Enantioselective quantification of omeprazole and its main metabolites in human serum by chiral HPLC-atmospheric pressure photoionization tandem mass spectrometry

Omeprazole is a proton pump inhibitor drug in widespread use for the reduction of gastric acid production. It is also proposed as a test substance for the phenotyping of cytochrome CYP3A4 and CYP2C19 enzyme activities. For this purpose, it is necessary to quantify, additionally to omeprazole, the two main metabolites 5-hydroxyomeprazole and omeprazole-sulfon in human plasma. Since omeprazole is a racemic mixture of two enantiomers and its enzymatic decomposition depends in part on its chiral configuration, full information about its metabolic breakdown can only be gained by enantioselective quantification of the drug and its metabolites. We introduce a new LC-MS/MS method that is capable to simultaneously quantify omeprazole and its two main metabolites enantioselectively in human serum. The method features solid-phase extraction, normal phase chiral HPLC separation and atmospheric pressure photoionization tandem mass spectrometry. As internal standards serve stable isotope labeled omeprazole and 5-hydroxyomeprazole. The calibration functions are linear in the range of 5-750 ng/ml for the omeprazole enantiomers and omeprazole-sulfon, and 2.5-375 ng/ml for the 5-hydroxyomeprazole enantiomers, respectively. Intra- and inter-day relative standard deviations are <7% for omeprazole and 5-hydroxyomeprazole enantiomers, and <9% for omeprazole-sulfon, respectively.     

Implications of chirality and geometric isomerism in some psychoactive drugs and their metabolites

Many drugs contain a chiral centre, or such a centre is introduced during metabolism of the drug in man and in animals. If a single chiral centre is present, the drug will normally exist as a mixture of two enantiomers, of which one may have quite different pharmacologic and/or toxic effects than the other. Chiral drugs that are used in psychiatry, and some other pharmacologically related drugs are identified, and the implications of the presence of one or two chiral centres in these drugs are discussed. Differences in pharmacologic properties of drug and metabolite enantiomers are identified and discussed. Also reviewed are the properties of some drugs used in psychiatry that both are chiral and display geometric isomerism.     

Chiral investigation of midodrine, a long-acting alpha-adrenergic stimulating agent

Midodrine hydrochloride is a peripheral alpha(1)-adrenoreceptor agonist that induces venous and arterial vasoconstriction. Midodrine, after oral or intravenous administration, undergoes enzymatic hydrolysis and releases deglymidodrine, a pharmacologically active metabolite. Midodrine and deglymidodrine have a chiral carbon in the 2-position. To investigate the bioactivity of racemates and enantiomers of the drug and metabolite, three chromatographic chiral stationary phases, Chiralcel OD-H, Chiralcel OD-R, and alpha(1)-AGP, were evaluated for enantiomeric resolution. Good enantioseparation of midodrine racemate was obtained using the Chiralcel OD-H column. This stationary phase was then used to collect separately the midodrine enantiomers. By alkaline hydrolysis of rac-midodrine and each separated enantiomer, rac-deglymidodrine and its enantiomers were prepared. The control of the enantiomeric purity was carried out by alpha(1)-AGP stationary phase, while the hydrolysis of rac-midodrine and its enantiomers was controlled by capillary electrophoresis using trimethyl-beta-cyclodextrin as chiral selector. The pharmacological activity of the two racemates and the two enantiomeric pairs was tested in vitro on a strip of rabbit descending thoracic aorta. The tests continued that the activity of the drug and metabolite is due only to the (-)-enantiomer because neither of the (+)-enantiomers is active.     

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.     

