Methods of analysis of chiral non-steroidal anti-inflammatory drugs

Although the analysis of the enantiomers of chiral non-steroidal anti-inflammatory drugs (NSAIDs) has been carried out for over 20 years, there often remains a deficit within the pharmaceutical and medical sciences to address this issue. Hence, despite world-wide therapeutic use of chiral NSAIDs the importance of stereoselectivity in pharmacokinetic, pharmacodynamic and pharmacological activity and disposition has often been ignored. This review presents both the general principles that allow separation of chiral NSAID enantiomers, and discusses both the advantages and disadvantages of the available chromatographic assay methods and procedures used to separately quantify NSAID enantiomers in biological matrices.     

Stereoselective degradation of fungicide benalaxyl in soils and cucumber plants

The enantioselective degradation of benalaxyl has been investigated to elucidate its behavior in several agricultural soils and plants (cucumber). Racemic benalaxyl was fortified into five types of agricultural soils and sprayed leaves of cucumber plants, respectively. The degradation kinetics and the enantiomer fraction (EF) were determined by normal-phase high-performance liquid chromatography (HPLC) with diode array detection (DAD) on the chiral column filled cellulose-tri-(3,5-dimethylphenylcarbamate)-based chiral stationary phase (CDMPC-CSP). The process of the degradation of benalaxyl enantiomers followed pseudo-first-order kinetics in cucumber plant. However, the dissipation phases of benalaxyl enantiomers in soils were biphasic ("slow-fast-slow" process). It has been shown that the degradation of benalaxyl was stereoselective. The results indicated that the (+)-S-benalaxyl showed a faster degradation in plants, while the (-)-R-benalaxyl showed a faster degradation in Soils 3, 4, and 5. No stereoselective degradation was observed in other soils.     

A new series of flavones,thioflavones, and flavanones as selective monoamine oxidase-B inhibitors

A new series of synthetic flavones, thioflavones, and flavanones has been synthesized and evaluated as potential inhibitors of monoamine oxidase isoforms (MAO-A and -B). The most active series is the flavanone one with higher selective inhibitory activity against MAO-B. Some of these flavanones (mainly the most effective) have been separated and tested as single enantiomers. In order to investigate the MAOs recognition of the most active and selective compounds, a molecular modeling study has been performed using available Protein Data Bank (PDB) structures as receptor models for docking experiments.     

Bi-directional chiral inversion of ketoprofen in CD-1 mice

The R enantiomers of some of the 2-arylpropionic acid non-steroidal antiinflammatory drugs (NSAIDs) are known to undergo metabolic chiral inversion to their more pharmacologically active antipodes. This process is drug and species dependent and usually unidirectional. The S to R chiral inversion, on the other hand, is rare and has been observed, in substantial extents, only for ibuprofen in guinea pigs and 2-phenylpropionic acid in dogs. After i.p. administration of single doses of racemic ketoprofen or its optically pure enantiomers to male CD-1 mice and subsequent study of the concentration time-course of the enantiomers, we noticed substantial chiral inversion in both directions. Following racemic doses, no stereoselectivity in the plasma-concentration time courses was observed. After dosing with optically pure enantiomer, the concentration of the administered enantiomer predominated during the absorption phase. During the terminal elimination phase, however, the enantiomers had the same concentrations. Our observation is suggestive of a rapid and reversible chiral inversion for ketoprofen enantiomers in mice. Chirality 9:29–31, 1997. © 1997 Wiley-Liss, Inc.     

Synthesis, chiral chromatographic separation, and biological activities of the enantiomers of 10,10-dimethylhuperzine A

(+/-)-10,10-Dimethylhuperzine A (2, DMHA) has been synthesized, and its enantiomers have been separated using chiral HPLC. (-)-DMHA inhibits AChE with a Ki value approaching that of (-)-huperzine A, whereas (+)-DMHA shows no AChE inhibitory activity. On the other hand, both enantiomers are equally potent against glutamate-induced neurotoxicity when tested in neurons.     

