Enantioselective recognition of mandelic acid based on γ-globulin modified glassy carbon electrode

A new chiral biosensor has been fabricated by immobilizing γ-globulin on gold nanoparticles modified glassy carbon electrodes, which could recognize and detect mandelic acid (MA) enantiomers. Differential pulse voltammetry, quartz crystal microbalance, ultraviolet-visible spectroscopy, and atomic force microscopy were used to characterize the enantioselectivity. The results exhibited that γ-globulin modified electrode could enantioselectively recognize MA enantiomers, and larger response signals were obtained from R-MA. The factors influencing the performance of the resulting biosensor were investigated. The enantiomeric composition of R- and S-MA enantiomer mixtures could be determined by measuring the current responses of the sample. The developed electrodes have the advantages of simple preparation, good stability, and rapid detection.     

Determination of the epimeric composition of ibuprofenyl-CoA

Ibuprofen [racemic2-(4-isobutylphenyl)propionic acid] is a 2-arylpropionic acid nonsteroidal anti-inflammatory drug which undergoes unidirectional, R to S chiral inversion in vivo. It has been proposed that this chiral inversion phenomenon occurs via a coenzyme A (CoA) thioester intermediate. To characterize the formation and metabolism of this metabolic intermediate, ibuprofenyl-CoA, reference standards were needed and thus the CoA derivatives of (R)-, (S)-, and racemic ibuprofen were chemically synthesized. An HPLC assay employing a C18 reverse-phase column was developed to quantitate "total" ibuprofenyl CoA. Samples collected from this assay were then analyzed for ibuprofenyl-CoA epimeric composition by chiral chromatography employing a Chiral-AGP alpha 1-acid glycoprotein column. The applicability of these methods was demonstrated by assessing (R)- and (S)-ibuprofenyl-CoA hydrolysis and epimerization following incubation with rat liver homogenates. Rat liver homogenate catalyzed the complete and rapid epimerization of ibuprofenyl-CoA and the rate constants for (R)- and (S)-ibuprofenyl-CoA hydrolysis were equal. ATP and CoA were found to inhibit rat liver-catalyzed ibuprofenyl-CoA hydrolysis by 70-80% with no effect on epimerization. Additionally, it was demonstrated that traditional indirect ibuprofenyl-CoA assays which employ basic hydrolysis result in erroneous epimeric ratio determinations due to chemical epimerization.     

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.     

Metabolic chiral inversion of ibuprofen in isolated rat hepatocytes

Ibuprofen was used to demonstrate that isolated rat hepatocytes offer a suitable in vitro model to investigate the metabolic chiral inversion of anti-inflammatory 2-arylpropionic acids (profens). The inversion of the pharmacologically inactive (-)-(R)-ibuprofen to the active (+)-(S)-ibuprofen was shown to obey apparent first-order kinetics during 5 h and to increase linearly with increasing hepatocyte concentration up to 4 x 10(5) cells/ml. No elimination of (R)-ibuprofen by routes other than inversion was seen, whereas the elimination of (S)-ibuprofen appeared to be saturable.     

Bi-directional chiral inversion of ketoprofen in CD-1 mice

The R enantiomers of some of the 2-arylpropionic acid non-steroidal antiinflammatory drugs (NSAIDs) are known to undergo metabolic chiral inversion to their more pharmacologically active antipodes. This process is drug and species dependent and usually unidirectional. The S to R chiral inversion, on the other hand, is rare and has been observed, in substantial extents, only for ibuprofen in guinea pigs and 2-phenylpropionic acid in dogs. After i.p. administration of single doses of racemic ketoprofen or its optically pure enantiomers to male CD-1 mice and subsequent study of the concentration time-course of the enantiomers, we noticed substantial chiral inversion in both directions. Following racemic doses, no stereoselectivity in the plasma-concentration time courses was observed. After dosing with optically pure enantiomer, the concentration of the administered enantiomer predominated during the absorption phase. During the terminal elimination phase, however, the enantiomers had the same concentrations. Our observation is suggestive of a rapid and reversible chiral inversion for ketoprofen enantiomers in mice. Chirality 9:29–31, 1997. © 1997 Wiley-Liss, Inc.     

Formation of glycine conjugate and (-)-(R)-enantiomer from (+)-(S)-2-phenylpropionic acid suggesting the formation of the CoA thioester intermediate of (+)-(S)-enantiomer in dogs.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.     

