Transient absorption spectroscopy for determining multiple site occupancy in drug-protein conjugates. A comparison between human and bovine serum albumins using flurbiprofen methyl ester as a probe

Laser flash photolysis (LFP) has been used to determine the degree of binding of (S)- or (R)-flurbiprofen methyl ester (FBPMe) to human and bovine serum albumins (HSA and BSA, respectively). Regression analysis of the triplet decay of the drug (lambda = 360 nm) in the presence of the proteins led to a satisfactory fitting when considering a set of three lifetimes; the corresponding Afree, AI and AII preexponential coefficients can be correlated with the presence of FBPMe in the bulk solution and within the two known binding sites. The most remarkable differences between HSA and BSA were found under nonsaturating conditions; thus, when the [FBPMe]/[SA] ratio was 1:1, all the drug was bound to HSA, whereas 20-30% of it remained free in the bulk solution in the presence of BSA. The LFP approach was also applicable to the study of more complex FBPMe/HSA/BSA mixtures; the obtained results were in good agreement with the previous findings in FBPMe/HSA and FBPMe/BSA systems. This suggests the possibility of making use of the transient triplet-triplet absorption for investigating the distribution of a drug between several compartments in different host biomolecules.     

Incorporation of a monolithic column into sequential injection system for drug-protein binding studies

A sequential injection analysis (SIA) manifold was incorporated with a monolithic strong anion-exchanger disk for on-line drug-protein interaction studies. The antibiotic ciprofloxacin (CF) was selected as a model drug compound. The separation principle was based on the strong retention of bovine serum albumin (BSA) on the monolithic strong anion-exchanger and the liberation/release of the free form of the drug. Elution of the retained BSA was easily achieved by delivering a different mobile phase via the SIA manifold. The type of functional group of the monolithic support, the breakthrough volume and the injected volumes of CF and BSA were studied and optimized. The influence of the variation of incubation time was studied in on-line binding assays. Scatchard plot was employed to obtain the number of binding sites and the equilibrium binding constants. For the off-line study of the CF-BSA binding, two binding classes were determined with constants of (3.16+/-0.21)x10(6)M(-1) and (1.27+/-0.48)x10(4)M(-1) and 6.1+/-1.3 and 17.8+/-3.9 binding sites per class, respectively. In non-equilibrium binding experiments the binding rate constant was k(1)=785 M(-1)min(-1). All measurements were monitored with fluorescence (lambda(ext)=300 nm, lambda(em)=460 nm) and spectrophotometric detection (lambda=280 nm). To evaluate the accuracy of the developed method the obtained results were compared versus ultrafiltration experiments and were found in good agreement.     

Fluorescent high-performance liquid chromatographic analysis of vigabatrin enantiomers after derivatizing with naproxen acyl chloride

Vigabatrin is widely used as an anticonvulsant in the treatment of seizures. Vigabatrin is usually supplied as racemate in formulation, but only the (S)-(+)-enantiomer of vigabatrin is pharmacologically active. A simple and sensitive liquid chromatographic method is described for the separation and quantification of vigabatrin enantiomers. The method is based on derivatizing racemic vigabatrin with a fluorescent chiral reagent (naproxen acyl chloride). The resulting diastereomeric derivatives are highly responsive to a fluorimetric detector (lambda(ex)=230 nm, lambda(em)=350 nm). The lower quantitation limit of the method is attainable at 25 nM for (S)-(+)-vigabatrin or (R)-(-)-vigabatrin with a detection limit of about 2.5 nM (S/N=3 with 10 microl injected). Application of the method to the analysis of vigabatrin in serum of dosed patients proved feasible.     

Determination of tertatolol enantiomers in biological fluids by high-performance liquid chromatography.

A stereospecific high-performance liquid chromatographic method for the quantification of (-)- and (+)-tertatolol in plasma and urine is described. The method involves solid-phase extraction followed by derivatization with S(+)-naphthylethylisocyanate to form the urea derivative, which is more sensitive to fluorescence detection. The separation of the diastereomeric derivatives was performed by reversed-phase high-performance liquid chromatography. Fluorimetric detection (lambda excitation = 220 nm, lambda emission = 320 nm) allows the quantification of tertatolol enantiomers down to 6 ng/ml. The assay was used to study the pharmacokinetic profile of tertatolol enantiomers following oral administration of racemic tertatolol; preliminary results suggest enantioselective absorption and/or disposition of tertatolol.     