Challenges and frustrations in the separation and analysis of chiral agrochemicals

The development of chiral HPLC methods and isolation techniques within Zeneca Agrochemicals (formerly ICI Agrochemicals) is reviewed. The use of low temperature to improve chiral separations has been successfully applied to production analysis, but although useful for some compounds it is regrettably not a universal panacea for all poor separations. The need to isolate small quantities of individual enantiomers from new compounds for research evaluation has led us to devise a more universal and cheap chiral stationary phase (CSP) for Preparative-LC. Joint academic research produced a CSP based on tartaric acid which was made commercially available and it was gratifying to find it was the only phase able to resolve a novel insecticide. However, as new CSPs emerged almost every month, our attention turned to using a universal chiral detector for analysis, rather than via separation of individual enantiomers. Diode laser-based polarimeters offered the opportunity of cheap, sensitive chiroptical detectors for HPLC and the ability to move away from chiral columns in both research and production analysis. Jointly sponsored research with a university has successfully explored the versatility of chiroptical detectors in agrochemical and food analysis. Comparison of chiral SFC with chiral HPLC and an extensive evaluation of established and research agrochemicals on a wide range of commercial CSPs have led to a revised method development strategy. Current work with high load displacement chiral chromatography will be described as a potential means of isolating pure enantiomers from racemates. © 1994 Wiley-Liss, Inc.     

Analysis of stereoisomers of chiral drug by mass spectrometry

Chiral molecules are of great importance in the life science since individual enantiomers may differ in biological activity, mechanism, and toxicity, making it necessary to explore efficient chiral analysis methods. Chromatography approaches are often used to differentiate enantiomers while mass spectrometry (MS) was thought to be blind in chiral analysis. With the development of MS technique, it began to play a more and more crucial part in chiral observation. In this review, we will give a detailed introduction of the analysis methods related to MS for chiral drugs, including its mechanism, applications, and future development.     

Synthesis, chiral separation, and absolute configuration of bis-(N-aryl) atropisomeric triads: 1,2-bis-[4-methyl-2-(thi)oxo-2,3-dihydrothiazol-3-yl]-benzene

In this work, a series of 1,2-bis-[4-methyl-2-(thi)oxo-2,3-dihydrothiazol-3-yl]-benzene has been prepared. These atropisomeric molecular triads were exclusively found to exist in the anti-form. They were separated into enantiomers by liquid chromatography on a chiral support. The absolute configurations of the enantiomers were determined using a chemical correlation method together with chiral chromatography. The barriers to interconversion of the enantiomers were determined.     

Enantioselective degradation of tebuconazole in cabbage, cucumber, and soils

The enantioselective degradation of tebuconazole has been investigated to elucidate the behaviors in agricultural soils, cabbage, and cucumber fruit. Rac-tebuconazole was fortified into three types of agricultural soils and sprayed foliage of cabbage and cucumber, respectively. The degradation kinetics, enantiomer fraction and enantiomeric selectivity were determined by reverse-phase high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) on a Lux amylose-2 chiral column. The process of the degradation of tebuconazole enantiomers followed first-order kinetic in the test soils and vegetables. It has been shown that the degradation of tebuconazole was enantioselective. The results indicated that the (+)-S-tebuconazole showed a faster degradation in cabbage, while the (-)-R-tebuconazole dissipated faster than (+)-S-form in cucumber fruit and the test soils.     

Enantiomeric separation of malathion and malaoxon and the chiral residue analysis in food and environmental matrix

Malathion is a widely used chiral phosphorus insecticide, which has a more toxic chiral metabolite malaoxon. In this work, the enantiomers of malathion and malaoxon were separated by high-performance liquid chromatography-mass/mass (HPLC-MS/MS) with chiral columns using acetonitrile/water or methanol/water as mobile phase, and the chromatographic conditions were optimized. Based on the chiral separation, the chiral residue analysis methods for the enantiomers in soil, fruit, and vegetables were set up. Two pairs of the enantiomers were better separated on CHIRALPAK IC chiral column, and baseline simultaneous separations of malathion and malaoxon enantiomers were achieved with acetonitrile/water (40/60, v/v) as mobile phase at a flow rate of 0.5 mL/min. The elution orders were −/+ for both malathion and malaoxon measured by an optical rotation detector. The chiral residue analysis in soil, fruit, and vegetables was validated by linearity, recovery, precision, limit of detection (LOD), and limit of quantification (LOQ). The LODs and LOQs for the enantiomers of malathion were 1 μg/kg and 3–5 μg/kg and 0.08 μg/kg and 0.20–0.25 μg/kg for malaoxon enantiomers. Good linear calibration curves for each enantiomer in the matrices were obtained within the concentration range of 0.02–12 mg/L. The mean recoveries of the enantiomers of malathion and malaoxon ranged from 82.26% to 109.04%, with RSDs of 0.71–8.63%.The results confirmed that this method was capable of simultaneously determining the residue of malathion and malaoxon in food and environmental matrix on an enantiomeric level.     

