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Serrano, Nadja Fernanda Gonzaga. "Produção de compostos antimicrobianos por Paenibacillus polymyxa RNC-D: otimização das condições de cultivo, purificação e caracterização dos bioprodutos." Universidade Federal de São Carlos, 2014. https://repositorio.ufscar.br/handle/ufscar/273.
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The increase in the production of antimicrobial metabolites by Paenibacillus polymyxa RNC-D was appraised through the study of cultivation variables. Two process variables, namely the glucose and inoculum concentrations, were evaluated in different levels (5 to 40 g/l, and 2.5% to 5.0% v/v, respectively), and their effects on biomass formation, minimal inhibitory concentration (MIC) against Escherichia coli and surface tension reduction (STR) were studied. The fermentation process was firstly carried out using non-optimized parameters, where the dependent variables biomass, MIC and STR reached the values of 0.6 g/l, 1.000,0 μg/ml and 18.4 mN/m, respectively. The optimum glucose (16 g/l) and inoculum concentrations (5.0% v/v) were defined in order to maximize the biomass formation, with low value of MIC and large STR of extract. Under these conditions, a biomass of 2.76 g/l, MIC of 15.8 μg/ml, and STR of 14.58 mN/m were predicted by the model. Data attained by experiments using optimized settings showed the following values: biomass 2.05 g/l; MIC 31.2 μg/ml; STR 10.7 mN/m. Thus, the percentage of improvement for each target response was: biomass 241.6%; MIC 96.88%; STR 41.85%. It was found that high concentrations of glucose substrate, although reflected in an increase in bacterial biomass, inhibited the microbial secondary metabolism, resulting in a low production of biomolecules associated with high values of MICs. Thus, initial concentrations of glucose and inoculum are shown as variables of strong influence in the production of antimicrobial metabolites by P. polymyxa RNC-D. Through the methods of experimental factorial design and surfaceresponse followed by graphical optimization it was possible to determine the optimum operating condition to achieve both maximum biomass and RTS as well as and lowest possible values of CIM. The validity of the proposed model was verified and confirmed. This is the first study on the optimization of culture conditions for the production of antimicrobial metabolites by P. polymyxa RNC-D, and constitutes an important step in the development of strategies to modulate the production of antimicrobial molecules by this microorganism in elevated levels. Novel antimicrobial compounds were isolated from the fermentation broth of P. polymyxa RNC-D, here named total extract (TE). It was possible to verify the presence of lipopeptide and peptide active compounds through enzymatic assays made with ET. Total extract was subjected to a two-phase system, resulting in lipopeptide extract (LPE) and aqueous fraction (AF). According to the results of bioassays, LPE has broad-spectrum activity against Gram-positive bacteria, Gram-negative bacteria and fungi. The mass spectrometry analysis of PLA revealed the existence of a novel compound that was named polycerradin. The purification of a novel antimicrobial peptide (AMP) from the AF was carried out by using chromatography. The compound was active against Gram-negative bacteria. Nterminal analysis determined the amino acid sequence, as well as MS / MS analysis confirmed the primary structure of this new compound. This research reports firstly the production of PAM PpRNCD that has an unusual amino acid in its constitution. It is an unprecedented fact considering the bacterial specie P. polymyxa. In terms of molecule size, PAM PpRNCD can be considered one of the smallest active natural peptide reported to date. It was also possible to isolate from FA the depsipeptides IL-F04a (m/z 883), LI-F04b (m/z 897), LI-F03a (m/z 947) and LI-F03b (m/z 961) previously described in the literature. The photoluminescence study of the LPE, TE, AF in both at room temperature (RT) and low temperature (T = 8K) was performed. In addition, this technique was applied to evaluate the action of the ELP on Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 29212, Shigella sonnei ATCC 1578 and Candida albicans ATCC 10231 in two different situations: (a) immediately after mixing LPE with the bacterial and fungus cell suspension, and (b) after thirty minutes. The photoluminescence emission was collected by a triple spectrometer (three diffraction gratings) T64000 model from Jobin Yvon, equipped with an optical microscope. For the detection of the radiation emitted by the sample we used a CCD camera (charge coupled device) cooled by liquid nitrogen. The slits of the spectrometer were adjusted to produce a spectral resolution of the order of 10-4 nm. The excitation source used was the line of 457 nm (violet) from an argon laser. The behaviors here observed indicate a strong potential for applications in biosensors as well as molecular markers.
Através do estudo de variáveis do cultivo pretendeu-se aumentar a produção de metabólitos antimicrobianos por Paenibacillus polymyxa RNC-D. Duas variáveis do processo - glicose e concentração de inóculo - foram avaliadas em diferentes níveis e seus efeitos na formação de biomassa, concentração inibitória mínima (CIM) contra Escherichia coli e redução na tensão superficial (RTS) foram estudados. Utilizando parâmetros não-otimizados as variáveis dependentes biomassa, CIM e RTS atingiram valores de 0,6 g/l, 1.000,0 μg/ml e 18,4 mN/m, respectivamente. As concentrações ótimas de glicose (16 g/l) e inóculo (5,0% v/v) foram definidas no sentido de maximizar a formação de biomassa e RTS do extrato, bem como diminuir o valor de CIM do extrato. Experimentalmente 2,05 g/l de biomassa; 31,2 μg/ml de CIM e 10,7 mN/m de RTS foram obtidos sob condições otimizadas. Foi constatado que altas concentrações do substrato glicose, embora refletissem em aumento de biomassa bacteriana, inibiram o metabolismo secundário microbiano, resultando em baixa produção de biomoléculas associada a altos valores de CIM. Através dos métodos de design fatorial experimental e superfície-resposta seguidos por otimização gráfica foi possível determinar a condição operacional ótima das concentrações iniciais de glicose e inóculo, as quais se demonstraram como variáveis de grande influência na produção de metabólitos antimicrobianos por P. polymyxa RNC-D. O extrato total (ET), proveniente do caldo de fermentação de P. polymyxa RNC-D, foi utilizado para pesquisa e isolamento de novos compostos antimicrobianos. Através de ensaios enzimáticos feitos com ET foi possível verificar a natureza lipopeptídica e peptídica dos compostos antimicrobianos. O ET foi submetido a um sistema de duas fases, separandose então em extrato lipopeptídico (ELP) e fração aquosa (FA). Resultados de bioensaios revelaram que o ELP apresenta amplo espectro de atividade contra bactérias Grampositivas, Gram-negativas e fungo. A análise por espectrometria de massas de ELP revelou a presença de um composto peptídico inédito o qual foi denominado polycerradin. A partir da fração aquosa (FA) foi possível a purificação de um novo peptídeo antimicrobiano (PAM) através de etapas cromatográficas. A bioatividade do composto foi avaliada e confirmada frente às bactérias Gram-negativas. A determinação da sequência de aminoácidos foi realizada por análise do N-terminal, e a confirmação da estrutura primária deste novo composto foi feita por MS/MS. O presente estudo relata pela primeira vez a produção do PAM PpRNCD que possui um aminoácido não usual em sua constituição, relato primeiramente aqui descrito considerando-se a espécie bacteriana P. polymyxa. Em termos de tamanho de molécula, pode-se considerar que o PAM PpRNCD é um dos menores peptídeos naturais ativos relatados até o momento. Utilizando-se a FA também foi possível o isolamento dos depsipeptídeos LI-F04a (m/z 883), LI-F04b (m/z 897), LI-F03a (m/z 947) e LI-F03b (m/z 961) previamente descritos na literatura. O estudo da fotoluminescência do ELP, do ET e da FA foi realizado tanto em temperatura ambiente (RT) quanto em baixa temperatura (T=8K). Também se estudou, através desta técnica, a ação do ELP sobre as bactérias Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 29212, Shigella sonnei ATCC 1578 e fungo Candida albicans ATCC 10231 em duas situações: (a) imediatamente após a mistura do ELP com a suspensão celular bacteriana, e (b) trinta minutos após a mistura. Detectou-se emissão fotoluminescente por ELP, ET e FA, e sinais de Raman a λ 699 nm (FA a baixa temperatura). Decorridos 30 min da mistura do ELP com as suspensões celulares microbianas houve alteração na emissão fotoluminescente, sendo que alguns sinais foram suprimidos (λ 470, 480 e 700 nm para S. sonnei, por exemplo). Isto evidencia a potencial aplicação destas frações (ELP, ET e FA) para a fabricação de sensores, detectores e marcadores moleculares.
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Zhang, Qunying. "Characterization of receptors for Escherichia coli 987P using competitive binding assays, thin-layer chromatography and gel filtration chromatography." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0023/MQ51825.pdf.
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Embree, Leanne. "Development of a high-performance liquid chromatographic assay for human chorionic gonadotropin as an alternative to the official United States pharmacopeial animal assay." Thesis, University of British Columbia, 1985. http://hdl.handle.net/2429/24637.
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Human chorionic gonadotropin (HCG), a glycoprotein hormone with two nonidentical subunits, is produced by chorionic tissue in pregnant women and by neoplastic tissue containing chorionic elements. It is used in the treatment of male hypogonadism and female sub-fertility.
Quantitation of HCG is used to monitor therapy, diagnose various disease states and diagnose and monitor pregnancy. Low levels of HCG in the early and late stages of pregnancy and in various disease states has prompted the development of extremely sensitive assay procedures. Clinically, radioimmunoassay methods are most frequently used due to their precision, sensitivity and cost. However, problems with specificity have been noted.
Commercial preparations of HCG must meet the standards outlined in the United States Pharmacopeia (USP). The assay procedure involves a rat uterine weight bioassay. This protocol is lengthy to perform (5 days), requires the sacrifice of a large number of animals (minimum of 60 female rats per assay) and may need to be repeated if the results do not meet the statistical requirements of the assay. Due to the use of animals and the animal care facilities required, this is an expensive assay. In addition, the bioassay is not specific for HCG. Therefore, this thesis reports the analysis of two commercial preparations of HCG, as well as USP Reference Standard HCG and commercially available purified intact HCG and purified individual subunits. Various HPLC assay procedures were evaluated to determine if HPLC would be a viable alternative to the official USP bioassay.
Size exclusion HPLC, using one Protein Pak 125 sw column and two Protein Pak 300 sw columns individually and in various combinations, was used to assess all the samples of HCG. Attempts to increase resolution of HCG from interfering components found in these preparations included using both 208 nm and 278 nm for ultraviolet detection, evaluation of 32 buffers as mobile phases with the Protein Pak 300 sw column, fluorescamine derivatization of HCG followed by fluorescence detection, connection of two size exclusion columns in series, and recycling on a Protein Pak 300 sw column. Further attempts to isolate HCG from its protein contaminants involved using ion exchange HPLC with a Protein Pak DEAE 5 pw column with 20 different buffers as mobile phases as well as reversed-phase HPLC with an Ultrasphere ODS column. The greatest resolution was obtained with one Protein Pak 300 sw column with a phosphate buffer (0.15 M, pH 7.0) for the mobile phase and ultraviolet detection.
Latex agglutination inhibition slide tests and electrophoresis techniques were used to evaluate commercial samples of HCG and chromatographic peak eluates.
Commercial HCG samples appear to contain the individual subunits of HCG and intact HCG along with impurities. The USP Reference Standard HCG contains intact HCG but also contains other ultraviolet absorbing components that were partially separated by HPLC. Electrophoresis also indicated that this HCG sample contained impurities. In addition, the purified intact HCG and purified subunit samples contained impurities, as shown by HPLC.
The size exclusion HPLC assay developed using one Protein Pak 300 sw column was unable to resolve intact HCG from the beta-subunit. This assay would be useful for a qualitative assay for purity of HCG preparations. However, at present, HPLC is not a viable alternative to the USP bioassay.Pharmaceutical Sciences, Faculty of
Graduate
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Fillmann, Gilberto. "Appraisal and validation of rapid, integrated chemical and biological assays of environmental quality." Thesis, University of Plymouth, 2001. http://hdl.handle.net/10026.1/2372.
