DETERMINATION OF SOTALOL, OXPRENOLOL AND LABETALOL IN BINARY MIXTURES AND IN SPIKED HUMAN SERUM BY DERIVATIVE SPECTROPHOTOMETRIC METHOD.

The usefulness of derivative spectrophotometry for the determination of labetalol, sotalol and oxprenolol in binary mixtures and in human spiked serum was checked. To this aim a spectrophotometric analysis of samples in the UV range was carried out and the obtained results revealed that derivative spectropho- tometry allows for the fast, accurate and precise determination of the tested substances in spite of their clear interference in the zero-order spectra. For quantitative determinations "zero-crossing" technique was used to establish wavelengths for zeros of specified component. In a mixture of labetalol and oxprenolol the following wavelengths were established: D1 λ = 245.32 nm and 266.03 nm, D2 λ = 243.30 nm and 301.09 nm. respectively. D3 derivative did not show zeros suitable for quantitative analysis. For the analysis of labetalol and sotalol mixture, D3 derivative spectrophotometry was used at the following wavelengths: = 246.03 nm and λ = 249.91 rum, respectively. In this case, the curves of Dl and D2 derivatives showed no zeros that can be used in quantitative analysis. To determine the concentration of the components in a mixture containing oxprenolol and sotalol the following wavelengths were selected: for oxprenolol DI λ = 245.32 nm, D2 λ = 240.18 run, D3 λ = 232.05 nm and for sotalol Dl λ = 230.56 nm, D2 Xλ= 232.65 nm and D3 X = 238.84 tm, respectively. The developed spectrophotometric method was characterized by high sensitivity and accuracy, LOD determined for sotalol was in the range of 0.21-1.88 µg/mL, for labetalol 1.00-3.43 µg/mL and for oxprenolol 0.16-2.06 µg/mL; LOQ determined for sotalol was in the range of 0.65-5.70 µg/mL, for labetalol 3.11-10.39 µg/mL and for oxprenolol 0.47-6.23 µg/mL, depending on the composition of the tested mixture and the order of the deriv- ative. The recovery of the individual components was within the range of 100 ± 5%. The linearity range was wide and estimated for sotalol in the range of 11.00-38.50 µg/mL, for labetalol 12.80-44.80 µg/mL and for oxprenolol 12.60-44.10 µg/mL with correlation coefficients in the range of 0.9977-0.9999.

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.

Quantitative determination of salbutamol in tablets by multiple-injection capillary zone electrophoresis.

A multiple-injection capillary zone electrophoresis (MICZE) method has been developed for the assay of salbutamol in Ventoline Depot tablets (GlaxoSmithKline). In the developed method, seven sample sets, each consisting of three samples, were sequentially injected into the capillary and analyzed within a single run. This enabled a total of twenty-one sequential injections, i.e., six standards and fifteen samples, containing salbutamol and the injection marker oxprenolol. The injected sample plugs were separated by plugs of background electrolyte, through application of a short-term voltage (30kV) over the capillary for different time periods, i.e., t(PE1) and t(PE2). The samples in each set were isolated from each other by partial electrophoresis for 2.35min (t(PE1)), while the sample sets were separated for 10.50min (t(PE2)). After the final injection, all the applied samples were subjected to electrophoresis for a time period corresponding to that in conventional single-injection CZE. The method was validated regarding linearity, accuracy, precision and robustness before it was applied to the determination of salbutamol in 15 tablets of Ventoline Depot with a labeled content of 8mg salbutamol. The average salbutamol content was determined to 7.8mg (+/-0.3mg) from simultaneous analyses of the 15 different tablets.

Enantiomeric separation by using nano-liquid chromatography with on-column focusing.

The present study shows a new nano-liquid chromatographic method for beta-blocker enantiomers' separation. This method consists of using a capillary column packed with silica particles which were chemically modified with vancomycin. On-column focusing allowed to inject relatively high sample volumes (1500 nL) increasing method sensitivity. The studied racemic compounds, namely atenolol, propranolol, oxprenolol, and metoprolol were dissolved in methanol and injected for chromatographic separation. The effect of injected sample volume was studied in the range of 50-2100 nL. Peak height of the two alprenolol enantiomers increased linearly up to 1500 nL. This volume was injected for validation and sample analysis. Under optimal experimental conditions, LODs and LOQs (LOD and LOQ for each alprenolol enantiomers) were 9.0 and 15.6 ng/mL, respectively. Calibration curves were linear in the studied range (9-250 ng/mL). The optimized method was applied to the analysis of a human plasma sample spiked with racemic alprenolol.

