Determination of cicletanine enantiomers in plasma by high-performance capillary electrophoresis.

A sensitive and selective high-performance capillary electrophoresis procedure was developed for the determination of S(+) and R(-) enantiomers of cicletanine in human plasma. The procedure consisted in extraction of the drug with diethyl ether and analysis by micellar electrokinetic capillary chromatography in a fused-silica capillary using gamma-cyclodextrins in the run buffers and ultraviolet detection. The method was linear from 10 to 500 ng/ml and the limit of detection was 10 ng/ml for each enantiomer in plasma samples. The within-run precision of the method, expressed as relative standard deviation, was 10.4 and 9.6% at 25 ng/ml for S(+) and R(-) cicletanine, and 4.2 and 4.6% at 500 ng/ml, respectively. This method has been used to follow the time course of the concentrations of the cicletanine enantiomers in human plasma after a single therapeutic dose of cicletanine given by mouth.     

Quantification of propranolol enantiomers in small blood samples from rats by reversed-phase high-performance liquid chromatography after chiral derivatization

A high-performance liquid chromatographic (HPLC) technique is described for quantification of R(+)- and S(-)-propranolol from 100-microliters rat blood samples. The procedure involves chiral derivatization with tert.-butoxycarbonyl-L-leucine anhydride to form diastereomeric propranolol-L-leucine derivatives which are separated on a reversed-phase HPLC column. The method as previously reported has been modified for assaying serial blood microsamples obtained from the rat for pharmacokinetic studies. An internal standard, cyclopentyldesisopropylpropranolol, has been incorporated into the assay and several derivatization parameters have been altered. Standard curves for both enantiomers were linear over a 60-fold concentration range in 100-microliters samples of whole rat blood (12.5-750 ng/ml; r = 0.9992 for each enantiomer). Inter- and intra-assay variability was less than 12% for each enantiomer at 25 ng/ml. No enantiomeric interference or racemization was observed as a result of the derivatization. No analytical interference was noted from endogenous components in rat blood samples. Preliminary data from two male Sprague-Dawley rats given a 2.0 mg/kg intravenous dose of racemic propranolol revealed differential disposition of the two enantiomers. R(+)-Propranolol achieved higher initial concentration but was eliminated more rapidly than S(-)-propranolol. Terminal half-lives of R(+)- and S(-)-propranolol were 19.23 and 51.95 min, respectively, in one rat, and 14.50 and 52.07 min, respectively, in the other.     

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.     

Stereospecific high-performance liquid chromatographic assay of acebutolol in human plasma and urine

A sensitive high-performance liquid chromatographic technique is described for the separation of R- and S-acebutolol in human plasma and urine. The procedure involves derivatization with the chiral reagent S-(+)-1-(1-naphthyl)ethyl isocyanate. The resulting diastereoisomers are quantified using normal-phase high-performance liquid chromatography with fluorescence detection (220/389 nm). Virtual baseline separation, free from interference, with achieved (resolution factor = 1.45). Excellent linearity (r greater than 0.998) was observed throughout the range 10-500 ng/l and 2-100 mg/l in plasma and urine, respectively. Inter-assay variability was less than 5% for each enantiomer at concentrations of 10 ng/ml. This method is applicable for the determination of the pharmacokinetics, in man, of acebutolol enantiomers in plasma and urine.     

Simultaneous determination of disopyramide and mono-N-dealkyldisopyramide enantiomers in human plasma by capillary electrophoresis

In this paper, a rapid method for the enantioselective analysis of the antiarrhythmic drug disopyramide and its main metabolite mono-N-dealkyldisopyramide in human plasma by capillary electrophoresis employing the cyclodextrin-modified electrokinetic chromatography mode is described. Sample clean-up was carried out by alkalinization with sodium hydroxide followed by liquid-liquid extraction with toluene. The complete enantioselective analysis was performed within less than 5 min using 20 mmol/L sodium acetate buffer, pH 5.0, containing 0.2% w/v sulfated beta-cyclodextrin as chiral selector. A 40 cm uncoated fused-silica capillary was used for the analysis, performed at a voltage of 15 kV and at 20 degrees C. The calibration curves were linear over the concentration range of 62.5-1850 ng/mL and 125-1850 ng/mL for each enantiomer of disopyramide and mono-N-dealkyldisopyramide. The mean recoveries for disopyramide and mono-N-dealkyldisopyramide enantiomers were up to 87 and 69%, respectively. All four enantiomers studied could be quantified at three different concentrations (200, 400 and 600 ng/mL) with coefficient of variation and % relative error not higher than 15%. The quantitation limit was 62.5 ng/mL for (+)-(S)-and (-)-(R)-disopyramide and (-)-(R)-mono-N-dealkyldisopyramide and 125 ng/mL for (+)-(S)-mono-N-dealkyldisopyramide, using 1 mL of human plasma.     

