The structure of isolated thalidomide as reference for its chirality-dependent biological activity: a laser-ablation rotational study.

Thalidomide is a drug that presents two enantiomers with markedly different pharmacological and toxicological activities. It is sadly famous due to its teratogenic effects mostly caused by the preferential docking of the (S)-enantiomer to the target protein cereblon (CRBN). To compare the structure of the bound CRBN thalidomide enantiomers with that of the isolated molecule, the rotational spectrum of laser-ablated thalidomide has been studied by chirp-pulsed Fourier transform microwave spectroscopy in supersonic jets complemented by theoretical computations. A new setup of the laser ablation nozzle used is presented. Two stable equatorial and axial conformers of thalidomide have been predicted corresponding to the two possible bent conformations exhibited by the glutarimide moiety. Only the most stable equatorial conformer has been detected. The comparison of its structure with those of the (S)- and (R)-enantiomers bound to CBRN shows that the bound (S) species is only slightly distorted. On the contrary, the bound (R)-enantiomer exhibits a highly distorted structure which affects the degree of puckering of the glutarimide ring and especially to the orientation of the phtalimide and glutarimide subunits. This is consistent with a less stable (R)-enantiomer and the known preference of (S)-thalidomide to bind CRBN, which starts the process leading to teratogenic effects.

Comparative Chiral Separation of Thalidomide Class of Drugs Using Polysaccharide-Type Stationary Phases with Emphasis on Elution Order and Hysteresis in Polar Organic Mode.

The enantioseparation of four phthalimide derivatives (thalidomide, pomalidomide, lenalidomide and apremilast) was investigated on five different polysaccharide-type stationary phases (Chiralpak AD, Chiralpak AS, Lux Amylose-2, Chiralcel OD and Chiralcel OJ-H) using neat methanol (MeOH), ethanol (EtOH), 1-propanol (PROP), 2-propanol (IPA) and acetonitrile (ACN) as polar organic mobile phases and also in combination. Along with the separation capacity of the applied systems, our study also focuses on the elution sequences, the effect of mobile phase mixtures and the hysteresis of retention and selectivity. Although on several cases extremely high resolutions (Rs > 10) were observed for certain compounds, among the tested conditions only Chiralcel OJ-H column with MeOH was successful for baseline-separation of all investigated drugs. Chiral selector- and mobile-phase-dependent reversals of elution order were observed. Reversal of elution order and hysteresis of retention and enantioselectivity were further investigated using different eluent mixtures on Chiralpak AD, Chiralcel OD and Lux Amylose-2 column. In an IPA/MeOH mixture, enantiomer elution-order reversal was observed depending on the eluent composition. Furthermore, in eluent mixtures, enantioselectivity depends on the direction from which the composition of the eluent is approached, regardless of the eluent pair used on amylose-based columns. Using a mixture of polar alcohols not only the selectivities but the enantiomer elution order can also be fine-tuned on Chiralpak AD column, which opens up the possibility of a new type of chiral screening strategy.

Surface-modified three-dimensional graphene nanosheets as a stationary phase for chromatographic separation of chiral drugs.

Carbon-based stationary phases for chromatographic separation have been commercially available since the 1980s. Porous graphitic carbon liquid chromatography columns are known to be highly resistant to aggressive mobile phases and extreme pH values of solvents and eluents, an important advantage compared to conventional silica-based alternatives. In our work, we demonstrate a new variant of carbon-based stationary phases for liquid chromatography, specifically developed for chiral separation. Mesoporous three-dimensional graphene nanosheets (3D GNS), functionalized with tetracyanoethylene oxide (TCNEO) and (S)-(+)-2-pyrrolidinemethanol, demonstrate pharmaceutical-grade chiral separation of model ibuprofen and thalidomide racemic mixtures when used as Chiral Stationary Phases (CSPs), with performance parameters comparable to currently commercially available CSPs. Simple covalent attachment of functionalization groups to the surface of mesoporous three-dimensional graphene nanosheets makes these carbon-based CSPs chemically stable and up to an order of magnitude less expensive than standard silica-based analogues.

A facile and efficient single-step approach for the fabrication of vancomycin functionalized polymer-based monolith as chiral stationary phase for nano-liquid chromatography.

