Stereoselective and species-dependent kinetics of reboxetine in mouse and rat

Reboxetine, (RS)-2-[(RS)-α-(2-ethoxyphenoxy)benzyl]morpholine methanesulphonate, is a racemic compound and consists of a mixture of the (R,R)- and (S,S)-enantiomers. In this study, brain and plasma levels of both enantiomers were determined in mice and rats after oral administration of reboxetine at doses (1.1 mg/kg, mouse; 20 mg/kg, rat) twice the respective ED₅₀ values in the antireserpine test. Plasma and brain concentrations of each enantiomer were measured up to 6 h postdosing using an HPLC method with fluorimetric detection after derivatization with a chiral agent (FLEC). In mice and rats, brain and plasma levels of the (R,R)-enantiomer were always higher than those of the (S,S)-enantiomer. After normalization for dose, the mean AUC_0-tz values of both the (R,R)- and (S,S)-enantiomers in mouse brain were about 23 and 32 times higher than in rat brain, respectively. In plasma, the corrected mean AUC_0-tz values were about 5 (R,R) and 10 (S,S) times higher in mice than in rats. These results provide evidence for the higher bioavailability and/or lower clearance of both enantiomers in mice than in rats, and for a higher penetration of both enantiomers into mouse brain compared to rat brain. © 1995 Wiley-Liss, Inc.     

Enantioselective gallopamil protein binding

The protein binding of the enantiomers of gallopamil has been investigated in solutions of human serum albumin, α₁-acid glycoprotein and serum. Over the range of concentrations attained after oral gallopamil administration, the binding of both enantiomers to albumin, α₁-acid glycoprotein, and serum proteins was independent of gallopamil concentration. The binding to both human serum albumin (40 g/liter) [range of fraction bound (f_b) R: 0.624 to 0.699; S: 0.502 to 0.605] and α₁-acid glycoprotein (0.5 g/liter) (range of f_b R: 0.530 to 0.718; S: 0.502 to 0.620) was stereoselective, favoring the (R)-enantiomer (predialysis gallopamil concentrations 2.5 to 10,000 ng/ml). When the enantiomers (predialysis gallopamil concentration 10 ng/ml) were studied separately in drug-free serum samples from six healthy volunteers the fraction of (S)-gallopamil bound (f_b: 0.943 ± 0.016) was lower (P < 0.05) than that of (R)-gallopamil (f_b: 0.960 ± 0.010). The serum protein binding of both (R)- and (S)-gallopamil was unaffected by their optical antipodes (f_b R: 0.963 ± 0.011; S: 0.948 ± 0.015) indicating that at therapeutic concentrations a protein binding enantiomer–enantiomer interaction does not occur. The protein binding of (R)- and (S)-gallopamil ex vivo 2 h after single dose oral administration of 50 mg pseudoracemic gallopamil (f_b R: 0.960 ± 0.010: predialysis [R] 6.9 to 35.3 ng/ml; S: 0.943 ± 0.016: predialysis [S] 9.5 to 30.7 ng/ml) was comparable to that observed in vitro in drug-free serum. Gallopamil metabolites formed during first-pass following oral administration, therefore, do not influence the protein binding of (R)- or (S)-gallopamil. © 1993 Wiley-Liss, Inc.     

Stereoselective pharmacokinetics of methadone in beagle dogs

The pharmacokinetics of methadone were studied in beagle dogs (n = 4) following intravenous administration of the racemate (0.5 mg/kg) and of the individual (R)-(0.25 mg/kg) and (S)-enantiomers (0.25 mg/kg) using a stereospecific HPLC assay. There was no significant difference between the pharmacokinetic parameters of (R)-methadone and (S)-methadone following administration of the individual enantiomers. Stereoselective differences were evident following administration of the racemate (P values for differences in AUC and CL were 0.001 and 0.046, respectively) and the clearance of the (S)-enantiomer was increased when administered as part of the racemate (316 ± 81 vs 487 ± 128 ml/min, P = 0.04). The data suggest that stereoselective disposition including potential enantiomer–enantiomer interactions should be considered in pharmacokinetic–pharmacodynamic studies of (R,S)-methadone. © 1994 Wiley-Liss, Inc.     