Global and target analysis of time-resolved fluorescence spectra of Di-9H-fluoren-9-yldimethylsilane: dynamics and energetics for intramolecular excimer formation

Dynamics and energetics for intramolecular excimer formation of a diarylsilane, di-9H-fluoren-9-yldimethylsilane (DFYDMS) have been investigated by means of ps time-resolved fluorescence spectroscopy and ab initio calculation. Multiple fluorescence decay curves were globally deconvolved to generate time-resolved fluorescence spectra and decay-associated spectra (DAS), from which species-associated spectra (SAS) were obtained. It is shown in the global analysis that there are at least three excited states: Two states are the locally excited (LE) states (lambda(max) approximately 320 nm) having lifetimes of 0.70 +/- 0.04 and 1.75 +/- 0.02 ns, and another is the excimer state (lambda(max) approximately 400 nm) having a lifetime of 7.34 +/- 0.02 ns. The species which decays with 0.70 ns evolves into a species with a red-shifted spectrum, which in turn decays in 7.34 ns. The experimental and ab initio results indicate that the rise time of 0.70 ns corresponds to the conversion of the initial S(1) LE state having a near sandwich geometry to the S(1) excimer state adopting a true sandwich geometry.     

Impurity profiling of ephedrines in methamphetamine by high-performance liquid chromatography

Separation of the enantiomers and diastereomers of ephedrines was investigated for impurity profiling of methamphetamine. We describe a method for the analysis of (1S,2R)-(+)-ephedrine, (1R,2S)-(-)-ephedrine, (1S,2S)-(+)-pseudoephedrine and racemic methylephedrine in bulk methamphetamine by HPLC using two different columns: a phenyl-beta-cyclodextrin-type column and an ODS-type column. The analytes were detected by UV absorbance measurement at 210 nm. As little as 0.05% of each ephedrine in bulk methamphetamine could be determined. In the impurity profiling of methamphetamine, the identification of ephedrines may provide valuable information about the precursor. This method was confirmed to be sufficiently sensitive to identify trace amounts of (1R,2S)-(-)-ephedrine and (1S,2S)-(+)-pseudoephedrine in bulk methamphetamine synthesized by the Emde method.     

Direct enantiomeric resolution of disopyramide and its metabolite using chiral high-performance liquid chromatography. Application to stereoselective metabolism and pharmacokinetics of racemic disopyramide in man

A method for the simultaneous determination of disopyramide and mono-N-desisopropyldisopyramide enantiomers extracted from human plasma and urine is presented. Separation and quantitation were carried out using two columns coupled in series, and UV detection at 254 nm. First, the racemates of the two compounds were separated using a reversed-phase column, and then the enantiomers were separated using a stereoselective column packed with human alpha 1-acid glycoprotein. The mobile phase was 8 mM phosphate buffer, pH 6.20-2-propanol (92:8, v/v). The coefficients of variation (%) for the plasma daily determination were 6.7% for R(-)- and S(+)-disopyramide at drug levels of 1.5 micrograms/ml, and 8.5% and 7.7% for R(-)- and S(+)-mono-N-desisopropyldisopyramide, respectively, at drug levels of 0.375 micrograms/ml. The method has allowed the study of stereoselective metabolism and pharmacokinetics of disopyramide after oral administration as a racemate.     

Stereoselective determination of R(-)- and S(+)-MK-571, a leukotriene D4 antagonist, in human plasma by chiral high-performance liquid chromatography.

