Evaluation of teicoplanin chiral stationary phases of 3.5 and 5 microm inside diameter silica microparticles by polar-organic mode capillary electrochromatography

Different types of fused-silica capillaries of 75 microm inside diameter (ID) were packed, namely type A and B, and evaluated for the direct resolution of racemates of several basic compounds by enantioselective capillary electrochromatography (e-CEC). Type A was packed with a chiral stationary phase (CSP) containing teicoplanin (TE) mixed with silica microparticles (3:1 w/w) while type B contained only the TE-CSP. In both cases, particles of different sizes (3.5 and 5 microm ID) were employed. A polar-organic mobile phase containing methanol-acetonitrile (60-40% v/v and 0.05% w/v ammonium acetate was used. Several beta-blockers (alprenolol, oxprenolol, metoprolol, pindolol, salbutamol, propranolol, atenolol, acebutolol) were baseline-enantioresolved with both capillary types, in very short times.     

Use of vancomycin chiral stationary phase for the enantiomeric resolution of basic and acidic compounds by nano-liquid chromatography

In this paper we studied the potentiality of nano-liquid chromatography (nano-LC) for the enantiomeric resolution of both basic and acidic compounds of pharmaceutical interest using a vancomycin modified silica stationary phase. Experiments were carried out in a fused silica capillary of 75 microm I.D. packed with chiral modified silica particles of 5 microm diameter, the detection, was done on-line at 195 nm. Enantiomeric resolution of alprenolol, atenolol, metoprolol, oxprenolol, pindolol, propranolol (basic compounds) and some acidic analytes, namely 2-[(5'-benzoyl-2'-hydroxy)phenyl]propionic acid (DF1738Y), 2-[(4'-benzoyloxy-2'-hydroxy)phenyl]propionic acid (DF1770Y), ketoprofen, indoprofen and suprofen was studied by nano-LC utilizing mobile phases containing methanol-acetonitrile-ammonium formate or acetate. The effect of mobile phase composition (buffer type and concentration, organic modifier type and concentration) on chiral resolution (Rs), retention factor (k) and retention time (tR) was also investigated. Good enantiomeric resolution was achieved for basic compounds utilizing the mobile phase containing 90% (MeCN-MeOH)/5% water/5% of 100 mM ammonium acetate pH 4.5. Acidic compounds such as DF1738Y and DF1770Y were better resolved at lower pH 3.5 while ketoprofen, indoprofen and suprofen exhibited the highest resolution at pH 4.5; in this case the mobile phase contained MeOH or MeCN (90%), 5% buffer and 5% of water. The nano-LC method was validated using R-(+)-propranolol as an internal standard finding good repeatability, detection limit, correlation coefficient and recovery and applied to the assay of a pharmaceutical formulation containing a racemic mixture of metoprolol.     

Separation of β-Blocker Enantiomers on Silica Modified with Gold Nanoparticles with Immobilized Macrocyclic Antibiotic Vancomicin

A new chiral sorbent based on mercaptosilica modified by gold nanoparticles, then treated by 3-mercaptopropionic acid and macrocyclic antibiotic vancomycin, was obtained. The enantioseparation of isomeric β-blockers (nadolol, atenolol, metoprolol, alprenolol, oxprenolol, and pindolol) by HPLC was studied on the synthesized sorbent. The effect of the composition of the mobile phase (nature and concentration of the organic solvent, concentration and pH of the buffer solution) on retention times of β-blocker enantiomers, selectivity of separation, and resolution of chromatographic peaks were studied. The best separation was achieved for pindolol and metoprolol. The procedure was used to determine the pindolol enantiomers in the preparation “Visken,” and metoprolol in the “Vazocardin” preparation.     

