A rapid, precise and specific hydrophilic interaction chromatography (HILIC) combined with postcolumn derivatization using o-phthaldialdehyde and fluorescence detection was developed and validated for the determination of selected pharmaceutically active thiols. The analysis was carried out on a Diol HILIC column using a mobile phase consisting of acetonitrile and solution of 10 mmol/L citric acid adjusted with 1-propylamine to pH 5.5 in ratio 75:25 (v/v) for separation of cysteine and homocysteine and in ratio 85:15 (v/v) for separation of N-acetyl-l-cysteine and captopril. The postcolumn derivatization reaction was performed at room temperature using reagent (5 mmol/L OPA in 0.05 mol/L 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid at pH 7) delivered at the flow rate of 0.3 mL/min. Fluorescence detection was carried out at excitation and emission wavelength of 345 nm and 450 nm, respectively. The effect of chromatographic conditions including acetonitrile content, salt concentration in the mobile phase and mobile phase pH on the retention of tested thiols was investigated. The postcolumn reaction conditions such as reaction temperature, derivatization reagent flow rate, o-phthaldialdehyde concentration and derivatization reagent pH were deeply studied. The developed method was validated in terms of linearity, accuracy, precision and selectivity according to the International Conference on Harmonisation guidelines. The HILIC method was successfully applied for the analysis of commercially available samples of pharmaceutically active thiols such as captopril, N-acetyl-l-cysteine (NAC) and cysteine.
- MeSH
- acetonitrily chemie MeSH
- acetylcystein analýza MeSH
- cystein analýza MeSH
- fluorescenční spektrometrie MeSH
- hydrofobní a hydrofilní interakce MeSH
- kaptopril analýza MeSH
- o-ftalaldehyd chemie MeSH
- propylaminy MeSH
- sulfhydrylové sloučeniny analýza chemie MeSH
- teplota MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
In this review article, CE methods for analysis of biologically important thiols are overviewed. The article covers the period from the previously published comprehensive review in 2004 until mid-2016, with emphasis on various detection modes, novel approaches for sample preconcentration, and applications in clinical practice. The most commonly used detection methods, such as conductometry or absorbance detection, although universally applicable and available in most commercial instruments have low sensitivity and have only limited use in thiol analysis. Amperometric and MS detection are more sensitive and have their steady place in thiol analysis, although the mainstay remains CE with LIF detection, reaching nanomolar concentration sensitivities for most of the thiols. Novel probes for CE-LIF have been developed and tested. The preconcentration approaches using modified gold nanoparticles reaching excellent sensitivities in the picomolar range and various sample stacking methods are also reviewed. Finally, significant clinical applications of the developed methods are discussed with critical insights into the future of CE analysis of thiols.
- MeSH
- elektrochemické techniky MeSH
- elektroforéza kapilární metody MeSH
- fluorescence MeSH
- fluorescenční barviva MeSH
- fluorescenční spektrometrie metody MeSH
- hmotnostní spektrometrie MeSH
- konformace proteinů MeSH
- kovové nanočástice MeSH
- lidé MeSH
- metabolomika MeSH
- oxidace-redukce MeSH
- proteiny chemie MeSH
- senzitivita a specificita MeSH
- sulfhydrylové sloučeniny analýza chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The aims of this study were to determine concentrations of total homocysteine, cysteine, cysteinylglycine and glutathione in spermatozoa, seminal fluid and blood plasma and to analyse their relationships with sperm parameters. For this reason, a new highly effective method of spermatozoa lysis was developed, using methanol, freezing and subsequent thawing in ultrasonic bath. An HPLC-FD assay was conducted on thiols concentrations in lysed spermatozoa, seminal fluid and blood plasma. Concentrations of thiols in spermatozoa were significantly lower in men with normozoospermia than in samples with pathological semen parameters. Statistical analysis found significant correlations between thiol concentrations in spermatozoa and semen parameters, while the same analysis with thiol concentrations in seminal fluid was substantially less powerful. Only cysteinylglycine concentrations in seminal fluid significantly correlated with pathological semen parameters. No significant differences or correlations were found with blood plasma concentrations.