Mutual inversion of flurbiprofen enantiomers in various rat and mouse strains

Flurbiprofen (F) is a nonsteroidal anti‐inflammatory drug (NSAID) used therapeutically as the racemate of (R)‐enantiomer and (S)‐enantiomer. The inversion of RF to SF and vice versa was investigated in C57Bl/6 and SJL mice and Dark Agouti and Lewis rats. The enzyme α‐methylacyl‐CoA racemase (AMACR) is involved in the chiral inversion pathway that converts members of the 2‐arylpropionic acid NSAIDs from the R‐enantiomer to the S‐enantiomer. We studied C57Bl/6 mice deficient in AMACR postulating that they should show reduced inversion of RF to SF. In line with the data of others in mice, (R)‐inversion to (S)‐inversion was relatively high in both the C57Bl/6 and SJL mice (fraction inverted, F_I = 37.7% and 24.7%, respectively). In contrast, in AMACR deficient mice, there was no measurable peak for SF after administration of RF. The results in both rat strains (Dark Agouti and Lewis rats, F_I = 1.4% and 4.1%, respectively) confirm the low chiral inversion of the enantiomers of flurbiprofen in the rat, as observed by other authors in the Sprague‐Dawley strain (<5%). From the present results, we conclude that for the study of flurbiprofen enantiomers, the rat is more suitable than the mouse as a model for the human in which (R)‐inversion to (S)‐inversion is negligible.     

Stereochemistry of methyl transfer catalyzed by tRNA (m5U54)-methyltransferase--evidence for a single displacement mechanism.

tRNA (m5U54)-methyltransferase (RUMT) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the 5-carbon of uridine 54 of tRNA. We have determined the steric course of methyl transfer, using (methyl-R)- and (methyl-S)-[methyl-2H1,3H]-AdoMet as the chiral methyl donors, and tRNA lacking the 5-methyl group at position 54 as the acceptor. Following methyl transfer, ribothymidine was isolated and degraded to chiral acetic acid for configurational analysis. Transfer of the chiral methyl group to U54 proceeded with inversion of configuration of the chiral methyl group, suggesting that RUMT catalyzed methyl transfer occurs by a single SN2 displacement mechanism.     

Evidence of absorption rate dependency of ibuprofen inversion in the rat

Ibuprofen (IB) is a chiral 2-arylpropionic acid derivative used as a nonsteroidal antiinflammatory drug (NSAID). It undergoes substantial R to S chiral inversion in humans and rats. In addition to systemic inversion, presystemic chiral inversion has been suggested for IB in humans but only after administration of formulations with slow absorption rates. In search for a suitable animal model, the absorption rate dependency of the extent of inversion was examined in male Sprague–Dawley rats given 20 mg/kg of racemic IB in aqueous solution (T_max, 0.6 h), suspension (T_max, 1 h) or as sustained release granules (T_max, 2.3 h). In addition, (R)-IB (5 mg/liter) was incubated in the presence of everted rat gut segments in an organ bath at 37°. After sustained release granules, the S:R AUC ratios (7.3 ± 1.5) were significantly higher than suspension (3.6 ± 1.1) and solution (3.5 ± 0.2). Accordingly, AUC_S and AUC_R, as percent of the total AUC (S + R), significantly increased and decreased, respectively, after administration of the sustained released granules as compared with the solution and suspension. A significant positive linear correlation was found between the S:R AUC ratios and the corresponding T_max for (R)-IB (r = 0.82). In vitro, (R)-IB was inverted by everted jejunum (12.2 ± 1.6%), ileum (14.2 ± 2.0%), and colon (4.4 ± 0.6%) segments. IB was also glucuronidated in the presence of the intestinal segments. Therefore, similar to earlier observations made in humans, in the rat, the S:R AUC ratio was positively and significantly correlated with the absorption rate from the dosage form. Rat small intestine was capable of inverting and conjugating (R)-IB. Hence, rat is a suitable model for studying the chiral inversion of IB. © 1994 Wiley-Liss, Inc.     

