Methadone Enantiomers Research Articles

Enantiomeric separation of methadone by cyclodextrin-based capillary and recycling isotachophoresis.

The separation of methadone enantiomers by cationic capillary isotachophoresis (CITP) and recycling isotachophoresis (RITP) having (2-hydroxypropyl)-beta-cyclodextrin (OHP-beta-CD) as chiral selector in the leading electrolyte is described. Sodium acetate/acetic acid (pH between 4 and 5) served as leading electrolyte (catholyte) and acetic acid as terminator (anolyte). Complete separation of the enantiomers was obtained by CITP in a 50 microm internal diameter (ID) fused-silica capillary and in a 500 microm ID Teflon capillary. In the first approach, enantiomeric separation could be monitored via UV absorbance detection at low wavelength. With the second instrumental setup, an additional conductivity sensor permitted the visualization of the enantiomeric separation and the characterization of the buffer system employed. A 10 mM sodium acetate/acetic acid leading buffer of pH 4.3, containing 5 mM OHP-beta-CD, was found to provide best enantiomeric separation and was thus chosen for RITP. With RITP processing of a few mg of racemic methadone, partial separation of methadone enantiomers was obtained. R-(-)-methadone and S-(+)-methadone were found to be significantly (up to about 80%) enriched at the front and back side, respectively, of the isotachophoretic zone. The enantiomeric composition of methadone in the collected fractions was assessed by chiral capillary zone electrophoresis (CZE) and circular dichroism spectroscopy. CZE was found to represent a simple and efficient method for the determination of the enantiomeric excess, whereas the latter technology was noted to be the superior approach for properly characterizing fractions that contain similar amounts of the two enantiomers. Furthermore, chiral RITP and analysis of the collected fractions by circular dichroism spectroscopy is shown to be potentially useful for identification of single enantiomers in absence of pure chiral standards.

Enantioselective high-performance liquid chromatography determination of methadone enantiomers and its major metabolite in human biological fluids using a new derivatized cyclodextrin-bonded phase

The simultaneous determination of methadone (Mtd) enantiomers and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in human urine and serum by enantioselective HPLC using a new Cyclobond I-2000 RSP column is described. After alkaline extraction from urine or serum with estazolam as an internal standard, Mtd enantiomers and its metabolite (EDDP) are separated on the previous column with reversed-mobile phase and detected at 210 nm. Peak resolutions are about 2.0 for Mtd enantiomers. The relative standard deviations (R.S.D.) of Mtd and EDDP standards are between 0.5 and 4.5%. Most drugs of abuse are shown not to interfere with this technique. The method has been applied to study levels of each Mtd enantiomer and of its racemic metabolite in urine and serum of patients under maintenance treatment for opiate dependence. In urine, R-(−)-Mtd levels are always higher (about 2±0.5-fold_ than those of S-(+)-Mtd and in most cases, metabolite concentrations are greater than those of global Mtd enantiomers. However, the R-(−) enantiomer levels of residual drug in serum of some patients were lower than those of its antipode. This method is suitable for pharmacokinetic and toxicological studies of Mtd enantiomers and its major metabolite in biological fluids.

Replacement of (R)-methadone by a double dose of (R,S)-methadone in addicts: interindividual variability of the (R)/(S) ratios and evidence of adaptive changes in methadone pharmacokinetics.

Twenty-two patients receiving (R)-methadone maintenance treatment were switched to a double dose of (R,S)-methadone: blood samples were collected before and after the change, and the concentrations of the enantiomers were measured. In the second period, during racemic methadone treatment, important interindividual variability in the stereoselective disposition of the enantiomers of methadone was measured, with (R)/(S) ratios ranging from 0.63 to 2.40. This point should be taken into account particularly with respect to therapeutic drug monitoring of racemic methadone. A significant decrease P < 0.005 in the mean serum concentration/dose ratios of the active (R)-enantiomer before and after the change was measured (mean 3.97 and 3.33). Although of small amplitude (16%), this decrease confirms previously described adaptive changes in methadone pharmacokinetics during racemic methadone maintenance treatment and may necessitate, in some patients, a dose adjustment.