Enantioselective Pharmacokinetics of Doxazosin and Pharmacokinetic Interaction Between the Isomers in Rats

In this study, the stereoselective pharmacokinetics of doxazosin enantiomers and their pharmacokinetic interaction were studied in rats. Enantiomer concentrations in plasma were measured using chiral high‐pressure liquid chromatography (HPLC) with fluorescence detection after oral or intravenous administration of (–)‐(R)‐doxazosin 3.0 mg/kg, (+)‐(S)‐doxazosin 3.0 mg/kg, and rac‐doxazosin 6.0 mg/kg. AUC values of (+)‐(S)‐doxazosin were always larger than those of (–)‐(R)‐doxazosin, regardless of oral or intravenous administration. The maximum plasma concentration (C_max) value of (–)‐(R)‐doxazosin after oral administration was significantly higher when given alone (110.5 ± 46.4 ng/mL) versus in racemate (53.2 ± 19.7 ng/mL), whereas the C_max value of (+)‐(S)‐doxazosin did not change significantly. The area under the curve (AUC) and C_max values for (+)‐(S)‐doxazosin after intravenous administration were significantly lower, and its Cl value significantly higher, when given alone versus in racemate. We speculate that (–)‐(R)‐doxazosin increases (+)‐(S)‐doxazosin exposure probably by inhibiting the elimination of (+)‐(S)‐doxazosin, and the enantiomers may be competitively absorbed from the gastrointestinal tract. In conclusion, doxazosin pharmacokinetics are substantially stereospecific and enantiomer–enantiomer interaction occurs after rac‐administration. Chirality 27:738–744, 2015. © 2015 Wiley Periodicals, Inc.     

Stereoselective analysis of ketorolac in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) analytical method is described for the quantification of the (R)- and (S)-enantiomers of ketorolac when present together in human plasma. The method involves derivatization with thionyl chloride/(S)-1-phenylethylamine and subsequent reversed-phase chromatography of the diastereomeric (S)-1-phenylethylamides of (R)- and (S)-ketorolac. The method is suitable for the analysis of large numbers of plasma samples and has been applied in this report to a pharmacokinetic study of ketorolac enantiomers upon intramuscular administration of racemic drug to a human subject. The limit of quantification for each enantiomer of ketorolac is 50 ng/ml (signal-to-noise ratio > 10). © 1993 Wiley-Liss, Inc.     

The disposition of venlafaxine enantiomers in dogs, rats, and humans receiving venlafaxine.

A stereospecific high-performance liquid chromatographic (HPLC) method was developed for the quantitation of the enantiomers of venlafaxine, an antidepressant, in dog, rat, and human plasma. The procedure involves derivatization of venlafaxine with the chiral reagent, (+)-S-naproxen chloride, and a postderivatization procedure. The method was linear in the range of 50 to 5,000 ng of each enantiomer per ml of plasma. No interference by endogenous substances or known metabolites of venlafaxine occurred. Studies to characterize the disposition of the enantiomers of venlafaxine were conducted in dog, rat, and human, following oral administration of venlafaxine. The Cmax, area under the curve (AUC) and (S)/(R) concentration ratios of the (R)- and (S)-enantiomers were compared. In rats, the mean plasma ratio of (S)-venlafaxine to that of (R)-venlafaxine over 0.5 to 6.0 h varied from 2.97 to 8.50 with a mean value of 5.51 +/- 2.45. The Cmax, AUC0-infinity, and t 1/2 values of the (R)- and (S)-enantiomers in dogs were not significantly different from one another (P greater than 0.1). The mean ratios [(S)/(R)] of enantiomers of venlafaxine in human over a 2 to 6 h interval ranged from 1.33 to 1.35 with an overall ratio of 1.34 +/- 0.26 (n = 12). These ratios of the enantiomers [(S)/(R)] were not statistically different from unity (P greater than 0.1) indicating that the disposition of venlafaxine enantiomers in humans is not stereoselective and is more similar to that in dogs than that in rats.     

