Hydroxypropyl ß-cyclodextrin nanohybrid monoliths for use in capillary electrochromatography with UV detection: application to the enantiomeric separation of adrenergic drugs, anticholinergic drugs, antidepressants, azoles, and antihistamine.

Two kinds of hydroxypropyl ß-cyclodextrin nanohybrid monoliths were synthesized and applied in capillary electrochromatography with UV detection. One column was fabricated by concurrently using glycidyl methacrylate-bonded hydroxypropyl ß-cyclodextrin (GMA-HP-ß-CD), sodium 3-mercaptopropanesulphonate, and alkoxysilanes in the "one-pot" process. The other was prepared by free radical polymerization of GMA-HP-ß-CD, vinylmethylcyclosiloxane, ethylene dimethacrylate, and 2-acrylamido-2-methyl propane sulfonic acid. Compared to the former hybrid monolith, the latter one displayed improved enantiomeric separation. For ten adrenergic drugs, six anticholinergic drugs, two antidepressants, six azoles, and one antihistamine enantiomeric separation was obtained on the monolith synthesized by free radical polymerization. Twelve out of twenty-five drugs were baseline-separated. Especially, anisodamine with two chiral centers was successfully separated with resolution values of 3.06, 2.11, and 2.17. The nanohybrid monoliths were characterized by optical microscopy, scanning electron microscopy, FT-IR, nitrogen adsorption analysis, and thermogravimetric analysis. Relative standard deviation values less than 5% were obtained through run-to-run, day-to-day, and column-to-column investigations (n = 3). Graphical abstract Schematic representation of two kinds of hydroxypropyl ß-cyclodextrin nanohybrid monoliths based on "one-pot" approach (route I) and free radical polymerization approach (route II), respectively.

In situ immobilization of sulfated-ß-cyclodextrin as stationary phase for capillary electrochromatography enantioseparation.

In this work, a novel sulfated-ß-cyclodextrin (S-ß-CD) coated stationary phase was prepared for open-tubular capillary electrochromatography (OT-CEC). The capillary was developed by attaching polydopamine/sulfated-ß-cyclodextrin (PDA/S-ß-CD) onto the gold nanoparticles (AuNPs) coated capillary which was pretreated with polydopamine. The results of scanning electron microscopy (SEM) and energy dispersive X-ray analysis spectroscopy (EDS) indicated that polydopamine/sulfated-ß-cyclodextrin was successfully fixed on the gold nanoparticles coated capillary. To evaluate the performance of the prepared open tubular (OT) column, the enantioseparation was carried out by using ten chiral drugs as model analytes. Under the optimal conditions, salbutamol, terbutaline, trantinterol, tulobuterol, clorprenaline, pheniramine, chlorpheniramine, brompheniramine, isoprenaline and tolterodine were baseline separated with the resolution (Rs) values of 3.25, 1.76, 2.51, 1.89, 3.17, 2.17, 1.99, 1.72, 2.01 and 3.20, respectively. Repeatability of the column was studied, with the relative standard deviations for run-to-run, day-to-day and column-to-column lower than 5.7%.

Chiral separation of brompheniramine enantiomers by recycling high-speed countercurrent chromatography using carboxymethyl-ß-cyclodextrin as a chiral selector.

A recycling high-speed countercurrent chromatography protocol was proposed for the enantioseparation of brompheniramine by employing ß-cyclodextrin derivatives as a chiral selector. The two-phase solvent system of n-hexane/isobutyl acetate/0.10 mol/L phosphate buffer solution with a volume ratio of 2:4:6 was selected by a series of extraction experiments. Factors that affected the distribution of the enantiomers over the two-phase system (e.g., the type and concentration of ß-cyclodextrin derivatives = pH value of the aqueous solution, and the separation temperature) were also investigated. In addition, the theory of thermodynamics is applied to verify the feasibility of the enantioseparation process and the corresponding results demonstrate that this separation process is feasible. The optimized conditions include carboxymethyl-ß-cyclodextrin concentration of 0.010 mol/L, pH of 7.5, and temperature of 5°C. Under the optimal conditions, the purities of both monomer molecules were over 99%, and the recovery yields were 88% for (+)-brompheniramine and 85% for (-)-brompheniramine, respectively.

Capillary electrophoretic enantioseparation of basic drugs using a new single-isomer cyclodextrin derivative and theoretical study of the chiral recognition mechanism.

