Simultaneous and sensitive capillary electrophoretic enantioseparation of three ß-blockers with the combination of achiral ionic liquid and dual CD derivatives.

Successful simultaneous enantioseparation and sensitive determination of three ß-blockers (PIN, OX and PRO), have been achieved by capillary electrophoresis using an achiral ionic liquid, [GTMA]Cl, as a modifier to cooperate with dual CDs containing DM-ß-CD and TM-ß-CD. The influence of aIL was investigated in details, including various aILs, the concentration of aIL and molar ratio of aIL to CD. The ratio of DM-ß-CD to TM-ß-CD in dual CDs was also discussed. DM-ß-CD and TM-ß-CD favor the enantioseparations of PIN/OX and PRO, respectively. Meanwhile, the presence of [GTMA]Cl was found to play a key role in enantioseparations, and it widened the scope of application of DM-ß-CD and TM-ß-CD. Furthermore, FESI as an effective on-line sample enrichment technique was developed to improve the detection sensitivity. Under the optimum conditions, the detection limits of the three pairs of enantiomers range from 0.10 to 0.65 nM, which are much lower than those in the conventional methods. Eventually, the proposed method was successfully applied to the analysis of spiked urine sample with good recoveries.

Determination of selected beta-receptor antagonists in biological samples by solid-phase extraction with cholesterolic phase and LC/MS.

A new method is presented for the determination of five selected beta-receptor antagonists by HPLC, which emphasizes sample preparation via retention on a new type of silica gel sorbent used for solid-phase extraction (SPE). Sorbents of this type were obtained by the chemical modification of silica gels of various porosities by cholesterol ligands. The cholesterol-based packing material was investigated by spectroscopic methods and elemental analysis. The recoveries obtained with the extraction procedure were optimum over a relatively broad sample pH range (3.08-7.50). Analytical factors such as the sample loading, the washing step and elution conditions, the concentration of beta-receptor antagonists to be extracted, and the type of sorbent were found to play significant roles in the sample preparation procedure and would therefore need to be controlled to achieve optimum recoveries of the analytes. Under optimum conditions, the recoveries of nadolol, acebutolol, esmolol, oxprenolol and propranolol from spiked buffers, blood and urine were reproducible and dependent on the polarity or hydrophilicity of the compounds. The above analytes were determined by reverse-phase high-performance liquid chromatography (HPLC) with UV and ESI-ion trap mass spectrometry (MS) detection. The described method was found to be suitable for the routine measurement of compounds that are both polar and basic, and can be applied for the analysis of biological samples such as urine and blood in clinical, toxicological or forensic laboratories. The recovery measurements were performed on spiked human urine and serum, and on real samples of mouse blood serum.

High sensitivity analysis of oxprenolol in urine by capillary electrophoresis with C18 packed on-line preconcentrator.

High sensitivity analysis of oxprenolol in spiked human urine has been performed by capillary zone electrophoresis (CZE) in ammonium formate buffer pH 2.5 using an uncoated capillary with 1cm length C18 on-capillary preconcentrator at the inlet side. The preconcentrator was fabricated in laboratory using the packing method and not encapped C18 5 microm particles as stationary phase material. The packed path was retained into the capillary by sintered stationary phase frits. Before running the CZE analysis, the oxprenolol was eluted from the preconcentrator by injecting a short plug of acetonitrile/water mixtures. With respect to classical CZE, the use of on-line preconcentrator widely increased the method sensitivity allowing the detection of the drug at 0.5 ng/mL (injected concentration). The method showed a linear response in the range of 1-150 ng/mL oxprenolol standard compound. The intra-day repeatability (n = 11) R.S.D. values for migration time, peak area and normalized peak area were 0.72%, 3.96% and 3.66%, respectively, while inter-day repeatability (n = 5 days) R.S.D. values were 2.74%, 9.41% and 9.83%, respectively. The method was successfully applied to the analysis of oxprenolol in extracted urine spiked at 250 pg/mL (oxprenolol LOQ concentration in urine).