- MeSH
- dospělí MeSH
- frakcionace buněk metody MeSH
- homocystein analýza krev MeSH
- intracelulární prostor chemie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužská infertilita krev metabolismus MeSH
- sperma chemie MeSH
- spermie chemie ultrastruktura MeSH
- sulfhydrylové sloučeniny analýza krev MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
In this work, we present a study on reusable thin metal film resistivity-based sensor for direct measurement of binding of thiol containing molecules in liquid samples. While in bulk conductors the DC current is not influenced by the surface events to a measureable degree in a thin metal layer the electrons close to the surface conduct a significant part of electricity and are influenced by the surface interactions. In this study, the thickness of the gold layer was kept below 100 nm resulting in easily measureable resistivity changes of the metal element upon a surface SH-groups binding. No further surface modifications were necessary. Thin film gold layers deposited on a glass substrate by vacuum sputtering were photolithographically structured into four sensing elements arranged in a Wheatstone bridge to compensate for resistance fluctuations due to the temperature changes. Concentrations as low 100 pM provided measureable signals. The surface after the measurement could be electrolytically regenerated for next measurements.
Separation technologies play an important role in revealing biological processes at various omic levels, in pharmacological and clinical research. In this context, CE is a strong candidate for analyses of samples with rapidly increasing complexity. Even though CE is well known for its many advantages in this regard, the sensitivity of CE analyses is insufficient for many applications. Accordingly, there are generally three main options for enhancing the sensitivity of CE analyses - using special detection techniques, using sample pre-concentration and derivatisation. Derivatisation is often the method of choice for many laboratories, since it is simple and provides several advantages such as small sample volume demand and the possibility of automation. Although it can be performed in different ways depending on where the reaction takes place, this article reviews one of the simplest and at the same time most useful approaches on-capillary derivatisation. Even if in many cases the use of on-capillary derivatisation alone is enough to improve the detection sensitivity, on other occasions it needs to be employed in combination with the other above-mentioned strategies. After a simple discussion of derivatisation in general, special attention is focused on the on-capillary approach and methodologies available for on-capillary reactant mixing. Its applications in various fields are also described.
The aim of this study was to investigate the effect of molds on levels of low molecular mass thiols in grasses. For this purpose, the three grass species Lolium perenne, Festulolium pabulare and Festulolium braunii were cultivated and sampled during four months, from June to September. The same species were also grown under controlled conditions. High-performance liquid chromatography with electrochemical detection was used for quantification of cysteine, reduced (GSH) and oxidized (GSSG) glutathione, and phytochelatins (PC2, PC3, PC4 and PC5). Data were statistically processed and analyzed. Thiols were present in all examined grass species. The effect of fungicide treatments applied under field conditions on the content of the evaluated thiols was shown to be insignificant. Species influenced (p < 0.05) PC3 and GSSG content. F. pabulare, an intergeneric hybrid of drought- and fungi-resistant Festuca arundinacea, was comparable in PC3 content with L. perenne and F. braunii under field conditions. Under controlled conditions, however, F. pabulare had higher (p < 0.05) PC3 content than did L. perenne and F. braunii. Under field conditions, differences between the evaluated species were recorded only in GSSG content, but only sampling in June was significant. F. pabulare had higher (p < 0.05) GSSG content in June than did L. perenne and F. braunii.
The oxidative effects of photodynamic therapy with 5,10,15,20-tetrakis(4-methoxyphenyl) porphyrin (TMP) and Zn-5,10,15,20-tetrakis(4-methoxyphenyl) porphyrin (ZnTMP) were evaluated in Wistar rats subcutaneously inoculated with Walker 256 carcinoma. The animals were irradiated with red light (? = 685 nm; D = 50 J/cm2; 15 min) 3 h after intra-peritoneal administration of 10 mg/kg body weight of porphyrins. The presence of free radicals in tumours after photodynamic therapy with TMP and ZnTMP revealed by chemiluminescence of luminol attained the highest level at 18 h after irradiation. Lipid peroxides measured as thiobarbituric-reactive substances and protein carbonyls, which are indices of oxidative effects produced on susceptible biomolecules, were significantly increased in tumour tissues of animals 24 h after photodynamic therapy. The levels of thiol groups and total antioxidant capacity in the tumours were decreased. The activities of antioxidant enzymes superoxide dismutase and glutathione peroxidase were also increased in tumour tissues after photodynamic therapy. Increased levels of plasma lipid peroxides as well as changes in the levels of erythrocyte antioxidant enzyme activities suggest possible systemic effects of photodynamic therapy with TMP and ZnTMP.