A novel single-isomer cyclodextrin derivative, heptakis {2,6-di-O-[3-(1,3-dicarboxyl propylamino)-2-hydroxypropyl]}-ß-cyclodextrin (glutamic acid-ß-cyclodextrin) was synthesized and used as a chiral selector in capillary electrophoresis for the enantioseparation of 12 basic drugs, including terbutaline, clorprenaline, tulobuterol, clenbuterol, procaterol, carvedilol, econazole, miconazole, homatropine methyl bromide, brompheniramine, chlorpheniramine and pheniramine. The primary factors affecting separation efficiency, which include the background electrolyte pH, the concentration of glutamic acid-ß-cyclodextrin and phosphate buffer concentration, were investigated. Satisfactory enantioseparations were obtained using an uncoated fused-silica capillary of 50 cm (effective length 40 cm) × 50 µm id with 120 mM phosphate buffer (pH 2.5-4.0) containing 0.5-4.5 mM glutamic acid-ß-cyclodextrin as background electrolyte. A voltage of 20 kV was applied and the capillary temperature was kept at 20°C. The results proved that glutamic acid-ß-cyclodextrin was an effective chiral selector for studied 12 basic drugs. Moreover, the possible chiral recognition mechanism of brompheniramine, chlorpheniramine and pheniramine on glutamic acid-ß-cyclodextrin was investigated using the semi-empirical Parametric Method 3.

Preparation of a ß-Cyclodextrin-Based Open-Tubular Capillary Electrochromatography Column and Application for Enantioseparations of Ten Basic Drugs.

An open-tubular capillary electrochromatography column was prepared by chemically immobilized ß-cyclodextrin modified gold nanoparticles onto new surface with the prederivatization of (3-mercaptopropyl)-trimethoxysilane. The synthesized nanoparticles and the prepared column were characterized by transmission electron microscopy, scanning electron microscopy, infrared spectroscopy and ultraviolet visible spectroscopy. When the column was employed as the chiral stationary phase, no enantioselectivity was observed for ten model basic drugs. So ß-cyclodextrin was added to the background electrolyte as chiral additive to expect a possible synergistic effect occurring and resulting in a better separation. Fortunately, significant improvement in enantioselectivity was obtained for ten pairs of drug enantiomers. Then, the effects of ß-cyclodextrin concentration and background electrolyte pH on the chiral separation were investigated. With the developed separation mode, all the enantiomers (except for venlafaxine) were baseline separated in resolutions of 4.49, 1.68, 1.88, 1.57, 2.52, 2.33, 3.24, 1.63 and 3.90 for zopiclone, chlorphenamine maleate, brompheniramine maleate, dioxopromethazine hydrochloride, carvedilol, homatropine hydrobromide, homatropine methylbromide, venlafaxine, sibutramine hydrochloride and terbutaline sulfate, respectively. Further, the possible separation mechanism involved was discussed.

An expanded cellular automata model for enantiomer separations using a ß-cyclodextrin stationary phase.

Chromatographic scale enantiomer separation has not been modeled using cellular automata (CA). CA uses easy to adjust equations to different enantiomers under various chromatographic conditions. Previous work has demonstrated that CA modeling can accurately predict the strength of one-to-one binding interactions between enantiomers and ß-cyclodextrin (CD) [1]. In this work, the model is expanded to a chromatographic scale grid environment in order to transform model output into HPLC chromatograms. The model accurately predicted the lack of chromatographic selectivity of mandelic enantiomers (1.05 published, 1.01 modeled) and the separation of brompheniramine enantiomers (1.13 published, 1.12 modeled) previously modeled in one-to-one interactions. By examining cyclohexylphenylglycolic acid (CHPGA) enantiomers, the model accurately predicted both the selectivity and resolution of the enantiomer peaks at varying chromatographic temperatures. Modeled changes in mobile phase pH agree with laboratory outcomes when examining peak resolution and selectivity. Changes in injection volume resulted in an increase in retention time of the modeled enantiomers as was observed in the published laboratory results.

The effect of achiral calixarenes on chiral separation of propranolol-HCl and brompheniramine maleate in capillary electrophoresis using cyclodextrin as chiral selector.