Role of chemical structure in stereoselective recognition of beta-blockers and H1-antihistamines by human serum transferrin in capillary zone electrophoresis

Studies on chiral resolution of beta-blocker and H1-antihistamine drugs by CZE using human serum transferrin are described. The drugs with different structures passed a pseudostationary protein zone in a coated capillary applying the partial filling method for the chiral separation. In this study we screened 15 compounds; most of them showed longer migration time, indicating an interaction with transferrin. Stereoselective interaction was observed only for five beta-blockers (celiprolol, talinolol, mepindolol, bopindolol, and oxprenolol) and for one H1-antihistamine (brompheniramine). The most important finding was that very small differences in the chemical structure of the drug resulted in significant changes in the stereoselective recognition. Resolution of mepindolol enantiomers was observed showing the essential role of one methyl group compared to pindolol, which is not resolved by transferrin. Bopindolol, also a derivative of pindolol having bigger difference in the structure, showed more experienced separation. The very slight difference between alprenolol and oxprenolol was also revealed with these methods, since only oxprenolol enantiomers, having an extra oxygen in the structure, are resolved. Determining the migration order of the eutomers and distomers (chlorpheniramine, brompheniramine) we can deduct conclusions about the role of serum proteins in the delivery of drugs within the body.     

Determination of β-adrenoreceptor antagonists in urine by high-performance liquid chromatography with diode-array spectrophotometric detection

The determination of pindolol, oxprenolol and propranolol in human urine is described. The drugs are isolated with a GDX-502 resin-packed column, separated on a C₁₈ (5-μm) reversed-phase column with methanol/aqueous acetic acid as mobile phase and quantified with diode-array spectrophotometric detector. The recovery was > 93%, and detection limits were 2 ng for pindolol, 12 ng for oxprenolol and 2 ng for propranolol. Results are given for urine from healthy volunteers who had received the drugs orally.     

Separation of the enantiomers of beta-receptor blocking agents and other cationic drugs using a CHIRAL-AGP column. Binding properties and characterization of immobilized alpha 1-acid glycoprotein

The effect of the immobilization procedure on the conformation of alpha 1-acid glycoprotein (AGP) was investigated by recording the fluorescence spectra of native and immobilized AGP. A 20-nm red shift was obtained for the immobilized form of AGP compared with the emission maximum of 338 nm obtained for native AGP. This demonstrates that the tryptophan residues are exposed on the protein surface after immobilization, indicating that the immobilized form of AGP has a more unfolded structure than the native AGP. The effect of N,N-dimethyloctylamine on the enantioselectivity for some fentiazine derivatives, observed with immobilized AGP, was equal to that obtained with AGP as a chiral complexing agent in the mobile phase. This demonstrates that even though the immobilization procedure affects the conformation of the protein there still exist large similarities between native and immobilized AGP concerning chiral recognition. The adsorption isotherm of (-)-terodiline was studied by use of the breakthrough technique. The adsorption isotherm indicates that (-)-terodiline is adsorbed to one site with high affinity and at least one more site with lower affinity. It was also observed that the enantiomers of amines, acids and non-protolytic compounds compete with the cationic compound, (-)-terodiline, for binding to the same sites. The beta-receptor blocking agents atenolol, metoprolol, pindolol, alprenolol, oxprenolol and propranolol were resolved on a CHIRAL-AGP column. The retention and enantioselectivity are highly influenced by the structure of the solute and the nature of the uncharged mobile phase additives. Separation factors of 1.2-1.8 were obtained for the beta-blockers under the studied conditions.     

Fluorescence studies on the interactions of barbaloin with bovine serum albumin

The fluorescence quenching reactions of barbaloin with bovine serum albumin (BSA) in pH 7.20 Tris-HCl buffer solution were studied. The quenching mechanism of BSA by barbaloin was interpreted using the Stern-Volmer (S-V) mechanism. The binding constant K values were 2.78 x 10(5) (293 K), 1.87 x 10(5) (310 K), 1.25 x 10(5) (318 K), and the number of binding sites (n) were 1.18, 1.14, and 1.09, respectively. In addition, the thermodynamic functions enthalpy (deltaH degrees ) and entropy (deltaS degrees ) for the reaction were also calculated according to Vant's Hoff equation were -23.7 kJ/mol and 23.6 J/mol, respectively. Plausible explanations of the quenching mechanism are discussed on the basis of a hydrophobic interaction between barbaloin and BSA.     

