Stereoselective cyclooxygenase inhibition in cellular models by the enantiomers of ketoprofen

The pharmacological activity of rac-ketoprofen and its enantiomers was investigated in vitro using different cellular models. The effect of these compounds on arachidonic acid metabolism was assessed by measuring the inhibition of prostanoid generation under the action of several agonists. Thus, we have evaluated the inhibition of (1) thromboxane B₂ synthesis in rabbit platelets and human polymorphonuclear leukocytes (PMNs), (2) prostaglandin E₂ synthesis in three cultured cells, namely human umbilical vein endothelial cells (HUVEC), human keratinocytes, and mouse macrophage-like P388D1 cells. The IC₅₀ values found for (+)-(S)-ketoprofen were in the range between 0.1 nM and 0.8 μM, being slightly lower in all models than those found for rac-ketoprofen (0.4 nM–3 μM). On the other hand, (?)-(R)-ketoprofen showed inhibition of cyclooxygenase only at concentrations two or three orders of magnitude higher than those required for the (+)-(S) enantiomer. These results, obtained with cell types of relevance for inflammatory processes and with compounds of high optical purity, demonstrate that the prostanoid biosynthesis inhibition caused by the drug rac-ketoprofen is exclusively due to its dextrorotatory enantiomer. © 1993 Wiley-Liss, Inc.     

Stereoselective inhibition of diphenylhydantoin metabolism by p-hydroxyphenyl-phenylhydantoin enantiomers in rats.

Different doses of rac-p-HPPH (0.4 and 4 mg/h) were given repeatedly to rats infused with [14C]phenytoin. The serum levels of 14C-labeled and unlabeled p-HPPH, and [14C]phenytoin were measured by an HPLC method and radiometric analysis. The clearance of phenytoin and p-HPPH was determined by rate of dosing divided by the steady-state concentration. The phenytoin clearance was significantly lower in the high dose p-HPPH injection group than in the low dose group (87 versus 262 ml/h), whereas p-HPPH clearance showed no difference. The formation clearance of [14C]p-HPPH was also significantly lower in rats injected with high dose of p-HPPH (35 versus 169 ml/h). The clearance of other elimination pathways was also lower in rats with high dose of p-HPPH (53 versus 89 ml/h). The serum protein binding of phenytoin was lower in rats injected with high dose of p-HPPH. The result indicated that injections of rac-p-HPPH mainly inhibited on the formation of p-HPPH itself. The formation of (R)-p-HPPH and (S)-p-HPPH in microsomal preparation was measured by a ligand-exchange chromatographic method. The formation of (S)-p-HPPH or (R)-p-HPPH was not only inhibited by the enantiomer itself, but also cross-inhibited by the other enantiomer. To the formation of either (S)-p-HPPH or (R)-p-HPPH, (S)-p-HPPH showed a higher inhibitory activity. The use of rac-p-HPPH to inhibit phenytoin metabolism in vivo involved several mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereoselective inhibition of human, mouse, and horse cholinesterases by bambuterol enantiomers

Bambuterol is a chiral carbamate and a selective inhibitor of butyrylcholinesterase (BChE, EC 3.1.1.8). In order to relate bambuterol selectivity and stereoselectivity of BChE and acetylcholinesterase (AChE, EC 3.1.1.7) of different species, we studied the inhibition of human, mouse, and horse BChE, as well as AChE of human and mouse by (R)- and (S)-bambuterol. AChE and BChE of all studied species were progressively inhibited by both bambuterol enantiomers, with a preference for the (R)-bambuterol whose inhibition rate constants were about five times higher than that of (S)-bambuterol. We observed no significant difference between human and mouse in bambuterol enantiomer BChE inhibition. However, (R)-bambuterol inhibited horse BChE about 14 times slower than human and mouse BChE, and the inhibition rate for (S)-bambuterol was about 18 times slower. Although the primary structure of horse BChE differs from the other two species in 15 amino acids, we presumed that differences in inhibition rates could be attributed to threonine at position 69 located close to the peripheral site of BChE. Since BChE inhibition by bambuterol enantiomers was at least 8000 times faster than that of AChE, both bambuterol enantiomers proved to be selective BChE inhibitors, as was previously shown for racemate.     

