Determination of bevantolol enantiomers in human plasma by coupled achiral-chiral high performance-liquid chromatography

A coupled achiral-chiral high performance liquid chromatographic method was developed and fully validated for the determination of bevantolol enantiomers, (-)-(S)-bevantolol and (+)-(R)-bevantolol, in human plasma. Plasma samples were prepared by solid phase extraction with Sep-Pak Plus C18 cartridges followed by HPLC. Bevantolol enantiomers and (+)-(R)-Propranolol as internal standard (IS) were preseparated from interfering components in plasma on a Phenomenex silica column and bevantolol enantiomers and IS were resolved and determined on a Chiralcel OJ-H chiral stationary phase. The two columns were connected by a switching valve equipped with silica precolumn. The Precolumn was used to concentrate bevantolol in the eluent from the achiral column before back flushing onto chiral phase. A detailed validation of the method was performed accordingly to FDA guidelines. For each enantiomer the assay was linear between 20 and 1600 ng/ml. The quantification limits of both bevantolol enantiomers were 20 ng/ml. The intraday variation was between 1.07 and 12.64% in relation to the measured concentration and the interday variation was 0.91 and 11.79%. The method has been applied to the determination of (-)-(S)- and (+)-(R)-bevantolol in plasma from healthy volunteers dosed with racemic bevantolol hydrochloride.     

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.     

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.     

Determination of the enantiomers of chlorpheniramine and its main monodesmethyl metabolite in urine using achiral–chiral liquid chromatography

The enantiomers of chlorpheniramine and its monodesmethyl metabolite were determined separately in urine by using a coupled achiral–chiral chromatographic system. The two enantiomers of the studied compound and the internal standard were separated from the biological matrix on a cyanopropyl column and reinjected into a chiral amylose AD column where the two enantiomers were separated and quantified by UV detection. The method was validated for chlorpheniramine and for the metabolite within the range 0–1000 ng/ml. It was also applied in a pilot pharmacokinetic study to samples from a volunteer given 8 mg of racemic chlorpheniramine by mouth.     

Stereoselective determination of benalaxyl in plasma by chiral high-performance liquid chromatography with diode array detector and application to pharmacokinetic study in rabbits

A chiral high-performance liquid chromatography method with diode array detector was developed and validated for stereoselective determination of benalaxyl (BX) in rabbit plasma. Good separation was achieved at 20 degrees C using cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase, a mixture of n-hexane and 2-propanol (97:3) as mobile phase at a flow rate of 1.0 ml/min. The assay method was linear over a range of concentrations (0.25-25 microg/ml) in plasma and the mean recovery was greater than 90% for both enantiomers. The limits of quantification and detection for both enantiomers in plasma were 0.25 and 0.1 microg/ml, respectively. Intra- and interday relative standard deviations (RSDs) did not exceed 10% for three-tested concentrations. The method was successfully applied to pharmacokinetic studies of BX enantiomers in rabbits. The result suggested that the pharmacokinetics of BX enantiomers was stereoselective in rabbits.     

Determination of felodipine, its enantiomers, and a pyridine metabolite in human plasma by capillary gas chromatography with mass spectrometric detection

Sensitive methods based on capillary gas chromatography (GC) with mass spectrometric (MS) detection in a selected-ion monitoring mode (SIM) for the determination of racemic felodipine, its enantiomers, and a pyridine metabolite in human plasma are described. Following liquid-liquid extraction from plasma, enantiomers of felodipine were separated on a chiral HPLC column (Chiralcel OJ) and fractions containing each isomer were collected on a continuous basis using a fraction collector. These fractions were later analyzed by GC-MS-SIM. A similar method based on GC-MS-SIM detection was developed for the determination of racemic felodipine and its pyridine metabolite with a minor modification of sample preparation. The limits of quantitation in plasma were 0.1 ng/ml for both the R(+)- and S(−)-enantiomers of felodipine and 0.5 ng/ml for both racemic felodipine and its pyridine metabolite. The stereoselective assay was used to support a clinical study with racemic felodipine, and was capable of analyzing more than 30 plasma samples per day.     