In this study we have examined the effect of achiral water soluble p-sulfonatocalixarenes (SCX[n]) on chiral separation propranolol-HCl and brompheniramine maleate. Several cyclodextrins (CDs) and cyclodextrin derivatives were examined as chiral selectors applying complete filling technique (CFT) accompanied with the partial filling technique PFT of (SCX[n]) as achiral modifier. Only with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) chiral separation could be achieved. The effect of the organic modifier on these chiral separations was examined. The results indicate that at pH 4.65, the use of HP-beta-CD (CFT) alone could not initiate chiral separations of both analytes and these chiral separations could be induced using HP-beta-CD (CFT) followed by SCX[n]/HP-beta-CD (PFT).

Possibilities of column coupling electrophoresis provided with a fiber-based diode array detection in enantioselective analysis of drugs in pharmaceutical and clinical samples.

The present work illustrated possibilities of column coupling electrophoresis combined with ionizable chiral selector and diode array detection (DAD) for the enantioselective analysis of trace drugs (pheniramine and its analogs) in pharmaceutical and clinical samples. Isotachophoresis (ITP), on-line coupled with capillary zone electrophoresis (CZE), served as an ideal injection technique (high sample load capacity, narrow and sharp drugs zones) of on-line pretreated samples (preseparation, purification and preconcentration of drugs) for the CZE stage. Enhanced (enantio)separation selectivity of CZE with ionizable chiral selector (carboxyethyl-beta-cyclodextrin recognized between drugs enantiomers on one hand as well as between drugs and sample matrix constituents on the other hand) enabled to obtain pure zones of the drugs enantiomers, suitable for their detection and quantitation. DAD in comparison with single wavelength UV detection enhanced value of analytical information verifying purity of drugs enantiomers zones (indicating interferents with different spectra to those of drugs). Obtained results indicated pure zones of interest confirming effective ITP-CZE (enantio)separation process. Distinguishing the trace analytes signals superposed on the baseline noise was provided with sufficient reliability (for this purpose the background correction and smoothing procedure had to be applied to the raw DAD spectra). The proposed ITP-CZE-DAD methods were characterized by favorable performance parameters (sensitivity, linearity, precision, recovery, accuracy, robustness, selectivity) and successfully applied for (i) enantiomeric purity testing of dexbrompheniramine in commercial pharmaceutical tablets and (ii) enantioselective metabolic study of pheniramine in human urine.

Potentialities of ITP-CZE method with diode array detection for enantiomeric purity control of dexbrompheniramine in pharmaceuticals.

The present work illustrates potentialities of on-line combined isotachophoresis-capillary zone electrophoresis (ITP-CZE) separation techniques coupled with on-capillary diode array detector (DAD) for enantiomeric purity testing of drugs in pharmaceuticals. The general advantages of the proposed method are its (i) high selectivity, (ii) low concentration limit of detection (LOD) obtainable, (iii) enhanced sample loadability, and (iv) enhanced reliability. For separation of brompheniramine (BP) enantiomers, serving as model analytes, carboxyethyl-beta-cyclodextrin (CE-beta-CD) was appropriate chiral selector providing complete enantioresolution. Given by a high sample load capacity (30 microl sample injection volume) and preconcentration of the analytes in ITP stage, concentration LOD of levobrompheniramine (LBP), serving as model impurity, was 2.5 ng/ml (8 x 10(-9)mol/l). Such separation and detection conditions enabled to easily determine LBP in samples containing a 10(3) excess of dexbrompheniramine (DBP). DAD detection in comparison with single wavelength detection can enhance value of analytical information when analytes and interferents have different spectra (distinguishing impurities in analyte zone, confirmation of migration positions of migrants). In this context purity of BP zones was confirmed with higher reliability in pharmaceutical sample. Moreover, distinguishing the trace analyte signal superposed on the baseline noise was provided with sufficient reliability (for this purpose the background correction and smoothing procedure had to be applied to the raw DAD spectra). Successful validation and application of the proposed ITP-CZE-DAD method suggest its routine use for the enantiomeric purity testing of pharmaceuticals.

Uniformly sized molecularly imprinted polymers for d-chlorpheniramine: influence of a porogen on their morphology and enantioselectivity.