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To assess the significance of pollutants released into the environment it is necessary to determine both the extent of contamination and the biological effects they give rise to. This research is based on a tiered system, which commences with conventional analytical chemistry (gas chromatography), followed by the development, evaluation and application of rapid and simple immunochemical techniques and, finally, the integration of chemical and biological markers to assess pollution. GC-ECD/FID/MS have been used to investigate the status of chemical contamination of the Black Sea by organochlorine residues, hydrocarbons and faecal sterols. Useful information is provided and problems with e.g. HCHs and sewage contamination are highlighted. Contamination by DDTs, PCBs, "total" hydrocarbons and PAHs is also reported. Next, these techniques are used to develop rapid screening methods. Four distinct applications of immunochemical techniques are presented. Initially, the BTEX RaPDD Assay® ELISA is evaluated to detect semi-volatile hydrocarbons in contaminated groundwater. Although overestimating concentrations when compared to GC-FID/PID, results are well correlated. Secondly, the effectiveness o f the BTEX and c-PAH RaPID Assay® to detect hydrocarbons in sediments is tested. Once again, good agreement with GC-FID/MS confirms the ELISA to be a useful screening protocol to focus more expensive high-resolution analytical techniques. The adaptability and applicability of an ELISA (PCB RaPID Assay®) method in measuring "total" PCB levels in mussel tissue is demonstrated. An underestimation of concentrations, despite of covariability between ELISA and cGC-ECD, is discussed. Next, ELISA (RaPID Assay®) and fluorometry were successfully applied to quantify PAH metabolites in crab urine as a measure of exposure. HPLC analyses indicated that conjugate PAH metabolites were dominant in urine of crabs exposed to pyrene. Differences could also be identified between crabs taken from clean and contaminated sites. Finally, an integration of chemical and biological techniques is used to investigate contamination and effects in mussels within a pollution gradient. Results indicate a correlation between micronucleus formation, heart rate and PCB and PAH level.
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Ahmed, Naila Masud. "Chromatographic assay of advanced glycation endproducts and application to the study of human disease." Thesis, University of Essex, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364507.
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Bhattacharjee, Rathindra Chandra. "Development of a sensitive and stereoselective high performance liquid chromatographic assay method for propafenone enantiomers in human plasma." Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/27800.
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Propafenone is a new class 1C antiarrhythmic agent with additional calcium antagonistic and beta-blocking activities. Clinically it is effective in the treatment of supraventricular and ventricular tachycardia, atrial and ventricular fibrillation, ventricular premature contractions and for the management of Wolf-Parkinson-White syndrome. In North America it is still an investigational drug.
Propafenone is a chiral drug and is used clinically in the racemic form. The enantiomers of numerous chiral drugs have been shown to differ in their disposition kinetics in the body due to their stereoselective pharmacokinetics and/or pharmacodynamic properties. Two enantiomers are thus often considered as two different entities. The relative antiarrhythmic activities of individual enantiomers of propafenone have not been studied, nor their pharmacokinetic parameters have been elucidated. In order to study the possible enantioselective role of propafenone in the body, a stereoselective assay method would be required. The present study describes the development of a sensitive and stereoselective chromatographic assay method for the simultaneous determination of the two enantiomers of propafenone in human plasma.
Attempts for direct separation of the enantiomers of propafenone included several GLC and HPLC chiral stationary phases. The chiral stationary phases were a Chirasil-Valʳ GLC stationary phase, a Pirkle 2,4 dinitro-(D)-phenylglycine HPLC stationary phase and a β-cyclodextrin HPLC stationary phase. Unfortunately, these did not resolve the enantiomers of propafenone.
Formation of the diastereomers with R(+)-⍺-methyl benzyl isocyanate and racemic propafenone were partially resolved on a reverse phase HPLC using a 5 u, 25 x 0.45 cm i.d. ODS column and methanol/water (70:30) as the mobile phase. However, due to the long retention time (42 min), incomplete resolution (RS=1.15) and poor sensitivity for detection (500 ng of each enantiomer injected) this method was not deemed suitable for the pharmacokinetic studies planned, since the therapeutic plasma concentration range of propafenone is 64-1044 ng/mL.
The second chiral derivatizing reagent, 2,3,4,6-tetra-0-acetyl-β-D-glucopyranosylisothiocyanate (GITC), was synthesized in our laboratory. This reagent gave better resolution of the enantiomers (RS=1.4) within 15 minutes with enhanced sensitivity for detection (150 ng of each enantiomer injected).
To further optimize the limit of detection for future pharmacokinetic studies of propafenone, R(-)-1 -(naphthyl) ethylisocyanate, a chiral derivatizing agent, was employed. This reagent reacted with racemic propafenone and permitted the resolution of both enantiomers within 24 minutes (R5=l.25) and the minimum level of detection was 100 ng (at the detector) for each enantiomer of propafenone. Using this method, linearity was established over the concentration range, 125-1000 ng for each enantiomer (injected) with a coefficient of determination (r²) of greater than 0.99.
Reproducibility and precision of this assay method was obtained with an average coefficient of variability of 4.5% for the R(-) enantiomer and 7.2% for S(+) enantiomer at concentrations of 125-1000 ng/mL. Below the lower quantity, the NEIC-propafenone reaction virtually stopped at the conditions set for derivatization. A similar lack of reactivity at low concentrations was also observed with the GITC-propafenone reaction.
The absence of an autocatalysing effect of propafenone at lower nanogram levels, as well as two possible conformational forms of propafenone were also investigated. The existence of two conformational isomers of propafenone, due to intramolecular hydrogen bonding in aprotic solvents, was chromatographically verified. In addition, chromatographic separation of all the proposed conformers was obtained, indicating that enantiomeric separation and quantitation of propafenone enantiomers as their urea derivatives is substantially hindered.
To eliminate hydrogen bonding interactions, the carbonyl group of propafenone was blocked with dansylhydrazine and subsequently derivatized with the chiral R(-)NEIC reagent. The HPLC resolution (RS=1.35) of this dual derivative was better than that using the R(-) NEIC reagent alone, and the minimum level of detection was 2.5 ng for each enantiomer. Unfortunately, this procedure still did not provide adequate assay precision and accuracy at the lower levels required for single dose pharmacokinetic studies.Pharmaceutical Sciences, Faculty of
Graduate
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CHILAKALA, SUJATHA. "DEVELOPMENT OF LIQUID CHROMATOGRAPHY-MASS SPECTROMETRIC ASSAYS AND SAMPLE PREPARATION METHODS FOR THE BIOLOGICAL SAMPLE ANALYSIS." Cleveland State University / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=csu1512927043412916.
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Lövgren, Ulf. "Enzyme immunoassay in combination with liquid chromatography for sensitive and selective determination of drugs in biosamples." Lund : Dept. of Analytical Chemistry, Lund University, 1997. http://books.google.com/books?id=Ju1qAAAAMAAJ.
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Kirk, Loren Madden, and Stacy D. Brown. "Beyond-Use Date Determination of Buprenorphine Buccal Solution Using a Stability-Indicating High-Performance Liquid Chromatographic Assay." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/5305.
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Objectives
The objectives of this study included developing and validating a stability-indicating high-performance liquid chromatographic (HPLC) method with ultraviolet (UV) detection for the determination of buprenorphine in a buccal solution for veterinary use, and applying that method to determine the stability of a 3 mg/ml buprenorphine preparation in room temperature and refrigerated storage conditions. This preparation, intended for buccal administration in feline patients, plays an important role in pain management in cats.
Methods
A stability-indicating HPLC method was developed and validated for system suitability, accuracy, repeatability, intermediate precision, specificity, linearity and robustness based on US Pharmacopeia (USP) General Chapter. The method was then applied to the study of potency changes over 90 days in a buccal buprenorphine solution stored at two temperatures.
Results
All HPLC-UV method data met acceptable criteria for the quantification of buprenorphine in a buccal solution formulation. The buprenorphine concentrations found in each stability sample remained within the 90–110% of label claim throughout the 90 days of study. All stability test bottles of the buprenorphine buccal solution retained their original appearance. For the room temperature bottles, some white particulate matter was noted in the threads of the container bottles starting at day 21. The pH of the preparations during the course of the study was in the range of 3.57–4.06 and 4.01–4.16 for the room temperature and refrigerated samples, respectively.
Conclusions and Relevance
Pharmacists have compounded a concentrated 3 mg/ml buccal solution to use easily in the home care or outpatient setting for treatment of feline pain. Prior to this investigation, pharmacists empirically assigned beyond-use dates to this formulation based on standards in USP General ChapterPharmaceutical Compounding – Nonsterile Preparations. This study of a 3 mg/ml buprenorphine buccal solution indicates stability through 90 days.
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Liu, Tina. "Development and validation of an HPLC assay for simethicone in pharmaceutical formulations." Master's thesis, Department of Pharmacy, 2001. http://hdl.handle.net/2123/12307.
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Lovering, A. M. "The development and use of high performance liquid chromatography (HPLC) : Methods in the rapid identification and characterisation of aminoglycoside-acetylating and -phosphorylating enzymes." Thesis, University of the West of England, Bristol, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.371406.
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Gaillard, Isabelle. "Mise en œuvre de la chromatographie liquide haute performance dans le cadre du suivi hors ligne et en ligne des protéines de cultures d'hybridomes : caracterisation de la méthode chromatographique et comparaison avec le test ELISA." Vandoeuvre-les-Nancy, INPL, 1993. http://www.theses.fr/1993INPL022N.
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Depuis la mise au point des techniques de formation des lignées d'hybridomes, des efforts importants sont fournis pour améliorer la productivité des anticorps monoclonaux et pour mieux comprendre le métabolisme cellulaire. Les méthodes analytiques jouent un rôle important dans le contrôle des procédés en permettant la détermination quantitative et qualitative de plusieurs paramètres et variables. L'objectif général de cette étude consiste à contribuer à l'amélioration de la maitrise des bioprocédés en proposant une méthode analytique capable de quantifier les protéines présentes dans les cultures d'hybridomes. Afin d'atteindre cet objectif, les potentialités de l'HPLC ont été testées. Les protéines considérées dans cette étude sont d'une part, les composes protéiques propres au milieu de culture et d'autre part, les anticorps monoclonaux secrétés par les cellules. La première partie de cette étude présente l'optimisation des conditions opératoires de la méthode chromatographique. Les deux modes chromatographiques mis à profit sont l'HPLC d'affinité et la RP-HPLC. Une étude comparative des performances de la méthode par HPLC et du test ELISA est exposée dans une seconde partie. La troisième partie concerne l'application de la méthode chromatographique aux suivis des protéines de cultures cellulaires d'hybridomes. Enfin, la réalisation couplage automatise réacteur-échantillonneur-HPLC et son application à la détermination en ligne de la concentration des IgG d'une culture discontinue, constituent la dernière partie de ce travail
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Baldassarre, Phyllis Ann. "Comparison of high-performance liquid chromatography (HPLC) and microbiological cylinder plate assays for the determination of erythromycin in uncoated entericoated pellets /." Staten Island, N.Y. : [s.n.], 1990. http://library.wagner.edu/theses/nursing/1990/thesis_nur_1990_balda_compa.pdf.
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Bedard, Pierre R. "Ion interaction liquid chromatography : energetics, mechanism and gradient design considerations for the assay of serum thyroid hormones." Thesis, McGill University, 1985. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=72024.
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The competition between two molecules of similar polarity for adsorption sites on the stationary phase is discussed in light of rapid kinetics of adsorption and desorption, and of the effects of temperature, acetonitrile, surfactant (cyclohexylaminopropane sulfonic acid, CAPS) and salt concentrations on the retention of the thyroid hormones (3,5-diiodothyronine, T2; 3,3',5-triiodothyronine, T3 and thyroxine, T4). A three parameter equation relates the surfactant concentration and ionic strength to the retention of the hormones and is analyzed in terms of the Stern-Gouy-Chapman theory. A second order polynomial describes the temperature dependency and permits the evaluation of the enthalpy, entropy and heat capacity, demonstrating a reduction in the molecular motion of the analyte with increasing surfactant and acetonitrile concentrations. The equation parameters for linear or non-linear equations, using data sets with or without homogeneous variances, are evaluated using a Simplex optimization procedure that uses one of two proposed optimization criteria. The construction and operation of a computer based gradient programmer for HPLC is described. A surfactant mediated gradient elution with electrochemical detection is examined for the analysis of serum thyroid hormones.
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Suen, Kin-wah, and 孫建華. "Validation of a HPLC assay for porphobilinogen synthase in human erythrocytes for use in the clinical laboratory." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B29624897.
