Quantification of trace levels of β-cyclodextrin and substitution patterns in hydroxypropyl-β-cyclodextrin using high-performance liquid chromatography with polarimetric detection

A high-performance liquid chromatographic (HPLC) method has been developed for separation and determination of components in hydroxypropyl-β-cyclodextrin (HP-β-CD). The method involves separation on an amino-bonded HPLC column using water–acetonitrile as a mobile phase with a polarimetric HPLC detector for quantification. It provides good selectivity and sensitivity and can also be used to compare different sources of HP-β-CD and to measure batch to batch variation. The similarity of the values of molar optical rotation for β-cyclodextrin (β-CD) and HP-β-CD suggests that a polarimetric HPLC detector may be used with a straightforward area normalization method, to quantify the proportion of β-CD in any HP-β-CD sample. Trace amounts of β-CD in HP-β-CD have been measured to a precision of 0.01%. © 1993 Wiley-Liss, Inc.     

Chiral discrimination by NMR spectroscopy of ephedrine and N-methylephedrine induced by β-cyclodextrin,heptakis(2,3-di-O-acetyl)β-cyclodextrin,and heptakis (6-O-acetyl)β-cyclodextrin

NMR spectroxcopy has been used to compare the interaction of ephedrine and N-methylephedrine with β-cyclodextrin, heptakis(2,3-di-O-acetyl)β-cyclodextrin, heptakis(6-O-acetyl)β-cyclodextrin. The stoichiometry of the complexes formed between all three cyclodextrins and N-methylephedrine was found to be 1:1 by UV spectroscopy by means of the Job technique. NMR spectra of the single enantiomers of ephedrine and N-methylephedrine in the presence of all three cyclodextrins gave information about the parts of the ligands which interact differently with the host molecules and may be responsible for the chiral discrimination. To quantify the complex stabilities, binding constants were calculated from the changes in the chemical shifts of the ligand signals upon complexation. Analyses of the coupling constants of both species showed that no significant conformational change occurs upon complexation. ROESY spectra of these optical isomers with all three cyclodextrins provided detailed information about the geometry of the complexes. Different intermolecular cross-peaks between the individual isomers of ephedrine and N-Methylephedrine were found for native β-cyclodextrin and its 2,3-diacetylated derivative but not for 6-acetyl cyclodextrin. Analyses of the intramolecular cross-signals of the ligands confirmed that no significant conformational change occurs upon complexation. Chirality 9:211–219, 1997. © 1997 Wiley-Liss, Inc.     

Non-aqueous capillary electrophoresis chiral separations with sulfated β-cyclodextrin

This paper reports the application of an anionic cyclodextrin (CD), sulfated β-cyclodextrin with a degree of substitution of four (β-CD-(SO₄⁻)₄, in chiral separations of pharmaceutical enantiomers by non-aqueous capillary electrophoresis (NACE). Upon complexation with the anionic CD, electrophoretic mobilities of the basic enantiomers decreased, however, both separation selectivity and resolution were enhanced. The advantage of NACE chiral separations over the aqueous CE with the charged CD is that higher electric field strength and higher ionic strength could be applied due to the characteristics of the solvent formamide. The higher ionic strength leads to stacking of peaks and reduces the electrodispersion caused by the mobility mismatch between β-CD-(SO₄⁻)₄–analyte complexes and the co-ions in the running buffer. As a result, better peak shapes and higher separation efficiency were obtained. Comparing with NACE chiral separations with neutral CDs, lower concentration of β-CD-(SO₄⁻)₄ was needed due to the fact that the electrostatic attraction caused stronger binding between β-CD-(SO₄⁻)₄ and the enantiomers. The effects of the experimental parameters, such as concentration of the CD, apparent pH (pH*), degree of substitutions of the CDs, percentage of water in mixed solvent systems, and type of solvents were also studied.     

Determination of 2-hydroxypropyl-γ-cyclodextrin in plasma of cynomolgus monkeys after oral administration by gas chromatography–mass spectrometry

This report describes a specific and highly sensitive gas chromatography–mass spectrometry (GC–MS) assay for the analysis of industrially produced 2-hydroxypropyl-γ-cyclodextrin, a heterogeneous mixture of homologues and isomers, in plasma of cynomolgus monkeys. Instead of analyzing the polysaccharide mixture as a whole, in a first step the HP-γ-cyclodextrin mixture, together with the internal standard (2,6-di-O-methyl-β-cyclodextrin), was deuteromethylated, and in a second step hydrolyzed with hydrochloric acid to the respective monosaccharides. The resulting reaction mixture was trimethylsilylated to 1,4-bis(O-trimethylsilyl)-2,3-bis-O-deuteromethyl-6-O-2′-deuteromethoxypropylglucose (representative for HP-γ-CD) and 1,4-bis-(O-trimethylsilyl)-bis-2,6-O-methyl-3-O-deuteromethylglucose (representative for the internal standard), respectively, and analyzed by GC–MS. The limit of quantification of this assay was 20 nmol/l.     