Development and validation of a chiral LC-MS method for the enantiomeric resolution of (+) and (−)-medetomidine in equine plasma by using polysaccharide-based chiral stationary phases

The detection and separation of medetomidine enantiomers from the complex biological matrices poses a great analytical challenge, especially in the field of forensic toxicology and pharmacology. Couple of researchers reported resolution of medetomidine using protein-based chiral columns, but the reported method is quiet challenging and tedious to be employed for routine analysis. This research paper reported a method that enables the enantio-separation of medetomidine by using polysaccharide cellulose chiral column. The use of chiralcel OJ-3R column was found to have the highest potential for successful chiral resolution. Ammonium hydrogen carbonate was the ideal buffer salt for chiral liquid chromatography (LC) with electrospray ionization (ESI)+ mass spectrometry (MS) detection for the successful separation and detection of racemic compound. The method was linear over the range of 0 to 20 ng/mL in equine plasma and the inter-day precisions of levomedetomidine, dexmedetomidine were 1.36% and 1.89%, respectively. The accuracy of levomedetomidine was in the range of 99.25% to 101.57% and that for dexmedetomidine was 99.17% to 100.99%. The limits of quantification for both isomers were 0.2 ng/mL. Recovery and matrix effect on the analytes were also evaluated. Under the optimized conditions, the validated method can be adapted for the identification and resolution of the medetomidine enantiomers in different matrices used for drug testing and analysis.     

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.     

Determination of warfarin enantiomers and hydroxylated metabolites in human blood plasma by liquid chromatography with achiral and chiral separation

An assay comprising two simple, selective and isocratic HPLC methods with UV detection was developed and validated for measuring warfarin enantiomers and all five warfarin monohydroxylated metabolites in patient blood plasma. Following liquid/liquid extraction from 1 ml of blood plasma a baseline separation of analytes was achieved on chiral (alpha(1) acid glycoprotein - AGP) and achiral (C(18)) column. Both methods were consistent (R.S.D.<6.9% for warfarin enantiomers and<8.9% for monohydroxylated metabolites) and linear (r>0.998). The limits of detection were 25 ng/ml for warfarin enantiomers, 25 ng/ml for 4'-, 10-, 6- and 7-hydroxywarfarin, 35 ng/ml for 8-hydroxywarfarin and 50 ng/ml for racemic warfarin. In a clinical study in 204 patients, it was confirmed that the assay is appropriate for evaluation of influences of genetic polymorphisms, demographic factors and concomitant drug treatment on warfarin metabolism.     

Quantification of the individual enantiomer plasma concentrations of the candidate antimalarial agent N-[2,6-dimethoxy-4-methyl-5-[(3-trifluoromethyl)phenoxyl]-8-quinolinyl]-1,4-pentanediamine (WR 238,605)

A high-performance liquid chromatographic method was developed to quantitate the plasma concentrations of the individual enantiomers of a candidate 8-aminoquinoline antimalarial agent WR 238,605 (I). The method employed one-step liquid extraction of a 0.5-ml plasma sample followed by direct injection of the extract through a chiral column and detection by fluorescence. Quantification was achieved using an internal standard. The limit of quantification was 10 ng/ml for each enantiomer. The method is sufficiently sensitive to quantitate the plasma concentrations of both enantiomers for 30 days following a single oral dose of 400 mg of the antimalarial agent administered as the racemic succinate salt to healthy human male volunteers. In nearly all samples taken 12 h to 30 days post-dose from three subjects, the difference in the plasma concentrations of the two enantiomers is less than 10%.     

