Rapid determination of methadone and its major metabolite in biological fluids by gas–liquid chromatography with thermionic detection for maintenance treatment of opiate addicts

A rapid gas–liquid chromatographic assay is developed for the quantification of methadone (Mtd) and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in biological fluids of opiate addicts. After alkaline extraction from samples with lidocaine hydrochloride as internal standard, Mtd and EDDP are separated on SP-2250 column at 220°C and detected with a thermionic detector. The chromatographic time is about 6 min. The relative standard deviations (R.S.D.) of Mtd and EDDP standards are between 1.5 and 5.5%. Most drugs of abuse (morphine, codeine, narcotine, cocaine, benzoylecgonine, cocaethylene, dextropropoxyphene etc) are shown not to interfere with this technique. The method has been applied to study the levels of Mtd and EDDP metabolite in serum, saliva and urine of patients under maintenance treatment for opiate dependence. EDDP levels were found higher than those of Mtd in urine samples from four treated patients, but lower in serum and undetectable in saliva. However, Mtd concentrations were higher in saliva than in serum.     

Quantitation of trimipramine enantiomers in human serum by enantioselective high-performance liquid chromatography and mixed-mode disc solid-phase extraction

A sensitive and stereospecific method for the quantitation of trimipramine enantiomers in human serum was developed. The assay involves the use of a novel mixed-mode disc solid-phase extraction for serum sample clean-up prior to HPLC analysis and is also free of interference from the enantiomers of desmethyltrimipramine, 2-hydroxytrimipramine, and 2-hydroxydesmethyltrimipramine, the three major metabolites of trimipramine. Chromatographic resolution of trimipramine enantiomers was performed on a reversed-phase cellulose-based chiral column (Chiralcel OD-R) under isocratic conditions using a mobile phase consisting of 0.3 M aqueous sodium perchlorate-acetonitrile (58:42, v/v) at a flow-rate of 0.5 ml/min. Recoveries for R- and S-trimipramine enantiomers were in the range of 93–96% at 25–185 ng/ml levels. Intra-day and inter-day precisions calculated as R.S.D. were in the ranges of 0.30-8.00% and 1.60-10.20% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percent error were in the 0.01–2.10% and 1.00–3.00% ranges for both enantiomers, respectively. Linear calibration curves were in the concentration range 15–250 ng/ml for each enantiomer in serum. The limit of quantification of each enantiomer was 15 ng/ml. The detection limit for each enantiomer in serum using a UV detector set at 210 nm was 10 ng/ml (S/N =2). In addition, separation of the enantiomers of desmethyltrimipramine, 2-hydroxytrimipramine, and 2-hydroxydesmethyltrimipramine were investigated. The desmethyltrimipramine enantiomers could be resolved on the Chiralcel OD-R column under the same chromatographic conditions as the trimipramine enantiomers, but the other two metabolite enantiomers required different mobile phases on the Chiralcel OD-R column to achieve satisfactory resolution with R_s values of 1.00.     

Selective antibodies to methadone enantiomers: synthesis of (R)- and (R,S)-methadone conjugates and determination by an immunoenzymatic method in human serum

Selective antibodies to (R)-methadone (Mtd) and to its racemate were produced in rabbits by immunization with conjugates of (R)- or (R,S)-hemisuccinyl-methadol-bovine serum albumin, respectively. A hapten was first prepared by reduction of (R)- or (R,S)-Mtd with sodium borohydride, followed by esterification with succinic anhydride. The conjugation of hapten with albumin was achieved by the mixed anhydride method. After immunization of rabbits, the titers and specificity of each antibody were determined by ELISA. The antibodies obtained were tested with (R)-, (S)-, (R,S)-Mtd, its major metabolite (EDDP), and some drugs of abuse (morphine, codeine, cocaine). The sensitivities of antibodies to (R)- and (R,S)-Mtd were about 1 and 2 ng/ml, respectively. Selective (R)-antibodies recognized (R)-Mtd about 40 times more avidly than the (S)-isomer, while an antiserum against (R,S)-Mtd recognized (R)- and (S)-isomers to about the same degree. Both selective antibodies showed little interference (about 0.5%) with EDDP metabolite and no crossreactivity with morphine, codeine, and cocaine. These two selective antibodies were used to develop an immunoenzymatic method (ELISA) for the determination of (R)- and (R,S)-Mtd in serum samples of patients under maintenance treatment for narcotic addiction.     

