Determination of bevantolol enantiomers in human plasma by coupled achiral-chiral high performance-liquid chromatography

A coupled achiral-chiral high performance liquid chromatographic method was developed and fully validated for the determination of bevantolol enantiomers, (-)-(S)-bevantolol and (+)-(R)-bevantolol, in human plasma. Plasma samples were prepared by solid phase extraction with Sep-Pak Plus C18 cartridges followed by HPLC. Bevantolol enantiomers and (+)-(R)-Propranolol as internal standard (IS) were preseparated from interfering components in plasma on a Phenomenex silica column and bevantolol enantiomers and IS were resolved and determined on a Chiralcel OJ-H chiral stationary phase. The two columns were connected by a switching valve equipped with silica precolumn. The Precolumn was used to concentrate bevantolol in the eluent from the achiral column before back flushing onto chiral phase. A detailed validation of the method was performed accordingly to FDA guidelines. For each enantiomer the assay was linear between 20 and 1600 ng/ml. The quantification limits of both bevantolol enantiomers were 20 ng/ml. The intraday variation was between 1.07 and 12.64% in relation to the measured concentration and the interday variation was 0.91 and 11.79%. The method has been applied to the determination of (-)-(S)- and (+)-(R)-bevantolol in plasma from healthy volunteers dosed with racemic bevantolol hydrochloride.     

Enantioselective analysis of citalopram and demethylcitalopram in human and rat plasma by chiral LC-MS/MS: application to pharmacokinetics

Citalopram (CITA) is available as a racemic mixture and as a pure enantiomer. Its antidepressive action is related to the (+)-(S)-CITA and to the metabolite (+)-(S)-demethylcitalopram (DCITA). In the present investigation, a method for the analysis of CITA and DCITA enantiomers in human and rat plasma was developed and applied to the study of pharmacokinetics. Plasma samples (1 ml) were extracted at pH 9.0 with toluene:isoamyl alcohol (9:1, v/v). The CITA and DCITA enantiomers were analyzed by LC-MS/MS on a Chiralcel OD-R column. Recovery was higher than 70% for both enantiomers. The quantification limit was 0.1 ng/ml, and linearity was observed up to 500 ng/ml plasma for each CITA and DCITA enantiomer. The method was applied to the study of the kinetic disposition of CITA administered in a single oral dose of 20 mg to a healthy volunteer and in a single dose of 20 mg/kg (by gavage) to Wistar rats (n = 6 for each time). The results showed a higher proportion of the (-)-(R)-CITA in human and rat plasma, with S/R AUC ratios for CITA of 0.28 and 0.44, respectively. S/R AUC ratios of DCITA were 0.48 for rats and 1.04 for the healthy volunteer.     

Determination of the enantiomers of metoprolol and its major acidic metabolite in human urine by high-performance liquid chromatography with fluorescence detection

Enantiomers of metoprolol and its acidic metabolite H 117/04 were determined in human urine by high-performance liquid chromatography (HPLC) with fluorometric detection after chiral derivatization. The carboxyl functional group of the major metabolite was blocked by esterification after solid-phase extraction, which helped to quantitate this compound from interfering substances. The assay method was validated. The recovery of (−)- and (+)-metoprolol from urine was 86.3–90.5%; and the recovery of the (−)- and (+)-acidic metabolite H 117/04 from urine was 74.4–83.9% at different concentrations.     

Probenecid-induced changes in the clearance of pranoprofen enantiomers

Probenecid is known to inhibit the elimination of several acidic drugs. Its influence on the pharmacokinetics of pranoprofen was investigated in rabbit after a single intravenous injection of racemic mixture (5 mg/kg). Levels of (-)-(R)- and (+)-(S)-pranoprofen and their glucuronide (after hydrolysis with sodium hydroxide) were determined in plasma, urine, and several tissues. The plasma concentration of the (+)-(S)-isomer was higher than that of the (-)-(R)-form. Oral coadministered probenecid (100 mg/kg) resulted in an increased plasma concentration of both enantiomers. Probenecid reduced the apparent total clearance and excretion of pranoprofen enantiomers in urine. It had a slight effect on the tissue distribution of pranoprofen at the dose used, but significantly reduced the formation of glucuronide for both enantiomers to the same extent in kidney microsomes. The differences caused by probenecid were significant with respect to its ability to inhibit glucuronidation in the kidney and subsequent excretion into urine, but enantioselective effects were negligible.     

