In this article, optimization of BGE for simultaneous separation of inorganic ions, organic acids, and glutathione using dual C4 D-LIF detection in capillary electrophoresis is presented. The optimized BGE consisted of 30 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, 15 mM 2-amino-2-hydroxymethyl-propane-1,3-diol, and 2 mM 18-crown-6 at pH 7.2 and allowed simultaneous separation of ten inorganic anions and cations, three organic acids and glutathione in 20 min. The samples were injected hydrodynamically from both capillary ends using the double-opposite end injection principle. Sensitive detection of anions, cations, and organic acids with micromolar LODs using C4 D and simultaneously glutathione with nanomolar LODs using LIF was achieved in a single run. The developed BGE may be useful in analyses of biological samples containing analytes with differing concentrations of several orders of magnitude that is not possible with single detection mode.

• Klíčová slova
• Capillary electrophoresis, Contactless conductivity detection, Dual detection, Laser induced fluorescence detection, Saliva,
• MeSH
• dechové testy metody   MeSH
• design vybavení MeSH
• elektrická vodivost MeSH
• elektroforéza kapilární metody   MeSH
• fluorescenční spektrometrie metody   MeSH
• glutathion analýza   izolace a purifikace   MeSH
• ionty analýza   izolace a purifikace   MeSH
• kyseliny karboxylové analýza   izolace a purifikace   MeSH
• lidé MeSH
• limita detekce MeSH
• lineární modely MeSH
• reprodukovatelnost výsledků MeSH
• slzy chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glutathion MeSH
• ionty MeSH
• kyseliny karboxylové MeSH

Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole), an alkaloid isolated from Apocynaceae plants, exhibits an antitumor activity, which is exceptionally high against several specific types of tumors. Ellipticine is also interesting as an anticancer drug as it has limited side effects and lacks of hematological toxicity. Various methods to study intercalating activity of this drug have been developed. However, to our best knowledge, capillary electrophoresis (CE) as a technique combining high separation resolution with various detection options has never been used for these purposes. In this study, a novel separation method based on CE with laser-induced fluorescence (CE-LIF) detection has been developed for the determination of ellipticine and for the monitoring of ellipticine-DNA interaction. Sodium acetate (50 mM, pH 4.5) was used as a background electrolyte and LIF detection at λ(ex) = 488 nm. The limit of detection for ellipticine was determined to be 5 × 10⁻⁸ M. A total of 20% dimethyl sulfoxide was found optimal as sample solvent. Additionally, intercalation of ellipticine into the double-stranded DNA was investigated. Signal corresponding to ellipticine was decreasing and a new peak appeared and was growing. It can be concluded that CE-LIF is a method applicable to in vitro studies of ellipticine-DNA complexes.

• MeSH
• dimethylsulfoxid chemie   MeSH
• DNA krev   chemie   izolace a purifikace   metabolismus   MeSH
• elektroforéza kapilární metody   MeSH
• elipticiny chemie   metabolismus   farmakologie   MeSH
• erytrocyty chemie   MeSH
• fluorescenční spektrometrie metody   MeSH
• kur domácí MeSH
• limita detekce MeSH
• lineární modely MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dimethylsulfoxid MeSH
• DNA MeSH
• elipticiny MeSH
• ellipticine MeSH Prohlížeč

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
• Názvy látek
• sulpirid MeSH
• tiapamil-hydrochlorid MeSH

Upconversion nanoparticles (UCNPs) are an emerging class of optical materials with high potential in bioimaging due to practically no background signal and high penetration depth. Their excellent optical properties and easy surface functionalization make them perfect for conjugation with targeting ligands. In this work, capillary electrophoretic (CE) method with laser-induced fluorescence detection was used to investigate the behavior of carboxyl-silica-coated UCNPs. Folic acid, targeting folate receptor overexpressed by wide variety of cancer cells, was used for illustrative purposes and assessed by CE under optimized conditions. Peptide-mediated bioconjugation of antibodies to UCNPs was also investigated. Despite the numerous advantages of CE, this is the first time that CE was employed for characterization of UCNPs and their bioconjugates. The separation conditions were optimized including the background electrolyte concentration and pH. The optimized electrolyte was 20 mM borate buffer with pH 8.