Quantitative determination of disopyramide, verapamil and flecainide enantiomers in rat plasma and tissues by high-performance liquid chromatography

Enantiomers of disopyramide (DP), flecainide (FLC) and verapamil (VP) were extracted from rat plasma and tissues (brain, lung, heart, liver, kidney and muscle), followed by quantitative determination using enantioselective high-performance liquid chromatography with chiral stationary-phase columns. The recoveries of S-(+)- and R-(−)-DP from tissues were higher than 69%, and the within- and between-day coefficients of variation were very low (0.5 – 5.7%). The lower limits of detection in each tissue were less than 289 ng/g tissue. The recoveries of S-(+)- and R-(−)-FLC from tissues were higher than 88%, and the within- and between-day coefficients of variation were 1.2–6.0%. The lower limits of detection in each tissue were less than 37 ng/g tissue. The recoveries of S-(−)- and R-(+)-VP from tissues were higher than 80%, and the within- and between-day coefficients of variation were 0.5–6.2%. The lower limits of detection in each tissue were less than 51 ng/g tissue. The analytical methods established in this study will be suitable for determining the concentrations of the enantiomers of these anti-arrhythmic agents in rat plasma and tissues.     

Enantiospecific high-performance liquid chromatographic assay with fluorescence detection for the monoamine oxidase inhibitor tranylcypromine and its applicability in pharmacokinetic studies

In order to be able to measure low concentrations of tranylcypromine enantiomers in biological material, chiral fluorescent derivatization and high-performance liquid chromatography (HPLC) were employed. The internal standard S-(+)-amphetamine and borate—sodium hydroxide buffer pH 11 were added to plasma or urine sample aliquots. o-Phthaldialdehyde was used for precolumn derivatization in combination with the chiral mercaptan N-acetylcysteine. HPLC resolution of the diastereoisomeric derivatives was possible on an octadecylsilane column. The mobile phase consisted of sodium phosphate buffer solution pH 6.5, methanol and tetrahydrofuran. The fluorescence of the eluate was monitored at 344/442 nm. The intra-day coefficients of variation were below 10%, the limit of determination was 0.5 ng/ml. The assay was found to be applicable for routine analyses in a preliminary pharmacokinetic study, in which an oral dose of 20 mg racemic tranylcypromine sulfate was administered to three healthy volunteers. The plasma concentrations were generally low, and those of S-(−)-tranylcypromine significantly exceeded those of the R-(+)-enantiomer. Average maximum concentrations were 57.5 and 6.3 ng/ml for S- and R-tranylcypromine, respectively. While S-tranylcypromine was well detectable within the whole study period (8 h), R-tranylcypromine concentrations fell below the detection limit after 4 h in two out of the three studied volunteers.     

Liquid chromatographic analysis of propranolol enantiomers in human blood using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate

A method for the determination of the R-(+) and S-(−) enantiomers of propranolol in blood was developed. After extraction with heptane—isopentanol and derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate, excess reagent was removed using solid-phase extraction. The enantiomers were separated on an achiral, reversed-phase, radially compressed column, and detected by fluorescence with excitation and emission wavelengths of 260 and 340 nm, respectively. The limit of quantification was 0.5 ng/ml. This method was used for pharmacokinetic analysis of propranolol enantiomers after administration of immediate-release (80 mg) or sustained-release (160 mg) racemic propranolol.     

