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.     

Determination of molindone enantiomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry using macrocyclic antibiotic chiral stationary phases

A sensitive and selective bioanalytical assay was developed and validated for the determination of enantiomeric molindone in human plasma using high-performance liquid chromatography-tandem mass spectrometry along with supported liquid extraction procedures. The chiral separation was evaluated and optimized on macrocyclic antibiotic type chiral stationary phases (CSPs) based on teicoplanin aglycone (Chirobiotic TAG) in polar organic, polar ionic, and reversed-phase mode chromatography, respectively. Complete baseline separation was achieved on a Chirobiotic TAG column under isocratic condition in reversed-phase chromatography. The method validation was conducted using a Chirobiotic TAG column (100 mm x 2.1 mm) over the curve range 0.100-100 ng/ml for each molindone enantiomer using 0.0500 ml of plasma sample. The flow rate was 0.8 ml/min and the total run time was 9 min. Supported liquid extraction in a 96-well plate format was used for sample preparation. Parameters including recovery, matrix effect, linearity, sensitivity, specificity, carryover, precision, accuracy, dilution integrity, and stability were evaluated. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels were RSD 相似文献   

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.     

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).     

Improved determination of the bisphosphonate alendronate in human plasma and urine by automated precolumn derivatization and high-performance liquid chromatography with fluorescence and electrochemical detection.

An improved method for the determination of 4-amino-1-hydroxybutane-1,1-bisphosphonic acid (alendronate) in human urine and an assay in human plasma are described. The methods are based on co-precipitation of the bisphosphonate with calcium phosphates, automated pre-column derivatization of the primary amino group of the bisphosphonic acid with 2,3-naphthalene dicarboxyaldehyde (NDA)-N-acetyl-D-penicillamine (NAP) or cyanide (CN-) reagents, and high-performance liquid chromatography (HPLC) with electrochemical (ED) or fluorescence detection (FD). The feasibility of ED of the NDA-CN- derivative of aldendronate has been demonstrated, and a HPLC-ED assay in human urine has been validated in the concentration range 2.5-50.0 ng/ml. In order to eliminate the cyanide ion from the assay procedure, several other nucleophiles in the NDA derivatization reaction were evaluated. An NDA-NAP reagent was found to produce highly fluorescent derivatives of alendronate. The assay in urine based on NDA-NAP derivatization and HPLC-FD has been developed and fully validated in the concentration range 1-25 ng/ml. Based on the same NDA-NAP derivatization, an assay in human plasma with a limit of quantification of 5 ng/ml has also been developed. Both HPLC-FD assays were utilized to support various human pharmacokinetic studies with alendronate.     

Determination of a cyclic heptapeptide, a novel fibrinogen receptor antagonist, in human plasma by high-performance liquid chromatography with automated pre-column derivatization, column switching and fluorescence detection.

A sensitive high-performance liquid chromatographic (HPLC) assay for the determination of the cyclic heptapeptide Ac-Cs-Asn-Dtc-Amf-Gly-Asp-Cys-OH (Dtc = beta,beta-dimethylthioproline, Amf = p-aminomethylphenylalanine) in human plasma has been developed. The key steps in the assay include: solid-phase extraction of the drug from plasma, chemical derivatization of the primary amino group with naphthalene-2,3-dicarboxyaldehyde in the presence of N-acetyl-D-penicillamine as a nucleophile to form a fluorescent benzo[f]isoindole derivative, and HPLC with column switching to provide the necessary chromatographic separation of the derivative from endogenous plasma components. The assay has been validated in the concentration range 1-10 ng/ml of plasma.     

High-performance liquid chromatographic method for the determination of finasteride in human plasma at therapeutic doses.

A high-performance liquid chromatographic (HPLC) method with ultraviolet detection for the determination of a novel 4-aza-steroidal inhibitor of 5 alpha-reductase in human plasma has been developed. The assay is based on a single solid-phase extraction and an efficient HPLC separation on two analytical columns in series. The assay has been fully validated and used to support Phase II and III clinical pharmacokinetic studies. The lowest limit of quantification was found to be at 1 ng/ml and allowed pharmacokinetic evaluation of the drug at doses down to 5 mg.     

