The biological profile of chiral pharmaceuticals often depends upon their three-dimensional nature and the environment in which they are present. Human body, being chiral, can differentiate a left-handed molecule from a right-handed molecule and may handle them differently. This chiral distinction capability may result in stereoselectivity in almost all the biological processes. Today, it is well documented that the component enantiomers of a racemic therapeutic may exhibit different pharmacokinetic, pharmacodynamic and toxicological profile. The prominence of drug chirality has been increasingly recognised and consequences of employing racemates or enantiopure molecules are frequently debated in pharmaceutical literature during recent years.
With increasing evidence of issues related to enantioselectivity in drug action, regulatory agencies also require stringent analytical characterisation of drug substances, including their isomeric forms. As a consequence, enantioselective analysis by high performance liquid chromatography (HPLC) has become the focus of intense research of separation scientists and this lead to the development of enantioselective HPLC method for the analysis of chiral drugs in formulation and biological matrices in recent years.
Enantioselective HPLC analysis
There are basically two options for chiral HPLC analysis namely direct and indirect approach. In the indirect approach, drug enantiomers are derivatised with an enantiopure chiral reagent to form a pair of diastereomers, which may be then separated on a conventional chromatographic column, since diastereomers exhibit different physicochemical properties. In the direct method, transient rather than covalent diastereomeric complexes are formed between the drug enantiomers and a chiral selector present either added to the mobile phase (CMPA) or coated/bonded to the surface of a silica support (CSP). The technique relying on chiral stationary phases (CSPs) are preferred as they offer specific advantages over indirect methods. There is no need to chemically manipulate the analytes, interference with sample matrix, chiral purity of the chiral stationary phase (CSP) does not need be known, fast analysis, method can be readily scaled to commercial production, online coupling with MS or NMR permits structure identification.
CSP
The principle behind the functioning of a chiral stationary phase for HPLC is that an enantiopure chiral molecule (chiral derivatising agent, CDA) is coated or immobilised on the surface of a solid support (usually silica microparticles). This selectively retains the enantiomers of analytes by forming transient diastereomeric adsorbates of different stabilities with them during elution process.
Classification of CSP
Wainer classified HPLC chiral stationary phases into five types. They are:
● Pirkle (Brush)CSPs
● Polysaccharide CSPs
● Cavity CSPs
● Ligand exchange CSPs
● Protein bound CSPs
Ligand exchange CSPs find limited application in pharmaceutical research and development environment.
Pirkle CSPs
These CSPs, developed and designed by Dr. W H Pirkle for HPLC separation, are based on ionic or covalent attachment of one enantiomer of an amino acid derivative (e.g., (R) -N-(3, 5-dinitrobenzoyl) phenylglycine) to aminopropyl silica. Chiral separation is based on preferential binding of one enantiomer to the CSP resulting in a diastereomeric complex. Transient diastereomeric complexes involve electron donor-acceptor (π- π) interactions, hydrogen bonding and dipole-dipole interactions.
Pirkle CSPs could be classified into three categories namely π-electron acceptor, π-electron acceptor phases have a dinitrobenzoyl derivatised phenylglycine or leucine group linked to a silica support. These will be separate compounds (e.g., aminoalochols, hydantoins, lactams), which possess an aromatic π-electron donating aromatic system. Conversely, π-electron donor phases incorporating, for example naphthylamine or naphthylurea phases, will separate π-electron acceptors such as suitably derivatised (e.g., with dinitrobenzoyl chloride or dinitrophenyl isocyanate) amines, alcohols and thiols. Derivatisation is generally required when separating analytes with strongly acidic or basic groups. Most revolutionary addition to Pirkle concept series is π-electron acceptor-donor phases. This concept is innovative incorporation of both π-electron acceptor-donor characteristics, resulting in a phase that can be used for the resolution of wide variety of compounds.
A number of Pirkle type CSPs are commercially available - Pirkle 1-J (π-electron acceptor phase), Naphthyl leucine (π-electron donor phase) and Whelk O-1 (π-electron acceptor-donor phases). They are used most often in the normal phase mode. The strengths of the Pirkle CSPS are:
● Robust and allows high sample load
● Better column durability due to covalent phase bonding
● Universal solvent. compatibility
● Allows inversion of elution order
● Excellent chromatographic efficiency
● Separation of enantiomers of a wide variety of compounds and
● Ability to invert elution order
Polysaccharide CSPs
Naturally occurring polysaccharide form an important class of chiral stationary phase. Polysaccharides such as cellulose and amylose consist of D-(+)-glucose units linked by - and -1, 4-glucosidic bonds, respectively, forming the natural polymers with a highly ordered helical structure. Presumably, a chiral cavity or space exists on or within these polymers accounts for their chiral distinction properties. Yoshio Okamoto invented amylose and cellulose CSPs. This technique was commercialised by Diacel Chemical Industries Ltd., Japan. To increase their chiral recognition capacity, these polysaccharides are converted into their triester and tricarbamate derivatives by modifying the 'R' group.
