A new type of high performance liquid chromatography (HPLC) stationary phase was prepared, and its chromatographic properties were evaluated. The sorbent was composed of metallacarborane covalently bound to silica. Because of the chemical structure of the immobilized metallacarborane, the synthesized stationary phase was able to interact with nonpolar analytes via hydrophobic interactions. The chromatographic behavior of several low-molecular-weight hydrocarbons on the sorbent under typical reversed-phase conditions was compared with octadecyl-, sulfo phenyl- and aminopropyl-modified silica stationary phases. Moreover, as a consequence of the synthetic protocol employed, the immobilization of the metallacarborane led to the development of a zwitterionic chemically bonded phase, which demonstrated excellent resistance to "phase collapse" in a 100% aqueous environment. Finally, preliminary experiments indicated that the new stationary phase has the potential for utilization in hydrophilic interaction chromatography (HILIC) mode for the separation of polar compounds.
- MeSH
- Acetonitriles chemistry MeSH
- Benzene Derivatives chemistry MeSH
- Boranes chemistry MeSH
- Chromatography, Reverse-Phase MeSH
- Hydrophobic and Hydrophilic Interactions MeSH
- Cobalt chemistry MeSH
- Linear Models MeSH
- Silicon Dioxide chemistry MeSH
- Spectrum Analysis, Raman MeSH
- Reproducibility of Results MeSH
- Chromatography, High Pressure Liquid instrumentation methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Capillary liquid chromatography (cLC) hyphenated with tandem mass spectrometry (MS-MS) was used to separate and quantitate trace concentrations of five estrogens in aqueous samples. New C(18)-based sorption materials bound to the silica support by monomeric and polymeric mechanisms were compared and tested for solid-phase extraction (SPE) of selected analytes with respect to optimization of their preconcentration yield. Application of an endcapped, monomer-bound preconcentration Discovery DSC-18Lt column under the optimized conditions provides yields in the range from 95 to 100% with a high repeatability (n=3, RSD≤7.2%). Using the electrospray ionization in the positive mode (ESI+), the cLC-MS-MS system (the Zorbax SB C18 capillary column and a binary mobile phase of acetonitrile and water containing 0.1% formic acid in both the components) was optimized to attain a sufficient retention of the early eluting estriol, a satisfactory resolution of the analytes and the maximum sensitivity of the determination. Both the isocratic and gradient elution were used and the optimized gradient method permitted analyses of aqueous environmental samples in 14 min within a linearity range from 6.1 to 25.0 (LOQ of analytes) to 500 ng/L and with a very good linearity (r>0.9981) for all the estrogens studied. The detection limits are in the range from 3.0 to 6.8 ng/L (1 μL injection volume). Six environmental water samples were analyzed and the studied estrogens were found in the Vltava river sample collected in Prague (13.2 ng/L for 17β-estradiol) and in the inlet to the wastewater treatment plant in Prague, at an overall concentration of 371.4 ng/L.
- MeSH
- Water Pollutants, Chemical analysis MeSH
- Estrogens analysis isolation & purification MeSH
- Solid Phase Extraction methods MeSH
- Spectrometry, Mass, Electrospray Ionization MeSH
- Capillary Electrochromatography methods MeSH
- Linear Models MeSH
- Rivers chemistry MeSH
- Reproducibility of Results MeSH
- Sensitivity and Specificity MeSH
- Tandem Mass Spectrometry methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Due to dramatic effects of even small changes in mobile phase composition on the retention, separations of high-molecular compounds are very difficult, if possible at all, at isocratic conditions and need gradient elution. The theory of gradient elution for small molecules is well established, however its applications to reversed-phase gradient separations of biopolymers are not straightforward because of specific problems, such as slow diffusion, limited accessibility of the stationary phase for larger molecules, or possible sample conformation changes during the elution. Theoretical prediction of gradient data needs the parameters of model retention equations to be known, which however cannot be determined at isocratic conditions. The present work overviews the attempts at implementation of the conventional gradient theory developed for low-molecular compounds to the description and prediction of gradient separations of peptides and proteins on various types of HPLC columns: conventional analytical columns packed with wide-pore fully porous, fused-core superficially porous and non-porous particles; silica-based monolithic columns and organic-polymer poly(alkylmethacrylate) and poly(styrene-divinylbenzene) monolithic columns in capillary and disc formats. The attention is focused on the determination of the parameters necessary to predict gradient retention times (volumes) and bandwidths using the theoretical model equations. The accuracy of the prediction of protein retention on totally porous columns improves if size exclusion effect is taken into account, but this is not necessary with non-porous or superficially porous particles. Band dispersion effects counteracting band compression in gradient elution depend on the type of column, on the protein and on the gradient volume (steepness) and complicate the prediction of band broadening in gradient chromatography of proteins, however the conventional gradient model can be employed to estimate the effects of changing gradient parameters on the bandwidths, as well as on the elution times (volumes) of proteins.
