pH Gradient linearity Dotaz Zobrazit nápovědu
Fourteen low molecular mass UV absorbing ampholytes containing 1 or 2 weakly acidic and 1 or 2 weakly basic functional groups that best satisfy Rilbe's requirement for being good carrier ampholytes (ΔpKa = pKamonoanion - pKamonocation < 2) were selected from a large group of commercially readily available ampholytes in a computational study using two software packages (ChemSketch and SPARC). Their electrophoretic mobilities were measured in 10 mM ionic strength BGEs covering the 2 < pH < 12 range. Using our Debye-Hückel and Onsager-Fuoss laws-based new software, AnglerFish (freeware, https://echmet.natur.cuni.cz/software/download), the effective mobilities were recalculated to zero ionic strength from which the thermodynamic pKa values and limiting ionic mobilities of the ampholytes were directly calculated by Henderson-Hasselbalch equation-type nonlinear regression. The tabulated thermodynamic pKa values and limiting ionic mobilities of these ampholytes (pI markers) facilitate both the overall and the narrow-segment characterization of the pH gradients obtained in IEF in order to mitigate the errors of analyte ampholyte pI assignments caused by the usual (but rarely proven) assumption of pH gradient linearity. These thermodynamic pKa and limiting mobility values also enable the reality-based numeric simulation of the IEF process using, for example, Simul (freeware, https://echmet.natur.cuni.cz/software/download).
A model was constructed which includes electron transport (linear and cyclic and Mehler type reaction) coupled to proton translocation, counter ion movement, ATP synthesis, and Calvin-Benson cycle. The focus is on modeling of the light-induced total electric potential difference (ΔΨ) which in this model originates from the bulk phase electric potential difference (ΔΨb), the localized electric potential difference (ΔΨc), as well as the surface electric potential difference (ΔΨs). The measured dual wavelength transmittance signal (ΔA515-560nm, electrochromic shift) was used as a proxy for experimental ΔΨ. The predictions for theoretical ΔΨ vary with assumed contribution of ΔΨs, which might imply that the measured ΔA515-560nm trace on a long time scale reflects the interplay of the ΔΨ components. Simulations also show that partitioning of proton motive force (pmf) to ΔΨb and ΔpH components is sensitive to the stoichiometric ratio of H(+)/ATP, energy barrier for ATP synthesis, ionic strength, buffer capacity and light intensity. Our model shows that high buffer capacity promotes the establishment of ΔΨb, while the formation of pHi minimum is not 'dissipated' but 'postponed' until it reaches the same level as that for low buffer capacity. Under physiologically optimal conditions, the output of the model shows that at steady state in light, the ΔpH component is the main contributor to pmf to drive ATP synthesis while a low ΔΨb persists energizing the membrane. Our model predicts 11mV as the resting electric potential difference across the thylakoid membrane in dark. We suggest that the model presented in this work can be integrated as a module into a more comprehensive model of oxygenic photosynthesis.
- MeSH
- biologické modely * MeSH
- časové faktory MeSH
- koncentrace vodíkových iontů účinky záření MeSH
- listy rostlin metabolismus účinky záření MeSH
- membránové potenciály účinky záření MeSH
- počítačová simulace MeSH
- protonmotorická síla účinky záření MeSH
- protony MeSH
- pufry MeSH
- světlo * MeSH
- transport elektronů MeSH
- tylakoidy metabolismus účinky záření MeSH
- uhlík metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
The stability of ramipril in the buffer solution with different pH and the influence of acid, alkaline and oxidative medium on ramipril stability were studied. The ramipril degradation products were determined by high-performance liquid chromatography (HPLC) method. Acetonitrile:sodium perchlorate was used as the mobile phase, at a flow rate of 1.0 ml/min (linear gradient elution). A Nucleosil 100-S 5 microm C18, 250 mm x 4.6 mm i.d. was utilized as stationary phase. Detection was affected spectrophotometrically at 210 nm. The drug substance was dissolved in the ammonium phosphate buffer (pH 3, 5 and 8) and these solutions were stored at 90 degrees C for 1 h. The other series of test solutions were prepared from stock solution (drug substance dissolved in solvent A of the mobile phase) by dilution in acid (0.1M HCl), alkaline (0.1M NaOH) and oxidative (hydrogen peroxide solution) medium. More then 0.2% of impurity D (ramipril-diketopiperazine) was detected in the buffer of pH 3 and pH 5. In the buffer of pH 8 there was detected more then 1% of impurity E (ramipril-diacid). No peaks for degradation products appeared in the chromatograms above limit of quantification. The alkaline medium has the greatest effect on degradation of ramipril into impurity E (more than 50%).
