Stereoselective metabolism of benalaxyl in liver microsomes from rat and rabbit

Benalaxyl (BX), methyl‐N‐phenylacetyl‐N‐2,6‐xylyl alaninate, is a potent acylanilide fungicide and consist of a pair of enantiomers. The stereoselective metabolism of BX was investigated in rat and rabbit microsomes in vitro. The degradation kinetics 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 t_1/2 of (?)‐R‐BX and (+)‐S‐BX in rat liver microsomes were 22.35 and 10.66 min of rac‐BX and 5.42 and 4.03 of BX enantiomers. However, the t_1/2 of (?)‐R‐BX and (+)‐S‐BX in rabbit liver microsomes were 11.75 and 15.26 min of rac‐BX and 5.66 and 9.63 of BX enantiomers. The consequence was consistent with the stereoselective toxicokinetics of BX in vitro. There was no chiral inversion from the (?)‐R‐BX to (+)‐S‐BX or inversion from (+)‐S‐BX to (?)‐R‐BX in both rabbit and rat microsomes. These results suggested metabolism of BX enantiomers was stereoselective in rat and rabbit liver microsomes. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Simultaneous determination of the enantiomers of esmolol and its acid metabolite in human plasma by reversed phase liquid chromatography with solid-phase extraction

A stereoselective RP-high performance liquid chromatography (HPLC) assay to determine simultaneously the enantiomers of esmolol and its acid metabolite in human plasma was developed. The method involved a solid-phase extraction and a reversed-phase chromatographic separation with UV detection (lambda = 224 nm) after chiral derivatization. 2,3,4,6-tetra-O-acetyl-beta-d-glucopyranosyl isothiocyanate (GITC) was employed as a pre-column chiral derivatization reagent. The assay was linear from 0.09 to 8.0 microg/ml for each enantiomer of esmolol and 0.07-8.0 microg/ml for each enantiomer of the acid metabolite. The absolute recoveries for all enantiomers were >73%. The intra- and inter-day variations were <15%. The validated method was applied to quantify the enantiomers of esmolol and its metabolite in human plasma for hydrolysis studies.     

Stereoselective determination of cisapride, a prokinetic agent, in human plasma by chiral high-performance liquid chromatography with ultraviolet detection: application to pharmacokinetic study

We have developed a simple, sensitive, specific and reproducible stereoselective high-performance liquid chromatography technique for analytical separation of cisapride enantiomers and measurement of cisapride enantiomers in human plasma. A chiral analytical column (ChiralCel OJ) was used with a mobile phase consisting of ethanol–hexane–diethylamine (35:64.5:0.5, v/v/v). This assay method was linear over a range of concentrations (5–125 ng/ml) of each enantiomer. The limit of quantification was 5 ng/ml in human plasma for both cisapride enantiomers, while the limit of detection was 1 ng/ml. Intra- and inter-day C.V.s did not exceed 15% for all concentrations except at 12.5 ng/ml for EII (+)-cisapride, which was 20 and 19%, respectively. The clinical utility of the method was demonstrated in a pharmacokinetic study of normal volunteers who received a 20 mg single oral dose of racemic cisapride. The preliminary pharmacokinetic data obtained using the method we describe here provide evidence for the first time that cisapride exhibits stereoselective disposition.     

A sensitive gas chromatographic/mass spectrometric method for the resolution and quantification of ethosuximide enantiomers in biological fluids

A modified specific, sensitive and reproducible chiral gas chromatographic (GC) method for the resolution and quantification of ethosuximide enantiomers in urine and plasma was developed. The samples were extracted by liquid-liquid extraction, using diethylether and the enantiomers were separated and quantified on a chiral gas chromatographic column (25QC2 / CYDEX- beta 0.25). The method involved the use of GC/MS instrumentation for the acquisition of data in the electron impact selective-ion monitoring mode, collecting ions characteristic of both ethosuximide and alpha, alpha - dimethyl - beta - methylsuccinimide, the internal standard and of mass-to-charge ratio (m/z) exactly equal to 55 and 70 units. The limit of quantitation of the method was 2.5 microg/ml for both urine and plasma with both enantiomers. The method proved to be linear, precise and reproducible in the 5-300 microg/ml concentration range for urine samples and in the 10-250 microg/ml concentration range for plasma samples. Future research work envisaged the application of this method in pharmacokinetic and pharmacodynamic studies.     