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(Uncorrected OCR)
Abstract
Porphobilinogen (PBG) synthase condenses two molecules of
aminolaevulinic acid (ALA) to form PBG in heme biosynthesis. The enzyme
activity is sensitive to inhibition by heavy metals such as lead. It can act as a biological indicator of chronic lead POis~r\g to identify the risk group,
especially in children, so that early treatment can be given to prevent possible
permanent damages. A reversed-phase ion-pair HPLC analytical method for
the assay of the PBG synthase activity based on detection of PBG production
has been validated. A Hypersil CN column (150 x 4.6 mm; 5 urn) was
employed together with a mixture of acetonitrile-40 mM phosphate buffer at pH
2.4 with 5 mM 1-heptanesulphonic acid (8:92, v/v). UV detection was
performed at 240 nm. PBG was eluted as a spectrally pure peak resolved from
its impurities in the methanol-inhibited enzyme reaction. The method was
sensitive with a limit of quantitation of 2 ~M. The within-run and between-run
precisions were 8.2% and 13.8% respectively. The recovery was 93.4 �7.1%
(n=6). The preliminary reference range of the PBG synthase activities in the
local pediatric population were from 21.5 to 26.3 ~mol/L RBC/min. Bland and Altman statistical analysis showed that the HPLC assay and the colorimetric assay could not be used interchangeably. The HPLC assay was an alternative way to assess the PBG synthase activities in the human erythrocyte samples.
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Medical Sciences
Master
Master of Medical Sciences
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Abi, Aad Elizabeth. "Impact de la Détente Instantanée Contrôlée (DIC) sur l’extraction des molécules bioactives de rhizomes de Rheum ribes L." Electronic Thesis or Diss., La Rochelle, 2023. http://www.theses.fr/2023LAROS008.
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Rheum ribes L. (la rhubarbe) est l'une des plantes médicinales méditerranéennes les plus importantes. La préparation des décoctions aqueuses des rhizomes de cette plante est devenue une pratique courante chez les patients souffrant de diverses maladies. La Détente Instantanée Contrôlée (DIC) est un traitement à haute température et à courte durée (HTST high-temperature/short-time) suivi d'une chute brutale de pression vers le vide (Pression absolue de 5 kPa environ). Le traitement DIC induit l’expansion et réduit la tortuosité de la matrice traitée, et assure ainsi une meilleure diffusivité des solvants d’extraction et par la suite une plus grande disponibilité des molécules actives. D’une part, nos travaux de recherche ont visé l’identification de l’impact du traitement par DIC des rhizomes d’une espèce libanaise de R. ribes L. Les évaluations ont été basées sur les teneurs en polyphénols totaux (TPC) et en flavonoïdes (TFC) ainsi que sur les pouvoirs antioxydants des extraits aqueux de rhizomes traités. Les résultats ont montré qu’une corrélation négative existe entre la pression de vapeur saturée appliquée et les valeurs de TPC, TFC et les activités antioxydantes des échantillons traités. Cependant, il n'y avait pas d'influence significative du temps de traitement par DIC sur les variables réponses. A ces deux facteurs opératoires de la DIC, pression et temps de traitement, s’ajoute la teneur en eau (W) des rhizomes. Les résultats ont montré qu’une augmentation ne provoque pas des meilleurs rendements en composés phénoliques. D’autre part, ce travail inclut également l’étude de l’impact du traitement par DIC sur les activités biologiques (effets antibactériens et antiprolifératifs) des extraits de rhizomes. Les résultats ont mis en évidence que les extraits aqueux de rhizomes traités par DIC présentent, aux concentrations testées, de très faibles pouvoirs antibactériens sur E. coli et S. aureus, avec des résultats légèrement supérieurs au niveau de l’inhibition de la croissance de S. aureus. En ce qui concerne le test de viabilité des cellules cancéreuses du mélanome, les résultats ont montré que le traitement par DIC a un impact positif sur le pouvoir antiprolifératif des échantillons traités en comparaison avec une matrice non-traitée par DIC et extraite par voie aqueuse. En particulier, deux échantillons parmi ceux testés, ont enregistré un pouvoir antiprolifératif de 60%. Les dosages par HPLC et UHPLC ont révélé la présence de trois anthraquinones (l’émodine, le chrysophanol et le physcion) dans les extraits de rhizomes ainsi qu’une vingtaine de composés de la famille des flavonoïdes. Cependant, les quantités d’anthraquinones détectés ne sont pas significativement plus élevées dans les extraits traités par DIC que dans ceux non-traités. Finalement, il a été constaté que la teneur phénolique totale d'un échantillon aqueux traité avec les paramètres optimaux de la DIC est égale à celle d'un point hydroalcoolique non traité. Des expérimentations supplémentaires seront nécessaires pour la validation de ce résultatRheum ribes L. (rhubarb) is one of the most important Mediterranean medicinal plants. Preparing its rhizomes’ aqueous decoctions has become common among patients suffering from various diseases. Instant Controlled Pressure Drop (DIC) is a High-Temperature Short-Time treatment (HTST) followed by an abrupt pressure drop towards the vacuum (approximately 5 kPa). This technology increases the expansion and reduces the tortuosity of the treated matrix to ensure better diffusivity of the extraction solvents and greater availability of the active molecules. On the one hand, our research aimed at the impact of DIC treatment on the rhizomes of a Lebanese species of R. ribes L. The evaluations were based on the contents of total phenolics (TPC) and flavonoids (TFC) as well as the antioxidant capacities of aqueous extracts of the treated rhizomes. The results showed a negative correlation between the saturated steam pressure applied and the values of TPC, TFC, and the antioxidant activities of the samples. However, DIC treatment time had no significant influence on the response variables. In addition to these two DIC operating factors (pressure and treatment time), the effect of the rhizomes water content (W) was also studied. The results showed that the increase of “W” does not lead to better yields of phenolic compounds. On the other hand, this study presents the impact of DIC treatment on the biological activities (antibacterial and antiproliferative effects) of rhizome extracts. The results revealed that the aqueous extracts of DIC-treated rhizomes show weak antibacterial powers on E. coli and S. aureus, with slightly superior results in inhibiting the growth of S. aureus. Regarding the viability test of melanoma cancer cells, the results showed that the DIC treatment positively impacts the antiproliferative power of the treated samples compared with the untreated point (aqueous MP). In particular, two samples among those tested recorded an antiproliferative power of 60%. The HPLC and UHPLC assays revealed the presence of three anthraquinones (emodin, chrysophanol, and physcion) in the rhizome extracts and about twenty compounds from the flavonoid family. However, the amounts of anthraquinones detected are not significantly higher in the extracts treated with DIC than in the untreated ones. Finally, it was found that the total phenolic content of an aqueous sample treated with the optimal DIC parameters is equal to that of an untreated hydroalcoholic sample. Further experiments will be necessary to validate this result
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Holman, Miranda K., Dorcas Frempong, Stacy Brown, Steven Dinh, and Ashana Puri. "Development and Validation of a Liquid Chromatography-Mass Spectrometry Based Analytical Assay for Determination of Cromolyn Sodium in Skin Permeation Studies." Digital Commons @ East Tennessee State University, 2021. https://dc.etsu.edu/asrf/2021/presentations/45.
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Cromolyn sodium (CS) is a mast cell stabilizer which has been used to treat systemic mastocytosis, allergic- and exercise-induced asthma, and allergic reactions induced by atopic dermatitis. Presently, CS is administered orally and intranasally, and with a short half-life and poor absorption, 4 to 8 doses are required daily for treatment. Developing a transdermal product for CS would eliminate such drawbacks that lead to inconsistent patient dosing and provide sustained therapeutic effects when administered via skin. Our long-term goal is to determine the feasibility of delivering CS through skin. However, a prerequisite for evaluating the performance of any transdermal system is to have a sensitive analytical method that can selectively detect and quantify the drug without any interference from compounds that may leach from skin during permeation studies. Therefore, our preliminary goal was to develop and validate such a method that can be employed for transdermal studies. The optimized liquid chromatography-mass spectrometry (LC-MS) method utilized a chromatographic separation which involved an isocratic mobile phase (10mM ammonium bicarbonate, pH 8.0, 90% and acetonitrile, 10%) at a flow rate of 0.2500 mL/min. Detection involved direct MS/MS channels with m/z 467.0255 (precursor) and m/z 379.0517 (fragment) with argon as the collision gas. CS calibrants were prepared in phosphate-buffered saline (PBS), pH 7.4, for validation (0.1, 0.25, 0.5, 0.75, 1, and 2.5 μg/mL). To ensure no skin interference, dermatomed porcine ear skin was minced, placed in PBS, and shaken for 15 hours to extract any possible interfering components. The extract was filtered and analyzed with the optimized LC-MS conditions. Calibrants were also analyzed over 3 days with each day examining 6 injections (20 μL) of each sample. Peak areas determined by LC-MS were used to construct calibration curves for CS and to calculate % error and % RSD to elucidate accuracy and precision of the method. Results showed CS retention time to be around 4.4 minutes with no interfering peak from skin extract, and linearity was observed between 0.1-2.5 μg/mL. Inter- and intra-day accuracy and precision of the method were within the acceptable limit of ±20% at the lower limit of quantitation and ±15% at other concentrations. Future studies will involve using the validated method for quantification of CS in skin permeation studies to investigate our long-term goal.
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Jones, David. "Synthesis, fractionation, characterisation and toxicity of naphthenic acids from complex mixtures." Thesis, University of Plymouth, 2013. http://hdl.handle.net/10026.1/1612.
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Amongst the polar organic compounds occurring in unrefined and refined crude oils and the associated polluted production waters, complex mixtures of acids, known historically as naphthenic acids (NAs), have achieved prominence. This is particularly because NAs have been designated a toxicant class of concern in the oil sands process-affected water (OSPW) that has accumulated in vast quantities following exploitation of the oil sands of Northern Alberta, Canada in recent years. However, though there have been calls for NAs to be added to pollutant inventories, at the initiation of the current study, little knowledge existed of the exact composition of refined or unrefined NAs. The overall aim of the current study was therefore to identify individual NAs in refined (commercial) and unrefined (e.g. oil sands process-derived) complex mixtures of acids and then to assess the toxicity of any identified NAs. Individual NAs were tentatively identified by interpretation of the electron ionisation mass spectra of methyl ester derivatives, following comprehensive multidimensional gas chromatography-mass spectrometry (GCxGC-MS). Reference acids were then either purchased, or more commonly, where they were not commercially-available, synthesised, mainly by micro-hydrogenation methods, for co-chromatography and comparison of mass spectra of methyl esters with those of unknowns. The synthetic NAs, purified to >97% were then subjected to toxicological assessments using the Microtox™ assay. In all, 34 compounds were obtained pure enough for testing. Microtox results revealed that the toxicity endpoint (50% Inhibition Concentration, IC50) was between 0.004 and 0.7 mM. Exponential and other correlations were noted between carbon number and toxicity in several of the structural groups of acids assayed, which may be beneficial for predictions of toxicity of non-synthesised acids. Although n-hexanoic acid (IC50 0.7 mM) had the lowest toxicity, adamantane-type acids were the least toxic as a group overall. Conversely, the decahydronaphthalene (decalin)-type acids had the largest range of toxicities (IC50 0.004 to 0.3 mM) and the most toxic acid assayed was 3-decalin-1-yl-propanoic acid. According to USEPA guidelines many individual acids can be said to show low to medium toxicity. Since the acids in commercial and unrefined NAs occur in complex mixtures, an attempt was also made to assess mixture toxicity. Mixtures of individual structural groups of acids (e.g. acyclic isoprenoid acids, n-acids) and a mixture of all 34 acids were assessed. Apart from the adamantane sub-group of acids, all of the mixtures showed toxicities lower than the sum of the parts when calculated using equations for Concentration Addition and Model Deviation Ratios (simply the predicted IC50/Observed IC50). A hypothesis that achievement of a critical micelle concentration is required to produce toxicity was proposed to explain the lower than expected results. Some of the mass spectra of NA present in the commercial and unrefined mixtures were inconsistent with those of any of the alicyclic acids synthesised or purchased. These were hypothesised to be aromatic acids. Fractionation experiments of the NA mixtures using silver ion thin layer chromatography and solid phase extraction (Ag+TLC and Ag+SPE) were carried out in order to provide further evidence for aromatic acids. Ag+TLC allowed separation of a methylated NA mixture from OSPW into three distinct fractions; Ag+SPE resulted in eleven fractions, through the use of a wider range of solvents and differential solvent ratios. Analysis of the fractions by GC-MS revealed that each fraction was largely still made up of unresolved acids (as esters), although one or two fractions revealed some resolved acids. Use of averaged mass spectra and mass chromatography on each fraction revealed further resolved chromatographic peaks and associated interpretable mass spectra. Each of eight of the eleven sub-fractions were examined by GC-MS, in some cases by GCxGC-MS, and all by infrared spectroscopy, ultraviolet visible spectrophotometry and elemental analysis. A number of structures were proposed for the aromatic acids, including those with sulphur-containing moieties. It was noted that far from being minor components, aromatic acids comprised ca.25-40% of the OSPW acid extracts.