Sample preparation prior to chromatographic separation plays an important role in the analytical process. To avoid time-consuming and manual handling sample-prep, automated on-line techniques such as on-line SPE-HPLC are therefore preferred. In this study, two different on-line extraction approaches for mycotoxin/endocrine disruptor zearalenone (ZEA) determination using either molecularly imprinted polymer (MIP) with selective cavities and binding sites for extraction or a reversed-phase sorbent C18 providing non-selective interactions have been developed, validated, and compared. The validation characteristics were compared and the two methods were evaluated as being almost equal in terms of linearity, repeatability, precision, and recovery. Recoveries were in the range of 99.0-100.1% and limits of detection were found the same for both methods (1.5 μg L-1). Method precision calculated for spiked beer samples was better for C18 sorbent (2.5 vs. 5.4% RSD). No significant differences in the selectivity of either extraction method were observed. The possible reasons and further details associated with this finding are discussed. Finally, both validated methods were applied for the determination of ZEA contamination in beer samples. Due to ZEA's native fluorescence, chromatographic separation with fluorimetric detection (λex = 270 nm and λem, = 458 nm) was selected. Graphical abstract Determination of zearalenone in beer using an on-line extraction chromatography system.
- MeSH
- Food Analysis methods MeSH
- Chromatography, Reverse-Phase methods MeSH
- Endocrine Disruptors analysis MeSH
- Solid Phase Extraction methods MeSH
- Limit of Detection MeSH
- Molecular Imprinting methods MeSH
- Mycotoxins analysis MeSH
- Estrogens, Non-Steroidal analysis MeSH
- Beer analysis MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Zearalenone analysis MeSH
- Publication type
- Journal Article MeSH
- Validation Study MeSH
A sensitive, specific, and rapid high-performance liquid chromatography (HPLC) method for the determination of ambrisentan enantiomers has been developed and validated. Six chiral columns were tested in a reversed-phase system. Excellent enantioseparation with the resolution more than 2.5 was achieved on Chiralcel OZ-3R (cellulose 3-chloro-4-methylphenylcarbamate) using mixture of 20 mM sodium formate (pH 3.0) with acetonitrile (55:45; v/v). Validation of the HPLC method including linearity, limit of detection, limit of quantification, precision, accuracy, and selectivity was performed according to the International Conference on Harmonisation (ICH) guidelines. The method has an advantage of a very quick chromatographic separation (less than 6 min) and therefore is highly suitable for routine determination of (R)-ambrisentan in enantiopure active pharmaceutical ingredient (S)-ambrisentan.
- MeSH
- Adsorption MeSH
- Cellulose chemistry MeSH
- Chromatography, Reverse-Phase instrumentation methods MeSH
- Phenylpropionates chemistry MeSH
- Pyridazines chemistry MeSH
- Stereoisomerism MeSH
- Chromatography, High Pressure Liquid instrumentation methods MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study 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
- Solid Phase Extraction MeSH
- Spectrometry, Fluorescence methods MeSH
- Microsomes, Liver metabolism MeSH
- Rats MeSH
- Humans MeSH
- Limit of Detection MeSH
- Linear Models MeSH
- Young Adult MeSH
- Reproducibility of Results MeSH
- Spectrophotometry, Ultraviolet methods MeSH
- Sulpiride blood MeSH
- Tiapamil Hydrochloride analogs & derivatives blood pharmacokinetics MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Multidimensional chromatography coupled to tandem mass spectrometry (MS/MS), including simple sample preparation with protein precipitation, anion conversion with ammonium hydroxide, and solid-phase extraction using mixed-mode anion exchange in a 96-well plate format, has been validated for rapid simultaneous analysis of human insulin and its six analogs (lispro, glulisine, glargine, degludec, detemir, and aspart) in human plasma. This method is critical for clinical diagnostics, forensic investigations, and anti-doping efforts due to the widespread use of these substances. In the present study, improved chromatographic resolution was achieved using a first-dimension trap-and-elute configuration with an XBridge C18 (2.1 × 20 mm, 3.5 μm) trap column combined with second dimension separation on a Cortecs Ultra-High-Performance Liquid Chromatography (UHPLC) C18+ (2.1 × 100 mm, 1.6 μm) analytical column implemented within a two-dimensional-LC-MS/MS system. The total chromatographic run time was 11 min. This setup increases both the resolution and sensitivity of the method. A mobile phase consisting of 0.8% formic acid (FA) in water and 0.7% FA in acetonitrile was used for gradient elution. Bovine insulin was used as the internal standard. MS detection was performed in positive electrospray ionization mode, and the ion suppression due to matrix effects was evaluated. Validation criteria included linearity, precision, accuracy, recovery, lower limit of quantitation, matrix effect, and stability tests with and without protease inhibitor cocktail under different conditions (short-term stability, long-term stability, and freeze-thaw stability). The concentration range for all insulins was 50-15 000 pg/mL, with limits of quantification below the therapeutic reference range for all analytes. Intra-run precision ranged from 1.1% to 5.7%, inter-run precision from 0.7% to 5.9%, and overall recovery from 96.9% to 114.3%. The validated method has been implemented successfully by the Department of Forensic Medicine at our hospital for the investigation of unexplained deaths.
