Development of a method to measure methadone enantiomers and its metabolites without enantiomer standard compounds for the plasma of methadone maintenance patients

A liquid chromatography–photodiode array (LC‐PDA) method using a chiral analytical column was developed to determine the plasma levels of enantiomers of methadone and its chiral metabolite, 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP), without the standard compounds of R‐form or S‐form enantiomers. This method was established by the characteristics of recombinant cytochrome P‐450 (CYP) isozymes, where CYP2C19 prefers to metabolize R‐methadone and CYP2B6 prefers to metabolize S‐methadone. We incubated the racemic methadone standard with either enzyme for 24 h. We identified the retention times of R‐ and S‐methadone to be around 10.72 and 14.46 min, respectively. Furthermore, we determined the retention times of R‐ and S‐EDDP to be approximately 6.76 and 7.72 min, respectively. No interferences were shown through the retention times of morphine, buprenorphine and diazepam. With the high recovery rate of a solid‐phase extraction procedure, this method was applied in analyzing plasma concentrations of seven methadone maintenance patients where R‐ and S‐methadone and R‐ and S‐EDDP were 233.4 ± 154.9 and 185.9 ± 136.3 ng/mL and 84.4 ± 99.4 and 37.6 ± 22.9 ng/mL, respectively. These data suggest that the present method can be applied for routine assay for plasma methadone and EDDP concentrations for patients under treatment. Copyright © 2009 John Wiley & Sons, Ltd.     

Stereoselective methadone disposition after administration of racemic methadone to anesthetized Shetland ponies assessed by capillary electrophoresis

The enantioselectivity of the pharmacokinetics of methadone was investigated in anesthetized Shetland ponies after a single intravenous (0.5 mg/kg methadone hydrochloride; n = 6) or constant rate infusion (0.25 mg/kg bolus followed by 0.25 mg/kg/h methadone hydrochloride; n = 3) administration of racemic methadone. Plasma concentrations of l -methadone and d -methadone and their major metabolites, l - and d -2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), respectively, were analyzed by CE with highly sulfated γ-cyclodextrin as chiral selector and electrokinetic analyte injection from liquid/liquid extracts prepared at alkaline pH. In both trials, the d -methadone concentrations were lower than those of l -methadone and the d -EDDP levels were lower than those of L-EDDP. For the case of a single intravenous bolus injection, the plasma concentration versus time profile of methadone enantiomers was analyzed with a two-compartment pharmacokinetic model. l -methadone showed a slower elimination rate constant, a lower body clearance, and a smaller steady-state volume of distribution than d -methadone. d -methadone and d -EDDP were eliminated faster than their respective l -enantiomers. This is the first study that outlines that the disposition of racemic methadone administered to anesthetized equines is enantioselective.     

Use of various β‐cyclodextrin derivatives as chiral selectors for the enantiomeric separation of ofloxacin and its five related substances by capillary electrophoresis

A capillary electrophoretic method for the enantioseparation of ofloxacin and its five related substances (potential impurities, indicated as impurities B–F) was developed using β‐cyclodextrin derivatives as chiral selectors. To our knowledge, there are no previous studies about using capillary electrophoresis for the separation of impurities B–D. Six β‐cyclodextrin derivatives including cationic (piperidine‐ and cyclohexylamine‐), neutral (dimethyl‐ and hydroxypropyl‐), and anionic (carboxymethyl‐ and sulfated‐) β‐cyclodextrin derivatives were tested and operational parameters such as buffer pH and concentration of β‐cyclodextrin derivatives were investigated. The best resolutions were all obtained with anionic β‐cyclodextrin derivatives: ofloxacin, impurities C–F could be best resolved with carboxymethyl‐β‐cyclodextrin at satisfactory resolutions of 8.27, 9.98, 5.92, 8.49 and 6.78, respectively, while for impurity B, a particularly impressive resolution value, up to 21.38, was observed using sulfated‐β‐cyclodextrin. The enhancement of enantioseparation observed for the tested analytes using anionic β‐cyclodextrin derivatives might be due to some favorable interaction between selectors and analytes. Given the fact that the selection of chiral selector depends on the structures of analytes, with the help of structural similarities and differences of the analytes, the structure–separation relationship was further discussed.     