- MeSH
- antioxidancia analýza metabolismus MeSH
- časové faktory MeSH
- fotochemoterapie MeSH
- fotosenzibilizující látky farmakologie metabolismus terapeutické užití MeSH
- karbonylace proteinů účinky záření MeSH
- karcinom Walkerův 256 farmakoterapie metabolismus MeSH
- krysa rodu rattus MeSH
- látky reagující s kyselinou thiobarbiturovou analýza metabolismus MeSH
- lipidové peroxidy analýza krev metabolismus MeSH
- luminiscence diagnostické užití MeSH
- luminol chemie MeSH
- oxidace-redukce účinky záření MeSH
- oxidoreduktasy analýza krev metabolismus MeSH
- porfyriny farmakologie metabolismus terapeutické užití MeSH
- potkani Wistar MeSH
- reaktivní formy kyslíku metabolismus MeSH
- spotřeba kyslíku účinky léků MeSH
- sulfhydrylové sloučeniny analýza krev metabolismus MeSH
- volné radikály analýza krev metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
OBJECTIVES: The aim of this study was to assess the effect of metals, with special attention of cadmium (Cd), content of the Svitava and Svratka rivers (Czech Republic) on levels of thiol compounds such as metallothioneins (MT), glutathione (GSH/GSSG), and cysteine (Cys) concentrations in chub (Leuciscus cephalus L.) liver and their use as biomarkers. DESIGN: Levels of thiol compounds in fish liver were determined by the differential pulse voltammetry Brdicka reaction. Cadmium content in L. cephalus liver was measured by atomic absorption spectometry (AAS) with electrothermal atomization. The contents of thiols and Cd were correlated with the Cd and metals content of river bottom sediments. RESULTS: Results were inconsistent with those of experimental studies. Correlations between liver Cd content and MT, Cys, GSSG were not significant in any locality (p > 0.05). Cd liver content was negatively (p< 0.05) correlated with GSH at one (Zidlochovice) of the seven tested sites. GSH and GSSG correlated negatively in five localities (p > 0.05). Correlations between Cd in sediment and Cys and GSSG (p > 0.05), and MT was negative (p< 0.05). The relationship between sum of metals value and MT was negative (p< 0.05), similarly with GSSG (p > 0.05). CONCLUSION: Many factors influence content of MT, GSH, and GSSG, and data for Cd were equivocal. Monitoring the aquatic ecosystem to identify metal content using thiol compounds in fish liver as biomarkers is complex. More relevant data including laboratory experiments will be required to assess most of the factors influencing thiols and their relevance to Cd contamination.
- MeSH
- biologické markery analýza metabolismus MeSH
- Cyprinidae metabolismus MeSH
- cystein metabolismus MeSH
- glutathion metabolismus MeSH
- játra chemie metabolismus MeSH
- kadmium analýza metabolismus MeSH
- kovy analýza metabolismus MeSH
- řeky chemie MeSH
- spektrofotometrie atomová MeSH
- sulfhydrylové sloučeniny analýza metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
The biosynthetic pathway of the clinically important antibiotic lincomycin is not known in details. The precise knowledge of the lincomycin biosynthesis is a prerequisite for generation of improved derivatives by means of combinatorial genetics. Methods allowing determination of the key intermediates are very important tools of the pathway investigation. Two new high-performance liquid chromatography methods with fluorescence detection for determination of lincomycin precursors in fermentation broth of Streptomyces lincolnensis and its lincomycin nonproducing mutants were developed. The first one enables simultaneous analysis of methylthiolincosamide (MTL) and N-demethyllincomycin (NDL), whereas the second one is suitable for 4-propyl-L-proline (PPL) assay. Both methods are based on the pre-column derivatization: MTL and NDL with 4-chloro-7-nitrobenzofurazan; PPL with o-phthaldialdehyde. The methods were validated with lower limit of quantification values of 2.50, 3.75, and 3.75 microg ml(-1) for MTL, NDL, and PPL, respectively. The inter- and intra-day accuracies and precisions were all within 12%. Stability of oxidized and derivatized analytes was investigated.
- MeSH
- amidy analýza MeSH
- fermentace MeSH
- financování organizované MeSH
- fluorescence MeSH
- linkomycin analogy a deriváty biosyntéza MeSH
- molekulární struktura MeSH
- prolin analogy a deriváty analýza MeSH
- reprodukovatelnost výsledků MeSH
- Streptomyces metabolismus MeSH
- sulfhydrylové sloučeniny analýza MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
