Determination of pindolol enantiomers in amniotic fluid and breast milk by high-performance liquid chromatography: applications to pharmacokinetics in pregnant and lactating women

A method for the determination of pindolol enantiomers in amniotic fluid and breast milk was developed, validated, and applied to the investigation of six pregnant women treated with rac-pindolol (10 mg/12 h). Biological samples were extracted with tert-methyl-butyl ether, and the pindolol enantiomers were resolved on a Chiralpak AD column. Amniotic fluid/plasma and milk/plasma concentrations ratios ranged from 0.4 to 4.5 and from 0.6 to 3.7, respectively, for (+)-R-pindolol and from 0.5 to 3.5 and from 1.1 to 2.8, respectively, for (-)-S-pindolol. Preliminary data suggest that amniotic fluid and breast milk are routes of fetal exposure to pindolol enantiomers.     

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.     

Nonlinear stereoselective pharmacokinetics of ketoconazole in rat after administration of racemate

The stereoselective pharmacokinetics of ketoconazole (KTZ) enantiomers were studied in rat after i.v. and oral administration of (+/-)-KTZ. Sprague-Dawley rats were administered racemic KTZ as 10 mg/kg i.v. or orally over the range 10-80 mg/kg as single doses. Serial blood samples were collected over a 24-h period via surgically placed jugular vein cannulae. Plasma was assayed for KTZ enantiomer concentrations using stereospecific HPLC. Enantiomeric plasma protein binding was determined using an erythrocyte partitioning method at racemic concentrations of 10 and 40 mg/L. Stereoselective metabolism was tested by incubating the racemate (0.5-250 microM) with rat liver microsomes. In all rats, (+)-KTZ plasma concentrations were higher (up to 2.5-fold) than (-)-KTZ. The clearance and volume of distribution of the (-) enantiomer were approximately twofold higher than antipode. Half-life did not differ between the enantiomers. After oral doses the t(max) was not stereoselective. For both enantiomers with higher doses the respective half-life were found to increase. The mean unbound fraction of the (-) enantiomer was found to be up to threefold higher than that of the (+) enantiomer. At higher concentrations nonlinearity in plasma protein binding was observed for both enantiomers. There was no evidence of stereoselective metabolism by liver microsomes. Stereoselectivity in KTZ pharmacokinetics is attributable to plasma protein binding, although other processes such as transport or intestinal metabolism may also contribute.     

Stereospecific disposition of fluvastatin in streptozotocin-induced diabetic rats

The study reports on the stereoselective pharmacokinetics of fluvastatin, a racemic mixture of (-)-(3S,5R)- and (+)-(3R,5S)-enantiomers, in streptozotocin-induced diabetic rats. Wistar (control) and streptozotocin-induced diabetic rats (n = 6/time point) received by oral gavage racemic fluvastatin (5 mg/kg), and blood samples were collected until 24 h. The enantiomers were analysed by chiral HPLC with fluorescence detection. The pharmacokinetic parameters were analysed by Wilcoxon and Mann-Whitney tests. The results are reported as means (95% CI). The following differences (p < 0.05) were observed between the control and diabetic groups, respectively: maximum plasma concentration (Cmax) of (-)-(3S,5R), 410.0 (310.0-510.0) versus 532.6 (463.5-601.8) ng x mL(-7); area under the plasma concentration versus time curve (AUC(0-infinity)) for (-)-(3S,5R), 4342A (3,775.7-4,909.0) versus 3025.2 (2,218.9-3,831.5) ng x h x mL(-1); apparent total clearance (Cl/f) of (-)-(3S,5R), 0.6 (0.5-0.7) versus 0.9 (0.6-1.1) L x h(-1) x kg(-1); AUC(0-infinity) for (+)-(3R,5S), 493.5 (376.9-610.1) versus 758.5 (537.1-980.0) ng x h x mL(-1); and Cl/f of (+)-(3R,5S), 5.3 (3.9-6.8) versus 3.5 (2.6-4.4) L x h(-1) x kg(-1). Streptozotocin-induced diabetes in rats alters the pharmacokinetics of fluvastatin in a stereoselective manner.     