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.     

Stereoselective pharmacokinetics of diniconazole enantiomers in rabbits

Diniconazole [(E)-(RS)-1-(2,4,-dichlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-triazole-1-yl)pent-1-en-3-ol)] is a potent triazole fungicide. The enantioselective pharmacokinetics of diniconazole enantiomers in rabbits was studied via intravenous (i.v.) injection. The pharmacokinetics and the enantiomer fraction (EF) were determined using normal high-performance liquid chromatography with diode array detection and a cellulose-tris-(3,5-dimethylphenylcarbamate)-based chiral stationary phase (CDMPC-CSP). The time-concentration curves in plasma were fitted by a two-compartment open mode. The results showed that the concentration of S-diniconazole in plasma decreased faster than that of R-diniconazole, and EFs increased with time after administration of racemic diniconazole (rac-diniconazole). The R-/S-enantiomer ratio of the area under the time-plasma concentration curve (AUC(0-infinity)) after administration was 1.52. The total plasma clearance value of S-enantiomer was 1.57-fold higher than that of the R-diniconazole. These results indicate substantial stereoselectivity in the kinetics of diniconazole enantiomers in rabbit.     

Stereoselective kinetic study of hexaconazole enantiomers in the rabbit

Hexaconazole [(RS)-2-(2,4-dichlorophenyl)-1-(1H-1,2,4-triazol-1-yl)hexan-2-ol] is a potent triazole fungicide. The (-) isomer accounts for most of the fungicidal activity. The stereo- and/or enantioselective kinetics of hexaconazole were investigated in rabbits by intravenous injection. The concentrations of (-)- and (+)-hexaconazole in plasma, liver, and kidney tissue were determined by HPLC with a cellulose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phase and by gas chromatography-mass spectrometry. After intravenous administration of racemic hexaconazole (rac-hexaconazole) at 30 mg/kg, plasma, liver, and kidney levels of the (+)-enantiomer decreased more rapidly than those of the (-)-enantiomer. The (-)-/(+)-enantiomer ratio of the area under the concentration-time curve (AUC(0-infinity)) was 1.35. The total plasma clearance value (CL) of (+)-enantiomer was more than 1.3-fold higher than that of the (-)-hexaconazole. The enantiomeric ratio (ER) increased with time in plasma, liver, and kidney. Other pharmacokinetic parameters of the enantiomers were also different. These results indicate substantial stereoselectivity in the kinetics of hexaconazole enantiomers in rabbits.     

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.     

Stereoselective effect of tranylcypromine enantiomers on brain serotonin

Brain levels of 5-HT, 5-HIAA and L-tryptophan were measured in reserpinized rats given the enantiomers of tranylcypromine (TCP). (+)-TCP (1.5-30 mg/kg) increased levels of 5-HT, whereas (-)-TCP failed to affect 5-HT levels. (+)-TCP at high dosage (30 mg/kg) decreased brain levels of 5-HIAA, whereas (-)-TCP failed to affect 5-HIAA levels. Neither (+)-TCP nor (-)-TCP influenced brain levels of L-tryptophan. The effects of (+)-TCP on behavior resembled those of drugs which influence 5-HT processes, whereas (-)-TCP failed to produce 5-HT-dependent behaviors. The findings support the notion that TCP enantiomers have stereoselective effects on serotonergic mechanisms.     

