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1
Bakirdere, Sezgin. "Speciation Studies Using Hplc-icp-ms And Hplc-es-ms". Phd thesis, METU, 2009. http://etd.lib.metu.edu.tr/upload/3/12611391/index.pdf.
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Knowledge about selenium content of foods containing selenium species is very important in terms of both nutrition and toxicity. Bioavailability of selenium species for human body is different from each other. Hence, speciation of selenium is more important than total selenium determination. In the selenium speciation study, chicken breast samples, selenium supplement tablets and egg samples were analyzed for their selenium contents. In chicken breast study, chickens were randomly categorized into three groups including the control group (25 chickens), inorganic selenium fed group (25 chickens) and organic selenium fed group (25 chickens). After the optimization of all the analytical parameters used throughout the study, selenomethionine, selenocystine, Se(IV) and Se(VI) were determined using Cation Exchange-HPLC-ICP-MS system. In selenium supplement tablet study, anion and cation exchange chromatographies were used to determine selenium species.
Arsenic is known as toxic element, and toxicity of inorganic arsenic species, As(III) and As(V), is much higher than organic arsenic species like arsenobetaine and arsenosugars. Hence, speciation of arsenic species in any matrix related with human health is very important. In the arsenic speciation study, Cation Exchange-HPLC-ICP-MS and Cation Exchange-HPLC-ES-MS systems were used to determine arsenobetaine content of DORM-2, DORM-3 and DOLT-4 as CRMs. All of the parameters in extraction, separation and detection steps were optimized. Standard addition method was applied to samples to eliminate or minimize the matrix interference.
Thiols play an important role in metabolism and cellular homeostasis. Hence, determination of thiol compounds in biological matrices has been of interest by scientists. In the thiol study, Reverse Phase-HPLC-ICP-MS and Reverse Phase-HPLC-ES-MS systems were used for the separation and detection of thiols. For the thiol determination, thiols containing &ndashS-S- bond were reduced using dithiothreitol (DTT). Reduction efficiencies for species of interest were found to be around 100%. Reduced and free thiols were derivatized before introduction on the column by p-hydroxymercuribenzoate (PHMB) and then separated from each other by using a C8 column. In the real sample measurement, yeast samples were analyzed using HPLC-ES-MS system.
2
Käsmä, S. (Salla). "(U)HPLC-MS/MS käyttö virtsanäytteiden steroidianalyyseissä". Bachelor's thesis, University of Oulu, 2019. http://urn.fi/URN:NBN:fi:oulu-201901121055.
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Kehossa esiintyy luontaisesti useita anabolisia androgeenisia steroidiyhdisteitä, joista lihasmassan kasvun ja sen ylläpidon kannalta merkittävin on testosteroni. Testosteronin voimakkaan anabolisen vaikutuksen takia siitä on tehty useita muunnelma, joilla on pyritty minimoimaan androgeenista vaikutusta, maksimoimaan anabolista tehoa ja esimerkiksi vaikuttamaan yhdisteen vapautumiseen kehossa. Luontaisetja synteettiset anaboliset androgeenit ovat sekä rakenteeltaan että toiminnaltaan testosteronin kaltaisia. Anabolisten androgeenien ja muiden steroidiyhdisteiden aineenvaihdunta on monimutkaista, sisältäen useita entsyymiavusteisia reaktioita, minkä johdosta steroidiyhdisteiden analysointi voi olla haastavaa ja tiettyä yhdistettä voidaan tutkia suoraan tai useiden eri markkerien tai metaboliittien avulla.
Steroidiyhdisteiden analytiikassa aineenvaihdunta huomioidaan näytteenkäsittelyssä. Virtsan sisältämät steroidit ovat suurilta osin glukuronidikonjugaatteja, joiden lisäksi virtsassa esiintyy sulfaattikonjugaatteja ja muita aineenvaihduntatuotteita. Näytteenkäsittelyssä glukuronidikonjugaatit yleensä hydrolysoidaan takaisin vapaaseen steroidimuotoon ja näytteet uutetaan poolittomien ja poolisten yhdisteiden erottamiseksi. Laitekomponenttien ja menetelmän kehittyminen on mahdollistanut myös suoraruiskutusmenetelmän soveltamisen.
Steroideja voidaan analysoida virtsasta perinteisen kaasukromatografia-massaspektrometrin lisäksi mm. korkean erotuskyvyn nestekromatografialla kytkettynä tandem massaspektrometriin. Menetelmää voidaan käyttää sekä kvantitatiivisiin että kvalitatiivisiin analyyseihin ja sen etu on lämpöherkkien yhdisteiden analysointi, herkkyys ja automaation helppous. Steroidianalyyseissä nestekromatografisessa erotuksessa käytetään yleisimmin käänteisfaasierotusta ja gradienttimenetelmää. Ionisointi suoritetaan sähkösumutusionisaatiolla tai kemiallisella ionisaatiolla normaali ilmanpaineessa, sillä kyseiset ionisaatiomenetelmät aiheuttavat vähän fragmentaatiota. Massa-analysaattorina käytetään yleisimmin kolmoiskvadrupolia, mutta myös ioniloukku- ja lentoaika-analysaattoreita yhdistettynä kvadrupoliin.
Korkean erotuskyvyn nestekromatografia kytkettynä tandem massaspektrometriin sisältää useita eri muuttujia, joita vaihtelemalla, säätämällä ja optimoimalla voidaan vaikuttaa analyysituloksiin. Menetelmän erilaisten mahdollisuuksien määrä mahdollistaa lukuisten erilaisten yhdisteiden analysoinnin ja menetelmän edelleen kehittymisen.
3
Berger, Irayani. "HPLC-MS/MS Analyse von Immunsuppressiva direkt in Vollblut". Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-127030.
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4
Zhong, Fanyi. "DEVELOPMENT AND APPLICATIONS OF HPLC-MS/MS BASED METABOLOMICS". Miami University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=miami1524792637748877.
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Berger, Irayani [Verfasser]. "HPLC-MS/MS Analyse von Immunsuppressiva direkt in Vollblut / Irayani Berger". München : Verlag Dr. Hut, 2011. http://d-nb.info/1010446894/34.
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6
Wollersen, Heike. "Bestimmung und Identifizierung von Flavonoiden in Gerste mit HPLC-DAD-MS/MS". [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971821453.
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Levaggi, Valeria <1991>. "Caratterizzazione chimica del tabacco lavorato (Nicotiana Tabacum, tipo Kentucky) attraverso un approccio analitico multi-metodologico (ICP-MS, GC-MS, HPLC-MS-MS)". Master's Degree Thesis, Università Ca' Foscari Venezia, 2019. http://hdl.handle.net/10579/15053.
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Il presente lavoro di tesi ha l’obiettivo di studiare dal punto di vista chimico, il tabacco “kentucky” coltivato e lavorato in alcune zone della Regione Veneto. Questo tipo di produzione agricola è considerata di nicchia e la caratterizzazione e quantificazione dei suoi principali costituenti chimici non è mai stata approfondita. Nel presente lavoro di tesi si sono individuati alcuni componenti chimici sviluppando e/o perfezionando metodi analitici utili all’individuazione di una "impronta digitale" chimica del tabacco Kentucky veneto.
Il Kentucky è un tabacco scuro appartenente alla classe dei cosiddetti fire cured (cura a fuoco), cioè i tabacchi seccati attraverso il fumo di legni speciali che, penetrando lentamente nelle foglie, ne conferiscono un particolare aroma e il colore scuro.
I metodi analitici sviluppati hanno reso possibile l’individuazione e la quantificazione di una serie di composti inorganici ed organici in foglie di tabacco kentucky dopo la cura a fuoco coltivato nel Veronese e in campioni di tabacco lavorato.
Il lavoro svolto in laboratorio prevede due fasi lavorative.
La prima, consiste nella quantificazione di analiti di tipo inorganico attraverso la digestione acida in microonde delle foglie di sigaro per poter analizzare in ICP-MS la quantità di metalli pesati presenti in essi (TE e REE), tenendo presente dei possibili effetti di frazionamento isotopico che incidono sui risultati finali di concentrazione.
La seconda fase invece include la quantificazione di analiti di tipo organico, nello specifico dell’alcaloide Nicotina e delle nitrosammine specifiche del tabacco NNN e NNK (noti anche come, rispettivamente, N-nitrosornicotina e 4-(metilnitrosamino)-1-(3-piridil)-1-butanone) tramite estrazione con MeOH dei campioni già polverizzati e successive analisi. La quantificazione è avvenuta via GC-MS per la nicotina e via HPLC-MS/MS per quanto riguarda le nistrosammine.
L'utilizzo di un materiale certificato di riferimento ha permesso di validare i metodi per la quantificazione degli analiti. I risultati ottenuti sono stati sottoposti ad una analisi statistica preliminare per valutare correlazioni utili a individuare una possibile indicazione di tracciabilità.
8
McCulloch, Melissa. "Development of Quantitative Bioanalytical Methods for the Measurement of Pharmaceutical Compounds via HPLC-UV and HPLC-MS/MS". Cleveland State University / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=csu1255046678.
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Mayton, Eric. "Characterization of Lysophosphatidic Acid Subspecies Using a Novel HPLC ESI-MS/MS Method". VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/2515.
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Lysophosphatidic acid (LPA) is a bioactive lipid with a plethora of biological functions, including roles in cell survival, proliferation, and migration. Although high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC ESI-MS/MS) technology has been used to measure the levels of LPA in human blood, serum and plasma, current methods cannot readily detect the minute levels of LPA from cell culture. In this study, a novel HPLC ESI-MS/MS method with enhanced sensitivity was developed which allows accurate measurements of LPA levels with a limit of quantitation at approximately 10 femtomoles. The method was validated by quantitation of LPA levels in the media of previously characterized cell lines ectopically expressing autotaxin. Autotaxin overexpression induced an increase in several subspecies of LPA while others remained unchanged. Lastly, this HPLC ESI-MS/MS method was validated via biological assays previously utilized to assay LPA production. Hence, this new HPLC ESI-MS/MS will allow researchers to measure in vitro LPA levels and also distinguish between specific LPA subspecies for the delineation of individual biological mechanisms.
10
Shah, Iltaf. "Determination of steroidal and nonsteroidal drugs in human body matrices by LC-MS/MS, GC-MS, HPLC and ELISA". Thesis, Kingston University, 2010. http://eprints.kingston.ac.uk/27839/.
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There is an ever-increasing need to develop analytical methods for the detection of steroids, NSAIDs, fibrates, iothalamates and fatty acids for therapeutic, forensic, medical and pharmaceutical applications. Hence, the primary aim of the project was to develop new methods for detection of these classes of drugs in different human. body matrices using chromatographic techniques like enzyme-linked immunosorbent assay [ELISA], HPLC-UVIDADÆLSD, LC-MS/MS and GC-MS/MS. The new methods developed were capable of detecting the drugs: prednisolone, cortisol, prednisone, nandrolone, stanozolol, testosterone, diclofenac, fenofibric acid, iothalamic acid, monoolein, diolein, triolein, palmatic acid, stearic acid and linolenic acid. The methods were validated for LLOD, specificity, accuracy, precision, linearity and accuracy according to FDA and ICH guidelines in a cGLP-accredited lab. The assay was linear in the range 1 to 400 pg/mg for stanozolol, 0.5 to 400 pg/mg for testosterone and 3 to 400 pg/mg for nandrolone. The linear range for the routine assays were from 1 to 25 ng/mI with no interference from matrices. The calibration standards and quality controls were prepared in fresh human matrices. New internal standards were developed and added to all aliquots of samples. All methods showed good precision, accuracy, recovery and linearity. LLODs for non-steroidal anti inflammatory drugs were found to be in the range of 0.02 ng/ml to 10 ng/mI in plasma, urine and synovial fluid. Corticosteroids were analysed in the range of 5 ng/ml to 50 ng/mI. The anabolic steroids assays were capable of detecting 0.25 pg testosterone, 0.5 pg stanozolol and 2.5 pg nandrolone as a LLOD per mg of hair when approximately 20 mg of hair was processed. The assay was linear in the range 1 to 400 pg/mg for stanozolol, 0.5 to 400 pg/mg for testosterone and 3 to 400 pg/mg for nandrolone. lothalamates were found to be in the range of 0.25 ug/ml to 0.5 ug/ml, fibrates 5 to 25 ng/mI and diuretics in 1-5 ng/ml range. The extended lower limits of detection and lower limits of quantification would help in the detection of these drugs even at very low concentrations in matrices. Hair, urine, plasma, synovial fluids etc from subjects that were found positive for steroidal, non-steroidal drugs, corticosterides and others by ELISA screens were successfully quantified on HPLC, GC-MS and LC-MS/MS. In conclusion, the results obtained demonstrate the application of hair analysis to detect steroids in line with WADA requirements at lower concentrations. These novel methods have been developed and validated in human plasma, urine, synovial fluids, gastrodeudenal fluids and hair using improved extraction techniques. In addition, new solvents have been added to the samples as a deproteination agent, which increased the recovery of most drugs. Narrow bore column technology has been used to detect lower levels of drugs in matrices. These revised processes allowed clean extraction and near-quantitative recovery of analyte (>95%).
