Drug-protein interaction studied by technique of microdialysis combined with high performance liquid chromatography

Drug-protein binding is an important process in determining the activity and fate of a pharmaceutical agent once it has entered the body. This review examines the method of microdialysis combined with high-performance liquid chromatography (HPLC) that has been developed;by ours to study such interactions, in which the microdialysis was applied to sample the free drug in the mixed solution of drug with protein, and HPLC to quantify the concentration of free drug in the microdialysate. This technique has successfully been used for determining various types of binding interactions between the low affinity drugs, high affinity drugs and enantiomers to HSA. For the case of competitive binding of two drugs to a protein in solution, a displacement equation has been derived and examined with four nonsteroidal anti-inflammatory drugs and HSA as model drugs and protein, respectively. Microdialysis with HPLC was adopted to determine simultaneously the free solute and displacing agent in drug-protein solutions. The method is able to locate the binding site and determine affinity constants even up to 10(7) L/mol accurately.

Theoretical study of the separation mechanism of ionizable compounds in capillary electrochromatography

The migration mechanism of ionizable compounds in capillary electrochromatography (CEC) is more complicated than in high performance liquid chromatography (HPLC) due to the involvement of electrophoresis and the second chemical equilibrium. The separation mechanism of ionizable compounds in CEC has been studied theoretically. The electrochromatographic capacity factors of ions (k *) in CEC and in the pressurized CEC are derived by phenomenological approach. The influence of pH, voltage, pressure on k* is discussed. in addition, the k * of weak acid and weak base are derived based on acid-base equilibrium and the influence of pH on k * is studied theoretically.

New method for analysis of Chinese herbal complex prescription and its application

Traditional Chinese medicine (TCM) is a great treasure of China, the analysis of which is an arduous task. The viewpoint that all chemical constituents of Chinese herbal complex prescription should be analyzed as a black box is elucidated for the first time. Intelligent multi-mode multi-column chromatographic system (IMMCC) with its hybrids is the basic method and HPLC Unified Method is the breakthrough for the black box analysis. Dang-Gui-Bu-Xue-Tang was selected as a typical TCM and a systematic separation method from non-aqueous mobile phase to pure water mobile phase was put forward in order to convert unknown sample to known sample. The a, c values and UV spectra of 66 components of Astragalus, 78 components of Angelica and 71 components of Dang-Gui-Bu-Xue-Tang were obtained. Intelligent optimization and peak identification method and software for complex samples were developed and the optimum multi-step multi-binary gradient curve of mobile phase for Astragalus was ascertained. The maximum error and minimum error of predicted retention time for all components of Astragalus are 8.62% and 0.05% respectively. All components of Astragalus were compared with those of Angelica and it is found that many components of Astragalus are the same as those of Angelica, while the contents of these components are different. Many components of Dang-Gui-Bu-Xue-Tang are also the same as those of Astragalus and Angelica with different contents.

Reversed-phase high-performance liquid chromatographic investigation of urinary normal and modified nucleosides of cancer patients

Post-transcriptional modifications in RNA give rise to free modified ribonucleosides circulating in the blood stream and excreted in urine. Due to their abnormal levels in conjunction with several tumor diseases, they have been suggested as possible tumor markers. The developed RP-HPLC method has been applied to analyze the urinary nucleosides in 34 urinary samples from 15 kinds of cancer patients. The statistical analyses showed the urinary nucleoside excretion, especially modified nucleoside levels, in cancer patients were significantly higher than those in normal healthy volunteers. Factor analysis was used to classify the patients with cancer and normal healthy humans. It was found that using 15 urinary nucleoside levels or only five modified nucleoside levels as data vectors the factor analysis plot displayed two almost separate clusters representing each group. (C) 1999 Elsevier Science B.V. All rights reserved.