A facile single-step preparation strategy for fabricating vancomycin functionalized organic polymer-based monolith within 100µm fused-silica capillary was developed. The synthetic chiral functional monomer, i.e 2-isocyanatoethyl methacrylate (ICNEML) derivative of vancomycin, was co-polymerized with the cross-linker ethylene dimethacrylate (EDMA) in the presence of methanol and dimethyl sulfoxide as the selected porogens. The co-polymerization conditions were systematically optimized in order to obtain satisfactory column performance. Adequate permeability, stability and column morphology were observed for the optimized poly(ICNEML-vancomycin-co-EDMA) monolith. A series of chiral drugs were evaluated on the monolith in either polar organic-phase or reversed-phase modes. After the optimization of separation conditions, baseline or partial enantioseparation were obtained for series of drugs including thalidomide, colchicine, carteolol, salbutamol, clenbuterol and several other ß-blockers. The proposed single-step approach not only resulted in a vancomycin functionalized organic polymer-based monolith with acceptable performance, but also significantly simplified the preparation procedure by reducing time and labor.

Chiral separation of lenalidomide by liquid chromatography on polysaccharide-type stationary phases and by capillary electrophoresis using cyclodextrin selectors.

Complementary techniques were applied for the investigation of the chiral recognition and enantiomeric resolution of lenalidomide using various cyclodextrins and polysaccharides as chiral selectors. The high-performance liquid chromatography enantioseparation of the anticancer drug was achieved using polysaccharide-type chiral stationary phases in polar organic mode. Elution order and absolute configuration were elucidated by combined circular dichroism spectroscopy and time-dependent density functional theory calculations after the isolation of pure enantiomers. Chiral selector dependent and mobile-phase dependent reversal of the enantiomer elution order was observed, and the nonracemic nature of the lenalidomide sample was also demonstrated. Eight anionic cyclodextrins were screened for their ability to discriminate between the uncharged enantiomers by using capillary electrophoresis. Only two derivatives presented chiral interactions, these cases being interpreted in terms of apparent stability constants and complex mobilities. The best results were delivered by sulfobutylether-ß-cyclodextrin, where quasi-equal stability constants were recorded and the enantiodiscrimination process was mainly driven by different mobilities of the transient diastereomeric complexes. The optimized high-performance liquid chromatography (Chiralcel OJ column, pure ethanol with 0.6 mL/min flow rate, 40°C) and capillary electrophoresis methods (30 mM sulfobutylether-ß-cyclodextrin, 30 mM phosphate pH 6.5, 12 kV applied voltage, 10°C) were validated for the determination of 0.1% (R)-lenalidomide as a chiral impurity, which could be important if a racemic switch is achieved.

Liquid chromatography with mass spectrometry enantioseparation of pomalidomide on cyclodextrin-bonded chiral stationary phases and the elucidation of the chiral recognition mechanisms by NMR spectroscopy and molecular modeling.

A sensitive and validated liquid chromatography with mass spectrometry method was developed for the enantioseparation of the racemic mixture of pomalidomide, a novel, second-generation immunomodulatory drug, using ß-cyclodextrin-bonded stationary phases. Four cyclodextrin columns (ß-, hydroxypropyl-ß-, carboxymethyl-ß-, and sulfobutyl-ß-cyclodextrin) were screened and the effects of eluent composition, flow rate, temperature, and organic modifier on enantioseparation were studied. Optimized parameters, offering baseline separation (resolution = 2.70 ± 0.02) were the following: ß-cyclodextrin stationary phase, thermostatted at 15°C, and mobile phase consisting of methanol/0.1% acetic acid 10:90 v/v, delivered with 0.8 mL/min flow rate. For the optimized parameter at multiple reaction monitoring mode 274.1-201.0 transition with 20 eV collision energy and 100 V fragmentor voltage the limit of detection and limit of quantitation were 0.75 and 2.00 ng/mL, respectively. Since enantiopure standards were not available, elution order was determined upon comparison of the circular dichroism signals of the separated pomalidomide enantiomers with that of enantiopure thalidomide. The mechanisms underlying the chiral discrimination between the enantiomers were also investigated. Pomalidomide-ß-cyclodextrin inclusion complex was characterized using nuclear magnetic resonance spectroscopy and molecular modeling. The thermodynamic aspects of chiral separation were also studied.

Single-step approach for fabrication of vancomycin-bonded silica monolith as chiral stationary phase.

