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.     

Chiral separation and determination of R-(-)- and S-(+)-baclofen in human plasma by high-performance liquid chromatography

The method presented here is a high-performance liquid chromatography (HPLC)-UV detection method for the determination of baclofen R-(-)- and S-(+)-enantiomers in human plasma using a chiral separation technique. Baclofen enantiomers were extracted from human plasma with a reversed-phase solid-phase extraction (SPE) cartridge. The extract was then injected onto a HPLC system with a UV detection system set at 220 nm. The separation was achieved by using a 150x4.6 mm, 5 microm Phenomenex chirex 3216 chiral column with a mobile phase consisting of 0.4 mM CuSO(4) in acetonitrile-20 mM sodium acetate (17:83). The calibration curves were linear for both R-(-)- and S-(+)-enantiomers of baclofen in the concentration range of 20-5000 ng/ml. The average regressions were 0.9980 and 0.9991 for R-(-)- and S-(+)-baclofen, respectively. Inter-day precision was 3.3-5.2% for R-(-)-baclofen and 3.5-3.9% for S-(+)-baclofen at a concentration range of 60-4000 ng/ml. Intra-day precisions were 0.6-4.4 and 0.5-3.5% for R-(-)-baclofen and S-(+)-baclofen, respectively. The average extraction recovery was 81.6% for R-(-)-baclofen, 83.0% for S-(+)-baclofen and 94.0% for the internal standard (p-aminobenzoic acid). The limit of quantitation for both R-(-)- and S-(+)-baclofen in human plasma was 20 ng/ml. The method is simple and easy to operate with accuracy and reproducibility and it is suitable for pharmacokinetic studies.     

Simultaneous enantiospecific separation and quantitation of mephenytoin and its metabolites nirvanol and 4'-hydroxymephenytoin in human plasma by liquid chromatography

A high-performance liquid chromatographic method for the enantiospecific quantitation of S- and R-mephenytoin and its metabolites S- and R-nirvanol and S- and R-4'-hydroxymephenytoin in plasma is described. The compounds were separated using a reversed-phase C(2) column in tandem with a chiral alpha(1)-acid glycoprotein column and were detected using ultraviolet detection at 205 nm. The lower limit of quantification was 10 ng/ml for all compounds using 0.5 ml human plasma (intra-day coefficient of variation <13%, accuracy <+/-20%). The method was validated for human plasma in the concentration range 10-2000 ng/ml for each of the six compounds. The method allows for the simultaneous characterisation of the metabolic capacity of two human drug-metabolising enzymes, CYP2C19 and CYP2B6, and may be used when investigating polymorphisms or changes in activity of these two enzymes.     

Quantitative determination of ondansetron in human plasma by enantioselective liquid chromatography-tandem mass spectrometry

A sensitive and enantioselective method was developed and validated for the determination of ondansetron enantiomers in human plasma using enantioselective liquid chromatography-tandem mass spectrometry. The enantiomers of ondansetron were extracted from plasma using ethyl acetate under alkaline conditions. HPLC separation was performed on an ovomucoid column using an isocratic mobile phase of methanol-5 mM ammonium acetate-acetic acid (20:80:0.02, v/v/v) at a flow rate of 0.40 mL/min. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, using the transitions of m/z 294-->170 for ondansetron enantiomers, and m/z 285-->124 for tropisetron (internal standard). The method was linear in the concentration range of 0.10-40 ng/mL for each enantiomer using 200 microL of plasma. The lower limit of quantification (LLOQ) for each enantiomer was 0.10 ng/mL. The intra- and inter-assay precision was 3.7-11.6% and 5.6-12.3% for R-(-)-ondansetron and S-(+)-ondansetron, respectively. The accuracy was 100.4-107.1% for R-(-)-ondansetron and 103.3-104.9% for S-(+)-ondansetron. No chiral inversion was observed during the plasma storage, preparation and analysis. The method was successfully applied to characterize the pharmacokinetic profiles of ondansetron enantiomers in healthy volunteers after an intravenous infusion of 8 mg racemic ondansetron.     

