Enantioseparations in nonaqueous capillary electrophoresis using charged cyclodextrins.

The enantioseparation of acidic and basic compounds can be successfully achieved in nonaqueous capillary electrophoresis using single-isomer charged ß-cyclodextrin (ß-CD) derivatives of opposite charge to that of the analytes. This chapter describes how to separate the enantiomers of three basic substances selected as model compounds, i.e., alprenolol, bupranolol, and terbutaline, using the negatively charged heptakis(2,3-di-O-acetyl-6-O-sulfo)-ß-CD. The enantiomers of three acidic drugs (tiaprofenic acid, suprofen, and flurbiprofen) are resolved using a monosubstituted amino ß-CD derivative, namely, 6-monodeoxy-6-mono(3-hydroxy)propylamino-ß-CD.

1H and 13C NMR characteristics of ß-blockers.

The ß-blockers are important drugs and decades of clinical experience proved their high medical status. However, to the best of our knowledge, there is no complete assignment of (1)H and (13)C NMR resonances of popular representatives: acebutolol, alpenolol, pindolol, timolol and propranolol and the published NMR data on carvedilol and atenolol are incorrect. Therefore, (1)H and (13)C NMR spectroscopy was applied for the characterization of a series of ß-adrenolytics: carvedilol (1), pindolol (2), alprenolol (3), acebutolol (4), atenolol (5), propranolol (6) and timolol (7). Two-dimensional NMR experiments (COSY, HMQC, HMBC, NOESY) allowed the unequivocal assignment of (1)H and (13)C spectra for solution (DMSO-d(6) ). Salts and bases can be easily distinguished based on (13)C chemical shifts which are within 65.0-65.5 ppm (OC2) and 46.9-47.0 (NC3) for hydrochlorides and larger, ca. 68.4 ppm (OC2) and 50.3-52.6 (NC3) for bases. NMR data of 1-7 should be included in pharmacopoeias.

Effect of radiation sterilization on alprenolol in the solid state studied by high-performance thin-layer chromatography.

The possibility of radiation sterilization of alprenolol (AL) has been studied. Irradiation of AL in solid form with a 25 kGy beam of electrons caused only an insignificant change in color that became more intense with increasing irradiation dose. Moreover, with increasing dose a decrease in pH, the content of water, and the degree of crystallinity were observed. AL in solid form was radiated with a high-energy electron beam (9.96 MeV) at doses from 25-400 kGy and analyzed by HPTLC using the mobile phase methanol-ammonia 25% (99 + 1, v/v). Densitometric analysis was carried out directly from chromatograms at 270 nm. The applied method was validated and characterized by good precision (RSD = 3.95%); good accuracy (80% level 100.15%, 100% level 99.99%, and 120% level 104.44%); and low LOD (LOD = 0.52 microg/zone and LOQ = 1.55 microg/zone). Chromatograms recorded for samples irradiated at the doses of 25 kGy were unchanged, but at higher doses (100-400 kGy) additional peaks corresponding to the radiodegradation products appeared (Rf = 0.24 and Rf = 0.40). The decrease in the concentration of AL was proportional to the applied radiation dose, and for 400 kGy the concentration of AL was 90.23%. The calculated radiolytic yield of the radiodegradation process was G(-AL) = 7.12 x 10(-7) mol/J.

Preparation and evaluation of propranolol molecularly imprinted solid-phase microextraction fiber for trace analysis of beta-blockers in urine and plasma samples.

An improved multiple co-polymerization technique was developed to prepare a novel molecularly imprinted polymer (MIP)-coated solid-phase microextraction (SPME) fiber with propranolol as template. Investigation was performed for the characteristics and application of the fibers. The MIP coating was highly crosslinked and porous with the average thickness of only 25.0 microm. Consequently, the adsorption and desorption of beta-blockers within the MIP coating could be achieved quickly. The specific selectivity was discovered with the MIP-coated fibers to propranolol and its structural analogues such as atenolol, pindolol, and alprenolol. In contrast, only non-specific adsorption could be shown with the non-imprinted polymer (NIP)-coated fibers, and the extraction efficiencies of propranolol and pindolol with the MIP-coated fibers were higher markedly than that with the commercial SPME fibers. A MIP-coated SPME coupled with high-performance liquid chromatography (HPLC) method for propranolol and pindolol determination was developed under the optimized extraction conditions. Linear ranges for propranolol and pindolol were 20-1000 microg L(-1) and detection limits were 3.8 and 6.9 microg L(-1), respectively. Propranolol and pindolol in the spiked human urine and plasma samples, extracted with organic solvent firstly, could be simultaneous monitored with satisfactory recoveries through this method.

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.

Insights into cyclodextrin interactions during sample stacking using capillary isotachophoresis with on-line microcoil NMR detection.