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.     

Enantioselective determination of triazole fungice tetraconazole by chiral high-performance liquid chromatography and its application to pharmacokinetic study in cucumber, muskmelon, and soils

A simple chiral high-performance liquid chromatography method with diode array detector was developed and validated for stereoselective determination of tetraconazole enantiomers in cucumber, muskmelon, and soils. Good separation was achieved at 20°C using cellulose tris-(4-methylbenzoate) as chiral stationary phase, a mixture of n-hexane and ethanol (90:10) as mobile phase at a flow rate of 0.8 ml/min. The assay method was linear over a range of concentrations (0.5-50 μg/ml) and the mean recoveries in all samples were more than 85% for the two enantiomers. The limits of detection for both enantiomers in plant and soil samples were 0.06 and 0.12 μg/g, respectively. Then, the proposed method was successfully applied to the study of enantioselective degradation of rac-tetraconazole in cucumber, muskmelon, and soils. The results showed that the degradation of two enantiomers of tetraconazole followed first-order kinetics and significantly stereoselective behavior was observed in cucumber, muskmelon, and Beijing soil. The preferential absorption and degradation of (-)-S-tetraconzole resulted in an enrichment of the (+)-R-tetraconazole residue in plant samples, whereas the (+)-R-tetraconazole showed a faster degradation in Beijing soil and the stereoselectivity might be caused by microorganisms. No stereoselective degradation was observed in Heilongjiang soil.     

Enantiomeric separation of norgestrel by reversed phase high-performance liquid chromatography using eluents containing hydroxypropyl-beta-cyclodextrin in stereoselective skin permeation study

The chiral separation of norgestrel enantiomers using reversed-phase high-performance liquid chromatography (RP-HPLC) was studied with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral mobile phase additive. The effect of mobile phase composition, concentration of HP-beta-CD and column temperature on enantioselective separation were investigated. The quantification properties of the developed RP-HPLC method were examined. A baseline separation of norgestrel enantiomers was achieved on a Agilent ZORBAX Eclipse XDB-C8 column (150 mm x 4.6 mm i.d., 5 microm). The mobile phase was a mixture of acetonitrile and phosphate buffer (pH 5.0, 20 mM) containing 25 mM HP-beta-CD (30:70, v/v) with a flow rate of 1.0 ml/min. The UV detector was set at 240 nm. Calibration curves were linear (n=8) in the range of 0.2-25 microg/ml, the limit of detection and quantitation were 0.10 and 0.20 microg/ml, respectively, for racemic norgestrel. The values of RSD of repeatability and intermediate precision for spiked sample were less than 4.8%. The method was successfully applied to the enantioselective determination of this drug in stereoselective skin permeation study.     

Influence of the route of administration on the pharmacokinetics of pirprofen enantiomers in the rat

The pharmacokinetics of the enantiomers of the non-steroidal anti-inflammatory drug pirprofen were studied in male Sprague-Dawley rats after oral and intravenous (iv) doses of the racemate. No significant differences were detected between the enantiomers after oral or iv dosing in t_½, Vd, or ∑Xu. However, the R:S area under the plasma concentration (AUC) ratio after oral doses (0.92 ± 0.13) was slightly but significantly lower than after matching iv doses (1.05 ± 0.036). The absolute bioavailability of the active S-enantiomer (78.5%) after oral doses was higher than the inactive R-enantiomer (69.3%). The plasma protein binding of both enantiomers was saturable over a fivefold range of plasma concentrations. At higher plasma concentrations, the S-enantiomer was less bound than the R-enantiomer. In an in vitro experiment using everted rat jejunum, no chiral inversion was discernible. The dependency of the AUC ratio of the enantiomers on the route of administration may be due to stereoselective first-pass metabolism. © 1993 Wiley-Liss, Inc.     