A vancomycin-bonded silica monolithic column for capillary electrochromatography (CEC) was prepared by a single-step in situ sol-gel approach. This sol-gel process incorporates a synthetic sol-gel precursor which contains a macrocyclic antibiotic, vancomycin, to form a porous silica network inside a fused-silica capillary. To avoid degradation of vancomycin during the column fabrication, a mild step was adopted into the sol-gel process. The performance of the vancomycin chiral stationary phase was investigated by CEC in both the reversed-phase mode and the normal-phase mode. The vancomycin chiral stationary phase was optimized with respect to vancomycin loading in the reversed-phase mode for chiral separation of thalidomide enantiomers. The best efficiency and resolution values of 94600plates/m and 5.79, respectively, were achieved. The optimized column was further applied to chiral separation of alprenolol enantiomers. A plate height of less than 7µm for the first eluted enantiomer of alprenolol was obtained in an aqueous mobile phase at a flow rate of 0.74mm/s. Using enantiomers of seven ß-blockers and some other basic enantiomers as test analytes, separation efficiencies of up to 148100plates/m in the reversed-phase mode and up to 138100plates/m in the normal-phase mode were achieved.

Development, validation and application of a sensitive LC-MS/MS method for the quantification of thalidomide in human serum, cells and cell culture medium.

A simple, robust, sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of thalidomide was developed and validated. The method was applied to thalidomide quantification in three different types of biological samples. Thalidomide was extracted from human serum (100 µL), cells (2.5 × 10(5)), or cell culture media (100 µL) by LLE and separated on a Prodigy C18 (150 mm × 4.0 mm, 5 µm i.d.) column with isocratic elution using water/acetonitrile (70/30, v/v) 0.1% formic acid, at a flow rate of 0.5 mL/min, with umbelliferone (600 ng/mL) as an internal standard. Thalidomide was quantified using a triple quadrupole mass spectrometer operated in multi-reaction-monitoring mode using positive electrospray ionisation. The method was validated in two separate thalidomide concentration ranges; human serum (0.05-20 µg/mL) and in vitro cells (0.78-50 ng) with an inter-day precision of 1.8% and 1.9% and average accuracy of 100% and 101% in serum and cells respectively. Despite the use of small sample volume, the limit of quantification for thalidomide in serum was determined to be 3 ng/mL. The method was successfully employed to measure levels of thalidomide in cancer patient serum and cell culture model systems. Although cellular levels were quantifiable, thalidomide was shown to be unstable under in vitro conditions with a half life of approximately 2 h. In patient samples, circulating serum levels showed a broad correlation with dose and uncovered some patient compliance issues.

Preparation and evaluation of a vancomycin-immobilized silica monolith as chiral stationary phase for CEC.

Enantiomeric separations in CEC with the macrocyclic antibiotic vancomycin immobilized silica monolith as a chiral stationary phase are presented. The monolithic silica capillary columns were prepared by a sol-gel process in fused-silica capillaries with an inner diameter of 50 mum and subsequently in situ immobilization of vancomycin as a chiral selector by reductive amination. Enantioselectivity was obtained for eight pairs of enantiomers in nonaqueous polar organic or aqueous mobile phases and most of them were baseline-separated with high column efficiencies. It was observed that the organic modifier ratio (MeOH/ACN) in the polar organic mobile phase played a significant role in controlling the resolution and efficiency of the enantiomers. In enantiomeric separation of propranolol, repeatability for column efficiency and resolution in the nonaqueous mobile phase was given in terms of RSD values at 1.1 and 2.3% (n = 5) for run-to-run injections and 7.2 and 9.6% (n = 5) for column-to-column testing while repeatability for the separation of thalidomide in the aqueous mobile phase was given in terms of RSD values at 1.5, 2.8% and 6.1, 10.5%, respectively.

Chiral recognition ability and solvent versatility of bonded amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase: enantioselective liquid chromatographic resolution of racemic N-alkylated barbiturates and thalidomide analogs.