On-line capillary isotachophoresis (cITP)-NMR experiments were used to probe the interactions of the pharmaceutical compounds S-alprenolol, S-atenolol, R-propranolol, R-salbutamol and S-terbutaline with beta-cyclodextrin (beta-CD) during cITP concentration. In cITP, ionic analytes are concentrated and separated on the basis of their electrophoretic mobility. Because neutral molecules have an electrophoretic mobility of zero, they are normally not concentrated or separated in electrophoretic experiments like cITP. Most of the analytes studied were concentrated by cITP sample stacking by a factor of around 300. For analytes that formed a strong inclusion complex, beta-CD co-concentrated during cITP sample stacking. However, once the focusing process was complete, a discrete diffusional boundary formed between the cITP-focused analyte band and the leading and trailing electrolyte, which restricted diffusion into and out of the analyte band.

[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.

Chiral separation of nanomole amounts of alprenolol with cITP/NMR.

On-line cITP-NMR with chiral selectors separates and concentrates analytes and identifies host-guest interactions of analytes with selectivity enhancers in the electrolyte. An NMR microcoil designed for a 200 microm i.d. capillary creates a high-mass-sensitivity 30 nL NMR cell and is used as an on-line detector for cITP. Using a mixture of 2 nmol racemic alprenolol in acetate buffer with alpha-cyclodextrin and sulfated beta-cyclodextrin at pD 6.0, cITP-NMR successfully separates and concentrates both R- and S-alprenolol. The concentration enhancement for the R isomer is 224-fold and for the S isomer is 200-fold. The estimated concentration at peak maximum for R-alprenolol is approximately 28 mmol L(-1) and a slightly lower concentration, 25 mmol L(-1) is achieved for S-alprenolol. These concentrations convert to placing 76% of the injected S-alprenolol and 84% of the R-alprenolol into the 30 nL detection cell at peak maximum. With on-flow cITP-NMR, intermolecular interactions between the cyclodextrins and the alprenolol are observed in the NMR spectra. Aromatic and methyl moieties of R- and S-alprenolol are identified as two important sites that bind with these particular cyclodextrins.

Electrophoretic behavior of alprenolol in mixed solvent electrolyte systems.

The electrophoretic mobilities of alprenolol have been determined in a mixed solvent background electrolyte system containing sodium acetate (40 mM)+acetic acid (40 mM) as buffering agent and different volume fractions of water, methanol and ethanol using capillary electrophoresis. The mobility of alprenolol has been used to test the prediction capability of a model trained by previously reported mobility data of five beta-blocker drugs at the same electrophoretic conditions. The average percentage mean deviations (APMD) between experimental and predicted values were used as an accuracy criterion. The APMD (+/-SD) obtained for alprenolol data in binary/ternary solvent electrolyte system employing the mobility values in mono-solvent buffers was 4.37 (+/-3.50)% and the corresponding value for an ab initio prediction method was 7.65 (+/-4.30)%.

Restricted access media-molecularly imprinted polymer for propranolol and its application to direct injection analysis of beta-blockers in biological fluids.

A restricted access media-molecularly imprinted polymer (RAM-MIP) for propranolol (PRP) has been prepared for direct injection analysis of beta-blockers in biological fluids. First, the MIP for PRP was prepared using methacrylic acid and ethylene glycol dimethacrylate as the functional monomer and cross-linker, respectively, by a multi-step swelling and polymerization method. Next, a 1:1 mixture of glycerol monomethacrylate and glycerol dimethacrylate was used for hydrophilic surface modification, and added directly to the MIP for PRP after 4 h from the start of polymerization. Then further polymerization was carried out for 20 h. The obtained RAM-MIP for PRP showed excellent molecular recognition ability for PRP, good ones for alprenolol (ALP) and pindolol, and fair ones for other beta-blockers. The RAM-MIP was applied for direct injection analysis of ALP enantiomers in a rat plasma sample by a column-switching HPLC system using a beta-cyclodextrin phenylcarbamate-bonded silica column as the analytical column. The calibration graph, constructed from peak area versus each ALP enantiomer concentration, was linear with a correlation coefficient of > 0.999 over the concentration ranges of 12.5-250 ng ml(-1). The limit of quantitation was 12.5 ng ml(-1) with a 50 microl injection. This method could be applicable for the assay of ALP enantiomers at the therapeutic plasma levels, and have wide applicability for the assay of beta-blockers in biological fluids.

On-line capillary electrophoresis-electrospray ionization mass spectrometry using a polymerized anionic surfactant.

On-line capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) has been used to determine the tricyclic antidepressant drugs (imipramine, doxepin, and amitriptyline) as well as the beta-adrenergic blocker drugs (propranolol and alprenolol). A CE-ESI-MS interface linking a manually operated CE system and a Finnigan MAT-900 sector mass spectrometer (with an Analytica electrospray ionization source) has been constructed in-house and employed for this study. Although a water/methanol based capillary zone electrophoresis (CZE) buffer was initially used to determine these analytes, enhanced resolution was obtained by addition of a polymerized surfactant, i.e., poly-sodium undecylenic sulfate (poly-SUS), into the electrokinetic chromatography (EKC) buffer. When a low concentration of this poly-SUS surfactant was added to a volatile EKC buffer, these structurally similar cationic drugs were EKC separated and on-line detected by ESI-MS.