Determination of saterinone enantiomers in plasma samples with an internal standard using a Chiralcel OD column, fractionation and reversed-phase chromatography

A specific and validated high-performance liquid chromatographic method was developed for the determination of the S-(-) and R-(+) enantiomers of saterinone. 1-[(4-cyano-1,2-dihydro-6-methyl-2-oxopyridin-5-yl)phenoxyl] -3-[4-(2- methoxyphenyl)piperazin-1-yl]propan-2-ol, in plasma at the low ng/ml level. The enantiomers of saterinone and an internal standard, 1-[(4-cyano-1,2-dihydro-6-methyl-2-oxo-pyridin-5-yl)phenoxy]-3-[4-(2- ethoxyphenyl)piperazin-1-yl]propan-2-ol, were chromatographed on a chiral Chiralcel OD stationary phase. However, the S-(-) enantiomers of saterinone and the internal standard were unresolved, as were the R-(+) enantiomers of both substances. Therefore, the two fractions were collected and each was separately resolved on an achiral Polyencap A reversed-phase column and quantified. The detection limit was 0.5 ng/ml of enantiomer, allowing the determination of plasma levels up to 36 h after oral administration of 90, 150 and 180 mg of saterinone to twelve subjects.     

Development of enantioselective gas chromatographic quantitation assay for dl-threo-methylphenidate in biological fluids

An enantioselective gas chromatographic quantitation assay was developed for the enantiomers of dl-threo-methylphenidate in plasma and urine. dl-threo-Methylphenidate and the internal standard were acylated with N-heptafluorobutyryl-1-prolylchloride under Schotten-Baumann conditions prior to gas chromatographic separation on achiral mixed stationary phases. The derivatives were detected by means of a nitrogen-phosphorus detector. Linear and reproducible calibration curves were obtained over the concentration ranges 0.43-43.25 and 2.16-216.25 ng/ml enantiomer in plasma or urine, respectively. This enantioselective gas chromatographic quantitation assay was applied in a single oral dose disposition study of dl-threo-methylphenidate in a healthy adult volunteer. Stereoselective differences were observed in the plasma concentration-time profiles and cumulative urinary excretion profiles following oral doses of 20 and 40 mg of dl-threo-methylphenidate hydrochloride. Only d-threo-methylphenidate was detectable in plasma after 4 h.     

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.     

Simultaneous determination of (R)- and (S)-celiprolol in human plasma and urine: high-performance liquid chromatographic assay on a chiral stationary phase with fluorimetric detection

The quantitative enantiospecific determination of the beta 1-selective adrenergic antagonist (R,S)-celiprolol in human plasma and urine is described. It involves a two-step liquid-liquid extraction of celiprolol from biological material and separation of the underivatized enantiomers by high-performance liquid chromatography on a chiral stationary phase (cellulose tris-3,5-dimethylphenyl carbamate, coated on silica gel) with fluorimetric detection. R-(+)-Propranolol was used as an internal standard. The detection limits of 1.5 ng/ml enantiomer in plasma and 2.5 ng/ml enantiomer in urine at signal-to-noise ratios higher than 3 permit the performance of pharmacokinetic studies after therapeutic doses.     