Separating enantiomers by HPLC on silica dynamically coated with a chiral stationary phase of permethylated β-cyclodextrin

The paper demonstrates that the technique of solvent generated liquid--solid chromatography can be used to create normal phase systems for chiral separations. The chiral adsorption layer was generated by pumping a binary hexane:ethanol eluent containing a small fraction of permethylated β-cyclodextrin through a column packed with microparticulate silica. This technique leads to columns with good time stability and reproducibility. The possibility of generating normal phase systems with permethylated β-cyclodextrin as chiral component via the mobile phase broadens the range of phase system which can be used to separate enantiomers by HPLC.     

Host–guest inclusion complex of mesalazine and β‐cyclodextrin and spectrofluorometric determination of mesalazine

The supramolecular interaction of mesalazine (MSZ) and β‐cyclodextrin (β‐CD) has been examined by ultraviolet–visible (UV–vis) light, infra‐red (IR) light and fluorescence spectroscopy. The formation of an inclusion complex has been confirmed based on the changes of the spectral properties. MSZ–β‐CD host–guest complex was formed in (1:1) stoichiometry and the inclusion constant (K = 1.359 × 10² L mol^–1) was ascertained by typical double reciprocal plots. Furthermore, the thermodynamic parameters (ΔG°, ΔH° and ΔS°) of (MSZ–β‐CD) were obtained. Based on the remarkable enhancement of the fluorescence intensity of MSZ produced through complex formation, a simple, accurate, rapid and highly sensitive spectrofluorometric method for the determination of MSZ in aqueous solution in the presence of β‐CD was developed. The measurement of relative fluorescence intensity was carried with excitation at 330 nm and emission 493 nm. All variables affecting the reactions were studied and optimized. Beer's law was obeyed in the concentration range 0.1–0.45 µg/mL. Absorbance was found to increase linearly with increasing concentration of MSZ, which is corroborated by the calculated correlation coefficient values of 0.99989. The molar absorptivity, Sandell's sensitivity, detection and quantification limits were calculated. The validity of the described methods was assessed, and the method was successfully applied to the determination of MSZ in its pharmaceutical formulation. In addition, a solid inclusion complex was synthesized by co‐precipitation method. Copyright © 2014 John Wiley & Sons, Ltd.     

Molecular modelling of the inclusion complexes between β-cyclodextrin and (R)/(S)-methylphenobarbitone and its application to HPLC

Molecular modelling of β-cyclodextrin and optimisation of its potential energy suggests that a favoured conformation is that distorted from a symmetrical torus. The inclusion of water molecules into the torus cavity simulates the increased stability in an aqueous solvent. Complexes of β-cyclodextrin with (R)- and (S)-enantiomers of methylphenobarbitone have been modelled and energetically optimised by the application of molecular mechanics. The simulations suggests that the guest molecules adopt an orientation in which the phenyl ring is projected into the torus cavity, with in each case the plane of the ring parallel to a longer axis of the distorted torus and slightly displaced from the axis through the torus cavity. It is suggested that the asymmetry in the macrocyclic ring contributes to chiral recognition as a result of additional discriminatory binding to the barbiturate ring residue of each enantiomer, which occupy different 3D geometries. The enantiomers form complexes of different minimum potential energies. The resulting difference in complex stability can be related to the behaviour of β-cyclodextrin, as a mobile phase additive in reverse-phase HPLC to effect chiral separation of rac-medthylphenobarbitone during chromatography. © 1994 Wiley-Liss, Inc.     

NMR investigation of the interaction of (+)- and (−)-flurbiprofen with β-cyclodextrin

The sodium salts of (+)-(S)- and (−)-(R)-2-(2-fluoro-4-biphenylyl)propionic acid (flurbiprofen, FBP) form 1:1 inclusion complexes with β-cyclodextrin (β-CD) having different association constants. Proton selective relaxation rate measurements revealed the existence of superior aggregated forms for both complexes (+)-FBP/β-CD and (−)-FBP/β-CD; information about their stereochemistry has been obtained by 2D ROESY analysis. © 1996 Wiley-Liss, Inc.     

Incorporation of the antibacterial agent, miconazole nitrate into a cellulosic fabric grafted with β-cyclodextrin

The aim of the study was to investigate the incorporation of the antibacterial agent, miconazole nitrate into cyclodextrin cavities covalently bonded onto cloth fibers. The cellulosic fabric was grafted with β-cyclodextrin molecules through reaction with monochlorotriaziny β-cyclodextrin (MCT-β-CD). The suitable bonded reaction conditions were found to be MCT-β-CD 60–100 g/L, catalyst Na₂CO₃ 50–60 g/L, the reaction temperature 150–160 °C and the reaction time 5–8 min.

