Kiralna separacija beta-blokatora tekućinskom kromatografijom visoke djelotvornosti i određivanje enantiomera bisoprolola u površinskim vodama

Beta-blockers are chiral compounds with enantiomers that have different bioactivity, which means that while one is active, the other can be inactive or even harmful. Due to their high consumption and incomplete degradation in waste water, they may reach surface waters and affect aquatic organisms. To address this issue we developed a chromatographic method suitable for determining beta-blocker enantiomers in surface waters. It was tested on five beta-blockers (acebutolol, atenolol, bisoprolol, labetalol and metoprolol) and validated on bisoprolol enantiomers. Good enantioseparation of all analysed beta-blockers was achieved on the Chirobiotic V column with the mobile phase composed of methanol/acetic acid/triethylamine (100/0.20/0.15 v/v/v) at a flow rate of 0.5 mL/min and column temperature of 45 °C. Method proved to be linear in the concentration range from 0.075 μg/mL to 5 μg/mL, and showed good recovery. The limits of bisoprolol enantiomer detection were 0.025 μg/mL and 0.026 μg/mL and of quantification 0.075 μg/mL and 0.075 μg/mL. Despite its limitations, it seems to be a promising method for bisoprolol enantiomer analysis in surface water samples. Further research could focus on waste water analysis, where enantiomer concentrations may be high. Furthermore, transferring the method to a more sensitive one such as liquid chromatography coupled with tandem mass spectrometry and using ammonium acetate as the mobile phase additive instead of acetic acid and triethylamine would perhaps yield much lower limits of detection and quantification.Beta-blokatori su kiralni spojevi s enantiomerima različite bioaktivnosti, dakle, dok je jedan enantiomer aktivan, drugi može biti neaktivan ili čak štetan za organizam. Zbog njihove visoke potrošnje i nepotpune razgradnje u pogonima za preradu otpadnih voda, postoji mogućnost da se pojave u prirodnim vodama i negativno utječu na vodene organizme. Stoga je u ovom radu razvijena kromatografska metoda za određivanje enantiomera beta-blokatora u prirodnim vodama. Metoda je testirana na pet beta-blokatora (acebutolol, atenolol, bisoprolol, labetalol i metoprolol) te validirana za enantiomere bisoprolola. Dobra enantioseparacija svih analiziranih beta-blokatora postignuta je na koloni Chirobiotic V sastava mobilne faze metanol/octena kiselina/trietilamin (100:0,2:0,15 v/v/v) pri protoku od 0,5 mL/min i temperaturi od 45 °C. Metodom je postignuta dobra linearnost u području od 0,075 μg/mL do 5 μg/mL s dobrim analitičkim povratom. Granice detekcije pojedinih enantiomera bisoprolola bile su 0,025 μg/mL i 0,026 μg/mL, a granice kvantifikacije 0,075 μg/ mL za oba enantiomera. Unatoč ograničenjima metode, pokazala se kao obećavajuća metoda analize enantiomera bisoprolola u površinskim vodama. Daljnja istraživanja mogla bi se izvoditi na otpadnim vodama, gdje bi koncentracije enantiomera mogle biti više. Također, korištenjem osjetljivije metode, primjerice vezanoga sustava tekućinske kromatografije i tandemne spektrometrije masa, te korištenjem amonijeva acetata kao aditiva mobilnoj fazi umjesto octene kiseline i trietilamina, mogle bi se postići znatno niže granice detekcije i kvantifikacije

Nonlinear phenomena in electrokinetic chromatography

Capillary electrophoresis often uses complexing agents since the interaction between the analyte and the complexing agent can result in achieving or improving the separation. Examples of such methods can be electrokinetic chromatography or affinity capillary electrophoresis (ACE). ACE is used to determine the complexing parameters. In case of chiral separation, this issue gets complicated, since the parameters of the two analytes (enantiomers) are not completely independent to one another. Therefore, a procedure has been proposed in this thesis, that should always be used to evaluate the complexing parameters of two enantiomers. Statistical evaluation of these parameters was assessed as well. This work also proposes a method that allows to determine the relative migration order of two enantiomers in two different complexing separation systems. The mathematical description of electrophoresis is based on continuity equations, that are inherently nonlinear. However, these equations can be linearized to obtain an approximate analytical solution. There was recently presented a generalized model, that enables inclusion of complete complexing equilibria in the theoretical description of electromigration. Thus, various phenomena, including nonlinear ones, associated with complexation can be predicted. This..