Stereoselective analysis of hydroxybupropion and application to drug interaction studies

A sensitive and stereoselective assay has been developed for the quantitation of the enantiomers of hydroxybupropion, an active metabolite of bupropion, in human plasma. The assay used liquid-liquid extraction and a Cyclobond I 2000 HPLC column with a mobile phase containing 3% acetonitrile, 0.5% triethylamine, and 20 mM ammonium acetate (pH 3.8). The technique was linear over the concentration range of 12.5-500 ng/ml for (2R,3R)- and (2S,3S)-hydroxybupropion. The method was reproducible as both interday and intraday variabilities were less than 10% for both hydroxybupropion enantiomers. Overall extraction recovery of hydroxybupropion enantiomers and the internal standard phenacetin from plasma was greater than 80% and reproducible over the concentration range of 12.5-500 ng/ml for each enantiomer. The limit of quantification (LOQ) of hydroxybupropion enantiomers was 12.5 ng/ml. The stereoselective pharmacokinetics of both (2R,3R)- and (2S,3S)-hydroxybupropion in healthy male subjects (n = 16) were investigated after a single dose of (rac)-bupropion either alone or during rifampicin administration. (2R,3R)-Hydroxybupropion was the predominant enantiomer present in plasma. A stereoselective effect of rifampicin on hydroxybupropion concentrations was observed, with rifampicin influencing the pharmacokinetics of each hydroxybupropion enantiomer in a different manner. The ratio of (2R,3R)-hydroxybupropion (AUC(0-24)) to (2S,3S)-hydroxybupropion (AUC(0-24)) increased from 4.9 +/- 1.6 to 8.3 +/- 1.9 during rifampicin administration (P < 0.001). A time-dependent change in the hydroxybupropion enantiomeric ratio was observed after (rac)-bupropion administration both before and during rifampicin coadministration, with an increase in the relative proportion of (2S,3S)-hydroxybupropion over the 24 h postdose period.     

Stereoselective Degradation of alpha‐Cypermethrin and Its Enantiomers in Rat Liver Microsomes

Alpha‐cypermethrin (α‐CP), [(RS)‐a‐cyano‐3‐phenoxy benzyl (1RS)‐cis‐3‐(2, 2‐dichlorovinyl)‐2, 2‐dimethylcyclopropanecarboxylate], comprises a diastereoisomer pair of cypermethrin, which are (+)‐(1R‐cis‐αS)–CP (insecticidal) and (?)‐(1S‐cis‐αR)–CP (inactive). In this experiment, the stereoselective degradation of α‐CP was investigated in rat liver microsomes by high‐performance liquid chromatography (HPLC) with a cellulose‐tris‐ (3, 5‐dimethylphenylcarbamate)‐based chiral stationary phase. The results revealed that the degradation of (?)‐(1S‐cis‐αR)‐CP was much faster than (+)‐(1R‐cis‐αS)‐CP both in enantiomer monomers and rac‐α‐CP. As for the enzyme kinetic parameters, there were some variances between rac‐α‐CP and the enantiomer monomers. In rac‐α‐CP, the V_max and CL_int of (+)‐(1R‐cis‐αS)–CP (5105.22 ± 326.26 nM/min/mg protein and 189.64 mL/min/mg protein) were about one‐half of those of (?)‐(1S‐cis‐αR)–CP (9308.57 ± 772.24 nM/min/mg protein and 352.19 mL/min/mg protein), while the K_m of the two α‐CP enantiomers were similar. However, in the enantiomer monomers of α‐CP, the V_max and K_m of (+)‐(1R‐cis‐αS) ‐CP were 2‐fold and 5‐fold of (?)‐(1S‐cis‐αR)‐CP, respectively, which showed a significant difference with rac‐α‐CP. The CL_int of (+)‐(1R‐cis‐αS)–CP (140.97 mL/min/mg protein) was still about one‐half of (?)‐(1S‐cis‐αR)–CP (325.72 mL/min/mg protein) in enantiomer monomers. The interaction of enantiomers of α‐CP in rat liver microsomes was researched and the results showed that there were different interactions between the IC₅₀ of (?)‐ to (+)‐(1R‐cis‐αS)‐CP and (+)‐ to (?)‐(1S‐cis‐αR)‐CP(IC_50(?)/(+) / IC_50(+)/(?) = 0.61). Chirality 28:58–64, 2016. © 2015 Wiley Periodicals, Inc.     

