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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
- acetonitrily chemie MeSH
- benzenové deriváty chemie MeSH
- borany chemie MeSH
- chromatografie s reverzní fází MeSH
- hydrofobní a hydrofilní interakce MeSH
- kobalt chemie MeSH
- lineární modely MeSH
- oxid křemičitý chemie MeSH
- Ramanova spektroskopie MeSH
- reprodukovatelnost výsledků MeSH
- vysokoúčinná kapalinová chromatografie přístrojové vybavení metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
- chemické látky znečišťující vodu analýza MeSH
- estrogeny analýza izolace a purifikace MeSH
- extrakce na pevné fázi metody MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- kapilární elektrochromatografie metody MeSH
- lineární modely MeSH
- řeky chemie MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
- analýza potravin metody MeSH
- chromatografie s reverzní fází metody MeSH
- endokrinní disruptory analýza MeSH
- extrakce na pevné fázi metody MeSH
- limita detekce MeSH
- molekulový imprinting metody MeSH
- mykotoxiny analýza MeSH
- nesteroidní estrogeny analýza MeSH
- pivo analýza MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zearalenon analýza MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie 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
- adsorpce MeSH
- celulosa chemie MeSH
- chromatografie s reverzní fází přístrojové vybavení metody MeSH
- fenylpropionáty chemie MeSH
- pyridaziny chemie MeSH
- stereoizomerie MeSH
- vysokoúčinná kapalinová chromatografie přístrojové vybavení metody MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie 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
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
- bor chemie MeSH
- chromatografie s reverzní fází MeSH
- diamant chemie MeSH
- elektrody MeSH
- imidazoly krev chemie MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- lineární modely MeSH
- piperaziny krev chemie MeSH
- puriny krev chemie MeSH
- senzitivita a specificita MeSH
- sulfony krev chemie MeSH
- triaziny krev chemie MeSH
- vysokoúčinná kapalinová chromatografie přístrojové vybavení metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
- anthrachinony analýza chemie MeSH
- chromatografie s reverzní fází MeSH
- fermentace MeSH
- Hypocreales chemie MeSH
- lineární modely MeSH
- methanol MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cíl práce: Analýza vztahů mezi farmakokinetikou, účinkem a nežádoucími účinky metotrexátu (MTX) v iniciální fázi systémové léčby středně těžké a těžké psoriázy. Metody: Souhrnná analýza třech prospektivních otevřených randomizovaných studií zahrnula 63 nemocných s psoriázou. Koncentrace MTX v plazmě byly stanoveny HPLC a farmakokinetické parametry byly vypočítány nekompartmentovými postupy v programu Kinetica (verze 4.0) v týdnu 1, 5 a 13. Stav postižení kůže byl hodnocen pomocí PASI skóre (Psoriasis area and severity index). Data byla testována analýzou rozptylu (ANOVA) a korelace byla hodnocena pomocí Spearmanova pořadového koeficientu. Výsledky: Terapeutický účinek se rozvíjel postupně během 120 dní a byl nejmenší po týdenní dávce 7,5 mg jednorázově a největší po týdenní dávce 15 mg podané rozděleně do 3 dávek po 12 h. Farmakokinetika MTX byla lineární v dávkovém rozmezí 2,5 mg až 15 mg. Byla nalezena vysoce signifikantní a těsná negativní korelace (rs = -0.82, p < 0.0001) mezi relativními hodnotami PASI po 13 týdnech (v % počáteční hodnoty) a hodnotami plochy pod křivkou koncentrací v intervalu 0 až 8h po podání (AUC0–8h) vyšetřenými na začátku studie. Při překročení hranice AUC0–8h = 700 nmol.h/l byla četnost dosažení PASI50 (50-procentní a větší pokles PASI) 100%, zatímco pod touto hranicí 33%. Mediány výsledků hematologických (hemoglobin, počet erytrocytů, leukocytů a trombocytů) a biochemických (ALT, AST, GMT, ALP, močovina, kreatinin) vyšetření krve se v období prvních 6 měsíců od zahájení farmakoterapie nezměnily s výjimkou koncentrace homocysteinu v plazmě (zvýšení o 35 %, p<0.005). Závěr. Úspěch iniciální fáze léčby psoriázy je závislý především na dosažení dostatečných koncentrací MTX v krvi (AUC0–8h > 700 nmol.h/l). Farmakokinetika MTX je interindividuálně variabilní, ale v průběhu terapie se nemění a intraindividuální kolísání hodnot AUC0–8h je nízké. Z těchto důvodů je indikováno terapeutické monitorování a individualizace dávky MTX podle AUC0–8h.