OBJECTIVES: The Caco-2 cell monolayer model is widely used as a standard screening tool for studying the mechanisms of cellular drug transport. Caffeine was chosen as a model drug and is supposed to be class I of the Biopharmaceutics Classification System (BCS). Our study was conducted 1) to characterize the mechanisms of caffeine transport across the intestinal barrier, 2) to classify caffeine according to BCS, 3) to predict drugs intestinal absorption in humans. METHODS: Caffeine transport (0.1, 0.3, 1 and 10 mmol/l) was studied in Caco-2 cell monolayer in apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction, under iso-pH 7.4 and pH-gradient (6/7.4) conditions. The relative contribution of the paracellular route was estimated using Ca2+- free transport medium (opening tight junctions). RESULTS: The caffeine transport was linear with time, transport direction and pH independent, displaying non-saturable (first-order) kinetics, with high permeability coefficient (Papp): in AP-BL direction Papp = 46.3-53.5 x 10-6 cm/s; in BL-AP direction Papp = 45.6-49.4 x 10-6 cm/s. Thus, the transport seems to be transcellular mediated by passive diffusion. Using Ca2+- free transport medium tight junctions were opened (confirmed by increased Papp of mannitol) but the caffeine Papp was not changed. Thus, the paracellular route is only a minor way of caffeine transport. CONCLUSION: High solubility and high permeability of caffeine rank it among class I of BCS and well absorbed compounds.
- MeSH
- biologický transport účinky léků fyziologie MeSH
- buněčné kultury MeSH
- Caco-2 buňky MeSH
- difuze MeSH
- intestinální absorpce MeSH
- kinetika MeSH
- kofein farmakokinetika farmakologie MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- lineární modely MeSH
- mannitol metabolismus MeSH
- permeabilita účinky léků MeSH
- střevní sliznice metabolismus MeSH
- těsný spoj účinky léků metabolismus MeSH
- viabilita buněk účinky léků MeSH
- xenobiotika farmakokinetika farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Caco-2 cell monolayer model is widely used as a standard screening tool for studying the mechanisms of cellular drug transport. Caffeine was chosen as a model drug and is supposed to be class I of the Biopharmaceutics Classification System (BCS). Our study was conducted 1) to characterize the mechanisms of caffeine transport across the intestinal barrier, 2) to classify caffeine according to BCS, 3) to predict drugs intestinal absorption in humans. METHODS: Caffeine transport (0.1, 0.3, 1 and 10 mmol/l) was studied in Caco-2 cell monolayer in apical to basolateral (AP-BL) and basolateral to apical (BL-AP) direction, under iso-pH 7.4 and pH-gradient (6/7.4) conditions. The relative contribution of the paracellular route was estimated using Ca2+- free transport medium (opening tight junctions). RESULTS: The caffeine transport was linear with time, transport direction and pH independent, displaying non-saturable (first-order) kinetics, with high permeability coefficient (Papp): in AP-BL direction Papp = 46.3-53.5 x 10-6 cm/s; in BL-AP direction Papp = 45.6-49.4 x 10-6 cm/s. Thus, the transport seems to be transcellular mediated by passive diffusion. Using Ca2+- free transport medium tight junctions were opened (confirmed by increased Papp of mannitol) but the caffeine Papp was not changed. Thus, the paracellular route is only a minor way of caffeine transport. CONCLUSION: High solubility and high permeability of caffeine rank it among class I of BCS and well absorbed compounds
- MeSH
- biologický transport fyziologie účinky léků MeSH
- buněčné kultury MeSH
- Caco-2 buňky MeSH
- difuze MeSH
- financování organizované MeSH
- intestinální absorpce MeSH
- kinetika MeSH
- kofein farmakokinetika farmakologie MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- lineární modely MeSH
- mannitol metabolismus MeSH
- permeabilita účinky léků MeSH
- střevní sliznice metabolismus MeSH
- těsný spoj metabolismus účinky léků MeSH
- viabilita buněk účinky léků MeSH
- xenobiotika farmakokinetika farmakologie MeSH
- Check Tag
- lidé MeSH
