Inclusional complex study between 6-p-toluidinylnaphthalene-2-sulfonate and 2-hydroxypropyl-beta-cyclodextrin

6-p-Toluidinylnaphthalene-2-sulfonate (TNS) is used as a fluorescent probe for exploring hydrophobic regions of several biological substances, such as proteins, and studying solution state folding behaviour. The current study examines the complexation of TNS with 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) in aqueous solution, mainly by ultraviolet spectrophotometry using various concentrations of HPbetaCD. The structure of HPbetaCD was confirmed by using positive-ion electrospray ionization (ESI+) mass spectrometry. The complex was examined for its stoichiometry applying the continuous variation (Job plot) method. Also, the kinetics of the complex formation was monitored and the determination of the stability constant was calculated. For this purpose, the spectrophotometric properties of TNS were observed in the presence of increasing concentrations of HPbetaCD applying the transformed Benesi-Hildebrand linear model as well as a nonlinear one. The results suggest that TNS forms a stable complex of 1:1 molar ratio, at least at the examined concentrations. Furthermore, from the measurements of H-1 nuclear magnetic resonance (H-1 NMR) spectra, interactions between protons of TNS with HPbetaCD were determined. (C) 2002 Elsevier Science B.V. All rights reserved

Effect of the luminol signal enhancer selection on the curve parameters of an immunoassay and the chemiluminescence intensity and kinetics

In the present study, three luminol signal enhancers 4-methoxyphenol, 4-hydroxybiphenyl and 4-(1H-pyrrol-1-yl)phenol were utilized in the chemiluminescence (CL) substrate solution of horseradish peroxidase (HRP). The latter was applied in a heterogenous enzyme immunoassay that has been previously described. The employment of these molecules greatly affected important assay parameters, such as detection limit and the range of the calibration curve and the results were compared with those obtained from other two similar enhancers that have been described from our group. Practically, the use of a novel enhancer, even if this is a slightly changed 4-substituted phenol derivative, can affect assay properties so dramatically, one can assume that another substrate/enzyme system was applied. Furthermore, the use of different luminol signal enhancers in the luminol/HRP/H2O2 system affected not only the intensity of the obtained signal, which is well known, but also its kinetics. It was monitored that the stronger intensity was combined with a more rapid decrease of the CL signal. © 2006 Elsevier B.V. All rights reserved

Employment of 4-(1-imidazolyl)phenol as a luminol signal enhancer in a competitive-type chemiluminescence immunoassay and its comparison with the conventional antigen-horseradish peroxidase conjugate-based assay

This study describes the employment of a novel imidazole-substituted phenol [4-(1-imidazolyl)phenol] as a highly potent signal enhancer in a horseradish peroxidase (HRP)-luminol chemiluminescence (CL) immunoassay. This competitive-type immunoassay for the model antigen fentanyl is based on the use of fentanyl polyclonal antibody immobilized on white microtiter plates and a biotinylated bovine serum albumin (BSA)-fentanyl derivative as a tracer. The latter was detected by means of streptavidin labeled with HRP, resulting in the generation of a high-intensity and relatively stable chemiluminescent signal, immediately after the addition of the substrate solution (NOAS). The developed method fulfilled the requirements of accuracy (percentage recovery ranged from 93.8 to 107%) and precision (intra- and inter-assay CVs were 2.5-5.2 and 4.5-11.9%, respectively). Its plasma detection limit (1.05pgml-1) was lower than those of previous immunoassays. The novel assay was compared in terms of sensitivity and concentration range with other common HRP substrate systems: luminol-p-iodophenol-H2O2 and TMB-H 2O2. Finally, the described method was compared with an HRP-fentanyl conjugate-based assay, similar to commercially available kits (SKIT), employing the novel substrate solution for both assays and the differences observed were explained by applying previously described models. The detection limit was 4.82pgml-1 for SKIT, recovery values were 94.2-105% and intra- and inter-assay CVs were 2.5-5.2 and 4.5-11.9%, respectively. In conclusion, the proposed assay could be utilized for a wide range of molecules and replace the existing enzyme-labeled antigen-based kits. © 2003 Elsevier B.V. All rights reserved

Improved performance of antigen-HRP conjugate-based immunoassays after the addition of anti-HRP antibody and application of a liposomal chemiluminescence marker

To overcome the sensitivity limit for small molecules (haptens) in immunoassays based on antigen-horseradish peroxidase (HRP) conjugates as labels, a novel approach was established that afforded very low detection limits. Biotinylated anti-HRP antibody was utilized in order to attach, via a streptavidin bridge, liposomaly entrapped HRP. Fentanyl, used as a model antigen, could be determined via the generation of a high-intensity and relatively stable chemiluminescence (CL) signal of a HRP-catalyzed luminol/H2O2enhancer system, immediately after the addition of a substrate solution. 4-(1-Imidazolyl)phenol (4-IMP) was used as an enhancer, and the outcome of this combination was a very low detection limit (0.895 pg mL-1) in plasma samples. The respective detection limit with the use of just the classical HRP-antigen conjugate was > 5-times higher. Intra- and inter-assay RSDs of the novel assay were 6.8-9.9 and 11-17%, respectively. The proposed method could be utilized for a wide range of molecules without replacing existing antigen-HRP based kits. © The Japan Society for Analytical Chemistry