Catalytic Enantioselective 1,3‐Alkyl Shift in Alkyl Aryl Ethers: Efficient Synthesis of Optically Active 3,3′‐Diaryloxindoles

Reported is the first organocatalytic asymmetric 1,3‐alkyl shift in alkyl aryl ethers for the synthesis of chiral 3,3′‐diaryloxindoles using a chiral Brønsted acid catalyst. Preliminary results showed that each enantiomer of the 3,3′‐diaryloxindole, and a racemic mixture, showed different antiproliferative activities against HeLa cell lines by using an MTT assay.     

The influence of the substituent position in monocarboxymethyl‐γ‐cyclodextrins on enantioselectivity in capillary electrophoresis

Three newly synthesized chiral selectors, namely, 2^I‐O‐, 3^I‐O‐, and 6^I‐O‐carboxymethyl‐γ‐cyclodextrin, native γ‐cyclodextrin, and commercially available carboxymethylated γ‐cyclodextrin with degree of substitution of 3–6 were used as additives in a background electrolyte composed of phosphate buffer at 20 mmol/L concentration and pH 2.5. This system was used for the analysis of several biologically significant low‐molecular‐mass chiral compounds by capillary electrophoresis. The results confirmed that the position of carboxymethyl group influences the enantioseparation efficiency of all the studied analytes. The 2^I‐O‐ and 3^I‐O‐ regioisomers provide a significantly better resolution than native γ‐cyclodextrin, while the 6^I‐O‐regioisomer gives only a slightly better enantioseparation than native γ‐cyclodextrin. The application of γ‐cyclodextrin possessing higher number of carboxymethyl groups led to the best resolution for the majority of the compounds analyzed.     

Optimization of the HPLC enantioseparation of 3,3′‐dibromo‐5,5′‐disubstituted‐4,4′‐bipyridines using immobilized polysaccharide‐based chiral stationary phases

The HPLC enantioseparation of nine atropisomeric 3,3′,5,5′‐tetrasubstituted‐4,4′‐bipyridines was performed in normal and polar organic (PO) phase modes using two immobilized polysaccharide‐based chiral columns, namely, Chiralpak IA and Chiralpak IC. The separation of all racemic analytes, the effect of the chiral selector, and mobile phase (MP) composition on enantioseparation and the enantiomer elution order (EEO) were studied. The beneficial effect of nonstandard solvents, such as tetrahydrofuran (THF), dichloromethane (DCM), and methyl t‐butyl ether on enantioseparation was investigated. All selected 4,4′‐bipyridines were successfully enantioseparated on Chiralpak IA under normal or PO MPs with separation factors from 1.14 to 1.70 and resolutions from 1.3 to 6.5. Two bipyridines were enantioseparated at the multimilligram level on Chiralpak IA. Differently, Chiralpak IC was less versatile toward the considered class of compounds and only five bipyridines out of nine could be efficiently separated. In particular, on these columns, the ternary mixture n‐heptane/THF/DCM (90:5:5) as MP had a positive effect on enantioseparation. An interesting phenomenon of reversal of the EEO depending on the composition of the MP for the 3,3′‐dibromo‐5,5′‐bis‐(E)‐phenylethenyl‐4,4′‐bipyridine along with an exceptional enantioseparation for the 3,3′‐dibromo‐5,5′‐bis‐ferrocenylethynyl‐4,4′‐bipyridine (α = 8.33, R_s = 30.6) were observed on Chiralpak IC.     

Approaching over 10 000‐fold sensitivity increase in chiral capillary electrophoresis: Cation‐selective exhaustive injection and sweeping cyclodextrin‐modified micellar electrokinetic chromatography

A novel and simple method that combines an online concentration technique with an enantioseparation technique for capillary electrophoresis—namely, cation‐selective exhaustive injection and sweeping cyclodextrin‐modified micellar electrokinetic chromatography (CSEI‐sweeping CD‐modified MEKC)—realizes the effective enantioseparation of cationic analytes while keeping a significant increase of detection sensitivity. This technique consists of a slight modification of the basic CSEI‐sweeping MEKC. The main idea is to simply add an anionic CD as a chiral selector into the micellar buffer including sodium dodecyl sulfate, but not to change any other buffers in order to preserve the online concentration mechanism. When applied to analysis of the street drug, methamphetamine, the method achieved not only a baseline enantioseparation but also limits of detection (LODs; S/N = 3) of 70–90 pg/mL (ppt) for each isomer. This translates to a more than 10 000‐fold improvement compared to the LODs by the usual injection method. The present technique, which was made from a slight modification of CSEI‐sweeping MEKC, would give an attractive approach that is applicable to almost any analytes for which CSEI‐sweeping MEKC is applicable; all that is required is the selection of an appropriate anionic CD to be added to the micellar buffer.     