The modified and unmodified fabrics were characterized by UV spectrophotometry. The level of miconazole nitrate entrapped in the fabrics were determined by HPLC and was founded to be much higher (0.458% w/w) for the textile functionalized with MCT-β-CD compared to the unmodified fabric (0.056% w/w). The antibacterial abilities measured by shaker flask method showed that the antibacterial property was markedly enhanced by impregnation with miconazole nitrate of the MCT-β-CD grafted textile. The finished fabric kept the antibacterial abilities more than 70% even after washing 10 times, while the antibacterial activity of the unmodified textile was almost lost.     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Enantiomeric separation of tetrahydroisoquinoline alkaloids by high-performance liquid chromatography with β-cyclodextrin as chiral selector

Tetrahydroisoquinoline alkaloids, which are known to be present not only in plants but also in animals, including mammals, can be considered as condensation products of 2-phenylethylamines (e.g., catecholamines) with aldehydes (e.g., acetaldehyde) or 2-oxo acids (e.g., pyruvic acid). In this study the possibility of separating the optical isomers of several tetrahydroisoquinolines by high-performance liquid chromatography was investigated. For isosalsoline, tetrahydropapaveroline and laudanosoline a good enantiomeric separation could be achieved by applying β-cyclodextrin-bonded silica as stationary phase in connection with various mobile phases. With respect to laudanosoline, the addition of β-cyclodextrin as chiral selector to the mobile phase using a C₁₈ reversed-phase column as stationary phase revealed an even higher resolution when compared with the chiral columns. All tested tetrahydroisoquinolines which could be well separated into enantiomers bear a hydroxyl group at carbon atom 7 as a common structural feature. Those alkaloids substituted with a methoxy group on position 7 instead of a hydroxyl group (e.g., salsolidine) failed to be resolved into their optical isomers. Therefore, the presence of a hydroxyl group on C7 of the aromatic ring seems to be conducive to steric discrimination. However, the separation results for 1-carboxysalsolinol were unsatisfactory although this molecule possesses a 7-hydroxyl group. In this case the existence of a carboxyl group on C1 reduced the chiral recognition and thus the enantiomeric resolution. © 1995 Wiley-Liss, Inc.     

Fed-batch production of (S)-flurbiprofen in lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin and its extractive purification

Optically active (S)-flurbiprofen was produced fed-batch-wisely in a lipase-catalyzed dispersed aqueous phase reaction system induced by succinyl β-cyclodextrin (suβ-CD). A highly concentrated 480 mM (S)-flurbiprofen, corresponding to 117.0 g/l, with an enantiomeric excess of 0.98 and conversion yield of 0.48 was obtained. (S)-Flurbiprofen produced in an inclusion complex form with suβ-CD was extractively purified using three-step procedures: decomplexation of (S)-flurbiprofen and residual (R)-flurbiprofen ethyl ester ((R)-FEE) using the ethyl acetate, dissolution of (S)-flurbiprofen from (R)-FEE using a sodium bicarbonate solution, and selective precipitation of (S)-flurbiprofen using 2-propanol. Consequently, an extremely high concentration of 420 mM (S)-flurbiprofen with an optical purity higher than 98% was recovered after purification.     

Solubility enhancement of mefenamic acid by inclusion complex with β-cyclodextrin: in silico modelling,formulation, characterisation,and in vitro studies

The aim of this study was to prepare and characterise inclusion complexes of a low water-soluble drug, mefenamic acid (MA), with β-cyclodextrin (β-CD). First, the phase solubility diagram of MA in β-CD was drawn from 0 to 21 × 10⁻³ M of β-CD concentration. A job’s plot experiment was used to determine the stoichiometry of the MA:β-CD complex (2:1). The stability of this complex was confirmed by molecular modelling simulation. Three methods, namely solvent co-evaporation (CE), kneading (KN), and physical mixture (PM), were used to prepare the (2:1) MA:β-CD complexes. All complexes were fully characterised. The drug dissolution tests were established in simulated liquid gastric and the MA water solubility at pH 1.2 from complexes was significantly improved. The mechanism of MA released from the β-CD complexes was illustrated through a mathematical treatment. Finally, two in vitro experiments confirmed the interest to use a (2:1) MA:β-CD complex.     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.     

Enhancement of the isolation selectivity of isoflavonoid puerarin using oligo-β-cyclodextrin coupled polystyrene-based media

The isolation selectivity of the isoflavonoid puerarin, a well-known traditional Chinese medicine, was studied when native and oligo-β-cyclodextrin (CDP) coupled polystyrene-based macroporous resins were used as adsorbents by static tests. The research results indicated that the CDP coupled resin HPD-100-CDP offered the best adsorption and desorption capacity for puerarin than others and its equilibrium adsorption data at 25 °C fit best to the Freundlich isotherm. The performance of separation of puerarin on HPD-100-CDP column in one step was evaluated. Based on the above experimental data, a novel medium PS-CDP was synthesized and its chromatographic retention behaviors were also explored. ESI-MS/MS, ¹H NMR spectroscopy and UV absorption spectrum were used for the detection and characterization of puerarin in isolated fraction. Under the optimum mobile phase, methanol/acetic acid/water = 5.0/6.6/88.4 (v/v/v), the purity and recovery of puerarin were 95.3% and 86.7%, respectively, by HPLC analysis. In conclusion, the PS-CDP medium can enhance the isolated selectivity of puerarin and it can be applied in preparative scale operations.