Chiral protein scissors activated by light: recognition and protein photocleavage by a new pyrenyl probe

Strong chiral discrimination and site-selective photocleavage of two model proteins, lysozyme and bovine serum albumin (BSA), by new pyrenyl probes are reported here. The enantiomeric pyrenyl probes D-phenylalanine-1(1-pyrene)methylamide (PMA- D-Phe) and L-phenylalanine-1(1-pyrene)methylamide (PMA- l-Phe) were synthesized by coupling the carboxyl function of D-phenylalanine or L-phenylalanine with the amino group of 1(1-pyrene)methylamine. Binding affinities of the two enantiomers with the proteins were quantitated in absorption titrations. BSA indicated 10-fold selectivity for PMA- D-Phe, and the binding constants for the L- and D-enantiomers were 3.8 x 10(5) and 4.0 x 10(6) M(-1), respectively. Lysozyme, similarly, indicated a 6-fold preference for PMA- D-Phe with binding constants of 3.3 x 10 (5) and 2.0 x 10(6) M(-1) for the L- and D-isomers, respectively. Such strong chiral discrimination illustrates the key role of the chiral center of the probe (Phe) in the binding interactions. The enantiomers were tested to examine how the chiral discrimination for their binding influences reactivity toward protein photocleavage. Irradiation of the probe-protein complexes, at 342 nm in the presence of hexammine cobalt(III) chloride, resulted in the cleavage of the protein backbone. Photocleavage did not proceed in the dark or in the absence of the pyrenyl probes. Both enantiomers indicated low reactivity with BSA (<5% yield), while large photocleavage yields ( approximately 57%) have been noted with lysozyme. This lysozyme photocleavage yield is a significant improvement over previous reports. However, both enantiomers cleaved lysozyme at the same location between Trp108-Val109, despite the strong chiral selectivity for binding. H-atom abstraction from Trp 108, accessible from the active site cleft, could initiate the observed peptide bond cleavage.     

Multivariate optimization approach for chiral resolution of drugs using human serum albumin in affinity electrokinetic chromatography-partial filling technique

The enantiomeric resolution of chiral compounds using HSA by means of affinity EKC (AEKC)-partial filling technique is the result of a delicate balance between different experimental variables such as protein concentration, running pH (background electrophoretic buffer, protein and compound solutions) and protein solution plug length. In this paper multivariate optimization approaches for chiral separation of four basic drugs (alprenolol, oxprenolol, promethazine and propranolol) using HSA as chiral selector in AEKC-partial filling technique are studied. The experimental conditions to achieve maximum resolution are optimized using the Box-Behnken experimental design. Partial least squares and pareto charts are used to analyse the main effects on the resolution. The experimental resolutions observed for all compounds studied in optimum conditions agree with the estimated values based on response surface models. The results obtained show that the range of experimental conditions that provided enantioresolution narrows as hydrophobicity of analytes decreases. This fact can be explained by assuming that hydrophobicity controls the interaction of basic compounds with HSA.     

Separation of β-blocker and amino acid enantiomers on a mixed chiral sorbent modified with macrocyclic antibiotics eremomycin and vancomycin

A mixed chiral sorbent based on silica with immobilized macrocyclic antibiotics eremomycin and vancomycin was synthesized. A possibility of the separation of enantiomers of β-blockers (metoprolol, pindolol, alprenolol, oxprenolol, labetalol, and atenolol) and amino acids (tryptophan, phenylalanine, DOPA, methionine, and acetyl glutamic acid) on this chiral sorbent by HPLC was studied. The influence of the composition of the mobile phase (pH of buffer solution, its concentration, content of organic modifier, and its nature) on the retention times of β-blocker and amino acid enantiomers, selectivity, and resolution of peaks was studied. It was shown that the mixed chiral sorbent has enantioselectivity to both classes of compounds, while silica modified with vancomycin has no ability to the separation of enantiomers of non-derivatized amino acids, and silica modified with eremomycin has no ability to the separation of β-blocker enantiomers. High values of resolution for amino acids (max Rs > 4) and β-blockers (max Rs > 1) were obtained.     