Infinite enantiomeric resolution of basic compounds using highly sulfated cyclodextrin as chiral selector in capillary electrophoresis

Four chiral basic analytes, namely methadone, fluoxetine, venlafaxine, and tramadol, were selected as model compounds for investigating their stereoselective separation with highly sulfated gamma-cyclodextrin (HS gamma-CD) by capillary electrophoresis (CE)-UV and CE-mass spectrometry (MS). At high concentration of chiral selector, the preferentially bonded enantiomer migrated faster in the anodic mode to the detector and high resolutions were obtained for all analytes. In the cathodic mode, at lower highly sulphated cyclodextrin (HS-CD) concentration, basic compounds could be detected, with the weakly bonded enantiomer migrating first (enantiomeric migration order inversion). It was also then possible, at intermediate HS-CD concentration, that only one enantiomer migrated to the detector as cation while the other enantiomer complexed with the CD was negatively charged and presented an opposite mobility. The latter never reached the detector achieving a perfect enantiomeric selectivity. Infinite chiral resolutions were thus achieved by CE-UV as well as by CE-electrospray ionisation (ESI)-MS where concentrations of HS-CD were adapted according to the negative contribution of the nebulization gas pressure of the interface.     

Computer simulation of the enantioselective separation of weak bases in an online capillary electrophoresis based microanalysis configuration comprising sulfated cyclodextrin as selector

Computer simulation was utilized to characterize the electrophoretic processes occurring after reactant mixing in an online assay format used for monitoring the enantioselective N‐demethylation of ketamine to norketamine in the presence of highly sulfated γ‐cyclodextrin (HS‐γ‐CD). The incubated reaction mixture (at pH 7.4 and without chiral selector) is bracketed by a low pH BGE containing 2% HS‐γ‐CD as chiral selector, thereby forming a discontinuous buffer system. Upon power application, simulation provides insight into the formation of moving boundaries and new zones together with the prediction of the behavior of ketamine and norketamine enantiomers. The analytes first migrate cationically in a zone electrophoretic manner until they come in contact with HS‐γ‐CD upon which enantioseparation is initiated. Complexation has a focusing effect and the electrophoretic transport becomes reversed, that is, toward the anode. Simulation revealed that the initial conditions for the chiral separation, including buffer components concentrations, pH, and ionic strength, are different than those in the BGE. As a consequence thereof, the experimentally determined complexation parameters for the BGE were unable to correctly describe the migration behavior of the analytes in this column section. An increase in the input binding constants by a factor of two to four, as a result of the decreased ionic strength, resulted in simulation data that agreed with experimental findings.     

Determination of the stereoisomers in aqueous medium and serum and validation studies of racemic aminoalkanol derivatives of 1,7‐dimethyl‐8,9‐diphenyl‐4‐azatricyclo[5.2.1.02,6]dec‐8‐ene‐3,5,10‐trione,potential new anticancer drugs,by capillary electrophoresis

A new method for the determination of the stereoisomers, in aqueous medium and serum, of the racemic aminoalkanol derivatives I and II of 1,7‐dimethyl‐8,9‐diphenyl‐4‐azatricyclo[5.2.1.0^2,6]dec‐8‐ene‐3,5,10‐trione, which were found in earlier studies to be potential anticancer drugs, was developed and validated. The optimized conditions included 25 mM phosphate buffer adjusted to pH 2.5, containing γ‐cyclodextrin at a concentration of 5% m/v, as background electrolyte, an applied voltage of +10 kV, and a temperature of 25°C. Separations were carried out using a fused‐silica capillary. The developed method of determining the enantiomers of compounds I(S), I(R) and II(S), II(R) was characterized by the following parameters: a detection time within 10.8 min, a detection limit in the range of 141.2–141.7 ng/mL using the UV absorption detection at 200 nm. Good linearity (R² = 0.9989–0.9998) was achieved within the range of concentrations studied. A very good extraction yield of 95.4–99.7% was achieved, and recoveries were carried out from both aqueous solutions and matrix serum. The repeatability of the method for peak areas with an accuracy of the determined concentrations of the analytes in the range of 1.43–1.89%, and limits of quantitation in the range of 432.4–436.3 ng/mL were achieved.     