Simultaneous analysis of verapamil and norverapamil enantiomers in human plasma by high-performance liquid chromatography

An improved HPLC method for the simultaneous determination of the enantiomers of verapamil (V) and its major metabolite norverapamil (NV) in human plasma samples is presented. NV is acetylated immediately to N-acetylnorverapamil (ANV) in the extraction solvent (2% butanol in hexane). Acetylation is so rapid that it does not delay sample processing. ANV and V enantiomers are then separated on an α₁-acid glycoprotein chiral column with a mobile phase of phosphate buffer (0.01 M, pH 6.65) and acetonitrile. The fluorescence detector wavelengths are set at 227 nm for excitation and 308 nm for emission. Introduction of the internal standard (I.S.) (+)-glaucine improves accuracy, precision and robustness of the method. The assay is sensitive and specific. Baseline separation is achieved for both V and ANV. Limits of quantitation are 3 ng/ml for V and 2 ng/ml for NV (single enantiomer) with precision and accuracy better than 15% at those levels. Detector response is linear in the range tested (3–200 ng/ml for V and 2–100 ng/ml for NV, single enantiomer). This assay has been applied to a clinical study of the pharmacodynamics of V involving six healthy volunteers.     

Stereoselective inhibition of rat brain cyclooxygenase by dexketoprofen

Although it has been assumed that the effects of nonsteroidal antiinflammatory drugs (NSAIDs) are mainly the result of their action on local synthesis of prostaglandins, there is growing evidence to suggest that they may also exert a central analgesic action. Some authors have suggested that inhibition of prostaglandin synthesis in the brain could contribute to the analgesic action. The effect of dexketoprofen trometamol (tromethamine salt of the enantiomer (+)-S-ketoprofen) on prostaglandin synthesis was investigated in rat brain fragments and in cyclooxygenase preparations from rat brain microsomes. Effects of the (-)-R-enantiomer and the racemic mixture were also evaluated. Significant levels of prostaglandin F_2α (PGF_2α) were synthesized in rat brain fragments after 10 min of incubation at 37°C. Dexketoprofen was found to be a potent inhibitor of this PGF_2α production in rat brain (IC₅₀ = 6.2 nM), and it completely suppressed PGF_2α production at 1 μM concentration. In addition, inhibition of PGF_2α synthesis by dexketoprofen was highly stereoselective since the enantiomer (-)-R-ketoprofen was significantly less potent (IC₅₀ = 294 nM); with this enantiomer, even at high concentrations such as 1 μM, less than 60% inhibition was achieved. These results correlated with those obtained in the study of racemic ketoprofen and its enantiomers on cyclooxygenase activity of rat brain microsomes, where dexketoprofen also inhibited enzymatic activity stereoselectively. IC₅₀ values obtained for dexketoprofen, (-)-R-ketoprofen, and rac-ketoprofen were 3.5 μM, 45.3 μM, and 5.8 μM, respectively. The above results could be related to the potent analgesic effect of dexketoprofen observed in vivo, which was also stereoselective. Taken together, these findings suggest that prostaglandin synthesis inhibition in rat brain by dexketoprofen could be associated, at least in part, with the analgesic effect of this NSAID. Chirality 9:281–285, 1997. © 1997 Wiley-Liss, Inc.     

In vitro inhibition of aromatase by the enantiomers of aminoglutethimide and analogs

The in vitro aromatase activity in microsomal fractions from rat ovary and its inhibition by enantiomers of aminoglutethimide (AG), rogletimide (RG), and cyclohexylaminoglutethimide (ChAG) were studied by analysing the [³H]H₂O released when [1β-³H]androstenedione was converted to estrone. Maximum velocity (V_max) and the Michaelis-Menten constant (K_m) of the microsomal aromatase enzyme were 17.40 ± 0.45 pmol/ml/mg protein/min and 1.02 ± 0.06 μM, respectively. The IC₅₀s for the enantiomers were similar for (+)-R-AG and (?)-R-ChAG (0.86 ± 0.06 and 0.89 ± 0.15 μM, respectively). (+)S-ChA'G was most potent with IC₅₀ of 0.075 ± 0.003 μM. The IC₅₀s for (?)-S-AG, (+)-R-RG, and (?)-S-RG were in the same range (23.15 ± 2.74, 24.58 ± 2.46, and 24.43 ± 2.20 μM, respectively). © 1994 Wiley-Liss, Inc.     