Enantioanalysis of bisoprolol in human plasma with a macrocyclic antibiotic HPLC chiral column using fluorescence detection and solid phase extraction

A sensitive, enantioselective, high-performance liquid chromatographic (HPLC) method was developed and validated to determine S-(-)- and R-(+)-bisoprolol in human plasma. Baseline resolution was achieved using the teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with a polar ionic mobile phase (PIM) consisting of methanol-glacial acetic acid-triethylamine (100 : 0.02 : 0.025, v/v/v) at a flow rate of 1.5 ml/min and fluorescence detection set at 275 nm for excitation and 305 nm for emission. All analyses with S-(-)-atenolol as the internal standard were conducted at ambient temperature. The assay involved the use of a solid-phase extraction procedure for human plasma samples prior to HPLC analysis. The C18 cartridge gave good recovery rates for both enantiomers without any interference. The method was validated over the range of 20-200 ng/ml for each enantiomer concentration. Recovery rates for S-(-)- and R-(+)-bisoprolol enantiomers were in the range of 95-102%. The method proved to be precise (within-run precision expressed as % RSD ranged from 1.0-6.2% and between-run precision ranged from 0.9-6.7%) and accurate (within-run accuracies expressed as percentage error ranged from 0.2-4.8% and between-run accuracies ranged from 0.3-1.7%). The limit of quantitation and limit of detection for each enantiomer in human plasma were 20 and 5 ng/ml, respectively.     

Enantiospecific determination of PNU-83894 and its major metabolite, PNU-83892, in plasma, and its application to the characterization of the enantioselective pharmacokinetics of PNU-83894 in the dog

A chiral method for the simultaneous analysis of the (+)- and (-)-enantiomers of PNU-83894 and its metabolite, PNU-83892, in plasma was developed to characterize the enantioselective pharmacokinetics of PNU-83894, a potential anticonvulsant candidate. The method involves solid-phase extraction (phenyl column) of the enantiomers from plasma followed by direct enantioselective separation on a beta-cyclodextrin HPLC chiral column and UV detection at 230 nm. The linear range for this method was found to be 12.5 ng/ml to 5.00 microg/ml and the intra- and inter-assay precision and accuracy for each enantiomer were <11% in all cases. The validity of this assay was also demonstrated by its application to the pharmacokinetic evaluation of PNU-83894 in the dog.     

Enantioselective determination of arotinolol in human plasma by HPLC using teicoplanin chiral stationary phase

A sensitive enantioselective high-performance liquid chromatography (HPLC) method was developed and validated to determine S-(+)- and R-(-)-arotinolol in human plasma. Baseline resolution was achieved by using teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with a polar organic mobile phase consisting of methanol:glacial acetic acid:triethylamine, 100:0.1:0.1, (v/v/v) at a fl ow rate of 0.8 mL/min and UV detection set at 317 nm. Human plasma was spiked with stock solution of arotinolol enantiomers and labetalol as the internal standard. The assay involved the use of liquid-liquid extraction procedure with ethyl ether under alkaline condition for human plasma sample prior to HPLC analysis. Recoveries for S-(+)- and R-(-)-arotinolol enantiomers were in the range 93-103% at 200-1400 ng/mL level. Intra-day and inter-day precision calculated as %RSD was in the ranges 1.3-3.4 and 1.9-4.5% for both enantiomers, respectively. Intra-day and inter-day accuracies calculated as percentage error were in the ranges 1.2-3.5 and 1.5-6.2% for both enantiomers, respectively. Linear calibration curves in the concentration range 100-1500 ng/mL for each enantiomer showed a correlation coefficient (r) of 0.9998. The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 100 and 50 ng/mL (S/N = 3), respectively.     

Determination of tolperisone enantiomers in plasma and their disposition in rats.

A stereoselective assay for the determination of tolperisone enantiomers in plasma by high performance liquid chromatography was developed. Calibration curves obtained for the enantiomers were linear over plasma concentrations of 0.1-3.0 micrograms/ml with a detection limit of 20 ng/ml. Following intravenous bolus administration of 10 mg/kg of racemic tolperisone to rats, stereoselective disposition of tolperisone enantiomers was observed, and plasma concentrations were significantly higher for l-tolperisone than for d-tolperisone at 5, 15 and 30 min after administration. When either enantiomer was administered alone to rats, both enantiomers were found in plasma, indicating that a mutual chiral inversion occurs in the body.     