• Klíčová slova
• Laser diode, Near-infrared excitation, Photon upconversion, Separation method,
• MeSH
• elektroforéza kapilární metody   MeSH
• fluorescenční barviva chemie   MeSH
• fluorescenční spektrometrie metody   MeSH
• kyselina listová chemie   MeSH
• limita detekce MeSH
• lineární modely MeSH
• nanokonjugáty chemie   MeSH
• protilátky chemie   MeSH
• reprodukovatelnost výsledků MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fluorescenční barviva MeSH
• kyselina listová MeSH
• nanokonjugáty MeSH
• protilátky MeSH

In this paper two fast and highly sensitive ultra-high performance liquid chromatography (UHPLC) methods for the determination of tetracycline antibiotics (oxytetracycline, tetracycline, doxycycline, demeclocycline, chlortetracycline, minocycline and degradation product epitetracycline) in surface waters have been developed using fluorescence (FL) and mass spectrometry (MS) detection. ACQUITY UPLC BEH C8 and ACQUITY CSH C18 columns were employed for FL and MS detection, respectively, both packed with 1.7μm particles. Mixed-mode separation mechanism of CSH (charged surface technology) sorbent was found particularly useful in analysis of TCs, which possess problematic amphoteric structures. The FL methodology was based on chelation of tetracyclines with calcium ions to perform on-column derivatisation. The developed methods were compared in the terms of validation parameters including linearity, sensitivity, precision and accuracy. The linearity range for FL detection was within 7ngmL(-1) to 50μgmL(-1) with method limit of detection (MLOD) as low as 0.2ngmL(-1) for most of the analytes. MS detection showed even higher sensitivity reaching MLOD of 0.003ngmL(-1), which is the highest sensitivity reported so far in analysis of TCs. Matrix matched calibration curves in the range of 0.01-50ngmL(-1) were used for quantification to compensate for matrix effects with the correlation coefficients demonstrating good linearity (0.9940-0.9999). The extraction of the antibiotics from surface waters was performed using solid phase extraction with Oasis HLB cartridges. Accuracy was expressed as recovery with values ranging from 96.52% to 127.30% and from 91.66% to 123.70% for FL and MS detection, respectively.

• MeSH
• antibakteriální látky analýza   MeSH
• chemické látky znečišťující vodu analýza   MeSH
• extrakce na pevné fázi MeSH
• fluorescenční spektrometrie MeSH
• lineární modely MeSH
• řeky chemie   MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• tetracykliny analýza   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• chemické látky znečišťující vodu MeSH
• tetracykliny MeSH

A 96-well microplate-based HPLC endpoint assay is described for the determination of NADPH-cytochrome P450 reductase (CPR) activity. Novel sampling of NADPH into microplates was optimized. Separation was performed on a Zorbax Eclipse XDB-C₁₈ analytical 4.6 × 150 mm, 5 µm column. To validate the method, recombinant human NADPH-P450 reductase and microsomes with cytochrome P450 CYP1A1 were used. The mobile phase consisted of 80% acetonitrile and 20% water at a flow-rate of 0.8 mL/min. The CPR activity was quantified using NADPH fluorescence at λ(Ex) = 340 nm and λ(Em) = 450 nm. Enzymatic activity was directly proportional to the decrease in NADPH fluorescence. This analytical process enables a highly sensitive endpoint determination for reductase activity in vitro and monitoring of the consumption of NADPH in enzymatic reactions. The method avoids the use of substrates and of organic solvents that may affect CPR and cytochrome P450 activity. In the reaction, molecular oxygen served as a proton source. The method can substitute spectrophotometric detection methods for its accuracy, high reproducibility (~100%) and sensitivity. The lower limit of detection, shown using the Agilent 1200 aparatus, is in the 250 nmol range. In addition, using this method it is possible to set up reactions in a high-throughput format.