Disposition and absorption of hydroxychloroquine enantiomers following a single dose of the racemate

The disposition of hydroxychloroquine enantiomers has been investigated in nine patients with rheumatoid arthritis following administration of a single dose of the racemate. Blood concentrations of (?)-(R)-hydroxychloroquine exceed those of (+)-(S)-hydroxychloroquine following both an oral and intravenous dose of the racemate. Maximum blood concentrations of (?)-(R)-hydroxychloroquine were higher than (+)-(S) -hydroxychloroquine after oral dosing (121 ± 56 and 99 ± 42 ng/ml, respectively, P = 0.009). The time to maximum concentration and the absorption half-life, calculated using deconvolution techniques, were similar for both enantiomers. The fractions of the dose of each enantiomer absorbed were similar, 0.74 and 0.77 for (?)-(R)- and (+)-(S)-hydroxychloroquine, respectively (P = 0.77). The data suggest that absorption of hydroxychloroquine is not enantioselective. The stereoselective disposition of hydroxychloroquine appears to be due to enantioselective metabolism and renal clearance, rather than stereoselectivity in absorption and distribution. © 1994 Wiley-Liss, Inc.     

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.     

Preliminary pharmacokinetic study of ibuprofen enantiomers after administration of a new oral formulation (ibuprofen arginine) to healthy male volunteers

The pharmacokinetics of ibuprofen enantiomers were investigated in a crossover study in which seven healthy male volunteers received single oral doses of 800 mg racemic ibuprofen as a soluble granular formulation (sachet) containing L-arginine (designated trade name: Spedifen®), 400 mg (-)R-ibuprofen arginine or 400 mg (+)S-ibuprofen arginine. Plasma levels of both enantiomers were monitored up to 480 minutes after drug intake using an enantioselective analytical method (HPLC with ultraviolet detection) with a quantitation limit of 0.25 mg/l. Substantial inter-subject variability in the evaluated pharmacokinetic parameters was observed in the present study. After (+)S-ibuprofen arginine, the following mean pharmacokinetic parameters ±SD were calculated for (+)S-ibuprofen: t_max 28.6 ± 28.4 min; C_max 36.2 ± 7.7 mg/l; AUC 86.4 ± 14.9 mg · h/l; t½ 105.2 ± 20.4 min. After (-)R-ibuprofen arginine, the following mean pharmacokinetic parameters were calculated for (+)S-ibuprofen and (-)R-ibuprofen, respectively: t_max 90.0 ± 17.3 and 50.5 ± 20.5 min; C_max 9.7 ± 3.0 and 35.3 ± 5.0 mg/l; AUC 47.0 ± 17.2 and 104.7 ± 27.7 mg · h/l; t_½ 148.1 ± 63.6 and 97.7 ± 23.3 min. After racemic ibuprofen arginine, the following mean pharmacokinetic parameters were calculated for (+)S- and (-)R-ibuprofen, respectively: t_max 30.7 ± 29.1 and 22.9 ± 29.8 min.; C_max 29.9 ± 5.6 and 25.6 ± 4.4 mg/l; AUC 105.1 ± 23.0 and 65.3 ± 15.0 mg · h/l; t_½ 136.6 ± 20.7 and 128.6 ± 45.0 min. T_max values of S(+)- and (-)R-ibuprofen after a single dose of 400 mg of each enantiomer did not differ significantly from the corresponding parameters obtained after a single dose of 800 mg of racemic ibuprofen arginine, indicating that the absorption rate of (-)R- and (+)S-ibuprofen is not different when the two enantiomers are administered alone or as a racemic compound. An average of 49.3 ± 9.0% of a dose of the (-)R-ibuprofen arginine was bioinverted into its antipode during the study period (480 minutes post-dosing). The percent bioinversion during the first 30 minutes after (-)R-ibuprofen arginine intake averaged 8.1 ± 3.9%. The mean AUC of (+)S-ibuprofen calculated after 800 mg racemic ibuprofen arginine (105.1 ± 23.0 mg · h/l) was lower than the mean AUC value obtained by summing the AUCs of (+)S-ibuprofen after administration of 400 mg (+)S-ibuprofen arginine and 400 mg (-)R-ibuprofen arginine (133.4 ± 26.6 mg · h/l). In conclusion, the administration of Spedifen® resulted in very rapid absorption of the (+)S-isomer (eutomer) with t_max values much lower than those observed for this isomer when conventional oral solid formulations such as capsules or tablets of racemic ibuprofen are administered. This characteristic is particularly favourable in those conditions in which a very rapid analgesic effect is required. Chirality 9:297–302, 1997. © 1997 Wiley-Liss, Inc.     

