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 determination of serine enantiomers in plasma using Mosher's reagent and stable isotope dilution gas chromatography-mass spectrometry

D-Serine is a co-agonist of the N-methyl-D-aspartate receptor in glutamate neurotransmission and has been proposed as a potential therapeutic agent for schizophrenia. However, D-serine also acts as a nephrotoxic substance in rats at high doses. To investigate the pharmacokinetics and toxicokinetics of D-serine, a method for the stereoselective determination of serine enantiomers in rat plasma was developed using GC-MS with selected ion monitoring (GC-MS-SIM). DL-[(2)H(3)]Serine was used as an internal standard to account for losses associated with the extraction, derivatization and chromatography. Serine enantiomers were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N,O-diacylation with optically active (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form epimeric amide. Quantitation was performed by SIM of the molecular-related ions of the epimers in the chemical ionization mode. The intra- and inter-day reproducibility of the assay was less than 5% for D-serine and 3% for L-serine. The method was successively applied to study the pharmacokinetics of D-serine in rats.     

Determination of 1,4-dioxane in cosmetic products by high-performance liquid chromatography

A rapid high-performance liquid chromatographic procedure has been developed for the assay of 1,4-dioxane in cosmetic products. After solid-phase extraction, using Bond Elut CN and Bond Elut C18 cartridges, samples were analysed directly on a LiChrospher CH-8 reversed-phase column with spectrophotometric detection at 200 nm and acetonitrile - water as eluent. Recovery of 1,4-dioxane from different cosmetic matrices was between 81.5 and 90.1% in the 30-90 microgram g(-1) range. The minimum quantifiable amount was 6.5 microgram g(-1). The method is simple, reproducible and specific and is suitable for routine analyses of commercial cosmetics.     

Sensitive procedure for the determination of reboxetine enantiomers in human plasma by reversed-phase high-performance liquid chromatography with fluorimetric detection after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate

A sensitive and selective high-performance liquid chromatographic method for the determination of reboxetine enantiomers in human plasma was developed. Although two chiral centres are present in reboxetine, its stereospecific synthesis leads to two rather than four possible enantiomers. After extraction from plasma and reaction with (+)-1-(9-fluorenyl)ethyl chloroformate, reboxetine enantiomers were separated as diastereoisomeric derivatives by reversed-phase high-performance liquid chromatography (HPLC) and determined by fluorimetric detection. The HPLC analysis time was about 90 min. The linearity, precision, accuracy and limit of quantification of the method were evaluated. No interference from blank plasma sample was observed. The suitability of the method for in vivo samples was assessed by the analysis of plasma samples obtained from a healthy male volunteer who had received a single oral dose of 4 mg of reboxetine in tablet form.     

Liquid chromatographic analysis of propranolol enantiomers in human blood using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate.

A method for the determination of the R-(+) and S-(-) enantiomers of propranolol in blood was developed. After extraction with heptane-isopentanol and derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate, excess reagent was removed using solid-phase extraction. The enantiomers were separated on an achiral, reversed-phase, radially compressed column, and detected by fluorescence with excitation and emission wavelengths of 260 and 340 nm, respectively. The limit of quantification was 0.5 ng/ml. This method was used for pharmacokinetic analysis of propranolol enantiomers after administration of immediate-release (80 mg) or sustained-release (160 mg) racemic propranolol.     

Chiral stability-indicating HPLC method for analysis of arotinolol in pharmaceutical formulation and human plasma