Characterization of the stereoselective metabolism of methadone and its primary metabolite via cyclodextrin capillary electrophoretic determination of their urinary enantiomers.

Using capillary zone electrophoresis with a phosphate buffer at pH 3, containing 4.3 mM hydroxypropyl-beta-cyclodextrin as chiral selector, the simultaneous separation of the enantiomers of methadone and its primary metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), is reported. After solid-phase extraction and analysis of the reconstituted extracts in a 60 cm fused-silica capillary of 50 microns ID within about 25 min, the mean S/R enantiomeric ratio of methadone in the urines of eight patients undergoing methadone therapy is shown to be 0.653 (range: 0.502-0.842). The mean enantiomeric ratio for the metabolite is 0.630 (range: 0.517-0.729). These data document the stereoselectivity of the methadone metabolism. Finally, the EDDP/ methadone ratio is shown to vary between 0.357 and 2.917 with a mean value of 1.731. The capillary electrophoretic assay described is simple, inexpensive and neither requires any sample derivatization, nor large amounts of organic solvents or expensive separation columns.

Sensitive high-performance liquid chromatographic assay with ultraviolet detection of methadone enantiomers in plasma

Methadone is being prescribed increasingly as an analgesic in palliative medicine. R-Methadone has been shown to be responsible for most of the pharmacological activity of this drug. Despite this, in most countries it is administered as the racemate. Few assay methods for the enantiomers are available; even fewer can determine accurately the low concentrations of enantiomers required to undertake pharmacokinetic studies in patients taking the drug in analgesic doses. We present here an HPLC method used to determine concentrations of the specific enantiomers of methadone as low as 5.0 ng/ml with adequate precision and accuracy. The mean R/ S ratio of the plasma concentrations was 0.80 ± 0.05 ( n = 3 samples) in one patient taking 25–27.5 mg daily and 1.21 ± 0.12 ( n = 6 samples) in another taking 10–20 mg daily. In the second patient, concentrations of the enantiomers ranged between 5.8 and 25.9 ng/ml. Tricyclic antidepressants did not interfere with the assay but dextropropoxyphene did. Its presence could be detected by dual wavelength monitoring.

Enantioselective high-performance liquid chromatographic method for the determination of methadone in serum using an AGP and a CN column as chiral and analytical column, respectively

A simple and sensitive HPLC method with ultraviolet absorption detection at 200 nm is described for the determination of methadone enantiomers in human serum, using dextropropoxyphene as an internal standard and organic solvent extraction. Separation was performed on two serially coupled columns, CN and Chiral AGP, with a mobile phase consisting of acetonitrile, dimethyloctylamine and phosphate buffer. Using 1.0 ml of serum, 5 nmol/1 of each enantiomer could be determined with an acceptable precision. No interactions from several drugs were observed. The method has been successfully used in a pharmacokinetic study. More than 2500 serum samples have been separated on the same AGP colunm with acceptable selectivity and resolution.

Stereoselective determination of the enantiomers of methadone in plasma using high-performance liquid chromatography

An enantioselective high-performance liquid chromatographic assay for the quantification of methadone in human and beagle plasma is described. The procedure involves extraction of methadone from alkalized plasma into hexane—isoamyl alcohol (99:1, v/v). Stereoselective separation was achieved with a silica column with covalently bound α 1-acid glycoprotein (Chiral-AGP) without any derivatization procedure. The detection wavelength was set at 215 nm. Using an internal standard provided reliable control of the extraction procedure as well as quantification of the enantiomers of methadone. The limit of quantification was found to be 2.5 ng/ml. The method was demonstrated to be sufficiently sensitive for stereoselective pharmacokinetic studies of methadone.

