Chiral discrimination and enantioselective analysis of drugs: an overview

Molecular recognition of different enantiomers of a drug has become of increasing importance in the last decade due to the racemic switch strategy adapted by the pharmaceutical industry. Different analytical techniques to carry out enantioselective analysis of chiral compounds have been suggested in the literature. In the following, a brief overview of different techniques used for enantioselective analysis is given. Challenging aspects of these techniques, such as the quality of analytical information received from each technique, advantages, and disadvantages are discussed. Alternatives (enantioselective membranes, amperometric biosensors, molecularly imprinted polymers (MIPs)), capable of meeting the requirements of industrial processes, in terms of productivity, cost-effectiveness, and environmental issues are critically reviewed.     

ENANTIOSELECTIVE DRUG ANALYSIS: PROBLEMS AND RESOLUTIONS

1. With the increasing appreciation that the enantiomers of a chiral drug can differ pharmacokinetically and/or pharmacodynamically, there is considerable interest in methods for the resolution and quantification of enantiomers. 2. Enantiomers possess identical physical and chemical properties in a symmetrical environment and, therefore, their resolution requires the introduction of an asymmetric or chiral environment allowing diastereomeric interactions. This can be achieved using a number of chromatographic techniques, of which the most developed and widely used is high-performance liquid chromatography (HPLC). 3. Resolution and quantification of enantiomers can be performed using HPLC by either converting the enantiomers to covalent diastereomers prior to chromatography or introducing a chiral environment to the chromatographic system, thereby allowing temporary diastereomeric interactions. 4. Antibodies are chiral molecules which can bind the enantiomers of a chiral drug in a differential manner. This is the basis of enantioselective immunoassay, which is a promising technique for the enantioselective analysis of drugs in biological fluids. 5. Each of the methods available has its limitations, advantages and potential applications in the pharmaceutical industry.     

Synthesis and applications of novel, highly efficient HPLC chiral stationary phases: a chiral dimension in drug research analysis

This review provides an overview of the synthesis and application of stable and versatile HPLC chiral stationary phases (CSPs), with emphasis placed on the binding strategies developed to anchor several structurally different chiral selectors to silica-gel microparticles. In addition, selected applications relating to the use of these CSPs for the direct resolution of racemates of biological and pharmaceutical relevance will be described. This review discusses enantioselective molecular recognition and dynamic stereochemistry of stereolabile compounds with reference to receptor-based chiral stationary phases (CSPs) and dynamic HPLC on CSPs, respectively.     

Chiral drugs: an industrial analytical perspective

In the pharmaceutical industry, chiral drug candidates introduce a unique set of challenges to all disciplines involved in the drug development process. For the analytical chemist in particular, the generation of relevant information about a variety of stereoisomeric issues is necessary. Chiral drug candidates, whether a single isomer or a mixture of isomers, require more analytical information than achiral drug candidates. This information can be derived from enantioselective spectroscopic and chromatographic techniques. Chiral analytical methods require proper development and validation to ensure accurate results. Issues related to method development and validation for complete stereochemical characterization are discussed, with primary emphasis on the generation of analytical data required for the registration of a chiral drug candidate. The presentation of pertinent analytical data depends on an awareness of the problems encountered during the development process and the appropriate use of methodology for the determination of stereoisomeric purity.     

Strategies for access to enantiomerically pure ecadotril,dexecadotril and fasidotril: a review

Ecadotril and dexecadotril are powerful and selective inhibitors of neprilysin (NEP, EC 3.4.24.11) and are being developed as therapeutic agents, since they behave as prodrugs of the enantiomers of thiorphan. They exhibit different pharmaceutical profiles (intestinal antisecretatory action for the (R) enantiomer, i.e. dexecadotril, and cardiovascular activity for the (S) enantiomer, i.e. ecadotril). Fasidotril is a related compound which has special interest as an equipotent dual inhibitor of NEP and ACE (EC 3.4.15.1). This behavior confers on fasidotril powerful pharmaceutical properties in the cardiovascular field. This review deals with various synthetic approaches, either published or patented, for access to the enantiomerically pure or highly enriched forms of these drugs. Thus, different methods have been studied, which are taken from different methodologies of resolution procedures and asymmetric synthesis, namely : i- Synthesis from a chiron from the chiral pool ii- Chemical resolution of racemic precursors iii- Enzymatic resolution and desymmetrization of meso starting materials iv- Asymmetric synthesis, including enantioselective catalytic hydrogenation, alkaloid catalyzed asymmetric Michael additions, and diastereoselective alkylation of a chiral derivative. Some of these methods are used in industrial processes leading to the indicated compounds.     