Enantioselective HPLC determination of E-6087, a new COX-2 inhibitor, in human plasma: Validation and pharmacokinetic application

E-6087 is a nonsteroidal anti-inflammatory compound that selectively inhibits cyclooxygenase-2. Because E-6087 has a chiral center, this compound is a racemic mixture of two stereoisomers, (+)-(R)-E-6087 (E-6231) and (-)-(S)-E-6087 (E-6232). A normal-phase liquid-chromatographic method for the enantioselective determination of E-6087 in human plasma was developed and validated. The samples were extracted using solid-phase extraction cartridges containing C(18) sorbent, and the extracts were redissolved in absolute ethanol and injected into the chromatographic system. The enantiomeric separation was achieved on a chiral stationary-phase column of derivatized amylose, and the enantiomers were quantified by fluorescence detection. The method was validated for drug concentrations ranging from 5 to 400 ng/ml for both enantiomers. No peaks interfering with the quantification of enantiomers were observed. The limit of quantification was 5 ng/ml, with precision expressed as a coefficient of variation lower than 10.6% and accuracy expressed as relative error lower than 12.2%. The utility of this method was demonstrated by analysis of plasma samples from healthy volunteers given an oral dose of rac-E-6087. Peak plasma levels of E-6231 were higher than levels obtained for E-6232. Results were consistent with those obtained with a conventional reversed-phase method used for determination of the racemic compound.     

Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of tetrahydropalmatine enantiomers in dogs

A selective chiral high performance liquid chromatographic (HPLC) method coupled with achiral column was developed and validated to separate and quantify tetrahydropalmatine (THP) enantiomers in dog plasma. Chromatography was accomplished by two steps: (1) racemic THP was separated from biological matrix and collected on a Kromasil C18 column (150 mmx4.6 mm, 5 microm) with the mobile phase acetonitrile-0.1% phosphoric acid solution, adjusted with triethylamine to pH 6.15 (47:53); (2) enantiomeric separation was performed on a Chiralcel OJ-H column (250 mmx4.6 mm, 5 microm) with the mobile phase anhydrous ethanol. The detection wavelength was set at 230 nm. (+)-THP and (-)-THP were separated with a resolution factor (Rs) of at least 1.6 and a separation factor (alpha) greater than 1.29. Linear calibration curves were obtained over the range of 0.025-4 microg/ml in plasma for each of (+)-THP and (-)-THP (R2>0.999) with a limit of detection (LOD) of 0.005 microg/ml and the recovery was greater than 88% for each enantiomer. The relative standard deviation (R.S.D.) and relative error values were less than 10% at upper and lower concentrations. The method was used to determine the pharmacokinetics of THP enantiomers after oral administration of racemic THP. The results presented herein showed the stereoselective disposition kinetics of THP in dogs and were a further contribution to the understanding of the kinetic behavior of THP analogues.     

Enantioselective liquid chromatographic-electrospray mass spectrometric assay of beta-adrenergic blockers: application to a pharmacokinetic study of sotalol in human plasma

An enantioselective high performance liquid chromatographic-electrospray ionization mass spectrometric (HPLC-ESI-MS) method for the direct determination of several beta-adrenergic blockers was developed and validated. The method is based on the direct separation of the enantiomers of drugs on a laboratory-made chiral stationary phase (CSP) containing covalently bonded teicoplanin (TE) as chiral selector. Detection of the effluent was performed by electrospray ionization mass spectrometry, run in the selected-ion recording (SIR) mode. The method was applied to the pharmacokinetic monitoring of sotalol (STL) in the plasma of five young healthy volunteers, dosed with racemic drug. The limits of quantitation (LOQ) reached 4 ng/ml for both sotalol enantiomers. Such a method, fully validated, offers a novel, fast and very efficient tool for the direct determination of sotalol enantiomers in human plasma, and can be generally applied to the beta-adrenergic blockers stereoselective pharmacokinetics.     