Enantioselective degradation of hexaconazole in rat hepatic microsomes in vitro

Hexaconazole [(RS)-2-(2,4-dichlorophenyl)-1-(1H-1,2,4-triazol-1-yl) hexan-2-ol] is a potent triazole fungicide and consists of a pair of enantiomers. Enantioselective degradation of hexaconazole was investigated in rat hepatic microsomes in vitro. Concentrations of (-)- and (+)-hexaconazole and enantiomer fraction were determined by high performance liquid chromatography with a cellulose-tris-(3,5-dimethylphenylcarbamate)-based chiral stationary phase. The t(1/2) of (-)-hexaconazole and (+)-hexaconazole were 23.70 and 13.95 min for rac- hexaconazole and 44.18 and 23.54 for enantiomers examined separately. Furthermore, hexaconazole is configurationally stable in rat hepatic microsomes, demonstrating no chiral inversion from the (-)-hexaconazole to (+)-hexaconazole or vice versa. Intrinsic metabolic clearance of (+)-hexaconazole is 1.12 times than that of (-)-hexaconazole. Interaction study revealed that there was competitive inhibition between (-)-hexaconazole and (+)-hexaconazole. In addition, there was a significant difference between the inhibitory concentration (IC(50)) of (-)- to (+)-hexaconazole and (+)- to (-)-hexaconazole [IC(50)(-)/(+)/IC(50)(+)/(-) = 1.88]. These results may have potential implications for better environmental and ecological risk assessment for hexaconazole.     

Stereoselective pharmacokinetics of flobufen in rats

Stereoselectivity of the pharmacokinetics of the nonsteroidal anti-inflammatory drug flobufen, 4-(2', 4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid, was studied in male Wistar rats after intravenous administration. Pharmacokinetic parameters and chiral inversion of flobufen enantiomers were studied after a bolus injection of the racemate and individual enantiomers (5 mg/kg). Determinations of the enantiomers in rat plasma were performed using chiral HPLC (terguride column). After i.v. administration of flobufen racemate, plasma levels of R-enantiomer decreased more rapidly. The S-/R-enantiomer ratio of AUCs after rac-flobufen was 13.3. The total plasma clearance value of S-flobufen was more than 10-fold lower than R-flobufen. The other pharmacokinetic parameters of the enantiomers were also significantly different. While only traces of R-enantiomer (less than 1%) were detected in rat plasma after S-flobufen administration, considerable conversion to the S-enantiomer was found after injection of R-flobufen (R-enantiomer AUC/S-enantiomer AUC = 0.52). The results indicate substantial stereoselectivity in the disposition of flobufen enantiomers in the rat, which is, at least in part, attributed to chiral bioconversion.     

Chiral inversion of RS-8359: a selective and reversible MAO-A inhibitor via oxido-reduction of keto-alcohol

RS-8359, (+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine is a selective and reversible MAO-A inhibitor. The (S)-enantiomer of RS-8359 has been demonstrated to be inverted to the (R)-enantiomer after oral administration to rats. In the current study, we investigated the chiral inversion mechanism and the properties of involved enzymes using rat liver subcellular fractions. The 7-hydroxy function of RS-8359 was oxidized at least by the two different enzymes. The cytosolic enzyme oxidized enantiospecifically the (S)-enantiomer with NADP as a cofactor. On the other hand, the microsomal enzyme catalyzed more preferentially the oxidation of the (S)-enantiomer than the (R)-enantiomer with NAD as a cofactor. With to product enantioselectivity of reduction of the 7-keto derivative, it was found that only the alcohol bearing (R)-configuration was formed by the cytosolic enzyme with NADPH and the microsomal enzyme with NADH at almost equal rate. The reduction rate was much larger than the oxidation rate of 7-hydroxy group. The results suggest that the chiral inversion might occur via an enantioselectivity of consecutive two opposing reactions, oxidation and reduction of keto-alcohol group. In this case, the direction of chiral inversion from the (S)-enantiomer to the (R)-enantiomer is governed by the enantiospecific reduction of intermediate 7-keto group to the alcohol with (R)-configuration. The enzyme responsible for the enantiospecific reduction of the 7-keto group was purified from rat liver cytosolic fractions and identified as 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) via database search of peptide mass data obtained by nano-LC/MS/MS.     

Studies on the in vitro inversion of flurbiprofen

This paper reports in vitro studies on the metabolic inversion of flurbiprofen (FL), an arylpropionic acid antiinflammatory agent (2-APA). The inversion was studied with both rac-FL and R-FL, by incubation with rat hepatic microsomes, in the presence of either CoASH and ATP or NADPH. The two isomers of the drug were separated as their (+)-(R)-1-phenylethylamides by direct phase high-performance liquid chromatography on a silica gel column with an achiral mobile phase. The inversion was more pronounced in the presence of CoASH and ATP for both the racemate and the R-isomer, which supports the key role of CoA thioesters in the metabolic inversion of profens. The inversion observed in the presence of NADPH suggests that, when the incubation is run with hepatic microsomes, a CYP450-mediated pathway is also active. In order to get more insight into the CYP450-mediated inversion pathway, we studied the effect of irradiating microsomes with a low dose of He-Ne laser radiation (0.2 J). Such irradiation caused a significant increase in inversion at all times studied and normalized the anomalous value of inversion observed at 15 min in his pathway. Chirality 9:317–319, 1997. © 1997 Wiley-Liss, Inc.     

