Stereoselective high-performance liquid chromatographic determination of the enantiomers of ketamine and norketamine in plasma.

An enantioselective high-performance liquid chromatographic assay for the quantitation of the enantiomers of ketamine and its major metabolite norketamine in human plasma is described (assay I). The procedure involved extraction of the compounds from alkalized plasma into cyclohexane. Stereoselective separation was achieved with a prepacked alpha 1-acid glycoprotein column without any derivatization procedure. A second assay using a conventional reversed-phase column to determine total (racemic) ketamine and norketamine is also described. Because of interfering plasma peaks (assay II) the cyclohexane solution was reextracted into 1 M hydrochloric acid. The detection wavelength was 215 nm for all substances. The limit of quantification of the method was ca. 40 ng/ml in plasma. The assays were sensitive and reproducible. The method was demonstrated to be sensitive for stereoselective pharmacokinetic studies of ketamine after clinical doses.     

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.     

Chiral analysis of methadone in plasma by high-performance liquid chromatography.

A method for the chiral high-performance liquid chromatographic analysis of methadone in plasma has been developed. The method employed organic solvent extraction, enantiomeric separation on a Chiral AGP column, and ultraviolet absorption detection at 212 nm. The intra-day variation in the quantification of methadone enantiomers was less than 9% at the 100 ng/ml level, and the values obtained correlated well with those from a gas chromatographic-mass spectrometric method. Results from patients indicate inter- and intra-individual differences in the ratio between l- and d-methadone in plasma during therapy with racemic methadone. In one patient, a higher level of d-methadone in plasma was caused by both faster elimination and lower bioavailability of l-methadone.     

Simultaneous determination of ketoprofen enantiomers and probenecid in plasma and urine by high-performance liquid chromatography.

A rapid, sensitive, stereospecific reversed-phase high-performance liquid chromatographic method was developed for simultaneous quantitation of ketoprofen enantiomers, probenecid and their conjugates in biological fluids. Following addition of the internal standard, indoprofen, the constituents were extracted into isooctane-isopropanol (95:5), water-washed, extracted with chloroform, then evaporated and the residue sequentially derivatized with ethyl chloroformate and L-leucinamide hydrochloride. The formed diastereomers were chromatographed on a reversed-phase column with a mobile phase of 0.06 M KH2PO4-acetonitrile-triethylamine (65:35:0.1) at a flow-rate of 1 ml/min and a detection wavelength of 275 nm. The minimum quantifiable concentration was 0.5 micrograms/ml in 100 microliters of rat plasma and urine samples. The intra- and inter-day coefficients of variation for this method are less than 10%. The assay is successfully applied to a pharmacokinetic study. The simultaneous analysis of probenecid with several other non-steroidal anti-inflammatory drugs was also successful.     

Simultaneous determination of reboxetine and O-desethylreboxetine enantiomers using enantioselective reversed-phase high-performance liquid chromatography

Current knowledge of stereoselective pharmacokinetics and different potencies of drug enantiomers requires the performance of stereoselective analysis during therapeutic drug monitoring in clinical practice. However, in the case of the new antidepressant drug reboxetine, no effort has been made so far to find a such a suitable system. Therefore, as a step towards developing an enantioselective bioanalytical method for reboxetine and the O-desethylreboxetine metabolite, three stereoselective chromatographic approaches have been investigated. Several chiral columns were tested, among them Chiral-AGP, ChiraGrom 2 and Chiral-CBH, which were able to simultaneously separate the two compounds into enantiomers in total running times of 28, 18 and 12 min, respectively.     

Direct injection analysis of ketoprofen enantiomers in plasma using column-switching high-performance liquid chromatography system

A high-performance liquid chromatography system was developed for the stereoselective determination of ketoprofen enantiomers in human plasma following direct sample injection. The system comprised of a pretreatment column and a chiral separation column connected in a series via a switching valve. When a 200 microliter portion of human plasma containing a therapeutic level of ketoprofen was directly applied to the system, ketoprofen was adsorbed in the pretreatment column, while plasma proteins were excluded. After the elution of proteins from the pretreatment column, the valve was switched and ketoprofen was desorbed and transferred to the chiral separation column where the enantiomers were separated and determined by ultraviolet-absorption. The mobile phase conditions for the pretreatment and chiral separation were optimized, which enabled rapid and complete recovery followed by satisfactory separation of the enantiomers. The calibration line for each enantiomer showed good linearity in the range of 0.25-5 micrograms/ml with a detection limit of 0.02 micrograms/ml (signal to noise ratio (S/N) greater than 3), which was sufficient for practical demands. The precision test indicated that the coefficient of variation for five repeated determinations of (-) ketoprofen was 5.4% at 0.1 microgram/ml and 1.4% at 1 microgram/ml.     