A stereoselective high-performance liquid chromatographic method that utilizes fluorescence detection was developed for the selective and sensitive quantification of R(-)- and S(+)-enantiomers of MK-571 (1), a potent and specific leukotriene D4 antagonist, in human plasma. Racemic 1 was isolated from the acidified plasma using solid-phase extraction and the resulting residue was successfully reacted with isobutyl chloroformate and R(+)-1-(1-naphthyl)ethylamine in triethylamine-acetonitrile medium to form the diastereomer of each enantiomer. A structural analogue of 1 was used as internal standard. The derivatized sample was dissolved in 1,1,2-trichlorotrifluoroethane and an aliquot was chromatographed on a (R)-urea chiral column using a mobile phase containing 89% triethylamine-pentane (3:1000, v/v), 10% 2-propanol, and 1% acetonitrile at a flow-rate of 1.5 ml/min. The fluorescence response (excitation wavelength, 350 nm; emission wavelength, 410 nm) was linear (r2 greater than 0.999) for concentrations of enantiomers of 1 from 0.05 micrograms/ml, the lowest quantitation limit, up to 2.5 micrograms/ml. Intra-day coefficients of variation at 0.05 microgram/ml were 2.4% for the R(-)-isomer and 2.0% for S(+)-isomer. The corresponding inter-day coefficients of variation for R(-)- and S(+)-1 were 2.6 and 3.6%, respectively. The utility of the methodology was established by analysis of plasma samples from male volunteers receiving single intravenous and oral doses of racemic 1.     

高效液相色谱法测定布诺酚的光学纯度

用正相高效液相色谱法配合衍生化法准确地测定了抗炎镇痛药布诺酚的光学纯度。方法的平均标准偏差为０．０６４％,准确度为９９．７１％～１００．２９％。     

Supramolecular photochirogenesis with biomolecules. Mechanistic studies on the enantiodifferentiation for the photocyclodimerization of 2-anthracenecarboxylate mediated by bovine serum albumin

Photophysics and photochemistry of 2-anthracenecarboxylate (AC) bound to bovine serum albumin (BSA) were investigated in detail for the first time by electronic absorption, circular dichroism (CD), steady-state and time-resolved fluorescence, fluorescence quenching, and product analysis studies. Through the spectroscopic investigations, it was revealed that the four independent binding pockets of BSA, which are known to accommodate 1, 3, 2, and 3 AC molecules in the order of decreasing affinity, are distinctly different in hydrophobicity, chiral environment, and accessibility. Interestingly, AC bound to site 1 gave highly structured fluorescence with dual lifetimes of 4.8 and 2.1 ns in an intensity ratio of 3:2, which may be assigned to the existence of two positional or orientational isomers within the very hydrophobic site 1. In contrast, the lifetime of AC in site 2 was much longer (13.3 ns), and ACs in sites 3 and 4 have broader fluorescence spectra with lifetimes that were practically indistinguishable from that in bulk water (15.8 ns). Although each of sites 2-4 simultaneously binds multiple AC molecules, no CD exciton coupling or static fluorescence quenching was detected, indicating that ACs bound to each site are not in close proximity to each other. Quenching studies with nitromethane further confirmed the significant difference in accessibility among the binding sites; thus, ACs bound to sites 1 and 2 are highly protected from the attack of the quencher, affording 32 and 10 times smaller rate constants than that for free AC in water. Product studies in the presence and absence of nitromethane more clearly revealed the photochirogenic performance of each binding site. Although the addition of nitromethane did not greatly alter the product distribution, the enantiomeric excesses (ee's) of chiral cycloadducts 2 and 3 were critically manipulated by selectively retarding the photoreaction occurring at the more accessible binding sites. Thus, the highest ee of 38% was obtained for 2 in the presence of 18 mM nitromethane, while the highest ee of 58% was attained for 3 in the absence of nitromethane, both at [AC]/[BSA]=3.6.     

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in plasma after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate.

A sensitive high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat plasma has been developed. Racemic atenolol and practolol (internal standard) were extracted from alkalinized plasma (pH 12) into dichloromethane containing 3% (v/v) heptafluoro-1-butanol, and the organic layer was evaporated. The samples were derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate at pH 8.5 for 30 min. After removal of excess reagent, the diastereomers were extracted into dichloromethane. The diastereomers were separated on a Microspher C18 column (3 microns) with a mobile phase of acetonitrile-sodium acetate buffer (0.01 M, pH 7) (50:50, v/v) at a flow-rate of 0.8 ml/min. Fluorescence detection (lambda ex = 227 nm, lambda em = 310 nm) was used. When 100 microliters of plasma were used, the quantitation limit was 10 ng/ml for the atenolol enantiomers. The assay was applied to measure concentrations of atenolol enantiomers in plasma after intravenous administration of racemic atenolol to rats.     