Enantioselective high-performance liquid chromatographic method for the determination of methadone and its main metabolite in urine using an AGP and a C8 column coupled serially

A simple and sensitive method for the enantioselective high-performance liquid chromatographic determination of methadone and its main metabolite, EDDP, in human urine is described. (−)-(R)-Methadone, (+)-(S)-methadone, (+)-(R)-EDDP, (−)-(S)-EDDP and imipramine as an internal standard are detected by ultraviolet detection at 200 nm. The enantiomers of methadone and EDDP were extracted from human urine by a simple liquid–liquid extraction procedure. The extracted sample was reconstructed in mobile phase and the enantiomers of methadone and EDDP were quantitatively separated by HPLC on a short analytical LiChrospher RP8 column coupled in series with a chiral AGP column. Determination of all four enantiomers was possible in the range of 0.03 to 2.5 μM. The recoveries of methadone enantiomers and EDDP enantiomers added to human urine were about 90% and 80%, respectively. The method was applicable for determination of methadone enantiomers and the enantiomers of its main metabolite in urine samples from methadone maintenance patients and patients suffering from severe chronic pain.     

Stereoselective determination of mepivacaine in human serum using a brush-type chiral stationary phase and solid-phase extraction

A stereoselective high-performance liquid chromatography assay method was developed for the quantitation of R-(+)- and S_-(−)-mepivacaine in human serum. The assay uses a Pirkle brush-type. ((S)-tert.-leucine, (R)-(-naphthyl)ethylamine stationary phase (Sumichiral OA-4700, 250×4 mm I.D.) at ambient temperature with a mobile phase of hexane-ethylenedichloride-absolutte methanol (85:10:5, v/v) for the separation of R-(+) and (S)-(−)-mepivacaine. The eluents were monitored using UV detection at 220 nm. Isolation of the analytes from serum was performed using a 1-ml C₁₈ solid-phase extraction cartridge with high recovery and selectivity. The detection limits were 100 ng/ml for each enantiomer and the limits of quantitation were 150 ng/ml for both enantiomers. Linear calibration curves in the 150–2400 ng/ml range showed good correlation coefficients (r>0.9994, N=3). Precision and accuracy of the method were within 2.1–5.3 and 2.0–3.6%, respectively, for (R)-(+)-mepivacaine and 2.7–5.7% and 1.7–4.2%, respectively, for S-(−)-mepivacaine.     

Validated chiral high-performance liquid chromatographic method for the determination of trans-(−)-paroxetine and its enantiomer in bulk and pharmaceutical formulations

A stereospecific high-performance liquid chromatography method for the determination of trans-(−)-paroxetine and its enantiomer in bulk raw material and pharmaceutical formulations was developed and validated. The enantiomeric separation was achieved, without any derivatization, on a carbamate derivative-based column (Chiralpak AD). The effect of the organic modifiers, 2-propanol and ethanol, in the mobile phases was optimised to obtain enantiomeric separation. Limits of detection and quantitation of 2 and 6 ng, respectively, were obtained for both of the enantiomers. The linearity was established in the range of 5–41 μg for trans-(−)-paroxetine and in the range of 10–160 ng for trans-(+)-paroxetine. The accuracy of the method was 102.3% (mean value) for trans-(−)-paroxetine and 99.9% (mean value) for trans-(+)-paroxetine. For the precision (repeatability), a relative standard deviation value of 1.5% (mean value) for trans-(−)-paroxetine and of 2.1% (mean value) for trans-(+)-paroxetine was found. The method is capable of determining a minimum limit of 0.2% of trans-(+)-isomer in commercial samples.     

High-performance liquid chromatographic separation of ondansetron enantiomers in serum using a cellulose-derivatized stationary phase and solid-phase extraction

R(−)-Ondansetron and S(+)-ondansetron in human serum were resolved and quantified using a stereospecific HPLC method. Each enantiomer and the internal standard prazosin were isolated from serum using a solid-phase extraction procedure on a cyanopropyl column. Recoveries of 97, 96 and 88% were obtained for the R(−)-enantiomer, the S(+)-enantiomer, and the internal standard, respectively. A cellulose-based chiral analytical column (Chiralcel OD) was used with a mobile phase consisting of hexane—95% ethanol—2-propanol—acetonitrile (65:25:10:1, v/v). Linear calibration curves were obtained for each enantiomer in serum in the concentration range 10–200 ng/ml. The limit of quantitation of each enantiomer was 10 ng/ml. The detection limit for each enantiomer in serum using UV detection at 216 nm was 2.5 ng/ml (signal-to-noise ratio of 3).     

