Chiral analysis
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.
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 (ADME) 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[23] plays a major role in the study of biological and environmental samples and also in the forensic field.
[25] There are number of articles, columns, and interviews in LCGC relating to emerging trends in chiral analysis and its application in drug discovery and development process.
Today wide range of CSPs are available commercially based on various chiral selectors including polysaccharides, cyclodextrins, glycopeptide antibiotics, proteins, Pirkle, crown ethers, etc.
[34] 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.
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 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.
[51] In this approach, an enantiomerically pure compound, the chiral selector, is added to the mobile phase and separation happens on a conventional achiral column.
[54] Under this model, for chiral recognition, and hence enantiomeric resolution to happen on a CSP one of the enantiomers of the analyte must be involved in three simultaneous interactions.
The success of chiral separation basically depends in manipulating the subtle energy differences between the reversibly formed non-covalent transient diastereomeric complexes.
Components of MP (such as bulk solvents, modifiers, buffer salts, additives) not only influence the conformational flexibility of CS and CA molecules but also their degree of ionization.
However, In late 1980s the subject of enantioselective chromatography attracted growing interest, particularly under the drive of the institution of Okamoto in Japan, the teams of Pirkle, and Armstrong in the US, Schurig and König in Germany, Lindner in Austria, and Francotte in Switzerland .
But the carbamate and benzoate derivatives of these polymers, especially amylose and cellulose, demonstrate excellent properties as chiral selectors for chromatographic separation.
More solvents to play with means better sample solubility, Improves resolution, and enables effective chiral method development.
The mechanism of Chiral discrimination is not well understood but believed to involve hydrogen bonding and dipole-dipole interaction between the analyte molecule and the ester or carbamate linkage of the CSP.
In this category, there are basically three types of cavity chiral selectors namely cyclodextrins,[68] crown ethers[69] and macrocyclic glycopeptide antibiotics.
Selectivity of a cyclodextrin phase is dependent on two key factors namely the size and structure of the analyte since it is based on a simple fit-unfit geometric criteria.
The aqueous compatibility of CD and its unique molecular structure make the CD- bonded phase highly suitable for use in chiral HPLC analysis of drugs.
A number of chiral pharmaceuticals has been resolved using derivatized CDs including ibuprofen, suprofen, flurbiprofen from NSAID category and b-blockers like metoprolol and atenolol.
These cyclic glycopeptides have multiple chiral centers and a cup-like inclusion area to which a floating sugar lid is attached.
Similar to protein chiral selectors, the amphoteric cyclic glycopeptides consist of peptide and carbohydrate binding sites leading to possibilities for different modes of interaction beside the formation of inclusion complexation.
In this chiral selector the cavities are shallower than that of CDs and hence the interactions are weaker, allows more rapid solute exchange between phases, higher column efficiency.
The complex structural nature of glycopeptide antibiotic class of CSP has made the understanding of the mechanism of chiral recognition at molecular level tricky.
Separation mechanism of proteins depends on unique combination of hydrophobic and polar interactions by which the analytes are oriented to chiral surfaces.
[84] α-AGP CSP (chiral AGP), has been employed for the quantification of atenolol enantiomers in biological matrices,[85] for pharmacokinetic investigation of racemic metoprolol.