Stereospecific gas chromatographic method for determination of methadone in serum.

rac-Methadone is used clinically for the chronic maintenance treatment of heroin addiction and for the relief of pain. As the pharmacological activity of methadone is due primarily to the (-)-(R)-enantiomer, stereospecific measurements of methadone serum concentrations in methadone-treated patients are expected to be more relevant for clinical studies than earlier described total drug measurements. This study describes a stereospecific gas chromatographic (GC) method for the determination of methadone in serum. The extracted methadone was derivatizised with (-)-menthyl chloroformate. The diastereometric derivatives were analysed by GC on a capillary column and detected with a nitrogen-phosphorus detector. The resolution factor obtained for the methadone enantiomers was 1.1 with a relatively short time of analysis (30 min). By analysing the pure (-)-(R)-enantiomer, no racemization was seen during the analysis. The lower limit of quantitation was 75 nmol/l for each enantiomer. Measurements of the ratio between (-)-(R)- and (+)-(S)-methadone concentrations in serum from five methadone-treated patients showed interindividual differences (range 0.5-1.1). The patient results correlated well with those from another GC method measuring total methadone.

Chiral analysis of methadone in plasma by high-performance liquid chromatography

A method for the chiral high-performance liquid chromatographic analysis of methadone in plasma has been developed. The method employed organic solvent extraction, enantiomeric separation on a Chiral AGP column, and ultraviolet absorption detection at 212 nm. The intra-day variation in the quantification of methadone enantiomers was less than 9% at the 100 ng/ml level, and the values obtained correlated well with those from a gas chromatographic—mass spectrometric method. Results from patients indicate inter- and intra-individual differences in the ratio between l- and d-methadone in plasma during therapy with racemic methadone. In one patient, a higher level of d-methadone in plasma was caused by both faster elimination and lower bioavailability of l-methadone.

Differential neuropharmacological effects of mu, kappa and sigma opioid agonists on cortical EEG power spectra in the rat: Stereospecificity and naloxone antagonism

The study was designed to determine and compare the acute effects of the enantiomers of mu, kappa and sigma opioid agonists on the cortical EEG with the spectral analysis technique. The relative ability of naloxone to antagonize such effects was also assessed. Adult female Sprague-Dawley rats were implanted with chronic cortical EEG and temporalis muscle EMG recording electrodes, and with permanent indwelling external jugular cannulae. (−)-Methadone (mu agonist) produced increases in spectral power over the zero to 10 Hz range, while (−)-ketocyclazocine (kappa agonist) produced increases in the 5–8 Hz band as a predominant peak. The (+)enantiomers of methadone and ketocyclazocine were inactive. The drug (+)-SKF-10,047 (sigma agonist), produced a predominant spectral peak in the 7–9 Hz band that was associated with behavior that suggested psychotomimetic effects. The effects of morphine (mu agonist) on EEG and EEG power spectra were more sensitive to antagonism by naloxone than those produced by ketocyclazocine. The effects of(±)-SKF-10,047 and (+)-SKF-10,047 were not antagonized by 10 mg/kg of naloxone, while the effects of (−)-SKF-10,047 were partially antagonized by lOmg/kg of naloxone. These findings further delineate the specificity of the differential effects of mu, kappa and sigma opioid agonists on the EEG and EEG power spectra in the rat.

Synthesis, X-ray crystallographic determination, and opioid activity of erythro-5-methylmethadone enantiomers. Evidence which suggests that mu and delta opioid receptors possess different stereochemical requirements.

Enantiomers of erythro-5-methylmethadone (3) were synthesized from optical antipodes of erythro-3-(dimethyl-amino)-2-butanol. X-ray crystallographic analysis of (-)-3 perchlorate revealed that it possesses the 5S,6S absolute configuration. It was found that (-)-3 is substantially more potent than its enantiomer (+)-3 as an opioid agonist in vivo and in vitro. In vitro tests (guinea pig ileal longitudinal muscle and mouse vas deferens preparations) suggest that (-)-3 mediates its effect chiefly through mu opioid receptors. On the other hand, (+)-3 and the more potent enantiomers of methadone, (-)-1, and isomethadone, (-)-2, appear to have less mu-receptor selectivity and interact with a greater fraction of delta receptors than does (-)-3. The fact that the solid-state conformation of (-)-3 differs from that of (-)-1 and (-)-2, which show great similarity in conformational features, suggests that mu and delta receptors have different conformational requirements. The possibility of different modes of interaction with a single opioid receptor population also is discussed.