Stereoselective determination of R-(+)- and S-(−)-remoxipride, a dopamine D2-receptor antagonist, in human plasma by chiral high-performance liquid chromatography

A stereoselective high-performance liquid chromatographic (HPLC) method is described for the selective and sensitive quantitation in human plasma of R-(+)- and S-(−)-enantiomers of remoxipride. Remoxipride was extracted from basified plasma into hexane-methyl-tert.-butyl ether (20:80, v/v), washed with sodium hydroxide (1.0 M), then back-extracted into phosphoric acid (0.1 M). A structural analog of remoxipride was used as an internal standard. The sample extracts were chromatographed using a silica-based derivatized cellulose chiral column, Chiralcel OD-R, and a reversed-phase eluent containing 30–32% acetonitrile in 0.1 M potassium hexafluorophosphate. Ultraviolet (UV) absorbance detection was performed at 214 nm. Using 0.5-ml plasma aliquots, the method was validated in the concentration range 0.02-2.0 μg/ml and was applied in the investigation of systemic inversion of remoxipride enantiomers in man.     

Development and application of a chiral high performance liquid chromatography assay for pharmacokinetic studies of methadone

Methadone enantiomers and EDDP, the main metabolite of methadone, were separated (R(s) = 2.0 for methadone enantiomers) following liquid-liquid extraction from human serum and urine followed by reverse-phase high-performance liquid chromatography on a derivatized beta-cyclodextrin column and quantified at therapeutic concentrations with ultraviolet detection. Detector response was linear (r(2) > 0.98) to 1,000 and 2,500 ng x mL(-1) for methadone enantiomers and EDDP, respectively. The limit of quantification from a 1-mL biological sample was 2.5 and 5 ng x mL(-1) for methadone enantiomers and EDDP, respectively. Interday variation was <13% and intraday variation was <8% for the analytes of interest. The assay was applied to plasma protein and erythrocyte binding studies and a 96-h pharmacokinetic study in two healthy female volunteers following oral dosing with rac-methadone. The binding of methadone to plasma proteins was enantioselective with the active (-)-(R) enantiomer having the highest free fraction (mean +/- SD: 21.2+/-7.6% vs. 13.3+/-6.2% for (+)-(S)-methadone, n = 8). Binding of methadone to erythrocytes was not apparently enantioselective (38.6+/-1.3% and 38.1+/-1.4% bound for (-)-(R)- and (+)-(S)-methadone, respectively). The pharmacokinetic study revealed enantioselective disposition of methadone in one volunteer but not in the other. EDDP was observed in urine but was only in small or undetectable concentrations in serum. The method is applicable to in vitro and pharmacokinetic studies of rac-methadone disposition in humans.     

Quantification of ketoprofen enantiomers in human plasma based on solid-phase extraction and enantioselective column chromatography

An HPLC method for the quantification of ketoprofen enantiomers in human plasma is described. Following extraction with a disposable C₁₈ solid-phase extraction column, separation of ketoprofen enantiomers and I.S. (3,4-dimethoxy benzoic acid) was achieved using a chiral column [Chirex 3005; (R)-1-naphthylglycine 3,5-dinitrobenzoic acid] with the mobile phase, 0.02 M ammonium acetate in methanol, set at a flow-rate of 1.2 ml/min. Baseline separation of ketoprofen enantiomers and I.S., free from interferences, was achieved in less than 20 min. The calibration curves (n = 14) were linear over the concentration range of 0.16 to 5.00 μg/ml per enantiomer [mean r² of 0.999 for both enantiomers, root mean square error were 0.015 for R(−) and 0.013 for S(+)]. The inter-day coefficient of variation for duplicate analysis of spiked samples was less than 7% and the accuracy was more than 93% over the concentration range of 0.2 to 4.0 μg/ml for individual enantiomer using 1 ml of plasma sample. This method has been applied to a pharmacokinetic study from healthy human volunteers following the administration of a ketoprofen extended release product (200 mg). This method is simple, fast and should find wide application in monitoring pharmacokinetic studies of ketoprofen.     