Stereoselective pharmacokinetics of tetrahydropalmatine after oral administration of (-)-enantiomer and the racemate

Tetrahydropalmatine (THP) is a biologically active ingredient isolated from a traditional Chinese herb Rhizoma corydalis (yanhusuo). THP is a racemic mixture which contains 50% of the (+) and 50% of (-) enantiomer. The (-) enantiomer accounts for most of the analgesic effects. Plasma concentrations of THP enantiomers were analyzed by chiral high-performance liquid chromatography (HPLC) on a Chiralcel OJ column with quantification by UV at 230 nm. The method was used to determine the pharmacokinetics of THP enantiomers in rats and dogs after oral administration of rac-THP or (-)-THP. The pharmacokinetic profiles of the two enantiomers after dosing with rac-THP were significantly different both in rats and dogs. The mean C(max) and AUC(0-infinity) values in rats were 1.93 +/- 0.36 microg/ml and 6.65 +/- 2.34 microg x h/ml for the (-) enantiomer, and 1.11 +/- 0.25 microg/ml and 2.03 +/- 0.45 microg x h/ml for the (+) enantiomer. The mean C(max) and AUC(0-infinity) in dogs were 1.60 +/- 0.81 microg/ml and 9.88 +/- 2.58 microg x h/ml for the (-) enantiomer, while 0.36 +/- 0.21 microg/ml and 1.22 +/- 0.40 microg x h/ml for the (+) enantiomer. rac-THP at 40 mg/kg and (-)-THP at 20 mg/kg had very similar plasma concentration-time profiles, and C(max), AUC(0-infinity), and t(1/2) of the (-) enantiomer in both rats and dogs, indicating that the two treatments were equivalent with respect to the pharmacokinetic properties of the (-) enantiomer.     

Bioanalytical chiral chromatographic technique and docking studies for enantioselective separation of meclizine hydrochloride: Application to pharmacokinetic study in rabbits

Enantiomeric resolution and molecular docking studies of meclizine hydrochloride on polysaccharide-based chiral stationary phase comprising cellulose tris(4-methylbenzoate) chiral selector (150 × 4.6 mm, 3.0 μm) were presented. The mobile phase used was acetonitrile:10mM ammonium bicarbonate (95:05, v/v). The developed technique was used to perform the enantioselective assay of meclizine hydrochloride in its marketed formulation. The elution order of meclizine hydrochloride enantiomers was determined by docking studies. Target compound was extracted from rabbit plasma using protein precipitation technique, followed by development of bioanalytical chiral separation method using the same matrix. Application of the method to determine pharmacokinetic parameters of meclizine hydrochloride enantiomers was performed using Phoenix WinNonlin 8.1 software. The results demonstrated stereoselective disposition of meclizine hydrochloride enantiomers in rabbits.     

Determination of Trantinterol Enantiomers in Human Plasma by High‐Performance Liquid Chromatography – Tandem Mass Spectrometry Using Vancomycin Chiral Stationary Phase and Solid Phase Extraction and Stereoselective Pharmacokinetic Application

A sensitive and enantioselective vancomycin chiral stationary phase high‐performance liquid chromatography–tandem mass spectrometry method was developed for the determination of trantinterol enantiomers in human plasma. Baseline resolution was achieved using the vancomycin chiral stationary phase known as Chirobiotic V with polar ionic mobile phase consisting of acetonitrile–methanol (60:40, v/v) containing 0.01% ammonia and 0.02% acetic acid at a flow rate of 1.0 mL/min. Waters Oasis HLB C18 solid phase extraction cartridges were used in the sample preparation of trantinterol samples from plasma. The detection was performed on a triple‐quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization. The calibration curve was linear in a concentration range from 0.0606 to 30.3 ng/mL in plasma, with the lower limit of quantification of 0.0606 ng/mL. The intra‐ and interday precision (relative standard deviation) values were within 9.7% and the accuracy (relative error) was from ?6.6 to 7.2% at all quality control levels. The method was successfully applied to a study of stereoselective pharmacokinetics in human. Chirality 27:327–331, 2015.© 2015 Wiley Periodicals, Inc.     

Stereospecific determination of mefloquine in biological fluids by high-performance liquid chromatography

A sensitive stereoselective HPLC method was developed for determination of mefloquine (MFQ) enantiomers in plasma, urine and whole blood. The assay involved liquid-liquid extraction of MFQ from biological fluids with a mixture of hexane and isopropanol in the presence of sodium hydroxide and derivatization of the residue by (+)-(S)-naphthylethylisocyanate (NEIC) as chiral derivatizing reagent. Separation of the resulting diastereomers was performed on a silica normal-phase column using chloroform-hexane-methanol (25:74:1) as the mobile phase with a flow-rate of 1 ml/min. Using 200 μl of plasma or whole blood, the limit of determination was 0.2 μg/ml with UV detection for both enantiomers. The limit of determination in 500 μl of urine was 0.08 μg/ml with UV detection.     