Analysis of enantiomers of chiral phenethylamine drugs by capillary gas chromatography/mass spectrometry/flame-ionization detection and pre-column chiral derivatization

Several important chiral phenethylamine agents such as mexiletine, fenfluramine, amphetamine, methamphetamine and N-n-propylamphetamine show stereoselective disposition in humans and large differences in therapeutic relevance and toxicity. To analyze the enantiomers of chiral amine drugs, stereoselective methods were developed to separate those enantiomers on an achiral capillary gas chromatography by pre-column chiral derivatization with S-(-)-N-(fluoroacyl)-prolyl chloride. The stereoselectivity and sensitivity can be improved by chiral derivatization. The methods established offer enantioselective, simple, flexible and economic approaches for the analysis of chiral amine drug enantiomers in biological fluids. The methods have been used to determine S-(+)-methamphetamine in human forensic samples and to analyze enantiomers of amphetamine and fenfluramine in rat liver microsomes.     

Toxicity of Binary Mixtures of Enantiomers in Chiral Organophosphorus Insecticides: The Significance of Joint Effects Between Enantiomers

The existence of enantiomer‐enriched mixtures of chiral pesticides in the environment is overwhelmingly positive. However, interactions between enantiomers have not been considered so far in risk assessments. Here, we chose three organophosphorus pesticides as representative chiral pesticides to investigate the possible interaction mode between each pair of enantiomers both in in vivo and in vitro. Data show that the enantiomers of methamidophos and profenofos have a simple additive effect, <zaq;1> whereas fensulfothion acts as an antagonist in AChE‐inhibition model. In contrast, enantiomers of methamidophos and fensulfothion had an additive effect in an acute toxicity test against Daphnia magna. A synergistic effect was observed in the joint toxicity of the profenofos enantiomers. The ability for enantiospecific biodegradation in the in vivo model contributed to the different interaction observed between the in vitro and in vivo models. Moreover, binding affinities were suspected as another reason for the different mode of action of mixture enantiomers. Our study recommends using a joint research model to treat chiral compounds in the real environment. Chirality 25:787–792, 2013. © 2013 Wiley Periodicals, Inc.     

Enantioselective LC/MS method for the determination of an antimalarial agent Fenozan B07 in dog plasma

A chiral liquid chromatography/mass spectrometry (LC/MS) bioanalytical procedure has been developed for the analysis of the antimalaric agent Fenozan B07 in dog plasma. Normal-phase chromatography involving a phenylcarbamate derivative of cellulose coated on silica gel as the chiral stationary phase was used to resolve (-)-(S,S)-B07 from (+)-(R,R)-B07. The enantiomers were detected by a mass spectrometer equipped with an atmospheric pressure chemical ionization (APCI) interface operated in the negative ion mode. A mass spectrum, characterized by a base peak of m/z 285, was obtained for each enantiomer. The m/z 285 ion was very specific for the analysis of both enantiomers in the plasma. The selected ion monitoring analysis of the plasma samples was therefore performed at m/z 285 for quantitative purposes. The enantiomers were extracted from the plasma in a basic medium and purified by solid-phase extraction using a hydrophilic-lipophilic balanced sorbent. A lower limit of quantification of 2 ng/mL in plasma was achieved for both enantiomers. The quantitative procedure reported in this study was highly specific and sensitive, and was validated according to the FDA guidance on bioanalytical method validation.     