Quantitative determination of oxprenolol and timolol in urine by capillary zone electrophoresis.

A simple capillary zone electrophoretic method with UV detection has been developed for the quantitative determination of the beta-adrenoreceptor antagonists (beta-blockers) oxprenolol and timolol in human urine, preceded by a solid-phase extraction step. The electrophoretic separation was performed on a 78 cm x 75 microm I.D. fused-silica capillary (effective capillary length: 70 cm). The electrolyte consisted of a Na2B4O7-H3BO3 (50 mM), pH 9. The introduction of the sample was made hydrostatically for 20 s and the running voltage 25 kV at the injector end of the capillary. Photometric detection was used at a wavelength of 229 nm for oxprenolol and 280 nm for timolol. Under these conditions oxprenolol migrated at 4.76+/-0.05 min and timolol at 4.97+/-0.05 min. The solid-phase extraction methods were optimised for each beta-blocker and provided recoveries of 72.8% for timolol and 94.52% for oxprenolol. Good resolution from the endogenous compounds present in the urine matrix were achieved for both compounds. The method was applied to the determination of both beta-blockers in pharmaceutical formulations and urine samples obtained from hypertensive patients after the ingestion of a therapeutic dose (in a 24-h time interval after the ingestion). The quantitative results were compared with results previously obtained at our laboratories by HPLC and were found to be in good agreement. Good reproducibility, linearity, accuracy and quantitation limits (in urine) of 0.19 microg/ml for timolol and 0.20 microg/ml for oxprenolol were obtained, allowing the method to be applied to pharmacokinetic studies of these compounds.

Disposition of human drug preparations in the horse. V. Orally administered oxprenolol.

Urinary concentrations of the beta-antagonist oxprenolol and some of its major human metabolites were determined following oral administration of a dose of 160 mg to five fasted horses. Quantitation was performed by gas chromatography-mass spectrometry (GC-MS) in the selected ion mode (SIM) by monitoring ion m/z 466 of the heptafluorobutyric derivatives. As early as 2 h after dosage oxprenolol could be detected in hydrolysed urine and remained detectable up to 24 h. Maximum urinary concentrations and excretion rates were obtained between 2 and 12 h. After 12 h only 2.8% of the administered dose was excreted as conjugates of oxprenolol and major human metabolites including 4-OH-oxprenolol and 5-OH-oxprenolol. These metabolites were detectable up to 48 h.

Detection of beta-blockers in urine by solid-phase extraction-supercritical fluid extraction and gas chromatography-mass spectrometry.

The most convenient way to perform supercritical fluid extraction (SFE) of liquid sample matrices is to combine it with solid-phase extraction (SPE). beta-Blockers from urine were collected on an Empore disc, which was then placed into an extraction cell for derivatization and SFE. SPE recovery was best at pH 10. Effects of temperature, pressure and volume of pyridine on the acetylation and SFE processes were studied. Without acetylation the beta-blockers were not significantly soluble in CO2. SFE temperatures of 70 degree C and 150 degree C together with 200 microliters of acetic anhydride and 400 microliters pyridine gave the best results. With the SPE-SFE-GC-MS method developed here, beta-blockers like oxprenolol, metoprolol and propranolol could easily be detected in urine samples, and the limit of detection (LOD) for these compounds was found to be 20 ng/ml, 30 ng/ml and 40 ng/ml, respectively.

Enantioselective determination of oxprenolol and its metabolites in human urine by cyclodextrin-modified capillary zone electrophoresis.

A stereospecific capillary electrophoresis assay for oxprenolol enantiomers and their basic metabolites in human urine has been developed using hydroxypropyl-beta-CD as a chiral selector in the mobile phase. The bioassay method has been validated and the detection limit from spiked urine samples is 0.2 micrograms/ml. The calibration curves are linear from 0.4 to 16 micrograms/ml. Extraction recovery ranged from 84.7 to 96.4% for all the compounds studied. The influence of various parameters on the chiral separation of oxprenolol and its basic metabolites have been investigated. Urinary excretion profiles of oxprenolol enantiomers and those of two metabolites have also been studied, following a single oral dose of racemic oxprenolol.