[Bromometric assay of alprenolol and oxprenolol]. / Zur bromometrischen Bestimmung von Alprenolol und Oxprenolol.

Alprenolol (1a) reacts with an excess of bromine to yield the tribromo derivative 3a by addition and monosubstitution, while applying oxprenolol (1b) the disubstituted tetrabromo derivative 2b is obtained. The N-dealkylated substance 3c was isolated as a by-product. Heating the compounds 2b and 3a with potassium hydroxide in acetone gives the 2-bromoallyl derivatives 5. Using potassium tert-butanolate the 2-propyne 7 is formed from 3a. The different colours, obtained from 1a, 1b, pindolol and propranolol with perchloric acid in acetic acid or conc. sulfuric acid, are suitable for the identification test in the European Pharmacopoeia.

Evaluation of the immobilized artificial membrane phosphatidylcholine. Drug discovery column for high-performance liquid chromatographic screening of drug-membrane interactions.

Chromatographic retention factors (k') of a series of eight beta-adrenoceptor antagonist compounds (beta-adrenolytic drugs) were determined employing an immobilized artificial membrane column (IAM.PC.DD). The influence of mobile phase pH, ionic strength, and organic modifier composition was studied in order to examine column performance. After the IAM.PC.DD columns were exposed to approximately 7000 column volumes of a 0.01 M PBS mobile phase, five out of six columns tested showed significant peak broadening and decreased k' values indicative of premature column failure. The data suggested that the immobilized phospholipids stationary phase was removed by the 0.01 M PBS mobile phase. The beta-adrenolytic drug's log k'IAM values obtained with an IAM.PC.DD column were compared to an esterIAM.PC.MG column for predicting drug membrane interactions. For the linear regression analysis between log k'IAM and the logarithm of the n-octanol-water partition coefficients (rIAM.PC.DD = 0.8710 vs. rIAM.PC.MG = 0.9538), the C18 HPLC retention factors (rIAM.PC.DD = 0.8408 vs. rIAM.PC.MG = 0.9380), the liposome partition coefficients (rIAM.PC.DD = 0.8887 vs. rIAM.PC.MG = 0.9187), and various pharmacokinetic parameters, significantly better correlations were obtained with the esterIAM.PC.MG column than the IAM.PC.DD column.

Determination of analytical error function for beta-blockers as a possible weighting method for the estimation of the regression parameters.

Three analytical methods have been developed and validated for the quantification of beta-blockers (celiprolol, bisoprolol and oxprenolol) using high performance liquid chromatography (HPLC) with UV detection. The methods were determined to be linear, precise and accurate (RSDs were lower than 5%), which allowed the quantitation of beta-blockers assayed at concentrations in the range 25-0.78 micrograms ml-1. After validation of reversed-phase HPLC methods, their analytical error functions were established by a rapid, simple and economical procedure. The discrimination of the best function for each active principle was performed by an appropriate polynomial statistical analysis, yielding SD (microgram ml-1) = 0.0295 + 0.0124C - 3.88 x 10(-4)C2 for celiprolol, 0.0199 + 0.011C - 1.27 x 10(-5)C3 for bisoprolol; and 0.0183 + 0.0089C - 9.68 x 10(-6)C3 for oxprenolol. These analytical error functions are an alternative to the weighting methods used in parameter estimation of beta-blockers.

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.

[Bromatometric determination of oxprenolol]. / Oxprenolol bromatometriás meghatározása.

A bromatometric method was elaborated for the quantitative determination of oxprenolol. 15 to 150 mg of oxprenolol dissolved in 10 cm3 of water is brominated by adding 20.00-40.00 cm3 of 0.02 or 0.1 N potassium bromate solution followed by 0.5-3 g of potassium bromide and 1-10 cm3 of 10% w/w hydrochloric acid. After a reaction time of 15 min. 2-10 cm3 of 10% w/w potassium iodide solution is added followed by 20-40 cm3 of chloroform. The liberated iodine is titrated with standard 0.1 N or 0.01 N sodium thiosulphate solution. A blank titration is also performed and the oxprenolol content is calculated from the difference of the two titrations. Under these conditions one oxprenolol molecule reacts with six bromine atoms. The relative standard deviation is 1.31%.