In 1961, Svensson described isoelectric focusing (IEF), the separation of ampholytic compounds in a stationary, natural pH gradient that was formed by passing current through a sucrose density gradient-stabilized ampholyte mixture in a constant cross-section apparatus, free of mixing. Stable pH gradients were formed as the electrophoretic transport built up a series of isoelectric ampholyte zones-the concentration of which decreased with their distance from the electrodes-and a diffusive flux which balanced the generating electrophoretic flux. When polyacrylamide gel replaced the sucrose density gradient as the stabilizing medium, the spatial and temporal stability of Svensson's pH gradient became lost, igniting a search for the explanation and mitigation of the loss. Over time, through a series of insightful suggestions, the currently held notion emerged that in the modern IEF experiment-where the carrier ampholyte (CA) mixture is placed between the anolyte- and catholyte-containing large-volume electrode vessels (open-system IEF)-a two-stage process operates that comprises a rapid first phase during which a linear pH gradient develops, and a subsequent slow, second stage, during which the pH gradient decays as isotachophoretic processes move the extreme pI CAs into the electrode vessels. Here we trace the development of the two-stage IEF model using quotes from the original publications and point out critical results that the IEF community should have embraced but missed. This manuscript sets the foundation for the companion papers, Parts 2 and 3, in which an alternative model, transient bidirectional isotachophoresis is presented to describe the open-system IEF experiment.
This study aimed i) to characterize the transepithelial transport of the mucolytic agent ambroxol hydrochloride across the intestinal barrier, ii) to classify the ambroxol according to Biopharmaceutics Classification System (BCS) and iii) to predict ambroxol absorption in humans. Transport of ambroxol (100, 300 and 1000 micromol/l) was studied in a human colon carcinoma cell line Caco-2 in apical to basolateral and basolateral to apical direction, under iso-pH 7.4 and pH-gradient (6 vs. 7.4) conditions. The relative contribution of the paracellular route was estimated using Ca2+-free transport medium. Ambroxol samples from receiver compartments were analysed by HPLC with UV detection (242 nm). Results showed that ambroxol transport is linear with time, pH-dependent and direction-independent, displays non-saturable (first-order) kinetics. Thus, the transport seems to be transcellular mediated by passive diffusion. Estimated high solubility and high permeability (P(app) = 45 x 10(-6) cm/s) of ambroxol rank it among well absorbed compounds and class I of BCS. It can be expected that the oral dose fraction of ambroxol absorbed in human intestine is high.
- MeSH
- absorpce MeSH
- ambroxol aplikace a dávkování farmakokinetika klasifikace MeSH
- biologické modely MeSH
- epitel metabolismus MeSH
- expektorancia aplikace a dávkování farmakokinetika klasifikace MeSH
- karcinom metabolismus MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- lineární modely MeSH
- nádorové buněčné linie MeSH
- nádory tračníku metabolismus MeSH
- permeabilita MeSH
- rozpustnost MeSH
- ultrafialové záření MeSH
- vápník nedostatek MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Agomelatine is one of the newest antidepressants. Due to a different mechanism of action it offers a completely new approach in the treatment of depressive disorders. Two chromatographic methods for determination of agomelatine and its impurities were developed. The separations on UHPSFC system were accomplished using stationary phase based on BEH 2-EP and gradient elution with CO2 and methanol containing 20mM ammonium formate and 5% of water. The UHPLC separations were performed on stationary phase BEH Shield RP18. The mixture of acetonitrile and methanol in ratio 1:1 and ammonium acetate buffer pH 9.5 were used as mobile phase. Both developed methods were properly validated in terms of linearity, sensitivity (LOD, LOQ), accuracy and precision according to ICH guidelines. The UHPSFC method was linear in the range 0.25-70μg/ml for all analytes with method accuracy ≥97.4% and ≥100.2% and method intra-day precision RSD ≤2.4 and ≤0.8 for impurities and API (active pharmaceutical ingredient), respectively. The UHPLC method was linear in the range 0.1-10μg/ml for all analytes except three impurities for which the linear range was larger 0.1-25μg/ml. Method accuracy ≥95.7% and ≥95.2% and method intra-day precision RSD ≤2.6 and ≤1.5 were found for impurities and API, respectively. The measurement of tablet samples was performed and the selected parameters of the methods were compared. In conclusion, both methods were appropriate for the determination of agomelatine and its impurities in pharmaceutical quality control (QC), although the UHPSFC method was found as more convenient especially in the method development phase. The advantages of newly developed UHPSFC-PDA (photo diode array detector) method were its environmental friendliness due to the mobile phase used, a very good resolution, selectivity and high speed of analysis (total time of separation was 4.1min).