The chiral recognition ability and solvent versatility of a new chiral stationary phase containing amylose 3,5-dimethylphenylcarabamate immobilized onto silica gel (CHIRALPAK IA) is investigated. Thus, the direct enantioselective separation of a set of racemic N-alkylated barbiturates and 3-alkylated analogs of thalidomide was conducted using different nonstandard solvents as eluent and diluent, respectively in high-performance liquid chromatography (HPLC). The separation, resolution, and elution order of the investigated compounds were compared on both immobilized and coated amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phases (Chiralpak IA and Chiralpak AD, respectively) using a mixture of n-hexane/2-propanol (90:10 v/v) as mobile phase with different flow-rates and fixed UV detection at 254 nm. The effect of the immobilization of the amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phase on silica (Chiralpak IA) on the chiral recognition ability was noted as the bonded phase (Chiralpak IA) was superior in chiral recognition and possesses a higher resolving power in most of the reported cases than the coated one (Chiralpak AD). A few racemates were not or poorly resolved on the immobilized Chiralpak IA or the coated Chiralpak AD when using standard solvents were most efficiently resolved on the immobilized Chiralpak IA upon using nonstandard solvents. Furthermore, the immobilized phase withstands the nonstandard (prohibited) HPLC solvents such as dichloromethane, ethyl acetate, tetrahydrofuran, methyl-tert-butyl ether, and others when used as eluents or as a dissolving agent for the analyte itself. The direct analysis of a real sample extracted from plasma using DCM on Chiralpak IA is also shown.

Thalidomide enantiomers: determination in biological samples by HPLC and vancomycin-CSP.

Thalidomide is a racemate with potentially different pharmacokinetics and pharmacodynamics of the component (+)-(R)- and (-)-(S)-thalidomide enantiomers. As part of a project on the adjunctive effects of thalidomide and cytotoxic agents, a method for the chiral separation and quantitation of thalidomide was developed and validated. Thalidomide in relevant serum and tissue homogenate samples was stabilized by buffering with an equal volume of citrate-phosphate buffer (pH 2, 0.2M), and stored at -80 degrees C pending assay. The thalidomide enantiomers, extracted from the samples with diethyl ether, were well separated on a chiral HPLC column of vancomycin stationary phase and a mobile phase of 14% acetonitrile in 20 mM ammonium formate adjusted to pH 5.4; their concentrations were determined with phenacetin as internal standard at 220 nm detection. Over a thalidomide concentration range of 0.1-20 microg/ml, assay precision was 1-5% (CV) for both enantiomers, and calibration curves were linear with all correlation coefficients being >0.99. The estimated limit of quantification for both enantiomers was 0.05 microg/ml with 0.2-0.6 ml serum samples. Thalidomide in rat and human serum, acidified and stored as described above, was found to be chemically and chirally stable over 1 year. The method has been successfully applied to serum samples from human patients undergoing thalidomide treatment for mesothelioma, and to serum, blood and tissue samples from a laboratory rodent model using transplanted 9l gliosarcoma. Enantioselectivity in thalidomide pharmacokinetics has been found, thereby reinforcing the need for considering the relevance of chirality in thalidomide pharmacology.

A simplified synthesis of polymeric nonparticulate stationary phases with macrocyclic antibiotic as chiral selector for capillary electrochromatography.

A simplified approach to synthesize nonparticulate (continuous or monolithic) beds with embedded vancomycin chiral selectors for capillary electrochromatography is proposed. In the present approach, N,N'-diallyltartardiamide monomer with diol functionality is used, which can be readily converted to aldehyde groups via periodate treatment. Parallel to the activation of the polymeric matrix for covalent attachment of vancomycin, the periodate treatment has shown secondary effects on the polymeric bed morphology, namely the increase of the average pore size and porosity of the skeleton. Inversed size-exclusion chromatography was applied to characterize porosimetric properties of the capillary columns before and after the periodate treatment. Electroosmotic and enantioselective properties of the nonparticulate beds synthesized are presented. The approach is of more general interest attaching different affinity groups to the polymeric matrix and/or enhancing the accessibility to the active sites, for instance, in the molecular imprinting technique.