Enantioselectivity of lipase-catalyzed hydrolysis of some 2-chloroethyl 2-arylpropanoates studied by chiral reversed-phase liquid chromatography

A technique based exclusively on chiral reversed-phase liquid chromatography has been shown to greatly facilitate studies of enantioselectivity in lipase-catalyzed hydrolysis of chiral organic esters. Only two sets of experimental data are needed to calculate the enantioselectivity (E) of a kinetically controlled enantiomer-differentiating reaction of this kind, viz. the enantiomeric excess of the product (ee_p) or substrate (ee_s), and the degree of substrate conversion (c). The product enantiomers are well separated on a BSA-based column, giving ee_p directly. In addition, separation of the (unresolved) ester substrate from the enantiomeric products gives c by integration. Via an optimization of the mobile phase used in the chiral chromatographic system, both these parameters can often be determined in a single run. Highly precise and detailed kinetic studies of the enzymatic reaction can thus be performed. In this way, E-values have been determined for a series of 2-chloroethyl 2-arylpropanoates hydrolyzed in the presence of a Candida cylindracea lipase at pH 6.0 and 7.1. Effects on the E-values from a partial purification and further processing of the lipase have also been studied.     

Enantioselective determination of oxprenolol and its metabolites in human urine by cyclodextrin-modified capillary zone electrophoresis

A stereospecific capillary electrophoresis assay for oxprenolol enantiomers and their basic metabolites in human urine has been developed using hydroxypropyl-β-CD as a chiral selector in the mobile phase. The bioassay method has been validated and the detection limit from spiked urine samples is 0.2μg/ml. The calibration curves are linear from 0.4 to 16 μg/ml. Extraction recovery ranged from 84.7 to 96.4% for all the compounds studied. The influence of various parameters on the chiral separation of oxprenolol and its basic metabolites have been investigated. Urinary excretion profiles of oxprenolol enantiomers and those of two metabolites have also been studied, following a single oral dose of racemic oxprenolol.     

Integration of metabolomics and in vitro metabolism assays for investigating the stereoselective transformation of triadimefon in rainbow trout

Triadimefon is a systemic agricultural fungicide of the triazole class whose major metabolite, triadimenol, also a commercial fungicide, provides the majority of the actual fungicidal activity, i.e., inhibition of steroid demethylation. Both chemicals are chiral: triadimefon has one chiral center with two enantiomers while its enzymatic reduction to triadimenol produces a second chiral center and two diastereomers with two enantiomers each. All six stereoisomers of the two fungicides were separated from each other using a chiral BGB‐172 column on a GC‐MS system so as to follow stereospecificity in metabolism by rainbow trout hepatic microsomes. In these microsomes the S‐(+) enantiomer of triadimefon was transformed to triadimenol 27% faster than the R‐(?) enantiomer, forming the four triadimenol stereoisomers at rates different from each other. The most fungi‐toxic stereoisomer (1S,2R) was produced at the slowest rate; it was detectable after 8 h, but below the level of method quantitation. The triadimenol stereoisomer ratio pattern produced by the trout microsomes was very different from that of the commercial triadimenol standard, in which the most rat‐toxic pair of enantiomers (known as “Diastereomer A”) is about 85% of the total stereoisomer composition. The trout microsomes produced only about 4% of “Diastereomer A”. Complementary metabolomic studies with NMR showed that exposure of the separate triadimefon enantiomers and the racemate to rainbow trout for 48 h resulted in different metabolic profiles in the trout liver extracts, i.e., different endogenous metabolite patterns that indicated differences in effects of the two enantiomers. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Chiral separation principles in chromatographic and electromigration techniques

Almost half of the drugs in use today are chiral. It is well established that the pharmacological activity is mostly restricted to one of the enantiomers (eutomer). There can be qualitative and quantitative differences in the activity of the enantiomers. In many cases, the inactive enantiomer (distomer) shows unwanted side effects or even toxic effects. Even if the side effects are not that drastic, the distomer has to be metabolized and this represents an unnecessary burden for the organism. Therefore, the development of methods for the separation of enantiomers, both on analytical and preparative scale, has become increasingly important. Chromatographic techniques such as thin layer chromatography (TLC), gas chromatography (GC), supercritical fluid chromatography (SFC), and above all high-performance liquid chromatography (HPLC) have been used for enantiomer separation for about two decades. More recently, electromigration techniques, such as capillary electrophoresis and capillary electrochromatography, have been shown to be powerful alternatives to chromatographic methods. This review gives a short overview of different chiral separation principles and their application. Several new developments are discussed.     