Determination of a neuroprotective agent (S)-(+)-BMS-204352 in human, rat and dog plasma by enantioselective liquid chromatography-tandem mass spectrometry

A sensitive liquid chromatography-electrospray ionization tandem mass spectrometry method (LC/ESI/MS/MS) for the enantioselective determination of (S)-(+)-BMS-204352, a potent and specific maxi-K channel opener, in human, rat and dog plasma was developed. (S)-(+)-BMS-204352, its enantiomer (R)-(--)-BMS-204353 and the internal standard (13C-deuterated racemate of (S)-(+)-BMS-204352) were extracted from plasma using toluene. Chromatographic separation for the enantiomers was achieved on a Chiralcel OD-H analytical column with a run time of 8 min. An aqueous mobile phase modifier was added post column to enhance the mass spectrometer sensitivity. ESI mass spectra were acquired in the negative mode with selected reaction monitoring. The limit of quantitation (LLOQ) is 0.10 ng/mL for human plasma assay. Samples from a clinical study and two animal studies were processed using these procedures. Based on the in vivo data, lack of inversion of (S)-(+)-BMS-204352 to (R)-(--)-BMS-204353 was demonstrated in human, rat and dog after administration of the drug. A sensitive non-enantioselective LC/ESI/MS/MS assay has also been developed for (S)-(+)-BMS-204352 which uses a similar extraction procedure with a C18 column with a limit of quantitation at 0.05 ng/mL. Human study samples were analyzed by both methods and the correlation coefficient between both sets of data is greater than 0.99.     

Pharmacokinetics of the enantiomers of verapamil in the dog

The intravenous (0.5 mg/kg) and oral (5 mg/kg) dose kinetics of verapamil were studied in 6 dogs during steady-state oral verapamil dosing (5 mg/kg every 8 h for 3 days). Racemic verapamil and norverapamil, a metabolite of verapamil, were quantitated in plasma by HPLC-fluorescence detection. The verapamil peaks eluting off the column were collected and rechromatographed on an Ultron-OVM column, which resolved the two verapamil enantiomers. After intravenous administration, the systemic clearance and apparent volume of distribution of (?)-(S)-verapamil were nearly twice that of the (+)-(R)-isomer. There was no difference in the elimination half-lives between the two isomers. After oral administration, the oral clearance of (?)-(S)-verapamil was 20 times that of the (+)-(R)-isomer. The apparent bioavailability of (+)-(R)-verapamil was over 14 times that of (?)-(S)-verapamil. The plasma protein binding of the (+)-(R)-isomer was slightly higher by 5% than (?)-(S)-verapamil; however, this effect was not enough to account for the difference between the apparent volume of distribution of the enantiomers, indicating that the tissue binding of (?)-(S)-verapamil was greater than that of the (+)-(R)-isomer. This data on the disposition of the enantiomers of verapamil in the dog is similar to that reported for man and demonstrates that the dog may be an appropriate animal model for man in future studies on the disposition of the enantiomers of verapamil. © 1993 Wiley-Liss, Inc.     

Determination of felodipine, its enantiomers, and a pyridine metabolite in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of racemic felodipine, its enantiomers, and a pyridine metabolite in human plasma are described. Following liquid-liquid extraction from plasma, enantiomers of felodipine were separated on a chiral HPLC column (Chiralcel OJ) and fractions containing each isomer were collected on a continuous basis using a fraction collector. These fractions were later analyzed by GC-MS-SIM. A similar method based on GC-MS-SIM detection was developed for the determination of racemic felodipine and its pyridine metabolite with a minor modification of sample preparation. The limits of quantitation in plasma were 0.1 ng/ml for both the R(+)- and S(−)-enantiomers of felodipine and 0.5 ng/ml for both racemic felodipine and its pyridine metabolite. The stereoselective assay was used to support a clinical study with racemic felodipine, and was capable of analyzing more than 30 plasma samples per day.     