Extent of chiral inversion of the 2-arylpropionic acids by Cordyceps militaris

The fungus Cordyceps militaris has been previously shown to be capable of inverting the chirality of 2-phenylpropionic acid from its (R)-enantiomer to its (S)-antipode. The structure of this compound is similar to the 2-arylpropionic acid non-steroidal anti-inflammatory drugs, which have also been reported to undergo a similar chiral inversion process in mammals and man. We report here an investigation into the substrate specificity of the enzyme system present in C. militaris using pure enantiomers and racemates of ibuprofen and ketoprofen and racemates of indoprofen, suprofen, flurbiprofen, and fenoprofen and the structurally related compounds 2-phenylbutyric acid and 2-phenoxypropionic acid as substrates, using optimised incubation conditions developed for the inversion of 2-phenylpropionic acid. The results demonstrated that C. militaris is capable of inverting the chirality of all the compounds investigated, which suggests that the active sites of the enzymes are very flexible with regard to the molecular dimensions of the substrate molecule and the spatial occupation of the groups surrounding the chiral centre. Metabolism of all the substrates was observed but the rate of metabolism varied extensively depending on the substrate. Achiral HPLC analysis was used to detect any potential metabolites and the results suggested that the site of the metabolism appeared to be at the aliphatic side groups only, with the aromatic ring being left intact in all cases. These results suggest that C. militaris could be a valuable tool in the investigation of the prospective metabolic fates of new 2-arylpropionic acids during their development. Chirality 10:528–534, 1998. © 1998 Wiley-Liss, Inc.     

D型氨基酸氧化酶活性对于D-硝基精氨酸手性转化的影响

D-硝基精氨酸(D-NNA)可在大鼠体内发生手性转化生成其L型异构体,即L-NNA,后者可抑制一氧化氮合酶活性,减少一氧化氮生成,升高动脉血压.研究了D型氨基酸氧化酶(DAAO)在D-NNA手性转化中的作用及DAAO对不同(包括已报道在体内可发生手型转化的)D型氨基酸的选择活性.体内实验显示,DAAO的选择性抑制剂苯甲酸钠(400mg/kg)或肌酐(400mg/kg)均可在不同程度上抑制D-NNA升压作用,进一步研究发现,肾脏或肝脏DAAO酶液在外加DAAO后可提高D-NNA的手性转化约2倍,表明DAAO对于D-NNA在体内的手性转化是必需的.DAAO酶液对可在体内发生手性转化且转化率相似(30%～50%)的D型氨基酸(D-Phe,D-Leu和D-NNA)的选择性表现出显著差异(Kcat/Km相差可达约15倍左右),这从另一方面表明体内D-硝基精氨酸氧化是其发生手性转化的前提条件但非决定因素.     

Estimation of the rate and extent of chiral inversion using linear systems analysis

An alternative method based on linear systems analysis is presented for the analysis of concentration-time data for the enantiomers of the 2-arylpropionic acids. This approach uses deconvolution to estimate the rate and extent of chiral inversion with respect to time, assuming linear pharmacokinetics and time invariance, without the need for complicated modelling procedures. Application to data for the chiral inversion of ibuprofen in the rat indicates that this approach provides a valid alternative to previous procedures for the analysis of chiral inversion data. © 1995 Wiley-Liss, Inc.     

Chiral aspects of metabolism of antiinflammatory drug flobufen in human hepatocytes