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Sealey-Voyksner, Jennifer A. Jorgenson James W. "Investigation of immunogenic gluten peptides identification using enzymatic tagging and HPLC-MSn; analysis and quantification using HPLC-MS/MS /". Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2009. http://dc.lib.unc.edu/u?/etd,2764.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.Title from electronic title page (viewed Mar. 10, 2010). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry." Discipline: Chemistry; Department/School: Chemistry.
12
Cartwright, Andrew James. "Analysis of pharmaceuticals and biomolecules using HPLC coupled to ICP-MS and ESI-MS". Thesis, University of Plymouth, 2005. http://hdl.handle.net/10026.1/1997.
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The work described within this thesis explores the use of HPLC coupled with ICPMS and ESI-MS in order to develop novel methods which overcome specific analytical challenges in the pharmaceutical industry. A membrane desolvation interface has been evaluated for coupling high performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry (ICP-MS). Desolvation of the sample prior to reaching the plasma was shown to facilitate a versatile coupling of the two instrumental techniques, enabling chromatographic eluents containing up to 100 % organic to be used. This interface also allowed gradient elution to be used with ICP-MS. Tris(2,4,6-trimethoxyphenyl)phosphonium propylamine bromide (TMPP) was used for the derivatisation of maleic, fumaric, sorbic and salicylic acids to facilitate determination by HPLC-electrospray ionisation tandem mass spectrometry (ESIMS/ MS) in positive ion mode. Improvements in detection limits post-derivatisation were achieved, and this method was successfully used for the determination of sorbic acid in a sample of Panadol™. HPLC coupled with sector field inductively coupled plasma mass spectrometry (SF-ICP-MS) has been used for the determination of maleic, sorbic and fumaric acids after derivatisation with TMPP. This allowed 31P+ selective detection to be performed for these compounds, which are normally undetectable by ICP-MS. Optimal reagent conditions for the derivatisation of 0.1 mM maleic acid were: 1 mM TMPP; 10 mM 2-chloro-1-methylpyridinium iodide (CMPI); 11 mM triethylamine. The efficiency of the derivatisation reaction was estimated to be between 10-20%. Detection limits, estimated as 3 times baseline noise, were 0.046 nmol for TMPP and 0.25 nmol for derivatised maleic acid, for a 5 f.JL injection. Following on from this, a novel derivatising reagent, tris(3,5-dibromo-2,4,6- trimethoxyphenyl) phosphonium propylamine bromide (BrTMPP), was synthesised and subsequently characterised by proton NMR spectroscopy and ESI-MS. This was utilised to derivatise maleic acid, with a 9-fold increase in sensitivity gained when analysed by bromine selective detection as apposed to phosphorus selective ICP-MS. This derivatising reagent (BrTMPP) was also utilised to determine the degree of phosphorylation on phosphorylated peptides. A phosphorus containing carboxylic acid was successfully derivatised and the correct Br:P ratio was determined for this compound by ICP-MS. However, phosphorylated peptides were not successfully derivatised by BrTMPP. A combination of UV and phosphorus selective ICP-MS was also used to distinguish between phosphorylated and un-phosphorylated peptides after HPLC separation.
13
Samano, Kimberly L. "Behavioral Assessment and HPLC/MS/MS Identification of the Synthetic Cannabinoid, CP47,497, in Mice". VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3328.
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CP47,497 and other synthetic cannabinoid compounds were incipiently synthesized as research tools to investigate the mechanisms by which marijuana affects the brain and to aid in the development of therapeutic agents. Recently, these cannabinoid compounds have resurfaced in the designer drug market, marketed as “herbal incense products” (HIPs). Their popular use has resulted in an alarming rate of reported adverse effects and toxicities. Current legislation classified CP47,497 and several other synthetic cannabinoids compounds as Schedule I agents, but abuse of these compounds persists with serious consequences to public health and safety. In vivo studies examining the behavioral consequences of abused synthetic cannabinoids are limited. As a result, the goals of this research were to elucidate the acute and chronic pharmacological effects of CP47,497 and to develop a bioanalytical method for CP47,497 drug detection in mice. Cannabimimetic effects were evaluated in well-established in vivo models, the tetrad paradigm and drug discrimination assay. The tetrad test is comprised of four outcome measures sensitive to the primary psychoactive cannabinoid present in marijuana, delta-9-tetrahydrocannabinol (THC): catalepsy (bar test), antinociception (tail withdrawal latency), hypothermia, and decreases in spontaneous locomotor activity. While many pharmacological agents can produce one or a subset of these tetrad effects, drugs that activate CB1 receptors produce characteristic effects in all four parameters. An HPLC/MS/MS method was developed and confirmed the presence of CP47,497 in brain. We investigated whether CB1 receptors mediate the pharmacological effects of CP47,497. Cumulative dose-response experiments determined CP47,497 is more potent than THC in vivo in using multiple behavioral assays. Complementary pharmacological (CB1 receptor antagonist, rimonabant) and genetic (CB1 (-/-) mice) approaches were used to investigate whether CB1 receptors mediate the effects of CP47,497. Rimonabant (3 mg/kg or 10 mg/kg, depending on independent measure) blocked all cannabinoid-like pharmacological effects of CP47,497. Supporting these findings, CB1(-/-) mice were resistant to cannabimimetic effects of CP47,497. CP47,497 fully substituted for THC in the drug discrimination assay, with a potency of more than 5 times that of THC. Collectively, these results indicate that CP47,497 is markedly more potent (i.e. 5-8 fold) than THC, and its repeated administration produces tolerance to the cataleptic, antinociceptive, hypothermic and hypolocomotor effects in mice, with significant presentation of somatic withdrawal signs (paw flutter and head shakes) upon drug cessation. These findings are consistent with the high incidence of adverse events in humans abusing synthetic cannabinoids.
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Eroglu, Ozcan Sefika. "Arsenic Speciation In Fish By Hplc-icp-ms". Master's thesis, METU, 2010. http://etd.lib.metu.edu.tr/upload/12612704/index.pdf.
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ABSTRACT
ARSENIC SPECIATION IN FISH BY HPLC-ICP-MS
ÖZCAN, Sefika Eroglu M.S., Department of Chemistry Supervisor: Prof. Dr. O. Yavuz ATAMAN September 2010, 103 pages Arsenic speciation in fish samples on the market was performed using isocratic elution with cation exchange column high performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry (ICP-MS) detection. Total As concentrations were found by ICP-MS using samples digested by nitric acid-hydrogen peroxide solution using microwave oven digestion
the results were in the range of 1.15-12.6 µ
g/g. Separation of organic arsenicals, namely arsenobetaine (AB), dimethylarsinic acid (DMA) and monomethylarsonic acid (MA), have been achieved in 12 minutes. Freeze-dried samples were extracted by deionized water with a shaker system
the concentrations of AB and DMA in the extract was then determined using HPLC-ICP-MS. The accuracy of the method for determining AB concentration was confirmed using certified reference material (CRM), DOLT 4 (dog fish liver)
for this CRM only preliminary data are available for AB. The arsenic compounds in 6 fish muscle samples were investigated. The predominant arsenic compound found in extracts was AB
the concentrations were in the range of 0.86-12.0 µ
g/g. DMA concentration was 0.40±
0.03 µ
g/g in one of the samples
in the others it was below the limit of quantation (0.21 µ
g/g).
15
Tevell, Åberg Annica. "Detection and Structure Elucidation of Drug Metabolites in Biological Samples using HPLC-MS/MS Techniques". Doctoral thesis, Uppsala universitet, Avdelningen för analytisk farmaceutisk kemi, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-98348.
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This thesis describes the structure elucidation of drug metabolites in biological samples by the use of high performance liquid chromatography (HPLC) atmospheric pressure ionization (API) tandem mass spectrometry (MS/MS). Due to their different advantages, various mass analyzers have been used in the different experiments. The metabolism of clemastine, flutamide, and meloxicam were studied in vitro and/or in vivo in different species such as humans, dogs, and horses. Accurate mass measurements with the quadrupole-time of flight mass spectrometer and MSn data supplied by the ion trap instrument were useful in the structural investigation of the product ions of the drugs and their metabolites. Different scan modes of the triple quadrupole mass spectrometer resulted in great flexibility, selectivity, and sensitivity in the qualitative and semi-quantitative studies. Additionally, hydrogen/deuterium exchange and experiments with atmospheric pressure chemical ionization were conducted, and the fungus Cunninghamella elegans was utilized to produce amounts of drug metabolites sufficient for structural investigation. Six isomers of oxidized clemastine were detected and characterized in C. elegans incubations and their retention times and mass spectral data were compared to the metabolites detected in urine samples. Two of the metabolites were concluded to be diastereomeric N-oxides. In urine from horses treated with meloxicam, the peak of 5'-hydroxymethylmeloxicam resulted in much higher intensity than the parent drug or the other metabolites, and it was detectable for at least 14 days after the last dose in some of the horses. That is useful information in the development of analytical methods for the detection of prohibited use of meloxicam. A mercapturic acid conjugate of hydroxyflutamide was detected in urine from cancer patients, which indicated that a reactive metabolite was formed. This metabolite could be responsible for the adverse events reported for flutamide. The results from the four papers included in the thesis clearly demonstrate the usefulness and the flexibility of the HPLC-API-MS/MS technique.
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Haddad, Renato. "Utilização da tecnica HPLC/APCI-MS/MS para determinação e identificação de danos ao DNA". [s.n.], 2004. http://repositorio.unicamp.br/jspui/handle/REPOSIP/313581.
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Orientadores: Nelci Fenalti Hoehr, Marcos Nogueira EberlinTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-04T01:26:19Z (GMT). No. of bitstreams: 1 Haddad_Renato_D.pdf: 3564615 bytes, checksum: 5d6ffc7c1ac5fa4d72b2c1664955439c (MD5) Previous issue date: 2004
Resumo: o acúmulo de danos oxidativos ao DNA vem sendo proposto como um dos responsáveis pelo envelhecimento e por doenças neurodegenerativas como a doença de A1zheimer. Estudos de danos oxidativos vêm sendo realizados com o objetivo de elucidar diversas doenças como a predisposição ao câncer e os processos de envelhecimento, uma vez que a formação de radicais livres dentro das células, causa lesões cuja natureza química ainda é pouco conhecida. Este estudo permitiu o desenvolvimento de uma nova metodologia de análise utilizando a cromatografia líquida de alta eficiência acoplada a espectrometria de massas sequencial com Ionização Química a Pressão Atmosférica (HPLC/APCI-MS/MS) para identificação das lesões oxidativas ao DNA. Foram adquiridos espectros de massas (APCI-MS e APCI-MS/MS) das bases oxidadas de DNA para verificar os padrões de fragmentação destas bases. Após a realização dos testes de linearidade, estudos iniciais com sistemas mais simples, oligonuc1eotídeos comerciais foram realizados para testar a aplicabilidade da técnica de HPLC/APCI-MS/MS. Estudos "in vivo" com culturas de células [PC-12, feocromocitoma de rato (fibroblastos)], foram realizados. Estas células foram irradiadas com Luz Fluorescente por 2, 4 e 6 horas e analisadas após exposição. Nós observamos a formação de cinco das seis bases de DNA foxidadas analisadas [8-0HGua, sss-Diamino-5-(formamido)-pirimidina (Fape-Ade), m5-Hidroxiuracila (5-0H -Uracila), 5-Hidroximetiluracila (5-0HMe- Uracila), Diidrotimina] lmostrando assim que a técnica desenvolvida é apropriada para este tipo de análise, recebendo bons níveis de detecção e sensibilidade
Abstract: Reactive oxygen species produce oxidized bases, deoxyribose lesions and DNA strand breaks in mammalian cells. Free radicaIs are produced in cells by cellular metabolism and by exogenous agents. These species react with biomolecules in cells, inc1udingDNA. The resulting damage to DNA, which is also called oxidative damage to DNA, is implicated in mutagenesis, carcinogenesis, and aging. We introduced a new methodology using Atmosphere Pressure Chemical Ionization (APCI) and HPLC/tandem mass spectrometry (HPLC/APCI-MS/MS). This technique for the measurement of modified nuc1eosides can simultaneously measure numerous products (8-hydroxyguanine, dihydrothymine, 4,6-diamino-5-(formamido)pyrimidine, isobarbituric acid (5-hydroxyuracil), 5-hydroxymethyluracil and thymine glycol), and provide positive identification and accurate quantification for monitoring in MRM mode. The responses for all damage bases and the four normal bases were highly linear with correlation coefficients 0.9950-0.9999 and detection limit infmols. We applied the method for quantification and identification ofthese bases in cultured PC-12 cells, a rat pheochromocytoma (fibroblasts) cellline irradiated for 2,4 and 6 hours following FL exposure
Doutorado
Ciencias Biomedicas
Doutor em Ciências Médicas
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LA, MAIDA NUNZIA. "Development and validation of analytical methods with hyphenated chromatographic techniques for the determination of NPS and drugs of abuse in biological matrices". Doctoral thesis, Università Politecnica delle Marche, 2022. https://hdl.handle.net/11566/299808.