Chiral separation of β-adrenergic blockers on CelluCoat column by HPLC

The chiral separation of 10 β-adrenergic blockers (acebutalol, alprenolol, bufuralol, bisoprolol, celiprolol, carazolol, indenolol, metoprolol, oxprenolol and propranolol) was achieved on CelluCoat column (250 mm × 4.6 mm, 5 μm particle size). The mobile phases used were (90:10:0.2, v/v/v) and (95:5:0.2, v/v/v) combinations of n-heptane-ethanol-diethylamine, respectively. The flow rates were 0.5, 1.0 and 2.0 mL min⁻¹ with detection at 225 nm. The capacity (k), selectivity (α) and resolution (R_s) factors were 0.44-12.91, 1.12-2.19 and 1.00-9.50, respectively. The proposed supra-molecular models indicated that the chiral resolution were governed by π-π interactions, hydrogen bondings and steric effect.     

Bovine serum albumin-mediated enantiodifferentiating photocyclodimerization of 2-anthracenecarboxylate

Enantiodifferentiating photocyclodimerization of 2-anthracenecarboxyalate (AC) was performed at 25 degrees C in aqueous buffer solution (pH 7) in the presence of bovine-serum albumin (BSA) to afford four [4 + 4] cyclodimers, i.e., anti- and syn-head-to-tail (HT) (1 and 2) and anti- and syn-head-to-head (HH) dimers (3 and 4), of which only 2 and 3 are chiral. We found that (1) BSA possesses four sets of binding sites for AC of different affinities, stoichiometries, and chiral environment for photoreaction, which bind 1, 3, 2, and 3 AC molecules with binding constants of 5.3 x 107, 1.3 x 105, 1.4 x 104, and 3.0 x 103 M-1, respectively, (2) the regioselectivity of photodimerization is switched from HT to HH by adding BSA (the HH/HT ratio varies from 0.28 to 4.3), (3) BSA-mediated photodimerization of AC affords optically active products 2 and 3 of up to 29% and 41% ee, respectively. It is emphasized that the selective excitation of bound substrate, utilizing the spectral shift upon complexation with BSA, is not a prerequisite for efficient photochirogenesis using biomolecules.     

Carboxymethyl β‐cyclodextrin as chiral selector in capillary electrophoresis: Enantioseparation of 16 basic chiral drugs and its chiral recognition mechanism associated with drugs' structural features

Herein we present the enantioseparation of 10 cardiovascular agents and six bronchiectasis drugs including propranolol, carteolol, metoprolol, atenolol, pindolol, esmolol, bisoprolol, bevantolol, arotinolol, sotalol, clenbuterol, procaterol, bambuterol, tranterol, salbutamol and terbutaline sulfate using carboxymethyl‐β ‐cyclodextrin (CM‐β ‐CD) as chiral selector. To our knowledge, there is no literature about using CM‐β ‐CD for separating carteolol, esmolol, bisoprolol, bevantolol, arotinolol, procaterol, bambuterol and tranterol. During the course of work, changes in pH, CM‐β ‐CD concentration, buffer type and concentration were studied in relation to chiral resolution. Excellent enantiomeric separations were obtained for all 16 compounds, especially for procaterol. An impressive resolution value, up to 17.10, was obtained. In particular, most of them achieved rapid separations within 20 min. Given the fact that enantioseparation results rely on analytes' structural characters, the possible separation mechanisms were discussed. In addition, in order to obtain faster separation for propranolol enantiomers in practical application, the effective length of capillary was innovatively shortened from 45 to 30 cm. After the validation, the method was successfully applied to the enantiomeric purity determination of propranolol in the formulation of drug substances.     