• MeSH
• acetonitrily chemie   MeSH
• fluorescenční spektrometrie metody   MeSH
• kalibrace MeSH
• lidé MeSH
• limita detekce MeSH
• lineární modely MeSH
• NADP analýza   chemie   metabolismus   MeSH
• NADPH-cytochrom c-reduktasa metabolismus   MeSH
• rekombinantní proteiny analýza   metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetonitrile MeSH Prohlížeč
• acetonitrily MeSH
• NADP MeSH
• NADPH-cytochrom c-reduktasa MeSH
• rekombinantní proteiny MeSH

A method is described for the determination of total glutathione (TGSH) in dried blood spot (DBS) samples using high-performance liquid chromatography with fluorescence detection. Whole blood and DBS samples were obtained from a group of blood donors. After GSH reduction with dithiothreitol and protein precipitation with ethanol, the samples were derivatized with naphthalene-2,3-dicarboxaldehyde to form a very stable, highly fluorescent derivative. For the separation, a reversed phase HPLC method was used. The mixture of ethanol and deionized water (8 : 92, v/v) was used as a mobile phase. The analytical performance of this method was satisfactory: the intra- and interassay coefficients of variation were below 10%. Quantitative recoveries from spiked DBS samples were between 98.3 and 103.6%. The presented method is inexpensive and suitable for clinical testing purposes.

• MeSH
• dospělí MeSH
• glutathion krev   chemie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• limita detekce MeSH
• lineární modely MeSH
• methanol MeSH
• mladiství MeSH
• mladý dospělý MeSH
• reprodukovatelnost výsledků MeSH
• test suché kapky krve metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glutathion MeSH
• methanol MeSH

There is a growing interest in evaluating molecular markers as predictors of response to new generation of targeted cancer therapies. One of such areas is biological therapy targeting epidermal growth factor receptor gene (EGFR) in lung cancer. The testing of tumor tissue is focused on specific EGFR mutations and EGFR gene amplification, since tumors exhibiting positivity of either of the two marker types are highly sensitive towards the treatment. Although traditional methods of DNA sequencing and fluorescence in situ hybridization are still in use for the detection of EGFR mutations and gene amplification, respectively, there is a need for new dedicated techniques with the primary emphasis on simplicity, sensitivity, speed and cost effectiveness. The main purpose of this work was to integrate diverse assays for both EGFR tests onto a single platform to eliminate the need for different instruments and separate processing. We demonstrate a chip capillary electrophoresis (chipCE) application for EGFR mutation detection by a combination of fragment analysis and denaturing CE along with multiplex ligation-dependent probe amplification (MLPA) for evaluation of EGFR amplification. All separations are carried out in denaturing sieving polymer on a modified Bioanalyzer 2100 chipCE instrument running at temperatures of up to 65°C. The main strength of the resulting high-resolution chipCE application is in its simplicity, speed of analysis and minimal amount of sample required for complete testing of EGFR status. Such an approach could potentially fit medium throughput laboratories providing molecular pathology services for clinical oncologists with fast turnaround times and limited consumption of tissue material.

• MeSH
• amplifikace genu * MeSH
• časové faktory MeSH
• elektroforéza mikročipová metody   MeSH
• erbB receptory genetika   MeSH
• genetická terapie MeSH
• geny erbB-1 * MeSH
• lidé MeSH
• lineární modely MeSH
• mutace * MeSH
• nádory plic enzymologie   genetika   terapie   MeSH
• polymerázová řetězová reakce metody   MeSH
• reprodukovatelnost výsledků MeSH
• sekvenční analýza DNA metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• EGFR protein, human MeSH Prohlížeč
• erbB receptory MeSH

