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.     

Determination of enantiomers of atenolol and propranolol in pharmaceutical formulations by HPLC

A validated HPLC-UV method was developed for the determination of R(-), S(+)-atenolol and R(-), S(+)-propranolol in pharmaceutical formulations. The proposed method required no elaborate sample preparation and was found to be selective, linear, and repeatable within the established ranges. Atenolol and propranolol isomers were separated using a Chirex 3022 (S) column with the mobile phases hexane-dichloromethane-methanol-trifluoroacetic acid (35 + 35 + 5 + 0.25, v/v/v/v) and hexane-dichloromethane-ethanol-trifluoroacetic acid (55 + 40 + 5 + 0.25, v/v/v/v), respectively. The LOD values of R(-) and S(+)-atenolol were 12.3 and 9.86 microg/mL, respectively, and 0.61 and 0.89 microg/mL, respectively, for R(-) and S(+)-propranolol. Retention times of R(-)-propranolol and S(+)-propranolol were 12.4 and 14.3 min, respectively, and 29.06 and 32.71 min, respectively, for (R)-atenolol and (S)-atenolol. The proposed method was applied to the determination of enantiomers in pharmaceutical formulations, and no interference from any excipients was found.     

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.     

Stereoselective analysis of the enantiomers of mexiletine by high-performance liquid chromatography using fluorescence detection and study of their stereoselective disposition in man

A sensitive, stereoselective high-performance liquid chromatographic assay was developed for the resolution of the enantiomers of mexiletine as their 2-naphthoyl derivatives on a Pirkle type 1A chiral phase column. Detection of the derivatives was accomplished with a fluorescent detector. Maximum recovery of the enantiomers from plasma was 83% and was observed when plasma proteins were precipitated with a mixture of barium hydroxide-zinc sulphate. The calibration curve in plasma was linear over the concentration range 5-750 ng/ml for each enantiomer (r2 = 0.999) and in urine the linear range was 0.25-7.5 micrograms/ml (r2 = 0.999) for each enantiomer. The minimum detectable quantity of each enantiomer in plasma was 5 ng/ml at a signal-to-noise ratio of 5:1, representing 100 pg injected. A preliminary pharmacokinetic study was undertaken in one healthy male volunteer following an oral dose of 300 mg of racemic mexiletine hydrochloride. The apparent elimination half-lives determined from the plasma data were 12.1 and 14.1 h for the R(-) and S(+) enantiomers, respectively. The cumulative urinary excretion amounts of R(-)- and S(+)-mexiletine were found to be 8.01 and 10.46 mg, respectively. The plasma data indicated that a cross-over of the enantiomer ratios occurred at approximately 8 h. The urinary excretion of the enantiomers was consistent with the pattern found in plasma.     

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

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

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.     

Recognition and simultaneous determination of ofloxacin enantiomers by synchronization-1st derivative fluorescence spectroscopy

Under controlling pH 3, R-(+)- and S-(-)-ofloxacin (OFLX) enantiomers can be well recognized and resolved by the synchronization-1st derivative fluorescence spectroscopic techniques, and the interference from urine blank also can be eliminated. The linear dynamic ranges are 0.36-2.16 (R), 0.36-2.89 and 3.16-31.6 mug/ml (S), respectively, for determining OFLX in urine samples. The limits of detection are 0.36 mug/ml (R) and the recoveries of R-(+)- and S-(-)-OFLX in urine samples are 97-104%. Relative standard deviation is <6.6%. Pharmacokinetic study of OFLX and levofloxacin shows that R-(+)- and S-(-)-ofloxacin reach their peak concentration in urine samples after a healthy subject has taken tablets for approximately 3 and 6 h, respectively. R-(+)-OFLX can be obviously detected in 5-6 h after a healthy subject has taken tablets, indicating the transformation of S-(-)- to R-(+)-OFLX enantiomer in human body (in vitro).     

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.     

