The present study describes a rapid and effective capillary electrophoresis (CE) method for the enantioseparation of pindolol using single-isomer octa(6-O-sulfo)-γ-cyclodextrin. The complexation parameters were determined under neutral and high pH conditions to identify optimal separation conditions using a theoretical model. Baseline separation of pindolol enantiomers was achieved within 6 min in a sodium/MOPS buffer, pH 7.2, with a selector concentration of 6 mM. The method was validated according to the ICH guidelines using imidazole as an internal standard. Low limits of detection and quantification were found, specifically 1.2 μg/mL and 4 μg/mL (0.6 μg/mL and 2 μg/mL per enantiomer), respectively. The calibration curves showed good linearity, with a coefficient of determination R2 ≥ 0.999 over a 5 - 55 μg/mL concentration range and over a 50 - 300 μg/mL concentration range of the racemic mixture. The relative standard deviations (%RSD) of intra-day and inter-day precision were lower than 8% at LOQ level, lower than 3% at 50 μg/mL level and lower than 1.5% at 300 μg/mL level. Accuracy ranged from 95 to 103% (106% at LOQ level). The proposed method was successfully tested on a medical formulation of Visken® Sandoz intravenous solution and Visken® Teofarma pills for oral use.
- MeSH
- časové faktory MeSH
- elektroforéza kapilární metody MeSH
- gama-cyklodextriny chemie MeSH
- kalibrace MeSH
- koncentrace vodíkových iontů MeSH
- limita detekce MeSH
- pindolol izolace a purifikace MeSH
- pufry MeSH
- reprodukovatelnost výsledků MeSH
- software * MeSH
- stereoizomerie MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
In this paper we determine acid dissociation constants, limiting ionic mobilities, complexation constants with β-cyclodextrin or heptakis(2,3,6-tri-O-methyl)-β-cyclodextrin, and mobilities of resulting complexes of profens, using capillary zone electrophoresis and affinity capillary electrophoresis. Complexation parameters are determined for both neutral and fully charged forms of profens and further corrected for actual ionic strength and variable viscosity in order to obtain thermodynamic values of complexation constants. The accuracy of obtained complexation parameters is verified by multidimensional nonlinear regression of affinity capillary electrophoretic data, which provides the acid dissociation and complexation parameters within one set of measurements, and by NMR technique. A good agreement among all discussed methods was obtained. Determined complexation parameters were used as input parameters for simulations of electrophoretic separation of profens by Simul 5 Complex. An excellent agreement of experimental and simulated results was achieved in terms of positions, shapes, and amplitudes of analyte peaks, confirming the applicability of Simul 5 Complex to complex systems, and accuracy of obtained physical-chemical constants. Simultaneously, we were able to demonstrate the influence of electromigration dispersion on the separation efficiency, which is not possible using the common theoretical approaches, and predict the electromigration order reversals of profen peaks. We have shown that determined acid dissociation and complexation parameters in combination with tool Simul 5 Complex software can be used for optimization of separation conditions in capillary electrophoresis.
- MeSH
- antiflogistika nesteroidní chemie MeSH
- beta-cyklodextriny chemie MeSH
- elektroforéza kapilární metody MeSH
- flurbiprofen chemie MeSH
- ibuprofen chemie MeSH
- ketoprofen chemie MeSH
- koncentrace vodíkových iontů MeSH
- magnetická rezonanční spektroskopie MeSH
- naproxen chemie MeSH
- osmolární koncentrace MeSH
- počítačová simulace MeSH
- software MeSH
- termodynamika MeSH
- viskozita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The FOXO forkhead transcription factors are potent transcriptional activators involved in a wide range of key biological processes. In this work, the real-time kinetics of the interaction between the FOXO4-DNA binding domain (FOXO4-DBD) and the DNA was studied by using surface plasmon resonance (SPR). SPR analysis revealed that the interaction between FOXO4-DBD and the double stranded DNA containing either the insulin-responsive or the Daf-16 family member-binding element is preferably described by using a conformational change model which suggests a structural change of FOXO4-DBD upon binding to the DNA. This was further confirmed by using the time-resolved tryptophan fluorescence anisotropy decay measurements which revealed profound reduction of segmental dynamics of FOXO4-DBD upon the complex formation. Alanine scanning of amino acid residues engaged in polar contacts with the DNA showed that certain non-specific contacts with the DNA backbone are very important for both the binding affinity and the binding specificity of FOXO4-DBD.