Stereospecific high-performance liquid chromatographic assay of ketoprofen in human plasma and urine

A high-performance liquid chromatographic (HPLC) assay suitable for the analysis of the enantiomers of ketoprofen (KT), a 2-arylpropionic acid (2-APA) non-steroidal antiinflammatory drug (NSAID), in plasma and urine was developed. Following the addition of racemic fenoprofen as internal standard (I.S.), plasma containing the KT enantiomers and I.S. was extracted by liquid-liquid extraction at an acidic pH. After evaporation of the organic layer, the drug and I.S. were reconstituted in mobile phase and injeted into the HPLC system. The enantiomers were separated at ambient temperature on a commercially available 250 × 4.3 mm amylose carbamate-packed chiral column (Chiralpak AD) column with hexane-isopropyl alcohol-trifluoroacetic acid (80:19.9:0.1, v/v/v) as the mobile phase pumped at 1.0 ml/min. The enantiomers of KT were quantified by ultraviolet detection with the wavelength set at 254 nm. The assay described allows for the direct quantification of KT enantiomers without pre-column derivatization, and is suitable for clinical studies of KT enantiomers in human plasma and urine after administration of therapeutic doses.     

Stereoselective metabolism of metoprolol: enantioselectivity of alpha-hydroxymetoprolol in plasma and urine

Direct stereoselective separation on chiral stationary phase was developed for HPLC analysis of the four stereoisomers of alpha-hydroxymetoprolol in human plasma and urine. Plasma samples were prepared using solid-phase extraction columns and urine samples were prepared by liquid-liquid extraction. The stereoisomers were separated on a Chiralpak AD column at 24 degrees C with fluorescence detection and a mobile phase consisting of a mixture of hexane:ethanol:isopropanol:diethylamine (88:10.2:1.8:0.2) for plasma samples and hexane:ethanol:diethylamine (88:12:0.2) for urine samples. Calibration curves for the individual stereoisomers were linear within the concentration range of 2.0-200 ng/ml plasma or 0.125-25 microg/ml urine. The methods were validated with intra- and interday variations less than 15%. The absolute configuration of the pure stereoisomers were assigned by circular dichroism spectra. The methods were employed to determine the concentrations of alpha-hydroxymetoprolol stereoisomers in a metabolism study of multiple-dose administration of racemic metoprolol to hypertensive patients phenotyped as extensive metabolizers of debrisoquine. We observed stereo-selectivity in the alpha-hydroxymetoprolol formation favoring the new 1'R chiral center from both metoprolol enantiomers (AUC(0-24) (1'R1'S) = 3.02). The similar renal clearances (Cl(R)) of the four stereoisomers demonstrated absence of stereoselectivity in their renal excretion. (-)-(S)-metoprolol was slightly more alpha-hydroxylated than its antipode (AUC(0-24) (2S/2R) = 1.19), suggesting that this pathway is not responsible for plasma accumulation of this enantiomer in humans.     

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.     

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.     

Analysis of Oxcarbazepine and the 10‐Hydroxycarbazepine Enantiomers in Plasma by LC‐MS/MS: Application in a Pharmacokinetic Study