Clinical pharmacokinetics of thalidomide

Thalidomide is a racemic glutamic acid derivative approved in the US for erythema nodosum leprosum, a complication of leprosy. In addition, its use in various inflammatory and oncologic conditions in being investigated. Thalidomide interconverts between the (R)- and (S)-enantiomers in plasma, with protein binding of 55% and 65%, respectively. More than 90% of the absorbed drug is excreted in the urine and faeces within 48 hours. Thalidomide is minimally metabolised by the liver, but is spontaneously hydrolysed into numerous renally excreted products. After a single oral dose of thalidomide 200mg (as the US-approved capsule formulation) in healthy volunteers, absorption is slow and extensive, resulting in a peak concentration (Cmax) of 1-2mg/L at 3-4 hours after administration, absorption lag time of 30 minutes, total exposure (AUCoo) of 18mg - h/L, apparent elimination half-life of 6 hours and apparent systemic clearence of 10 L/H. Thalidomide pharmacokinetics are best described by a one-comportment model with first-order absorption and elimination. Because of the low solubility of the drug in the gastrointestinal tract, thalidomide exhibits absorption rate-limited pharmacolinetics (the 'flip-flop' phenomenon), with its elimination rate being faster than in absorption rate. The apparent elimination half-life of 6 hours therefore represents absorption, not elimination. The 'true' apparent volume of distribution was estimated to be 16L by use of the faster elimination-rate half-life. Multiple doses of thalidomide 200 mg/day over 21 days cause no change in the pharmacokinetics, with a steady-state Cmax (Cssmax) of 1.2 mg/L. Simulation of 400 and 800 mg/day also shows no accululation, with Css of 3.5 and 6.0 mg/L, respectively. Multiple-dose studies in cancer patients show pharmacokinetics comparable with those in healthy populations at similar dosages. Thalidomide exhibits a dose-proportional increase in AUC at doses from 50 to 400mg. Because of the low solubility of thalidomide Cmax is less than proportional to dose, and tmax is prolonged with increasing dose. Age, sex and smoking have no effect on the pharmacokinetics of thalidomide, and the effect of food is minimal. Thalidomide does not alter the pharmacokinetics of oral contraceptives, and is also unlikely to interact with warfarin and grapefruit juice. Since thalidomide is mainly hydrolysed and passively excreted, its pharmacokonetics are not expected to change in patients with impaired liver...

Recent results of biotransformation of drugs: investigation of the in vitro biotransformation of thalidomide using a dual cyclodextrin system in capillary electrophoresis.

A previously developed capillary electrophoresis method for the simultaneous separation and enantioseparation of thalidomide (TD) and its hydroxylated metabolites was extended to one additional biotransformation product. The dual chiral selector system using native beta-cyclodextrin (beta-CD) and the negatively charged sulfobutyl-beta-CD (SBE-beta-CD) was slightly modified up to a concentration of 12 mg/ml running buffer of each CD. The carrier mode in which these buffer additives transport the neutral compounds to the detector as well as the use of a polyacrylamide-coated capillary were necessary to achieve reproducible enantioseparations of all eight analytes.

Continuous beds with vancomycin as chiral stationary phase for capillary electrochromatography.

Enantiomeric separations in capillary electrochromatography (CEC) carried out using a continuous-bed chiral stationary phase (CSP) based on the macrocyclic antibiotic, vancomycin, is presented. The continuous beds were prepared from methacryloxypropyl modified fused silica capillaries (100 microm ID) by in situ copolymerization of N-(hydroxymethyl)acrylamide and piperazine diacrylamide with vinyl sulfonic acid comonomer used to introduce ionic functionality and thus a strong electroosmotic flow (EOF). The CSP was subsequently prepared by immobilizing the vancomycin stationary phase by reductive amination. Preliminary results have indicated that an extremely strong EOF is obtained in both the nonaqueous polar organic (15.2 x 10(-5) cm2 V(-1) s(-1) and the aqueous reversed-phase modes of operation (8.5 x 10(-5) cm2 V(-1) s(-1)). Enantioselectivity was obtained for four racemic compounds, the best of which was in the case of thalidomide which was separated in 10 minutes with high resolution (Rs = 2.5) and efficiency (120,000 plates meter(-1)) values.

Enantioseparation of aminoglutethimide and thalidomide by high performance liquid chromatography or supercritical fluid chromatography on mono-2 and mono-6-O-pentenyl-beta-cyclodextrin-based chiral stationary phases.

Mono-2 and mono-6-O-pentenyl-beta-cyclodextrin (mono-2-pent-beta-CD and mono-6-pent-beta-CD), covalently linked to mercaptopropylsilica gel (thiol-Si) through thioether or sulfone linkage, reveal differentiated enantioselectivities in the separation of piperidine-2,6-dione-related drugs, namely aminoglutethimide and thalidomide, in supercritical fluid conditions. Supercritical fluid chromatographic resolution on completely defined mono-cyclodextrin derivative-based chiral stationary phases (CSP) is a method of choice for the separation of aminoglutethimide but not effective for thalidomide. For both high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) conditions, the impact of the position, imposed to be 2 or 6 in our synthetic pathway, of the pentenyl moiety on one of the glucopyranosidics of the CD cage is of crucial importance in the chiral discrimination phenomenon. Additionally, the nature of the heteroatom present in the spacer arm between the CD and the silica gel, in this case thioether or sulfone functionality, is also essential for the chiral recognition mechanism(s) for the solute enantiomer.