Separation and quantitation of (R)- and (S)-atenolol in human plasma and urine using an alpha 1-AGP column

A method for the determination of (R)- and (S)-atenolol in human plasma and urine is described. The enantiomers of atenolol are extracted into dichloromethane containing 3% heptafluorobutanol followed by acetylation with acetic anhydride at 60 degrees C for 2 h. The acetylated enantiomers were separated on a chiral alpha 1-AGP column. Quantitation was performed using fluorescence detection. A phosphate buffer pH 7.1 (0.01 M phosphate) containing 0.25% (v/v) acetonitrile was used as mobile phase. The described procedure allows the detection of less than 6 ng of each enantiomer in 1 ml plasma. The relative standard deviation is 4.4% at 30 ng/ml of each enantiomer in plasma. The plasma concentration of (R)- and (S)-atenolol did not differ significantly in two subjects who received a single tablet of racemic atenolol. The R/S ratio of atenolol in urine was approximately 1.     

Simple and efficient method for enantioselective determination of omeprazole in human plasma

A practical and selective HPLC method for the separation and quantification of omeprazole enantiomers in human plasma is presented. C18 solid phase extraction (SPE) cartridges were used to extract the enantiomers from plasma samples and the chiral separation was carried out on a Chiralpak AD column protected with a CN guard column, using ethanol:hexane (70:30) as the mobile phase, at a flow rate of 0.5 ml/min. The detection was carried out at 302 nm. The method proved to be linear in the range of 10-1000 ng/ml for each enantiomer, with a quantification limit of 5 ng/ml. Precision and accuracy, demonstrated by within-day and between-day assays, were lower than 10%.     

Enantioselective determination of mexiletine and its metabolites p-hydroxymexiletine and hydroxymethylmexiletine in rat plasma by normal-phase liquid chromatography-tandem mass spectrometry: application to pharmacokinetics

Mexiletine (MEX), hydroxymethylmexiletine (HMM) and p-hydroxymexiletine (PHM) were analyzed in rat plasma by LC-MS/MS. The plasma samples were prepared by liquid-liquid extraction using methyl-tert-butyl ether as extracting solvent. MEX, HMM, and PHM enantiomers were resolved on a Chiralpak(R) AD column. Validation of the method showed a relative standard deviation (precision) and relative errors (accuracy) of less than 15% for all analytes studied. Quantification limits were 0.5 ng ml(-1) for the MEX and 0.2 ng ml(-1) for the HMM and PHM enantiomers. The validated method was successfully applied to quantify the enantiomers of MEX and its metabolites in plasma samples of rats (n = 6) treated with a single oral dose of racemic MEX.     

Stereoselective HPLC analysis of tertatolol in rat plasma using macrocyclic antibiotic chiral stationary phase

Enantiomeric resolution of teratolol was achieved on a vancomycin macrocyclic antibiotic chiral stationary phase known as Chirobiotic V with UV detection set at 220 nm. The polar ionic mobile phase (PIM) consisted of methanol-glacial acetic acid-triethylamine (100:0.01:0.015, v/v/v) has been used at a flow rate of 0.8 ml min(-1) . The calibration curves in plasma were linear over the range of 5-500 ng ml(-1) for each enantiomer with detection limit of 2 ng ml(-1) . The proposed method was validated in compliance with the international conference on harmonization (ICH) guidelines. The developed method applied for the trace analyses of tertatolol enantiomers in plasma and for the pharmacokinetic study of tertatolol enantiomers in rat plasma. The assay proved to be suitable for therapeutic drug monitoring and chiral quality control for tertatolol formulations by HPLC.     

Plasma determination of the enantiomers of SL 84.0418, a new antihyperglycaemic drug, by HPLC on a chiral alpha 1-AGP column.