Enantioselective drug monitoring of (R)- and (S)- propranolol in human plasma via derivatization with optically active (R,R)-O,O-diacetyl tartaric acid anhydride

A sensitive high-performance liquid chromatographic method was developed for the stereoselective assay of (R)- and (S)-propranolol in human plasma. The method involves diethyl ether extraction of the drugs and a racemic internal standard, N-tert.-butylpropranolol, followed by derivatization of the compounds with the chiral reagent (R,R)-O,O-diacetyl tartaric acid anhydride. The resulting diastereomeric derivatives were separated isocratically on a reversed-phase column. Quantitation was achieved by the peak-height ratio method with reference to the internal standard. The assay was accurate and reproducible in the concentration range 1-100 ng of (R)- and (S)-propranolol per ml plasma, using fluorescence detection at lambda ex 290 nm and lambda em 335 nm. The applicability of this method was demonstrated for the determination of concentration-time profiles of propranolol enantiomers in the course of comparative pharmacokinetic studies.     

Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug-protein interactions

In this study, sequential injection affinity chromatography was used for drug-protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy DeltaG, enthalpy DeltaH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 x 10(6)M(-1) at pH 7.4 and 39 degrees C for a single binding site. The associated change in enthalpy (DeltaH) was -27.36 kcal mol(-1) and the change in entropy (DeltaS) was -73 cal mol(-1)K(-1) for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (lambda(ext)=230 nm, lambda(em)=350 nm) and spectrophotometric detection (lambda=280 nm).     

Simultaneous stereoselective analysis of tramadol and its primary phase I metabolites in plasma by liquid chromatography. Application to a pharmacokinetic study in humans

This paper describes a bioanalytical method involving a simple liquid-liquid extraction for the simultaneous HPLC determination of the enantiomers of tramadol, the active metabolite O-desmethyltramadol (M1), and the other main metabolite N-desmethyltramadol (M2) in biological samples. Chromatography was performed at 5 degrees C on a Chiracel OD-R column containing cellulose tris(3,5-dimethylphenylcarbamate) as chiral selector, preceded by a achiral end-capped C8 column (LiChrospher 60-RP-selected B 5 microm, 250 mm x 4 mm). The mobile phase was a mixture of phosphate buffer containing sodium perchlorate (1 M) adjusted to pH 2.5-acetonitrile-N,N-dimethyloctylamine (74.8:25:0.2). The flow rate was 0.5 ml/min. Fluorescence detection (lambda(ex) 200 nm/lambda(em) 301 nm) was used. Fluconazol was selected as internal standard. The limit of quantitation of each enantiomer of tramadol and their metabolites was 0.5 ng/ml (sample size = 0.5 ml). The chiral conditions and the LC optimisation were investigated in order to select the most appropriate operating conditions. The method developed has also been validated. Mean recoveries above of 95% for each enantiomer were obtained. Calibration curves for tramadol enantiomers (range 1-500 ng/ml), M1 enantiomers (range 0.5-100 ng/ml), and M2 enantiomers (range 0.5-250 ng/ml) were linear with coefficients of correlation better than 0.996. Within-day variation determined on four different concentrations showed acceptable values. The relative standard deviation (R.S.D.) was determined to be less than 10%. This method was successfully used to investigate plasma concentration of enantiomers of tramadol, O-desmethyltramadol and N-desmethyltramadol in a pharmacokinetic study.     