Enantioselective high-performance liquid chromatographic method for the determination of baclofen in human plasma

A new analytical method for the separation and determination of R-(−)- and S-(+)- baclofen enantiomers in human plasma by high-performance liquid chromatography (HPLC) with UV detection was developed. Enantioselective resolution of the baclofen enantiomers was achieved by using teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with a polar ionic mobile phase (PIM) consisting of methanol: glacial acetic acid: triethylamine, 100:0.1:0.1, (v/v/v) at a flow rate of 0.5 ml min⁻¹ and UV detection set at 220 nm. The analytes of interest with S-(+)-sulpiride as the internal standard were extracted from human plasma using liquid-liquid extraction procedure with ethyl ether under alkaline condition prior to HPLC analysis. Recoveries for R-(−)- and S-(+)-baclofen enantiomers were in the ranges of 96-103% at 60-2500 ng ml⁻¹ level. Intra-day and inter-day precision calculated as %RSD was in the ranges of 1.2-5.2 and 1.3-4.3% for both enantiomers, respectively. Intra-day and inter-day accuracy calculated as percentage error were in the ranges of 1.2-3.9 and 1.1-3.9% for both enantiomers, respectively. Linear calibration curves in the concentration ranges of 20-3000 ng ml⁻¹ for each enantiomer showed correlation coefficient (r) of 0.9997. The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 20 and 10 ng ml⁻¹ (S/N=3) respectively.     

Restricted access media-molecularly imprinted polymer for propranolol and its application to direct injection analysis of beta-blockers in biological fluids

A restricted access media-molecularly imprinted polymer (RAM-MIP) for propranolol (PRP) has been prepared for direct injection analysis of beta-blockers in biological fluids. First, the MIP for PRP was prepared using methacrylic acid and ethylene glycol dimethacrylate as the functional monomer and cross-linker, respectively, by a multi-step swelling and polymerization method. Next, a 1:1 mixture of glycerol monomethacrylate and glycerol dimethacrylate was used for hydrophilic surface modification, and added directly to the MIP for PRP after 4 h from the start of polymerization. Then further polymerization was carried out for 20 h. The obtained RAM-MIP for PRP showed excellent molecular recognition ability for PRP, good ones for alprenolol (ALP) and pindolol, and fair ones for other beta-blockers. The RAM-MIP was applied for direct injection analysis of ALP enantiomers in a rat plasma sample by a column-switching HPLC system using a beta-cyclodextrin phenylcarbamate-bonded silica column as the analytical column. The calibration graph, constructed from peak area versus each ALP enantiomer concentration, was linear with a correlation coefficient of > 0.999 over the concentration ranges of 12.5-250 ng ml(-1). The limit of quantitation was 12.5 ng ml(-1) with a 50 microl injection. This method could be applicable for the assay of ALP enantiomers at the therapeutic plasma levels, and have wide applicability for the assay of beta-blockers in biological fluids.     

Validation of a high-performance liquid chromatographic method for the enantiospecific quantitation of zopiclone in plasma

Zopiclone is a hypnosedative with clinical effects similar to benzodiazepines but thought to have less potential for rebound insomnia and withdrawal effects. Zopiclone is administered as a racemic mixture, and an enantiospecific method of analysis of zopiclone in plasma is desirable in the study of pharmacokinetic drug interactions. We report a modification of an HPLC method reported by Foster et al. using a closely related structural analogue of zopiclone as internal standard. Zopiclone was detected at 306 nm and linear calibration curves were constructed in the range of 1.0-250 ng/mL for each enantiomer. The % CV at 2.5 ng/mL was 12.0% for (-)-zopiclone and 14.3% for (+)-zopiclone, and the limit of quantification of each enantiomer was 2.5 ng/mL. At higher concentrations, the coefficient of variation was less than 10%. The nominal concentration of quality control samples was predicted with an accuracy within a range of +/-11.6%. The method was used in the analysis of plasma obtained from psychiatric patients. One sample obtained following a non-fatal overdose with zopiclone contained the metabolites (-)-N-oxide zopiclone and both enantiomers of desmethyl zopiclone. The metabolite enantiomers were resolved on the column with retention times similar to zopiclone. The N-oxide metabolite co-eluted with internal standard.     