SL 84.0418 is a new antihyperglycaemic drug. It is an orally active and selective alpha 2-adrenoceptor antagonist. This molecule, a pyrroloindole derivative, contains an asymmetric center yielding two enantiomers. In order to evaluate the pharmacokinetic profile of both enantiomers following oral administration of the racemate we have developed an HPLC method for their separation and quantification. The liquid-liquid extraction involved three steps with two salting-out procedures at pH 11.5 before and after a back-extraction with 0.005 M H2SO4. The enantiomers were separated by HPLC on a stainless-steel column (100 x 4.0 mm) packed with a chiral alpha 1-acid. The UV response was linear from 1 to 250 ng/ml for both enantiomers. The relative standard deviation (RSD) for reproducibility was below 10.7%. Using quality control samples, precision was found below 7.8% and accuracy was 108%. Extraction recoveries were ca. 60% for both enantiomers and 94% for the internal standard. Column life was brought up to 2 months, which corresponds to about 1,000 injections of biological extract. No guard column was used, but a daily back-flush was carried out. This method is suitable for routine analysis and 30 to 40 plasma samples a day can be processed. This method allows the definition of the pharmacokinetic profile of both enantiomers of SL 84.0418 in human plasma after single oral administration of doses as low as 20 mg of the racemic drug.     

Automated online dual-column extraction coupled with teicoplanin stationary phase for simultaneous determination of (R)- and (S)-propranolol in rat plasma using liquid chromatography-tandem mass spectrometry

An automated online sample extraction method for rat plasma was developed and validated for the quantification of (R)- and (S)-propranolol following the intravenous administration of either the racemate or the individual enantiomers at 5 mg/kg. A dual-column extraction system coupled to a chiral stationary phase (CSP) was used in conjunction with liquid chromatography-tandem mass spectrometry. In this method, two Oasis HLB extraction columns (50x1.0 mm) in parallel were used for online plasma sample purification and teicoplanin CSP (Chirobiotic T) was used for the enantiomeric separation. This method allowed the use of one of the extraction columns for purification while the other was being equilibrated. Hence, the time required for re-conditioning the extraction columns did not contribute to the total analysis time per sample, which resulted in a relatively shorter run time and higher throughput. The lower limit of detection was 0.5 ng/ml and the lower limit of quantification was 2 ng/ml for each enantiomer using 25 microl of rat plasma. The method was validated with a linear calibration curve between 2 and 2000 ng/ml for (R)- and (S)-propranolol, respectively. The intra- and inter-day precision (C.V.) was no more than 7.6% and the accuracy of the assay was between 92 and 103%. The teicoplanin CSP proved to be rugged with excellent reproducibility of chromatographic parameters.     

Determination of fluoxetine, norfluoxetine and their enantiomers in rat plasma and brain samples by liquid chromatography with fluorescence detection

Fluoxetine (FLX) and norfluoxetine (NFLX) racemic mixtures were determined by reversed-phase liquid chromatography with fluorescence detection (lambda(exc)=227 nm, lambda(em)=305 nm). The calibration curves prepared from drug-free plasma and brain were linear in the range of 5-1000 ng ml(-1) and 100-40,000 ng g(-1) for doped samples, with detection limits of 3.2 and 2.1 ng ml(-1) in plasma and 31.5 and 26.1 ng g(-1) in brain tissue for FLX and NFLX, respectively. Enantiomer determination was carried out through normal phase HPLC-FD (lambda(exc)=224 nm, lambda(em)=336 nm) after precolumn chiral derivatization with R-1-(1-naphthyl)ethyl isocyanate. Standard curves also prepared in a drug-free matrix were linear for each enantiomer over the range of 2-1000 ng ml(-1) and 20-7000 ng g(-1) with detection limits for the four compounds ranging between 0.2 and 0.5 ng ml(-1) in plasma and between 3.0 and 8.2 ng g(-1) in brain tissue. In both methods the analytes were isolated from the biological matrix by a new solid-phase extraction procedure with recovery in plasma and brain over 90 and 87%, respectively. The repeatability of this extraction procedure was satisfactory within-day and between-day with CV<9.1%. This study also offered the opportunity to obtain an assessment of the potential relationships between the concentration of individual enantiomers of FLX and NFLX in plasma and brain tissue after chronic treatment with racemic FLX at a dose intended to mimic the human plasma concentration of FLX in standard clinical conditions, and therefore should make for more reliable extrapolation of neurochemical findings in other species.     