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Sin dal 1990 l'apparizione sul mercato di nuove sostanze psicoattive (NPS) è un fenomeno mondiale sempre crescente. Ogni anno vengono introdotte nuove sostanze nel mercato della droga come alternative legali alle sostanze controllate dalla legge. L’identificazione di queste nuove sostanze in matrici biologiche rappresenta una continua sfida per i laboratori di analisi. È estremamente importante sviluppare metodologie analitiche in grado di identificare sostanze sconosciute consentendone il monitoraggio e fornendo informazioni scientifiche riguardo la farmacocinetica, possibili range di tossicità ed effetti farmacologici. Oggi, le tecniche cromatografiche ifenate (tecniche basate sull’accoppiamento di più strumentazioni) sono dei mezzi molto utilizzati nella tossicologia clinica e forense. Il seguente progetto di ricerca ha lo scopo di sviluppare e validare nuovi metodi analitici basati su tecniche cromatografiche accoppiate alla spettrometria di massa e spettrometria di massa tandem per la determinazione delle NPS e delle classiche droghe d’abuso in matrici biologiche. In particolare, considerando la poliassunzione di droghe, è stato sviluppato un metodo di screening utilizzando la cromatografia liquida ad alta prestazione abbinata alla spettrometria di massa tandem (HPLC-MS-MS) per la determinazione di 77 NPS (appartenenti a diverse classi di sostanze), 24 droghe classiche e 18 metaboliti nel sangue, in urina e saliva. Attualmente, i cannabinoidi sintetici sono il più grande gruppo di NPS monitorato dall’Agenzia Europea per le Droghe (EMCDDA ). Due nuovi metodi analitici sono stati sviluppati per la determinazione dei cannabinoidi sintetici JWH-122, JWH-210 e UR-144 in saliva: un metodo in gascromatografia accoppiata alla spettrometria di massa (GC-MS) e un metodo complementare che utilizza la cromatografia liquida ad alta prestazione abbinata alla spettrometria di massa ad alta risoluzione (UHPLC-HRMS) per la quantificazione sia dei parent drugs che dei loro metaboliti. Infine, in risposta alla crescente diffusione di nuovi catinoni sintetici, viene presentato un metodo mirato per la loro identificazione e quantificazione mediante cromatografia liquida ad alte prestazioni e spettrometria di massa ad alta risoluzione (UHPLC-HRMS). Tutti i metodi qui descritti sono stati validati con successo e applicati a campioni reali (sangue, urina, saliva, capelli) fornendo risultati affidabili e dimostrando di essere metodologie sensibili utilizzabili in laboratori clinici e forensi.The emergence of new psychoactive substances (NPS) has been a growing global phenomenon since 1990. Every year new substances appear into the drug market as legal alternatives to illicit drugs. In this regard, the identification in biological matrices of emerging synthetic compounds represents a continuous challenge for analytical toxicologists. It is extremely important to develop suitable drugs analysis methodologies which can provide information on pharmacokinetics, pharmacodynamics effects and possible toxic concentration ranges, and on the diffusion of these unknown substances. Hyphenated chromatographic techniques have become powerful tools largely used in clinical and forensic toxicology. This research project aimed to develop and validate new analytical methodologies based on hyphenated chromatographic techniques for the determination of NPS and classical drugs of abuse in biological matrices. Considering the polydrug consumption, a comprehensive high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS-MS) screening method was developed for the determination of 77 NPS (belonging to different chemical classes), 24 classic drugs and 18 related metabolites in blood, urine, and oral fluid. Currently, synthetic cannabinoids are the largest group of NPS monitored by the European Monitoring Centre for Drugs and Drug Addiction and to investigate the oral fluid pharmacokinetics of JWH-122, JWH-210 and UR-144 two new methodologies were developed. A gas chromatography-mass spectrometry (GC-MS) method for the determination of parent compounds and a complementary ultra-high performance liquid chromatography high resolution mass spectrometry (UHPLC-HRMS) confirmatory method for the quantification also of their metabolites. Finally, to keep in pace with the increasing spread of new synthetic cathinones an ultra-high performance liquid chromatography high resolution mass spectrometry (UHPLC-HRMS) method is presented for their simultaneous targeted screening and quantification in hair samples of consumers. All methods described here have been successfully validated and applied to authentic samples (blood, urine, oral fluid, hair) providing reliable results and proving to be sensitive methodologies suitable for use in clinical and forensic laboratories.
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Bellinaso, Laura <1988>. "Determinazione degli amminoacidi presenti nei leganti di natura proteica utilizzati nella pittura a tempera. Confronto tra GC-MS e HPLC-MS/MS". Master's Degree Thesis, Università Ca' Foscari Venezia, 2012. http://hdl.handle.net/10579/2292.
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Nello svolgimento di questa tesi sperimentale sono state affrontate alcune problematiche analitiche relative alla caratterizzazione di leganti proteici in campioni provenienti da dipinti murali quali, la presenza di miscele complesse e degradate, le interferenze dovute alla presenza di specie inorganiche, le piccole dimensioni dei campioni, il rischio di contaminazione. La procedura pre-analitica ha previsto la determinazione della composizione amminoacidica mediante estrazione, purificazione, idrolisi e derivatizzazione del campione al fine di caratterizzarlo sfruttando le potenzialità analitiche degli strumenti GC-MS e HPLC-MS/MS. Questo procedimento ha permesso di valutare analogie e differenze tra i risultati ottenuti sia nel caso della validazione della metodica analitica che successivamente dall’analisi di campioni reali.
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Wang, Lanquing. "Characterization of selenide drugs and their metabolites by hydride generation ICP-MS and HPLC/ICP-MS". Diss., Georgia Institute of Technology, 1995. http://hdl.handle.net/1853/28041.
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Umbach, Frank [Verfasser]. "Stabilitäts- und Metabolismusuntersuchungen neuer purinerger Wirkstoffe und Ectonucleotidase-Inhibitoren mittels HPLC-DAD-MS(/MS) / Frank Umbach". Bonn : Universitäts- und Landesbibliothek Bonn, 2014. http://d-nb.info/1047622815/34.
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Tu, Travis Y. "HPLC and MS/MS Method for the Separation and Identification of Inositol Phosphates in C. Reinhardtii". Scholarship @ Claremont, 2016. http://scholarship.claremont.edu/cmc_theses/1306.
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Inositol phosphates (IPs) have important cellular functions from teleomere maintenance (IP7 and IP8) to Ca2+ signaling pathways (1, 4, 5-IP3). Yet there is no robust, quantitative method to separate all the inositol phosphate isomers from IP1 to IP8. Four findings contributed heavily towards the development of a robust, quantitative IP isomer separation and identification method on the EskpertTM MicroLC 200+ QTrap 6500 system with a SelexIonTM DMS attachment. 1) TCA from inositol phosphate algal extractions was removed by elution with 100 mM ammonium carbonate, ammonium formate, or ammonium bicarbonate or by immobilized metal affinity chromatography (Fe-NTA columns). 2) A 250 mM ammonium carbonate and 25% methanol gradient was run on a weak anion exchange column to separate all the inositol phosphates from each other (IP1 through IP8. 3) Using ten different inositol phosphate isomer standards and fluoro- IP3 as an internal standard for future quantitation and recovery studies, isomer separation was obtained using SelexIonTM DMS with a 2- propanol modifier. 4) Ion suppression of inositol phosphate signals caused by 250 mM ammonium carbonate can be alleviated with a post-column dilution. The final assembled EskpertTM MicroLC 200+ QTrap 6500 system with a SelexIonTM DMS attachment and post-column dilution method was able to separate out IP isomers from IP1 to IP6 and detect IP7 and IP8. Once further optimized using the full compensation voltage range and a more polar modifier such as methanol, this method will allow the lipid biosynthesis pathways of C. reinhardtii, a promising candidate for algal biofuels, to be better studied.
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Jungfer, Elvira [Verfasser]. "Authentizitätsbestimmung ausgewählter Vaccinium-Spezies mittels HPLC-MS / Elvira Jungfer". München : Verlag Dr. Hut, 2013. http://d-nb.info/1045988286/34.
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Zonaro, Victor Alexandre [UNESP]. "Análise de estirilpironas de Cryptocarya por HPLC-DAD-MS". Universidade Estadual Paulista (UNESP), 2016. http://hdl.handle.net/11449/147999.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
As 5,6-diidro-2-pironas 6-substituídas, também conhecidas como estirilpironas, são uma importante classe de metabólitos secundários presentes em plantas. São substâncias biologicamente ativas, apresentando como, por exemplo, atividade anticâncer, antioxidante, antifúngica e antiviral. O gênero Cryptocarya, pertence à família Lauraceae e apresenta diversas estirilpironas em suas mais diversas partes: folhas, cascas, sementes e raízes. Foram selecionadas para análise as espécies C. mandioccana, C. moschata e C. botelhensis. Foram utilizadas apenas as folhas, por apresentarem facilidade para coleta e abundância. Este trabalho tem como objetivo a identificação das estirilpironas presentes em três espécies brasileiras de Cryptocarya com o uso das técnicas HPLC-DAD-MS. Foi desenvolvido método cromatográfico para análise do extrato hidroalcoólico das folhas de espécies de Cryptocarya por HPLC-DAD-MS utilizando etanol como fase orgânica na fase móvel. Com os espectros no UV foi possível identificar que as classes de metabólitos presentes nas amostras eram alcaloides, flavonoides e estirilpironas. Utilizando os dados de MS e MS/MS, foi possível a caracterização de estirilpironas presentes nos extratos, assim como a sugestão de suas fragmentações por espectrometria de massas, além da identificação dos íons m/z 163 e m/z 189 característicos da fragmentação das estirilpironas. Foi detectado o íon m/z 423 no extrato de C. moschata, referente a um possível dímero da goniotalamina, sem relato anterior para as espécies de Cryptocarya. Com o auxilio de pironas purificadas obtidas pelo grupo, foi possível a comparação de seus tempos de retenção com os dados obtidos a partir dos extratos de folhas das espécies de Cryptocarya ajudando na identificação das substâncias presentes. Além das estirilpironas, foram identificados os alcalóides menisperina e xantoplanina nas três espécies estudadas. Nota-se uma grande diferença na quantidade de metabólitos entre as espécies analisadas, sendo que a C. mandioccana é a mais rica em estirilpironas.
The 6-substituted 5,6-dihydro-2-pyrones, also known as styrylpyrones, are an important class of secondary metabolites present in plants. They are biologically active substances, presenting, for example, anticancer, antioxidant, antifungal and antiviral activity. The genus Cryptocarya belongs to the family Lauraceae and presents several styrylpyrones in its most diverse parts: leaves, barks, seeds and roots. The species C. mandioccana, C. moschata and C. botelhensiswere selected for analysis. We chose to use only leaves, because they are easy to collect and abundant. The objective of this work is to identify the styrylpyrones present in three Brazilian Cryptocarya species using the HPLC-DAD-MS techniques. A chromatographic method was developed to analyze the hydroalcoholic extract from leavesCryptocarya species by HPLC-DAD-MS using ethanol as the organic solvent in the mobile phase. With the UV spectra were possible to identify the classes of metabolites present in the samples as alkaloids, flavonoids and styrylpyrones. Addtitionaly, with the MS and MS2 data, was possible to characterize the styrylpyrones present in the extracts, as well as the suggestion of their fragments by mass spectrometry, in addition to the identification of íons m/z 163 and m/z 189 characteristics of the fragmentation of styrylpirones. The m/z 423 ion detected in C. moschata extract was tentatively attributed to a goniotalamine dimer, with no previuous reports for Cryptocarya species. With the help of purified pirones obtained by the group, it was possible to compare their retention times with the data obtained from leaf extracts of the Cryptocarya species, helping to identify the substances present. Besides the styrylpyrones, the alkaloids menisperin and xantoplanin were also identified in the three species studied. There is a great difference in the amount of metabolites among the analyzed species, with C. mandioccana being the richest in styrylpyrones.