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.     

Stereoselective regulation of MDR1 expression in Caco-2 cells by cetirizine enantiomers

MDR1-encoded P-glycoprotein (P-gp) is a drug efflux transporter mainly expressed in liver, kidney, intestine, brain (at the level of the blood-brain barrier), and placenta. It thus plays important roles in drug absorption, distribution, and excretion. Cetirizine is a second-generation nonsedating antihistamine used to treat allergic disease of respiratory system, skin and eyes. To evaluate P-gp expression and function in Caco-2 cells pretreated with cetirizine enantiomers, we assessed the sensitivity of Caco-2 cells to paclitaxel using the MTT assay and the polarized transport of rhodamine-123 and doxorubicin across Caco-2 monolayers. RT-PCR and flow cytometry were used to assay MDR1 mRNA and P-gp protein respectively. The sensitivity of Caco-2 cells to paclitaxel decreased significantly after cells were pretreated with 100 microM R-cetirizine but increased upon treatment with S-cetirizine. The efflux of rhodamine-123 and doxorubicin was enhanced significantly after Caco-2 monolayers were pretreated with 100 microM R-cetirizine but was reduced by S-cetirizine. The MDR1 mRNA and P-gp levels in Caco-2 cells were increased by 100 microM R-cetirizine and decreased by 100 microM S-cetirizine. These results suggest that R-cetirizine up-regulates MDR1 expression while S-cetirizine down-regulates MDR1 expression.     

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.     

Stereoselective inhibition of glutamate carboxypeptidase by chiral phosphonothioic acids

A series of phosphonothioic acid derivatives of (S)-2-hydroxyglutarate with various alkyl or aryl ligands to the central phosphorus atom was examined for stereoselective inhibition of the glutamate carboxypeptidase, carboxypeptidase G. The inhibitory potencies of these stereoisomers were compared to corresponding synthetic phosphonic acid analogues in order to reveal the significance of the sulfur ligand of the phosphonothioic acid motif upon the inhibition of this metallopeptidase. The acquisition of the individual phosphonothioic acid diastereomers was achieved through the resolution of the respective phosphonate ester precursors. In all cases, the (+)p-diastereomers of these phosphonothioic acid derivatives of (S)-2-hydroxyglutarate were found to be more potent inhibitors of glutamate carboxypeptidase than the corresponding (-)p antipodes with the most dramatic difference observed for the butyl isomers (13.6-fold). Based upon Ki values obtained, the most potent inhibitor of the series by nearly an order of magnitude was the (+)p-n-butylphosphonothioic acid derivative, revealing specific structural and stereochemical requirements by this glutamate carboxypeptidase. With the exception of the (+)p-n-butyl analogue, the isosteric replacement of oxygen with sulfur of the phosphonic acid moiety did not enhance inhibitory potency.     

Stereoselective disposition and tissue distribution of carvedilol enantiomers in rats.

After intravenous bolus injection of rac-carvedilol at 2 mg/kg to the rat, the (+)-(R)- and (-)-(S)-enantiomer levels in the blood and tissues (liver, kidney, heart, muscle, spleen, and aorta) were measured by stereospecific HPLC assay. As compared with the (+)-(R), the (-)-(S) had a larger Vdss (3.32 vs. 2.21 liter/kg), MRT (33.4 vs. 25.6 min), and CLtot (96.1 vs. 83.8 ml/min/kg). AUC comparison after iv and po administration showed systemic bioavailability of the (-)-(S) to be about half that of its antipode, explained by the fact that the free fraction of the (-)-(S) in blood was 1.65-fold greater than that of the (+)-(R). Tissue-to-blood partition coefficient values for the (-)-(S) were 1.6- to 2.1-fold greater than those for the (+)-(R) in all tissues, showing that the (-)-(S) accumulates more extensively in the tissues. These results were consistent with the greater Vdss for the (-)-(S) estimated from systemic blood data. The stereoselective tissue distribution of carvedilol enantiomers results from an enantiomeric difference in plasma protein binding rather than in tissue binding.     