Chiral recognition based on enantioselective interactions of propranolol enantiomers with cyclosophoraoses isolated from Rhizobium meliloti

Cyclosophoraoses isolated from Rhizobium meliloti, as an NMR chiral shift agent, were used to discriminate propranolol enantiomers. Continuous variation plot made from the complex of cyclosophoraoses with propranolol showed that the diastereomeric complex had predominantly 1:1 stoichiometry through UV spectroscopic analysis. The chiral recognition of propranolol enantiomers by cyclosophoraoses was investigated through the determination of binding constant based on the (13)C NMR chemical shift changes. The averaged K(obs) values from the plots were 55.7 M(-1) for (R)-(+)-propranolol and 36.6 M(-1) for (S)-(-)-propranolol, respectively. Enantioselectivity (alpha = K(R+)/K(S(-)) of 1.52 was then obtained. Computational calculation also revealed that (R)-(+) propranolol was more tightly bound with cyclosophoraose than (S)-(-)-propranolol due to the enhanced van der Waals interaction.     

Assay of physiological levels of 2,3-butanediol diastereomers in blood and urine by gas chromatography-mass spectrometry

We present an assay for 2,3-butanediol by gas chromatography-mass spectrometry of its trimethylsilyl ethers. 2R,3R- and/or 2S,3S-2,3-butanediol and meso-2,3-butanediol are quantitated with corresponding internal standards of [2,3-2H2]butanediol. Limits of detection are 1 and 0.1 microM for split and splitless injections, respectively. Blood concentrations of 2,3-butanediol in nonalcoholics are 0.5 +/- 0.3 (SD) microM for 2R,3R- and/or 2S,3S-2,3-butanediol and 0.8 +/- 0.4 microM for meso-2,3-butanediol (n = 9). Two hours after alcohol ingestion, blood levels had risen in eight of nine subjects to 1.2 +/- 0.7 microM for 2R,3R-/2S,3S-2,3-butanediol and to 1.2 +/- 0.6 microM for meso-2,3-butanediol. Baseline urinary excretion of 2,3-butanediol is 0.4 +/- 0.2 mumol/mmol creatinine for 2R,3R-/2S,3S-2,3-butanediol and 0.9 +/- 0.5 mumol/mmol creatinine for meso-2,3-butanediol.     

Simultaneous determination of d- and l-propranolol in human plasma by high-performance liquid chromatography

A method for the determination of d- and l-propranolol in human plasma is described. The method involves extraction of propranolol from plasma, and the formation of diastereomeric derivatives with the chiral reagent N-trifluoroacetyl-1-prolylchloride. Separation and quantitation of the diastereomeric propranolol derivatives are carried out by a reversed-phase high-performance liquid-chromatographic system with fluorimetric detection. The reproducibility in the determination of d- and l-propranolol in human plasma was 4.5% (relative standard deviation) at drug levels of 10 ng/ml.In two subjects who received a single 40-mg tablet of racemic propranolol the plasma levels of the d-isomer were lower than of the l-propranolol. The half-lives of d- and l-propranolol were similar.     