Oxcarbazepine is a second‐generation antiepileptic drug indicated as monotherapy or adjunctive therapy in the treatment of partial seizures or generalized tonic–clonic seizures in adults and children. It undergoes rapid presystemic reduction with formation of the active metabolite 10‐hydroxycarbazepine (MHD), which has a chiral center at position 10, with the enantiomers (S)‐(+)‐ and R‐(?)‐MHD showing similar antiepileptic effects. This study presents the development and validation of a method of sequential analysis of oxcarbazepine and MHD enantiomers in plasma using liquid chromatography with tandem mass spectrometry (LC‐MS/MS). Aliquots of 100 μL of plasma were extracted with a mixture of methyl tert‐butyl ether: dichloromethane (2:1). The separation of oxcarbazepine and the MHD enantiomers was obtained on a chiral phase Chiralcel OD‐H column, using a mixture of hexane:ethanol:isopropanol (80:15:5, v/v/v) as mobile phase at a flow rate of 1.3 mL/min with a split ratio of 1:5, and quantification was performed by LC‐MS/MS. The limit of quantification was 12.5 ng oxcarbazepine and 31.25 ng of each MHD enantiomer/mL of plasma. The method was applied in the study of kinetic disposition of oxcarbazepine and the MHD enantiomers in the steady state after oral administration of 300 mg/12 h oxcarbazepine in a healthy volunteer. The maximum plasma concentration of oxcarbazepine was 1.2 µg/mL at 0.75 h. The kinetic disposition of MHD is enantioselective, with a higher proportion of the S‐(+)‐MHD enantiomer compared to R‐(?)‐MHD and an AUC^0‐12 _{S‐(+)/R‐(?)} ratio of 5.44. Chirality 25:897–903, 2013. © 2013 Wiley Periodicals, Inc.     

Determination of cyclophosphamide enantiomers in plasma by LC-MS/MS: Application to pharmacokinetics in breast cancer and lupus nephritis patients

This article describes the enantioselective analysis of cyclophosphamide (CPA) in human plasma using LC-MS/MS. CPA enantiomers were extracted from plasma using a mixture of ethyl acetate and chloroform (75:25, v/v). The enantiomers were separated on a Chiralcel(R) OD-R column, with the mobile phase consisting of a mixture of acetonitrile and water (75:25, v/v) plus 0.2% formic acid. The protonated ions and their respective product ions were monitored using two functions, 261 > 141 for CPA enantiomers and 189 > 104 for the internal standard (antipyrine). Recovery rates were higher than 95% and the quantification limit was 2.5-ng/ml plasma for both enantiomers. The coefficients of variation and the relative errors obtained for the validation of intra- and interassay precision and accuracy were less than 10%. The method was applied for the investigation of the enantioselective pharmacokinetics of CPA in a lupus nephritis patient treated with 1 g CPA infused over 2 h and in a breast cancer patient treated with 0.9 g infused over 1 h. No stereoselectivity in the pharmacokinetic parameters was observed for either patient. Clearance values of 2.63 and 2.93 l/h and of 3.36 and 3.61 l/h for (-)-(S) and (+)-(R)-CPA were obtained for the breast cancer and lupus nephritis patient, respectively.     

Pharmacokinetics of fexofenadine enantiomers in healthy subjects

Fexofenadine, a substrate of P-glycoprotein and an organic anion transporter polypeptide, is commonly used to assess P-glycoprotein activity in vivo. The purpose of this study was to elucidate the pharmacokinetics of each fexofenadine enantiomer. After a single oral dose of racemic fexofenadine (60 mg), the plasma and urine concentrations of fexofenadine enantiomers were measured over the course of 24 h in six healthy subjects. The mean plasma concentration of R(+)-fexofenadine was higher than that of S(-)-fexofenadine. The area under the plasma concentration-time curve (AUC(0-infinity)) and the maximum plasma concentration (C(max)) of R(+)-fexofenadine were significantly greater than those of the S(-)-enantiomer (P = 0.0018 and 0.0028, respectively). The R/S ratios of AUC and C(max) of fexofenadine were 1.75 and 1.63, respectively. The oral clearance and renal clearance of S(-)-fexofenadine were significantly greater than that of R(+)-fexofenadine (P = 0.0074 and 0.0036). On the other hand, the stereoselective metabolism of fexofenadine using recombinant CYP3A4 was investigated; however, fexofenadine enantiomers were not metabolized by CYP3A4. Fexofenadine is transported by both P-glycoprotein and OATP and is not metabolized by intestinal CYP3A. Our findings suggest that the affinity of P-glycoprotein for S(-)-fexofenadine is greater than its affinity for the R(+)-enantiomer. Thus, P-glycoprotein is likely to have chiral discriminatory abilities.     

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.     