Steady-state pharmacokinetics of the enantiomers of citalopram and its metabolites in humans

The steady-state pharmacokinetics in serum and urine of the enantiomers of citalopram and its metabolites, demethylcitalopram (DCT) and didemethylcitalopram (DDCT), were investigated after multiple doses of rac-citalopram for 21 consecutive days (40 mg per day) to healthy human subjects who were extensive metabolisers of sparteine and mephenytoin. Comparable pharmacokinetic variability was noted for (+)-(S)-, (−)-(R)- and rac-citalopram. Enantiomeric (S/R) serum concentration ratios for citalopram were always less than unity and were constant during the steady-state dosing interval. A modest, but statistically significant, stereoselectivity in the disposition of citalopram and its two main metabolites was observed. Serum levels of the (+)-(S)-enantiomers of citalopram, DCT, and DDCT throughout the steady-state dosing interval investigated were 37 ± 6%, 42 ± 3% and 32 ± 3%, respectively, of their total racemic serum concentrations. The (+)-(S)-enantiomers of citalopram, DCT, and DDCT were eliminated faster than their antipodes. For (−)-(R)- and (+)-(S)-citalopram, respectively, the serum t½ averaged 47 ± 11 and 35 ± 4 h and AUC_ss averaged 4,193 ± 1,118 h · nmol/l and 2,562 ± 1,190 h · nmol/l. The observed enantiospecificities were apparently more related to clearance, rather than to distributional mechanisms. Chirality 9:686–692, 1997. © 1997 Wiley-Liss, Inc.     

Pharmacokinetics and pharmacodynamics of ketoprofen enantiomers in calves

The pharmacokinetics (PK) and pharmacodynamics (PD) of (S)- and (R)- ketoprofen (KTP) enantiomers were studied in calves after intravenous administration of each enantiomer at a dose of 1.5 mg/kg. Pharmacodynamic properties were evaluated using a model of acute inflammation, comprising subcutaneously implanted tissue cages stimulated by intracaveal injection of carrageenan. Chiral inversion of (R)-KTP to the (S)-antipode occurred. The R:S ratio in plasma was 33:15 min after administration, decreasing to 1:1 at 8 h. The calculated extent of inversion was 31 ± 7%. The R:S ratio in inflammatory exudate was of the order 3:1 at all the sampling times and the ratio in transudate was approximately 2:1 for 6 h, declining to 1:1 at 30 h. Only (S)-KTP was detected in biological fluids after administration of this enantiomer. Elimination half-life was longer for the (S) (2.19 h) than the (R)-enantiomer (1.30 h) and volume of distribution was also somewhat higher for the (S)-enantiomer. Body clearance values were 0.119 1/kg/h for (S)-KTP and 0.151 1/kg/h for the (R)-antipode. For (R)-KTP effects obtained were considered as a hybrid, since they potentially reflect the actions of both enantiomers. Concentrations of LTB₄ and the cytokines interleukin-1, interleukin-6, and tumor necrosis factor alpha, in exudate were not significantly affected by either (R)- or (S)-KTP treatments. Inhibition of ex vivo thromboxane B₂ (TxB₂) synthesis, exudate prostaglandin E₂ (PGE₂) synthesis, β-glucuronidase release (β-glu), and bradykinin-induced skin swelling was significant in both treated groups. PK/PD modelling was applied to the (S)-KTP treatment only. EC₅₀ values for inhibition of serum TxB₂, exudate PGE₂ and β-glu and BK-induced swelling were 0.047, 0.042, 0.101, and 0.038 μg/ml, respectively. It is concluded that the low EC₅₀ values for inhibition of TxB₂ and PGE₂ by (S)-KTP are likely to explain the effects produced by (R)-KTP administration, since concentrations of (S)-KTP in exudate of these calves following chiral inversion were at least 5 times higher than the EC₅₀ at all sampling times. The data for β-glu and bradykinin-induced swelling inhibition indicate possible inhibitory actions of (R)-KTP as well as (S)-KTP. © 1995 Wiley-Liss, Inc.     