Toxic doses of rac-, (−)-(S)- and (+)-(R)-propranolol in rats and rabbits

The contribution of the individual enantiomers ([+]-[R]- and [−]-[S]-propranolol) to rac-propranolol intoxication was studied in anaesthetized, spontaneously breathing (SB) rats and artificially ventilated (AV) rats and rabbits. In the SB rat, propranolol (30 mg.kg⁻¹.h⁻¹ i.v.) decreased heart rate and mean arterial blood pressure and caused hypoventilation, serious hypoxaemia, respiratory acidosis, and death by respiratory arrest. Survival time (ST) in the (+)-(R)-propranolol group (ST 91 ± 5 min) was significantly longer than in the rac-propranolol group (ST 68 ± 6 min). In AV rats and rabbits toxic doses of rac-, (−)-(S)- and (+)-(R)-propranolol, 30 mg.kg⁻¹.h⁻¹ and 15 mg.kg⁻¹.h⁻¹ i.v., respectively, induced comparable effects on haemodynamic variables as in the SB rat. Artificial ventilation lengthened ST by a factor of three to four in rats. In the AV rat, ST's were not significantly different between the rac-, (−)-(S)- and (+)-(R)-propranolol groups. In the rabbit, as in the SB rat, ST in the (+)-(R)-propranolol group was significantly longer than ST's in the rac- and (−)-(S)-propranolol groups. The acute respiratory acidosis in SB rats and the prolonged ST in AV rats suggest that respiratory failure is the primary and cardiovascular failure the secondary cause of death in propranolol intoxication. The potentiation of the toxic effect of the enantiomers observed after dosing the racemate instead of the pure enantiomers could not be explained by a stereoselective difference in plasma propanolol concentration. © 1996 Wiley-Liss, Inc.     

Enantiospecific quantification of hexobarbital and its metabolites in biological fluids by gas chromatography/electron capture negative ion chemical ionization mass spectrometry.

A highly sensitive and specific assay based on gas chromatography/electron capture negative ion chemical ionization mass spectrometry has been developed for the analysis of the enantiomers of hexobarbital and its major metabolites in human urine and plasma. S-(+)-(5-2H3)hexobarbital and R-(-)-(5-2H3)hexobarbital were synthesized for clinical studies along with (+/-)-(1,5-2H6)hexobarbital and the deuterated major metabolites for use as internal and reference standards. Hexobarbital enantiomers and their metabolites were analyzed after pentafluorobenzyl and trimethylsilyl derivatization, following solid-phase extraction from plasma and urine. Intense negative ion spectra were observed for all of the derivatives. The base peak in the spectra corresponded to the M-pentafluorobenzyl anion [M-PFB]- except for 1,5-dimethylbarbituric acid, where M-. was the most abundant ion. The applicability of the method was demonstrated by following the plasma concentration-time profiles and urinary excretion in a male extensive metabolizer of mephenytoin who was given a pseudoracemic oral dose of hexobarbital containing equal 50 mg amounts of S-(+)-2(H0)hexobarbital and R-(-)-(2H3)hexobarbital. Marked stereoselective disposition was observed, with the R-(-)-enantiomer being more efficiently metabolized, primarily by alicyclic oxidation and ring cleavage.     

Simultaneous stereoselective analysis of venlafaxine and O-desmethylvenlafaxine enantiomers in human plasma by HPLC-ESI/MS using a vancomycin chiral column

A high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI/MS) method for simultaneous stereoselective analysis of venlafaxine (VEN) and its major metabolite O-desmethylvenlafaxine (ODV) enantiomers in human plasma has been developed and validated. Chiral chromatography is performed on the CHRIOBIOTIC V (5 microm, 250 mm x 4.6 mm) column with mobile phase constituted of 30 mmol/l ammonium acetate-methanol (15:85, pH 6.0) at a flow rate of 1.0 ml/min and a postcolumn splitting ratio of 3:1. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and detected using the selected ion recording (SIR) mode. Calibration curves obtained from spiked plasma were linear in the range of 5.0-400 ng/ml for S-(+)-VEN and R-(-)-VEN, 4.0-280 ng/ml for S-(+)-ODV and R-(-)-ODV, respectively, with linear correlation coefficient all above 0.999. The average extraction recoveries for all the four analytes were above 76%. The methodology recoveries were higher than 92%. The limit of detection were 1.0 ng/ml for S-(+)-VEN and R-(-)-VEN, 1.5 ng/ml for S-(+)-ODV and R-(-)-ODV, respectively. The intra- and inter-day variation coefficients were less than 9%.     