Achiral-chiral LC/LC-MS/MS coupling for determination of chiral discrimination effects in phenprocoumon metabolism

Many physiological processes show a high degree of stereoselectivity, including the metabolism of xenobiotics as catalyzed by cytochrome P450 enzymes. An analysis of these chiral discrimination effects in drug metabolism is essential for an in-depth understanding of metabolic pathways that differ between enantiomers of a given chiral drug or metabolite thereof. Achiral chromatographic separation and structural identification followed by chiral analysis of metabolites from blood specimens usually requires a time-consuming multistage analytical technique. In an effort to optimize such a complicated analytical scheme, a novel two-dimensional online achiral-chiral liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) coupling method was developed by using a peak parking technique in combination with a makeup flow system. Metabolites were separated in the first dimension using a C18 reversed-phase system. A makeup eluent of water/methanol (95/5) was split into the flow before storing the metabolites separately on chiral cartridges. Subsequently, the metabolite enantiomers were eluted backward onto the analytical chiral column and separated, and the ratio of enantiomers was determined. The method was successfully validated with respect to limit of detection, linearity, intra- and interday accuracy, and precision. In the course of a human volunteer study investigating the influence of CYP (cytochrome) 2C9 genetic polymorphism on phenprocoumon (PPC) metabolism, we used this new two-dimensional online analytical technique for the analysis of PPC metabolites in plasma. The enantiomeric forms of 4'-, 6-, and 7-hydroxy-PPC metabolites as well as two novel metabolites were identified, and the ratio of the enantiomers was calculated. We found that the enantiomeric ratio for the different metabolites in the plasma sample of each measured individual differs markedly from a nearly 100% chiral discrimination for the two new putative metabolites. This new analytical coupling method possesses general utility in the analysis of chiral discrimination effects, particularly as it relates to pharmacokinetics and dynamics, a scientific field that is rapidly becoming an area of concern and interest.     

Cyclodextrins and enantiomeric separations of drugs by liquid chromatography and capillary electrophoresis: basic principles and new developments

Investigation of individual drug enantiomers is required in pharmacokinetic and pharmacodynamic studies of drugs with a chiral centre. Cyclodextrins (CDs) are extensively used in high-performance liquid chromatography as stationary phases bonded to a solid support or as mobile phase additives in HPLC and capillary electrophoresis (CE) for the separation of chiral compounds. We describe here the basis for the liquid chromatographic and capillary electrophoretic resolution of drug enantiomers and the factors affecting their enantiomeric separation. This review covers the use of CDs and some of their derivatives in studies of compounds of pharmacological interest.     

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.     

The different dissipation behavior of chiral pesticide paclobutrazol in the brine during Chinese cabbage pickling process

Paclobutrazol (PBZ) is a kind of chiral pesticide, which is a plant growth regulator and has fungicidal activity. Because of the steric‐hindrance effect, there are two enantiomers (2S, 3S; 2R, 3R) in the production. This research studied on the dissipation behavior of chiral pesticide PBZ in the brine during the Chinese cabbage pickled process by phase column‐high performance liquid chromatography (HPLC). The result demonstrated the PBZ enantiomers had the different dissipation in the brine. The study on the behavior of chiral pesticide PBZ in food may provide more sufficient data and information for understanding the potential risk in food and evaluating the environmental pollution at the enantiomer level.     

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.     

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.     

A fluorescent chiral chemosensor for the recognition of the two enantiomers of chiral carboxylates

A new 7-nitrobenz-2-oxa-1,3-diazole (NBD)-based fluorescent chiral chemosensor (NBD-1) was prepared and applied to the recognition of the two enantiomers of the tetrabutylammonium salts of N-t-Boc-α-amino acids and chiral carboxylic acids including naproxen. In particular, the chiral recognition by the new fluorescent chiral chemosensor for the two enantiomers of N-t-Boc-threonine (tetrabutylammonium salt) was quite excellent, the Stern-Volmer constant ratio (K(D)/K(L)) for the two enantiomers being as high as 4.89.     