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Shah, Heta N. "INVESTIGATION OF PHENYLEPHRINE SULFATION AND INHIBITION USING A NOVEL HILIC ASSAY METHOD." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3952.
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Phenylephrine (PE) is the most commonly used over-the-counter nasal decongestant. The problem associated with phenylephrine is that it undergoes extensive first pass metabolism in the intestinal gut wall leading to its poor and variable oral bioavailability.
This research project aims at developing strategies in order to increase the oral bioavailability of PE by co-administration of GRAS compounds. A HILIC assay method was developed to detect the parent drug, phenylephrine (PE) and its sulfate metabolite (PES).The enzyme kinetic studies were done with phenolic dietary or GRAS compounds using LS180 human intestinal cell model, recombinant SULT enzymes and human intestinal cytosol (HIC). From the screening studies done, one inhibitor was selected in order to study the mechanism of inhibition. In conclusion the studies done in vitro provided a basis in order to predict in vivo intrinsic clearance through the sulfation pathway.
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Liu, Xiangli [Verfasser], Alfred Akademischer Betreuer] Fahr, Rolf [Akademischer Betreuer] Schubert, and Gert [Akademischer Betreuer] [Fricker. "High-Throughput Chromatographic Approaches to Assess Drug Partition into Biomembranes / Xiangli Liu. Gutachter: Alfred Fahr ; Rolf Schubert ; Gert Fricker." Jena : Thüringer Universitäts- und Landesbibliothek Jena, 2011. http://d-nb.info/1017078939/34.
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Kovach, Jessica Lynn. "USING ANALYTICAL METHODOLOGIES TO ASSESS THE ORGANOLEPTIC CHARACTER OF CITRUS ESSENTIAL OIL." Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/590546.
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ChemistryPh.D.
Essential oils are natural products used to flavor food and beverages. With the increase in nutrition conscious consumers, manufacturers of food additives and food products are faced with the challenge of making healthy alternatives. In particular, food products going to market with label claims stating reductions in sugar and salt, organic certified, organic compliant, and all natural; moreover the ingredients used in flavors must meet these label claims as well. More often than not, the challenge in using ingredients that follow these requirements is the pricing, the sourcing and the variability among those sources. Variability is common in the ingredients coming from nature such as fruits and plants because the area of cultivation can vary by the soil at the sight of planting and/or the climate in the region. Pricing is also problematic in naturally grown ingredients because it is a matter of supply and demand. Stock could be depleted from natural disasters, disease carrying pest(s), pests that consume the crop, and/or other causes for scarce supply of crop(s). Essential oils are natural byproducts of fruit, peels, and leaves from plants that contribute to flavor formulae for a large variety of food products. Because the essential oils are a crop based commodity, every variety has inherent differences based on the growing conditions and their ripening stages [1]. Nevertheless, each type of oil has marker chemicals that make up the majority of its composition; these marker chemicals have the tendency to degrade over time based on their interaction with light, oxygen exposure, and temperature. For companies that manufacture flavorings, understanding the variability among sources of essential oils as well as the possible degradants of essential oils is valuable information to obtain because it is possible the variants and degradants will negatively impact the flavor profile. Flavor is without question the most important attribute of the food we consume and by default stability of said flavor(s) need to be understood [30]. The content in this dissertation involves the stability analysis of a common essential oil, Oil Mandarin Italian Select, from Citrus Reticulata Blanco. It has known off notes that form from unknown causes. Most common is the plastic note that has formed in carbonated products like soda. Studying this particular essential oil in various conditions is intended to shed light on what those degradants are and under which conditions they form to give mandarin oil an off-note when applied to high acid and carbonated beverage applications. Once the note is reproduced, a correlation between analytical data and sensory interpretation of the oil will be developed. Mandarin essential oil being in the Citrus genus is traditionally analyzed via gas chromatography (GC) because of the high quantities of volatile constituents that give an oil high aroma activity. The volatile fraction of mandarin oil to be studied includes stability of methyl-N-methylanthranilate (MNMA), a major component giving mandarin its distinct grapey character, as well as gamma terpinene, thymol, sinensal, alpha pinene, beta pinene, myrcene, para cymene, alpha terpineol, and beta caryophyllene. Each of these ten compounds contributes to the unique flavor profile of mandarin oils when compared to orange and tangerine essential oils [1]. It was the common knowledge that para cymene can be perceived as rancid in aroma and the many interconversions the terpenes make that cause para cymene formation in Citrus oils, which made monitoring the changes of this chemical in the three stability environments crucial. Attention is being paid to para-cymene, as a specific marker of degradation in Citrus. The data obtained from the applied stability studies were challenging to understand as the marker chemicals are volatile and sensitive to chemical change. In this work the chemical changes and trends were analyzed under various storage conditions. Significant statistical analyses were employed to help define criteria of usability. The analyses were required because of natural variants and apparent inconsistencies of the data. Dixon Q Test and the Z Test were applied to determine outliers. Additionally, the Bland Altman method was applied to compare storage conditions and to determine if this statistical approach could be used to define significant changes in the marker chemical stability. The Bland Altman plots suggest that each component met the statistical limits of agreement, meaning the samplings were not significantly changing, statistically speaking. A final approach to assess the analytical data of the mandarin oil for significant change was the mass balance of each marker chemical from week 0 to week 24. Instrumental fluctuations have an acceptable range of +/- 20% in the industry; hence, a significant change criterion for a chemical in the mass balance must be one that exceeded +/- 20%. Unlike classical statistic methods, the mass balance was indicative that significant change had occurred to the compounds in the three studies. Upon sensory analysis of the oil samples, display of plastic note, oxidation, and overall loss of characteristic mandarin notes, the mass balance was found to correlate best to the significant change detected by sensory evaluation of the oil samplings. Due to the inadequate number of validated methods on Citrus essential oil research and the absence of large groupings of terpenes validated in a unified methodology, reconciliation of mass balance is an underutilized method of assessment in the literature. As a final assessment of the GC method validated, a product containing the selected mandarin oil was analyzed to evaluate the ability of the method to separate the oil components within a significantly more complicated matrix than the initial samples. The method was successful though not all marker chemicals were detected due to their low formulation concentration being below the level of detection of the method. This should not be seen as a failure of the method. For the major components of the essential oil studied, the method was quantitatively successful, meeting industry requirements.
Temple University--Theses
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Harun, Norlida. "Application of molecularly imprinted solid phase extraction, enzyme-linked immunosorbent assay and liquid chromatography tandem mass spectrometry to forensic toxicology." Thesis, University of Glasgow, 2010. http://theses.gla.ac.uk/1992/.
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The rapid growth of ketamine and amphetamine misuse worldwide has led to the development of methods for the detection and analysis of ketamine and amphetamines in biological specimens. Most methods previously developed in forensic toxicology for the detection of ketamine and amphetamines used GC-MS. The present work developed alternative methods based on LC-MS/MS. Ketamine was chosen as the drug of interest because there are no data currently available on the extent of ketamine abuse in Malaysia even though a large amount of ketamine has been seized by the Malaysian Royal Police, while amphetamines are the most widely abused synthetic drugs in South East Asia including Malaysia. The study addressed some of the challenges facing the forensic toxicologist, such as the need to use new technology (LC-MS/MS) and improve sensitivity and selectivity in forensic toxicology analysis through efficient sample preparation techniques. The general requirements of method validation, including as the parameters linearity, limit of detection (LOD) and Lower Limit of Quantification (LLOQ), recovery, precision and matrix effects were observed. Three main techniques were used in the study: enzyme-linked immunosorbent assay (ELISA), liquid chromatography tandem mass spectrometry (LC-MS/MS) and molecularly imprinted polymer solid phase extraction (MISPE). MISPE is a new extraction technique in forensic toxicology applied to biological samples. Initially work was carried out on the optimization, development and validation of the Neogen® ELISA for screening ketamine and norketamine in urine. The Neogen® ketamine ELISA kit was found to be adequately sensitive and precise for ketamine screening at a cut-off concentration of 25 ng/mL. The ELISA test was shown to be highly specific to ketamine and demonstrated minimal (2.1%) cross-reactivity to its main metabolite norketamine compared to ketamine. Subsequently, an LC–MS/MS confirmation method for ketamine and norketamine in urine samples was developed and validated with application of the method to urine samples from chronic ketamine users in Malaysia. The method demonstrated good linearity, LOD, LOQ, accuracy and precision and had acceptable matrix effects. The efficiency of ELISA as a screening method at cut-off of 25 ng/ml and LC-MS/MS as a confirmation method at 2 ng/ml was evaluated. These methods complemented each other and both ELISA and LC-MS methods were 100% sensitive and specific with no false positive results for ketamine and norketamine in urine samples. The results demonstrated that a combination of these two methods can be reliably used for routine screening and confirmation of ketamine and norketamine in urine specimens. Preliminary data from this study provided information on the concentrations of ketamine and norketamine typically found in urine samples collected from individuals frequenting pubs in Malaysia. The main work in this thesis involved molecularly imprinted polymer materials which were used as sorbents in solid phase extraction (MISPE). Ketamine was used as a model substance for novel in-house synthesised MIPs as no anti-ketamine MIP have previously been reported and because the ketamine structure is suitable for the synthesis of molecularly imprinted polymers. The study was intended to improve the selectivity and sensitivity of the extraction method (MISPE) prior to LC-MS/MS analysis. Evaluation of polymer imprinting was carried out using HPLC-UV. MIP extraction and LC-MS/MS analysis were applied to the determination of ketamine and norketamine in hair samples and compared with a conventional SPE-based method. MISPE extraction was selective and sensitive with fewer matrix effects than the conventional SPE method and could also be applied to norketamine, the principal metabolite of ketamine, due to the group-selective binding nature of the MIP, but not to structurally dissimilar analytes such as PCP and tiletamine. MISPE was superior to conventional SPE for trace detection of ketamine and norketamine in hair, in terms of improved sensitivity, lower limits of detection and reduced matrix effects. In addition, the commercial product Amphetamine SupelMIPTM was evaluated for identification of amphetamines in post mortem blood coupled with LC-MS/MS analysis. This work assessed whether the MIP, sold by the manufacturer for the extraction of amphetamines in urine, could also be used for whole blood. The results demonstrated that the MIP can be used successfully for the determination of amphetamines in post mortem blood. The recoveries of five amphetamines were lower than with a comparable GC-MS method but the LODs and LLOQs of the LC-MS/MS method were better and suitable for detection of low levels amphetamines in post mortem blood. Further optimisation is needed to develop an improved protein precipitation method prior to MISPE. Liquid Chromatography Electro-Spray Ionization Mass Spectrometry (LC–ESI-MS) was used with the MISPE and SPE methods for detection and quantification of ketamine, norketamine and amphetamines in urine, whole blood and hair samples. LC-ESI-MS was found to be easy to use and could detect lower concentrations of drugs and gave reproducible results for all the methods developed in this thesis.
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Miller, Eleanor Isabel. "The detection of drugs of abuse in biological matrices using enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry." Thesis, University of Glasgow, 2007. http://theses.gla.ac.uk/1516/.
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The aim of this study was to investigate the potential use of ELISA and LC-MS-MS in combination and as individual techniques, for the detection of drugs of abuse in biological matrices. Overall the LC-MS-MS method showed good correlation results for opiates compared to the GC-MS method. 6-MAM was however detected in more root segments and segments excluding roots by LC-MS-MS. Morphine was detected in a greater number of root segments by LC-MS-MS compared to GC-MS. However, morphine was detected in a greater number of segments excluding roots by GC-MS. Codeine and dihydrocodeine were also detected in a greater number of root segments and segments excluding roots by GC-MS. The cocaine results showed excellent qualitative correlation between the LC-MS-MS and GC-MS methods for cocaine and benzoylecgonine. The GC-MS method did not however extract greater concentrations of cocaine and its metabolites compared to LC-MS-MS due to the higher recovery of the drug group specific GC-MS method. Cocaethylene and EME were detected in some samples by LC-MS-MS method for opiates and cocaine and its metabolites compared to the GC-MS method; there may be some cases where the GC-MS method would detect the analytes where the LC-MS-MS method would not. This has been demonstrated in 3 samples for morphine and in 6 samples for codeine. The LC-MS-MS method analysed for and detected amphetamines in samples that were not tested for amphetamines by GC-MS. In one sample that was tested by both methods, amphetamine was detected in the root sample by LC-MS-MS where GC-MS failed to detect it. Also a greater concentration of amphetamine was extracted using the LC-MS-MS method in the segment without roots. The LC-MS-MS method was useful for the analysis of 17 drugs of abuse in post-mortem hair samples in forensic toxicology cases. Using this method, it is possible to obtain maximum information from one hair sample which is extremely useful when the sample weight is limited. The ability of the LC-MS-MS method to extract and analyse a greater number of drug groups from one hair sample highlights the advantages of using this method over GC-MS which targets individual drug groups and requires splitting of the sample. This method is particularly applicable for implementation in the forensic toxicology laboratory at the University of Glasgow where currently GC-MS methods that target individual drug groups are used for routine hair screening and confirmation.