Determination of oxprenolol and its glucuronide metabolite in plasma and urine using high-performance liquid chromatography.

A HPLC assay is presented for the determination of oxprenolol (1) and its glucuronic acid conjugate (2) in human plasma and urine. The procedure employs a selective re-extraction using alprenolol (3) as the internal standard, followed by reversed-phase chromatography and UV-detection. The minimal detectable concentration is 10 ng/ml in plasma and 50 ng/ml in urine, using 1.0 and 0.5 ml of plasma and urine, respectively. Within-run and day-to-day variations are below 10% at all concentrations examined. Plasma and urine samples of either healthy volunteers or patients with renal failure are free of interferences from endogenous compounds and drugs frequently used in these patients. The glucuronic acid conjugate of oxprenolol is determined as the parent compound after hydrolytic cleavage with beta-glucuronidase/arylsulfatase. The specificity and selectivity of this cleavage are also demonstrated.

Rapid determination of the enantiomers of metoprolol, oxprenolol and propranolol in urine.

A method is described that makes possible the rapid determination of the enantiomers of beta-blocking agents. After extraction from urine samples (at pH 9.9) using toluene, the enantiomers are derivatised with S-(+)-benoxaprofen chloride. The chromatographic separation can be performed on thin-layer plates with toluene-acetone as mobile phase. The derivatives can be detected by measuring the fluorescence (lambda ex = 313 nm,lambda em = 365 nm).

The disposition and metabolism of 14C-oxprenolol.HCl in man.

The disposition and metabolism of oxprenolol have been investigated in two healthy male volunteers, following a single 160 mg oral dose of racemic 14C-labelled oxprenolol. Absorption was rapid and complete. Peak blood concentrations of total radioactivity were 8.83 and 8.21 nmol X g-1 after 1 and 1.5 h in the two subjects. After 4 days 93.4 and 81.9% of the dose was excreted in urine, and a total of 96.6 and 84.5% found in the excreta. Mean peak blood concentrations of unchanged R(+)- and S(-)- oxprenolol were 0.83 and 0.81 nmol X g-1. Maximal concentrations of the glucuronides of the R(+)- and S(-)- isomers were 1.98 and 3.51 nmol X g-1. The mean half-lives of both oxprenolol enantiomers were 1.8 h, those of their glucuronides were 3.2 h (R(+] and 4.6 h (S(-]. Unchanged oxprenolol and the oxprenolol glucuronides constituted 11.4 and 66.5% of the area under the blood concentration-time curve (AUC, 0-24 h) of total radioactivity. The AUC-ratio of R(+) to S(-) was 1.19 for free oxprenolol and 0.36 for the glucuronides. Free metabolites II-X represented together 4.3% of 14C-AUC, and their glucuronides 15.2%. In urine, 1.8 and 1.0% of the total radioactivity was present as unchanged R(+)- and S(-)- oxprenolol, respectively. The glucuronides of the enantiomers accounted for 24.5 and 26.5%. The percentages of free 4- and 5-hydroxy oxprenolol were 0.7 and 2.4% while those of their glucuronides were 12.3 and 7.5%. Metabolites IV-X constituted together 6.2% in free form and 5.3% in conjugated form. In conclusion, the good mass balances in blood and urine has enabled the comprehensive and quantitative description of the metabolic fate of oxprenolol in man. Oxprenolol is extensively metabolized, direct O-glucuronidation being the major metabolic pathway and oxidative reactions minor ones. The disposition of the oxprenolol enantiomers revealed no remarkable stereoselective differences.

The development of reliable compliance tests for antihypertensive drugs.