Stereoselective pharmacokinetics of clausenamide enantiomers and their major metabolites after single intravenous and oral administration to rats

The pharmacokinetics of clausenamide (CLA) enantiomers and their metabolites were investigated in Wistar rat. After intravenous and oral administration at a dose of 80 and 160 mg/kg each enantiomer, plasma concentrations of (-)- or (+)-CLA and its major metabolites were simultaneously determined by reverse-phase HPLC with UV detection. Notably, stereoselective differences in pharmacokinetics were found. The mean plasma levels of (+)-CLA were higher at almost all time points than those of (-)-CLA. (+)-CLA also exhibited greater t(max), C(max), t(1/2beta), AUC(0-12h), and AUC(0--> infinity) and smaller CL (or CL/F) and V(d) (or V(d)/F), than its antipode. The (+)/(-) isomer ratios for t(1/2beta), t(max), AUC(0-12 h), and AUC(0--> infinity), which ranged from 1.26 to 2.08. The ratio for CL (or CL/F) was about 0.5, and there were significant differences in these values between CLA enantiomers (P < 0.05), implying that the absorption, distribution, and elimination of (-)-CLA were more rapid than those of (+)-CLA. Similar findings for (-)-7-OH-CLA, the major metabolite of (-)-CLA, and (+)-4-OH-CLA, the major metabolite of (+)-CLA, can be also seen in rat plasma. The contributing factors for the differences in stereoselective pharmacokinetics of CLA enantiomers appeared to be involved in their different plasma protein binding, first-pass metabolism and interaction with CYP enzymes, especially with their metabolizing enzyme CYP 3A isoforms.     

Enantioselective kinetic disposition of albendazole sulfoxide in patients with neurocysticercosis

The enantioselectivity of the kinetic disposition of albendazole sulfoxide (ASOX) was investigated in 18 patients with neurocysticercosis treated with a multiple dose regimen of albendazole for 8 days (5 mg/kg every 8 h). Serial blood samples were collected on the eighth day of treatment during the last dose interval, with prorogation up to 12 h. Albendazole sulfone (ASON) and enantiomers of ASOX were analyzed in plasma samples by HPLC using a Chiralpak AD column and detection by fluorescence. The pharmacokinetic parameters showing statistically significant differences between the (+) ASOX and (-) ASOX enantiomers are presented as respective means (95% CI) as follows: maximum plasma concentration, Cmax = 301.6 (179.7-423.5) vs 54.9 (21.9-87.9) ng.ml-1; elimination half-life, t1/2 = 5.2 (4.1-6.3) vs 3.3 (2.8-3.8) h, area under the plasma concentration-time curve, AUCss0-8 = 1719.2 (978.6-2459.8) vs 261.4 (102.9-419.8) ng.h.ml-1 and apparent clearance, Cl/fm = 5.8 (3.8-7.8) vs 54.0 (35.2-72.7) l.h-1.kg-1. The mean value of 9.2 (7.6-10.9) for the AUC0-8(+)-ASOX/AUC0-8(-)-ASOX ratio demonstrated plasma accumulation of the (+) enantiomer. Sulfone formation capacity, expressed by the AUCss0-8 ratio ASON/ASOX + ASON, was 8.0 (7.0-8.9). The present data indicate enantioselectivity in the kinetic disposition of ASOX in patients with neurocysticercosis.     