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Klassen, Cherie Leanne. "The development and application of an electron-capture gas chromatographic assay procedure for the simultaneous determination of sertraline and N-desmethylsertraline in biological samples." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ40071.pdf.
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Wang, Ding. "Application of mass spectrometry in enzyme deficiency assay for newborn screening purpose /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/11557.
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Ho, Crystal. "The study of organofluorine analysis applied to totaloxidizable precursor (TOP) assay to understand perandpolyfluoroalkyl substances (PFASs)." Thesis, Örebro universitet, Institutionen för naturvetenskap och teknik, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-69083.
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Per- and polyfluoroalkyl substances (PFASs) are synthetic chemicals which are used in a wide range of applications such as aqueous film forming foam (AFFF) for firefighting, paint, coating and cosmetics. For almost a decade, PFASs have received worldwide attention because of the ubiquitous detection of PFAS in the environment and their bioaccumulative, persistent and give toxic effects on animals and humans. A number of study have shown many unknown organofluorine present in environmental samples. The total oxidizable precursor (TOP) method is an oxidation method to convert any precursor compounds of PFASs into measurable perfluorinated carboxylic acids (PFCAs) or sulfonic acids (PFSAs). The aims of this project are to evaluate if the use of total oxidizable precursor (TOP) assay may help improve the mass balance analysis between the quantifiable PFASs and extractable organofluorine in samples. In this study, three water samples: landfill leachate water, contaminated groundwater and adiluted Sthamex AFFF with tap water were undergone TOP assay to reveal the present of any unidentified precursor compounds. To obtain the extractable organofluorine and quantifiable PFAS levels in the samples, combustion ion chromatography (CIC) and liquid chromatography – tandem mass spectrometry (LC-MS/MS) were used, respectively. Results suggested the presence of unidentified precursor compounds in the samples with the aid of TOP assay reaction. Although after the oxidative conversion, a large portion of unidentified PFASs compounds were still present in the samples. The quantifiable PFASs in the samples were up to 7 % of the EOF; in other word, there were still around 93 % of unidentified compounds present in the samples. Further development of analytical method for characterization or identification of these unknown PFAS compounds is needed.
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Summers, Jay W. "Feathers as bioindicators of PCB exposure in clapper rails /." View online, 2009. http://repository.eiu.edu/theses/docs/32211131565121.pdf.
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Embree, Leanne. "Development of a sensitive, quantitative high-performance liquid chromatographic assay for the measurement of digoxin in patient groups with high levels of digoxin-like immunoreactive substances." Thesis, University of British Columbia, 1989. http://hdl.handle.net/2429/29093.
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Digoxin is the most commonly used digitalis glycoside for the treatment of congestive heart failure and certain disturbances of cardiac rhythm. The low therapeutic index observed for digoxin and the clinical significance of digoxin therapy have necessitated the development of sensitive analytical methods for the quantitation of digoxin in biological samples. Digoxin may be analysed by several methods including immunoassays, chromatographic procedures and various biological and chemical methods.
Immunoassays, both radioimmunoassay (RIA) and fluorescence polarization immunoassay (FPIA) procedures, are used in the clinical laboratory because of their speed, precision, sensitivity and relatively low cost. However, reaction of the digoxin antibodies used in the immunoassay methods with digoxin metabolites, endogenous compounds such as digoxin-like immunoreactive substances (DLIS), and other drugs that may be co-administered with digoxin continues to be a major problem.
The lack of specificity of the immunoassay methods for digoxin has led to difficulties in interpretation of assay values. Attempts to compensate for this lack of specificity have included the use of chromatographic systems as elaborate sample handling methods prior to immunoassay. However, since an immunoassay was used for detection of digoxin in these techniques, the specificity may still be quest ionable.
A sensitive and specific assay for digoxin using physico-chemical methods for measurement is therefore needed. A method was developed using pre-column
derivatization of digoxin and its metabolites with 3,5-dinitrobenzoyl chloride followed by HPLC analysis with electrochemical detection. A maximum sensitivity of 0.883 ng of 3,5-dinitrobenzoyl digoxin (0.394 ng digoxin) was observed using dual electrode detection in the redox mode. Although resolution between derivatized digoxin and its metabolites was obtained, the low yield of the digoxin derivative and the formation of metabolites when small (ng) samples were derivatized made this method unsuitable for evaluating patient samples.
A high-performance liquid chromatographic (HPLC) assay using post-column derivatization of digoxin, which separated digoxin from its metabolites and some commonly coadministered drugs, was developed. Post-column (PC) derivatization of digoxin with concentrated hydrochloric acid and dehydroascorbic acid, followed by fluorescence detection, allowed for quantitation within the therapeutic range of digoxin.
Steroids which have been reported to cross-react with digoxin antisera were assayed using the HPLC-PC method developed in this study. The steroid samples either did not elute from the HPLC system or did not produce a fluorescent product under these conditions.
Serum samples from digitalized patients were evaluated using both the HPLC-PC and the FPIA methods. When compared to the HPLC procedure, the FPIA assay results gave, on average, higher digoxin levels. This may have been due to the inclusion of digoxin metabolites or endogenous compounds with the FPIA assay.
Serum samples from undigitalized patient groups where high DLIS levels have been reported were also evaluated. These included umbilical cord blood samples and samples from hypertensive patients, renal failure patients and hepatic failure patients. Comparison of the HPLC-PC and FPIA methods demonstrated that the HPLC-PC assay gave fewer false positive results than the FPIA.
The HPLC-PC assay developed for analysis of digoxin was unaffected by the presence of digoxin metabolites, numerous steroids, co-administered drugs and endogenous compounds, most of which have been reported to give false positive results with the FPIA.Pharmaceutical Sciences, Faculty of
Graduate
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Yalo, Masande Nicholas. "An investigation of the natural products composition of Porphyra capensis (a red seaweed)." University of the Western Cape, 2017. http://hdl.handle.net/11394/6354.
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Magister Scientiae - MSc (Chemistry)Plants have been widely used in traditional medicine for a number of ailments, among which may be included infectious diseases such as colds, influenza, chicken pox, TB, etc. as well as lifestyle diseases such as diabetes and cancer. Seaweeds have also been shown to contribute to the maintenance of health through their nutritional and medicinal properties and recently, a great deal of interest has developed towards the isolation of bioactive compounds from marine sources due to their numerous health benefits. Furthermore, marine algae are valuable sources of structurally diverse metabolites with scientifically proven therapeutic claims. Chemical constituents of red seaweed, Porphyra capensis was investigated in this present study along with subsequent brine shrimp lethality assay analysis of the crude extracts. The compounds isolated from the plant were from the hexane (6) and butanol (2) extracts. These compounds were all isolated and purified by various chromatographic techniques, namely silica gel chromatography, Sephadex LH-20 gel as well as C18 reversed phase silica gel. The structures of the isolated compounds were analysed and characterised by NMR, GC-MS, ESI MS and FTIR spectroscopy. Eight compounds were isolated and identified as phytol, desmosterol, 9-eicosenoic acid, 5,8,11,14,17-eicosapentanoic acid, palmitic acid, methyl (E)-hexadec-9-enoate, glycerol and compound 1 (novel compound). All the compounds were isolated from Porphyra capensis for the first time. The hexane, butanol and methanol extracts were found to be non-toxic with the brine shrimp test LC50 value at least two times greater than ?g/ml.
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Cockcroft, Jennifer Jean. "The development, implementation and validation of a plasma-based high performance liquid chromatographic assay for Isoniazid and N-Acetylisoniazid: an acetylator status population study at Brewelskloof Hospital." Master's thesis, University of Cape Town, 2001. http://hdl.handle.net/11427/26764.
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A novel high performance liquid chromatographic assay has been developed for the simultaneous determination of isoniazid and N-acetylisoniazid in plasma. Solid phase extraction involving C18 columns is used to extract the drug and the metabolite from 0.5 ml plasma. The analyte peaks are resolved using a CB Spherisorb analytical column and ultraviolet detection at 270 nm. The assay is specific to the compounds, with consistent recovery of greater than 75% for isoniazid and over 90% for N-acetylisoniazid. The limits of detection in plasma are 300 ng/ml and 150 ng/ml for isoniazid and N-acetylisoniazid respectively. Linearity was conserved down to these concentrations. This assay was used to generate pharmacokinetic data on 114 tuberculosis patients recruited for this study at Brewelskloof hospital, Worcester, South Africa. Using these data, various markers were investigated for the determination of acetylator phenotype, namely isoniazid half-life, isoniazid plasma level at three hours, and the ratio of metabolite to drug at three hours. The ratio of metabolite to drug at three hours proved to be the most reliable method for phenotype classification, this being confirmed during the genotypic portion of the study. Trimodality was evident, although the nondiscrete separation of intermediate and rapid acetylators made this tentative. The mean values of area under the concentration-time curve for each acetylator type were found to be significantly different, with rapid acetylators being potentially compromised in terms of exposure to isoniazid (slow 32.39 mg. l⁻¹.hr, intermediate 21.25 mg. l⁻¹.hr and rapid 16.04 mg. l⁻¹.hr). Other pharmacokinetic parameters were bimodally distributed, homozygous and heterozygous rapid acetylators forming a single acetylator group. Codominance of the rapid and slow alleles was confirmed, the estimation of a mean intermediate elimination rate constant being within 7% of the observed mean. The correlation of genotype to phenotype was found to be 88.2% and the allelic distribution was determined to be acceptable using the Hardy Weinberg equation. The incidence of raised liver enzyme levels was low in the study population with no relation to acetylator phenotype. Age and weight gain after two months of daily therapy did not correlate with phenotype. The slow acetylator population comprised of a greater proportion of men, while women exhibited twice the number of rapid acetylators. No patient factors could be implicated in the apparent discordance of phenotype with genotype, and this suggests that there may be new allelic variants in this population. This report provides validation and proves the usefulness of a novel HPLC plasma-based assay for determining isoniazid and N-acetylisoniazid levels in patients with tuberculosis.
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El-Khoury, Joe M. "Chronic Kidney Disease: Vitamin D Treatment Regimens and Novel Assay Development for Kidney and Cardiovascular Function Biomarkers." Cleveland State University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=csu1343914060.
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Seno, Hiroshi, Osamu Suzuki, Akira Ishii, Kei Zaitsu, Hideki Hattori, Tadashi Ogawa, and Masae Iwai. "SIMPLE AND RAPID ASSAY METHOD FOR SIMULTANEOUS QUANTIFICATION OF URINARY NICOTINE AND COTININE USING MICRO-EXTRACTION BY PACKED SORBENT AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY." Nagoya University School of Medicine, 2013. http://hdl.handle.net/2237/18475.
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Schirmacher, Anastasiya. "Modification of transmembrane peptides to probe SNARE-induced membrane fusion and cross-presentation of membrane-buried epitopes." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://hdl.handle.net/21.11130/00-1735-0000-0005-1576-F.
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GRANDI, F. DE. "DETERMINATION OF LUTEINIZING HORMONE (LH) AND FOLLICLE STIMULATING HORMONE (FSH) IN BOVINE PLASMA: DEVELOPMENT AND VALIDATION OF SPECIES-SPECIFIC MONOCLONAL ANTIBODY-BASED ENZYME-LINKED IMMUNOSORBENT ASSAYS (ELISA)." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/246436.
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Maximizing livestock reproductive potential is of primary importance in business farming, and it’s the reason for the increasing interest in the development of innovative bio-techniques addressed to the improvement of in vivo and in vitro farm animal fertility. A better knowledge of gonadotropin (i.e.: luteinizing hormone (LH) and follicle stimulating hormone (FSH)) plasma patterns can have a positive impact on animal reproductive efficiency, for example by helping detection of dysfunction of the pituitary-ovarian axis, diagnosis of reproductive disorders, monitoring of antifertility programs or superovulatory responses for embryo transfer.
Gonadotropins in bovine plasma are currently measured only in a limited number of specialized laboratories. The first reason is that immunoassay for detecting bovine LH (bLH) and bovine FSH (bLH) are not easily accessible, being all in-house developed methods relying on custom reagents. Second, FSH and LH are species-specific (both amino acid and oligosaccharides differs among species), limiting the use for such assays to homologous reagents. Finally, hormone standards as well as anti-bFSH and anti-bLH antibodies are difficult to produce.