Application of avidin-biotin technology for the characterization of a model hapten-protein conjugate

A simple method was developed for the rapid characterization of the covalent binding of haptens to proteins such as enzymes, bovine serum albumin (BSA), and other carrier-proteins and antibodies. In the present study, a commercially available fentanyl-BSA conjugate was characterized by a 4′-hydroxyazobenzene-2-carboxylic acid (HABA) dye assay that followed a biotinylation reaction. This protocol allowed the indirect observation of the average hapten number per BSA molecule. Such measurement is useful for optimizing reaction conditions to yield a more precisely defined product for immunological applications. The obtained result was within the limits suggested by the manufacturer of the conjugate. Copyright © Taylor & Francis, Inc

Efficient determination and evaluation of model cyclodextrin complex binding constants by electrospray mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is now routinely used for the detection of cyclodextrin noncovalent complexes, complementing previously established methods. Host-guest complexes formed in solution are also stable for characterization by ESI in the gas phase. This paper reports the first investigations to characterize the stability of three inclusion complexes between β-cyclodextrin (β-CD) and three model "guest" molecules, by determining the cyclodextrin compound complex stability constant (Kst) with the use of mass spectrometric studies. The relative signal intensity of the complexes were monitored in the positive ion mode by mixing each "guest" molecule with an up to 50-fold molar excess of βCD. A novel linear equation, similar to Benesi-Hildebrand, was derived allowing the determination of Kst for 1:1 stoichiometry in all complexes. These values were compared with the Kst obtained by spectrophotometric experiments and they were evaluated to be slightly different, indicating the validity of the described method. © 2003 American Society for Mass Spectrometry

Kinetic degradation study of insulin complexed with methyl-beta cyclodextrin. Confirmation of complexation with electrospray mass spectrometry and 1H NMR

Hydrolysis of insulin has been studied during storage of various preparations at different temperatures. Deamidation is the predominant degradation process in acid solution resulting in a desamido product. The current study examines whether the interaction of insulin with methyl-beta cyclodextrin (metβCD) improves its stability. Hydrolysis of insulin was monitored by an HPLC assay with ultraviolet detection. The stability constant of insulin-metβCD complex was calculated by Lineweaver-Burke linear equation. Furthermore, the complexation of insulin with metβCD was characterized by 1H NMR and Electrospray Mass Spectrometry (ESI-MS). MetβCD had a stabilizing effect on insulin degradation according to the kinetic parameters, leading to a decreased chemical deterioration. Furthermore, the stability constant Kst and the activation energy Ea were calculated by fitting the kinetic results to Lineweaver-Burke and to Arrhenius linear equations, respectively. Finally, the complexation of insulin with metβCD was characterized in aqueous media by 1H NMR chemical shift displacements of assignable aromatic protons of specific amino acids upon the addition of the cyclodextrin, as well as by ESI-MS, since additional m/z peaks, which were attributed to insulin-metβCD complex, were detected. It is concluded that addition of metβCD resulted in a significant increase in the stability of complexed insulin compared with free insulin. © 2002 Elsevier Science B.V. All rights reserved

Desirability based optimization of new mesalazine modified release formulations: Compression coated tablets and mini tablets in capsules

Background: Mesalazine (5-aminosalicylic acid, 5-ASA) is a drug substance with an anti-inflammatory activity, which is mainly used in the symptomatic treatment of diseases, such as Ulcerative Colitis, the Crohn's disease and the idiopathic inflammatory bowel disease. Mesalazine exerts its effect locally in the inflamed area of the intestine and not through systematic absorption, therefore the investigation of its release characteristics from solid pharmaceutical formulations is of great importance. Objective: The development of novel mesalazine modified release formulations with improved properties, regarding drug release in the gastrointestinal tract, by utilisation of the Design of Experiments (DoE) approach. Methods: D-optimal experimental design was applied. A Simplex Lattice mixture design was used for the development of suitable capsules containing 4 mini tablets and a D-optimal mixture design was used for compression-coated tablets, with the following characteristics: ≤10% release in 2 h, to minimize its degradation in the upper gastrointestinal tract, 20-40% release in 5 h for mesalazine administration in the small intestine, and quantitative release in 12 h for colonic delivery. The dissolution experiments were conducted in gastrointestinal-like fluids and pectinases to simulate the pectinolytic enzymes present in the colon. Results: The optimal compositions were reached via the desirability function, as a compromise to the different responses. The optimal solutions for both formulations led to colon-specific delivery of the active substance with minimal 5-ASA release in the upper gastrointestinal tract and appeared to conform with the pre-determined characteristics. Hard gelatin capsules, when filled with mini-tablets led to the aimed modified release profile, having sigmoidal characteristics and compression coated tablets led to colonic delivery. Conclusion: Two novel mesalazine formulations were developed with the desirable colonic release, by conducting a minimal number of experiments, as suggested by DoE experimental design. © 2020 Bentham Science Publishers