Enantioselective renal excretion of albendazole metabolites in patients with neurocysticercosis

The present study investigates the urinary excretion of the enantiomers of (+)- and (-)-albendazole sulfoxide (ASOX) and albendazole sulfone (ASON) in 12 patients with neurocysticercosis treated with albendazole for 8 days (7.5 mg/kg/12 h). Serial blood samples (0-12 h) and urine (three periods of 8 h) were collected after administration of the last dose of albendazole. Plasma and urine (+)-ASOX, (-)-ASOX, and ASON metabolites were determined by HPLC using a chiral phase column (Chiralpak AD) with fluorescence detection. The pharmacokinetic parameters (P < 0.05) for (+)-ASOX, (-)-ASOX, and ASON metabolites are reported as means (95% CI); amount excreted (Ae) = 3.19 (1.53-4.85) vs. 0.72 (0.41-1.04) vs. 0.08 (0.03-0.13) mg; plasma concentration-time area under the curve, AUC(0-24) = 3.56 (0.93-6.18) vs. 0.60 (0.12-1.08) vs. 0.38 (0.20-0.55) microg x h/ml, and renal clearance Cl(R) = 1.20 (0.66-1.73) vs. 2.72 (0.39-5.05) vs. 0.25 (0.13-0.37) l/h. Sulfone formation capacity, expressed as the Ae ratio ASON/ASOX + ASON, was 2.21 (1.43-2.99). These data point to enantioselectivity in the renal excretion of ASOX as a complementary mechanism to the metabolism responsible for the plasma accumulation of (+)-ASOX. The results also suggest that the metabolite ASON is partially eliminated as a reaction product of the subsequent metabolism.     

Stereoselective plasma protein binding and target tissue distribution of clausenamide enantiomers in rats

Stereoselective differences in pharmacokinetics between clausenamide (CLA) enantiomers have been found after intravenous and oral administration of each enantiomer to rats. The differences could be associated with protein binding of CLA enantiomers. By equilibrium dialysis methods, the binding of CLA enantiomers to rat plasma protein was investigated. The results showed that mean percentages of (-) and (+)CLA in the bound form were 28.5% and 38.0%, respectively, indicating that the unbound fraction of (-)CLA was higher than that of (+)CLA, which provided an explanation for stereoselective pharmacokinetics of CLA enantiomers in rats. The results also showed that there were species differences in plasma protein binding of (-)-isomer between rats (28.5%) and rabbits (47.2%). Furthermore, effects of plasma protein binding on the distribution of CLA enantiomers to their possible target tissues were observed. The amount of (-)CLA in brain was greater than that of (+)CLA 15 min after administration of each enantiomer to rats. But the results were reverse at 4 h postdose. Further studies in distributional kinetics showed that (-)CLA had a more rapid absorption and distribution to hippocampus, cortex, and cerebellum than (+) CLA. (+)CLA had greater values for T(max), t(1/2) (beta), and AUC(0) (-->infinity), and smaller ones for CL/F and V(d)/F than its antipode. The data indicated that the distribution of (-) and (+)CLA in their target tissues was stereoselective. The stereoselective distribution might be involved in the metabolism and transport of two enantiomers in the central nerve system.     

Stereoselective toxicokinetics and tissue distribution of ethofumesate in rabbits

The stereoselective toxicokinetics of ethofumesate enantiomers following a single intravenous (i.v.) administration at doses of 30 mg/kg were investigated in rabbits. Plasma concentrations of (+)- and (-)-ethofumesate were analyzed by a validated chiral HPLC method that involved extraction of plasma with organic solvent followed by separation on a cellulose-Tris-(3,5-dimethylphenylcarbamate)-based chiral column and quantification by UV absorbance at 230 nm. Plasma concentration-time curves after i.v. administration were best described by an open two-compartment model. The concentration of the (-)-enantiomer decreased more rapidly than that of the (+)-enantiomer. Significant differences in toxicokinetic parameters between the two enantiomers indicated that stereoselective behavior occurred with the (-)-enantiomer being preferentially metabolized and eliminated.     