Toxicokinetics of a single intravenous dose of rac-propranolol versus optically pure propranolol in the rat

Conscious male Wistar SPF Riv:TOX rats were dosed intravenously with 2.5, 5, or 10 mg/kg rac-propranolol·HCl, or with 5 mg/kg of either (-)-(S)- or (+)-(R)-propranolol·HCl. Disposition of (-)-(S)- and (+)-(R)-propranolol after dosing of rac-propranolol was linear in the dose range examined. Total plasma clearance was not changed in animals dosed with the individual enantiomers compared to the animals that were dosed with rac-propranolol. However, for (-)-(S)-propranolol both volume of distribution and elimination half-life decreased, whereas for (+)-(R)-propranolol increases were observed for these characteristics, in animals dosed with the individual enantiomers. Our observations suggest that the (+)-(R)-enantiomer competes with (-)-(S)-propranolol for plasma protein binding sites, resulting in lower plasma protein binding of the (-)-(S)-enantiomer when the racemate is administered. From recent toxicological experiments, it was concluded that rac-propranolol is more toxic than the individual enantiomers in the rat, when dosed iv at the same total mass. It is concluded that the observed potentiation of toxic effects of propranolol enantiomers when administered as a racemate can at least partly be explained by a pharmacokinetic interaction. © 1995 Wiley-Liss, Inc.     

Stereoselective degradation of benalaxyl in tomato, tobacco, sugar beet, capsicum, and soil

The stereoselective degradation of the racemic benalaxyl in vegetables such as tomato, tobacco, sugar beet, capsicum, and the soil has been investigated. The two enantiomers of benalaxyl in the matrix were extracted by organic solvent and determined by validated chiral high-performance liquid chromatography with a cellulose-tris-(3, 5-dimethylphenylcarbamate)-based chiral column. Rac-benalaxyl was fortified into the soil and foliar applied to vegetables. The assay method was linear over a range of concentrations (0.5-50 microg ml(-1)) and the mean recoveries in all the samples were more than 70% for the two enantiomers. The limit of detection for both enantiomers was 0.05 microg g(-1). The results in soil showed that R-(-)-enantiomer dissipated faster than S-(+)-enantiomer and the stereoselectivity might be caused by microorganisms. In tomato, tobacco, sugar, beet, and capsicum plants, there was significantly stereoselective metabolism. The preferential absorption and degradation of S-(+)-enantiomer resulted an enrichment of the R-(-)-enantiomer residue in all the vegetables.     

Bisanthraquinone glycosides of Hypericum perforatum with binding inhibition to CRH-1 receptors

Four new bisanthraquinone glycosides, S-(+)-skyrin-6-O-beta-glucopyranoside (1), R-(-)-skyrin-6-O-beta-glucopyranoside (2), S-(+)-skyrin-6-O-beta-xylopyranoside (3) and S-(+)-skyrin-6-O-beta-alpha-arabinofuranoside (4), have been isolated from an ethanol-water (1:1, v/v) dry extract of the aerial parts of Hypericum perforatum L. The structures were elucidated by spectroscopic methods, mainly NMR and mass spectrometry. Circular dichroism was used to determine their axial stereochemistry revealing 1 and 2 to be atropisomers. 1 and 2 inhibited [125I]sauvagine binding to corticotropin releasing hormone (CRH-1) receptors.     

Side chain hydroxylations in bile acid biosynthesis catalyzed by CYP3A are markedly up-regulated in Cyp27-/- mice but not in cerebrotendinous xanthomatosis.