Quantitation of Methadone Eenantiomers in Humans Using Stable Isotope-labeled [2H3]-, [2H5]-, and [2H8]methadone

A new technique for simultaneous stereoselective kinetic studies of methadone enantiomers was developed using three deuterium-labeled forms of methadone and GLC-chemical-ionization mass spectrometry. A racemic mixture (1:1) of (R)-(-)-[2H5]methadone (l-form) and (S)-(+)-[2H3]methadone (d-form) was administered orally in place of a single daily dose of unlabeled (±)-[2H8]methadone in longterm maintenance patients. Racemic (±)-[2H8] methadone was used as an internal standard for the simultaneous quantitation of [2H0]-, [2H3]-, and [2H5] methadone in plasma and urine. A newly developed extraction procedure, using a short, disposable C18 reversed-phase cartridge and improved chemical-ionization procedures employing ammonia gas, resulted in significant reduction of the background impurities contributing to the ions used for isotopic abundance measurements. These improvements enabled the measurement of labeled plasma methadone levels for 120 hr following a single dose. This methodology was applied to the study of methadone kinetics in two patients; in both patients, the analgesically active l-enantiomer of the drug had a longer plasma elimination half-life and a smaller area under the plasma disappearance curve than did the inactive d-form.

ChemInform Abstract: STEREOCHEMICAL STUDIES ON MEDICINAL AGENTS. 21. INVESTIGATION OF THE ROLE OF CONFORMATIONAL FACTORS IN THE ACTION OF DIPHENYLPROPYLAMINES. SYNTHESIS AND ANALGETIC POTENCY OF 5‐METHYLMETHADONE DIASTEREOMERS

AbstractKonformative Einflüsse auf die pharmakologische Wirkung von Diphenylpropylaminen werden untersucht und hierzu entsprechende, z.Tl. stereospezifische Synthesen beschrieben.

Adsorption and conduction-block of opiate narcotics on nerve membranes: identical effects of levo- and dextro-forms.

(1) In order to detect the possible existence of stereospecific binding sites for the opiate narcotics, the adsorption and conduction-blocking action of the enantiomers of methadone and levorphanol were tested on nerve membranes.(2) The adsorption of radioactive dextro- and levo-methadone to homogenates of guinea pig brains were identical. The addition of nonradioactive levo-methadone displaced equal amounts of the radioactive dextro- and levo-methadones.(3) Simultaneous adsorption of 3H-levo-methadone and 14C-dextro-methadone to homogenates of various brain regions also did not reveal any stereoselective adsorption of the levo-isomer.(4) In vivo perfusion of guinea pig brain with 3H-levo-methadone simultaneously with 14C-dextro-methadone did not reveal any methadone uptake which was stereoselective for the levo-isomer and which was significantly different from skeletal muscle. In vivo perfusion with 3H-dextro-methadone simultaneously with 14C-levo-methadone also did not reveal stereoselectivity for the levo-form.(5) The synaptosome membrane/buffer partition coefficients for dextro- and levo-methadone were identical, having a value of 300 at 22°, and about 430 at 37° (in 10 mM sodium phosphate, pH 7). In 0.9% NaCl, the methadone partition coefficient fell to 32 (at 22 °C). The adsorption isotherm indicated only one set of binding sites.(6) The minimum concentrations required to block impulse conduction in rat phrenic nerve were 9.5 × 10−6 M for both dextro- and levo-methadone, 2 × 10−4 M for levorphanol, and 3 × to 4 × 10−4 M for dextrorphan.(7) It is possible that the binding is not stereospecific but that it is only the efficacy on the receptor which is stereospecific.