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Zonaro, Victor Alexandre. "Análise de estirilpironas de Cryptocarya por HPLC-DAD-MS /". Araraquara, 2016. http://hdl.handle.net/11449/147999.
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Orientador: Alberto José CavalheiroBanca: Cíntia Duarte de Freitas Milagre
Banca: Marcelo Telascrêa
Resumo: As 5,6-diidro-2-pironas 6-substituídas, também conhecidas como estirilpironas, são uma importante classe de metabólitos secundários presentes em plantas. São substâncias biologicamente ativas, apresentando como, por exemplo, atividade anticâncer, antioxidante, antifúngica e antiviral. O gênero Cryptocarya, pertence à família Lauraceae e apresenta diversas estirilpironas em suas mais diversas partes: folhas, cascas, sementes e raízes. Foram selecionadas para análise as espécies C. mandioccana, C. moschata e C. botelhensis. Foram utilizadas apenas as folhas, por apresentarem facilidade para coleta e abundância. Este trabalho tem como objetivo a identificação das estirilpironas presentes em três espécies brasileiras de Cryptocarya com o uso das técnicas HPLC-DAD-MS. Foi desenvolvido método cromatográfico para análise do extrato hidroalcoólico das folhas de espécies de Cryptocarya por HPLC-DAD-MS utilizando etanol como fase orgânica na fase móvel. Com os espectros no UV foi possível identificar que as classes de metabólitos presentes nas amostras eram alcaloides, flavonoides e estirilpironas. Utilizando os dados de MS e MS/MS, foi possível a caracterização de estirilpironas presentes nos extratos, assim como a sugestão de suas fragmentações por espectrometria de massas, além da identificação dos íons m/z 163 e m/z 189 característicos da fragmentação das estirilpironas. Foi detectado o íon m/z 423 no extrato de C. moschata, referente a um possível dímero da goniotalamina, sem rela... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The 6 - substituted 5,6 - dihydro - 2 - pyrones, also known as styryl py rone s, are an important class of secondary metabolites present in plants. They are biologically active substances, presenting, for examp le, anticancer, antioxidant, antifungal and antiviral activity. The genus Cryptocarya belongs to the family Lauraceae and presents several styrylpyrones i n its most diverse parts: leaves, barks, seeds and roots. The species C. mandioccana, C. moschata and C. botelhensis were selected for an alysis. We chose to use only leaves, because they are easy to collect and abu n dant . The objective of this work is to identify the styrylpyrones present in three Brazilian Cryptocarya species using the HPLC - DAD - MS technique s. A chromatographic method was developed to analyze the hydroal coholic extract from leaves Cryptocarya species by HPLC - DAD - MS using ethanol as the organic solvent in the mobile phase. With the UV spectra were possible to identify the classes of metabolites present in the samples as alkaloids, flavonoids and styrylpyrones . Addtitionaly, with the MS and MS 2 data, was possible to characterize the styrylpyrones present in the extracts, as well as the suggestion of their fragments by mass spectrometry, in additi on to the identification of íons m/z 163 and m/z 189 characteristics of the f ragmentation of styry lpi rones. The m/ z 423 ion detected in C. moschata extract was tentatively attributed to a goniotalamine dimer, with no previuous reports for Cryptocarya species. W ith the help of purified pirones obtained by the group, it was possible to compare their retention times with the data obtained from leaf extracts of the Cryptocarya species, helping to identify the substances present. Besides the styrylpyrones, the alkaloids menisperin and xantoplanin were also identified in the three species studied. There is a great difference in the amount of...
Mestre
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Jungfer, Elvira [Verfasser]. "Authentizitätbestimmung ausgewählter Vaccinium-Spezies mittels HPLC-MS / Elvira Jungfer". Bonn : Universitäts- und Landesbibliothek Bonn, 2013. http://d-nb.info/1045872040/34.
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Schuhmann, Imelda. "Aufbau einer HPLC-UV-ESI-MS-MS-Datenbank und ihre Anwendung im Screening arktischer und antarktischer Meeresbakterien". Doctoral thesis, [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=977037568.
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Egle, Hannes. "Schnelles und zuverlässiges therapeutisches Drug-Monitoring mit HPLC-UV und LC-LC, MS-MS aus komplexen Matrizes". [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=974035548.
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Hernández, Moreno Luis Gustavo Eduardo. "Implementación de una Metodología Analítica para la Cuantificación de Acrilamida en Papas Chips por HPLC MS/MS". Tesis, Universidad de Chile, 2007. http://www.repositorio.uchile.cl/handle/2250/105663.
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Memoria para optar al título de Químico FarmacéuticoEn el año 2002 la Administración Nacional de Alimentos de Suecia detectó altas concentraciones de acrilamida que se formaba durante el calentamiento a altas temperaturas de alimentos ricos en almidón, como las papas fritas, pan, galletas, alimentos extruídos, etc. La acrilamida es genotóxico y ha sido clasificada como “probable carcinogénico para humanos” (Grupo 2A) por la Agencia Internacional de Investigación sobre Cáncer IARC, y esta exposición causa daño al sistema nervioso en seres humanos y animales Con motivo de este hallazgo, surgió el requerimiento urgente de desarrollar un método analítico sensible, robusto y de un costo razonable, que pueda cuantificar acrilamida en diferentes matrices de alimentos con bajos niveles de detección (Mg/Kg). Durante el desarrollo de esta memoria se implementó en el Centro de Investigación y Desarrollo en Grasas y Aceites (CIDGRA), Departamento de Ciencia de los Alimentos y Tecnología Química de la Facultad de Ciencias Químicas y Farmaceúticas de la Universidad de Chile, la metodología analítica para cuantificar acrilamida por HPLC MS/MS. Se determinó la precisión y exactitud en el análisis de acrilamida por HPLC MS/MS. Finalmente se aplicó la metodología para el análisis de papas fritas chips comerciales y elaboradas en el laboratorio dentro del Proyecto Heatox 506820. En conclusión se puede decir que se logró una respuesta total a los objetivos planteados siendo implementado en el Centro de Investigación y Desarrollo en Grasas y Aceites (CIDGRA), la metodología analítica para la extracción de acrilamida en papas chips y su cuantificación por HPLC MS/MS. Lo cual abre las puertas, para el comienzo de la investigación, de los alimentos de consumo habitual en nuestro país y que pudieran contener acrilamida, y así tomar las medidas necesarias en cuanto a políticas de salud, para el bienestar de nuestra población
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Bandeira, Nelson Miguel Grubel. "Determinação multiclasse de resíduos de medicamentos veterinários em músculo ovino por HPLC-FD E UHPLC-MS/MS". Universidade Federal de Santa Maria, 2015. http://repositorio.ufsm.br/handle/1/10603.
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Conselho Nacional de Desenvolvimento Científico e TecnológicoThe application of good livestock practices in the sheep industry is a growing concern on food security due to the increase of financial transactions in this sector in Brazil, mainly due to imports. To perform the monitoring Maximum Residue Limits (MRLs) are adopted such as suitable matrices for monitoring. The deposition of residues of veterinary drugs in sheep muscle is verified by appropriate analytical methods. In this work it were developed two methods aiming to determine avermectins residues in ovine muscle by HPLC-FD using QuEChERS method and also a method for the determination of different classes of veterinary drugs using UHPLC-MS / MS. The extraction was optimized by comparing the three most common variants of QuEChERS method and also the application of experimental design to select the best composition of the clean-up stage, varying amounts of PSA and C18. Some parameters such as derivatization step, the mobile phase composition, mobile phase flow, and the conditions of ionization source and ions entrance of the system were also optimized. The developed methods were validated by reference to Decision 657 of 2002 published by the European Community. The following parameters were evaluated: selectivity; linearity and working range; matrix effect; limits of detection and quantification; decision limit and detection capability; accuracy; and precision. The results were satisfactory, with the values of recovery in the concentration ranges required for each rated level, determination coefficients for curves prepared in solvent blank extract and white matrix were above 0.99 (except for ciprofloxacin) and linear working range was from 5 to 200 μg kg-1 for UHPLC-MS / MS and 2.5 to 112.5 μg kg-1 for HPLC-FD. There were no significant matrix effects only sulfamethoxazole, trichlorfon and albendazole by determining UHPLC-MS / MS. The limits of detection for HPLC-FD ranged from 4 to 18.4 μg kg-1 and decision limits from 10.7 to 59.4 μg kg-1 for UHPLC-MS / MS detection limits ranged from 1.7 to 33.3 μg kg-1 and for decision threshold from 5 to 100 μg kg-1. The method was considered suitable for routine analysis, and the confirmation was only possible using UHPLC-MS / MS, and the method was applied for determination of veterinary drug residues in sheep muscle in 11 samples by HPLC-FD and 36 UHPLC MS / MS
A aplicação das boas práticas agropecuárias na ovinocultura é uma preocupação crescente na segurança alimentar em virtude do aumento das movimentações financeiras neste setor no Brasil, principalmente em função das importações. Para realização do monitoramento são adotados Limites Máximos de Resíduo (LMR) e matrizes adequadas para monitoramento. A deposição de resíduos de medicamentos veterinários em músculo ovino é verificada através de métodos analíticos adequados. Neste trabalho desenvolveram-se dois métodos um buscando a determinação de resíduos de avermectinas em músculo ovino por HPLC-FD, utilizando o método QuEChERS e, também, um método para determinação de diferentes classes de medicamentos veterinários utilizando UHPLC-MS/MS. A extração foi otimizada pela comparação das 3 variações mais usuais do método QuEChERS e, também, aplicação de planejamento experimental para escolha da melhor composição da etapa de clean-up, variando as quantidades de PSA e C18 aplicadas. Alguns parâmetros como etapa de derivatização, composição da fase móvel, vazão da fase móvel e condições da fonte de ionização e entrada de íons no sistema também foram otimizadas. Os métodos desenvolvidos foram validados tomando como referência a Decisão 657 de 2002 publicada pela Comunidade Europeia. Foram avaliados os seguintes parâmetros: seletividade; linearidade e faixa de trabalho; efeito matriz; limites de detecção e quantificação; limite de decisão e capacidade de detecção; exatidão; e precisão. Os resultados obtidos foram satisfatórios, tendo como valores de recuperação nas faixas de concentração exigidas para cada nível avaliado, os coeficientes de determinação para curvas preparadas em solvente, em extrato branco e matriz branco ficaram acima de 0,99 (com exceção de ciprofloxacin) e a faixa linear de trabalho foi de 5 a 200 μg kg-1 para UHPLC-MS/MS e de 2,5 a 112,5 μg kg-1 para HPLC-FD. Não foram observados efeitos de matriz significativos apenas para sulfametoxazol, triclorfon e albendazol na determinação por UHPLC-MS/MS. Os limites de detecção para HPLC-FD variaram de 4 a 18,4 μg kg-1 e os limites de decisão de 10,7 a 59,4 μg kg-1, para UHPLC-MS/MS os limites de detecção variaram de 1,7 a 33,3 μg kg-1 e para limite de decisão de 5 a 100 μg kg-1. O método foi considerado adequado para análises de rotina, sendo que a confirmação só foi possível utilizando UHPLC-MS/MS, e o método foi aplicado para determinação de resíduos de medicamentos veterinários em músculo ovino em 11 amostras por HPLC-FD e 36 por UHPLC-MS/MS.
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Samson-Thibault, François. "Analyse des modifications de la cytosine après oxydation de l'ADN par digestion enzymatique et HPLC-MS/MS". Mémoire, Université de Sherbrooke, 2012. http://hdl.handle.net/11143/6359.