An enantioselective stability-indicating high performance liquid chromatographic method was developed for the analysis of arotinolol in standard solution. The degradation behaviour of arotinolol was investigated under different stress conditions recommended by International Conference on Harmonization (ICH). Resolution of the drug and complete separation from its degradation products were successfully achieved on a Chirobiotic V column, using UV detector set at 315 nm, polar organic mobile phase (POM) consisting of methanol:glacial acetic acid:triethylamine, 100:0.02:0.03, (v/v/v), and a flow rate of 1 ml/min. The drug was subjected to oxidation, hydrolysis, photolysis, and heat to apply stress conditions. The drug was found to degrade in alkaline, acidic, oxidative conditions and when exposed to heat. The drug was stable to sunlight. The method reported here has also been successfully applied to pharmaceutical formulation and to human plasma that spiked with stock solutions of arotinolol enantiomers.Arotinolol enaniomers were recovered from plasma by using liquid–liquid extraction procedure with ethyl ether. The method was highly specific, where degradation products and coformulated compounds did not interfere, and was sensitive with good precision and accuracy and was linear over the range of 50–400 ng/ml (R² > 0.9981) with a detection limit of 20 ng/ml for each enantiomer. The mean extraction efficiency for arotinolol was in the ranges 96–104% for each enantiomer. The mean relative standard deviation (RSD) of the results of within-day precision and accuracy of the drug were ?7.1%. There was no significant difference between inter- and intra-day studies for each enantiomers which confirmed the reproducibility of the assay. The overall recoveries of arotinolol enantiomers from pharmaceutical formulations were in the ranges 97.6–101.8%.     

Enantioselective determination of trantinterol in rat plasma by ultra performance liquid chromatography-electrospray ionization mass spectrometry after derivatization

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the determination of trantinterol enantiomers in rat plasma. Diphenhydramine was employed as the internal standard. The plasma samples were prepared using liquid-liquid extraction with n-hexane-dichloromethane-isopropanol (20:10:1, v/v/v) as the extractant. Trantinterol enantiomers after pre-column derivatization using diacetyl-l-tartaric anhydride (DATAAN) were separated on a C18 column using a gradient solvent programme. The mobile phase was composed of 3 mM ammonium acetate and acetonitrile. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI). Linear calibration curve for each enantiomer was obtained in the concentration range of 1-80 ng/mL, with limit of quantification (LOQ) of 1 ng/mL. The intra- and inter- precision (R.S.D.) values were below 9.6% and accuracy (R.E.) was from −2.4 to 6.2% at all quality control (QC) levels. The developed method was applied to the enantioselective pharmacokinetic study of trantinterol in rats.     

Determination of tertatolol enantiomers in biological fluids by high-performance liquid chromatography.

A stereospecific high-performance liquid chromatographic method for the quantification of (-)- and (+)-tertatolol in plasma and urine is described. The method involves solid-phase extraction followed by derivatization with S(+)-naphthylethylisocyanate to form the urea derivative, which is more sensitive to fluorescence detection. The separation of the diastereomeric derivatives was performed by reversed-phase high-performance liquid chromatography. Fluorimetric detection (lambda excitation = 220 nm, lambda emission = 320 nm) allows the quantification of tertatolol enantiomers down to 6 ng/ml. The assay was used to study the pharmacokinetic profile of tertatolol enantiomers following oral administration of racemic tertatolol; preliminary results suggest enantioselective absorption and/or disposition of tertatolol.     

High-performance liquid chromatographic method for simultaneous determination of enantiomers of 5-dimethylsulphamoyl-6,7-dichloro-2-dihydrobenzofuran-2, carboxylic acid and its N-monodemethyl metabolite in monkey plasma and urine after chiral derivatization

A quantitative method for the simultaneous high-performance liquid chromatographic (HPLC) resolution and determination of the enantiomers of 5-dimethylsulphamoyl-6,7-dichloro-2,3-dihydrobenzofuran-2-carboxyl ic acid, a new diuretic, and its N-monodemethylated metabolite in monkey plasma and urine is described. The method includes diethyl ether extraction of the samples and S-(-)-alpha-methylbenzylamide derivatization of the extract, followed by reversed-phase solid-phase extraction and injection of the resulting diastereoisomers onto a reversed-phase HPLC column. Baseline separation was obtained. The assay showed linearity over the range 0.1-50 micrograms/ml of plasma and 0.25-500 microliters of urine, with a lower limit of detection of ca. 0.01 micrograms/ml for each of the enantiomers. The method is adequate for pharmacokinetic and enantioselective disposition studies of both the diuretic and its metabolite.     