The aim of the present study was therefore to develop and validate two novel species-specific immunoassay for measuring bLH or bFSH in bovine plasma, based on reagents (antibody and reference standard) produced in our laboratory. Specifically, the objectives were: (1) to produce a panel of anti-bLH and anti-bFSH mAbs by immunizing with standard hormones obtained from the USDA; (2) to select mAbs suitable for immuno-affinity chromatography purification of bLH and bFSH from pituitary glands; (3) to combine the mAbs and the purified gonadotropins to develop two enzyme-linked immunosorbent assay (ELISA) for bLH and bFSH detection.
Several anti-bFSH and anti-bLH mAbs were produced and characterized. On the basis of their affinity and specificity we selected: two mAbs recognizing non-overlapping epitopes on the β-subunit of the bLH molecule; one mAbs binding to an epitope localized on the β-subunit of the bFSH molecule; and one mAb binding to the pituitary glycoprotein common α-subunit.
An anti-bLH β-subunit and an anti-bFSH β-subunit mAb were used to develop immunoaffinity chromatography protocols for the one-step extraction of bLH and bFSH from pituitary glands. The methods resulted efficient, and ensure the supply of substantial amounts of highly purified biologically active bLH and bFSH. The purity of the hormones was assessed by SDS-electrophoresis, isoelectric focusing, western blotting, amino acid composition, amino acid sequence, nanoLC-ESI-MS/MS. The biological activity was measured by in vitro specific bioassays based on cell lines expressing LH and FSH receptors.
The purified gonadotropins and the anti-bFSH and anti-bLH mAbs were combined to develop two ELISAs to detect plasma bLH and bFSH in cattle. These sandwich ELISAs with biotin-avidin amplification showed a good sensitivity, ranging from 0.05 to 2.5 ng/ml for bLH and from 0.25 to 10 ng/ml for bFSH. Cross-reactivity, recovery and reproducibility tests confirmed the accuracy and precision of the assays to measure gonadotropins in bovine plasma without any prior treatment of samples. The analytical specificity was finally validated in vivo by measuring plasma bLH and bFSH patterns after provocative test with gonadotropin releasing hormone (GnRH) in heifers.
In conclusion, the ELISA developed satisfy all the criteria required to investigate LH and FSH secretory patterns in the bovine species. The fact that both assays are based on mAbs, together with the easiness of the one-step production of reference bLH and bFSH ensure long-term continuity in large-scale measurements of LH and FSH. These methods allow the rapid, inexpensive and quantitative measurement of the hormone and are therefore valuable tools to further our knowledge on the reproductive physiology of the bovine species.
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Moysan-Le, Meur Annie. "Caracterisation de dosage des produits de photoaddition de psoralene dans l'adn "in vitro" et dans l'adn cellulaire." Paris 6, 1987. http://www.theses.fr/1987PA066021.
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Cazedey, Edith Cristina Laignier [UNESP]. "Análise químico-farmacêutica de cloridrato de ciprofloxacino em solução oftálmica." Universidade Estadual Paulista (UNESP), 2009. http://hdl.handle.net/11449/91687.
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O cloridrato de ciprofloxacino, um antibacteriano quinolônico, apresenta amplo espectro de ação e é eficaz in vitro contra praticamente todos os patógenos Gram-negativos, incluindo Pseudomonas aeruginosa. Mostra-se também eficaz contra microrganismos Gram-positivos, como estafilococos e estreptococos. A importância de desenvolver e validar métodos analíticos para este fármaco é justificada por seu potencial terapêutico, grande emprego em terapias microbianas e baixo custo, assim como pelo conhecimento de que a baixa qualidade dos produtos anti-infecciosos está relacionada ao desenvolvimento de cepas resistentes, como consequência da administração de doses subterapêuticas. Por tal razão, é de enorme importância o desenvolvimento de métodos analíticos eficazes e confiáveis para o controle de qualidade dos medicamentos comercializados. Neste trabalho foram desenvolvidos métodos de análise para o cloridrato de ciprofloxacino em solução oftálmica. Os métodos desenvolvidos e validados foram: (i) doseamento microbiológico, método turbidimétrico na faixa de concentração de 14,0 a 56,0 μg/mL, utilizando Staphylococcus epidermidis ATCC 12228 IAL 2150, com exatidão de 99,71% e teor de 102,27%; (ii) método espectrofotométrico na região do UV a 275 nm com faixa de concentração de 2,0 a 7,0 μg/mL, utilizando água como solvente, com exatidão de 101,51% e teor de 99,79%; (iii) método espectrofotométrico derivativo na região do visível a 386,4 nm, na primeira derivada, com faixa de concentração de 50,0 a 100,0 μg/mL, utilizando cloreto férrico 1,0% como reagente, com exatidão de 99,83% e teor de 106,72%; (iv) método por cromatografia líquida de alta eficiência com detector UV a 275 nm, com fase móvel composta por ácido acético 2,5% v/v, metanol e acetonitrila (70:15:15, v/v/v) e faixa de concentração de 1,0 a 6,0 μg/mL, exatidão...
Ciprofloxacin hydrochloride, a quinolone antibiotic, presents a wider spectrum of activity and is effective against practically all Gram-negative pathogens, including Pseudomonas aeruginosa. It is potent against Grampositive microorganisms, as Staphylococcus and Streptococcus. Analytical methods for quantitative determination of ciprofloxacin hydrochloride is important due to its therapeutic potential, wide use in antimicrobial therapy and low cost. Moreover, it is known that the poor quality antibiotic product is direct related development of resistant strains, as consequence of subtherapeutic doses administration. Thus, it is important to develop efficient analytical methods for quality control commercialized products. In this work, analytical methods for determination of ciprofloxacin hydrochloride were validated: (i) microbiological assay, turbidimetric method at concentration range 14.0 to 56.0 μg/mL, using Staphylococcus epidermidis ATCC 12228 IAL 2150 as indicator microorganism, accuracy 99.71% and quantitation of 102.27; (ii) UV spectrophotometry at 275 nm with concentration range of 2.0 to 7.0 μg/mL, using water as solvent, with accuracy of 101.51% and quantitation of 99.79%; (iii) Derivative visible spectrophotometric method at 386.4 nm, in first derivate, with concentration range of 50.0 a 100.0 μg/mL, using 1.0% ferric chloride as reagent, with accuracy of 99.83% and quantitation of 106.72%; (iv) HPLC method with UV detector at 275 nm using 2.5 M acetic acid (v/v), methanol and acetonitrile (70: 15: 15, v/v/v) as mobile phase and concentration range of 1.0 to 6.0 μg/mL, accuracy of 100.11%, quantitation 103.25% and mean retention time of 2.6 minutes; (v) Indirect titrimetric method using bromate/bromide solution in acid medium as reagent in concentration range of 1.0 to 11.0 mg/mL, with accuracy of 100.28% and quantitation of 98.97%.
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Cazedey, Edith Cristina Laignier. "Análise químico-farmacêutica de cloridrato de ciprofloxacino em solução oftálmica /." Araraquara : [s.n.], 2009. http://hdl.handle.net/11449/91687.
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Orientador: Hérida Regina Nunes SalgadoBanca: Magali Benjamin de Araújo
Banca: Hérida Regina Nunes Salgado
Banca: Greici Cristina Gomes Tozo
Resumo: O cloridrato de ciprofloxacino, um antibacteriano quinolônico, apresenta amplo espectro de ação e é eficaz in vitro contra praticamente todos os patógenos Gram-negativos, incluindo Pseudomonas aeruginosa. Mostra-se também eficaz contra microrganismos Gram-positivos, como estafilococos e estreptococos. A importância de desenvolver e validar métodos analíticos para este fármaco é justificada por seu potencial terapêutico, grande emprego em terapias microbianas e baixo custo, assim como pelo conhecimento de que a baixa qualidade dos produtos anti-infecciosos está relacionada ao desenvolvimento de cepas resistentes, como consequência da administração de doses subterapêuticas. Por tal razão, é de enorme importância o desenvolvimento de métodos analíticos eficazes e confiáveis para o controle de qualidade dos medicamentos comercializados. Neste trabalho foram desenvolvidos métodos de análise para o cloridrato de ciprofloxacino em solução oftálmica. Os métodos desenvolvidos e validados foram: (i) doseamento microbiológico, método turbidimétrico na faixa de concentração de 14,0 a 56,0 μg/mL, utilizando Staphylococcus epidermidis ATCC 12228 IAL 2150, com exatidão de 99,71% e teor de 102,27%; (ii) método espectrofotométrico na região do UV a 275 nm com faixa de concentração de 2,0 a 7,0 μg/mL, utilizando água como solvente, com exatidão de 101,51% e teor de 99,79%; (iii) método espectrofotométrico derivativo na região do visível a 386,4 nm, na primeira derivada, com faixa de concentração de 50,0 a 100,0 μg/mL, utilizando cloreto férrico 1,0% como reagente, com exatidão de 99,83% e teor de 106,72%; (iv) método por cromatografia líquida de alta eficiência com detector UV a 275 nm, com fase móvel composta por ácido acético 2,5% v/v, metanol e acetonitrila (70:15:15, v/v/v) e faixa de concentração de 1,0 a 6,0 μg/mL, exatidão... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Ciprofloxacin hydrochloride, a quinolone antibiotic, presents a wider spectrum of activity and is effective against practically all Gram-negative pathogens, including Pseudomonas aeruginosa. It is potent against Grampositive microorganisms, as Staphylococcus and Streptococcus. Analytical methods for quantitative determination of ciprofloxacin hydrochloride is important due to its therapeutic potential, wide use in antimicrobial therapy and low cost. Moreover, it is known that the poor quality antibiotic product is direct related development of resistant strains, as consequence of subtherapeutic doses administration. Thus, it is important to develop efficient analytical methods for quality control commercialized products. In this work, analytical methods for determination of ciprofloxacin hydrochloride were validated: (i) microbiological assay, turbidimetric method at concentration range 14.0 to 56.0 μg/mL, using Staphylococcus epidermidis ATCC 12228 IAL 2150 as indicator microorganism, accuracy 99.71% and quantitation of 102.27; (ii) UV spectrophotometry at 275 nm with concentration range of 2.0 to 7.0 μg/mL, using water as solvent, with accuracy of 101.51% and quantitation of 99.79%; (iii) Derivative visible spectrophotometric method at 386.4 nm, in first derivate, with concentration range of 50.0 a 100.0 μg/mL, using 1.0% ferric chloride as reagent, with accuracy of 99.83% and quantitation of 106.72%; (iv) HPLC method with UV detector at 275 nm using 2.5 M acetic acid (v/v), methanol and acetonitrile (70: 15: 15, v/v/v) as mobile phase and concentration range of 1.0 to 6.0 μg/mL, accuracy of 100.11%, quantitation 103.25% and mean retention time of 2.6 minutes; (v) Indirect titrimetric method using bromate/bromide solution in acid medium as reagent in concentration range of 1.0 to 11.0 mg/mL, with accuracy of 100.28% and quantitation of 98.97%.
Mestre
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Duvic, Bernard. "Purification et etude de la proteine de transport de la 20-hydroxyecdysone chez un insecte : locusta migratoria." Université Louis Pasteur (Strasbourg) (1971-2008), 1988. http://www.theses.fr/1988STR13160.
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La purification du transporteur est realisee a partir d'hemolymphe par differentes techniques (coagulation a temperature ambiante; chromatographie sur colonne echangeuse d'anions; chromatographie sur colonne de gel permeation; hplc sur phase echangeuse d'anions). Le transporteur est une glycoproteine acide (pi=5,6) de 280 kda. C'est un homodimere a 2 sous-unites de 140 kda dont la sequence n-terminale a ete determinee. Un polyserum de lapin et 2 anticorps monoclonaux de souris ont ete obtenus et permettent un dosage elisa du transporteur. Des dosages chez l. M. Montrent que le transporteur et l'ecdysone varient dans le meme sens. L'injection massive d'ecdysone suggere une synthese du transporteur sous le controle des ecdysteroides
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Eriste, Elo. "Purification, structure and function of bioactive peptides /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-853-X/.
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Yamamoto, Célia Hitomi. "Aspectos da qualidade da tetraciclina em preparações farmacêuticas sólidas. Correlação entre os métodos de dosagem por cromatografia líquida de alta eficiência e turbimético." Universidade de São Paulo, 1999. http://www.teses.usp.br/teses/disponiveis/9/9139/tde-05102011-150147/.