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Résumé: Les dommages à la cytosine, spontanés et induits par des oxydants, sont probablement la principale cause des transitions GC vers AT, la mutation du génome la plus commune chez les organismes aérobiques. Ces dommages sont impliqués dans le processus de mutagenèse, dans le vieillissement et dans le cancer. Les dommages à la cytosine par les oxydants et les radicaux libres sont nombreux et ont été découverts et étudiés dans les monomères de cytosines et dans de courts oligonucléotides. Dans cette étude, nous avons développé une méthode d'analyse par HPLC-MS/MS des plus importants produits d'oxydation de la cytosine dans l'ADN. Cette méthode permet l'analyse de la formation de 5-hydroxy-2'-désoxycytidine (5-OH-dC), de 5,6-dihydroxy-5,6-dihydro-2'-désoxyuridine (dUg), de 1-(2-désoxyribose)-5-hydroxyhydantoïne (HdU) et de 3-(2-désoxyribose)-1-carbamoy1-4,5-dihydroxy-2-oxo-imidazolidine (C422-dC) lors d'irradiation aux rayons gamma par réaction de type fenton et par ozonolyse. Les résultats de l'irradiation de l'ADN aux rayons gamma (en modifications/106bases/Gy) sont de 1.62 pour la 5-OH-dC, de 1.48 pour le dUg, de 7.43 pour la HdU et de 1.38 pour la C422-dC. La réaction de type fenton avec le cuivre donne une formation de dommages environ 25 fois plus grande qu'avec le fer et les deux types (cuivre et fer) donnent des ratios de produits semblables à ceux par les rayons gamma avec une augmentation de la 5- OH-dC et une diminution de la HdU. L'exposition .de l'ADN à l'ozone donne une très grande formation de la HdU et une faible formation des autres produits d'oxydation.//Abstract: The damages to cytosine, spontaneous and inducted by oxidants, are probably the principal cause of the GC to AT transition, the most important mutation of the genome for the aerobic organisms. Those damages are involved in the process of mutagenesis, aging and cancer. The damages to cytosine by oxidants and fre radicals are numerous and have been discovered and studied in monomers of cytosine and in short oligonucleotides. In this study, we have developed a analysis method of the most important oxidation products of cytosine in DNA by HPLC-MS/MS. This method allows the analysis of the formation of 5-hydroxy-2'-deoxycytidine (5-OH-dC), de 5,6-dihydroxy-5,6-dihydro-2'-desxyuridine (dUg), de 1-(2-deoxyribose)-5-hydroxyhydantoin (HdU) et de 3-(2-deoxyribose)- 1-carbamoyl-4,5-dihydroxy-2-oxo-imidazolidine (C422-dC) after irradiation by gamma rays, by fenton type reaction and ozonolysis. The results of the irradiation of DNA by gamma rays (in modifications10[indice supérieur 6] bases/Gy) are of 1.62 for 5-OH-dC, of 1.48 for dUg, of 7.43 for HdU and of 1.38 for C422-dC. The fenton type reaction with copper gives a formation of damages about 25 times higher than with ferrous and both kind gives a ratio of formation similar to the the ones by gamma rays with a increase of 5-OH-dC and a decrease of HdU. The exposition of DNA to ozone gives a strong formation of HdU and a small formation of the other modifications. [symboles non conformes]
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Schütze, Gregor. "Entwicklung, Validierung und Anwendung einer HPLC-MS/MS-Methode zur quantitativen Bestimmung von Tryptophan und seinen Metaboliten". Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-175326.
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Die vorliegende Arbeit beschäftigt sich mit der Entwicklung, Validierung und Anwendung einer HPLC-MS/MS-Methode zur quantitativen Bestimmung von Tryptophan und seinen Metaboliten. Der Stoffwechsel der proteinogenen, essentiellen Aminosäure Tryptophan umfasst nicht nur den Neurotransmitter Serotonin und das in der Epiphyse gebildete Hormon Melatonin, sondern er schließt auch den Kynurenin Pathway mit ein. Die neurobiochemische Bedeutung dieses Stoffwechselweges wird bereits durch die Tatsache offensichtlich, dass im zentralen Nervensystem mehr als 95% des Tryptophans über diesen Weg metabolisiert werden. Die Intermediate des Kynurenin Pathways spielen eine bedeutende Rolle als Mediatoren zwischen Immun- und Nervensystem. Dabei nehmen sie potenziell Einfluss auf höhere zentralnervöse Funktionen, wie Kognition, Verhalten und Affekt, weshalb sie von herausragender Bedeutung für das Forschungsgebiet der Psychoneuroimmunologie sind.
Auf molekularer Ebene wird Tryptophan im ersten Schritt durch die beiden Schlüsselenzyme Tryptophan-2,3-dioxygenase und Indoleamin-2,3-dioxygenase zu N-Formylkynurenin oxidiert. Durch Kynureninformidase erfolgt eine Metabolisierung von N-Formylkynurenin zu Kynurenin - der Ausgangssubstanz des gleichnamigen Stoffwechselweges. Während die Tryptophan-2,3-dioxygenase ausschließlich Tryptophan oxidiert, ist die Indolamin-2,3-dioxygenase nicht nur für den Abbau von Tryptophan spezifisch, sondern für alle Indolamine, inklusive Serotonin und Melatonin. Die Aktivität der Tryptophan-2,3-dioxygenase wird dabei durch Glucocorticoide, die der Indolamine-2,3-dioxygenase durch bestimmte Zytokine reguliert. Die Indolamin-2,3-dioxygenase spielt ebenfalls eine wichtige Rolle in der Modulation der T-Zell-Toleranz. Des Weiteren werden die Aktivitätszustände diverser Zellen des angeborenen und des erworbenen Immunsystems durch Metabolite des Kynurenins gesteuert. Einige Bestandteile des Kynurenin Pathways besitzen neuroprotektive oder neurotoxische Eigenschaften. So ist beispielsweise die neuroprotektive Kynureninsäure der bisher einzige bekannte endogene Antagonist des NMDA-Rezeptors und zusätzlich ein nichtkompetitiver Antagonist des α7-nikotinischen Acetylcholinrezeptors. Dieser Substanz steht der alternative Kynurenin-Metabolit, das exzitotoxische Neurotoxin Chinolinsäure gegenüber, welcher ein Agonist des NMDA-Rezeptors ist.
Bei Untersuchungen zum Tryptophanstoffwechsel ist es daher von entscheidender Bedeutung nicht nur einzelne Substanzen sondern die Gesamtheit der Metabolite zu quantifizieren. Nur so kann die Balance zwischen neuroprotektiven und neurodegenerativen Substanzen, aber auch die Balance zwischen inhibierenden und aktivierenden Mediatoren des Immun- und Nervensystems dargestellt werden. In klinischen Studien können so Dysbalancen mit der Pathogenese einzelner Erkrankungen assoziiert werden, wodurch die Möglichkeit besteht, dass Biomarker zur frühzeitigen Diagnose identifiziert und neue Therapieansätze postuliert werden können.
In dieser Arbeit wird eine neue HPLC-MS/MS-Methode vorgestellt, mit der eine bisher unerreichte Vielfalt an Tryptophan-Metaboliten quantifiziert werden kann. Die Probenvorbereitung ist eine einfache und kosteneffiziente Proteinfällung in zwei Stufen, wobei unterschiedliche Matrices, wie beispielsweise Serum oder Liquor cerebrospinalis, als Probenmaterial verwendet werden können. Um Analyte mit sehr niedrigen physiologischen Konzentrationen zu detektieren, wurde für diese eine Derivatisierung entwickelt. Die Methode benötigt ein sehr geringes Probenvolumen, ist durch die Verwendung der HPLC-MS/MS-Technologie äußerst sensitiv und spezifisch und umfasst alle bedeutenden Metabolite des Tryptophanstoffwechsels mit nur einer Probenvorbereitung. Die Robustheit wurde in einer ausführlichen Validierung bestätigt. Dabei wurden auch die analytischen Grenzwerte und Kennzahlen ermittelt, sowie die Stabilität der Proben untersucht. In Versuchen zu präanalytischen Einflüssen wurde festgestellt, dass unterschiedliche Zeitpunkte der Blutentnahme, unterschiedliche Blutentnahmesysteme und die Nahrungsaufnahme zu gravierenden Änderungen der Konzentrationen der Analyte führen. So führt die Verwendung unterschiedlicher Blutentnahmesystem beispielsweise bei der Quantifizierung des Metaboliten Picolinsäure zu Ergebnissen, die sich um bis zu 60% unterscheiden. Es wurde nachgewiesen, dass es postprandial zu starken interindividuell unterschiedlichen Änderungen der Konzentrationen einzelner Intermediate kommt. Dabei verhalten sich die freien und proteingebundenen Metabolite ebenfalls unterschiedlich. Deshalb muss für Studien eine normierte Probengewinnung mit möglichst exakten Bedingungen eingeführt werden. Auch konnte gezeigt werden, dass die Metabolisierung des Tryptophans in vivo einer circadianen Rhythmik unterliegt. Aufbauend auf diesen Erkenntnisse wurden in einem in vitro Experiment tageszeitabhängige Schwankungen nachgewiesen und der Einfluss einer LPS-Stimulation auf den Kynurenin Pathway untersucht.
Durch den Einsatz dieser neuen HPLC-MS/MS-Methode besteht die Möglichkeit, die funktionellen Zusammenhänge zwischen Intermediaten des Kynurenin Pathways und diversen immunologischen, neurochemischen und anderen pathophysiologischen Vorgängen zu untersuchen. In der Literatur gibt es eine Vielzahl an Hinweisen darauf, dass der Tryptophanstoffwechsel an der Pathogenese unterschiedlichster Erkrankungen beteiligt ist. Jedoch bedarf es der genauen Klärung der Zusammenhänge und der Möglichkeit, die Gesamtheit einer potentiellen Störung des Tryptophanstoffwechsels zu erfassen. So sind im Bereich psychiatrischer und neurologischer Erkrankungen beispielsweise Schizophrenie, Alzheimer-Krankheit, Chorea Huntington und Epilepsie zu nennen. Hier wurden in publizierten Studien häufig nur einzelne Metabolite quantifiziert und isoliert betrachtet. Darüber hinaus konnten unterschiedliche Arbeitsgruppen zeigen, dass eine Aktivierung des Kynurenin Pathways auch bei der Pathogenese von Tumoren beteiligt ist, weshalb auch hier die von uns entwickelte Methode für weitere Studien von großem Nutzen ist.
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Myers, Tanya R. "New Techniques for the Qualitative and Quantitative Measurement of Naturally-Ocurring Gonadotropin-Releasing Hormone Analogues by Mass Spectrometry". Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/chemistry_diss/12.
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GnRH peptides have been discovered in a wide variety of vertebrate and invertebrate organisms, and work is ongoing to characterize additional unique isoforms. This dissertation describes the investigation of reversed-phase chromatographic and mass spectrometric behavior of GnRH peptides, the development and application of an LC-MS/MS method for qualitative identification of GnRH peptides, and the comprehensive validation of an LC-MS/MS method for simultaneous, quantitative measurement of hydroxyproline9GnRH (Hyp9GnRH) and mammalian GnRH (mGnRH) in rat brain tissues. Chromatographic and mass spectrometric behavior of GnRH isoforms was characterized for six GnRH model peptides. Using reversed-phase high performance liquid chromatography (HPLC), nearly complete separation of the model GnRH peptides was achieved. Evaluation of electrospray source conditions indicated that certain parameters can be adjusted to affect the abundance of selected charge states and improve response. Using the conditions found to be optimal for GnRH peptides in general, a method was developed to facilitate characterization of novel GnRH isoforms or confirm the identity of known isoforms. Fragmentation patterns for six model GnRH isoforms were examined to determine what portion of the primary sequence could be elucidated by de novo sequencing, and a simple solid phase extraction protocol was developed to isolate the model GnRH compounds from tissue samples. Application of the method to rat brain samples resulted in successful isolation and structural confirmation of hydroxyproline9GnRH and mammalian GnRH. A quantitative method for the determination of concentrations of hydroxyproline9GnRH and mammalian GnRH in rat brain tissue was developed and rigorously validated. Guinea pig brains were found to be a suitable substitute matrix for rat brains, and accuracy and precision were determined after four validation runs. Stability of both peptides in samples over long-term storage and under experimental conditions were evaluated, and the LC-MS/MS method was compared to an enzyme-linked immunoassay. Thirty-one brains from Sprague-Dawley rats were analyzed using the LC-MS/MS procedure and compared to published results for Hyp9GnRH and mGnRH.
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Armando, Yara Popst. "Avaliação da bioequivalência entre comprimido convencional e comprimido de desintegração oral contendo 8 mg de ondansetrona". Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/9/9139/tde-08102009-190008/.