In vitro release and stereoselective disposition of flurbiprofen loaded to poly(D,L-lactide- co-glycolide) nanoparticles in rats

Flurbiprofen (FL) is a chiral 2-arylpropionate used clinically as the racemate (rac-FL). This study was undertaken to investigate the influence of sustained release formulation on the pharmacokinetics of flurbiprofen enantiomers (-) -R-FL and (+)-S-FL. Therefore, a stereoselective high-performance liquid chromatographic (HPLC) method was developed and validated for the rapid, quantitative determination of (-)-R-FL and (+)-S-FL in rat plasma. Flurbiprofen-loaded poly(D,L-lactide-co-glycolide) nanoparticles (rac-FL-PLGA) were prepared by in emulsion-solvent evaporation technique. Optimum conditions for rac-FL-PLGA nanoparticle preparation were considered, and the in vitro release of rac-FL, R-FL, and S-FL were followed up to 48 h in phosphate buffer (pH 7.4). The three tested formulations revealed approximately zero-order release of either (-)-R-FL or S-FL up to 24 h with r >/= 0.97.Surprisingly, there was no significant difference between t(50%) of the three formulations (21.6 +/- 1.1 h). The stereoselective disposition of the sustained release rac-FL deliverv system was investigated in rats. There was a rapid release of R-FL, S-FL, or rac-FL followed by a slower one and C(max) values were observed after 2.5 +/- 2.5, 8.3 +/- 3.4 and 8.86 +/- 3.6 h of (-)-R-FL, (+)-S-FL, and rac-FL, respectively, after nanoparticle administration. PLGA nanoparticles increased the mean retention time (MRT) of S-FL by 2.7-fold, from 6.8 to 16.3 h, compared to rac-FL. Although the dose of rac-FL-PLGA nanoparticles was only 2.5 times higher than that of the drug in the suspension, the mean (+)-S-FL concentration after 12 h was 3.4 times higher in the case of nanoparticles than after the free form, 10.35 +/- 1.6 and 3.04 +/- 1.1 mg/l, respectively. The area under the concentration-time curve (AUC) values of (+)-S-FL and rac-FL were about 2.5-fold higher after the nanoparticles compared to suspension, while the AUC of the (-)-R-FL was about 3.5 times higher. This difference may indicate that the two enantiomers have different absorption kinetics. The present study provides evidence that the sorption of racemic flurbiprofen to PLGA nanoparticles was successful in maintaining (at least up to 12 h) elevated plasma drug concentrations of (+)-S-FL in rats. Chirality 16:119-125, 2004.     

Pharmacokinetics of (R)- and (S)-cyclophosphamide and their dechloroethylated metabolites in cancer patients

The complete pharmacokinetics (PK) of (R)- and (S)-cyclophosphamide (CP) and their dechloroethylated (DCE) metabolites have not been reported to date. We collected plasma and urine samples from 12 cancer patients and determined concentrations of both enantiomers of CP and DCE-CP using a chiral GC-MS method. All concentrations of (R)-CP, (S)-CP, (R)-DCE-CP, and (S)-DCE-CP were simultaneously modeled using an enantiospecific compartmental PK model. A population PK analysis was performed. Enantiospecific differences between (R)- and (S)-CP were found for the formation clearance of CP to the DCE metabolites (Clf: 0.25 (R) vs. 0.14 (S) L/h). No difference was found between enantiomers for Cl40H, Cld, Cl(m)R, ClT, or T1/2. In contrast to the adolescent and adult group of patients, a child (6 years old) appeared to have a very different PK and metabolic profile (Bayesian control analysis). Proportions of the (R,S)-CP doses transformed to the (R)-DCE- and (S)-DCE-CP were much higher (R: 25 vs. 1.9%, and S: 38 vs. 3.6%), while formation of active metabolites was much lower (R: 42 vs. 74%, and S: 48 vs. 77%). CP appears to be enantioselectively metabolized to the DCE metabolites. This PK model can evaluate the proportion of a CP dose that is transformed to toxic or active metabolites. It may therefore be used to optimize CP treatment, to identify important drug interactions and/or patients with an abnormal metabolic profile.     