Enantioselective determination of FCE 28833, a new potential antiischemic agent,in gerbil plasma using column switching HPLC with UV detection

A sensitive and selective high performance liquid chromatographic method using an automated column switching technique for the determination of FCE 28833 enantiomers in gerbil plasma was developed. After solid-liquid extraction using a Supelcosil C₁₈ cartridge FCE 28833 was eluted on a clean-up column (Spherisorb CN) and the enantiomers were separated using an analytical chiral column (Crownpack CR(+)). The mobile phase (15% methanol in HClO₄ 1 mM) was directed through the columns at a flow rate of 1 ml/min and the fraction eluted between 13 and 40 min was transferred from the clean-up column into the analytical column. FCE 28833 enantiomers were monitored at 257 nm. The limit of quantitation of the method was 20 ng/ml plasma for both enantiomers and proved to be linear, precise, and accurate for the assay of both enantiomers in the 20–6,000 ng/ml concentration range. No interference from the blank gerbil plasma sample was observed. The suitability of the method was assessed using plasma samples obtained from male gerbils treated with a single oral dose (400 mg/kg) of FCE 28833. Chirality 9:133–138, 1997. © 1997 Wiley-Liss, Inc.     

Assay of the enantiomers of ibutilide and artilide using solid-phase extraction,derivatization, and achiral-chiral column-switching high-performance liquid chromatography

Ibutilide fumarate and artilide fumarate, new drugs for the treatment of cardiac arrhythmias, each contain a stereogenic center bearing a secondary alcohol group. Reversed-phase achiral-chiral column-switching HPLC separations of the enantiomers of each compound were developed and validated for quantitation in plasma and other biofluids. The key component of the method was derivatization with 1-naphthyl isocyanate, which enhanced the sensitivity of fluorescence detection and enabled the enantiomers to be separated on a Pirkle column (covalent 3,5-dinitrobenzoyl-d-phenylglycine stationary phase). The lower limit of quantitation of ibutilide fumarate was typically 0.1 ng/ml or less of each enantiomer in 1 ml of plasma. Two of the special features of the column-switching system included operation with two samples in the system at one time, which reduced analysis time to 16 min/sample for ibutilide and 19 min/sample for artilide, and a relay-operated switching of detector outputs, which allowed achiral and chiral column chromatographic data to be gathered from two detectors into a single data acquisition channel.     

HPLC separation technique for analysis of bufuralol enantiomers in plasma and pharmaceutical formulations using a vancomycin chiral stationary phase and UV detection

A sensitive and selective high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of bufuralol enantiomers in plasma and pharmaceutical formulations. Enantiomeric resolution was achieved on a vancomycin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic V with UV detection set at 254 nm. The polar ionic mobile phase (PIM) consisting of methanol-glacial acetic acid-triethylamine (100:0.015:0.010, v/v/v) has been used at a flow rate of 0.5 ml/min. The method is highly specific where other coformulated compounds did not interfere. The stability of bufuralol enantiomers under different degrees of temperature was also studied. The results showed that the drug is stable for at least 7 days at 70 degrees C. The method was validated for its linearity, accuracy, precision and robustness. An experimental design was used during validation to evaluate method robustness. The calibration curves in plasma were linear over the range of 5-500 ng/ml for each enantiomer with detection limit of 2 ng/ml. The mean relative standard deviation (RSD) of the results of within-day precision and accuracy of the drug were 0.05) between inter- and intra-day studies for each enantiomer which confirmed the reproducibility of the assay method. The mean extraction efficiency for S-(-)- and R-(+)-bufuralol from plasma was in the range 97-102% at 15-400 ng/ml level for each enantiomer. The overall recoveries of bufuralol enantiomers from pharmaceutical formulations was in the range 99.6-102.2% with %RSD ranging from 1.06 to 1.16%. The assay method proved to be suitable as chiral quality control for bufuralol formulations by HPLC and for therapeutic drug monitoring.