A new method for determination of fumonisins in corn samples was developed and validated. The mycotoxins were extracted by a mixture of methanol-acetonitrile-water (1:1:2, v/v/v) and determined on a liquid chromatograph with mass spectrometric detection. The separation was performed on Zorbax XDB-C(18) column (150 x 4.6 mm; 5 microm) with a Metaguard ODS-2 precolumn (30 x 4.6 mm; 5 mum) using gradient elution with mobile phase consisting of acetonitrile and 5 mmol/L ammonium acetate (adjusted by acetic acid to pH 3.0). For detection of (M+H(+)) ions, a quadrupole mass spectrometer in single ion monitoring mode was applied. Developed method showed very good linearity in a tested range of concentration. Detection limit is 62.0 microg FB(1)/kg and 58.5 microg FB(2)/kg of maize grains. Because the detection limits lie under the maximum permitted EU levels, the method is suitable for determination of fumonisins in milled corn grains.
- MeSH
- acetáty MeSH
- chemická frakcionace MeSH
- fumonisiny analýza chemie MeSH
- hmotnostní spektrometrie metody MeSH
- kukuřice setá chemie MeSH
- lineární modely MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
New bioanalytical SPE-HPLC-PDA-FL method for the determination of the neuroleptic drug tiapride and its N-desethyl metabolite was developed, validated and applied to xenobiochemical and pharmacokinetic studies in humans and animals. The sample preparation process involved solid-phase extraction of diluted plasma spiked with sulpiride (an internal standard) using SPE cartridges DSC-PH Supelco, USA. Chromatographic separation of the extracts was performed on a Discovery HS F5 250 mm × 4 mm (Supelco) column containing pentafluorophenylpropylsilyl silica gel. Mobile phase (acetonitrile-0.01 M phosphate buffer pH=3, flow rate 1 ml min(-1)) in the gradient mode was employed in the HPLC analysis. Tandem UV photodiode-array→fluorescence detection was used for the determination of the analytes. Low concentrations of tiapride and N-desethyl tiapride were determined using a more selective fluorescence detector (λ(exc.)/λ(emiss.)=232 nm/334 nm), high concentrations (500-6000 pmol ml(-1)) using a UV PDA detector at 212 nm with a linear response. Each HPLC run lasted 15 min. Lower limits of quantification (LLOQ) for tiapride (N-desethyl tiapride) were found to be 8.24 pmol ml(-1) (10.11 pmol ml(-1)). The recoveries of tiapride ranged from 89.3 to 94.3%, 81.7 to 86.8% for internal standard sulpiride and 90.9 to 91.8% for N-desethyl tiapride.
- MeSH
- extrakce na pevné fázi MeSH
- fluorescenční spektrometrie metody MeSH
- jaterní mikrozomy metabolismus MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- limita detekce MeSH
- lineární modely MeSH
- mladý dospělý MeSH
- reprodukovatelnost výsledků MeSH
- spektrofotometrie ultrafialová metody MeSH
- sulpirid krev MeSH
- tiapamil-hydrochlorid analogy a deriváty krev farmakokinetika MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