Chiral separation of enantiomers of amino acid derivatives by high-performance liquid chromatography on a norvancomycin-bonded chiral stationary phase

A novel norvancomycin-bonded chiral stationary phase (NVC-CSP) was synthesized by using the chiral selector of norvancomycin. The chiral separation of enantiomers of several dansyl-amino acids by high-performance liquid chromatography (HPLC) in the reversed-phase mode is described. The effects of some parameters, such as organic modifier concentration, column temperature, pH and flow rate of the mobile phase, on the retention and enantioselectivity were investigated. The study showed that ionic, as well as hydrophobic interactions were engaged between the analyte and macrocycle in this chromatographic system. Increasing pH of buffers usually improved the chiral resolution for dansyl-alpha-amino-n-butyric acid (Dns-But), dansyl-methionine (Dns-Met) and dansyl-threonine (Dns-Thr), but not for dansyl-glutamic acid (Dns-Glu) which contains two carboxylic groups in its molecular structure. The natural logarithms of selectivity factors (In alpha) of all the investigated compounds depended linearly on the reciprocal of temperature (1/T), most processes of enantioseparation were controlled enthalpically. Interestingly, the process of enantioseparation for dansyl-threonine was enthalpy-controlled at pH of 3.5, while at pH of 7.0, it was entropy-controlled according to thermodynamic parameters Delta(R,S)DeltaHdegrees and Delta(R,S)DeltaSdegrees afforded by Van't Hoff plots. In order to get baseline separation for all the solutes researched, norvancomycin was also used as a chiral mobile phase additive. In combination with the NVC-CSP remarkable increases in enanselectivity were observed for all the compounds, as the result of a "synergistic" effect. (C) 2003 Elsevier B.V. All rights reserved.

Discriminating the xanthones in an extract of Swertia franchetiana by retention parameters

The quantitative structure-retention relationship is one of the most actively studied topics in the field of chromatography. In this paper, retention parameters of components were used to discriminate the xanthones in a methanol extract of Swertia franchetiana. The extract was analysed by HPLC under two different multistage linear gradient conditions and the retention parameters calculated from these retention data. It was found that the retention parameters of xanthones are in a specific region in the plot of log k(w) vs. S and the xanthones in the extract could be distinguished from other components by this feature. Furthermore, xanthone aglycones and xanthone glucosides could also be discriminated by retention parameters. Copyright (C) 2005 John Wiley Sons, Ltd.

Quantitative and qualitative analysis of flavonoids in leaves of Adinandra nitida by high performance liquid chromatography with UV and electrospray ionization tandem mass spectrometry detection

The extract of Adinandra nitida leaves, named as Shiyacha in China, was studied by high performance liquid chromatography (HPLC)-ultraviolet detection-electrospray ionisation (ESI) tandem mass spectrometry (MS). Under the optimized condition, the analysis could be finished in 45 min on a Hypersil C18 column combined with negative ion detection using information-dependent acquisition (IDA) mode of a Q TRAP (TM) instrument. Six flavonoids were identified as epicatechin, rhoifolin, apigenin, quercitrin, camellianin A, and camellianin B among which rhoifolin was for the first time found in Shiyacha. And the fragment pathways of these flavonoids were elucidated. Furthermore, with epicatechin, rhoifolin, and apigenin as markers, the quality control method for Shiyacha and its relevant product was firstly established. Calibration linearity was good (R-2 > 0.9992) over a three to four orders of magnitude concentration range with an S/N = 3 detection limit of 2.5 ng. (c) 2004 Elsevier B.V. All rights reserved.

Analysis of the estrogenic components in kudzu root by bioassay and high performance liquid chromatography

The estrogenic activity of the Chinese herb kudzu root was investigated by a recombinant yeast screening assay (YES). Isoflavones are the main components in the plant, of which puerarin is the most abundant one. The kudzu root extract was separated into four fractions according to the polarity. The crude extract and its sub-fractions, except the water fraction, showed clear estrogenic activity and the potencies were in the range of 10(-3) to 10(-1) g/l. The ligand potency was used to compare the estrogenic activity of these fractions. The crude extract and its sub-fractions were further analyzed by high performance liquid chromatography (HPLC) to correlate the activity and the active components. Bioassay and chemical analysis showed that theoretical estrogenic activity expressed as equivalent 17 beta-estradiol concentration or the cumulative effects are comparable to that experimentally determined by YES. The results showed that the high content of isoflavones as well as the high estrogenic activity could make kudzu root extract an interesting candidate for hormone replacement therapy. (c) 2005 Elsevier Ltd. All rights reserved.