Chiral separation of lipoxygenase metabolites utilizing high-performance liquid chromatography

An isocratic, reversed-phase HPLC assay has been developed for the separation of the enantiomers of four lipoxygenase metabolites, without the need for a derivatization step. Separation of the enantiomers was studied on a polysaccharide type chiral stationary phase column. Upon determination of suitable mobile phase composition, the assay was evaluated at various temperatures. In all cases the R enantiomer eluted before the S enantiomer. The best separations were observed at 0 degrees C.     

Stereoselective high-performance liquid chromatographic assay for pirmenol enantiomers in dog plasma.

Pirmenol enantiomers in dog plasma were quantified using a stereospecific high-performance liquid chromatographic method with ultraviolet detection at 262 nm. Racemic pirmenol and internal standard, (+)-propranolol, were isolated from dog plasma by a three-step extraction procedure using toluene, 0.1 M hydrochloric acid and hexane, respectively. A chiral analytical column (Chiralcel OJ) was used with a mobile phase consisting of hexane-isopropanol-diethylamine (98.9:1.0:0.1). Linear calibration curves were obtained in the concentration range 0.0200-5.00 micrograms/ml for each enantiomer. Precision of the method, expressed as coefficient of variation for nine quality control samples, was 7.1% for (+)-pirmenol and 6.4% for (-)-pirmenol. Bias was +/- 2.2% for (+)-pirmenol and +/- 1.5% for (-)-pirmenol in quality control samples.     

Determination of (S)-(-)-cathinone and its metabolites (R,S)-(-)-norephedrine and (R,R)-(-)-norpseudoephedrine in urine by high-performance liquid chromatography with photodiode-array detection.

A high-performance liquid chromatographic (HPLC) procedure with photodiode-array detection (DAD) is described for the determination of (S)-(-)-cathinone (S-CA) and its metabolites (R,S)-(-)-norephedrine (R-NE) and (R,R)-(-)-norpseudoephedrine (R-NPE) in urine. Extraction and clean-up of 1-ml urine samples were performed on a cyano-bonded solid-phase column using (+/-)-amphetamine as internal standard. The concentrated extracts were separated on a 3-microns ODS-1 column with acetonitrile-water-phosphoric acid-hexylamine as the mobile phase. Peak detection was done at 192 nm. The detection limits for S-CA and R-NE/R-NPE in urine were 50 and 25 ng/ml, respectively. The differentiation of the enantiomers of cathinone and norephedrine was achieved by derivatization with (S)-(-)-1-phenylethyl isocyanate to the corresponding diastereomers followed by HPLC-DAD on a 5-microns normal-phase column. The R and S enantiomers of norpseudoephedrine were determined by gas chromatography-mass spectrometry after on-column derivatization with (S)-(-)-N-trifluoroacetylprolyl chloride. Following a single oral dose of 0.5 mg/kg of S-CA, the concentrations found in urine ranged from 0.2 to 3.8 micrograms/ml of S-CA, from 7.2 to 46.0 micrograms/ml of R-NE and from 0.5 to 2.5 micrograms/ml of R-NPE.     

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.     

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.     

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   

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.     

Development of direct stereoselective and non-stereoselective assays in biological fluids for the enantiomers of a thieno[2,3-b]thiopyran-2-sulfonamide, a topically effective carbonic anhydrase inhibitor

A stereoselective assay for the optical isomers [(S) and (R)] of 5,6-dihydro-4-[(2-methylpropyl)amino]-4H-thieno[2,3-b]thiopyran-2- sulfonamide-7,7-dioxide in human whole blood has been developed. The assay is based on direct enantiomer separation on a chiral stationary phase column of bovine serum albumin attached to silica. The effect of pH, ionic strength, column length and organic modifier on chiral separation has been studied. The assay methodology, based on high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection (252 nm), has been fully validated in the concentration range 25-250 ng/ml of each enantiomer. Since no interconversion of the isomers was observed in vivo for the clinical studies involving the single (S)-enantiomer, a more sensitive (2.5 ng/ml), non-stereoselective assay has been developed. This method, also based on HPLC with UV detection, was fully validated in whole blood, plasma and urine in the concentration range 2.5-100 ng/ml. The details of these assays, together with some representative data from a pilot human study, are also presented.     