基于L-谷氨酸的手性硅胶球的制备及其应用

无机介孔硅球因其具有足够的机械强度、热稳定性,以及适应多种流动相的优点,成为高效液相色谱(HPLC)柱填料中使用最广泛和最重要的材料.但在此研究领域中,并未见球形的全无机手性硅胶用作HPLC手性固定相.该文以无机球形介孔硅胶作为研究对象,通过堆砌硅珠法,以硅溶胶为原料,L-谷氨酸(L-Glu)为手性源,在手性环境中制造...     

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.     

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.     

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.     

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.     

Validated high-performance liquid chromatographic enantioseparation of selenomethionine using isothiocyanate based chiral derivatizing reagents

A high-performance liquid chromatographic (HPLC) method for enantioseparation of selenomethionine (SeMet) was developed using two isothiocyanate-based chiral derivatizing reagents [(R)-methyl benzyl isothiocyanate (MBIC) and (S)-1-(1-naphthyl) ethyl isothiocyanate (NEIC)] and UV detection. Diastereomers of selenomethionine were synthesized either via stirring (using MBIC) or by microwave irradiation (using NEIC). Derivatization conditions were optimized and the synthesized diastereomers were successfully resolved using triethyl ammonium phosphate buffer and acetonitrile on a reversed-phase column. The method was validated for accuracy, precision and limit of detection. The mechanism of separation is also discussed.     

Direct chiral resolution of cloquintocet-mexyl and its application to in vitro degradation combined with clodinafop-propargyl

A simple chiral high‐performance liquid chromatography (HPLC) method with ultraviolet (UV) detection was developed and validated for measuring Cloquintocet‐mexyl (ClM) enantiomers and clodinafop‐propargyl (CP) using cellulose tris‐(3,5‐dimethylphenylcarbamate) (CDMPC) as chiral stationary phase (CSP). The effects of mobile phase composition and column temperature on the ClM enantiomer separation were investigated. Good separation was achieved by using a mixture of n‐hexane and n‐propanol as mobile phase. Based on the chiral HPLC method, enantioselective quantitative determination analysis methods for this herbicide combined with CP in diluted plasma were developed and validated. The assay method was linear over a range of concentrations (0.5–100 µg/mL) in diluted plasma and the mean recovery was greater than 80% for both enantiomers and CP. The limits of quantification and detection for both ClM enantiomers and CP were 0.5 and 0.2 µg/mL, respectively. Intra‐ and interday relative standard deviations did not exceed 10% for three tested concentrations. The result suggested that the degradation of ClM enantiomers was stereoselective in rabbit plasma, and both rac‐ClM and CP degraded quickly in plasma, showing that the main existing forms with biological effect in animals are their metabolites. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of metrifonate enantiomers in blood and brain samples using liquid chromatography on a chiral stationary phase coupled to tandem mass spectrometry

A novel enantioselective assay is described for the simultaneous determination of the metrifonate enantiomers BAY z 7216 and BAY z 7217 in extracts of whole blood samples obtained from rats, mice, rabbits and Beagle dogs as well as in rat brain tissue using liquid chromatography tandem mass spectrometry (LC/MS/MS) with thermally and pneumatically assisted electrospray ionization (TurboIonSpray(R)). Chromatographic separation is achieved on a chiral normal phase column with a mobile phase containing 0.25% water only. The total run time per sample is 11.0 min giving chromatographic base line separation of the enantiomers. Compared with previous methods this assay offers a higher sample throughput, excellent ruggedness and higher sensitivity. The limits of quantification for each enantiomer are 5.00 microg/L from 0.5 mL whole blood and 7.50 ng/g (ppb) using 0.333 g brain tissue, respectively. Similar assay specifications have been derived for the two enantiomers. The method has been validated for the analysis of blood samples from low and high dosed preclinical pharmacokinetic and toxicokinetic studies, corresponding to two analytical working ranges like e.g. 5.00 to 1000 microg/L and 200 to 40000 microg/L (0. 200 to 40.0 mg/L). For rat brain tissue the validated concentration range is 7.50 to 750 ng/g (ppb).     