The accumulation of various 25-hydroxylated C(27)-bile alcohols in blood and their excretion in urine are characteristic features of cerebrotendinous xanthomatosis (CTX) a recessively inherited inborn error of bile acid synthesis caused by mutations in the mitochondrial sterol 27-hydroxylase (CYP27) gene. These bile alcohols may be intermediates in the alternative cholic acid side chain cleavage pathway. The present study was undertaken to identify enzymes and reactions responsible for the formation of these bile alcohols and to explain why Cyp27(-/-) mice do not show CTX-related abnormalities. Microsomal activities of 5beta-cholestane-3alpha,7alpha,12alpha-triol 25- and 26-hydroxylases, 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol 23R-, 24S-, and 27-hydroxylases and testosterone 6beta-hydroxylase, a marker enzyme for CYP3A, in Cyp27(-/-) mice livers were markedly up-regulated (5.5-, 3.5-, 6.5-, 7.5-, 2.9-, and 5.4-fold, respectively). In contrast, these enzyme activities were not increased in CTX. The activities of 5beta-cholestane-3alpha,7alpha,12alpha-triol 25- and 26-hydroxylases and 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol 23R-, 24R-, 24S-, and 27-hydroxylases were strongly correlated with the activities of testosterone 6beta-hydroxylase in control human liver microsomes from eight unrelated donors. Troleandomycin, a specific inhibitor of CYP3A, markedly suppressed these microsomal side chain hydroxylations in both mouse and human livers in a dose-dependent manner. In addition, experiments using recombinant overexpressed human CYP3A4 confirmed that these microsomal side chain hydroxylations were catalyzed by a single enzyme, CYP3A4. The results demonstrate that microsomal 25- and 26-hydroxylations of 5beta-cholestane-3alpha,7alpha,12alpha-triol and microsomal 23R-, 24R-, 24S-, and 27-hydroxylations of 5beta-cholestane-3alpha,7alpha,12alpha,25-tetrol are mainly catalyzed by CYP3A in both mice and humans. Unlike Cyp27(-/-) mice, CYP3A activity was not up-regulated despite marked accumulation of 5beta-cholestane-3alpha,7alpha,12alpha-triol in CTX.     

Enantioselective binding to the human organic cation transporter-1 (hOCT1) determined using an immobilized hOCT1 liquid chromatographic stationary phase

A liquid chromatography stationary phase containing immobilized membranes obtained from a cell line that expresses the human organic cation transporter (hOCT1-IAM) has been used to study the binding of the enantiomers of propranolol, atenolol, pseudoephedrine, and alpha-methylbenzylamine to the immobilized hOCT1. Frontal displacement chromatography was used to determine the binding affinities (K(d) values), and the data demonstrate that there was an enantioselective difference in the K(d) values of the enantiomers of propranolol, atenolol, and pseudoephedrine, while alpha-methylbenzylamine did not significantly bind to the transporter. Competitive inhibition studies with the cell line used to create the chromatographic column demonstrated that, for the enantiomers of propranolol, the ratio of the chromatographically determined K(d) values [K(d (+)-(R)-propranolol)/K(d (-)-(S)-propranolol) = 2.98] reflected an enantioselective difference in the functional activity of the two enantiomers [IC(50 (+)-(R)-propranolol)/IC(50 (-)-(S)-propranolol) = 2.75]. The chromatographically determined K(d) values were used to construct an initial pharmacophore which contains a hydrogen bond donating site that appears to be responsible for the observed enantioselectivity.     

Synthesis and characterization of stereoisomers of 5,6-dihydro-5,6-dihydroxy-thymidine

Six products were isolated by reverse phase HPLC from the reaction of thymidine with osmium tetroxide. Four of the products were identified as stereoisomers of 5,6-dihydro-5,6-dihydroxy-thymidine (TG). The absolute configurations of these four compounds (from the shortest to the longest HPLC retention times) were determined by two-dimensional nuclear magnetic resonance spectroscopy to be (-)-trans-5S,6S-, (+)-trans-5R,6R-, (-)-cis-5R,6S-, and (+)-cis-5S,6R-5,6-dihydro-5,6-dihydroxy-thymidine. The other two products were dimers with unknown linking sites. Parameters of the mass and nuclear magnetic resonance spectra are reported and discussed.     