Enantioseparation of 2‐(substituted phenyl)propanoic acids by high‐speed countercurrent chromatography and investigation of the influence of substituents in enantiorecognition

This paper concentrates on the enantioseparation of racemic 2‐(substituted phenyl)propanoic acids by high‐speed countercurrent chromatography with substituted β‐cyclodextrin as the chiral selector, and an investigation of the influence of the substituent on the benzene ring in enantiorecognition between the chiral selector and enantiomer of each racemate is presented. This is an extension research of our previous work on the enantioseparation of 2‐phenyl propanoic acid derivatives, to investigate the relationship between the value of enantioseparation factor and the different substituent on the benzene ring. In total, ten racemic 2‐(substituted phenyl)propanoic acids were investigated, of which four including 2‐(4‐nitrophenyl)propanoic acid, 2‐(4‐methylphenyl)propanoic acid, 2‐(4‐hydroxyphenyl)propanoic acid, and 2‐(4‐chlorophenyl)propanoic acid, were studied by countercurrent chromatography for the first time, and two racemates were successfully enantioseparated. The distribution ratio and enantioseparation factor for all the ten racemates were determined by enantioselective liquid–liquid extraction. The results showed that an electron‐donating group on the benzene ring presents a higher enantiorecognition induced by chiral selector than that of racemates with an electron‐withdrawing group on the benzene ring.     

Enantioseparation of pheniramine enantiomers by high‐speed countercurrent chromatography using β‐cyclodextrin derivatives as a chiral selector

The enantioselective separation of pheniramine was studied by a high‐speed countercurrent chromatography method using β‐cyclodextrin derivatives as a chiral selector. Several key variables, for instance, type of organic solvent and chiral selector, concentration of chiral selector, pH value of aqueous phase, and temperature on the enantioselectivity, were investigated systematically by liquid–liquid extraction experiments. Combining the results of extraction experiments and high‐speed countercurrent chromatography, the most suitable conditions for separation of pheniramine enantiomers were obtained with the two‐phase system that consisted of isobutyl acetate/aqueous phase, containing 0.02 mol/L carboxymethyl‐β‐cyclodextrin, pH 8.50 at 278.15 K. Under the optimal conditions, pheniramine enantiomer was successfully resolved after four cycles of high‐speed countercurrent chromatography. By using high‐performance liquid chromatography to analyze the fractions, the purities of both (+)‐pheniramine and (–)‐pheniramine were over 99% and the recovery of this method was up to 85–90%.     

Separations of antifungal compounds in capillary electrophoresis with two anionic cyclodextrins

CD‐CZE methods were developed for complete stereoisomeric separations of a series of six γ‐lactam analogues, of which some were neutral, or cationic depending on the background electrolyte nature. The tested cyclodextrin was the versatile sulfobutylether‐ β‐CD, used either in a phosphate buffer using capillaries dynamically coated with polyethylene oxide or in a borate buffer using uncoated capillaries. Long‐end and short‐end modes and concentration variations of chiral selectors allowed finding conditions of complete separation of four out of the six derivatives (i.e., 1 , 2 , 3, and 4 ) in short run times, confirming their broad range of applications. To separate the two last compounds, the highly sulfated‐ γ‐CD was examined as chiral selector in acidic phosphate conditions. The enantiomers of the γ‐lactam analogues 5 and 6 were baseline resolved with 5.5 and 4%, respectively as concentration in the buffer.     