Many tests for measuring compliance have been proposed, but in most cases compliance rates have been determined without taking into account the factors influencing the interval during which a drug can be detected by a qualitative test after having been taken by the patient. The drug half-life, often used for determining the time at which the sample is collected, is inadequate for obtaining conclusive test results. A procedure is described for the determination of urine collection intervals during which reliable information on compliance can be obtained, using oxprenolol, hydrochlorothiazide, and pindolol as examples.

Multiple inverse isotope dilution assay for oxprenolol and nine metabolites in biological fluids.

An isotope dilution assay for the specific determination of 14C-labelled oxprenolol and nine of its metabolites in the same biological sample is described. After addition of unlabelled carriers to the sample, oxprenolol and the metabolites were isolated by base- and acid-specific extraction and separated by normal-phase high-performance liquid chromatography using two different mobile phases. Quantitation of the various peaks was performed by on-line ultraviolet detection at 275 nm and off-line radiometry by liquid scintillation counting. Endogenous compounds and unknown metabolites did not interfere in the assay. The analysis of rat and dog blood, plasma and urine samples spiked with [14C]oxprenolol hydrochloride, showed mean recoveries between 98.7 and 99.8%. The assay was used to investigate the metabolic fate of [14C]oxprenolol in the dog. Analyses of blood and urine demonstrated the quantitative significance of the various metabolites in the biotransformation of oxprenolol.

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

An isotope dilution assay has been developed for the determination of both oxprenolol enantiomers in biological samples after administration of the racemic 14C-labelled mixture. The enantiomers were reacted with optically pure S(-)-1-phenylethyl isocyanate and the diastereoisomeric urea derivatives formed were separated by normal-phase high-performance liquid chromatography. Quantitation was performed by on-line ultraviolet detection at 275 nm and off-line radiometry. Endogenous compounds and oxprenolol metabolites did not interfere with the assay. Analysis of water and blood, plasma and urine samples of rats and dogs spiked with [14C]oxprenolol hydrochloride showed mean recoveries for R(+)-oxprenolol hydrochloride of 99.2% (water), 99.3% (blood), 99.1% (plasma) and 97.9% (urine), and for S(-)-oxprenolol hydrochloride of 99.7% (water), 98.1% (blood), 98.6% (plasma) and 96.9% (urine). In a pilot study, the presented method was used to investigate the metabolic fate of the enantiomers in two dogs dosed orally with racemic [14C]oxprenolol hydrochloride (3 mg/kg). The results show that conjugation of R(+)-oxprenolol exceeded that of S(-)-oxprenolol.

The effect of age on the pharmacokinetics of oxprenolol.

The pharmacokinetics of oxprenolol have been studied in young and healthy elderly volunteers. Drug concentrations in plasma and urine have been measured after a single 80-mg dose of oxprenolol and again after repeated administration (80 mg twice daily) for a period of 1 week. It has been shown that multiple dosing does not result in oxprenolol accumulation in either group and that age has no effect on the pharmacokinetics of this drug.

Propranolol, alprenolol and oxprenolol metabolism in the dog. Identification of N-methylated metabolites.

The metabolism of propranolol, alprenolol and oxprenolol was studied in the dog and rat; propranolol in five additional species, including man. Basic, phenolic and neutral metabolites were extracted from urine at pH 9.6 after enzymatic hydrolysis. Separation and identification of parent drug and seven metabolites each for propranolol, alprenolol and oxprenolol in the dog were accomplished by gas chromatography mass spectrometry as the trifluoroacetyl derivatives. A very uniform and predictable fragmentation pattern was observed for all 24 compounds. Seven new metabolites were identified. The metabolism of all three drugs was qualitatively the same, including N-dealkylation followed by N-methylation or deamination of the primary amines. The parent drugs as well as all of their sidechain metabolism products were also partially ring hydroxylated. N-Methylation was only found in the dog and is a minor metabolic pathway. The stereochemical composition of N-methyldesisopropylpropranolol and its immediate precursor N-desisopropylpropranolol showed a marked enrichment of the (+)-isomer.