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As tetraciclinas são encontradas no mercado sob várias formas farmacêuticas, sendo provenientes de diversos laboratórios farmacêuticos. Com o objetivo de avaliar a qualidade destes medicamentos, foram realizados os ensaios de dissolução in vitro e determinação quantitativa. Um total de 38 amostras de cápsulas de cloridrato de tetraciclina, fosfato de tetraciclina e cloridrato de oxitetraciclina e drágea de cloridrato de doxiciclina, englobando 12 fabricantes, foram analisadas. A dissolução do princípio ativo foi determinada para todas as amostras, conforme o método recomendado pela USP XXIII. Das amostras, duas foram reprovadas e outras quatro foram aprovadas após o reteste. A variação dos valores individuais obtidos no ensaio de dissolução para cada amostra, foi significativa, apresentando coeficiente de variação de até 14,2 %. A determinação quantitativa através do método microbiológico turbidimétrico empregando Staphylococcus aureus ATCC 29737, resultou em duas amostras de um mesmo fabricante com potência muito abaixo do limite especificado de 90 a 125 % do valor rotulado, com 55.5 e 68,7 %. Estudo comparativo desta metodologia com o método de cromatografia líquida de alta eficiência (CLAE) foi realizado. Para isto, o método da USP XXIII foi escolhida após o ensaio preliminar, seguido de determinação dos parâmetros de validação e adequação do método. O sistema cromatográfico estabelecido consistiu de coluna de fase reversa SYMMETRYTM C8 e fase móvel composta de oxalato de amônio 0,09 M, dimetilformamida e fosfato dibásico de amônio 0.18 M (64:32:4.7) com pH entre 7,6 e 7.7. Os resultados para determinação de cloridrato de tetraciclina confirmaram os valores obtidos no ensaio microbiológico, sendo reprovadas duas amostras. O teor máximo encontrado de 4-epianidrotetraciclina foi de 0,5 %, abaixo do limite de 3 % especificado na USP XXIII. Na comparação entre os dois métodos, foram observados resultados sempre superiores para o método microbiológico. A análise estatística destes resultados mostrou diferença significativa entre as médias das determinações obtidas a partir de cada método.Tetracyclines are avaiable under several pharmaceutical forms and manufactured by different laboratories. Aiming at the evalution of the quality of these medicines, assays of in vitro dissolution and quantitative determination have been performed in 38 samples of tetracycline hydrochloride and phosphate and oxytetracycline hydrochloride capsules and docycycline hydrochloride coated tablet taken from 12 manufactures. The range of dissolution of the substance was determined in all the samples, according to the method recommended by USP XXIII. On the whole, only two of samples were rejected and all the others approved without restrictions, except four of them, wich required a retest. The evaluation of the individual values obtained in the assay of dissolution in each sample was significant, with a variation coefficient of up to 14.2%. The quantitative determination through the turbidimetric microbiological method employing Staphylococcus aureus ATCC 29737, resulted in two samples of tetracycline hydrochloride from the same manufatures with potency of 55.5 % and 68.7 % below the specified limit from 90 % to 125 % the labeled value. A comparative study of this method and the HPLC one was then performed. The USP XXIII method was chosen after a preliminary assay, followed by the determination of its validation parameters and system suitability. The established chromatographic method employed reversed phase column SYMMETRYTM C8 (octylsilane chemically bounded to totally porous sílica particles) and mobile phase consisting of ammonium oxalate 0.09 M, dimethyformamide and dibasic ammonium phosphate 0.18 M (63.9:32:4.7) adjusted to pH 7.6-7.7. The results confirmed the values obtained in the microbiological assay. When this method (HPLC) was used for the determination of 4-epianidrotetracycline, a maximum of 0.5 % was found, below the limit of 3% specified in the USP XXIII. The comparison between both methods reavealed constant superior results in the microbiological one, and the difference between the averages of the determinations from the methods was meaningful.
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Ngwalero, Precious. "Development and validation of liquid chromatography mass spectrometry (L/C/MS/MS) assay for the determination of plasma 4betahydroxycholestrol and cholesterol in HIV infected children in Africa." Master's thesis, University of Cape Town, 2015. http://hdl.handle.net/11427/16654.
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Includes bibliographical references4β-hydroxycholesterol (4β-OHC) is a metabolite of cholesterol formed by Cytochrome (CYP) 3A4/5/7 enzymes. It has recently been proposed as an endogenous biomarker forCYP3A4/5/7 activity. This may be useful in prediction of drug-drug interactions and other metabolic processes affected by regulators of CYP3A activity. The aim of this study was to develop and validate an LC/MS/MS assay for the determination of 4β-OHC in human plasma and use 4β-OHC as a biomarker of CYP3A4/5/7 metabolism in HIV-infected children with and without treatment in Africa. Determination of 4β-OHC from plasma was performed by saponification and derivatisation reaction processes followed by high performance liquid chromatography with MS/MS detection on an AB Sciex Qtrap 5500 mass spectrometer. Since 4β-OHC is an endogenous metabolite in human plasma, a stable isotope labelled (SIL) analogue, 4β-OHC-D7, was used as a surrogate analyte for the preparation of calibration standards and quality controls. A second SIL analogue, 4β-OHC-D4 was used as the internal standard. The transitions of the protonated derivatised products were monitored atm/z 613, 620 and 617 to the product ions m/z 490, 497 and 494 for 4β-OHC, 4β-OHC-D7and 4β-OHC-D4 respectively. The calibration curve fitted a quadratic (weighted by1/concentration2) regression over the range 2-500 ng/ml. Validation accuracy and precision statistics summary for three consecutive runs were between 98.9% and 103%, and 3.5%and 12% respectively of all quality controls. The assay's recovery, selectivity and analyte stability were established. The validated assay was successfully applied on clinical samples, where 4β-OHC was used as a biomarker to investigate the levels of CYP3A induction in HIV-infected children with and without treatment containing non-nucleoside reverse transcriptase inhibitors (NNRI).It was found that plasma 4β-OHC concentrations at baseline were significantly lower in children belonging to the naïve group compared to nevirapine (NVP) and efavirenz (EFV)groups. When NVP and EFV groups were compared at non-baseline treatment weeks, the median 4β-OHC concentrations were significantly higher in EFV group than the NVP group. Regarding the effect of time on treatment, a significant increase in 4β-OHC concentrations was observed from baseline to each of the non-baseline weeks in naïve group. Conversely, in the NVP group, there was a significant decrease in 4β-OHC concentrations from baseline to each of the non-baseline weeks. Time did not show any significant effect on 4β-OHC concentrations in EFV group. Furthermore, at baseline, age, sex and weight did not affect 4β-OHC concentrations in all the three groups. This study has provided a method that would be utilised to determine plasma 4β-OHC concentrations using relatively small volumes - typical of samples taken from children. The results of this study suggest that children on antiretroviral therapy (ART) are at risk of effects of CYP3A induction, as indicated by the increase of 4β-OHC concentrations in the NVP and EFV groups. Additionally, prolonged use of the ART may activate some nuclear receptors that regulate CYP3A enzyme activity thereby negatively affecting, for example, the regulation of lipid and glucose metabolism. The developed method may therefore be useful in predicting drug-drug interactions in the context of multiple therapy and may also be used in predicting other metabolic processes affected by regulators of CYP3A activity. Further prospective studies with larger sample sizes are required to confirm and build on the evidence shown in this study.
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Ballentine, Regina. "Chemical Characterization of Pseudognaphalium obtusifolium by Gas Chromatography - Mass Spectrometry (GC-MS) to Assess Potential Therapeutic Phytochemicals and Toxicological Concerns Using Simulated Use Conditions." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/6052.
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Chemical Characterization of Pseudognaphalium obtusifolium by Gas Chromatography – Mass Spectrometry (GC-MS) to Assess Potential Therapeutic Phytochemicals and Toxicological Concerns Using Simulated Use Conditions
By Regina Ballentine
Virginia Commonwealth University, 2019
Director: Sarah C. Rutan, Professor, Department of Chemistry
Currently, there is an increasing demand for natural therapies and herbal products to treat various ailments. It is generally believed that natural therapies have fewer side-effects than traditional western medicine; however, they are often used in different strengths and formulations without consistency of the levels of target compounds or knowledge about toxicity. Due to this growing trend, a comprehensive chemical evaluation of plants used for medicinal purposes is necessary.
Pseudognaphalium obtusifolium is a plant that has been used historically by Native Americans as an herbal medicine. It is a flowering plant belonging to the Asteraceae family indigenous to the Eastern United States. There are documented accounts of the Native Americans using the herb therapeutically. Reportedly, they used the plant to prepare tea and as filler for bedding. Additionally, they smoked the plant material.
To date, there has been little research published on the chemical composition of this plant. Thus, the objective of this work was to conduct a chemical survey of P. obtusifolium using methodologies that would simulate the three historical routes of administration (tea, bedding material, and smoke inhalation).
To determine the types of compounds that may be found in the plant, initial experiments using pressurized solvent extraction (PSE) with an ethanolic solvent were performed followed by analysis using gas chromatography – mass spectrometry (GC-MS) in scan mode. This extraction technique enabled a broad range of compounds to be identified.
For the analysis of the tea, the leaves and the flowers were ground and analyzed separately. The “tea” simulation was then performed using a water extraction which was then back extracted into dichloromethane for GC-MS analysis in Selected Ion Monitoring (SIM) mode. Seventeen target compounds (terpenes, terpinoids, flavanoids, etc.) were quantified using this method.
A bedding material simulation was performed using headspace solid phase micro-extraction (HS-SPME) to collect the volatile and/or semi-volatile components of the headspace. The compounds collected on the SPME fiber were then analyzed by GC-MS in scan and SIM modes to qualitatively and quantitatively determine the types of chemical compounds (most of which were terpenes) that may be off-gassed from bedding material. This analysis compared levels of compounds in two different crop years and four terpene compounds were quantified.
To simulate smoking of the plant material, the leaves and flowers were fashioned into smoking articles. Sample collection was performed by a smoking machine and smoke condensate was collected. The smoke condensate was then analyzed by GC-MS in scan mode. As combustion and pyrolysis of plant material are known to produce toxic products, specific potentially harmful compounds were investigated and quantified.
This chemical analysis of P. obtusifolium identified target compounds that can be found in the three simulated usage forms. Identification of these compounds gives insight on why the Native Americans may have used P. obtusifolium as an herbal medicine. Among the detected compounds, there were many unknowns. Elucidating these unknown compounds will be important in the effort to understand the full chemical profile of this plant.
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Parbhunath, Olivia Leshia. "Validation of two bio-analytical assays for the measurement of hydrophilic antioxidant in several food and beverage commodities in accordance with ISO 17025 regulatory guidelines." Thesis, Cape Peninsula University of Technology, 2013. http://hdl.handle.net/20.500.11838/1495.
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Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Biomedical Technology in the Faculty Health and Wellness Sciences at the Cape Peninsula University of Technology, 2013The accurate and consistent measurement of antioxidants is crucial to evaluating their biological role in the prevention and delay of cancer and other pathological conditions. Hence, the performance of the analytical method utilized should be evaluated for acceptable levels of accuracy, precision and other performance parameters according to internationally accepted standards. Additionally, the measure and influence of existing errors should be evaluated and the method optimized to reduce such errors. In furtherance of this vital aim, this research project sought out to optimize and validate two bio-analytical assays for the measurement of total antioxidant capacity and L-ascorbic acid (L-AA), respectively in food commodities. The validation procedure was performed in accordance with ISO 17025 international standard. The first study in this thesis evaluated, optimized and validated the hydrophilic oxygen radical absorbance capacity (H-ORACFL) assay using fluorescein for total antioxidant capacity in various food and beverage products. The assay demonstrated good results with regard to accuracy, precision, linearity, specificity, limits of detection (LOD) and quantification (LOQ) and robustness. The extraction solvent (60% ethanol) recovered excellent antioxidant yields for most samples tested. The optimization of the method in terms of temperature and sample usage on the micro-plate significantly (p<0.05) reduced errors and subsequently improved precision substantially.
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Kamble, Ujjwala Kerba. "Use of liquid chromatography for assay of flavonoids as key constituents and antibiotics as trace elements in propolis : investigation into the application of a range of liquid chromatography techniques for the analysis of flavonoids and antibiotics in propolis, and extraction studies of flavonoids in propolis." Thesis, University of Bradford, 2016. http://hdl.handle.net/10454/14564.