Stereoselective determination of fenfluramine enantiomers in rat liver microsomal incubates

An enantioselective assay for l- and d-fenfluramine in rat liver microsomal incubates was developed. The method involves extraction of fenfluramine from the microsomal incubates, and formation of fenfluramine diastereomeric derivatives with the chiral reagent S-(−)-N-trifluoroacetyl prolyl chloride. Separation and quantitation of the diastereomeric fenfluramine derivatives are carried out by a capillary gas chromatographic system with flame ionization detection. The assay is linear from 1 to 50 μg/ml for each enantiomer. The analytical method affords average recoveries of 92.28 and 96.44% for l- and d-fenfluramine, respectively. The limits of detection and quantitation for the method are 0.1 and 1.0 μg/ml for the l- and d-fenfluramine isomers, respectively. The reproducibility of the assay was <10% (RSD). The method allowed study of the depletion of l- and d-fenfluramine in rat liver microsomal incubates. The stereoselectivity of fenfluramine phase I metabolism was observed.     

Stereoselective inhibition of human placental aromatase

We have synthesized the (19R)- and (19S)-isomers (2 and 3 respectively) of 10 beta-oxiranylestr-4-ene-3,17-dione. The configurations and conformations of these compounds were established by X-ray crystallographic analysis. Each of these compounds is a powerful competitive inhibitor of human placental microsomal aromatase, and stereoselectivity of inhibition was observed (Ki values for 2 and 3 were 7 and 75 nanomolar, respectively). Spectroscopic studies with purified aromatase indicate that the inhibition process involves reversible binding of oxirane oxygen to the heme iron of the enzyme. The (19R)- and (19S)-10 beta-thiiranes (6 and 7) corresponding to 2 and 3 have been synthesized from the oxiranes by a stereospecific process. The thiiranes are very effective competitive inhibitors of placental aromatase, and show even greater stereoselectivity in binding than the oxiranes (Ki values for 6 and 7 were 1 and 75 nanomolar, respectively). Spectroscopic studies with purified aromatase indicate that the inhibition process involves reversible binding of thiirane sulfur to heme iron.     

Stereoselective binding of ketoprofen enantiomers to human serum albumin studied by high-performance liquid affinity chromatography

A chiral stationary phase based on immobilized human serum albumin (HSA) was used to study the stereoselective binding of ketoprofen enantiomers by means of high-performance liquid affinity chromatography. The technique of zonal elution was applied together with a novel mathematical approach describing attachment to more than one type of binding site. Phenylbutazon (PBZ) and diazepam (DAZ) were used as markers for the major believed binding regions on HSA. Both R- and S-ketoprofen (KTR and KTS) display high affinity to the primary PBZ- and DAZ-binding sites and low-affinity to the secondary DAZ sites. The binding to high-affinity regions is accepted to be a stepwise process initiated by the binding to the primary DAZ sites and followed by the attachment to the primary PBZ sites. The chiral recognition is attributed to the high-affinity PBZ-binding sites and to the low-affinity DAZ-binding sites.     

Stereoselective high-performance liquid chromatographic assay for propranolol enantiomers in serum

A simple and sensitive stereoselective high-performance liquid chromatographic assay for the quantitation of propranolol enantiomers in serum is described. The method involves conversion of the propranolol enantiomers to diastereomeric urea derivatives by reaction with the chiral reagent (+)-phenylethylisocyanate, followed by chromatographic separation of the diastereomeric products. Conditions of the derivatization reaction were optimized to achieve rapid and quantitative yield with either of the enantiomers. Baseline resolution of the diastereomers was achieved on a reversed phase C₈ column with an isocratic mobile phase. Fluorescence detection afforded an absolute on-column detection limit of 100 pg. The assay has been applied to pharmacokinetic studies in humans and small laboratory animals.