In Vivo Absorption Studies of Insulin from an Oral Delivery System

The objectives of our study were two fold: to examine enantioselective release of controlled delivery granules based on molecularly imprinted polymers (MIPs) for various racemic drugs, including ibuprofen and ketoprofen (NSAIDs) and propranolol (β-blockers); to evaluate the use of controlled delivery granules containing a combination of different MIPs for the multiple simultaneous enantioselective-controlled delivery of mixed racemic drugs. In this work, the MIP beads selective to S-Ibuprofen, S-ketoprofen, and R-propranolol were prepared using multistep swelling and thermal polymerization method. Afterward, the MIP beads were formulated with racemate of the chiral drugs and a binder and followed by granulation. Then, the enantioselective release of racemic drugs from the prepared MIP granules was investigated by an in vitro dissolution test using a chiral HPLC for assays of enantiomers. The influence of drug/polymer ratio and medium pH on the selective enantiomeric release of MIP granules was explored. Further, the release of the enantiomers of racemic ibuprofen and racemic ketoprofen from the granule containing two MIPs - S-ibuprofen MIP and S-ketoprofen MIP - was examined. The release profiles of both S-ibuprofen MIP granule and R-propranolol MIP granule exhibited differential release of enantiomers. Also, the findings indicated the stereoselective retardation of those controlled delivery granules as well as the influence of MIP formulation on enantioselective release mechanism. The enantioselective release of S-ibuprofen MIP granule and R-propranolol MIP granule appeared to depend on polymer loading and medium pH. In this case, the drug/polymer ratio of 1:25 showed the best enantioselective release with initial enantiomeric excess of 100%. On the other hand, the enantioselectivity of both granules was the greatest in buffer pH 7.4. Furthermore, the efficiency in enantioselective release of the combined MIP granule was higher than its relative single MIP granules, as a result of the cross-reactivities of the MIPs. In our study, controlled delivery granules based on MIPs demonstrated significant enantioselective release for several chiral drugs, and thus it may be developed as a tool to administer chiral pharmaceutical as a single enantiomer.     

Enantioselective release of controlled delivery granules based on molecularly imprinted polymers

The objectives of our study were two fold: to examine enantioselective release of controlled delivery granules based on molecularly imprinted polymers (MIPs) for various racemic drugs, including ibuprofen and ketoprofen (NSAIDs) and propranolol ( β-blockers); to evaluate the use of controlled delivery granules containing a combination of different MIPs for the multiple simultaneous enantioselective-controlled delivery of mixed racemic drugs. In this work, the MIP beads selective to S-Ibuprofen, S-ketoprofen, and R-propranolol were prepared using multistep swelling and thermal polymerization method. Afterward, the MIP beads were formulated with racemate of the chiral drugs and a binder and followed by granulation. Then, the enantioselective release of racemic drugs from the prepared MIP granules was investigated by an in vitro dissolution test using a chiral HPLC for assays of enantiomers. The influence of drug/polymer ratio and medium pH on the selective enantiomeric release of MIP granules was explored. Further, the release of the enantiomers of racemic ibuprofen and racemic ketoprofen from the granule containing two MIPs - S-ibuprofen MIP and S-ketoprofen MIP - was examined. The release profiles of both S-ibuprofen MIP granule and R-propranolol MIP granule exhibited differential release of enantiomers. Also, the findings indicated the stereoselective retardation of those controlled delivery granules as well as the influence of MIP formulation on enantioselective release mechanism. The enantioselective release of S-ibuprofen MIP granule and R-propranolol MIP granule appeared to depend on polymer loading and medium pH. In this case, the drug/polymer ratio of 1:25 showed the best enantioselective release with initial enantiomeric excess of 100%. On the other hand, the enantioselectivity of both granules was the greatest in buffer pH 7.4. Furthermore, the efficiency in enantioselective release of the combined MIP granule was higher than its relative single MIP granules, as a result of the cross-reactivities of the MIPs. In our study, controlled delivery granules based on MIPs demonstrated significant enantioselective release for several chiral drugs, and thus it may be developed as a tool to administer chiral pharmaceutical as a single enantiomer.     