Synthesis and structural and photoswitchable properties of novel chiral host molecules: axis chiral 2,2'-dihydroxy-1,1'-binaphthyl-appended stiff-stilbene

Novel photoswitchable chiral hosts having an axis chiral 2,2'-dihydroxy-1,1'-binaphthyl (BINOL)-appended stiff-stilbene, trans-(R,R)- and -(S,S)-1, were synthesized by palladium-catalyzed Suzuki-Miyaura coupling and low-valence titanium-catalyzed McMurry coupling as key steps, and they were fully characterized by various NMR spectral techniques. The enantiomers of trans-1 showed almost complete mirror images in the CD spectra, where two split Cotton effects (exciton coupling) were observed in the beta-transitions of the naphthyl chromophore at 222 and 235 nm, but no Cotton effect was observed in the stiff-stilbene chromophore at 365 nm. The structures of (R)-10 and trans-(R,R)-1 were confirmed by X-ray structural analysis. The optimized structure of cis-1 by MO calculations has a wide chiral cavity of 7-8 A in diameter, whereas trans-1 cannot form an intramolecular cavity based on the X-ray data. Irradiation of (R,R)-trans-1 with black light (lambda = 365 nm) in CH3CN or benzene at 23 degrees C led to the conversion to the corresponding cis-isomer, as was monitored by 1H NMR, UV-vis, and CD spectra. At the photostationary state, the cis-1/trans-1 ratio was 86/14 in benzene or 75/25 in CH3CN. On the other hand, irradiation of the cis-1/trans-1 (75/25) mixture in CH3CN with an ultra-high-pressure Hg lamp at 23 degrees C (lambda = 410 nm) led to the photostationary state, where the cis-1/trans-1 ratio was estimated to be 9/91 on the basis of the 1H NMR spectra. The cis-trans and trans-cis interconversions could be repeated 10 times without decomposition of the C=C double bond. Thus, a new type of photoswitchable molecule has been developed, and trans-1 and cis-1 were quite durable under irradiation conditions. The guest binding properties of the BINOL moieties of trans- and cis-(R,R)-1 with F-, Cl-, and H2PO4- were examined by 1H NMR titration in CDCl3. Similar interaction with F- and Cl- was observed in trans-1 (host/guest = 1/1, Kassoc = (1.0 +/- 0.13) x 103 for F- and (4.6 +/- 0.72) x 102 M-1 for Cl-) and cis-1 (host/guest = 1/1, Kassoc = (1.0 +/- 0.13) x 103 for F- and (5.9 +/- 0.69) x 10 M-1 for Cl-), but H2PO4- interacted differently: the cis-isomer formed the 1/1 complex (Kassoc = (9.38 +/- 2.67) x 10 M-1), whereas multistep equilibrium was expected for the trans-isomer.     

Laser flash photolysis of tert-butyl aroylperbenzoates: kinetics of the singlet and triplet states and the aroylphenyl radicals

tert-Butyl aroylperbenzoates (1-4) were studied by laser flash photolysis (LFP). LFP (380 nm, pulse width approximately 350 fs) of 2 and 3 allowed direct observation of their singlet states, which showed broad absorption (lambda(max) approximately 625 nm; tau approximately 20 and approximately 7.9 ps, respectively). The triplet state of each (lambda(max) approximately 530-560 nm) rapidly dissociates by O-O cleavage as indicated by the short triplet lifetimes (e.g., triplet lifetime of 3 approximately 0.74 ns). The approximately 550 nm absorption obtained from the 355 nm LFP (pulse width approximately 7 ns) of 1, 2, and 4 has been assigned to the corresponding aroylphenyl radicals. Two representative radicals (4-benzoylphenyl 5 and 3-(4'-methylbenzoyl)phenyl 6) investigated in detail showed solvent-dependent lifetimes. Absolute bimolecular rate constants of reactions of these radicals with various quenchers including double-bond-containing monomers have been observed to range from 7.56 x 10(7) to 1.68 x 10(9) M(-1) s(-1) in CCl(4) at room temperature. A possible structure of the aroylphenyl radicals and the transition responsible for the 550 nm absorption are discussed.     