Enantiomeric separation of basic drugs with partially filled serum albumin as chiral selector in capillary electrophoresis.

A reliable method is presented for the chiral separation of three basic drugs (mexiletine, chlorpheniramine and propranolol) with serum albumins (human and porcine, HSA and PSA) as chiral selectors by capillary electrophoresis in combination with the partial filling technique. Based on the systematic optimization of operation variables, the chiral separation of mexiletine, chlorpheniramine and propranolol was achieved in the pH 7.4 phosphate buffer by using HSA, PSA and PSA as selectors, respectively. The chiral recognition ability of HSA and PSA was compared. HSA and PSA show a different chiral recognition ability for each of the three drugs. In addition, the association constants between enantiomeric drugs and proteins were determined to be 2.00 and 3.80 x 10(2) M(-1) for mexiletine and HSA, 0.59 and 1.12 x 10(3) M(-1) for chlorpheniramine and PSA, and 0.87 and 1.42 x 10(3) M(-1) for propranolol and PSA. The method for the chiral separation and determination of association constants possesses the advantages of simple performance, effective avoiding of the interference of the UV detection from protein, and lowering of the reagent consumption.     

Indirect resolution of beta-blocker agents by reversed-phase capillary electrochromatography

The indirect resolution of five beta-adrenoreceptor blocking agents (propranolol, oxprenolol, pindolol, metoprolol, and atenolol) using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC), and capillary electrochromatography (CEC) is reported. Three octadecylsilanized (ODS) silica gel-based stationary phases, differing in particle diameter and carbon surface density for suitability of determination of the enantiomeric composition of these substances in drugs and biological fluids, were studied. Under optimum separation conditions, all the investigated compounds were baseline-resolved within acceptable analysis times (i.e., between 10-16 min). The resolution values ranged between 1.80 and 1.14, and the separation factors were in no case less than 1.07. The possible use of the developed CEC method for the determination of enantiomeric composition of beta-adrenoreceptor blocking agents in drug substances was investigated. A study, in which the enantiomers of metoprolol were examined regarding specificity, reproducibility, limit of detection, and ruggedness, was developed in accordance with some analytical procedures for method validation.     

Synthesis and application of clindamycin succinate as a novel chiral selector for capillary electrophoresis

A novel chiral selector, clindamycin succinate, was synthesized and first used as a chiral selector in capillary electrophoresis (CE). The chiral resolution ability of this kind of clindamycin derivation was studied by CE using some racemic drugs as model analytes. From the experimental results, it was found that both resolution and selectivity of the selector were dependent on the following parameters: concentration of chiral selectors, pH of the running buffer, temperature of the capillary column, applied voltage and organic modifier used. The results show that the chiral selector possesses high resolution toward some racemic drugs, including ofloxacin, chlorphenamine, tryptophan, propranolol, sotalol and metoprolol. Excellent chiral resolution of these tested drugs was achieved under the optimal conditions of 50 mM clindamycin succinate, 10% MeOH v/v, 50 mM Tris buffer, pH 4.0, at 22 kV and 20 °C within 25 min.     