Investigation of the stereoselective in vitro biotransformation of thalidomide using a dual cyclodextrin system in capillary electrophoresis.

A previously developed capillary electrophoresis method for the simultaneous separation and enantioseparation of thalidomide (TD) and its hydroxylated metabolites was extended to one additional biotransformation product. The dual chiral selector system using native beta-cyclodextrin (beta-CD) and the negatively charged sulfobutyl ether-beta-CD (SBE-beta-CD) was slightly modified up to a concentration of 12 mg/mL running buffer of each CD. The carrier mode in which these buffer additives transport the neutral compounds to the detector as well as the use of a polyacrylamide-coated capillary were necessary to achieve reproducible enantioseparations of all eight analytes. The optimized method was applied to the analysis of the in vitro biotransformation of TD by rat liver microsomes. The S-enantiomer undergoes metabolism preferentially by hydroxylation in the phthalimide ring, whereas R-(+)-TD is mainly transformed to diastereomeric 5'-hydroxythalidomide (5'-OH-TD) pairs. The chiral capillary electrophoresis of incubation samples of TD enantiomers in combination with X-ray diffraction data allowed us to determine the absolute configuration of all metabolites and furthermore to follow the enantio- and stereoselective effects of metabolism in detail.

Simultaneous separation and enantioseparation of thalidomide and its hydroxylated metabolites using high-performance liquid chromatography in common-size columns, capillary liquid chromatography and nonaqueous capillary electrochromatography.

The separation of thalidomide (TD) and its hydroxylated metabolites including their simultaneous enantioseparation was studied using three different polysaccharide-type chiral stationary phases (CSPs) in combination with polar organic mobile phases. Three different techniques, high-performance liquid chromatography in common-size columns, capillary LC and nonaqueous capillary electrochromatography were compared in terms of separation. As this study illustrates, polar organic mobile phases represent a valuable extension for less polar and polar aqueous-organic mobile phases in combination with polysaccharide CSPs. Chiralpak AD consisting of 25% of amylose-tris(3,5-dimethylphenylcarbamate) coated on wide-pore aminopropylsilanized silica gel exhibited higher resolving ability compared to the similar cellulose derivative (Chiralcel OD) as well as to cellulose-tris(4-methylbenzoate) (Chiralcel OJ) CSPs for this particular set of chiral analytes. Baseline separation and simultaneous enantioseparation of all three compounds could be achieved under optimized separation conditions.

Selected applications of capillaries with dynamic or permanent anodal electroosmotic flow in chiral separations by capillary electrophoresis.

The techniques for a dynamic and permanent reversal of the electroosmotic flow (EOF) were used for the reversal of the enantiomer migration order (EMO) of neutral and cationic analytes in chiral capillary electrophoresis (CE). Native beta-Cd and an anionic CD derivative, CM-beta-CD were used in both, bare silica- and positively coated capillaries. Advantages and disadvantages of a dynamic and permanent modification of the capillary inner surface are briefly discussed.

Investigation of the in vitro biotransformation and simultaneous enantioselective separation of thalidomide and its neutral metabolites by capillary electrophoresis.

Reversed-phase liquid chromatography is a long established method for the analysis of drug metabolism. The current investigation demonstrates that micellar electrokinetic capillary chromatography can be an attractive alternative. Two methods were developed using sodium dodecyl sulfate and hexadecyltrimethylammonium bromide for the determination of possible hydroxylated metabolites of the former sedative drug thalidomide (Contergan) in order to study the in vitro metabolism of the drug by incubation with rat liver microsomes. The biotransformation was found to be stereoselective: S-(-)-thalidomide mainly formed 5-hydroxythalidomide, whereas R-(+)-thalidomide was preferentially transformed to two metabolites tentatively assigned to be diastereomers of 5'-hydroxythalidomide. Furthermore, the simultaneous enantioseparation of thalidomide and two of its possible hydroxylated metabolites was achieved using capillary electrophoresis with negatively charged carboxymethyl-beta-cyclodextrin. The dependencies of the selectivity of the enantioseparation on the concentration of the chiral additive and the pH of the run buffer were investigated.