Multiple inverse isotope dilution assay for the stereospecific quantitative determination of R(-) and S(+)-oxaprotiline in biological fluids

An isotope dilution assay for the determination of both oxaprotiline enantiomers in biological samples after administration of the racemic mixture has been developed. The enantiomers were reacted with synthetically prepared, optically pure N-trifluoroacetyl-S(-)-prolyl chloride, followed by high-performance liquid chromatographic separation of the diastereoisomers formed. Quantitation was performed by on-line UV detection at 260 nm and off-line radiometry by liquid scintillation counting. Endogenous compounds and metabolites do not interfere in the assay. Analysis of water and the blood and urine of rats spiked with [14C]oxaprotiline X HCl showed recoveries for S(+)-oxaprotiline X HCl (mean +/- coefficient of variation, n = 4-6) of 98.0 +/- 1.0% (water), 100.5 +/- 0.6% (blood) and 101.5 +/- 2.0% (urine), and for R(-)-oxaprotiline X HCl of 101.3 +/- 2.0% (water), 102.2 +/- 2.1% (blood) and 103.2 +/- 0.2% (urine). A pilot study to determine blood levels of the two enantiomers in two rats dosed with racemic [14C]oxaprotiline X HCl (10 mg/kg i.v.) was carried out to test the method. The results indicated stereoselective disposition of oxaprotiline enantiomers in the rat. The ratio of the areas under the blood concentration curves for R(-)-to S(+)-oxaprotiline X HCl was 1.14.     

Stereospecific high-performance liquid chromatographic determination of tocainide

A sensitive high-performance liquid chromatographic assay was developed for the determination of tocainide enantiomers in plasma. Following extraction of tocainide from plasma, the enantiomers were derivatized with S-(+)-1-(1-naphthyl)ethylisocyanate. The resulting diastereomers were separated and quantified using normal-phase chromatography with fluorescence detection set at 220/345 nm (excitation/emission). The peaks, resolved with a resolution factor greater than 1.5, were free from interference. Linearity was established over the concentration range 0.25-10.0 mg/l for each enantiomer in plasma (r2 greater than 0.998). The inter-assay variability was less than 10% at all concentrations examined. The method can be used to determine the pharmacokinetics of tocainide enantiomers in man.     

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.     

Simultaneous determination of fluoxetine and norfluoxetine enantiomers in biological samples by gas chromatography with electron-capture detection.

An electron-capture gas chromatographic procedure was developed for the simultaneous analysis of the enantiomers of fluoxetine and norfluoxetine. The assay involves basic extraction of these enantiomers from the biological samples, followed by their conversion to diastereoisomers using the chiral derivatizing reagent (S)-(-)-N-trifluoroacetylprolyl chloride. The method was utilized to detect and measure the quantity of these enantiomers in plasma and urine of patients and in liver and brain tissue of rats treated with (R,S)-fluoxetine.     

Determination of eugenol in rat plasma by liquid chromatography-quadrupole ion trap mass spectrometry using a simple off-line dansyl chloride derivatization reaction to enhance signal intensity

A rapid, selective and sensitive method was developed for the determination of eugenol concentration using an off-line dansyl chloride derivatization step to enhance signal intensity. The method consisted of a protein precipitation extraction followed by derivatization with dansyl chloride and analysis by full scan liquid chromatography electrospray quadrupole ion trap mass spectrometry (LC-ESI-QIT). The separation was achieved using a 100 x 2 mm C(8) analytical column combined with an isocratic mobile phase composed of 75:25 acetonitrile: 0.1% formic acid in water set at a flow rate of 0.25 mL/min. Signal intensity of the eugenol-dansyl chloride derivative was increased up to 100-fold as compared with the underivatized eugenol in positive electrospray mode. An analytical range of 100-20,000 ng/mL was used in the calibration curve of plasma and blood samples. The LOD observed was 0.5 pg injected on column. The novel method met all requirements of specificity, sensitivity, linearity, precision, accuracy and stability. In conclusion, a rapid and sensitive LC-ESI/MS/MS method using a derivatization agent was developed to enhance signal intensity of eugenol.     

Determination of S-carboxymethyl-L-cysteine and some of its metabolites in urine and serum by high-performance liquid chromatography using fluorescent pre-column labelling.