To cite a few commercially available polysaccharide based on CSPs (coated on silica gel) are cellulose triacetate (chiralcel OA), cellulose tribenzoate (chiralcel OB) and cellulose triphenylcarbamate (chiralcel OC), amylose tris (3, 5-dimethylphenyl carbamate (chiralpak AD).
Polysaccharide derivatives coated on silica matrix have been extensively used as CSPs for their high selectivity and loading capacity in enantioseparation by HPLC. Immobilisation of the polymeric chiral selector on the support has been considered as direct approach to confer a universal solvent compatibility to this kind of CSP, thereby broadening the choice of solvents able to be used as mobile phases. In this context, Diacel Chemical Industries Ltd has recently developed a new generation of CSPs for HPLC using a novel immobilisation technology, Chiralpak IA, a 3,5-dimethylphenyl carbamate derivative of amylose, immobilised onto silica, is the first of this series of CSPs to become commercially available. The second member of this series is of the same nature as in chiralcel OD. Chiralpak IA has been employed for the HPLC separation of Hexobarbital, Bupivacaine, Naproxen, Suprofen, etc. and Chiralpak IB for the separation of Propranolol, Oxprenolol, Hydroxycine etc.
Cavity CSPs
Another strategy for chiral discrimination on a stationary phase is creation of chiral cavities, in which enantioselective guest-host interactions govern the resolution. The first important consideration for chiral distinction in this kind of CSPs is the proper fit of the analyte in the chiral cavity, structure of the analyte with reference to the stereogenic centre and the interaction between the analyte and chiral selector. This category of stationary phases includes cyclodextrins, macrocyclic antibiotics/glycopeptides and crown ethers.
Cyclodextrins: Commercially available cyclodextrins (CDs) are cyclic oligosaccharides of six, seven or eight glucose units designated as , and -cyclodextrin, respectively. They are chiral due to inherent chirality of building glucose units. These cyclodextrins are chiral molecules that resemble truncated cone. The interior surface of the cone forms hydrophobic chiral cavity rimmed by the secondary 2- and 3- hydroxyl groups at the larger opening and by the primary 6-hydroxyl groups at the smaller orifice.
Cyclodextrin can form inclusion complexes with molecules of the appropriate size and configuration. The stability of these complexes depends on the goodness of fit of the relatively nonpolar (preferably aromatic) side chain of the analyte and the hydrophobic cavity of the cyclodextrin.
Hydrogen bonding interaction between the substituent at or near the analyte chiral center and the hydroxyl group at the entrance of the cavity contribute to inclusion complex stability and to enantioselectivity.
The chiral cavity of -cyclodextrin is of appropriate size for the formation of inclusion complexes with a number of pharmaceuticals. Cyclodextrins covalently linked to silica via a spacer arm by Armstrong process provide stable CSPs suitable of reversed phase HPLC use. Armstrong is considered the father of Cyclodextrin based CSPs.
The strength of CS based CSPS include:
■ Aqueous compatibility of CDs and its unique molecular structure make the CD based CSPs highly promising for use in chiral separation of drugs
■ They are relatively cheaper than other CSPs
■ Reversed phase conditions can be applied. The major weakness is that this kind of CSPs is limited to compounds that can enter into CD cavity
Cyclodextrin CSPs are marketed by Diacel Chemical Industries, Japan and Astec, USA.
Protein bound CSPs
Proteins are complex and high molecular weight biopolymers. They are composed of L-amino acids and possess ordered three-dimensional structure. They are known to bind and interact stereoselectively with small molecules reversibly making them versatile CSPs for chiral separation of pharmaceuticals. Lots of CSPs have been developed by immobilising proteins or enzymes. Protein polymer remains in twisted form because of the different intramolecular bonding. These bonding are responsible for different chiral loops/grooves present in the protein molecule. Separation mechanism of proteins rely on unique combination of hydrophobic and polar interactions by which the analytes reoriented to the chiral surfaces. Hydrogen bonding and charge transfer may also contribute to enantioselectivity.
Albumin (Resolvosil), 1-Acid Glyco-Protein (enantiopac), Cellulose Bio-Hydrolase (Chiral CBH), Human Serum Albumin (Chiral HSA) and Vomucoid (Ultron Es -OVM) CSPs are commercially available. Protein based CSPs has been successfully employed for the chiral resolution of a number of pharmaceuticals viz. omeprazole, mephenytoin and atenolol.
Major limitations of protein based CSPs are (i) protein phases are expensive (ii) low loading capacity (iii) extremely fragile and delicate to handle (iv) low efficiency and (v) cannot invert elution order.
Today, the significant difference in the pharmacokinetic, pharmacodynamic and toxicological profiles of the enantiomers of a racemic therapeutic is well appreciated. Further, regulatory agencies insist on enantiospecific data if the molecule under investigation is chiral. Hence there is a need for analytical tool to quantify enantiomers of chiral drug in formulations and biological fluids, study stereochemical stability during formulation and production, carry out enantiospecific bioavailability and bioequivalence assessment, control enantiomeric purity in chiral synthesis, and check for racemisation process and chiral impurity profiling. It is in this context chiral HPLC becomes a valuable tool in drug research analysis.
(The author is professor with Department of Pharmacy, Annamalai University)