Quantitation of 1,3-butanediol and its acidic metabolites by gas chromatography-mass spectrometry

A number of problems present themselves during the gas chromatographic-mass spectrometric assay of R,S-1,3-butanediol as its bis-tert-butyldimethylsilyl ether. To circumvent these problems, three labeled internal standards were synthesized: (i) R,S-1,3-[3,4-13C2]-butanediol, (ii) R,S-1,3-[1,1,3-2H3]butanediol, and (iii) R,S-1,3-[1,1,3-2H3,3,4-13C2]butanediol. The availability of internal standards with different degrees of labeling allows (i) assaying of either unlabeled or 13C-labeled R,S-1,3-butanediol and (ii) analysis of 1,3-butanediol in either blood or urine samples. Reproducible standard curves were obtained using both electron impact and ammonia chemical ionization modes. The latter provides greater sensitivity and a lower limit of detection (5 microM). We have also designed an indirect assay of S-3-hydroxybutyrate, a catabolite of R,S-1,3-butanediol, which is difficult to analyze by conventional methods. This assay relies on the difference between (i) the concentration of R,S-3-hydroxybutyrate assayed by gas chromatography-mass spectrometry and (ii) the concentration of R-3-hydroxybutyrate assayed enzymatically.     

Stereoselective determination of the CYP2C19 probe drug mephenytoin in human urine by gas chromatography-mass spectrometry

A sensitive, specific and reproducible gas chromatographic assay utilizing mass-selective detection has been developed for the stereoselective determination of mephenytoin (MP) in human urine. Following extraction of urine samples using methyl tert.-butyl ether, separation of R- and S-MP was achieved with a chiral capillary column; detection and quantitation were accomplished by mass spectrometry in the single ion monitoring mode (m/z 104 and 189). Excellent linearity was observed for both enantiomers over the concentration range of 5-1000 ng/ml with corresponding correlation coefficients (r)>0.99. The intra- and inter-day precision and accuracy were within +/-5%. This method employs a simplified processing procedure, demonstrates improved extraction recovery, and provides at least 5-fold greater sensitivity than previously reported assays. This method is well suited for the phenotypic evaluation of CYP2C19 activity using mephenytoin.     

Stable expression of monkey cytochrome P-450IA1 cDNA in Chinese hamster CHL cells and its application for detection of mutagenicity of aflatoxin B1.

A monkey cytochrome P-450IA1 cDNA (MKah1) was transfected into Chinese hamster CHL cells using a vector containing the SR alpha promoter, and sublines stably expressing P-450IA1 were established. The cells showed 25-fold higher sensitivity to the cytotoxic effect of aflatoxin B1 than the parental CHL cells. This hypersensitivity was almost completely suppressed by alpha-naphthoflavone, which is a known specific inhibitor of P-450IA. The cells expressing P-450IA1, but not CHL cells, showed a positive response to aflatoxin B1 in an assay for mutagenicity at the HGPRT locus.     

Comparison of interactions of R-(+)- and S-(-)-isomers of beta-adrenergic partial agonists, befunolol and carteolol, with high affinity site of beta-adrenoceptors in isolated rabbit ciliary body and guinea-pig taenia caeci.

The stereoselectivities of beta-adrenergic partial agonists for the high affinity binding site of beta-adrenoceptors in the rabbit ciliary body and the guinea-pig taenia caeci were studied. The pA2 values of the S-(-)-isomers of befunolol and carteolol against S-(-)-isoprenaline, which were calculated from the shift of each concentration - response curve in increasing cyclic AMP levels, were significantly larger than those of the R-(+)-isomers in the guinea-pig taenia caeci, while the pA2 values of the S-(-)-isomers were not significantly larger than those of the R-(+)-isomers in the rabbit ciliary body. The pK1 values determined from the binding experiments were in good agreement with the pA2 values from the increases in cyclic AMP levels. These results suggest that the high affinity binding site of beta-adrenoceptors in the guinea-pig taenia caeci may be able to discriminate stereoselectively between the R-(+)- and S-(-)-isomers, while in the rabbit ciliary body there is no stereo-selectivity between the two enantiomers.