毛细管区带电泳法测定4种抗HIV-1活性的化合物与牛血清白蛋白的结合常数

采用毛细管区带电泳紫外检测方法,在pH 8.0、浓度50 mmol/L的磷酸盐缓冲溶液及运行电压15 kV的条件下,测定了4种新合成的具有抗HIV-1活性的化合物(IG3,iso-C3,C3,MC3)与牛血清白蛋白(BSA)相互作用的结合常数。在缓冲溶液中加入不同浓度的BSA,通过测定化合物迁移时间的变化,计算得到了上述4种化合物与BSA的结合常数分别为1.07×104, 1.34×104, 8.51×103和9.45×103 L/mol。该方法简单、快捷,可用于研究结合比为1∶1的小分子与生物大分子的     

Electrochemical studies of the interaction of clarithromycin with bovine serum albumin

The interaction of clarithromycin (CAM) with bovine serum albumin (BSA) was investigated in pH 4.5 - 8.0 phosphate buffer solutions in which three irreversible reduction waves P(1), P(2) and P(3) of CAM appeared on linear-sweep voltammetry on a static dropping mercury working electrode. In the acidic media, with the addition of BSA into the CAM solution, a new electrochemically active complex was formed and there was interaction between the carbonyl group C=O in the C-9 position of CAM and BSA. It was found that electrostatic and hydrophobic forces played an important role in the binding reaction. However, new electrochemically non-active complexes were formed at physiological pH condition. The study showed that the formation constant and formation ratio of the interaction between CAM and BSA were 1.51 x 10(12) and 3:1 for P(2) wave, and 4.53 x 10(5) and 1:1 for P(3) wave, respectively. The ion strength enhanced the hydrophobic interaction between CAM and BSA.     

Chiral complexation and aggregation of bilirubin with serum albumin at a liquid/liquid interface

The chiral complexation of bilirubin (BR) with bovine and human serum albumin (BSA and HSA), and the aggregation of the complexes at the heptane+chloroform(5:1)/water interface were studied via UV/Vis absorption and circular dichroism (CD) measurements in combination with the centrifugal liquid membrane (CLM) method. The interfacial adsorptivities of BR, BSA and their complexes were also studied by performing interfacial tension measurements at the interface. The changes in the absorbances and the induced CD amplitudes of the interfacial BR-BSA complex provided insights into the mechanism of the conformational enantioselective complexation at the interface, and indicated that the chiral conversion induced by the complexation with BSA was from the P(+) form to the M(-) form of BR. The broadening of the 450 nm band and the appearance of a new shoulder at 474 nm further supported the formation of aggregates of the complexes at the interface. The dependence of the CD amplitude on the molar ratio of BSA to BR revealed that the composition of the complex was 1:1 BSA:BR. The probable interfacial reaction scheme was proposed, and the affinity constant of BR-BSA at the interface was found to be 4.67 x 10(8) M(-2). The interfacial complexation and aggregation of BR and HSA were weaker than those of the BR-BSA complex due to the different BR binding positions adopted for BSA and HSA and the binding effect of chloroform.     

Improvement of peak shape and separation performance of beta-blockers in conventional reversed-phase columns using solvent modifiers

A comparative study of peak shape, elution behavior, and resolution of 16 beta-blockers (acebutolol, alprenolol, atenolol, bisoprolol, carteolol, celiprolol, esmolol, labetalol, metoprolol, nadolol, oxprenolol, pindolol, practolol, propranolol, sotalol, and timolol) chromatographed with hybrid mobile phases of triethylamine (TEA)-acetonitrile and sodium dodecyl sulfate (SDS)-propanol is performed using conventional reversed-phase columns and isocratic elution. Both solvent modifiers (TEA and SDS) prevent the interaction of the basic drugs with the alkyl-bonded phase. However, the protection mechanisms of silanols on the packing are different. Whereas TEA associates with the silanol sites (blocking ion-exchange processes or repelling the solutes), the long hydrophobic chain of SDS is inserted in the bonded organic layer with the sulfate group protruding outside, which makes the stationary phase negatively charged. The effects of TEA, acetonitrile, SDS, and propanol on the elution strength, efficiency, peak asymmetry, and resolution are examined under an experimental design basis that is assisted by computer simulation to reach more general conclusions. The combination of improved peak shapes, larger selectivity, and a smaller range in retention among compounds of extreme polarity leads to the observation that a greater number of beta-blockers can be resolved with a hybrid micellar system.