Nonlinear phenomena in electrokinetic chromatography

V kapilární elektroforéze se často využívají komplexující činidla, protože interakce mezi analytem a komplexující látkou může vést k dosažení či zlepšení separace. Mezi metody, které komplexaci využívají, se řadí i elektrokinetická chromatografie a afinitní kapilární elektroforéza (ACE). ACE se využívá ke stanovování komplexačních konstant mezi analytem a komplexujícím činidlem. To se komplikuje v případě chirální separace, kdy na sobě parametry dvou analytů (enantiomerů) nejsou zcela nezávislé. V rámci práce byl proto navržen postup, který by při vyhodnocování komplexačních parametrů dvou enantiomerů měl být vždy používán. Současně bylo posouzeno i statistické vyhodnocení stanovených parametrů. Práce se dále zabývá stanovením metody, která umožňuje určit relativní migrační pořadí dvou enantiomerů ve dvou odlišných separačních systémech s komplexujícími činidly. Matematický popis elektroforézy je založený na rovnicích kontinuity, které jsou z definice nelineární. Rovnice je však možné linearizovat a získat tak aproximativní analytické řešení. Z tohoto přístupu vychází i nový generalizovaný model elektromigrace, který umožňuje zahrnout kompletní komplexační rovnováhy do teoretického popisu elektroforézy. Tak je možné predikovat různé jevy, včetně nelineárních, spojené s komplexací. Práce se zaměřuje...Capillary electrophoresis often uses complexing agents since the interaction between the analyte and the complexing agent can result in achieving or improving the separation. Examples of such methods can be electrokinetic chromatography or affinity capillary electrophoresis (ACE). ACE is used to determine the complexing parameters. In case of chiral separation, this issue gets complicated, since the parameters of the two analytes (enantiomers) are not completely independent to one another. Therefore, a procedure has been proposed in this thesis, that should always be used to evaluate the complexing parameters of two enantiomers. Statistical evaluation of these parameters was assessed as well. This work also proposes a method that allows to determine the relative migration order of two enantiomers in two different complexing separation systems. The mathematical description of electrophoresis is based on continuity equations, that are inherently nonlinear. However, these equations can be linearized to obtain an approximate analytical solution. There was recently presented a generalized model, that enables inclusion of complete complexing equilibria in the theoretical description of electromigration. Thus, various phenomena, including nonlinear ones, associated with complexation can be predicted. This...Katedra fyzikální a makromol. chemieDepartment of Physical and Macromolecular ChemistryPřírodovědecká fakultaFaculty of Scienc

Generalized model of the linear theory of electromigration and its application to electrokinetic chromatography: Capillary zone electrophoretic systems with complex-forming equilibria

We discuss several possible phenomena in electrophoretic systems with complexing agents present in the background electrolyte. In our previous work, we extended the linear theory of electromigration with the first-order nonlinear term, which originally applied to acid-base equilibria only, by generalizing it to any fast chemical equilibria. This extension provides us with a fresh insight into the well-established technique of elecktrokinetic chromatography (EKC). We combine mathematical analysis of the generalized model with its solution by means of the new version of our software PeakMaster 6, and experimental data. We re-examine the fundamental equations by Wren and Rowe and Tiselius in the frame of the generalized linear theory of electromigration. Besides, we show that selector concentration can increase inside the interacting-analyte zone due to its complexation with the analyte, which contradicts the generally accepted idea of a consumption of a portion of the selector inside the zone. Next, we focus our discussion on interacting buffers (i.e., buffer constituents that form a complex with the selector). We demonstrate how such side-interaction of the selector with another buffer constituent can influence measuring analyte-selector interactions. Finally, we describe occurrence and mobilities of system peaks in these EKC systems. We investigate systems with fully charged analytes and neutral cyclodextrins as selectors. Although the theory is not limited in terms of the charge and/or the degree of (de)protonation of any constituent, this setup allows us to find analytical solutions to generalized model under approximate, yet realistic, conditions and to demonstrate all important phenomena that may occur in EKC systems. An occurrence of system peaks in a system with fully charged selector is also investigated.Fil: Dovhunová, Magda. Charles University; República ChecaFil: Malý, Michal. Charles University; República ChecaFil: Dubský, Pavel. Charles University; República ChecaFil: Gerlero, Gabriel Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones en Métodos Computacionales. Universidad Nacional del Litoral. Centro de Investigaciones en Métodos Computacionales; ArgentinaFil: Kler, Pablo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones en Métodos Computacionales. Universidad Nacional del Litoral. Centro de Investigaciones en Métodos Computacionales; Argentin

Generalized model of the linear theory of electromigration and its application to electrokinetic chromatography: Theory and software PeakMaster 6—Next Generation

The linear theory of electromigration, including the first-order nonlinear approximation, is generalized to systems with any equilibria fast enough to be considered instantaneous in comparison with the timescale of peak movement. For example, this theory is practically applied in the electrokinetic chromatography (EKC) mode of the CZE. The model enables the calculation of positions and shapes of analyte and system peaks without restricting the number of selectors, the complexation stoichiometry, or simultaneous acid–base equilibria. The latest version of our PeakMaster software, PeakMaster 6—Next Generation, implements the theory in a user-friendly way. It is a free and open-source software that performs all calculations and shows the properties of the background electrolyte and the expected electropherogram within a few seconds. In this paper, we mathematically derive the model, discuss its applicability to EKC systems, and introduce the PeakMaster 6 software.Fil: Malý, Michal. Charles University; República ChecaFil: Dovhunová, Magda. Charles University; República ChecaFil: Dvořák, Martin. Charles University; República ChecaFil: Gerlero, Gabriel Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones en Métodos Computacionales. Universidad Nacional del Litoral. Centro de Investigaciones en Métodos Computacionales; ArgentinaFil: Kler, Pablo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Centro de Investigaciones en Métodos Computacionales. Universidad Nacional del Litoral. Centro de Investigaciones en Métodos Computacionales; ArgentinaFil: Hruška, Vlastimil. Agilent Technologies Deutschland GmbH & Co. KG; AlemaniaFil: Dubský, Pavel. Charles University; República Chec