The metabolism of the nonsteroidal antiinflammatory drug flobufen, 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid, was studied in primary cultures of human hepatocytes prepared by two-step collagenase perfusion of livers from four donors. Racemic flobufen or its individual enantiomers, R-(+)- and S-(-)-flobufen were used as substrates. Aliquots of culture medium were collected during 24-h incubation. The time-dependent disappearance of flobufen enantiomers and the formation of metabolites (stereoisomers of dihydroflobufen (DHF)) in hepatocytes were measured by chiral HPLC. The reduction of flobufen in human hepatocytes was stereoselective ((+)-R-flobufen was preferentially metabolized) and stereospecific ((2R;4S)-DHF and (2S;4S)-DHF stereoisomers were mostly formed). Although the structure of flobufen is different from the profens (2-arylpropionates), flobufen undergoes chiral inversion in human hepatocytes. The inversion of R-(+)-flobufen to S-(-)-flobufen predominates. The individual DHF stereoisomers were incubated in hepatocyte cultures and their biotransformation studied. The unidirectional chiral inversion of (2S;4S)-DHF to (2R;4S)-DHF and (2R;4R)-DHF to (2S;4R)-DHF was observed. Stereoselective oxidation of the DHFs to flobufen was also detected. Thus, flobufen metabolism in primary cultures of human hepatocytes is much more complicated (via chiral inversion and DHF re-oxidation) than was presumed from a preliminary achiral point of view.     

Application of antibody-mediated extraction for the stereoselective determination of the active metabolite of loxoprofen in human and rat plasma.

Antibody-mediated extraction followed by chiral high-performance liquid chromatography (HPLC) was applied to stereoselective determination in human and rat plasma of the active metabolite [(2S,1'R,2'S)-trans-alcohol] with three chiral centers of Loxoprofen, a 2-arylpropionic acid antiinflammatory agent after oral administration. Antiserum against the (1'R,2'S)-cyclopentanol moiety was obtained from a rabbit immunized with bovine serum albumin conjugate linked to the propionic acid moiety, in which another chiral center is located. Then, the immunoglobulin G purified by a protein A column was coupled to BrCN-activated Sepharose 4B. Plasma samples were applied to the immobilized antibody column. After washing the column to remove unrequired stereoisomers, a mixture of two diastereomers whose configurations were 1'R,2'S in the cyclopentanol moiety was extracted with 95% methanol. The solvent was evaporated and the residue was derivatized with (+)-(R)-1-(1-naphthyl)ethylamine as a chiral reagent to separate the diastereomers by HPLC. This combined analytical method showed the stereoselective metabolism of Loxoprofen in human, that is, 64% of the total amount of four trans-alcohol stereoisomers was in the 2S,1'R,2'S form, which is the active metabolite. This phenomenon was also observed in rats given Loxoprofen and its (2S)- and (2R)-isomers, and is explained by stereoselective ketone reduction of Loxoprofen to the (1'R,2'S)-trans-alcohol and inversion from 2R to 2S in the propionic acid moiety. Antibody-mediated extraction should be a selective and simple clean-up method for determining haptens with complicated structures, combined with an appropriate analytical method.     

Topical administration of racemic ibuprofen

In vitro experiments to investigate possible stereoselective aspects of the topical administration of ibuprofen have been conducted. Incubation of ibuprofen with rat skin homogenates in the presence of coenzyme A, ATP, and magnesium provided no evidence for the formation of ibuprofenyl coenzyme A (the initial intermediate in the metabolic inversion of [R]- to [S]-ibuprofen). Similar incubation studies gave no indication of a change in the enantiomeric ratios of ibuprofen over the time course of the experiments. Percutaneous penetration studies of ibuprofen gel through porcine skin indicated that the ibuprofen enantiomer levels in the reservoir solutions were consistent with racemic ibuprofen having traversed the skin with no metabolic inversion. These results suggest that, in the models studied, skin metabolism does not result in the chiral inversion of (R)- to (S)-ibuprofen and that the topical administration of ibuprofen will result in the delivery of 50% “isomeric ballast.” Chirality 9:313–316, 1997. © 1997 Wiley-Liss, Inc.     

Development and validation of a direct enantiomeric separation of pregabalin to support isolated perfused rat kidney studies

Pregabalin (Lyrica) is the first compound approved to treat the neural pain associated with fibromyalgia. Pregabalin is the S-enantiomer of a gamma-amino acid analogue and chiral separation from its R-enantiomer must be achieved to support metabolic studies. The direct chiral separation of pregabalin from its R-enantiomer has been developed and HPLC/MS/MS assays have been validated to support isolated perfused rat kidney studies. The separation was developed through serial coupling of various macrocyclic glycopeptide stationary phases until partial separation of the enantiomers was achieved. Identification of the resolving stationary phase followed by optimization of the mobile phase enabled the baseline resolution of the enantiomers using mass spectrometry compatible solvents and modifiers. Assays were developed and validated for quantitation of the enantiomers from rat urine, isolated rat kidney perfusate, and isolated rat kidney perfusate ultrafiltrate to support pregabalin metabolic studies.