Development of a capillary electrophoresis method for the determination of the chiral purity of dextromethorphan by a dual selector system using quality by design methodology

Dextromethorphan is a centrally acting antitussive drug, while its enantiomer levomethorphan is an illicit drug with opioid analgesic effects. As capillary electrophoresis has been proven as an ideal technique for enantiomer analysis, the present study was conducted in order to develop a capillary electrophoresis‐based limit test for levomethorphan. The analytical target profile was defined as a method that should be able to determine levomethorphan with acceptable precision and accuracy at the 0.1 % level. From initial scouting experiments, a dual selector system consisting of sulfated β‐cyclodextrin and methyl‐α‐cyclodextrin was identified. The critical process parameters were evaluated in a fractional factorial resolution IV design followed by a central composite face‐centered design and Monte Carlo simulations for defining the design space of the method. The selected working conditions consisted of a 30/40.2 cm, 50 μm id fused‐silica capillary, 30 mM sodium phosphate buffer, pH 6.5, 16 mg/mL sulfated β‐cyclodextrin, and 14 mg/mL methyl‐α‐cyclodextrin at 20°C and 20 kV. The method was validated according to ICH guideline Q2(R1) and applied to the analysis of a capsule formulation. Furthermore, the apparent binding constants between the enantiomers and the cyclodextrins as well as complex mobilities were determined to understand the migration behavior of the analytes.     

Facile routes for the preparation of 3,4‐disubstituted 1,3‐oxazolidines and 1,2,5‐trisubstituted imidazolidin‐4‐ones

Facile, alternative synthetic routes to 6 , (R)‐6 , and (S)‐6 ‐3‐benzyl‐N‐(2,6‐dimethylphenyl)‐1,3‐oxazolidine‐4‐carboxamides ( 6 ), a chiral oxazolidine derivative of tocainide, are reported. The synthetic routes described herein also afforded 11 ‐, (R)‐11 ‐, and 12 , which present the imidazolidin‐4‐one core and belong to a class of compounds interesting for their biological activities. All the final compounds and intermediates were fully characterized. Enantiomeric excesses of homochiral 6 and 11 were determined by capillary electrophoresis analysis using 2‐hydroxypropyl‐β‐cyclodextrin or highly sulfated γ‐cyclodextrin as chiral selectors. J. Heterocyclic Chem., (2010)     

Influence of substituent position and cavity size of the regioisomers of monocarboxymethyl‐α‐, β‐, and γ‐cyclodextrins on the apparent stability constants of their complexes with both enantiomers of Tröger's base

Enantiomers of Tröger's base were separated by capillary electrophoresis using 2^I‐O‐, 3^I‐O‐, and 6^I‐O‐carboxymethyl‐α‐, β‐, and γ‐cyclodextrin and native α‐, β‐, and γ‐cyclodextrin as chiral additives at 0–12 mmol/L for β‐cyclodextrin and its derivatives and 0–50 mmol/L for α‐ and γ‐cyclodextrins and their derivatives in a background electrolyte composed of sodium phosphate buffer at 20 mmol/L concentration and pH 2.5. Apparent stability constants of all cyclodextrin–Tröger's base complexes were calculated based on capillary electrophoresis data. The obtained results showed that the position of the carboxymethyl group as well as the cavity size of the individual cyclodextrin significantly influences the apparent stability constants of cyclodextrin–Tröger's base complexes.     

Capillary electrophoresis evidence of the stereoselective ketoreduction of mebendazole and aminomebendazole in echinococcosis patients

An assay for the simultaneous determination of the enantiomers of hydroxymebendazole (OH-MBZ) and hydroxyaminomebendazole (OH-AMBZ) together with aminomebendazole (AMBZ) in human plasma is described for the first time. It is based upon liquid-liquid extraction at alkaline pH from 0.5 mL plasma followed by analysis of the reconstituted extract by CE with reversed polarity in the presence of a 50 mM, pH 4.2 acetate buffer containing 15 mg/mL sulfated beta-CD as chiral selector. For all compounds, detection limits are between 0.01 and 0.04 microg/mL, and intraday and interday precisions evaluated from peak area ratios are <6.9 and <8.5%, respectively. Analysis of 39 samples of echinoccocosis patients undergoing pharmacotherapy with mebendazole (MBZ) revealed that the ketoreduction of MBZ and AMBZ is highly stereoselective. One enantiomer of each metabolite (firstly detected peak in both cases) could only be detected. The CE data revealed that OH-MBZ (mean: 0.715 microg/mL) is the major metabolite followed by AMBZ (mean: 0.165 microg/mL) and OH-AMBZ (mean: 0.055 microg/mL) whereas the MBZ plasma levels (mean: 0.096 microg/mL, levels determined by HPLC) were between those of AMBZ and OH-AMBZ.     