Resonance double light scattering method for the determination of proteins with morin-CTMAB

A new determination method of proteins with the limit of determination at nanogram levels is proposed by using a common spectrofluorimeter to detect intensity of resonance double line scattering (RDLS). Proteins including bovine serum albumin (BSA), human serum albumin (HSA) can combine with morin and cetyltrimethylammonium briomide (CTMAB) in the pH range 7.0-8.0 and produce enhanced RDLS signal at lambda(ex)/lambda(em) 305.0/610.0 nm. Optimization conditions for the morin-protein-CTMAB interaction were tested. In the studied system, BSA/CTMAB/morin = 1:2:3. The association constant of morin with BSA is 5.2 x 10(4). Under the optimum conditions, the linear range is 7.5 x 10(-8)-1.0 x 10(-5) g/ml for BSA, 2.5 x 10(-8)-5.0 x 10(-6) g/ml for HSA. The detection limits (S/N = 3) are 66.0 ng/ml for BSA and 23.0 ng/ml for HSA, respectively. Four synthetic samples were analyzed satisfactorily.     

Studies on the constituents of Gentiana species. II. A new triterpenoid,and (S)-(+)- and (R)-(-)-gentiolactones from Gentiana lutea

A new triterpenoid, 12-ursene-3beta, 11alpha-diol 3-O-palmitate (1), has been isolated from the rhizomes and roots of Gentiana lutea, together with the artificial diene derivative, 9 (11), 12-ursadien-3beta-ol 3-O-palmitate (1a) and five known compounds (3-7). Their structures were established on the basis of spectral analysis. In addition, (+/-)-gentiolactone [(+/-)-2], isolated from this plant, was successfully separated into its enantiomers [(+)-2, (-)-2] for the first time, and the absolute configurations at C-9 of (+)-2, (-)-2 were assigned as S and R, respectively, from the optical rotations and the circular dichroism (CD) spectral data.     

Comprehensive Characterisation of the Ketoprofen-β-Cyclodextrin Inclusion Complex Using X-ray Techniques and NMR Spectroscopy

Racemic ketoprofen (KP) and β-cyclodextrin (β-CD) powder samples from co-precipitation (1), evaporation (2), and heating-under-reflux (3) were analysed using X-ray techniques and nuclear magnetic resonance (NMR) spectroscopy. On the basis of NMR studies carried out in an aqueous solution, it was found that in the samples obtained by methods 1 and 2, there were large excesses of β-CD in relation to KP, 10 and 75 times, respectively, while the sample obtained by method 3 contained equimolar amounts of β-CD and KP. NMR results indicated that KP/β-CD inclusion complexes were formed and the estimated binding constants were approximately 2400 M⁻¹, showing that KP is quite strongly associated with β-CD. On the other hand, the X-ray single-crystal technique in the solid state revealed that the (S)-KP/β-CD inclusion complex with a stoichiometry of 2:2 was obtained as a result of heating-under-reflux, for which the crystal and molecular structure were examined. Among the methods used for the preparation of the KP/β-CD complex, only method 3 is suitable.     

Multiple inverse isotope dilution assay for the stereospecific quantitative determination of R(-) and S(+)-oxaprotiline in biological fluids

An isotope dilution assay for the determination of both oxaprotiline enantiomers in biological samples after administration of the racemic mixture has been developed. The enantiomers were reacted with synthetically prepared, optically pure N-trifluoroacetyl-S(-)-prolyl chloride, followed by high-performance liquid chromatographic separation of the diastereoisomers formed. Quantitation was performed by on-line UV detection at 260 nm and off-line radiometry by liquid scintillation counting. Endogenous compounds and metabolites do not interfere in the assay. Analysis of water and the blood and urine of rats spiked with [14C]oxaprotiline X HCl showed recoveries for S(+)-oxaprotiline X HCl (mean +/- coefficient of variation, n = 4-6) of 98.0 +/- 1.0% (water), 100.5 +/- 0.6% (blood) and 101.5 +/- 2.0% (urine), and for R(-)-oxaprotiline X HCl of 101.3 +/- 2.0% (water), 102.2 +/- 2.1% (blood) and 103.2 +/- 0.2% (urine). A pilot study to determine blood levels of the two enantiomers in two rats dosed with racemic [14C]oxaprotiline X HCl (10 mg/kg i.v.) was carried out to test the method. The results indicated stereoselective disposition of oxaprotiline enantiomers in the rat. The ratio of the areas under the blood concentration curves for R(-)-to S(+)-oxaprotiline X HCl was 1.14.