Electroencephalographic effects of thiopentone and its enantiomers in the rat

Electrophysiological studies with some chiral barbiturates have shown that one enantiomer can be excitant while the other is depressant. Thiopentone, a chiral barbiturate, has both differences in potency between enantiomers and biphasic effects on the electroencephalogram (EEG). This study investigated whether a differential EEG activity between the enantiomers of thiopentone could account for the biphasic effects. Rats were administered rac-, R- or S-thiopentone to determine the nature and time course of quantitative EEG effects. Two studies using computer-controlled i.v. infusions of the three drugs were performed in groups of animals previously prepared with EEG electrodes and/or arterial blood sampling cannulae. Study 1 used several stepwise increments in plasma drug concentration over 35 min, followed by washout. Study 2 used a 4 min period of constant plasma drug concentration, followed by washout. In both studies, both enantiomers and racemate caused an initial EEG activation followed by deactivation. Quantitative enantioselectivity was found for depression. The extent of depression was significantly less for R-thiopentone (P=0.008) and racthiopentone (P=0.038) than for S-thiopentone; recovery from depression appeared to be faster for R-thiopentone than either rac- or S-thiopentone. Fatality was only found with S-thiopentone (3/7 animals in Study 2). R-thiopentone plasma concentrations were approximately 8% less than those of S-thiopentone in rats treated with racthiopentone. Although small differences in clearance between enantiomers were found that may influence recovery, they were not large enough to account for the reported differences in potency between the two enantiomers.     

Enantioselective synthesis and teratogenicity of propylisopropyl acetamide,a CNS‐active chiral amide analogue of valproic acid

Propylisopropyl acetamide (PID), an amide analogue of the major antiepileptic drug valproic acid (VPA), possesses favorable anticonvulsant and CNS properties. PID contains one chiral carbon atom and therefore exists in two enantiomeric forms. The purpose of this work was to synthesize the two PID enantiomers and evaluate their enantiospecific teratogenicity. Enantioselective synthesis of PID enantiomers was achieved by coupling valeroyl chloride with optically pure (4S)‐ and (4R)‐benzyl‐2‐oxazolidinone chiral auxiliaries. The two oxazolidinone enolates were alkylated with isopropyl triflate, hydrolyzed, and amidated to yield (2R)‐ and (2S)‐PID. These two PID enantiomers were obtained with excellent enantiomeric purity, exceeding 99.4%. Unlike VPA, both (2R)‐ and (2S)‐PID failed to exert teratogenic effects in NMRI mice following a single 3 mmol/kg subcutaneous injection. From this study we can conclude that individual PID enantiomers do not demonstrate stereoselective teratogenicity in NMRI mice. Due to its better anticonvulsant activity than VPA and lack of teratogenicity, PID (in a stereospecific or racemic form) has the potential to become a new antiepileptic and CNS drug. Chirality 11:645–650, 1999. © 1999 Wiley‐Liss, Inc.     

Stereospecificity and stereoselectivity of flobufen metabolic profile in male rats in vitro and in vivo: phase I of biotransformation.

Flobufen (F) is the original nonsteroidal antiinflammatory drug (NSAID) containing two enantiomers. The aim of this investigation was to elucidate the biotransformation pathway of F at chiral level in phase I of biotransformation. Stereoselectivity and stereospecificity of the respective enzymes were studied in male rats in vitro (microsomal and cytosolic fractions, hepatocytes suspension) and in vivo. The rac-F, (+)-R-F and (-)-S-F were used as substrates. Amounts of F enantiomers, 4-dihydroflobufen diastereoisomers (DHF) and other metabolites (M-17203, UM) were determined with a chiral HPLC method in two chromatographic runs on R,R-ULMO and allyl-terguride bonded columns. Stereoselective biotransformation of the two enantiomers of F was observed at all tested levels and significant bidirectional chiral inversion of enantiomers of F was observed in hepatocytes. Mean enantiomeric ratios of F concentrations (S-/R-), after rac-F incubations, ranging from 1.09 in cytosolic fraction to 18.23 in hepatocytes. Stereospecificity of the respective F reductases was also observed. (2R;4S)-DHF and (2S;4S)-DHF are the principal metabolites of F in microsomes and hepatocytes. Neither DHF diastereoisomers nor M-17203 were found in cytosolic fraction. Only the nonchiral metabolite, M-17203, was found in all urine and feces samples after oral administration of F.     