Environmentally friendly analytical method to assess enantioselective behaviour of pharmaceuticals and pesticides in river waters

Reported herein is the first enantioselective method for simultaneous separation of chiral pharmaceuticals and pesticides using ultra-high-performance liquid chromatography-tandem mass spectrometry. Separation was achieved using a ChiralPak IG-U® column (amylose tris(3-chloro-5-methylphenylcarbamate) stationary phase) and ethanol as a ‘green’ mobile phase organic modifier. Minimum enantiomer resolutions ranged from 0.5 to 6.9 for 28 pharmaceuticals, herbicides, fungicides and insecticides. The total run time was 26 min and is considerably shorter than other multi-residue enantioselective methods for similar numbers of pesticides, and is the first to facilitate simultaneous pharmaceutical separation. Direct injection of river water samples enabled omission of acetonitrile and methanol from the sample treatment step (and the whole methodology). This approach was considerably faster than enantioselective methodologies that rely on solid phase extraction and avoids the need for large sample volumes for analysis. The suitability of this approach was demonstrated by the method's sensitivity with enantiomer method quantitation limits in the range 0.005–0.6 μg L^-1. The new method was applied to river water microcosms to investigate enantiospecific transformation of racemic pharmaceuticals and pesticides. The pharmaceutical omeprazole, fungicide prothioconazole and insecticide profenofos were all subject to enantioselective transformation under biotic conditions, represented by a change in enantiomeric fraction of ≥0.1 units. Individual enantiomer microcosms revealed chiral inversion of R-omeprazole to S-omeprazole in the environment for the first time. In conclusion, this method offers comparatively fast enantioselective analysis for a high number of pharmaceuticals and pesticides in river water, and is achieved in an environmentally friendlier way than previously reported liquid chromatography methods.     

Penicillin G acylase-based stationary phases: analytical applications

A review of Penicillin G Acylase (PGA)-based stationary phases is given, focusing on immobilisation methods, selection of immobilisation material and applications in chiral liquid chromatography. Two immobilization methods, namely "in situ" and "in batch" techniques, are described for the immobilisation of PGA on silica supports. Microparticulate and monolithic silica, both functionalized with aminopropyl- and epoxy-groups, were used in the development of the PGA immobilised enzyme reactor (IMER). The best results, in terms of PGA immobilised amount and enzyme activity, were obtained with the "in situ" immobilisation on epoxy monolithic silica. The use of PGA columns as enzyme reactors for the preparation of 6-APA and for the production of enantiomeric pure drugs in a one-step reaction in described. The review also covers the application of PGA-columns as chiral stationary phases for the separation of acidic enantiomers. An on-line chromatographic system based on the PGA-IMER combined with a switching valve to an analytical column is also described as a highly efficient tool to study the enantioselective hydrolyses properties of PGA. Finally a molecular modelling study is reported with the aim to give more insights into PGA-substrates interactions and to expand the application of these stationary phases as a chiral biocatalysts for pharmaceutical processes.     