Direct enantiospecific HPLC bioanalysis of (R,S)-atenolol on a chiral stationary phase

The simultaneous determination of the enantiomers of the β₁-selective adrenergic antagonist atenolol in human plasma and urine is described. After an alkaline preextraction atenolol is extracted from biological material at pH 12.3 using dichloromethane/propan-2-ol. The separation of the underivatized enantiomers is achieved by high-performance liquid chromatography on a chiral stationary phase (Chiralcel OD, cellulose tris-3, 5-dimethylphenylcarbamate, coated on silica gel) with fluorimetric detection. (?)-(S)-Pindolol is used as an internal standard. The detection limits of 5 ng/ml enantiomer in plasma and 50 ng/ml enantiomer in urine are sufficient for pharmacokinetic studies after therapeutic doses. © 1993 Wiley-Liss, Inc.     

Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of tetrahydropalmatine enantiomers in dogs

A selective chiral high performance liquid chromatographic (HPLC) method coupled with achiral column was developed and validated to separate and quantify tetrahydropalmatine (THP) enantiomers in dog plasma. Chromatography was accomplished by two steps: (1) racemic THP was separated from biological matrix and collected on a Kromasil C18 column (150 mmx4.6 mm, 5 microm) with the mobile phase acetonitrile-0.1% phosphoric acid solution, adjusted with triethylamine to pH 6.15 (47:53); (2) enantiomeric separation was performed on a Chiralcel OJ-H column (250 mmx4.6 mm, 5 microm) with the mobile phase anhydrous ethanol. The detection wavelength was set at 230 nm. (+)-THP and (-)-THP were separated with a resolution factor (Rs) of at least 1.6 and a separation factor (alpha) greater than 1.29. Linear calibration curves were obtained over the range of 0.025-4 microg/ml in plasma for each of (+)-THP and (-)-THP (R2>0.999) with a limit of detection (LOD) of 0.005 microg/ml and the recovery was greater than 88% for each enantiomer. The relative standard deviation (R.S.D.) and relative error values were less than 10% at upper and lower concentrations. The method was used to determine the pharmacokinetics of THP enantiomers after oral administration of racemic THP. The results presented herein showed the stereoselective disposition kinetics of THP in dogs and were a further contribution to the understanding of the kinetic behavior of THP analogues.     

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

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

Stereoselectivity in the placental transfer and kinetic disposition of racemic bupivacaine administered to parturients with or without a vasoconstrictor

The aim of the present study was to investigate the stereoselectivity in the kinetic disposition and the transplacental distribution of bupivacaine in term parturients during labor. Maternal age ranged from 18-37 years and fetal gestational age from 37.6-41.5 weeks. Healthy parturients (n = 23) received epidural 0.5% racemic bupivacaine alone (group A) or combined with epinephrine (group B). Maternal venous blood was sampled at regular intervals until 8 h after drug administration and umbilical venous blood was obtained at delivery. Bupivacaine enantiomers were determined in plasma samples by HPLC using a Chiralcel(R) OD-R column and a UV detector. One- or two-compartment models were fitted to data and differences between the (+)-(R) and (-)-(S) enantiomers were compared with the paired Wilcoxon test (P< 0.05). The influence of epinephrine was evaluated using the unpaired Mann-Whitney test (P< 0.05). The disposition of bupivacaine in maternal plasma was stereoselective, with higher V(d/f) (140.60 vs. 132.81 L for group A and 197.86 vs. 169.46 L for group B) and C(l/f) (29.00 vs. 25.43 L/h for group A and 33.15 vs. 26.39 L/h for group B) and lower t(1/2)beta (3.24 vs. 3.30 h for group A and 4.36 vs. 4.45 h for group B) being observed for (+)-(R)-bupivacaine. The combined administration of epinephrine resulted in higher V(d/f) (197.86 vs. 140.60 L for (+)-(R) and 169.46 vs. 132.81 L for (-)-(S)) and t(1/2)beta values (4.36 vs. 3.24 h for (+)-(R) and 4.45 vs. 3.30 h for (-)-(S)). The transplacental distribution of bupivacaine was stereoselective only when bupivacaine was administered without epinephrine (group B), with a higher cord blood/maternal blood ratio being observed for (-)-(S)-bupivacaine (0.40 vs. 0.35). Chirality 16:65-71, 2004.     

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.