Inverse cationic ITP for separation of methadone enantiomers with sulfated β‐cyclodextrin as chiral selector

Chiral ITP of the weak base methadone using inverse cationic configurations with H⁺ as leading component and multiple isomer sulfated β‐CD (S‐β‐CD) as leading electrolyte (LE) additive, has been studied utilizing dynamic computer simulation, a calculation model based on steady‐state values of the ITP zones, and capillary ITP. By varying the amount of acidic S‐β‐CD in the LE composed of 3‐morpholino‐2‐hydroxypropanesulfonic acid and the chiral selector, and employing glycylglycine as terminating electrolyte (TE), inverse cationic ITP provides systems in which either both enantiomers, only the enantiomer with weaker complexation, or none of the two enantiomers form cationic ITP zones. For the configuration studied, the data reveal that only S‐methadone migrates isotachophoretically when the S‐β‐CD concentration in the LE is between about 0.484 and 1.113 mM. Under these conditions, R‐methadone migrates zone electrophoretically in the TE. An S‐β‐CD concentration between about 0.070 and 0.484 mM results in both S‐ and R‐methadone forming ITP zones. With >1.113 mM and < about 0.050 mM of S‐β‐CD in the LE both enantiomers are migrating within the TE and LE, respectively. Chiral inverse cationic ITP with acidic S‐β‐CD in the LE is demonstrated to permit selective ITP trapping and concentration of the less interacting enantiomer of a weak base.     

Ultrashort partial-filling technique in capillary electrophoresis for infinite resolution of tramadol enantiomers and its metabolites with highly sulfated cyclodextrins

The possibility to enhance resolution to infinite value in chiral capillary electrophoresis is attained as soon as the apparent mobility of one enantiomer becomes opposite to the other. This could be achieved on the basis of the carrier ability of multiple charged chiral selectors such as highly sulfated cyclodextrin (HS-CD). With tramadol and its phase I metabolites selected as model compounds, the HS-gamma-CD was found to be the most appropriate chiral selector. The CD concentration was determined where one enantiomer still migrated as a cation while the other migrated in the opposite side. Besides the chiral selector concentration, secondary parameters such as buffer concentration appeared to be critical to reach infinite resolution. The latter was achieved with partial filling technique using ultrashort separation zones (a few mm). In order to better understand the interaction mechanism between the selected CD and the analytes, the classical affinity capillary electrophoresis method, although not fully satisfactory because of ionic strength variations within a series of mobility shift measurements, was applied to estimate complexation constants and complex mobilities. The results obtained point to the prevailing role of complex mobility differences in the enantioselectivity mechanism.     

Simultaneous determination of disopyramide and mono-N-dealkyldisopyramide enantiomers in human plasma by capillary electrophoresis

In this paper, a rapid method for the enantioselective analysis of the antiarrhythmic drug disopyramide and its main metabolite mono-N-dealkyldisopyramide in human plasma by capillary electrophoresis employing the cyclodextrin-modified electrokinetic chromatography mode is described. Sample clean-up was carried out by alkalinization with sodium hydroxide followed by liquid-liquid extraction with toluene. The complete enantioselective analysis was performed within less than 5 min using 20 mmol/L sodium acetate buffer, pH 5.0, containing 0.2% w/v sulfated beta-cyclodextrin as chiral selector. A 40 cm uncoated fused-silica capillary was used for the analysis, performed at a voltage of 15 kV and at 20 degrees C. The calibration curves were linear over the concentration range of 62.5-1850 ng/mL and 125-1850 ng/mL for each enantiomer of disopyramide and mono-N-dealkyldisopyramide. The mean recoveries for disopyramide and mono-N-dealkyldisopyramide enantiomers were up to 87 and 69%, respectively. All four enantiomers studied could be quantified at three different concentrations (200, 400 and 600 ng/mL) with coefficient of variation and % relative error not higher than 15%. The quantitation limit was 62.5 ng/mL for (+)-(S)-and (-)-(R)-disopyramide and (-)-(R)-mono-N-dealkyldisopyramide and 125 ng/mL for (+)-(S)-mono-N-dealkyldisopyramide, using 1 mL of human plasma.