Solid-phase extraction of methadone enantiomers and benzodiazepines in biological fluids by two polymeric cartridges for liquid chromatographic analysis

The aim of this work was to present the advantages of two polymeric cartridges (Oasis HLB from Waters and Abselut Nexus from Varian) for the solid-phase extraction of methadone enantiomers and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and of some benzodiazepines (diazepam, flunitrazepam, nitrazepam, oxazepam) in serum and urine in comparison with classical C18-bonded-silica cartridges or liquid extraction. After addition of serum or urine samples, these two cartridges were washed with a water-methanol mixture (95:5, v/v) and eluted with diethylether. After rapid evaporation, the residue was regenerated with mobile phase and injected either in a chiral column (Cyclobond I-2000 RSP) for methadone enantiomers and its metabolite or in a reversed-phase column (Symmetry Shield RP8) for benzodiazepines. The results showed that the chromatograms of blank serum and urine were cleaner than those obtained from classical solid-phase extraction or liquid extraction. The recoveries from these two polymeric cartridges were higher (95-102%) than those obtained by the two previous classical methods and the total time for extraction and solvent evaporation was also shorter (about 6-7 min). For methadone and benzodiazepine extraction, the use of acidic or alkaline buffer was not necessary.     

Determination of total dopamine, R- and S-salsolinol in human plasma by cyclodextrin bonded-phase liquid chromatography with electrochemical detection

A reliable and sensitive high-performance liquid chromatographic (HPLC) method is presented for the determination of total (free and conjugated) plasma dopamine and the enantiomers R- and S-salsolinol. Plasma is purified on two cartridges, containing primary and secondary amines and phenylboronic acid. Dopamine, R- and S-salsolinol are then separated by HPLC using a β-cyclodextrin-OH phase column. The eluate is monitored electrochemically, without further purification nor derivatization. The method is suited for routine analysis. It allows the detection of total (free and conjugated) dopamine and R- and S-salsolinol in human plasma in concentrations as low as 0.02 ng/ml plasma. The sensitivity is sufficient to measure the naturally occurring levels of salsolinol.     

Chiral phase analysis of warfarin enantiomers in patient plasma in relation to CYP2C9 genotype

A direct chiral-phase high-performance liquid chromatographic method for measuring the ratio of S-warfarin/R-warfarin in patient plasma is described. Plasma samples are first extracted using solid-phase C₁₈ extraction columns, and the concentrated extracts analyzed using an (R,R) Whelk-O 1 column with a mobile phase of 0.5% glacial acetic acid in acetonitrile. The resulting chromatography provides baseline resolution of the warfarin enantiomers and internal standard (racemic ethylwarfarin), and is free from interference from other plasma components. Calibration curves were linear (mean r² of 0.999 for both enantiomers) over the concentration range 0.25–1.5 μg/ml. The intra-day and inter-day coefficients of variation for analysis of plasma spiked with 0.33 μg/ml S-warfarin and 0.67 μg/ml R-warfarin (S/R=0.5:1) was less than 7% for each enantiomer, with an accuracy of more than 93%. Plasma extracts from thirty-one patients homozygous for wild-type CYP2C9*1 provided an S/R ratio of 0.51±0.15. Two warfarin patients homozygous for the mutant CYP2C9*2 and CYP2C9*3 alleles exhibited elevated S/R ratios relative to the mean for individuals homozygous for the wild-type CYP2C9*1 allele. This method is suitable for population studies aimed at establishing the effect of polymorphic expression of CYP2C9 alleles on S-warfarin elimination in humans.     

Enantioselective determination of metoprolol acidic metabolite in plasma and urine using liquid chromatography chiral columns: applications to pharmacokinetics