Pre-column labelling techniques are described for the determination of S-carboxymethyl-L-cysteine (CMC) and its metabolites in urine and plasma samples by high-performance liquid chromatography (HPLC) without prior extraction. All substances containing an amino group were converted into fluorescent fluorenylmethyl derivatives with 9-fluorenylmethyloxycarbonyl chloride (FMOC). Deaminated or N-acetylated carbocysteine metabolites were coupled with 1-pyrenyldiazomethane (PDAM) to give fluorescent PDAM esters. Similar results were obtained with the two commercially available and stable diazomethane derivatives PDAM and 9-anthryldiazomethane (ADAM). Following double derivatization with PDAM and FMOC, in a single chromatographic run with two fluorescence detectors connected in series, amines and amino(carboxylic) acids could be detected by their FMOC residues and, simultaneously, carboxylic acids were detected as fluorescent PDAM esters. The (R) and (S) enantiomers of the sulphoxides of CMC, of methylcysteine and of N-acetyl CMC were separated, although the reversed-phase HPLC system did not contain a chiral additive or stationary phase designed for the separation of enantiomers. The methods do not include liquid extraction steps and can therefore be performed either manually or automatically using an HPLC autosampler. These methods were used for the investigation of a disputed pharmacogenetic polymorphism of S-oxidation of CMC in humans, which until now has most often been studied using paper chromatography. The described techniques were applied to the determination of CMC and its metabolites in human urine and plasma samples.     

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.     

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.     

Stereospecific high-performance liquid chromatographic assay of metoprolol.

A valid, sensitive high-performance liquid chromatographic technique is reported for the separation of the two enantiomers of metoprolol in human plasma. The procedure involves pre-column derivatization with the homochiral reagent S-(+)-1-(1-naphthyl)ethyl isocyanate. Once formed, the diastereomers are separated using normal-phase high-performance liquid chromatography. Fluorescence detection (220 nm excitation; no emission filter) was utilized, resulting in baseline resolution (Rs greater than 1.5). The peaks corresponding to metoprolol enantiomers were free from interference throughout the examined range of 5-500 ng/ml; accuracy and precision were within approximately 10%. Analysis of a plasma sample collected from a healthy volunteer demonstrated that the assay is applicable to clinical studies.     

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in plasma after chiral derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate.

A sensitive high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat plasma has been developed. Racemic atenolol and practolol (internal standard) were extracted from alkalinized plasma (pH 12) into dichloromethane containing 3% (v/v) heptafluoro-1-butanol, and the organic layer was evaporated. The samples were derivatized with (+)-1-(9-fluorenyl)ethyl chloroformate at pH 8.5 for 30 min. After removal of excess reagent, the diastereomers were extracted into dichloromethane. The diastereomers were separated on a Microspher C18 column (3 microns) with a mobile phase of acetonitrile-sodium acetate buffer (0.01 M, pH 7) (50:50, v/v) at a flow-rate of 0.8 ml/min. Fluorescence detection (lambda ex = 227 nm, lambda em = 310 nm) was used. When 100 microliters of plasma were used, the quantitation limit was 10 ng/ml for the atenolol enantiomers. The assay was applied to measure concentrations of atenolol enantiomers in plasma after intravenous administration of racemic atenolol to rats.     

High-performance liquid chromatographic method for the determination of tris(hydroxymethyl)aminomethane (tromethamine) in human plasma.

Determination of tris(hydroxymethyl)aminomethane (tromethamine) in human plasma involved derivatization of the amino and hydroxyl groups with a ultraviolet-absorbing chromophore followed by extraction into an organic phase. Reversed-phase high-performance liquid chromatography with gradient elution was used for the separation of the analyte from the internal standard (2,3-butanediol). The assay was linear in the range 1.0-1000.0 micrograms/ml of plasma and the coefficient of variation varied between 9.6 and 16.3% whereas the accuracy varied between 90 and 108%. The limit of detection for the assay was 0.282 micrograms/ml. Stability of tris(hydroxymethyl)aminomethane in human plasma frozen at -20 degrees C was studied over a period of three month and the data indicated no significant change.