Age-dependence of urinary normal and modified nucleosides in childhood as determined by reversed-phase high-performance liquid chromatography

Modified nucleosides have been characterized as tumor markers for a number of malignant diseases. In order to use these markers in children, the age-dependence of the nucleoside levels in healthy children has to be established and taken into account in diagnostic decisions. In this study, the levels of 12 normal and modified nucleosides in urine of 166 healthy children and adolescents with an age between 1 day and 19 years are determined by reversed-phase HPLC, and age-dependent reference ranges are defined. The urinary nucleoside concentrations are related to the creatinine concentrations, which allows the use of randomly collected urine samples. All nucleoside levels in urine of children decrease with age, most pronounced during the first 4 years of life, and the age-dependence of the reference values of the individual nucleosides can be approximated by a mathematical function y = b(0) + b(1) (1/x) with the regression coefficients b(0) and b(1), the nucleoside levels y and the age x between 1 year and 19 years. In the very young children, the shifts in the nucleoside concentrations are more differentiated. Starting with low levels on the first day of life, the concentrations of all studied nucleosides rise up to an age of 1-2 months, when they reach their absolute maximum for all age periods, and then decrease. (C) 2004 Elsevier B.V. All rights reserved.

Purification and characterization of recombinant truncated human interleukin-11 expressed as fusion protein in Escherichia coli

Mature human interleukin-11 (HuIL-11) is a cytokine consisting of 178 amino acid residues that results from scission of the N-terminal signal peptide, consisting of 21 amino acid residaues, from the corresponding nascent polypeptide. A DNA fragment encoding a truncated HuIL-11 (trHuIL-11), with an additional 5 amino acid residues removed from the N-terminus, was cloned into vector pGEX-2T between the BamHI site and the EcoRI site. Upon transformation with Escherichia coli BL21, the construct over-produced a glutathione S-transferase (GST)-fused protein in a soluble form after IPTG induction. The fusion protein was initially fractionated with butyl-Sepharose 4 fast flow column and by affinity chromatography using a GSH-Sepharose 4B column. On-site enzymatic release with thrombin gave the target protein at 96% purity as judged by SDS-PAGE and HPLC. Expression of the interleukin as a GST-fused protein thus greatly improved downstream processing. Subsequent biological activity assay suggested that trHuIL-11 had similar activity profile to the naturally produced sample and may be a promising candidate for further development as biopharmaceutical.

Rapid protein identification using monolithic enzymatic microreactor and LC-ESI-MS/MS

A monolithic enzymatic microreactor was prepared in a fused-silica capillary by in situ polymerization of acrylamide, glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, followed by ammonia solution treatment, glutaraldehyde activation and trypsin modification. The choice of acrylamide as co-monomer was found useful to improve the efficiency of trypsin modification, thus, to increase the enzyme activity. The optimized microreactor offered very low back pressure, enabling the fast digestion of proteins flowing through the reactor. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at high flow rate. The digests were then characterized by CE and HPLC-MS/MS with the sequence coverage of 57.7%. The digestion efficiency was found over 230 times as high as that of the conventional method. in addition, for the first time, protein digestion carried out in a mixture of water and ACN was compared with the conventional aqueous reaction using MS/MS detection, and the former solution was found more compatible and more efficient for protein digestion.