Uniformly sized molecularly imprinted polymers (MIPs) for d-chlorpheniramine have been prepared by a multi-step swelling and polymerization method using methacrylic acid (MAA) or 2-(trifluoromethyl)acrylic acid (TFMAA) as a functional monomer and toluene, phenylacetonitrile, benzylacetonitrile or chloroform as a porogen. From measurement of their scanning electron microscopy images and physical properties in the dry state, the MIP prepared using TFMAA and chloroform as the functional monomer and porogen, respectively, seemed to be non-porous and had extremely low specific surface areas and pore volumes, while the other MIPs were porous beads with high specific surface areas and pore volumes. All the MIPs prepared were evaluated using hydro-organic mobile phases in HPLC. As a result, they showed the similar retentive and enantioselective properties for chlorpheniramine, brompheniramine and pheniramine. This result suggests the presence of enantioselective binding sites in the swollen state for all the MIPs.

pH-independent large-volume sample stacking of positive or negative analytes in capillary electrophoresis.

In capillary electrophoresis, the short optical path length associated with on-column UV detection imposes an inherent detection problem. Detection limits can be improved using sample stacking. Recently, large-volume sample stacking (LVSS) without polarity switching was demonstrated to improve detection limits of charged analytes by more than 100-fold. However, this technique requires suppression of the electroosmotic flow (EOF) during the run. This necessitates working at a low pH, which limits using pH to optimize selectivity. We demonstrate that LVSS can be performed at any buffer pH (4.0-10.0) if the zwitterionic surfactant Rewoteric AM CAS U is used to suppress the EOF. Sensitivity enhancements of up to 85-fold are achieved with migration time, corrected area, and peak height reproducibility of 0.8-1.6%, 1.3-3.7%, and 0.8-4.9%, respectively. Further, it is possible to stack either positively or negatively charged analytes using zwitterionic surfactants to suppress the EOF.

Separation of brompheniramine enantiomers by capillary electrophoresis and study of chiral recognition mechanisms of cyclodextrins using NMR-spectroscopy, UV spectrometry, electrospray ionization mass spectrometry and X-ray crystallography.

Opposite migration order was observed for the enantiomers of brompheniramine [N-[3-(4-bromphenyl)-3-(2-pyridyl)propyl]-N,N-dimethylamine] (BrPh) in capillary electrophoresis (CE) when native beta-cyclodextrin (beta-CD) and heptakis(2,3,6-tri-O-methyl)-beta-CD (TM-beta-CD) were used as chiral selectors. NMR spectrometry was applied in order to obtain information about the stoichiometry, binding constants and structure of the selector-selectand complexes in solution. The data were further confirmed by UV spectrometry and electrospray ionization mass spectrometry. The structure of the complexes in the solid state was determined using X-ray crystallography performed on the co-crystals precipitated from the 1:1 aqueous solution of selector and selectand. This multiple approach allowed an elucidation of the most likely structural reason for a different affinity (binding strength) of BrPh enantiomers towards beta-CD and TM-beta-CD. However, the question about a force responsible for the opposite affinity pattern of BrPh enantiomers towards these CDs could not be answered definitely.

Application of heparin to chiral separations of antihistamines by capillary electrophoresis.

A study of the chiral separations of antihistamines, including pheniramine, chlorpheniramine, brompheniramine, carbinoxamine and doxylamine in capillary electrophoresis (CE) was accomplished using heparin as a chiral additive (CA) and phosphate buffer as the background electrolyte. Several factors were shown to affect both the selectivity and the migration time, including concentration of heparin, concentration of buffer, and the pH. A dual mechanism involving both inclusion complexation and ionic interactions with heparin is thought to be responsible for the chiral recognition. In the pH range of 2.6-3.5 and reversed polarity, baseline resolutions were obtained using a wide range of buffer and heparin concentrations. Typically, chiral resolution was obtained within 50 min.

Liquid chromatography of antihistamines using post-column tris(2,2'-bipyridine) ruthenium(III) chemiluminescence detection.

The separation and detection of five antihistamine drugs commonly found within over-the-counter allergy and cold pharmaceutical products was performed by HPLC with chemiluminescence (CL) detection. Comparable detection limits at 5-10 pmol were found for the antihistamines by both UV at 214 nm and tris(2,2'-bipyridine) ruthenium(III) CL. However, urine samples were found not to generate as large an unretained peak by CL detection as compared to those peaks by UV detection at 214 and 254 nm. For example, the pheniramine peak representing 0.15 microgram/ml was almost totally obscured at 214 nm. Quantitative results received for three antihistamine commercial samples ranged from 4 to 8% error in accuracy when an internal standard was used to compensate for short term detector drift.