An evaluation of four commercial HPLC chiral detectors: a comparison of three polarimeters and a circular dichroism detector

With increasing frequency, new drug candidates being introduced into pharmaceutical drug pipelines are chiral. Often only one enantiomer exhibits the desired biological activity and the other enantiomer may exhibit undesired side effects, thereby making chiral purity an important parameter. The introduction of chiral analysis adds additional complications in drug development. The pharmaceutical industry is constantly striving to streamline processes and improve efficiencies in an effort to move molecules to market quickly. In order to simplify the process of chiral method development, chiral screening can be set up, however a successful chiral screen depends on optimizing two factors: the column and the detector. The following work investigated the second factor and evaluated two types of commercially available chiral detectors for their possible use in chiral method development and screening: polarimeters and circular dichroism (CD) detectors. Linearity, precision, and the limit of detection (LD) of six compounds (trans-stilbene oxide, ethyl chrysanthemate, propranolol, 1-methyl-2-tetralone, naproxen, methyl methionine) on four commercial detectors (three polarimeters and one CD detector) were determined experimentally and the limit of quantitation (LQ) calculated from the experimental LD. Trans-stilbene oxide worked well across all the detectors, showing good linearity, precision and low detection limits. However, the other five compounds proved to be more discriminating and showed that the circular dichroism detector performed better as a detector for chiral screens, over the polarimeters.     

Enantioselective quantification of chiral drugs in human plasma with LC-MS/MS

Today, approximately 60% of synthetic drugs are chiral and 88% of these chiral synthetic drugs are used therapeutically as racemates. However, for many racemic drugs, their stereospecific plasma pharmacokinetics in humans are not known due to the limitations of the analytical methods. Nowadays, liquid chromatography (LC)-tandem mass spectrometry (MS/MS) methods based on various chiral stationary phases (CSPs), with a high degree of specificity and sensitivity, have been widely used in enantioselective determination of chiral drugs and/or their metabolites in human plasma. The technologies and issues when coupling chiral chromatography with MS/MS detection in bioanalytical methods will be reviewed herein. The introduction and applications of various CPSs, including polysaccharide-, macrocyclic glycopeptide-, protein- and cyclodextrin-based phases, are described here. This review also includes a discussion of interface and matrix effects in enantioselective LC-MS/MS methods.     

Microbial/enzymatic synthesis of chiral pharmaceutical intermediates

Chirality is a key factor in the efficacy of many drugs; thus, the production of single enantiomers of drug intermediates has become increasingly important in the pharmaceutical industry. Chiral intermediates and fine chemicals are in high demand from the pharmaceutical and agrochemical industries for the preparation of bulk drug substances and agricultural products. There has been an increasing awareness of the enormous potential of microorganisms and enzymes for the transformation of synthetic chemicals with high chemo-, regio- and enantioselectivity. In this article, biocatalytic processes for the synthesis of chiral pharmaceutical intermediates are described.     

Direct HPLC enantioseparation of omeprazole and its chiral impurities: Application to the determination of enantiomeric purity of esomeprazole magnesium trihydrate

Analytical and semipreparative high-performance liquid chromatography (HPLC) enantioseparation of the proton-pump inhibitor omeprazole (OME) and its potential organic chiral impurities were accomplished on the immobilised-type Chiralpak IA chiral stationary phase (CSP) under both polar organic and normal-phase conditions. The (S)-enantiomers were isolated with a purity of >99% ee and their absolute configuration was empirically assigned by circular dichroism (CD) spectroscopy. A chemo- and enantioselective HPLC method was validated to control the enantiomeric purity of the (S)-enantiomer of OME (ESO), an active ingredient contained in drug products, in the presence of chiral and achiral related substances. The precision, linearity and accuracy of the determination of the (R)-impurity as well as the recovery of ESO from a pharmaceutical preparation were determined.     

Enantioselective analysis of amisulpride in pharmaceutical formulations by means of capillary electrophoresis

A capillary electrophoretic method has been developed for the enantioselective analysis of amisulpride in pharmaceutical formulations, using β-cyclodextrin sulfate as the chiral selector. Several parameters, such as cyclodextrin type and concentration, buffer concentration and pH and capillary temperature were investigated for method optimisation. Baseline enantioseparation of the racemic compound was achieved in less than 10 min using a fused silica capillary (50 μm i.d. and 33.0, 8.5 cm, total and effective length, respectively), filled with a background electrolyte consisting of a 10 mM citrate buffer at pH 3.5 supplemented with 0.22% (w/v) β-cyclodextrin sulfate at 20 °C and applying a voltage of +15 kV. Formulation analysis was carried out after analyte extraction by methanol. The method was fully validated, with good results in terms of precision, selectivity, accuracy and amount of drug found with respect to the label claim. Thus, the method seems to be suitable for the enantiomeric analysis of amisulpride in pharmaceutical formulations.     