The stereoselective metabolism of halofantrine to desbutylhalofantrine in the rat: evidence of tissue-specific enantioselectivity in microsomal metabolism

The pharmacokinetics of the antimalarial drug (+/-)-halofantrine are stereoselective in humans and rats. To better understand the stereoselective metabolism of the drug to its primary metabolite, desbutylhalofantrine (DHF), a series of in vitro and in vivo experiments were undertaken in the rat. Formation of (-)-DHF exceeded that of (+)-DHF in liver microsomes [(-):(+) ratio of intrinsic formation clearances = 1.4]. In contrast, in intestinal microsomes no significant stereoselectivity was noted in the formation of the DHF enantiomers. Intestinal microsomes were also less efficient at producing the DHF enantiomers than were liver microsomes. Based on kinetic analysis of the DHF formation, there appeared to be more than one enzyme involved in the biotransformation. (+/-)-Ketoconazole (KTZ) effectively inhibited the formation of both DHF enantiomers by both liver and intestinal microsomes, although the reduction was more marked in liver microsomes. Through a combination of the use of CYP antibodies and recombinant CYP isoenzymes, the involvement of CYP 2B1/2, 3A1, 3A2, 1A1, 2C11, 2C6, 2D1, and 2D2 were implicated in the metabolism of halofantrine to DHF. Of these, CYP3A1/2 and CYP2C11 appeared to be the primary isoenzymes involved, although CYP2C11 showed greater (+)-DHF than (-)-DHF formation, whereas for CYP3A1 it was similar to the isolated rat liver microsomes. In vivo, oral (+/-)-KTZ caused significant increases in plasma halofantrine and decreases in DHF enantiomer plasma concentrations.     

Stereoselective distribution of hydroxychloroquine in the rabbit following single and multiple oral doses of the racemate and the separate enantiomers

Hydroxychloroquine (HCQ) stereoselective distribution was investigated in rabbits after 20 mg/kg po of racemic-HCQ (rac-HCQ) and 20 mg/kg po of each enantiomer, 97% pure (?)-(R)-HCQ and 99% pure (+)-(S)-HCQ. Concentrations were 4 to 6 times higher in whole blood than in plasma. Melanin did not affect plasma and whole blood levels since concentrations did not differ between pigmented and nonpigmented animals. After single and multiple doses of the separate enantiomers, only 5–10% of the antipode could be measured, in blood or plasma. Therefore, there was no significant interconversion from one enantiomer into the other. Following rac-HCQ, plasma (+)-(S)-levels always surpassed (?)-(R)-ones while in whole blood, (?)-(R)-HCQ concentrations were always the highest. When the enantiomers were administered separately, blood concentrations achieved after (?)-(R)-HCQ were higher, especially after multiple doses. These observations suggest that (?)-(R)-HCQ is preferentially concentrated by cellular components of blood. This enantioselective distribution of HCQ could be secondary to a stereoselective protein binding to plasma proteins, although a more specific binding of (?)-(R)-HCQ to blood cells cannot be ruled out. Since in whole blood (?)-(R)-HCQ is retained in cellular components, metabolism would favour the more available (+)-(S)-enantiomer. © 1994 Wiley-Liss, Inc.     

Steady-state pharmacokinetics of indobufen enantiomers in patients with obliterative atherosclerosis

Steady-state pharmacokinetics of indobufen (INDB) enantiomers administered as racemic INDB (rac-INDB) tablets and bleeding time were studied in patients. Two-hundred mg INDB tablets (Ibustrin) were administered twice daily for 7 days to obliterative atherosclerosis patients. Enantiospecific reversed phase (RP) HPLC with UV detection (lambda = 275 nm) was used for determination of INDB enantiomers in serum of patients. The ratio AUCR:AUCS equalled 1.7 +/- 0.2 as a result of higher (-)-R-enantiomer serum levels. The (+)-S-enantiomer was more rapidly eliminated (oral clearance, Cl = 1.1 +/- 0.3 L/h) than its (-)-R-antipode (Cl = 0.7 +/- 0.2 L/h). Therefore, the mean steady/state levels of (-)/R/enantiomer (13.5 +/- 3.8 mg/L) exceeded those of its (+)-S-enantiomer (7.8 +/- 1.8 mg/L). Furthermore, half-life (t1/2) was significantly shorter for (+)-S-INDB (t1/2 = 4.5 +/- 1.2 h as compared to (-)-R-INDB (t1/2 = 7.4 +/- 2.4 h). However, no significant differences were observed in the respective Vd values. The bleeding time of patients was not significantly extended. The above pharmacokinetic data provide a rationale for potential future replacement of INDB racemic tablets with tablets of its (+)-S-enantiomer.     