Stereoselective disposition of tiaprofenic acid enantiomers in rats

The pharmacokinetics and metabolic chiral inversion of the S(+)‐ and R(−)‐enantiomers of tiaprofenic acid (S‐TIA, R‐TIA) were assessed in vivo in rats, and in addition the biochemistry of inversion was investigated in vitro in rat liver homogenates. Drug enantiomer concentrations in plasma were investigated following administration of S‐TIA and R‐TIA (i.p. 3 and 9 mg/kg) over 24 hr. Plasma concentrations of TIA enantiomers were determined by stereospecific HPLC analysis. After administration of R‐TIA it was found that 1) there was a time delay of peak S‐TIA plasma concentrations, 2) S‐TIA concentrations exceeded R‐TIA concentrations from ∼2 hr after dosing, 3) C_max and AUC_(0‐∞) for S‐TIA were greater than for R‐TIA following administration of S‐TIA, and 4) inversion was bidirectional but favored inversion of R‐TIA to S‐TIA. Bidirectional inversion was also observed when TIA enantiomers were incubated with liver homogenates up to 24 hr. However, the rate of inversion favored transformation of the R‐enantiomer to the S‐enantiomer. In conclusion, stereoselective pharmacokinetics of R‐ and S‐TIA were observed in rats and bidirectional inversion in rat liver homogenates has been demonstrated for the first time. Chiral inversion of TIA may involve metabolic routes different from those associated with inversion of other 2‐arylpropionic acids such as ibuprofen. Chirality 11:103–108, 1999. © 1999 Wiley‐Liss, Inc.     

Determination of the enantiomers of mianserin,desmethylmianserin, and 8-hydroxymianserin in the plasma and urine of mianserin-treated patients

An HPLC method is presented which allows the measurement in the same run of the enantiomers of mianserin, desmethylmianserin, and 8-hydroxymianserin in plasma and urine of mianserin-treated patients. Limits of quantitation for the (S)- and (R)- enantiomers of mianserin and desmethylmianserin were 4 and 2.5 ng/ml, respectively, in plasma, and for the (S)- and (R)-enantiomers of mianserin, desmethylmianserin, and 8-hydroxymianserin 5, 2.5, and 5 ng/ml, respectively, in urine. The measured ratios of (S)-mianserin/(R)-mianserin and (S)-desmethylmianserin/(R)-desmethylmianserin in the plasmas of 10 mianserin-treated patients, all extensive metabolizers of debrisoquine as determined by CYP2D6 genotyping, varied, respectively, from 1.0 to 4.06 and from 0.19 to 0.64. As the enantiomers of mianserin differ in their pharmacological profile, these results could partially explain why, until now, no consistent relationship has been established between the therapeutic response and total [(S) + (R)] plasma levels of this antidepressant. © 1994 Wiley-Liss, Inc.     

Simple and sensitive liquid chromatography-tandem mass spectrometry method for determination of the S(+)- and R(-)-enantiomers of baclofen in human plasma and cerebrospinal fluid

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method to determine the enantiomers of the muscle relaxant baclofen in human plasma and cerebrospinal fluid (CSF) has been developed. A commercially available ultrafiltration membrane is used to prepare the sample. A chiral CROWNPAK CR(+) stationary phase column is then used to perform complete resolution of the S(+)- and R(-)-enantiomers of baclofen. This method was used to analyze human plasma and CSF spiked with baclofen, and the calibration curves for both biologic samples were linear over a concentration range of 0.15-150 ng enantiomer/ml. The lower limit of quantification was 0.15 ng enantiomer/ml in both fluids. Finally, the method was tested with an artificial CSF as an alternative to authentic human CSF. The results showed that no matrix effects and no interfering peaks were observed using this artificial CSF.     