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Propolis is an approved food additive containing flavonoids as a major active constituent. Variability has been found in the composition of propolis in distinctive regions and it was noticed that there are limitations in the analysis of propolis. In this study, the identification of ten flavonoids and residual antibiotics in propolis was investigated by using several liquid chromatography techniques, including reversed-phase high-performance liquid chromatography (RP-HPLC), microemulsion LC (MELC) and ultra-performance LC (UPLC). The ten flavonoids that were selected for this research include rutin, myricetin, quercetin, apigenin, kaempferol, pinocembrin, CAPE, chrysin, galangin and acacetin while chlortetracycline, oxytetracycline and doxycycline were selected to examine the residual antibiotics in propolis. For the analysis of the selected flavonoids, routine RP-HPLC method was found to be the best method, while MELC technique was found more efficient for the analysis of the selected antibiotics. Solid phase extraction with HLB sorbent was utilised in the analysis of antibiotics for clean-up of propolis. In method development studies for flavonoids and antibiotics, one-factor-at-a-time (OFAT) approach was followed. The final optimised method for the analysis of flavonoids as well as the method. for the analysis of antibiotics was validated using the ICH guidelines, and various aspects, such as the linearity, selectivity, accuracy, recovery, robustness and stability parameters, were examined. Development of efficient conventional method for the extraction of flavonoids from propolis was studied extensively in the present research work using different extraction techniques such as maceration, hot extraction, ultrasound assisted extraction. Among all extraction experiments, ethanolic extraction using ultrasound extraction method was the best efficient approach. This thesis shows that, in general, the performance of O/W MELC is superior to that of conventional HPLC for the determination of residual antibiotics in propolis. UPLC was not suitable for the analysis of flavonoids and antibiotics. The conventional LC was the only technique to separate the ten flavonoids but MELC was able to separate nine of the flavonoids with faster analysis time. This work also showed that MELC uses cheaper solvents. This considerable saving in both cost and time will potentially improve efficiency within quality control.
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Haynes, Christopher Allen. "Development of an assay for fatty acyl-CoAs using liquid chromatography-electrospray ionization-tandem mass spectrometry and its application to the stable isotope labeling and quantitation of sphingolipid metabolism." Diss., Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/37171.
Full textAbstract:
Fatty acyl-Coenzyme As are metabolites of lipid anabolism and catabolism. A method was developed for their quantitation in extracts of cultured mammalian cells using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Palmitoyl-CoA (C16:0-CoA) is utilized for de novo sphingolipid biosynthesis catalyzed by serine palmitoyltransferase (SPT), which condenses palmitoyl-CoA and serine to form 3-ketosphinganine. After reduction to form sphinganine (Sa), dihydroceramide synthase (CerS) can N-acylate the Sa using a second fatty acyl-CoA molecule, forming dihydroceramide (DHCer). The CerS enzyme family utilizes different acyl chain lengths of fatty acyl-CoAs in an isoform-specific manner, resulting in DHCer with N-acyl chains ranging from C16 to C26 [and even longer] in mammalian tissues. DHCer is trans-4,5-desaturated to yield ceramide, which is further metabolized by the addition of moieties at the 1-O-position, forming sphingomyelin (SM) and ceramide monohexose (CMH).
The rates of fatty acyl-CoA and sphingolipid biosynthesis were determined using stable isotope-labeling and LC-ESI-MS/MS analysis of the analyte isotopologues and isotopomers. Isotopic labeling of palmitoyl-CoA with [U-13C]-palmitate in HEK293 and RAW264.7 cells was robust and rapid (~ 60% labeling of the metabolite pool in 3 hr). Isotopic labeling of sphingolipids indicated utilization of [M + 16]-palmitoyl-CoA by SPT and CerS isoforms in both cell types. Metabolic flux modeling was applied to the data for [U-13C]-palmitate activation to [M + 16]-palmitoyl-CoA and its subsequent utilization in de novo sphingolipid biosynthesis, and this analysis indicated rapid turn-over rates for palmitoyl-CoA and ceramide in both cell types.
Palmitate treatment of cultured cells alters their metabolic status and gene expression, therefore labeling of palmitoyl-CoA by treatment with [1-13C]-acetate was employed. A distribution of mass-shifted palmitoyl-CoA species (isotopologues) is observed based on the number of incorporations of [1-13C]-acetate during de novo biosynthesis, requiring computational analysis to derive two parameters: the isotopic enrichment of the precursor pool, and the fraction of palmitoyl-CoA that was biosynthesized during the experiment. Previous reports by others describe mass isotopomer distribution analysis (MIDA) and isotopomer spectral analysis (ISA) for this purpose, and both calculation approaches indicated concurrent results.
In summary, the quantitation of fatty acyl-CoAs and their isotopic enrichment during stable isotope-labeling studies of lipid metabolism can provide data that significantly change the interpretation of analyte quantitation in these experiments, as demonstrated here for investigations of de novo sphingolipid biosynthesis.
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Nixon, Christopher E. "2ND TIER ASSAY FOR THE DETECTION OF CONGENITAL ADRENAL HYPERPLASIA BY VIRGINIA’S NEWBORN SCREENING LABORATORY: STEROID PROFILE BY HPLC-MS/MS." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/6075.
Full textAbstract:
Congenital Adrenal Hyperplasia (CAH) encompasses several disorders related to disruptions in the adrenal steroid production pathway. These disruptions may cause virilization of the external female sex organs, incorrect gender assignment, precocious puberty, and in the most severe form, may cause life-threatening salt wasting and adrenal crisis if not detected and treated early in the newborn period.
17α-Hydroxyprogesterone (17-OHP) is the primary target for immunofluorescence detection of CAH from dried blood spots in newborn screening (NBS). Unfortunately, current immunoassay techniques for the detection of CAH suffer from high false positive rates. The primary factors contributing to false positive determinations can include the natural increase of 17-OHP due to stress stimuli as well as cross-reactivity of the immunoassay antibody with other hormones and endogenous compounds in blood.
Analysis of the adrenal steroid profile and corresponding analyte ratios using high performance liquid chromatography (HPLC)or ultra-high pressure liquid chromatography (UHPLC)combined with tandem mass spectrometry (MS/MS) has been shown to be a sensitive and selective technique for the significant reduction of the false positive reporting rate for CAH in newborn screening.
In working toward optimization, validation, and implementation of an HPLC-MS/MS steroid profile for use by Virginia’s Newborn Screening laboratory as a 2nd tier analysis for CAH screening, a commercially-available core-shell HPLC column with a biphenyl stationary phase was determined to offer adequate retention and selectivity to achieve baseline resolution of isobaric target analytes under rapid reversed phase gradient conditions. Method linearity, precision, and accuracy were assessed using enriched dried blood spot materials. Double-blinded analyses of over 300 newborn dried blood spot specimens were used to determine clinical sensitivity and specificity of the assay, which is projected to substantially reduce the false positive reporting rate for CAH screening while meeting target sample turnaround times.
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Menou, Sophie. "Comparaison de méthodes d'analyses de traces de pesticides organiques dans les productions végétales, par chromatographies et immunodosages (ELISA)." Angers, 1995. http://www.theses.fr/1995ANGE0005.
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Les travaux consignés dans ce mémoire avaient pour objectif de situer les méthodes immunoenzymatiques ELISA dans l'ensemble des procédés d'analyse de résidus de pesticides dans les végétaux : limite de détection, sensibilité, étendue du domaine d'utilisation, sélectivité, reproductibilité et répétabilité, précision et dispersion des résultats, commodité d'emploi, perspectives de perfectionnement dans une première partie, on a rappelé quelques généralités relatives aux résidus de produits phytosanitaires et à leur dosage par les techniques chromatographiques conventionnelles. Dans une deuxième partie, on a consigne les définitions et les concepts de l'immunologie utiles pour son application à l'analyse de traces. Dans la partie expérimentale, on a rapporté la comparaison d'analyses chromatographiques et immunoenzymatiques de résidus de plusieurs pesticides contenus dans des matrices végétales. Les produits phytosanitaires étudiés sont des insecticides et des fongicides : le carbofuran, le métalaxyl, le méthoprène, le carbendazim, le bénomyl, l'aldicarbe et l'endosulfan. Notre conclusion est qu'il semble difficile que les techniques Elisa rivalisent avec les techniques chromatographiques en matière d'analyse quantitative. Les cas que nous avons examinés montrent que, d'une part, elles n'éliminent pas les difficultés dues à la préparation des échantillons, et que d'autre part, leur sensibilité et leur sélectivité sont limitées. Les effets de matrice et les réactions croisées sont les principales sources de dispersion des résultats dans l'analyse ELISA. Néanmoins, sa limite de détection très faible et sa commodité d'emploi font de l'analyse immunoenzymatique une analyse de première intention intéressante lorsqu'il s'agit de contrôler de nombreux échantillons parce qu'elle permet de sélectionner ceux qui présentent un risque de résidus.
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Letourneur, Didier. "Polymères fonctionnels phosphorylés : interactions biospécifiques avec des protéines humaines, anticorps anti-adn, antiphospholipides et facteurs de transcription." Paris 13, 1988. http://www.theses.fr/1988PA132010.
Full textAbstract:
Des résines dérivées du polystyrene réticule ont été synthétisées. Ces polymères sont fonctionnalisés par des groupements chimiques analogues à ceux de l'adn et des phospholipides. L'affinité biospécifique de ces resines pour des protèines humaines qui admettent pour substrat l'adn et les phospholipides : anticorps anti adn, anti phospholipide, facteurs de transcription de l'adn. Les anticorps qui sont présents dans le sang des patients atteints de lupus érythemateux dissemine (l. E. D) ont montré une affinité spécifique tres élevée pour les dérivés fonctionnels du polystyrene;ces interactions impliquent le fragment fab des immunoglobulines lupiques et des sites distribués de façon statistique à la surface des polymères. Les polymères fonctionnels phosphoryles permettent en tant que phase stationnaire la résolution chromatographique d'extraits cellulaires contenant des facteurs de transcription de l'adn
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Zech, Julie [Verfasser], Leif-Alexander [Akademischer Betreuer] Garbe, Hajo [Gutachter] Haase, Juri [Gutachter] Rappsilber, and Sascha [Gutachter] Rohn. "Analysis of bisphenols and bisphenol A diglycidyl ethers by stable isotope dilution assay liquid chromatography-tandem mass spectrometry / Julie Zech ; Gutachter: Hajo Haase, Juri Rappsilber, Sascha Rohn ; Betreuer: Leif-Alexander Garbe." Berlin : Technische Universität Berlin, 2016. http://d-nb.info/1156011728/34.
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Magnér, Jörgen. "Methodologies to assess the fate of polar organic compounds in aquatic environments." Doctoral thesis, Stockholms universitet, Institutionen för tillämpad miljövetenskap (ITM), 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-35552.
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Polar organic compounds (POCs) are chemicals with polar functional groups in their structure. The functional groups make the compounds hydrophilic and less prone to partition with biota. However, the knowledge of their fate is limited due to difficulties associated with their measurements. Although, the persistence of POCs in the environment is generally low, they are considered to be semi-persistent compounds due to their continuous introduction to the environment via wastewater. Studies have shown that complex mixtures of POCs of different classes may have synergistic toxic effects on biota at environmental concentration levels. Therefore, it is important to develop analytical methods in order to establish the occurrence and fate of POCs in aquatic environments. In Study I, a positive correlation between the sorption of a novel poly(ethylene-co-vinyl acetate-co-carbon monoxide) (PEVAC) material and the theoretical logarithmic dissociation partition coefficient (Log D) for seven POCs was observed. The PEVAC material showed an enhanced sorption of the POCs compared to the silicone material. Study II, demonstrated that the PEVAC sampler assess the freely dissolved concentration of POCs in aquatic environments. The results showed that the PEVAC polymer is an attractive alternative to silicone for mimicing the biological uptake of POCs in aquatic environments. Additionally, Study II showed that total extraction is appropriate for determination of the freely dissolved concentration of uncharged POCs with Log KOW < 2.67 in natural water. In study III, a novel bag-solid phase extraction (bag-SPE) technique was compared to a conventional SPE-technique. Despite that the extraction efficiencies for POCs in wastewater were lower using the bag-SPE method, the two methods showed similar detection limits due to the lower ion-suppression experienced with the bag-SPE. In study IV the bag-SPE method was further developed with the aim of lowering the detection limits for POCs. Detection limits (LOD) below 13 ng/L showed that the bag-SPE method was suitable for determination of POCs in surface sea water.This research was financially supported by European Union (European Commission, FP6 Contract No. 003956) “Novel Methods for Integrated Risk Assessment of Cumulative Stressors in the Environment” (NoMiracle) and by the Swedish research council Formas.