Stereoselective pharmacokinetics of ornidazole after intravenous administration of individual enantiomers and the racemate

The pharmacokinetics of ornidazole (ONZ) were investigated following i.v. administration of racemic mixture and individual enantiomers in beagle dogs. Plasma concentrations of ONZ enantiomers were analyzed by chiral high-performance liquid chromatography (HPLC) on a Chiralcel OB-H column with quantification by UV at 310 nm. Notably, the mean plasma levels of (-)-ONZ were higher in the elimination phase than those of (+)-ONZ. (-)-ONZ also exhibited greater t1/2, MRT, AUC(0-t) and smaller CL, than those of its antipode. The area under the plasma concentration-time curve (AUC(0-t)) of (-)-ONZ was about 1.2 times as high as that of (+)-ONZ. (+)-ONZ total body clearance (CL) was 1.4 times than its optical antipode. When given separately, there were significant differences in the values of AUC(0-infinity) and CL between ONZ enantiomers (P < 0.05), indicating that elimination of (+)-ONZ was more rapid than that of (-)-ONZ. No significant differences were found between the estimates of the pharmacokinetic parameters of (+)-ONZ or (-)-ONZ, obtained following administration as the individual and as a racemic mixture. This study demonstrates that the elimination of ONZ enantiomers is stereoselective and chiral inversion and enantiomer/enantiomer interaction do not occur when the enantiomers are given separately and as racemic mixture.     

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 disposition of fenoprofen in plasma and synovial fluid of patients with rheumatoid arthritis

The simultaneous disposition of fenoprofen enantiomers in synovial fluid and plasma was studied in 11 patients with arthritis and chronic knee effusions treated with a single oral dose of 600 mg rac-fenoprofen. A plasma sample and a synovial fluid sample were collected simultaneously from each patient up to 16 h after the administration of fenoprofen. A stereospecific assay for fenoprofen using LC-MS-MS was developed and applied successfully to the analysis of the enantiomers in plasma (LOQ = 10 ng of each enantiomer/ml) and synovial fluid (LOQ = 25 ng of each enantiomer/ml). The values of the area under the curve (AUC) for the S-(+)-fenoprofen eutomer were approximately 2.5 times higher in plasma than in synovial fluid (256 vs 104 microg h/ml), while the values for the R-(-)-fenoprofen distomer were about four times higher in plasma than in synovial fluid (42.5 vs 10.5 microg h/ml). These data demonstrate accumulation of the S-(+)-fenoprofen eutomer in plasma and in synovial fluid, with concentrations versus time AUC (+)/(-) ratios of 6.0 in plasma and 9.9 in synovial fluid, suggesting a greater accumulation of the eutomer at the active site represented by synovial fluid than in plasma. This result demonstrates the importance of enantioselective methods and of analysis of synovial fluid rather than plasma in studies of the pharmacokinetics-pharmacodynamics of fenoprofen.     

