Aleš Mádr

On‐line drug metabolites generation and their subsequent target analysis by capillary zone electrophoresis with UV‐absorption detection

This study presents the in‐capillary enzymatic biotransformation of dextromethorphan, an antitusive drug and opioid receptor antagonist, and subsequent electrophoretic separation of its products. The study includes the optimization of separation parameters to fulfill the requirements of an online microreaction. The analyses were performed in a bare fused‐silica capillary using 100 mM sodium tetraborate (pH 10.0) mixed with linear polyacrylamide (20%, v/v) and 2‐propanol (10%, v/v). This BGE was suitable for monitoring both off‐line and in‐capillary incubations. The partial filling technique enabled the enzymatic reaction to be carried out in its optimal environment (20 mM sodium phosphate, pH 7.4). Finally, in‐capillary microreaction in the presence of cytochrome P450 3A4 gave satisfactory outcomes.

Electrophoretically mediated microanalysis for simultaneous on-capillary derivatization of standard amino acids followed by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

Amino acids are crucial compounds involved in most biochemical processes essential for life. Since their dynamic turnover reflects the actual physiology of the cell/organism, a turnover assessment may provide valuable information related to multiple physiological and pathophysiological conditions. The sensitive determination of amino acids is predominantly associated with their derivatization which might be laborious, time-consuming and difficult to standardize. However, capillary electrophoresis offers the automatic injection and mixing of reactants, incubation of the reaction mixture, separation and detection of the reaction products in one on-capillary procedure. Among the on-capillary mixing strategies, electrophoretically mediated microanalysis (EMMA) is superior in terms of mixing efficiency. In this paper, we present an optimization of EMMA for the simultaneous derivatization of standard amino acids by naphthalene-2,3-dicarboxaldehyde/NaCN and its application to targeted human embryo metabolomics. For such a purpose, novel separation conditions were developed involving the background electrolyte, comprised of 73mM sodium dodecyl sulfate, 6.7 % (v/v) 1-propanol, 0.5mM (2-hydroxypropyl)-β-cyclodextrin and 135mM boric acid/sodium hydroxide buffer (pH 9.00). Finally, the optimized EMMA was compared to a fundamentally different mixing strategy, namely the transverse diffusion of laminar flow profiles, and proved to be also suitable for human plasma analysis.

MEKC LIF method for analysis of amino acids after on-capillary derivatization by transverse diffusion of laminar flow profiles mixing of reactants for assessing developmental capacity of human embryos after in vitro fertilization

Evaluating the physiological state of an organism is of clinical importance. In assisted reproduction, knowledge of the embryos physiology is crucial for selecting the embryo with the highest developmental capacity to ensure high pregnancy rates. Amino acids (AAs) are involved in many biochemical processes during embryo development, which means that the determination of AA fluctuations in the embryos surroundings can determine the embryos physiological state. Since current embryo selection methods are mainly based on visual assessment, which lacks proper accuracy, a novel method for the analysis of AAs in the embryos surroundings was developed. AAs were analyzed by means of MEKC‐LIF after on‐capillary derivatization by naphthalene‐2,3‐dicarboxaldehyde. The reactants were injected under the three zone arrangement and mixed using the transverse diffusion of laminar flow profiles methodology. The resulting derivatives of all the standard AAs were baseline resolved in the BGE comprised of 35 mM sodium tetraborate, 55 mM SDS, 2.7 M urea, 1 mM BIS‐TRIS propane, and 23 mM NaOH within 50 min. The method was applied on an analysis of spent culture media used in assisted reproduction to culture embryos after in vitro fertilization.

Determination of pyruvate and lactate as potential biomarkers of embryo viability in assisted reproduction by capillary electrophoresis with contactless conductivity detection

Human‐assisted reproduction is increasing in importance due to the constantly rising number of couples suffering from infertility issue. A key step in in vitro fertilization is the proper assessment of embryo viability in order to select the embryo with the highest likelihood of resulting in a pregnancy. This study proposes a method based on CE with contactless conductivity detection for the determination of pyruvate and lactate in spent culture media used in human‐assisted reproduction. A fused‐silica capillary of 64.0 cm total length and 50 μm inner diameter was used. The inner capillary wall was modified by the coating of successive layers of the ionic polymers polybrene and dextran sulfate to reverse EOF. The BGE was composed of 10 mM MES/lithium hydroxide, pH 6.50. The sample was injected by pressure 50 mbar for 18 s, separation voltage was set to −24 kV, and capillary temperature to 15°C. The presented method requires only 2 μL of the culture medium, with LODs for pyruvate and lactate of 0.03 and 0.02 μM, respectively. The results demonstrated the methods suitability for the analysis of spent culture media to support embryo viability assessment by light microscopy, providing information about key metabolites of the energy metabolism of a developing embryo.

Study of metabolic activity of human embryos focused on amino acids by capillary electrophoresis with light-emitting diode-induced fluorescence detection

Assisted reproduction is a quickly developing field of reproductive medicine whose importance is growing every year due to the increasing number of patients suffering from infertility. As a result, there is a need for the continuous development and/or improvement of assisted reproductive technologies. This paper presents a new method for the in vitro measurement of the amino acid turnover of developing embryos based on capillary electrophoresis with light‐emitting diode‐induced fluorescence detection. Amino acids were derivatized with naphthalene‐2,3‐dicarboxaldehyde/NaCN, and the resulting fluorescent derivatives were baseline resolved within 25 min in a background electrolyte comprised of 50 mM sodium tetraborate, 73 mM sodium dodecyl sulphate, 5 mM sodium deoxycholate and 2.5 mM (2‐hydroxypropyl)‐β‐cyclodextrin (pH ≈ 9.3). The migration time and the peak area repeatability (n = 10) were below 0.5 and 4.3%, respectively. The limits of detection ranged from 12.6 nM (histidine) to 39.3 nM (taurine). The developed method, which only requires 2 μL of raw sample, was successfully applied for determining the metabolic activity of human embryos exposed to different environmental stress conditions.

Determination of Caffeine and its Metabolites in Saliva and Urine as a Measure of CYP1A2 Metabolic Activity

Byla vyvinuta nova citliva a robustni RP-HPLC metoda pro stanoveni kofeinu, 1,7 dimethylxanthinu, 1,7-dimethylmocove kyseliny, 5-acetylamino-6-formylamino-3-methyluracilu, 5-acetylamino-6-amino-3-methyluracilu, 1-methylxanthinu a 1-methylumocove kyseliny ve slinach a moci. Metoda slouži ke stanoveni metabolicke aktivity CYP1A2.