A stereospecific high-performance liquid chromatographic assay for the determination of ketoconazole enantiomers in rat plasma

A stereospecific high-performance liquid chromatographic assay was developed for the quantitation of ketoconazole enantiomers (KTZ) in rat plasma. After protein precipitation of 100 microL plasma using acetonitrile, a wash step was performed using hexane. The supernatant was removed and KTZ enantiomers and amiodarone, the internal standard, were extracted using liquid-liquid extraction with tert-butyl methyl ether. After transfer and evaporation of the organic layer, the residue was reconstituted in mobile phase and injected into the HPLC through a chiral column. The mobile phase consisted of hexane:ethanol:2-propanol with diethyl amine, pumped at 1.5 mL/min. All components eluted within 18 min. KTZ enantiomers were baseline resolved and peaks were symmetrical in appearance with no interferences. Calibration curves were linear over the range 62.5-5000 ng/mL of enantiomer. The intraday and interday CV% assessments were 相似文献   

Analysis of metoprolol enantiomers in human serum by liquid chromatography on a cellulose-based chiral stationary phase

Metoprolol is a lipophilic, cardioselective beta-adrenergic blocking agent commercially available as a racemic compound. A normal phase high-performance liquid chromatographic method was developed to directly determine individual enantiomeric concentrations of metoprolol in human serum. Separation of the enantiomers was accomplished by a cellulose-tris(3,5-dimethylphenylcarbamate) chiral stationary phase. Metoprolol enantiomers were detected by means of fluorescence with excitation and emission wavelengths of 275 and 315 nm, respectively. Standard curves were linear over the concentration range 12.5-400 ng/ml for each enantiomer. Within-day coefficient of variation was less than 15% at all concentrations and the between-day coefficient of variation ranged from 4.1 to 11.2%. The limit of detection was determined to be 5 ng/ml for each enantiomer and the stereoselective resolution (alpha) of R- and S-metoprolol was 3.08. The assay was employed to determine enantiomeric serum concentrations of metoprolol in healthy male volunteers.     

Determination of thyroxine enantiomers in pharmaceutical formulation by high-performance liquid chromatography-mass spectrometry with precolumn derivatization

A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method for the separation and analysis of d- and l-thyroxine was developed using R(−)/S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole, [R(−)/S(+)-DBD-PyNCS] as a chiral derivatization reagents. The T₄ derivatives with R(−)-DBD-PyNCS were efficiently separated on a reversed-phase column with water-acetonitrile containing 0.1% formic acid (41:59, v/v) as the eluent and analyzed using ESI-MS with negative selected ion monitoring (SIM) mode. The calibration curves of both the d-T₄ and l-T₄ were linear over the concentration range of 0.13-13 μg/ml. The detection limits (S/N = 3) were 28 ng/ml for d-T₄ and 40 ng/ml for l-T₄, respectively. The relative standard deviations (RSD, n = 5) were less than 3.6% at 1.3 μg/ml for both T₄ enantiomers. The proposed method was applied to the determination of l-T₄ enantiomer in a pharmaceutical formulation.     

Determination of (R)- and (S)-phenprocoumon in human plasma by enantioselective liquid chromatography/electrospray ionisation tandem mass spectrometry

Phenprocoumon is a commonly used oral anticoagulant of the coumarin type, and has found extensive clinical use in the treatment of thrombophlebitis, pulmonary embolism and atrial fibrillation. In the course of a clinical study to investigate the influence of genetic polymorphisms of the CYP2C9 enzyme on phenprocoumon metabolism, we developed a new enantioselective liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/MS/MS) method to quantify (R)- and (S)-phenprocoumon in human plasma. HPLC separation of the enantiomers was achieved on a Chira-Grom-2 column under isocratic conditions using a water/acetonitrile/formic acid eluent. For detection and quantification a triple-quadrupole MS system was used in the selected reaction monitoring (SRM) mode. As an internal standard the structurally homologous compound warfarin was chosen. The detector response was linear with a correlation coefficient of 0.988-0.999 for (R)-phenprocoumon and 0.989-0.999 for (S)-phenprocoumon in the investigated concentration range between 62.5 and 1000 ng/mL (per enantiomer). The limit of detection (LOD) was 12.5 ng/mL.