Fiber-based liquid-phase micro-extraction of mebeverine enantiomers followed by chiral high-performance liquid chromatography analysis and its application to pharmacokinetics study in rat plasma

The applicability of two-phase liquid-phase micro-extraction (LPME) in porous hollow polypropylene fiber for the sample preparation and the stereoselective pharmacokinetics of mebeverine (MEB) enantiomers (an antispasmodic drug) in rat after intramuscular administration were studied. Plasma was assayed for MEB enantiomer concentrations using stereospecific high-performance liquid chromatography with ultraviolet detection after a simple, inexpensive, and efficient preconcentration and clean-up hollow fiber-based LPME. Under optimized micro-extraction conditions, MEB enantiomers were extracted with 25 μl of 1-octanol within a lumen of a hollow fiber from 0.5 ml of plasma previously diluted with 4.5 ml alkalized water (pH 10). The chromatographic analysis was carried out through chiral liquid chromatography using a DELTA S column and hexane-isopropyl alcohol (85:15 v/v) containing 0.2% triethylamine as mobile phase. The mean recoveries of (+)-MEB and (-)-MEB were 75.5% and 71.0%, respectively. The limit of detection (LOD) was 3.0 ng/ml with linear response over the concentration range of 10-2500 ng/ml with correlation coefficient higher than 0.993 for both enantiomers. The pharmacokinetic studies showed that the mean plasma levels of (+)-MEB were higher than those of (-)-MEB at almost all time points. Also, (+)-MEB exhibited greater t(max) (peak time in concentration-time profile), C(max) (peak concentration in concentration-time profile), t(1/2) (elimination half-life), and AUC(0-240 min) (area under the curve for concentration versus time) and smaller CL (clearance) and V(d) (apparent distribution volume) than its antipode. The obtained results implied that the absorption, distribution, and elimination of (-)-MEB were more rapid than those of (+)-MEB and there were stereoselective differences in pharmacokinetics.     

Stereoselective RP-HPLC determination of esmolol enantiomers in human plasma after pre-column derivatization

A stereoselective reversed-phase HPLC assay to determine S-(-) and R-(+) enantiomers of esmolol in human plasma was developed. The method involved liquid-liquid extraction of esmolol from human plasma, using S-(-)-propranolol as the internal standard, and employed 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate as a pre-column chiral derivatization reagent. The derivatized products were separated on a 5-microm reversed-phase C18 column with a mixture of acetonitrile/0.02 mol/L phosphate buffer (pH 4.5) (55:45, v/v) as mobile phase. The detection of esmolol derivatives was made at lambda=224 nm with UV detector. The assay was linear from 0.035 to 12 microg/ml for each enantiomer. The analytical method afforded average recoveries of 94.8% and 95.5% for S-(-)- and R-(+)-esmolol, respectively. For each enantiomer, the limit of detection was 0.003 microg/ml and the limit of quantification for the method was 0.035 microg/ml (RSD<14%). The reproducibility of the assay was satisfactory.     

Stereoselective metabolism of tetrahydropalmatine enantiomers in rat liver microsomes

Tetrahydropalmatine (THP), with one chiral center, is an active alkaloid ingredient in Rhizoma Corydalis. The aim of the present paper is to study whether THP enantiomers are metabolized stereoselectively in rat, mouse, dog, and monkey liver microsomes, and then, to elucidate which Cytochrome P450 (CYP) isoforms are predominately responsible for the stereoselective metabolism of THP enantiomers in rat liver microsomes (RLM). The results demonstrated that (+)-THP was preferentially metabolized by liver microsomes from rats, mice, dogs, and monkeys, and the intrinsic clearance (Cl(int)) ratios of (+)-THP to (-)-THP were 2.66, 2.85, 4.24, and 1.67, respectively. Compared with the metabolism in untreated RLM, the metabolism of (-)-THP and (+)-THP was significantly increased in dexamethasone (Dex)-induced and β-naphthoflavone (β-NF)-induced RLM; meanwhile, the Cl(int) ratios of (+)-THP to (-)-THP in Dex-induced and β-NF-induced RLM were 5.74 and 0.81, respectively. Ketoconazole had stronger inhibitory effect on (+)-THP than (-)-THP, whereas fluvoxamine had stronger effect on (-)-THP in untreated and Dex-induced or β-NF-induced RLM. The results suggested that THP enantiomers were predominately metabolized by CYP3A1/2 and CYP1A2 in RLM, and CYP3A1/2 preferred to metabolize (+)-THP, whereas CYP1A2 preferred (-)-THP.