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A ondansetrona (1,2,3,9-tetrahidro-9-metil-3-[(2-metil-1H-imidazol-1-il)metil]-4H-carbazol-ona) é o primeiro fármaco da classe dos antagonistas seletivos dos receptores de serotonina 5-HT3. É utilizada na prevenção de náusea e vômito induzidos por agentes quimioterápicos. O objetivo deste estudo foi avaliar a equivalência terapêutica através da análise da bioequivalência de dois produtos contendo 8 mg de ondansetrona, sendo um sob a forma de comprimidos de liberação convencional e outro sob a forma de comprimidos de desintegração oral, produzidos por laboratórios distintos. O ensaio de bioequivalência entre o produto teste (Vonau® flash) e o produto referência (Zofran®) foi do tipo randomizado, cruzado e aberto. Os produtos foram administrados por via oral aos voluntários em dose única de 8 mg de ondansetrona. Amostras de sangue foram coletadas até 24 horas após a administração e analisadas através de método de cromatografia líquida de alta eficiência acoplada a um detector de massas. As curvas médias de decaimento plasmático dos produtos teste (Vonau® flash) e referência (Zofran®) e as médias dos parâmetros farmacocinéticos Cmax (referência: 31,88 ng/mL; teste: 30,42 ng/mL); tmax (referência: 1,99 h; teste: 2,15 h), ASC0-t (referência: 227,66 ng×h/mL; teste: 223,68 ng×h/mL) e ASC0- (referência: 252,76 ng×h/mL; teste: 248,22 ng×h/mL) apresentaram-se semelhantes. O intervalo de confiança 90 % para a razão de Cmax (87,5 % - 103,8 %), ASC0-t (89,3 % - 107,2 %) e ASC0- (89,7 106,0 %) encontram-se entre 80 125 %, dentro dos limites propostos pela ANVISA. Conclui-se que os produtos teste e referência são bioequivalentes, podendo ser administrados de forma intercambiável, sem prejuízo do efeito terapêutico.Ondansetron (1,2,3,9-tetrahydro-9-methyl-3-[2-methyl-1H-imidazol-1yl)methyl]-4H-carbazol-one is the first drug of the class of antagonists selective receptor 5-HT3. It is used in the prevention of nausea and vomiting caused by chemotherapy agents. The purpose of this study was to evaluate the therapeutic equivalence examining the bioequivalence of two products containing 8 mg ondansetron, one in the conventional release tablet, and other in oral disintegration tablet produced by different laboratories. The bioequivalence assay between the test product (Vonau® flash) and reference product (Zofran®) was randomized, crossover and open study. The medication was administered in a single dose of 8 mg of ondansetron. Blood samples were collected until 24 hours after administration and analyzed using a validated high-performance liquid chromatographic method with mass spectrometer detection. The average plasmatic decay curves obtained for the test product (Vonau® flash) and reference product (Zofran®) and the averages of pharmacokinetics parameters Cmax (reference: 31.88 ng/mL; test: 30.42 ng/mL); tmax (reference: 1.99 h; test: 2.15 h), AUC0-t (reference: 227.66 ng×h/mL; test: 223.68 ng×h/mL) and AUC0- (reference: 252.76 ng×h/mL; test: 248.22 ng×h/mL) has been similar. The 90 % confidence intervals for the ratio of Cmax (87.5 % - 103.8 %), AUC0-t (89.3 % - 107.2%) and AUC0- (89.7 % - 106.0 %) values for the test and reference products are within the 80 125 % interval proposed by ANVISA. It was concluded that the test and reference products are bioequivalent and can be considered interchangeable in medical practice, without prejudice to the therapeutic effect.
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Brioschi, Tatiane Maria de Lima Souza. "Avaliação da bioequivalência de comprimidos contendo 10 mg de cloridrato de ciclobenzaprina". Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/9/9139/tde-13122006-091912/.
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A ciclobenzaprina é um relaxante muscular de ação central estruturalmente similar aos antidepressivos tricíclicos. O objetivo deste trabalho foi avaliar a bioequivalência de comprimidos contendo 10 mg de cloridrato de ciclobenzaprina em voluntários sadios. O estudo de bioequivalência entre o produto teste (Miosan®) e referência (Flexeril®) foi do tipo randomizado, aberto e cruzado. Os produtos foram administrados por via oral aos voluntários em dose única de 10 mg de cloridrato de ciclobenzaprina. Amostras de sangue foram coletadas até 240 horas após a administração do fármaco e quantificadas por método previamente validado através de cromatografia líquida de alta eficiência acoplada a um detector de massas. As curvas médias de decaimento plasmático dos produtos teste (Miosan®) e referência (Flexeril®) foram semelhantes ASC0-t (teste: 193,00 ngxh/mL; referência: 191,66 ngxh/mL) e ASC0∞ (teste: 211,34 ngxh/mL; referência: 209,35 ngxh/mL). Assim como os parâmetros farmacocinéticos relativos à absorção de ciclobenzaprina, Cmax (teste: 7,16 ng/mL; referência: 6,95 ng/mL), tmax (teste: 4,61 h; referência: 4,48 h), Ka (referência: 0,79; teste: 0,67) e t(1/2)a (referência: 1,79 h; teste: 2,02 h). Os parâmetros farmacocinéticos relativos à eliminação plasmática de ciclobenzaprina Cl (teste: 31,15 L/h; referência: 31,73 L/h), Vd (teste: 1378,54 L e referência (1357,87 L), kß (referência: 0,08; teste: 0,08), t(1/2)ß (referência: 9,43 h; teste: 9,20 h), kγ (referência: 0,02; teste: 0,02) e t(1/2)γ (referência: 32,92 h; teste: 31,67 h) também apresentaram-se semelhantes entre os dois produtos. A análise multivariada realizada por meio da análise de variância (ANOVA), para a avaliação dos efeitos produto, grupo e período, revelou a ausência destes efeitos, indicando que o delineamento do estudo foi adequado. Os resultados do intervalo de confiança (I.C. 90 %) para a razão de Cmax (93,0 % - 112,0 %), ASC0-t(92,6 % - 111,1 %) e ASC0∞ (93,1 % - 110,4 %), encontram-se dentro dos limites estabelecidos pela ANVISA e FDA (80 - 125 %). A análise estatística dos parâmetros Cmax, ASC0-t e ASC0-∞ indicam que não há diferenças entre os dois produtos contendo 10 mg de cloridrato de ciclobenzaprina. Com base nos resultados deste estudo, conclui-se que os produtos avaliados são bioequivalentes e podem ser considerados intercambiáveis na terapêutica.Cyclobenzaprine is a centrally acting muscle relaxant that has similarity with a tricyclic antidepressant. The purpose of this study was to evaluate the bioequivalence of two brands of cyclobenzaprine 10 mg tablets in healthy volunteers. The procedure of bioequivalence between test product (Miosan®) and reference product (Flexeril®) was a randomized, open and crossover study. The products were administered in a single oral dose of 10 mg of cyclobenzaprine hydrochloride to healthy volunteers. Blood samples were collected until 240 hours after administration and quantified by validated method using high-pressure liquid chromatography with mass spectrometric detection. The average plasmatic decay curves of test (Miosan®) and reference (Flexeril®) products were similar ASC0-t (test: 193,00 ngxh/mL; reference: 191,66 ngxh/mL), in the same way that absorption parameters Cmax (test: 7,16 ng/mL; reference: 6,95 ng/mL), tmax (test: 4,61 h; reference: 4,48 h), Ka (reference: 0,79; test: 0,67) e t(1/2)a (reference: 1,79 h; test: 2,02 h). The elimination parameters Cl (test: 31,15 L/h; reference: 31,73 L/h), Vd (test: 1378,54 L e reference (1357,87 L), kΒ (reference: 0,08; test: 0,08), t(1/2)β (reference: 9,43 h; test: 9,20 h), kγ (reference: 0,02; test: 0,02) e t(1/2)γ (reference: 32,92 h; test: 31,67 h) were similar between products too. The multivariate analysis accomplished trough analysis of variance (ANOVA), for assessment of product, group and period effects, revealed the absence of any of these effects in the present study, indicating that the crossover design was properly performed. The 90 % confidence intervals for the ratio of Cmax(93,0 % - 112,0 %), AUC0-t(92,6 % - 111,1 %) and AUC0∞ (93,1 % - 110,4 %) values for the test and reference products are within the 80 - 125 % interval proposed by ANVISA e FDA. Statistical analysis of Cmax, AUC0-t e AUC0-∞ parameters indicated no significant difference between two brands of 10 mg cyclobenzaprine hydrochloride products. Based in the results of this study, we can conclude that the two products are bioequivalent and can be considered interchangeable in the medical practice.
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Nascimento, DemÃtrius Fernandes do. "Nimodipine determination in human plasma by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS-MS)". Universidade Federal do CearÃ, 2005. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=31.
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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgicoA rapid, specific and highly sensitive liquid chromatography-tandem mass spectrometry method was developed to determine nimodipine in human plasma using dibucaine as the internal standard (IS) is described. The analyte (m/z 418,6 > 342,6) and IS (m/z 344,2 > 271,0) were extracted from plasma samples by liquid-liquid extraction using hexane-ethyl acetate (1:1v/v). Chromatography was performed on a Varian Polaris C18 analytical column (3 micrometer, 50 x 2,0 mm) and pre-column SecurityguardTM C18 (4,0 x 3,0 mm). The phase mobile consisted of Acetonitrile-Ammonium acetate 0.02 ml/L (80:20v/v). The method had a chromatographic run time of 4.5 min and linear calibration curve over the range 0.1- 40 ng/mL (r2 > 0.9938). The limit of quantification (LQ) was 0.1 ng/mL. The intra-day precision for the limit quantification was 0.00% (batch 01), 5.71% (batch 02) and 5.27% (batch 03); for the quality controls low (QCL), middle (QCM) and high (QCH) the results were respectively 8.57, 0.81 and 1.37%. The inter-day precision for LQ and QCL, QCM and QCH were respectively: 7% and 5.46, 4.12 and 3.37%. The intra-day accuracy for LQ was 110, 96 and 104%; for QCL, QCM and QCH the results were100.67, 109.09 and 109.72% respectively. The results of the inter-day accuracy for LQ, QCL, QCM and QCH were respectively and 110.0, 96.0, 104.0% for the limit of quantification and 8.57, 0.81, 1.37% and 100.67, 109.09, 109.72% respectively: 103% e 102.89, 106.60, 109.69%. This validated method was successfully applied for the pharmacokinetic profiles of nimodipine tablets administered to 24 healthy volunteerâs participant of bioavailability comparative study. Geometric mean of Test formulation/Refernce formulation individual percent ratio was 104,56% for AUC0-48h and 55,73% for Cmax. The 90% for the confidence intervals (CI) were 94,80-115,32% e 44,73-69,42%, respectively. The values of half-life and Cmax for test formulation and reference formulation were 27,83;32,78h and 9,48;18,76ng/mL, respectively. The values of Tmax were 2,34;0,98h for the formulations test and reference respectively. Since the 90% CI for Cmax and AUC0-48h, were within the 80-125% interval proposed by the âFood and Drug Administrationâ and ANVISA, it was concluded that the two formulations of nimodipine 30mg tablets were not bioequivalent, according to the rate of absorption after single dose administration.
Um mÃtodo rÃpido, sensÃvel e especÃfico de Cromatografia LÃquida de Alta EficiÃncia acoplada à Espectrometria de Massa (LC-MS-MS) foi desenvolvido para determinar nimodipino (analito) em plasma humano usando dibucaÃna como padrÃo interno (PI). O analito (m/z 418,6 > 342,6) e o PI (m/z 344,2 > 271,0) foram extraÃdos de amostras de plasma atravÃs de extraÃÃo lÃquido-lÃquido utilizando hexano-acetato de etila (1:1v/v). As corridas cromatogrÃficas foram executadas utilizando-se uma coluna analÃtica Varian Polaris C18 (3 micrÃmetros, 50 x 2,0 mm) e uma prÃ-coluna SecurityguardTM C18 (4,0 x 3,0 mm). A fase mÃvel consistiu de acetonitrila-soluÃÃo de acetato de amÃnio 0,02 mol/L (80:20v/v). O mÃtodo teve um tempo total de corrida de 4,5 min e uma curva de calibraÃÃo linear que variou de 0,1-40 ng/mL. O limite de quantificaÃÃo de 0,1 ng/mL. A precisÃo intra-ensaio para o limite de quantificaÃÃo (LQ) foi 0,00% (lote 01), 5,71% (lote 02) e 5,27% (lote 03); para os controles de qualidade baixo (CQB), mÃdio (CQM) e alto (CQA) os resultados foram respectivamente: 8,57, 0,81 e 1,37%. A precisÃo interensaio para o LQ e os CQB, CQM e CQA foram respectivamente de: 7% e 5,46, 4,12 e 3,37%. A exatidÃo intra-ensaio para o LQ foi 110, 96 e 104%; para CQB, CQM e CQA os resultados foram 100,67, 109,09 e 109,72% respectivamente. Os resultados da exatidÃo interensaio para o LQ, CQB, CQM e CQA foram respectivamente de: 103% e 102,89, 106,6, 109,69%. Este mÃtodo foi aplicado para a avaliaÃÃo do perfil farmacocinÃtico do nimodipino administrado em 24 voluntÃrios sadios participantes de um estudo de biodisponibilidade comparativa. A mÃdia geomÃtrica da FormulaÃÃo teste/FormulaÃÃo referÃncia para as porcentagens individuais foi 104,56% para ASC0-48h e 55,73% para Cmax. Os intervalos obtidos a partir do intervalo de confianÃa (IC) de 90% foram 94,80-115,32% e 44,73-69,42% respectivamente. Os valores de meia-vida e Cmax para as formulaÃÃes teste e referÃncia foram de 27,83;32,78h e 9,48;18,76ng/mL, respectivamente. Os valores de Tmax foram de 2,34;0,98h para as formulaÃÃes teste e referÃncia, respectivamente. Considerando o IC de 90% para Cmax e ASC0-48h dentro da variaÃÃo de 80-125% proposto pelo Food and Drug Administration e ANVISA, as duas formulaÃÃes de nimodipino 30mg nÃo sÃo bioequivalentes quanto à taxa de absorÃÃo (Cmax) apÃs uma Ãnica administraÃÃo.