Separation and quantitation of (R)- and (S)-atenolol in human plasma and urine using an alpha 1-AGP column

A method for the determination of (R)- and (S)-atenolol in human plasma and urine is described. The enantiomers of atenolol are extracted into dichloromethane containing 3% heptafluorobutanol followed by acetylation with acetic anhydride at 60 degrees C for 2 h. The acetylated enantiomers were separated on a chiral alpha 1-AGP column. Quantitation was performed using fluorescence detection. A phosphate buffer pH 7.1 (0.01 M phosphate) containing 0.25% (v/v) acetonitrile was used as mobile phase. The described procedure allows the detection of less than 6 ng of each enantiomer in 1 ml plasma. The relative standard deviation is 4.4% at 30 ng/ml of each enantiomer in plasma. The plasma concentration of (R)- and (S)-atenolol did not differ significantly in two subjects who received a single tablet of racemic atenolol. The R/S ratio of atenolol in urine was approximately 1.     

Enantiomeric bioanalysis of simendan and levosimendan by chiral high-performance liquid chromatography

rac-Simendan, (±)-(R, S)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)-phenyl]hydrazono]propanedinitrile, and the levorotatory enantiomer levosimendan, are drug candidates intended for the treatment of congestive heart failure. An enantiospecific high-performance liquid chromatographic (HPLC) method suitable for determination of the ratio of the enantiomer concentrations in blood plasma samples was developed. Direct resolution of the enantiomers was achieved by using a chiral β-cyclodextrin stationary phase in reversed phase mode. With an eluent containing 24–33% of methanol in a 0.5% (v/v) triethylammonium acetate buffer, pH 6.0, and a flow rate of 1 ml/min, a resolution (1.2–1.6) adequate for the determinations was achieved. By using UV detection, the relative concentration of the enantiomers in plasma was assessed down to 10 ng/ml. For the racemate, the results indicated a slightly enantioselective disposition and plasma protein binding in rat, dog, and man. The pure enantiomer, levosimendan, was found not to isomerize in vivo. © 1996 Wiley-Liss, Inc.     

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.     

AMPA receptor agonists: Resolution,configurational assignment,and pharmacology of (+)-(S)- and (-)-(R)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-isoxazol-4-yl]-propionic acid (2-Py-AMPA)

We have previously shown that whereas (RS)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid (APPA) shows the characteristics of a partial agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, (S)-APPA is a full AMPA receptor agonist and (R)-APPA a weak competitive AMPA receptor antagonist. This observation led us to introduce the new pharmacological concept, functional partial agonism. Recently we have shown that the 2-pyridyl analogue of APPA, (RS)-2-amino-3-[3-hydroxy-5-(2-pyridyl)isoxazol-4-yl]propionic acid (2-Py-AMPA), is a potent and apparently full AMPA receptor agonist, and this compound has now been resolved into (+)- and (-)-2-Py-AMPA (ee ≥ 99.0%) by chiral HPLC using a Chirobiotic T column. The absolute stereochemistry of the enantiomers of APPA has previously been established by X-ray analysis, and on the basis of comparative studies of the circular dichroism spectra of the enantiomers of APPA and 2-Py-AMPA, (+)- and (-)-2-Py-AMPA were assigned the (S)- and (R)-configuration, respectively. In a series of receptor binding studies, neither enantiomer of 2-Py-AMPA showed detectable affinity for kainic acid receptor sites or different sites at the N-methyl-D-aspartic acid (NMDA) receptor complex. (+)-(S)-2-Py-AMPA was an effective inhibitor of [³H]AMPA binding (IC⁵⁰ = 0.19 ± 0.06 μM) and a potent AMPA receptor agonist in the rat cortical wedge preparation (EC₅₀ = 4.5 ± 0.3 μM) comparable with AMPA (IC₅₀ = 0.040 ± 0.01 μM; EC₅₀ = 3.5 ± 0.2 μM), but much more potent than (+)-(S)-APPA (IC₅₀ = 5.5 ± 2.2 μM; EC₅₀ = 230 ± 12 μM). Like (-)-(R)-APPA (IC₅₀ > 100 μM), (-)-(R)-2-Py-AMPA (IC₅₀ > 100 μM) did not significantly affect [³H]AMPA binding, and both compounds were week AMPA receptor antagonists (K_i = 270 ± 50 and 290 ± 20 μM, respectively). Chirality 9:274–280, 1997. © 1997 Wiley-Liss, Inc.