Epstein-Barr virus (EBV) is associated with approximately one-third of Hodgkin lymphoma (HL) cases. EBV-DNA is often present in the plasma and whole blood of EBV-associated HL patients. However, the significance of EBV-DNA monitoring is debated. In a cohort of 165 adult HL patients, EBV-DNA viral load was prospectively monitored both in the plasma and whole blood. Diagnostic tissue samples of all patients were histologically reviewed; in 72% nodular sclerosis was detected, 24% presented with mixed cellularity (MC), and 5% had other type of HL. Tissues from 150 patients were also analyzed for the presence of latent EBV infection using in situ hybridization for EBV-encoded RNA (EBER) and immunohistochemistry for latent membrane protein (LMP1). Using these methods, 29 (19%) patients were classified as EBV positive. Using real-time quantitative PCR, 22 (76%) of EBV-positive HL patients had detectable EBV-DNA in the plasma and 19 (66%) patients in whole blood prior to therapy. In the group of EBV-negative HL cases, three (2%) patients had detectable plasma EBV-DNA and 30 (25%) patients whole blood EBV-DNA before treatment. EBV-positive HL was significantly associated with EBV-DNA positivity both in the plasma and whole blood in pretreatment samples, increasing age and MC subtype. Serial analysis of plasma EBV-DNA showed that response to therapy was associated with decline in viral load. Moreover, significantly increased plasma EBV-DNA level recurred before disease relapse in one patient. Our results further suggest that the assessment of plasma EBV-DNA viral load might be of value for estimation of prognosis and follow-up of patients with EBV-positive HL.

• MeSH
• DNA virů krev   MeSH
• dospělí MeSH
• Hodgkinova nemoc krev   patologie   virologie   MeSH
• hybridizace in situ fluorescenční MeSH
• imunohistochemie MeSH
• infekce virem Epsteina-Barrové krev   komplikace   epidemiologie   virologie   MeSH
• kohortové studie MeSH
• lidé středního věku MeSH
• lidé MeSH
• logistické modely MeSH
• mladiství MeSH
• mladý dospělý MeSH
• prospektivní studie MeSH
• proteiny virové matrix krev   MeSH
• RNA virová krev   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• virová nálož MeSH
• virus Epsteinův-Barrové genetika   izolace a purifikace   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• DNA virů MeSH
• EBV-associated membrane antigen, Epstein-Barr virus MeSH Prohlížeč
• Epstein-Barr virus encoded RNA 1 MeSH Prohlížeč
• Epstein-Barr virus encoded RNA 2 MeSH Prohlížeč
• proteiny virové matrix MeSH
• RNA virová MeSH

Chromothripsis is a one-step genome-shattering catastrophe resulting from disruption of one or few chromosomes in multiple fragments and consequent random rejoining and repair. This study defines incidence of chromothripsis in 395 newly diagnosed adult acute myeloid leukemia (AML) patients from three institutions, its impact on survival and its genomic background. SNP 6.0 or CytoscanHD Array (Affymetrix®) were performed on all samples. We detected chromothripsis with a custom algorithm in 26/395 patients. Patients harboring chromothripsis had higher age (p = 0.002), ELN high risk (HR) (p < 0.001), lower white blood cell (WBC) count (p = 0.040), TP53 loss, and/or mutations (p < 0.001) while FLT3 (p = 0.025), and NPM1 (p = 0.032) mutations were mutually exclusive with chromothripsis. Chromothripsis-positive patients showed a worse overall survival (OS) (p < 0.001) compared with HR patients (p = 0.011) and a poor prognosis in a COX-HR optimal regression model. Chromothripsis presented the hallmarks of chromosome instability [i.e., TP53 alteration, 5q deletion, higher mean of copy number alteration (CNA), complex karyotype, alterations in DNA repair, and cell cycle] and focal deletions on chromosomes 4, 7, 12, 16, and 17. CBA. FISH showed that chromothripsis is associated with marker, derivative, and ring chromosomes. In conclusion, chromothripsis frequently occurs in AML (6.6%) and influences patient prognosis and disease biology.

• MeSH
• akutní myeloidní leukemie diagnóza   genetika   mortalita   terapie   MeSH
• chromothripsis * MeSH
• dospělí MeSH
• genetická predispozice k nemoci MeSH
• hybridizace in situ fluorescenční MeSH
• jednonukleotidový polymorfismus MeSH
• kruhové chromozomy MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mutace MeSH
• nádorové biomarkery MeSH
• nukleofosmin MeSH
• prognóza MeSH
• proporcionální rizikové modely MeSH
• protokoly protinádorové kombinované chemoterapie škodlivé účinky   terapeutické užití   MeSH
• pruhování chromozomů MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• výsledek terapie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nádorové biomarkery MeSH
• NPM1 protein, human MeSH Prohlížeč
• nukleofosmin MeSH