Aim: To evaluate the relationship between pharmacokinetics (PK) and pharmacodynamics (PD, therapeutic and adverse effects) during the initial phase of psoriasis treatment with methotrexate (MTX). Methods: Analysis of the results obtained in three prospective randomized trials enrolled 63 patients with moderate-severe and severe psoriasis. In weeks 1, 5 and 13, plasma concentrations of MTX were assayed using HPLC and pharmacokinetic parameters were evaluated using standard noncompartmental methods in the software Kinetica, version 4.0. The Psoriasis Area and Severity Index (PASI) was obtained. Data were tested using analysis of variance (ANOVA) and Spearman rank correlation. Results: Therapeutic effect developed gradually over 120 days since the start of therapy. The least effective dose was 7.5 mg as a bolus, the highest effect was observed after 15 mg given in three doses at 12h intervals. MTX pharmacokinetics were linear in the range of 2.5 mg to 15 mg. PK/PD analysis revealed a highly significant inverse relationship between the PASI at week 13 (in % of the initial value) and the area under concentration-time curve AUC0–8h at week 1 (rho = -0.82, p < 0.0001. All patients with the AUC0–8h > 700 nmol.h/l attained the PASI50 (a drop in PASI ≥ 50%), while those with lower values attained it in only 33% of cases. There were no significant changes in the results of routine hematology and clinical biochemistry tests with the exception of plasma homocysteine which increased by 35 % (p<0.005). Conclusion: The results of this study suggest that the AUC0–8h > 700 nmol.h/l is associated with a high skin-clearing effect of antipsoriatic therapy with MTX. MTX pharmacokinetics is characterized by a high interindividual variability, no changes in time and low within-subject fluctuation. Therefore, therapeutic monitoring and dose individualization at the start of therapy are strongly recommended.
- MeSH
- analýza rozptylu MeSH
- aplikace orální MeSH
- farmakokinetika MeSH
- finanční podpora výzkumu jako téma MeSH
- hematologické testy metody statistika a číselné údaje MeSH
- homocystein krev MeSH
- lidé MeSH
- methotrexát aplikace a dávkování farmakologie krev MeSH
- prospektivní studie MeSH
- psoriáza diagnóza farmakoterapie patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- randomizované kontrolované studie MeSH
Direct analysis of complex samples is demonstrated by the at-line coupling of hollow fiber liquid-phase microextraction (HF-LPME) to capillary electrophoresis (CE). The hyphenation of the preparative and the analytical technique is achieved through a 3D-printed microextraction device with an HF located in a sample vial of a commercial CE instrument. The internal geometry of the device guides the CE separation capillary into the HF and the CE injection of the HF-LPME extract is performed directly from the HF lumen. The 3D-printing process ensures uniform dimensions of the devices, their constant position inside the sample vial, and excellent repeatability of the HF-LPME as well as the CE injection. The devices are cheap (∼0.01 €) and disposable, thus eliminating any possible sample-carryover, moreover, the at-line CE analysis of the extract is performed fully autonomously with no need for operator's intervention. The developed HF-LPME/CE-UV method is applied to the determination of acidic drugs in dried blood spot and wastewater samples and is characterized by excellent repeatability (RSD, 0.6-9.6%), linearity (r2, 0.9991-0.9999), enrichment (EF, 29-97), sensitivity (LOD, 0.2-3.4 μg/L), and sample throughput (7 samples/h). A further improvement of selected characteristics of the analytical method is achieved by the at-line coupling of HF-LPME to capillary isotachophoresis (ITP) with electrospray ionization-mass spectrometry (ESI-MS). The HF-LPME/ITP-ESI-MS system facilitates enhanced selectivity, matrix-free analytical signals, and up to 34-fold better sensitivity due to the use of ESI-MS detection and additional on-capillary ITP preconcentration of the HF-LPME extracts.