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.     

Development and validation of a stereoselective liquid chromatography-tandem mass spectrometry assay for quantification of S- and R-metoprolol in human plasma

A stereoselective liquid chromatography-tandem mass spectrometry assay was developed and validated for quantification of S- and R-metoprolol at concentrations of 0.5-50 microg/L in human plasma. Metoprolol was extracted from plasma by liquid-liquid extraction with ethyl acetate (82% recovery). Chromatographic separation of the enantiomers was achieved on a chiral Chirobiotic T column using an isocratic mobile phase consisting of methanol/acetic acid/ammonia (100/0.15/0.15, v/v/v). An ion trap mass spectrometer with an electrospray interface was used for detection in the positive mode, monitoring the m/z transition 268-->191 for metoprolol. Standard curves for S- and R-metoprolol fitted quadratic functions (r(2)>or=0.9995) over the range 0.5-50 microg/L in plasma, with 0.5 microg/L representing the limit of quantification. In this range, relative standard deviations were <6% for intra-day precision and <10% for inter-day precision. The accuracy was within the range of 92-105%.     

Enantioselective determination of dencichine in rabbit plasma by high-performance liquid chromatography-electrospray mass spectrometry

An analytical method was developed for the determination of enantiomers of dencichine in plasma. Sample extraction from plasma was achieved by a solid-phase extraction (SPE) procedure using a C(18) cartridge, with carbocisteine as the internal standard. Plasma was deproteinized using inorganic acid and derivatizated before the SPE. Chiral separation of dencichine enantiomers was achieved by pre-column derivatization using o-phthaldialdehyde (OPA) and the chiral thiol N-isobutanoyl-L-cysteine (NIBC) to form diastereoisomeric isoindole derivatives that were separable by ODS column using a gradient solvent programme. The column eluent was monitored using mass spectrometry (MS). The conditions of MS detection were optimized, and selected ion monitoring was used to selectively detect D-dencichine and its arrangement isomer. High sensitivity and selectivity were obtained using this method. The limit of detection was determined to be 10 ng/ml for D-dencichine and 8 ng/ml for L-dencichine in plasma. The linearity was demonstrated over a wide range of concentrations, from 0.5 to 50 microg/ml for both enatiomers. The intra- and inter-day precision (C.V.), studied at four concentrations, was less than 7.0%. No interferences from endogenous amino acids and isomers of dencichine were found. The method was suitable for pharmacokinetic studies of dencichine enantiomers.     

Stereoselective toxicokinetics and tissue distribution of ethofumesate in rabbits

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

Enantiomeric determination of pantoprazole in human plasma by multidimensional high-performance liquid chromatography

Multidimensional HPLC is a powerful tool for the analysis of samples of a high degree of complexity. This work reports the use of multidimensional HPLC by coupling a RAM column with a chiral polysaccharide column to the analysis of Pantoprazole in human plasma by direct injection. The enantiomers from the plasma samples were separated with high resolution on a tris(3,5-dimethoxyphenylcarbamate) of amylose phase after clean-up by a RAM BSA octyl column. Water was used as solvent for the first 5 min in a flow-rate of 1.0 ml/min for the elution of the plasmatic proteins and then acetonitrile-water (35:65 v/v) for the transfer and analysis of pantoprazole enantiomers, which were detected by UV at 285 nm. Analysis time was 28 min with no time spent on sample preparation. A good linear relationship was obtained in the concentration range of 0.20 to 1.5 microg/ml for each enantiomer. Inter and intra-day precision and accuracy were determined by one low (0.24 microg/ml), one medium (0.70 microg/ml) and one high (1.3 microg/ml) plasma concentration and gave a C.V. varying from 1.80 to 8.43% and accuracy from 86 to 92%. Recoveries of pantoprazole enantiomers were in the range of 93.7-101.2%. The validated method was applied to the analysis of the plasma samples obtained from ten Brazilian volunteers who received an 80 mg oral dose of racemic pantoprazole and was able to quantify the enantiomers of pantoprazole in all clinical samples analyzed.