Enantioselective separations on chiral stationary phases with or without derivatization were developed and compared for the HPLC analysis of (+)-(R)- and (-)-(S)-metoprolol acidic metabolite in human plasma and urine. The enantiomers were analysed in plasma and urine without derivatization on a Chiralcel OD-R column, and in urine after derivatization using methanol in acidic medium on a Chiralcel OD-H column. The quantitation limits were 17 ng of each enantiomer/ml plasma and 0.5 microgram of each enantiomer/ml urine using both methods. The confident limits show that the methods are compatible with pharmacokinetic investigations of the enantioselective metabolism of metoprolol. The methods were employed in a metabolism study of racemic metoprolol administered to a patient phenotyped as an extensive metabolizer of debrisoquine. The enantiomeric ratio (+)-(R)/(-)-(S)-acid metabolite was 1.1 for plasma and 1.2 for urine. Clearances were 0.41 and 0.25 l/h/kg, respectively, for the (+)-(R)- and (-)-(S)-enantiomers. The correlation coefficients between the urine concentrations of the acid metabolite enantiomers obtained by the two methods were >0.99. The two methods demonstrated interchangeable application to pharmacokinetics.     

Enantiomers of cis- and trans-3-(4-propyl-cyclopent-2-enyl) propyl acetate. A study on the bioactive conformation and chiral recognition of a moth sex pheromone component

The enantiomers of cis- and trans-3-(4-propyl-cyclopent-2-enyl) propyl acetate, which are conformationally constrained analogues of (Z)-5-decenyl acetate (1), a sex pheromone component of the turnip moth, Agrotis segetum, have been synthesized and tested using the electrophysiological single-sensillum technique. The analogues mimic a cisoid and transoid conformation of 1, respectively. In addition, the enantiomers of each of the cis- and trans-isomers are conformationally constrained analogues of enantiomeric cisoid and transoid conformations of 1. Thus, the compounds prepared and tested are well suited to investigate the nature of the bioactive conformation of the natural pheromone component 1 and the chiral sense of its interaction with the receptor. Electrophysiological single-sensillum recordings show that the activity of the most active cis-isomer, which has a (1S,4R)-configuration, is more than two orders of magnitude higher than that of the most active trans-isomer. Furthermore, the (1S,4R)-isomer is at least 100 times more active than its enantiomer. These results strongly support a previously proposed cisoid bioactive conformation of 1. Furthermore, the (1S,4R)-configuration of most active stereoisomer identifies the chiral sense of the interaction between the natural pheromone component 1 and its receptor.     

Enantiomeric Composition of Trans-Dihydrodiols Formed from Meso-K-Region Arene Oxides by Microsomal Epoxide Hydrolase

Absolute configurations for the enantiomers of trans-4,5-dihydroxy-4,5-dihydrobenzo[e]pyrene were determined by the exciton chirality method and by correlation of physical properties of their (−)-(menthyloxy)acetyl diesters. Microsomal epoxide hydrolase catalyzed the hydrolysis of K-region arene oxides of benzo[e]pyrene, pyrene, and phenanthrene to trans-dihydrodiols containing 83 %, 86 %, and 42 % of the R,R enantiomer, respectively.     

Stereoselective analysis of bupropion and hydroxybupropion in human plasma and urine by LC/MS/MS

A sensitive, stereoselective assay using solid phase extraction and LC-MS-MS was developed and validated for the analysis of (R)- and (S)-bupropion and its major metabolite (R,R)- and (S,S)-hydroxybupropion in human plasma and urine. Plasma or glucuronidase-hydrolyzed urine was acidified, then extracted using a Waters Oasis MCX solid phase 96-well plate. HPLC separation used an alpha(1)-acid glycoprotein column, a gradient mobile phase of methanol and aqueous ammonium formate, and analytes were detected by electrospray ionization and multiple reaction monitoring with an API 4000 Qtrap. The assay was linear in plasma from 0.5 to 200 ng/ml and 2.5 to 1000 ng/ml in each bupropion and hydroxybupropion enantiomer, respectively. The assay was linear in urine from 5 to 2000 ng/ml and 25 to 10,000 ng/ml in each bupropion and hydroxybupropion enantiomer, respectively. Intra- and inter-day accuracy was >98% and intra- and inter-day coefficients of variations were less than 10% for all analytes and concentrations. The assay was applied to a subject dosed with racemic bupropion. The predominant enantiomers in both urine and plasma were (R)-bupropion and (R,R)-hydroxybupropion. This is the first LC-MS/MS assay to analyze the enantiomers of both bupropion and hydroxybupropion in plasma and urine.