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Mataveli, Lidiane Raquel Verola 1983. "Metalômica comparativa de soja [Glycine max (L.) Merrill] transgênica e não-transgênica utilizando sistema multidimensional de separação". [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/248607.
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Orientadores: Marco Aurélio Zezzi Arruda, Ljubica TasicTese (doutorado) - Universidade Estadual de Campinas, Instituto de Química
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Resumo: Este trabalho consistiu em estudos de metalômica comparativa de sementes de soja transgênica (T) e não-transgênica (NT). Para tanto, as sementes de soja T e NT foram comparadas, em um primeiro, momento levando-se em conta: concentração total dos elementos, comportamento dos elementos durante extração utilizando-se fracionamento sequencial, e bioacessibilidade dos elementos após procedimento de digestão gastrointestinal simulada (in vitro). As análises preliminares foram feitas utilizando-se ICP-MS com analisador de massas quadrupolar, e as análises posteriores utilizando espectrometria de massas de alta resolução com plasma acoplado indutivamente (HR-ICP-MS). Foram determinados 25 elementos em concentrações variando de ng g até %. Foi observado que as sementes de soja T e NT exibem diferenças estatisticamente significantes nas concentrações de Cu, Fe e Sr, sendo os dois primeiros apresentando maiores concentrações na semente T, e, o último, com maior concentração nas sementes de soja NT. Estes resultados também se refletiram nos conteúdos desses elementos em extratos aquosos e resíduos obtidos por meio de fracionamento sequencial. Ainda, os experimentos de bioacessibilidade realizados mostraram que as frações bioacessíveis de Cu, Fe, e outros elementos (Mn, S, Zn) contribuíram em maior porcentagem para a concentração total dos elementos nas sementes de soja T do que para as sementes de soja NT. Posteriormente, foi dada continuidade aos estudos utilizando cromatografia líquida bidimensional off line para as amostras de sementes de soja, sendo a primeira dimensão constituída de cromatografia de exclusão por tamanho (SEC,) e a segunda dimensão constituída de cromatografia de troca aniônica (AEX). Na primeira dimensão cromatográfica foram identificadas três frações contendo metais por meio da hifenação SEC-ICP-MS: a primeira correspondendo a massas molares entre 38,1 e 181,1 kDa, a segunda correspondendo a massas molares entre 8,2 e 17,2 kDa, e a terceira fração correspondendo a massas molares entre 0,4 e 3,8 kDa. As três frações identificadas foram separadas, liofilizadas, e separadas novamente utilizando a cromatografia de troca aniônica. Foram detectados metais em todas as frações separadas por SEC: três sub-frações da primeira fração, uma sub-fração na segunda fração e três sub-frações na terceira fração. Os eluatos foram coletados, liofilizados, digeridos e levados ao espectrômetro de massas com fonte de ionização por electrospray (ESI-MS) para identificação de proteínas. Foram identificadas 33 e, entre elas, duas proteinas previamente relacionadas a metais foram encontradas: seed lipoxygenase 1 e b-conglycinin. Após a separação na segunda dimensão cromatográfica, as sub-frações resultantes foram liofilizadas e submetidas a uma terceira dimensao de separacao, utilizando a eletroforese em gel de poliacrilamida unidimensional (SDS-PAGE). As bandas obtidas por SDS-PAGE foram recortadas e digeridas a fim de analisar os metais presentes nas mesmas, destacando os resultados obtidos para Fe, onde o mesmo foi quantificado nas bandas da sub-fração onde o pico deste elemento foi encontrado nas análises por AEX-ICP-MS. Ainda, as bandas foram digeridas tripticamente a fim de identificar as proteínas presentes, e, novamente, proteínas associadas a metais foram identificadas: chain A lipoxygenase-3 (Soybean) complex with 13(S)-hydroperoxy- 9(Z),11(E)-octadecadienoic acid; beta-amylase [Glycine max]; seed lipoxygenase-1, lipoxygenase [Glycine max], seed lipoxygenase-2 (PISUM SATIVUM) e beta-conglycinin
Abstract: This work consisted of comparative metallomics studies of transgenic (T) and nontransgenic (NT) soybean seeds. To that end, T and NT seeds were compared at first taking into account: the total concentration of the elements, the elements behavior during extraction using sequential fractionation, and bioacessibility of the elements after simulated gastrointestinal digestion procedure (in vitro). Preliminary analyzes were done using ICP-MS with quadrupole mass analyzer, and the subsequent analysis using high-resolution inductively coupled plasma mass spectrometry, (HR-ICP-MS). 25 elements were determined at concentrations ranging from 1 ng g up to %. It was observed that T and NT soybean seeds exhibit statistically significant differences in the concentrations of Cu, Fe and Sr, the first two having higher concentrations in the T seeds, and the last with the highest concentration in soybeans NT. These results were also reflected in the contents of these elements in aqueous extracts and residues obtained through sequential fractionation. Also, the contributions of bioaccessible fractions of Cu, Fe and other elements (Mn, S, Zn) to the total content of the elements in T soybean seeds were higher than those found in NT soybean seeds. Subsequently, studies were continued using bidimensional liquid chromatography, the first dimension consisting of size exclusion chromatography (SEC) and the second dimension of anion exchange chromatography (AEX). In the first chromatographic dimension three fractions containing metals were identified using hyphenation SEC-ICP-MS, the first corresponding to molar masses between 38.1 and 181.1 kDa, the second corresponding to molar masses between 8.2 and 17.2 kDa and the third fraction corresponding to molar masses between 0.4 and 3.8 kDa. The three identified fractions were separated and lyophilized, and again separated using anion exchange chromatography (AEX). Metals were found in all the separated fractions by SEC: three sub-fractions of the first fraction, a subfraction in the second fraction and three sub-fractions in the third fraction. These peaks were collected, lyophilized, digested and taken to the mass spectrometer for protein identification. 33 proteins were identified, and, among them, two proteins previously related to metals were found: seed lipoxygenase 1 and b-conglycinin. After chromatographic separation in the second dimension, the resultant subfractions were lyophilized and subjected to a third separation dimension using onedimensional polyacrylamide gel electrophoresis (SDS-PAGE). After the separation, the bands were cut out and digested to examine the metals contained therein, highlighting the results obtained for Fe, which was quantified in the bands of the sub-fraction where the peak of the element is found in the analysis by ICP-AEX - MS. Also, the bands were digested triptically to identify the proteins, and once again proteins associated to metals were identified: 3-lipoxygenase A chain (Soybean) complex with 13 (S)-Hydroperoxy-9 (Z), 11 ( E)-octadecadienoic acid, beta-amylase [Glycine max]; seed lipoxygenase-1, lipoxygenase [Glycine max] seed lipoxygenase-2 (Pisum sativum) and beta-conglycinin
Doutorado
Quimica Analitica
Doutora em Ciências
37
Jones, Owen G. "Comparison of analytical techniques for quantification of 8-iso-PGF2[alpha] using HPLC-MS-MS and enzyme immunoassay". Connect to this title online, 2005. http://hdl.handle.net/1811/347.
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Thesis (Honors)--Ohio State University, 2005Title from first page of PDF file. Document formattted into pages: contains, 16 p.; also includes graphics. Includes bibliographical references (p. 15-16) Available online via Ohio State University's Knowledge Bank.
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Persson, Josefin. "Development and evaluation of methods for analysis of TBECH and HBCD using HRGC/HRMS and HPLC/MS/MS". Thesis, Örebro University, School of Science and Technology, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-7695.
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The two additive brominated flame retardants, tetrabromoethylcyclohexane (TBECH) and hexabromocyclododecane (HBCD) are used to prevent fire to start and spread. They are simply mixed with material and are most likely to leach out in the environment, because of non-covalently binding to the material. TBECH can exist as four pairs of enantiomers, α-, β-, γ- and δ-TBECH. The technical HBCD can exist as three pairs of enantiomers, α-, β- and γ-HBCD and two meso forms δ- and ε-HBCD. None of these compounds are produced in Sweden, but they are imported to industries. TBECH has been found in Beluga blubber and can accumulate in zebrafish. HBCD has been found in water environments and can be toxic to and bioaccumulate in water-living animals.
In this study, a method was developed for separation and detection of α-, β-, γ- and δ-TBECH on HRGC/HRMS. All TBECH-isomers could be separated with the developed method. How much of the TBECH isomers that were recovered after applying existing extraction and clean-up procedures, normally applied for clean-up and extraction of PCBs and PCDD/Fs, was evaluated. Low recovered amounts (6.8-35.5 %) of TBECH-isomers added in known amounts to three different whale samples indicate severe evaporation losses and possibly photolytic degradation. None of the four enantiomers were detected in the three whale samples. For HBCD analysis, both the chromatography and MS/MS parameters were optimised for δ- and ε- HBCD yielding good chromatography and sensitivity. However, due to technical difficulties during the time-period of this project, no whale samples could be analysed for HBCD on UPLC/MS/MS.
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Rivas-Urraca, Cristina. "Environmental speciation of tin and lead by HPLC-ICP-MS". Thesis, University of Plymouth, 1996. http://hdl.handle.net/10026.1/459.
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New methodologies have been developed for the determination of organotin and organolead compounds in environmental samples. Several high performance liquid chromatographic separations of organotin compounds have been tested and the best system (cation-exchange chromatography with methanol and a citrate buffer) employed for the determination of tributyltin (TBT), triphenyltin (TPhT), dibutyltin (DBT) and monobutyltin (MBT) in environmental samples. The coupling between high performance liquid chromatography (HPLQ and the inductively coupled plasma-mass spectrometer (ICP-MS) for this application has been modified to yield limits of detection of 0.44,0.26,1.4 and 0.23 ng. g-' as Sri for TBT, TPhT, DBT and MBT respectively. Different extraction procedures have been tested for the determination of organotin species in samples of environmental interest, such as sediments and biological materials. The values obtained for TBT, TPhT and DBT in the analysis of a mussel candidate reference material, CRM 477, have been incorporated in the certification campaign of this material. A liquid chromatographic separation for trimethyllead (TML) and triethyllead (TEL) has also been developed. Artificial rain water has been analysed for TML. The system proved to be valid for the determination of TML in this sample, even in the presence of high amounts of inorganic lead. Finally, isotope dilution analysis (IDA) was incorporated in the method. Tributyltin iodide (TBTI) and trimethyllead chloride (TMLCI), isotopically enriched in "Sn and "Pb, respectively, were synthesised. The mussel tissue CRM 477 was analysed with IDA-HPLC-ICPMS for TBT. As for the analysis without isotope dilution, the result obtained was incorporated in the certification campaign. The analysis with this methodology gave a better precision in the overall determination than external calibration analysis. Artificial rain water, at two different concentration levels, was analysed for TML with IDA-HPLC-ICP-MS. Better precision and accuracy was obtained for the analysis of this material with this method than when external calibration procedures were employed. IDA-HPLC-ICP-MS has proved to be a valid technique for the analysis of environmental samples. The technique simplifies the procedure, compensates for different sources of variability and, thus, the overall precision obtained in the analysis is improved compared to other calibration techniques.