Optimization of a capillary electrophoresis method to determine the chiral purity of a drug

Capillary electrophoresis (CE) using hydroxypropyl-β-cyclodextrin (HP-β-CD) in the separation buffer was investigated to determine the overall chiral purity of a drug containing a single stereogenic center. The effects of primary factors —pH, buffer components, buffer concentration, cyclodextrin concentration and sample amount (concentration and injection volume) — on the resolution of the enantiomers were investigated. Secondary factors such as the HP-β -CD source, lot and degree of substitution that were expected to affect the robustness of the assay were investigated also. The linearity, quantitation limit for the trace enantiomer and the precision of the measurements were determined. This study shows that understanding and optimizing the assay conditions leads to a chiral CE separation that is comparable to that obtained by chiral HPLC. However, chiral CE separations achieved with buffer additives have the advantages of shorter run times, higher numbers of theoretical plates (greater resolution), smaller amounts of chiral additive (less cost) and greater ruggedness (separation virtually independent of column properties unlike HPLC).     

A new application of stopped-flow chiral HPLC: inversion of enantiomer elution order

A newly developed procedure to reverse the enantiomer elution order of compounds resolved on chiral stationary phases (CSPs) for HPLC is presented. The optimized analytical protocol is based on the effect of temperature on enantioselectivity and does not involve any changing in mobile phase composition or type of CSP. In essence, the approach entails variable temperature chromatography at two temperatures. The enantiomer separation is performed at a low column temperature, with stopping the flow prior to elution of the less retained enantiomer. Then, the column temperature is changed with the peaks trapped inside the column, followed by elution with the same mobile phase in reverse direction. Under these conditions, the more pronounced loss in free energy of binding for the more strongly bound enantiomer results in an inversion of the elution order. This procedure may be applied to each enantiomer pair that is separated by chiral HPLC under an appreciable enthalpy-control.     

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.     

Stereospecific analysis of loxoprofen in plasma by chiral column liquid chromatography with a circular dichroism-based detector

The chiral separation of loxoprofen was achieved on a chiral column with UV and circular dichroism (CD) detection. The good resolution of four loxoprofen stereoisomers was obtained. The column used for the chiral separation was Chiralcel OJ column (250 x 4.6 mm) using hexane-2-propanol-trifluoroacetic acid (95:5:0.1), as an eluent. The flow-rate was 1.0 ml/min and the detection was at 225 nm. In addition, CD and UV spectra were obtained by stopped flow scanning. The method allows the determination of the stereoisomers of loxoprofen in human plasma after the administration of therapeutic dose of the racemic drug, thus HPLC with CD detector is useful for the stereospecific determination of loxoprofen products in biological samples.     

Use of dabsylation, column switching and chiral separation for the determination of a renin inhibitor in rat, marmoset and human plasma.

A high-performance liquid chromatographic method with column switching was developed for the determination of the renin inhibitor Ro 42-5892/001, (S)-alpha-[(S)-alpha-[(tert.-butylsulphonyl)methyl]hydrocinnama mido]-N- [(1S,2R,3S)-1-(cyclohexylmethyl)-3-cylcopropyl-2, 3-dihydroxypropyl]imidazole-4-propionamide methanesulphonate (1:1), in rat, marmoset and human plasma, using a Nucleosil C8 120 (3 microns) stationary phase. Since the analyte and the internal standard are optical isomers, beta-cyclodextrin was used as a mobile phase constituent for their chiral separation. The method took advantage of the characteristics of dabsyl chloride derivatives, namely sensitivity, specificity and, particularly, stability, providing a quantification limit of 5 ng/ml. The accuracy (range of inaccuracy 1-13%) and the inter-assay precision (coefficient of variation range 1.8-9.1%) were acceptable. The method was successfully applied to toxicokinetic studies in rats and marmosets.