Quantitative determination of clenbuterol enantiomers in human plasma by high-performance liquid chromatography using the macrocyclic antibiotic chiral stationary phase teicoplanin

We report a method for the high-performance liquid chromatographic (HPLC) chiral separation of racemic clenbuterol in human plasma. Human plasma was spiked with stock solutions of clenbuterol hydrochloride and practolol as the internal standard. Following a liquid-liquid extraction procedure with 10% (+/-)-2-butanol/isopropyl ether under alkaline conditions, the dried samples were reconstituted in methanol and chromatographed using a macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T(trade mark) (teicoplanin). The mobile phase composition was methanol:acetonitrile (70:30, v/v), containing 0.3% (v/v) acetic acid and 0.2% (v/v) triethylamine. The resulting chromatogram achieved baseline separation for the clenbuterol enantiomers. Calibration curves (peak area ratio vs plasma concentration, n = 10) were constructed for the (-)-R-and (+)-S-clenbuterol enantiomers with a plasma concentration range of 0. 25-10 microM. The correlation coefficient (r) range was 0.99988-0. 99999 (mean = 0.99999). The lowest concentration measured was 0.25 microM. Inter- and intra-assay variation was determined for the lowest, medium and highest plasma concentration (0.25, 2 and 10 microM) by calculating the analytical recoveries with a range of 96-104%. The percentage recoveries for the clenbuterol enantiomers were 88.4-102% over the concentration range used. Detailed methodology is presented.     

Stereoselective determination of p-hydroxyphenyl-phenylhydantoin enantiomers in rat liver microsomal incubates by reversed-phase high-performance liquid chromatography using beta-cyclodextrin as chiral mobile phase additives

An analytical method was developed for determination of p-hydroxyphenylphenylhydantoin enantiomers in rat liver microsome by using reversed-phase high-performance liquid chromatography. A 50 mm C(8) column was used as the analytical column. The mobile phase was made up of 8.8 mmol/L beta-cyclodextrin, 0.25 mol/L urea and 0.05 mol/L ammonium acetate in water. The assay was linear from 2.05 to 410.0 micromol/L for each enantiomer. The limits of detection and of quantitation for the method were 0.90 and 2.05 micromol/L for each enantiomer, respectively. The analytical method afforded average recoveries of 93.59 +/- 2.75% and 94.72 +/- 1.78% for S- and R-p-hydroxyphenylphenylhydantoin, respectively. The method allowed study of the in vitro glucuronidation of p-hydroxyphenylphenylhydantoin in rat liver microsomal incubates. The stereoselectivity of p-hydroxyphenylphenylhydantoin phase II metabolism was observed.     

Rapid determination of propyphenazone in plasma by high-performance liquid chromatography.

A rapid and simple high-performance liquid chromatographic assay for the determination of propyphenazone in plasma is described. Phenylbutazone was used as the internal standard. Plasma proteins were precipitated with acetonitrile before injection onto a 3-microns Supelcosil LC-18 column. The mobile phase, ethanol containing 0.2% (v/v) heptylamine-0.005 M potassium dihydrogenphosphate (30:70, v/v), was used at a flow-rate of 1.3 ml/min. The quantitation was performed by ultraviolet detection at a wavelength of 270 nm. The chromatographic time was 7 min. The within- and between-day coefficients of variation were less than 6% and the recoveries close to 100% for concentrations between 0.4 and 22 mumol/l. The limit of quantitation was 0.4 mumol/l (ca. 100 ng/ml).     

Stereoselective high-performance liquid chromatographic assay for propranolol enantiomers in serum

A simple and sensitive stereoselective high-performance liquid chromatographic assay for the quantitation of propranolol enantiomers in serum is described. The method involves conversion of the propranolol enantiomers to diastereomeric urea derivatives by reaction with the clinical reagent (+)-phenylethylisocyanate, followed by chromatographic separation of the diastereomeric products. Conditions of the derivatization reaction were optimized to achieve rapid and quantitative yield with either of the enantiomers. Baseline resolution of the diastereomers was achieved on a reversed phase C8 column with an isocratic mobile phase. Fluorescence detection afforded an absolute on-column detection limit of 100 pg. The assay has been applied to pharmacokinetic studies in humans and small laboratory animals.     

Quantitation of clobazam in human plasma using high-performance liquid chromatography.

A rapid and simple reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of clobazam concentrations in human blood samples is developed and validated. Solid-phase column extraction is performed to clean up blood samples before running the analytical HPLC system. The chromatography is isocratic with a mobile phase consisting of acetonitrile (20%, v/v), methanol (23%, v/v), and 0.1 M potassium hydrogen phosphate buffer (pH 3.6; 57%, v/v) at a constant flow rate of 2 mL/min. Clobazam is detected at 226 nm. Chromatography is completed within less than 25 min. The recovery rate is greater than 95% and linear over a wide range of drug concentrations. The intra-assay coefficient of variation percentage varies between 4.3 and 12. This method is used for therapeutic drug monitoring in patients undergoing antiepileptic therapy with clobazam. Plasma levels of clobazam ranged from 21 to 663 ng/mL. Other antiepileptic compounds, such as clonazepam and phenobarbital, did not interfere with the detection of clobazam.     

Simultaneous determination of propranolol and 4-hydroxypropranolol enantiomers after chiral derivatization using reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method is described, which allows the simultaneous quantification of propranolol and 4-hydroxypropranolol enantiomers in human plasma. After extraction from plasma (pH 10.5) using ethyl acetate, the enantiomers are derivatized with R-(+)-phenylethylisocyanate as chiral derivatization reagent and triethylamine as basic catalyst in chloroform. Ascorbic acid is used to prevent 4-hydroxypropranolol from oxidation during the extraction. Chromatographic separation on ODS columns and fluorescence detection (228 nm/greater than 340 nm) allows sensitive quantitation of all derivatives. Incubation of the plasma samples with beta-glucuronidase/arylsulfatase and the use of the specific beta-glucuronidase inhibitor saccharo-1,4-lactone allows the quantitation of both the sulfate and glucuronide conjugates of the enantiomers. The method was applied to human plasma samples from a subject after administration of 60 mg racemic propranolol three times daily.