A simple, fast and sensitive HPLC method with electrochemical detection employing boron-doped diamond electrode (BDD) for the determination of sildenafil (Viagra™), vardenafil (Levitra™) and their main metabolites, N-desmethyl sildenafil and N-desethyl vardenafil in human plasma is presented. The assay involved drug extraction by tert-butyl methyl ether and isocratic reversed-phase liquid chromatography with amperometric detection. Complete separation of all analytes was achieved within 12 min. The mobile phase consisted of 20mM sodium dihydrogen phosphate with 40 mM sodium perchlorate/acetonitrile (70:30, v/v), pH 3.5. The electrode working potential was +1520 mV (vs. Pd/H(2)). Calibration curves were linear over the concentration range of 10-400 ng mL(-1). Phloretin was used as an internal standard. The limits of detection (LOD) and quantification (LOQ) for the studied analytes were within the range of 2-4 ng mL(-1) and 7.0-13.4 ng mL(-1), respectively. The developed method was applied to human plasma samples spiked with analytes at therapeutic concentrations. The study confirms the method's suitability for both pharmacokinetic studies and therapeutic monitoring.
- MeSH
- Boron chemistry MeSH
- Chromatography, Reverse-Phase MeSH
- Diamond chemistry MeSH
- Electrodes MeSH
- Imidazoles blood chemistry MeSH
- Hydrogen-Ion Concentration MeSH
- Humans MeSH
- Linear Models MeSH
- Piperazines blood chemistry MeSH
- Purines blood chemistry MeSH
- Sensitivity and Specificity MeSH
- Sulfones blood chemistry MeSH
- Triazines blood chemistry MeSH
- Chromatography, High Pressure Liquid instrumentation methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Lilac coloured species of Geosmithia lavendula produce a mixture of polyhydroxylated anthraquinones under condition of submerged fermentation. Three pigments had been isolated and identified earlier as a 1,3,6,8-tetrahydroxyanthraquinone (compound 7), rhodolamprometrin (1-acetyl 2,4,5,7-tetrahydroxyanthraquinone; compound 5), and 1-acetyl 2,4,5,7,8-penthahydroxyanthraquinone (compound 4). A new HPLC method was developed for the separation of three known and ten new anthraquinone pigments. In addition, five new pigments were determined by FTMS as coeluting impurities. The analyses were performed on a reversed phase column using gradient elution with a mobile phase system consisting of phosphate buffer (50 mM; pH=2.0) and acetonitrile. The structure evaluation was based namely on FTMS and UV-VIS spectrometry. The developed procedure was used for the determination of individual anthraquinones in fermentation broth of G. lavendula after 14 days of cultivation. The extractable amount and LOQ (both in μg ml(-1)) for the two main pigments from G. lavendula are 50.02 and 2.15 for compound 4, and 63.77 and 2.75, for compound 5, respectively.
- MeSH
- Anthraquinones analysis chemistry MeSH
- Chromatography, Reverse-Phase MeSH
- Fermentation MeSH
- Hypocreales chemistry MeSH
- Linear Models MeSH
- Methanol MeSH
- Reproducibility of Results MeSH
- Sensitivity and Specificity MeSH
- Tandem Mass Spectrometry methods MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The fluorescent molecule diphenylhexatriene (DPH) has been often used in combination with fluorescence anisotropy measurements, yet little is known regarding the non-linear optical properties. In the current work, we focus on them and extend the application to fluorescence, while paying attention to the conformational versatility of DPH when it is embedded in different membrane phases. Extensive hybrid quantum mechanics/molecular mechanics calculations were performed to investigate the influence of the phase- and temperature-dependent lipid environment on the probe. Already, the transition dipole moments and one-photon absorption spectra obtained in the liquid ordered mixture of sphingomyelin (SM)-cholesterol (Chol) (2:1) differ largely from the ones calculated in the liquid disordered DOPC and solid gel DPPC membranes. Throughout the work, the molecular conformation in SM:Chol is found to differ from the other environments. The two-photon absorption spectra and the ones obtained by hyper-Rayleigh scattering depend strongly on the environment. Finally, a stringent comparison of the fluorescence anisotropy decay and the fluorescence lifetime confirm the use of DPH to gain information upon the surrounding lipids and lipid phases. DPH might thus open the possibility to detect and analyze different biological environments based on its absorption and emission properties.
- MeSH
- Cholesterol chemistry MeSH
- Diphenylhexatriene chemistry MeSH
- Fluorescent Dyes chemistry MeSH
- Fluorescence Polarization MeSH
- Lipid Bilayers chemistry MeSH
- Molecular Conformation MeSH
- Sphingomyelins chemistry MeSH
- Molecular Dynamics Simulation MeSH
- Transition Temperature MeSH
- Structure-Activity Relationship MeSH
- Phase Transition MeSH
- Publication type
- Journal Article MeSH
