using enantiomerically pure chiral derivatizing agent. The diastereomers thus formed unlike enantiomers, exhibit different physicochemical properties in an achiral environment and are eventually separated as a result of differential retention time on a stationary phase. The success of this approach depends on the availability of stable enantiopure chiral derivatizing agent and on the presence of a suitable reactive functional group in the chiral drug molecule for covalent formation of diastereomeric derivative. The reaction of a racemic, - Drug with a chirally and chemically pure chiral derivatizing agent, -CDA, will afford diastereomeric products, -Drug--CDA + -Drug-- CDA. The chiral derivatization reaction scheme is illustrated in the box on the right hand side.
In contrast to enantiomers, diastereomers have different physicochemical properties that make them separable on regular achiral stationary phases. The major benefit of the indirect methodology is that conventional achiral stationary phase/mobile phase system may be used for the separation of the generated diastereomers. Thus, considerable flexibility in chromatographic conditions is available to achieve the desired separation and to eliminate interferences from metabolites and endogenous substances. Moreover, the sensitivity of the method can be enhanced by sensible choice of the CDA and the chromatographic detection system. But this indirect approach to enantiomeric analysis has some potential problems. These include availability of a suitable functional group on the enantiomer for derivatization, enantiomeric purity of the CDA, racemization of the CDA during derivatization, and racemization of the analyte during the derivatization. Currently, however, the application of indirect analytical approaches is in decline.