Enantioselective chromatography in drug discovery

Molecular chirality is a fundamental consideration in drug discovery, one necessary to understand and describe biological targets as well as to design effective pharmaceutical agents. Enantioselective chromatography has played an increasing role not only as an analytical tool for chiral analyses, but also as a preparative technique to obtain pure enantiomers from racemates quickly from a wide diversity of chemical structures. Different enantioselective chromatography techniques are reviewed here, with particular emphasis on the most widespread high performance liquid chromatography (HPLC) and the rapidly emerging supercritical fluid chromatography (SFC) techniques. This review focuses on the dramatic advances in the chiral stationary phases (CSPs) that have made HPLC and SFC indispensable techniques for drug discovery today. In addition, screening strategies for rapid method development and considerations for laboratory-scale preparative separation are discussed and recent achievements are highlighted.     

Chirality and its implications in transdermal drug development

Today more than 50% of marketed drugs are chiral. It has been well recognized that the stereochemistry of chiral drugs has a major influence on their pharmacological, pharmacokinetic, and toxicological actions. Studies on enantiomeric differences in the percutaneous permeation of chiral compounds have been actively pursued in recent years. Stratum corneum, the rate-limiting barrier in transdermal permeation, is made up of keratin and ceramide, which could potentially provide chiral environment. Transdermal delivery is often facilitated by the presence of penetration enhancers, which act primarily by altering the diffusion by disrupting the highly ordered membrane structure or by affecting the partitioning behavior of the diffusant molecules. Enantioselective permeation was observed with some chiral excipients including terpene enhancers. While studies on crystalline structures of pure enantiomers and racemates are helpful in understanding the basis for differentiation of physicochemical properties, prediction and control of permeability of enantiomers and racemates based on the physicochemical characteristics would be highly beneficial. The flux characteristics of enantiomers and racemates appear to be dependent upon their thermodynamic properties. Such analyses have a predictive value and are useful in the transdermal drug product development of chiral molecules. While the decision to market either an individual enantiomer or racemate lies strictly under the control of the sponsors, the guidance of the Food and Drug Administration is very helpful. This review presents an overview of the skin structure and transport and a concise review of enantioselective permeation with or without chiral enhancers. Regulatory perspectives on chiral drug product development are also discussed.     

Process simulation in the pharmaceutical industry: a review of some basic physical models

This study reviews process modeling efforts which have been developed to elucidate the fundamental physical process underlying the manufacture and delivery of pharmaceutical dosage forms. Within the pharmaceutical industry, process models have been applied to a diverse array of physical processes at length and time scales that vary by orders of magnitude. As such, both large-scale continuum and particle-scale discrete approaches will be discussed in this study. Challenges associated with the practical application of process models within the pharmaceutical industry will be discussed, and opportunities for future research will be identified.     

RP－HPLC手性流动相添加剂法分析尿中氧氟沙星对映体

以Ｌ－苯丙氨酸为配合剂，Ｃｕ２＋为配合离子，建立了一种简便的手性配合交换ＲＰ－ＨＰＬＣ拆分氧氟沙星对映体的方法，该法已用于人尿中氧氟沙星对映异构体的分析。     

手性药物代谢研究中的对映体分析方法

生物体是一个复杂的手性系统,其手性特征促使了它们与手性化合物的立体选择性相互作用。手性药物的两个对映体在吸收、分布、代谢、排泄过程中存在着动力学差异,尤其是代谢行为。因此,在手性药物代谢的研究中,同时测定对映体是极为重要的。本文通过综述近年来发表的众多文献,详细论述了在药物代谢中应用广泛的手性分析方法,包括高效液相色谱、高效液相色谱-质谱联用、气相色谱和毛细管电泳等。