Enantioseparation of amfepramone (rac-diethylpropion): preparative separation of the enantiomers and enantioselective analysis

The enantiomers of the anorectic drug amfepramone [rac-diethylpropion, rac-2-(diethylamino)-1-phenyl-1-propanone; rac-DEP] were separated in the preparative scale by crystallization. With enantiopure di-O-benzoyltartaric acid as salt-forming chiral selector, diastereoisomeric salts of DEP enantiomers with a final purity of more than 97.5% were obtained. Analytical liquid chromatographic and electrophoretic methods for the control of the enantiomeric purity and the stoichiometry of the salts were developed. The enantioseparation of rac-DEP by capillary electrophoresis (CE) using hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral discriminator and phosphate buffer (pH 3.3) as run buffer led to good separations. HPLC methods were developed using polysaccharide chiral stationary phases (CSP). The separation of the two enantiomers and the two main degradation products (1-phenyl-1,2-propanedione and propiophenone), known from solid and liquid pharmaceutical preparations, was attained in one run on the silica-based CSP cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD). The conditions which might affect the enantioselectivity and the quality of the enantiomeric separation were investigated for Chiralcel OD and the related CSP amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD). Both CSPs showed very similar chromatographic properties. The separation factors could be influenced significantly by varying the polar organic modifier added to the mobile phase.     

Biocatalysis for synthesis of pharmaceuticals

Chirality is a key factor in the safety and efficacy of many drug products and thus the production of single enantiomers of drug intermediates and drugs has become important and state of the art in the pharmaceutical industry. There has been an increasing awareness of the enormous potential of microorganisms and enzymes (biocatalysts) for the transformation of synthetic chemicals with high chemo-, regio- and enatioselectivities providing products in high yields and purity. In this article, biocatalytic processes are described for the synthesis of key chiral intermediates for development pharmaceuticals.     

Enabling chiral separations in discovery chemistry with open‐access chiral supercritical fluid chromatography

Work from this paper details a novel walk‐up open‐access (OA) approach to enable chiral analytical method development and preparative separation of enantiomers in early discovery chemistry using supercritical fluid chromatography (SFC). We have demonstrated the success of this OA approach using immobilized chiral stationary phases (CSPs). After screening a diverse set of racemic drug candidates, we have concluded that a simplified OA chiral SFC platform can successfully purify approximately 60% of the analysed racemates. This streamlined OA workflow enables medicinal chemists with limited expertise in chiral method development to successfully and rapidly purify enantiomers for their projects using Waters UPC² and Prep100‐SFC instrumentation.     

Chiral assay methods for lifibrol and metabolites in plasma and the observation of unidirectional chiral inversion following administration of the enantiomers to dogs

Lifibrol, a new drug for the treatment of hypercholesterolemia, contains a stereogenic center bearing a secondary alcohol group. A normal-phase achiral–chiral HPLC separation of the enantiomers of lifibrol and two of its metabolites was developed and validated for quantitation in dog plasma. A silica and a Chiralcel OD-H column were operated in series and all six enantiomeric components and internal standard were directly separated. An initial solid-phase extraction (phenyl) clean-up step and a column-switching step to eliminate late-eluting compounds were also utilized. The solid-phase extraction step was automated using a robotic system. Assay development, validation, and application of the method to a bioavailability study of the racemate and enantiomers of lifibrol in dogs are described. The lower limit of quantitation was 0.0125 μg/ml for each enantiomer of lifibrol using 200 μl of dog plasma with UV detection (255 nm). In dog plasma following oral or intravenous administration of the racemate, the (R)/(S) ratio of the enantiomers of lifibrol was greater than one and increased with time. Following administration of the individual enantiomers, chiral inversion of the (S)-enantiomer but not the (R)-enantiomer was observed. © 1994 Wiley-Liss, Inc.     

Preparative chiral chromatographic resolution of enantiomers in drug discovery

A brief account is given of the role of preparative chromatography for the direct separation of enantiomers in the drug discovery process. Although it is not yet possible to predict the outcome of a chromatographic resolution attempt, and the optimisation procedure sometimes might be time-consuming, the technique is still indispensable as a means to obtain both enantiomers in pure form from a drug racemate for biological testing. The most suitable types of chiral stationary phases (CSPs) available for this purpose are discussed with special reference to loadability and compatibility with different mobile phase systems.