Caratterizzazione proteomica di fluidi e tessuti in diverse condizioni fisio-patologiche

This thesis has been focused on the proteomic characterization of human saliva from donors of different ages, starting from birth up to adult age, and pediatric brain tumor tissues. The first study has been performed in order to compare the acid-insoluble fraction of saliva from preterm with at-term newborns and adults and establish if differences exist. In the second study medulloblastoma and pilocytic astrocytoma pediatric brain tumor extracts have been compared. In both studies 2- DE analysis was coupled with high resolution tandem mass spectrometry (MS/MS). The proteomic characterization of the acid-insoluble fractions of saliva from preterm newborns allowed to integrate data previously obtained on the acid-soluble fraction by HPLC-electrospray ionization (ESI)-mass spectrometry (MS), and to evidence several differences between preterm newborns, at-term newborns and adults. Spots differentially expressed between the three groups, according to image analysis of the gels, were submitted to in-gel tryptic digestion and the peptide mixture analyzed by high performance HPLC-ESI-MS/MS for their characterization. By this strategy, we identified three over-expressed proteins in atterm newborns with respect to preterm newborns and adults (BPI fold-containing family A member 1, two proteoforms of annexin A1, and keratin type 1 cytoskeletal 13), and several over-expressed proteins in adults (fatty acid-binding protein, S100A6, S100A7, two proteoforms of S100A9, several proteoforms of prolactin-inducible protein, Ig kappa chain, two proteoforms of cystatin SN, one proteoform of cystatin S and several proteoforms of α-amylase 1). Moreover, for the first time, it was possible to assign by MS/MS four spots of human saliva 2-DE, already detected by other authors, to different proteoforms of S100A9. The strategy applied used a sequential staining protocol to the 2-DE gels, first with Pro-Q Diamond, that allows specific detection of phosphoproteins, and successively with total protein SYPRO Ruby stain. In the second study, proteomic analysis of two pediatric brain tumor tissues pointed out differences between medulloblastoma, the prevalent malignant tumor in childhood, and pilocytic astrocytoma, the most common, that only rarely shows a malignant progression. Due to the limited availability of bioptic tissue, the study was performed on pooled tumor tissues, and was focused on acid-insoluble fraction to integrate the characterization performed by a group of colleagues in Rome on the acid-soluble fraction by high performance HPLC-ESI-MS/MS. The results indicated that the two tumors exhibit different proteomic profiles and evidenced interesting differential expression of several proteins. Among them, peroxiredoxin- 1, peptidyl-prolyl cis–trans isomerase A, heterogeneous nuclear ribonucleoproteins A2/B1, mitochondrial isoform of malate dehydrogenase, nucleoside diphosphate kinase A, glutathione S-transferase P and fructose bisphosphate aldolase A resulted significantly over-expressed in medulloblastoma while glial fibrillary acidic protein, serotransferrin, α crystallin B chain, ferritin light chain, annexin A5, fatty acid-binding protein (brain), sorcin and apolipoprotein A-I resulted significantly over-expressed in pilocytic astrocytoma. In conclusion, the work done allowed to evidence the usefulness of using an integrated bottom-up/top-down approach, based on 2-DE-MS analysis and high performance MS in order to obtain a complete characterization of the proteome under investigation, revealing and identifying, not only peptides and small proteins, but also proteins with higher MW, that often it is not possible to identify by using exclusively a top-down ESI-MS approach.

Analiza specjacyjna arsenu w wodzie techniką wysokosprawnej chromatografii cieczowej połączonej ze spektrometrią mas z jonizacją w indukowanej plazmie

Wydział Chemii: Pracownia Analizy Spektroskopowej Pierwiastków

Badania właściwości fotochemicznych azydonukleozydów purynowych w kontekście ich potencjalnego wykorzystania jako czynników fotozszywających w dwuniciowych układach oligonukleotydowych

Wydział Chemii: Zakład Fizyki Chemicznej

Estudo do efeito protector da espécie Cydonia oblonga Miller na danificação oxidativa em eritrócitos humanos

Monografia apresentada à Universidade Fernando Pessoa para obtenção do grau de Licenciada em Ciências Farmacêuticas.