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

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

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

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

Quantitative structure-chemiluminescence intensity relationships of 4-substituted phenols acting as luminol signal enhancers

Luminol-peroxidase-H2O2 is among the most popular systems in chemiluminescence-based analytical applications. The addition of a forth compound into this system has been thoroughly explored over the years, namely an enhancer promoting potency in the signal attained. Improvements in this regard serve to increase sensitivity and applicability of various biochemical methods. Studies exploring the ability of compounds to act as enhancers have proposed several chemical groups as possible candidates, amongst which 4-substituted phenols have been widely employed. Although some studies have explored the effect of the substituent on enhancer potency, no quantitative structure - property relationships (QSPR) employing molecular descriptors for this group of compounds have been presented. Current study provides two such cross- and externally validated models, constructed by the projection to latent structures by means of partial least squares (PLS) multivariate linear regression method: i) a PLS-DA model contributing to the classification of such compounds into classes (relative to the signal), and ii) a PLS model for predicting the signal acquired. Validation was based on statistical metrics Q2ext(F3) for the test set and Roy's metrics r2m(Av) & r2m(δ), assessing both predictive stability and internal validity. Applied in tandem, those models can assist in the recognition of available compounds as potential enhancers or inspire the synthesis of novel analogues. The significance of some characteristics governing the compounds' behaviour are also discussed based on the molecular descriptors chosen. © 2015 Elsevier B.V.

Turbulent flow and ternary column-switching on-line clean-up system for high-throughput quantification of risperidone and its main metabolite in plasma by LC-MS/MS. Application to a bioequivalence study

A high-throughput LC-MS/MS method was developed for the simultaneous determination of Risperidone and 9-OH-risperidone in human plasma. A semi-automated sample preparation procedure was applied, including protein precipitation after addition of ACN, via a robotic system, and subsequent sub-zero temperature extraction of the latter. Injections of the ACN extractants were performed on a turbulent flow ternary column-switching system, consisted of dual extraction columns in parallel for on-line purification of samples and an analytical column. Toggling with the assistance of two valves provided a run cycle time of 3 min and the whole procedure minimized carry-over effect. On-line clean-up procedure along with sub-zero temperature extraction increased sample purification and extended column life. The analytical range of the method was 0.1-200 ng mL-1 for both analytes with excellent linearity and very good accuracy and precision. The proposed method was employed in a bioequivalence study after per os administration of a 2 mg tablet of risperidone and allowed the completion of the study (>1400 samples) in only 4 days time. © 2007 Elsevier B.V. All rights reserved