A novel procedure for the determination of stability constants in systems with neutral analytes and charged complexation agents by affinity capillary electrophoresis was established. This procedure involves all necessary corrections to achieve precise and reliable data. Temperature, ionic strength, and viscosity corrections were applied. Based on the conductivity measurements, the average temperature of the background electrolyte in the capillary was kept at the constant value of 25°C by decreasing the temperature of the cooling medium. The viscosity correction was performed using the viscosity ratio determined by an external viscosimeter. The electrophoretical measurements were performed, at first, at constant ionic strength. In this case, the increase of ionic strength caused by increasing complexation agent concentration was compensated by changing of the running buffer concentration. Subsequently the dependence of the analyte effective mobility on the complexation agent concentration was measured without the ionic strength compensation (at variable ionic strength). The new procedure for determination of the stability constants even from such data was established. These stability constants are in a very good agreement with those obtained at the constant ionic strength. The established procedure was applied for determination of the thermodynamic stability constants of (R, R)-(+)- and (S, S)-(-)-hydrobenzoin and R- and S-(3-bromo-2-methylpropan-1-ol) complexing with 6-monodeoxy-6-mono(3-hydroxy)propylamino-β-cyclodextrin hydrochloride.
Simul 5 Complex is a one-dimensional dynamic simulation software designed for electrophoresis, and it is based on a numerical solution of the governing equations, which include electromigration, diffusion and acid-base equilibria. A new mathematical model has been derived and implemented that extends the simulation capabilities of the program by complexation equilibria. The simulation can be set up with any number of constituents (analytes), which are complexed by one complex-forming agent (ligand). The complexation stoichiometry is 1:1, which is typical for systems containing cyclodextrins as the ligand. Both the analytes and the ligand can have multiple dissociation states. Simul 5 Complex with the complexation mode runs under Windows and can be freely downloaded from our web page http://natur.cuni.cz/gas. The article has two separate parts. Here, the mathematical model is derived and tested by simulating the published results obtained by several methods used for the determination of complexation equilibrium constants: affinity capillary electrophoresis, vacancy affinity capillary electrophoresis, Hummel-Dreyer method, vacancy peak method, frontal analysis, and frontal analysis continuous capillary electrophoresis. In the second part of the paper, the agreement of the simulated and the experimental data is shown and discussed.
The complete mathematical model of electromigration dispersion in systems that contain a neutral complex forming agent and a fully charged analyte was introduced in the previous part of this series of papers (Part III - Theory). The model was implemented in the newest version of our simulation program PeakMaster 5.3 that calculates the effective mobility of the analyte and its nonlinear electromigration mobility slope, S(EMD), in the presence of a complex forming agent in the background electrolyte. The mathematical model was verified by both experiments and simulations, which were performed by our dynamic simulator Simul 5 Complex. Three separation systems differing in the chiral selector used (having different values for the complexation constant and the mobility of the complex) were chosen for the verification. The nonlinear electromigration mobility slope values were calculated from the simulations and the experiments that were performed at different complex forming agent concentrations. These data agree very well with those predicted by the mathematical model and provided the foundation for the discussion and explanation of the electromigration dispersion process that occurs in systems which contain a complex forming agent. The new version of PeakMaster 5.3 was shown to be a powerful tool for optimization of the separation conditions by minimizing electromigration dispersion which improves the symmetry of the analyte peaks and their resolution.
The stability (affinity, association, binding, complexation, formation) constant characterizes binding interaction between the analyte and the complexing agent. Knowledge of the stability constant makes possible the prediction and estimation of the binding behavior of constituents (amino acids, peptides, proteins, drugs, antibiotics, enzymes, enantiomers) to their partners, and the finding of a suitable partner for the given analyte to form a stable complex. The present paper summarizes the stability constant determination methods and the approaches used to evaluate the experimental data. Further, the paper recapitulates the published stability constant values determined, mainly, by capillary electrophoretic methods, taken from the Web of Science database covering the last decade. Details of the experimental conditions employed for the determination of the stability constants are also given. The review attempts to give a critical evaluation of the problems that accompany the determination of stability constant and discusses their solution.