Development and Validation of a Chiral Liquid Chromatographic Assay for Enantiomeric Separation and Quantification of Verapamil in Rat Plasma: Stereoselective Pharmacokinetic Application

A novel, fast and sensitive enantioselective HPLC assay with a new core–shell isopropyl carbamate cyclofructan 6 (superficially porous particle, SPP) chiral column (LarihcShell-P, LSP) was developed and validated for the enantiomeric separation and quantification of verapamil (VER) in rat plasma. The polar organic mobile phase composed of acetonitrile/methanol/trifluoroacetic acid/triethylamine (98:2:0.05: 0.025, v/v/v/v) and a flow rate of 0.5 mL/min was applied. Fluorescence detection set at excitation/emission wavelengths 280/313 nm was used and the whole analysis process was within 3.5 min, which is 10-fold lower than the previous reported HPLC methods in the literature. Propranolol was selected as the internal standard. The S-(−)- and R-(+)-VER enantiomers with the IS were extracted from rat plasma by utilizing Waters Oasis HLB C18 solid phase extraction cartridges without interference from endogenous compounds. The developed assay was validated following the US-FDA guidelines over the concentration range of 1–450 ng/mL (r² ≥ 0.997) for each enantiomer (plasma) and the lower limit of quantification was 1 ng/mL for both isomers. The intra- and inter-day precisions were not more than 11.6% and the recoveries of S-(−)- and R-(+)-VER at all quality control levels ranged from 92.3% to 98.2%. The developed approach was successfully applied to the stereoselective pharmacokinetic study of VER enantiomers after oral administration of 10 mg/kg racemic VER to Wistar rats. It was found that S-(−)-VER established higher C_max and area under the concentration-time curve (AUC) values than the R-(+)-enantiomer. The newly developed approach is the first chiral HPLC for the enantiomeric separation and quantification of verapamil utilizing a core–shell isopropyl carbamate cyclofructan 6 chiral column in rat plasma within 3.5 min after solid phase extraction (SPE).     

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.     

Liquid chromatographic analysis of propafenone enantiomers in human plasma

A convenient and sensitive high-performance liquid chromatographic method for analysis of the enantiomers of propafenone (PPF) in human plasma was developed. Racemic propafenone and (-)-ephedrine (internal standard) were first extracted from plasma samples into a mixture of hexane-2-propanol-heptafluorobutanol (95:5:1.25, v/v). After evaporation of the organic layer, the samples were derivatized with R(-)-naphthylethyl isocyanate. The derivatization reached its maximum within 30 s at room temperature with an efficiency of 93.9 +/- 2.8% (mean +/- S.D.). The formed diastereomers were subsequently separated on a silica column with a mobile phase of hexane-2-propanol-isobutanol (96:2:2, v/v) at a flow-rate of 1.5 ml/min. The ultraviolet detection wavelength was set at 220 nm. Using 1 ml plasma, the detection limit was 6.25 ng/ml for the propafenone enantiomers. The assay was successfully employed to measure propafenone enantiomers in plasma samples of a healthy subject after oral administration of a single 150-mg dose of the racemate.     

Stereoselective determination of demethyl- and didemethyl-citalopram in rat plasma and brain tissue by liquid chromatography with fluorescence detection using precolumn derivatization

A rapid and sensitive HPLC enantioselective method with fluorescence detection was developed to determine (-)-(R) and (+)-(S) enantiomers of the metabolites of citalopram, demethyl- and didemethyl-citalopram in plasma and brain tissue. This assay involves pre-column chiral derivatization with (-)-(R)-1-(1-naphthyl)ethyl isocyanate followed by separation on a normal-phase silica column. The developed liquid-liquid extraction procedure permits quantitative determination of analytes with recoveries ranged between 81 and 88% with intra- and inter-day relative standard deviations less than 10.5%. Linearity was obtained over the concentration range 5-1000 ng/mL and 100-10,000 ng/g for spiked drug-free plasma and brain tissue, respectively, with detection limits lower than 2.1 ng/mL and 42.8 ng/g.