Stereochemical facets of clinical β-blockers: An overview

The modern β-adrenergic agonists (β-blockers) possess one or more than one chiral center in their structure. Two enantiomers exhibit distinct pharmacodynamic and pharmacokinetic behaviors. Current progress in drug designing has resulted in the ability to understand the role of chirality in modern therapeutics. Furthermore, with a greater understanding of the molecular structure of precise drug targets, development of new drugs is directed towards the pure enantiomers instead of its racemates. The present review deals with a discussion on the stereochemical facets of chiral clinical β-blockers. This review provides details of stereo-selectivity in the pharmacological behavior of some of β-blockers and their metabolites. An effort has been made on highlighting the distinction between the therapeutic behavior of the racemic mixtures and pure enantiomers.     

Enantioseparation of Racemic Flurbiprofen by Aqueous Two‐Phase Extraction With Binary Chiral Selectors of L‐dioctyl Tartrate and L‐tryptophan

A novel method for chiral separation of flurbiprofen enantiomers was developed using aqueous two‐phase extraction (ATPE) coupled with biphasic recognition chiral extraction (BRCE). An aqueous two‐phase system (ATPS) was used as an extracting solvent which was composed of ethanol (35.0% w/w) and ammonium sulfate (18.0% w/w). The chiral selectors in ATPS for BRCE consideration were L‐dioctyl tartrate and L‐tryptophan, which were screened from amino acids, β‐cyclodextrin derivatives, and L‐tartrate esters. Factors such as the amounts of L‐dioctyl tartrate and L‐tryptophan, pH, flurbiprofen concentration, and the operation temperature were investigated in terms of chiral separation of flurbiprofen enantiomers. The optimum conditions were as follows: L‐dioctyl tartrate, 80 mg; L‐tryptophan, 40 mg; pH, 4.0; flurbiprofen concentration, 0.10 mmol/L; and temperature, 25 °C. The maximum separation factor α for flurbiprofen enantiomers could reach 2.34. The mechanism of chiral separation of flurbiprofen enantiomers is discussed and studied. The results showed that synergistic extraction has been established by L‐dioctyl tartrate and L‐tryptophan, which enantioselectively recognized R‐ and S‐enantiomers in top and bottom phases, respectively. Compared to conventional liquid–liquid extraction, ATPE coupled with BRCE possessed higher separation efficiency and enantioselectivity without the use of any other organic solvents. The proposed method is a potential and powerful alternative to conventional extraction for separation of various enantiomers. Chirality 27:650–657, 2015. © 2015 Wiley Periodicals, Inc.     

Stereoselective regulations of P-glycoprotein by ginsenoside Rh2 epimers and the potential mechanisms from the view of pharmacokinetics

Chirality is an interesting topic and it is meaningful to explore the interactions between chiral small molecules and stereoselective biomacromolecules, with pre-clinical and clinical significances. We have previously demonstrated that 20(S)-ginsenoside Rh2 is an effective P-glycoprotein (P-gp) inhibitor in vitro and in vivo. Considering the stereochemistry of ginsenoside Rh2, in our present study, the regulatory effects of 20(R)-Rh2 on P-gp were assayed in vivo, and the differential regulations of P-gp by ginsenoside Rh2 epimers in vivo were compared and studied. Results showed that 20(S)-Rh2 enhanced the oral absorption of digoxin in rats in a dose-dependent manner; 20(R)-Rh2 at low dosage increased the oral absorption of digoxin, but this effect diminished with elevated dosage of 20(R)-Rh2. Further studies indicated stereoselective pharmacokinetic profiles and intestinal biotransformations of Rh2 epimers. In vitro studies showed that Rh2 epimers and their corresponding deglycosylation metabolites protopanaxadiol (Ppd) epimers all exhibited stereoselective regulations of P-gp. In conclusion, in view of the in vitro and in vivo dispositions of Rh2 and the regulations of P-gp by Rh2 and Ppd, it is suggested that the P-gp regulatory effect of Rh2 in vivo actually is a double actions of both Rh2 and Ppd, and the net effect is determined by the relative balance between Rh2 and Ppd with the same configuration. Our study provides new evidence of the chiral characteristics of P-gp, and is helpful to elucidate the stereoselective P-gp regulation mechanisms of ginsenoside Rh2 epimers in vivo from a pharmacokinetic view.