Spectrophotometric determination of oxprenolol hydrochloride as its Fe (III) complex.

A spectrophotometric determination of oxprenolol hydrochloride in pharmaceutical preparations is described. The method is based on the reaction of oxprenolol hydrochloride with Fe (III) ion in the presence of ammonium thiocyanate, in acid media. The complex formed between oxprenolol hydrochloride and Fe (III) ion was extracted with chloroform and assayed spectrophometrically at 477 nm. The results obtained are reproducible and hence the method is suitable for the determination of oxprenolol hydrochloride in pharmaceutical dosage forms.

[Resolution of the enantiomers of some beta-amino alcohols using chiral derivatization and reversed-phase liquid chromatography].

A convenient and rapid procedure for enantiomeric resolution of beta-amino alcohol drugs, such as oxprenolol, propafenone, timolol and phenylephrine, is described. The method is based on derivatization with the chiral reagent 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC), and resolution of the resulting diastereomeric thioureas by reversed-phase HPLC using methanol-aqueous monobasic ammonium phosphate mixture as the mobile phase. The salt forms of these drugs can be directly derivatized with GITC reagent instead of being converted into their free bases and without protection of the phenolic hydroxyl group of phenylephrine.

Analysis of oxprenolol in formulations by high-performance liquid chromatography.

A simple, accurate, and rapid high-performance liquid chromatographic method for the analysis of oxprenolol in commercial formulations is described. The analysis was performed on a cyano radial compression cartridge, with 0.0539 M, pH 3 phosphate buffer-acetonitrile-methanol (76: 15.6:8.4) as the mobile phase. The flow rate was 5 mL/min, with detection at 272 nm; the mobile phase was employed for extraction. The assay was applied to the content uniformity test of three oxprenolol hydrochloride tablet formulations of different strengths and the contents of a 2-mg dry ampule for intravenous/intramuscular injection. The percent of label claim for each formulation tested was within 91.7-110%. The applicability of this assay to the analysis of some other beta-blocking drugs was investigated. It was found that under the above conditions, atenolol, metoprolol, oxprenolol, and propranolol can be fully resolved in less than 3 min.

Aromatic hydroxylation of oxprenolol. Quantitation and stereoselectivity in the formation of 4'- and 5-hydroxyoxprenolol in vivo in the rat.

Aromatic hydroxylation of oxprenolol administered ip to rats produced 4'- and 5-hydroxyoxprenolol in 9.4% and 2.8% of the administered dose, in a ratio of ca. 3.5 to 1, as determined after deconjugation of the urinary metabolites by 8-glucuronidase. After HPLC separation, the metabolites were determined by mass spectral (CIMS) techniques using specifically deuterated 4'- and 5'-hydroxyoxprenolol-d2 as standards. From in vivo metabolic experiments using a pseudoracemate of oxprenolol prepared from (2R)-oxprenolol-d2 and (2S)-oxprenolol-d0, greater 4'-hydroxylation of the (2R)-enantiomer was shown to occur by 3.1 fold, and greater 5'-hydroxylation also of the (2R)-enantiomer occured by 1.4 fold. These results could occur because of stereoselectivity in the metabolic hydroxylation process and/or could reflect possible enantiomeric enrichment of the oxprenolol presented to the liver due to stereoselective tissue binding of the (2S)-enantiomer or could be due to stereoselectivity in the metabolism of oxprenolol by one or more competing pathways.

Quantitative determination of propranolol, propranolol glycol and N-desisopropylpropranolol in brain tissue by electron capture gas chromatography.

A method for the quantitative determination of propranolol and two of its active metabolites, 3-(alpha-naphthoxy)-1,2-propanediol (propranolol glycol) and N-des-isopropylpropranolol, in brain tissue of mice is described. Tissues are homogenized in perchloric acid-acetonitrile. Propranolol and its metabolites are isolated from the supernatant by solvent extraction and separated and detected as their trifluoroacetyl derivatives by electron capture gas chromatography. Chemical structures of the derivatives were confirmed by gas chromatography-mass spectrometry. The electron capture detector response of all three compounds is high, 0.7-2.0 X 10(-16) moles/sec. Brain levels of 10-250 ng/g can be detected of all three compounds with high specificity and good precision.