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.

Enantioselective quantitation of (R)- and (S)-alprenolol by gas chromatography-mass spectrometry in human saliva an plasma.

An enantioselective gas chromatographic-mass spectrometric assay is developed for alprenolol and its metabolite, 4-hydroxy-alprenolol, in saliva and plasma. The procedure is based on a two-step derivatization technique with N-heptafluorobutyryl-l-prolylchloride and N-methyl-N-trimethylsilyl-trifluoroacetamide, followed by a gas chromatographic separation with mass spectrometric detection of the diastereomeric derivatives. A selected ion chromatogram extracted from full scan data shows that the respective enantiomers of alprenolol, 4-hydroxy-alprenolol, and the internal standard (ions at m/z 481) are well-separated in saliva and plasma. Linear and reproducible calibration curves are obtained over the concentration ranges 1.67-13.33 ng/mL and 2.50-20.00 ng/mL enantiomer in saliva and plasma, respectively. The performance of the method for alprenolol, in terms of accuracy and precision, fits well within the generally accepted criteria for validation. The enantioselective assay is successfully used in a study involving a single oral dose of alprenolol administered to two healthy volunteers. Stereoselective differences are observed in the saliva and plasma concentrations following an oral dose of 50 mg (R,S)-alprenolol hydrochloride.

[Application of Pirkle-type chiral column to the resolution of enantiomeric drugs: resolution and identification of alpha-naphthyl isocyanate derivatives of alprenolol and some other beta-blockers].

alpha-Naphthyl isocyanate (NI) as a derivatizing agent to separate several beta-blockers was described. This procedure is simple, rapid and gives good resolution. Experiments with NI derivatives of alprenolol showed that the isocyanate group reacted with the amino group on the analyte to form urea under mild conditions and with the hydroxyl group to form carbamate under relatively vigorous conditions. The results also indicated that the derivatized reactions is 1:1 between NT and analytes.

Influence of solute polarity in column-switching chromatography for the assay of drugs in plasma and urine.

The polarity of a drug is one of the most important parameters for the elaboration of switching systems. If the polarity of the drug is low or medium, "reversed-phase" chromatography is well adapted. The plasma or urine sample is diluted with water, centrifuged and injected first into a column of medium polarity (C2, CN or diol bonded phases). The compounds of interest are stopped on the top of the column and rinsed with water, then eluted and chromatographed on a C8 or C18 analytical column. A third column of still lower polarity can be added to improve the specificity of the system. In each successive step, the polarities of the mobile phases and columns should be decreased to reconcentrate the sample and reduce the band broadening that occurred in the previous step. Compounds of high polarity show almost no retention on reversed-phase columns, and normal-phase chromatography should be used. Aqueous solutions cannot be injected into polar bonded-phase columns as they lead to excessive band broadening. This problem can be solved by diluting plasma or urine with a large volume of a water-miscible organic solvent and injecting the clear supernatant. The compounds to be assayed are first reconcentrated on a polar column (NH2 or N(CH3)2 bonded phase) and then eluted. The selected "heart cut" of the eluate is chromatographed on another, more polar column. The influence of the polarity of drugs on the choice of switching systems is exemplified by assay methods for drugs of low, medium and high polarity.

NMR determination of isosorbide dinitrate and beta-adrenergic blocking agents in tablets.

An NMR spectroscopic method for the determination of isosorbide dinitrate, alone or together with alprenolol or propranolol, is described. Spectra are determined in dimethyl sulfoxide-d6 containing maleic acid or 1,4-dinitrobenzene as internal standards. Both synthetic mixtures and commercial formulations were assayed, and the results were compared using compendial procedures.

Separation of enantiomeric amines by ion-pair chromatography.

A high-performance liquid chromatographic method for the separation of optical isomers (enantiomers) of amines is described. It is based on ion-pair chromatography with a chiral counter ion in a system with an organic mobile phase and an adsorbing stationary phase. The method has been applied to enantiomers of 1-aryloxy-3-isopropylamine-2-propanol derivatives (alprenolol, metoprolol, propranolol) which are completely resolved with (+)-10-camphorsulphonate as the counter ion. Studies of the influence of the counter-ion structure and the mobile phase composition are presented.

The central action of drugs affecting beta-adrenergic receptor. IV. The influence of intraventricularly administered drugs affecting beta-adrenergic receptor on blood pressure in rats.

Isoprenaline, propranolol, alprenol and sotalol administered intraventricularly (ivc) to anaesthetized rats induce hypotension. Propanolol and alprenolol given ivc to rats pretreated with isoprenaline increase blood pressure up to the values equal to those, previous to isoprenaline administration; another isoprenaline dose acts slightly hypotensively.