40
Ghebreamlak, Weyni. "Identification of Trypsin Digested Transferrin using HPLC and MALDI-MS". Thesis, KTH, Skolan för kemi, bioteknologi och hälsa (CBH), 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-266157.
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In this project, separation of trypsin digested transferrin (Tf) has been studied, using a RP HPLC- UV system equipped with a C18 column. 0.1% TFA/MQ-water and 90% MeOH were used as mobile phase A and mobile phase B, respectively. For economic reasons, the protein cytochrome c (cyt-C) was used to optimize the digestion procedure and LC system, before analysis of Tf. Four digestion methods were applied for analyzing cyt-C and Tf. The first method was digestion with no denaturing, reducing or alkylating agent. The other digestion methods used urea or heating as a denaturing agent, and lastly dithiothreitol (DTT) and iodoacetamide (IAA) as reducing and alkylating agent, respectively. The results from HPLC-UV showed that a gradient elution with a high concentration of organic solvent is favorable for the separation of cyt-C peptides. MALDI-MS was used to identify peptides, and the outcomes showed that denaturation by heat before digestion gave the best results.I detta projekt har separation av trypsin-klyvt transferrin (Tf) studerats, med användning av ett RP HPLC-UV system, som bestod av en C18 kolonn. 0,1% TFA/MQ-vatten och 90% MeOH användes som mobilfas A respektive mobilfas B. Av ekonomiska skäl användes proteinet cytokrom c (cyt-C) före analys av Tf för att optimera klyvningsprocessen och LC systemet. Fyra klyvningsmetoder studerades för analysering av cyt-C och Tf. Den första metoden innehöll inget denaturerande, reducerande eller alkylerande medel. De andra klyvningsmetoderna innehöll urea eller värme som denaturerande medel, och slutligen ditiotreitol (DTT) och jodacetamid (IAA) som reducerande respektive alkylerande medel. Resultaten från HPLC-UV visade att en gradienteluering med en hög koncentration av den organiska lösningen är gynnsam för separationen av peptiderna från cyt-C. MALDI-MS användes för att identifiera peptiderna, och resultaten visade att denaturering med värme före klyvning gav bäst resultat.
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Silva, Elidiane Gomes da 1983. "Combinação de técnicas para a determinação de espécies de selênio". [s.n.], 2012. http://repositorio.unicamp.br/jspui/handle/REPOSIP/248605.
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Orientadores: Marco Aurélio Zezzi Arruda, Fábio AugustoTese (doutorado) - Universidade Estadual de Campinas, Instituto de Química
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Resumo: No Capítulo 1, é proposta a combinação das técnicas de microextração em fase sólida com a espectrometria de absorção atômica em forno de grafite para extração de Se(IV) derivado, seguida de detecção por GC-MS. O método é aplicado com sucesso na determinação de Se total em materiais de referência certificado (BCR-414 and SRM 1643e), sendo obtidas recuperações de 97% para a água (SRM 1643e) e 101% para o plâncton (BCR-414). O método também é aplicado para determinação de selênio em amostra de medicamento manipulado a base de quelato de selênio. No Capítulo 2, a técnica de HPLC com troca aniônica acoplada a ICP-MS, é usada para identificação e determinação de quatro espécies de selênio (Se(IV), Se(VI), selenometionina e selenocistina) em amostras biológicas (plâncton, castanha-do-pará, urina e folhas de girassol). A extração das espécies, presente em plâncton (BCR-414), castanha-do-pará e folhas de girassol, é realizada com água, e um teste de simulação de digestão gastrointestinal também é aplicado para identificar as espécies de selênio bioacessíveis em castanha do Pará. O Se(IV) é a principal espécie extraída no plâncton, com maior concentração de selênio no extrato. A castanha-do-pará possui, principalmente, selenocistina e selenometionina, sendo que somente a selenometionina é identificada como bioacessível. O estudo de especiação de selênio por HPLC-ICP-MS sugere a presença de selenocistina em urina de homens e mulheres. As folhas de girassol, de plantas cultivadas com solução de selenito apresentam um maior número de espécies de selênio, em comparação a planta controle
Abstract: In Chapter 1, the combination of the techniques of solid phase microextraction with atomic absorption spectrometry graphite furnace is proposed, using an SPME fiber device and graphite furnace (GF) for extracting Se compounds. The extracted compounds are separated and detected by GC-MS. The method is successfully applied to the total Se determination in certified reference materials (BCR-414 and SRM 1643e). The SPME-GF method combined with GC-MS is also applied to the determination of total selenium in a commercial drug sample (selenium chelate). In Chapter 2, the anion-exchange HPLC technique coupled to ICP-MS is used for identification and determination of four selenium species (Se(IV), Se(VI), selenomethionine and selenocystine) in biological samples (plankton, Brazil nuts, urine and sunflower leaves). The extraction of the species is carried out using water for plankton (BCR-414), Brazil nuts and sunflower leaf. A simulated gastrointestinal digestion is also used to identify bioaccessible selenium in Brazil nuts. The Se(IV) is the predominant specie in the plankton, with the highest selenium concentration in the extract. The Brazil nuts contain selenomethionine and selenocystine species, but only selenomethionine is identified as bioaccessible. The study of selenium speciation by HPLC-ICP-MS suggests the presence of selenocystine in the urine of women and men. The sunflower leaves of plants cultivated with selenite have a higher number of selenium species compared to the control plants
Doutorado
Quimica Analitica
Doutora em Ciências
42
Rösemann, G. M. "Analysis of pyrrolizidine alkaloids in Crotalaria species by HPLC-MS/MS in order to evaluate related food health risks". Electronic thesis, 2006. http://upetd.up.ac.za/thesis/available/etd-08032007-170633/.
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43
Lorenzetti, Raquel. "Metodo de quantificação de nucleotideos por HPLC-MS/MS e avaliação da atividade de analogos de sildenafil sobre fosfodiesterase". [s.n.], 2007. http://repositorio.unicamp.br/jspui/handle/REPOSIP/309493.
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Orientador: Gilberto de NucciTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: No presente trabalho foi padronizado um novo método para a dosagem da atividade de fosfodiesterase in vitro, por HPLC-MS/MS. Este novo método conseguiu apresentar exatidão, precisão, sensibilidade e rapidez nas análises; monitorando os nucleotídeos (AMP, GMP, AMPc e GMPc). O desenvolvimento de novos fármacos derivados de um protótipo aponta para a obtenção de moléculas com um melhor perfil farmacocinético ou uma melhor relação estrutura-atividade. Atualmente o sildenafil é considerado o principal fármaco para o tratamento de disfunção erétil. Neste trabalho, avaliaram-se novos compostos denominados análogos de sildenafil (carbonato de lodenafil, dímero uréia e dímero uretana). Os análogos foram analisados quanto à atividade em PDE5 e agregação plaquetária humana, in vitro. Foi determinada a estabilidade destes compostos, em meio ácido e plasma humano, in vitro, além de seus possíveis metabólitos em microssomas e hepatócitos de rato in vitro, e os seus parâmetros farmacocinéticos via intravenosa e oral, em cão, in vivo. Os resultados mostraram que os análogos de sildenafil inibem a atividade de PDE e não inibem a agregação plaquetária do mesmo modo que o sildenafil in vitro, no entanto potencializam a ação do doador de NO (SNP). Os análogos de sildenafil foram estáveis em meio ácido e em plasma humano. No ensaio de metabolização, os dímeros uréia e uretana não foram metabolizados, entretanto o carbonato de lodenafil foi metabolizado principalmente em lodenafil, in vitro. O carbonato de lodenafil é rapidamente biotransformado em lodenafil, após administração v.i. e v.o. em cão. Concluiu-se que este trabalho apresenta um novo método de dosagem de PDEs e uma nova perspectiva terapêutica para a disfunção erétil, representada pelo carbonato de lodenafil, o qual inibe concentração-dependente a atividade de PDE5
Abstract: In the present work a new method for the dosage of the activity of phosphodiesterase was standardized in vitro, for HPLC-MS/MS. This new method obtained to present exactness, precision, sensitivity, and rapidity in the analyses; monitoring the nucleotides (AMP, GMP, cAMP and cGMP). The development of new drug derived from an archetype points with respect to the molecule attainment with one better pharmacokinetic profile or one better relation structure-activity. Currently the sildenafil is considered the main drug for the treatment of erectile dysfunction. In this work, we evaluate new analogous called composites of sildenafil (carbonate of lodenafil, dimer urea and dimer uretana). The analogous ones had been analyzed how much the activity in PDE5 and platelet aggregation human being, in vitro. The stability of these composites was determined, in human acid way and plasma, in vitro, beyond its possible metabolites in microsomes and hepatocytes of rat in vitro, and its pharmacokinetic profile after intravenous and oral, in dog, in vivo. The results had shown that the analogous of sildenafil inhibit the activity of PDE and they do not inhibit the platelet aggregation in a similar way that the sildenafil in vitro, however potencializam the action of the giver of NO (SNP). The analogous ones of sildenafil are presented steady in human acid way and plasma. In the metabolization assay, metabolization of dimer urea and dimer uretana was not observed, however the lodenafil carbonate was metabolizado mainly in lodenafil, in vitro. The lodenafil carbonate quickly is biotransformation in lodenafil, after administration v.i. and v.o. in dog. We conclude that this work presents a new method for analyze activity of PDEs and a new therapeutically perspective for the erectile dysfunction, represented for lodenafil carbonate, which inhibits concentration-dependent the activity of PDE5
Doutorado
Doutor em Farmacologia
44
Rosemann, G. M. (Gertruida Magdalena). "Analysis of pyrrolizidine alkaloids in Crotalaria species by HPLC-MS/MS in order to evaluate related food health risks". Thesis, University of Pretoria, 2007. http://hdl.handle.net/2263/26960.
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Pyrrolizidine alkaloids (PAs) are one of the most significant groups of plant toxins in the world and are an important cause of poisoning in livestock, resulting in significant financial and production losses each year (Kellerman et al. 1996). Pyrrolizidine alkaloids may also enter the human food chain as contaminants of grains, via animal products such as milk, eggs and honey or may be consumed as constituents of herbal medicines (ANZFA 2001). Not all PAs are toxic. Pyrrolizidine alkaloids affecting human health are the esters of 1,2-unsaturated hydroxymethyl dehydropyrrolizidines (DHP). Before it can be converted to DHP, PAs need to have certain essential features, which include an unsaturated 3-pyrrole ring, one or two hydroxyl groups attached to the ring, one or two ester groups and a branched acid moiety (Mattocks 1986). These compounds can be metabolized in the liver to nucleophillic pyrroles which cause damage to hepatocytes (Winter and Segall 1989). Although the involvement of PAs in the development of hepatic veno-occlusive disease is well established (Bras et al. 1961), there is still uncertainty concerning the consequences of long-term, low-dose exposure in humans. Exposure to PAs through the use of herbal remedies may also be a contributing factor to the high rates of liver cancer and cirrhosis seen in Africa (Steenkamp et al. 2000). Crotalaria spp. are known to contain toxic PAs and various incidences of human poisoning through contaminated grains have been recorded in the scientific literature (IPCS 1989). Legislation controlling the allowable levels of toxic seeds in grains in South Africa is generally much stricter than in many other grain producing countries. The Soybean and Sunflower Forum recently commissioned a study (Eloff et al. 2003) to review published and unpublished information on toxic seed that could affect human health in South Africa and to make recommendations accordingly. Crotalaria sphaerocarpa is one of the problem plants discussed in the review and is apparently the only species which regularly contaminate grain in certain areas in South Africa. There is uncertainty at present about the number of these seeds that should be allowed in grains and the threat that this may pose to human health. Based on the review a provisional recommended level of 10 seeds of C. sphaerocarpa per 10 kg of grain was proposed as an approximated safe level in the report. As emphasized by the authors (Eloff et al. 2003), this absolute level is based on assumptions that must still be tested. As a follow-up on the report, a sensitive LC-MS/MS method for the determination of toxic PAs in plants was developed in this study. The characteristic fragments produced by 1,2-unsaturated necine bases under specific MS/MS conditions were used to discriminate between the toxic and non-toxic PAs. The concentration of these PAs were then determined using multi-reaction-mode experiments. Quantitative results were calculated against a retrorsine calibration curve and expressed as µg retrorsine equivalents per gram plant material. Various extraction methods described in the literature were investigated. A final liquid-liquid extraction method was used to extract unsaturated PAs from small amounts (about one gram) of milled plant samples. Recoveries from spiked lucerne samples were 98% for retrorsine and 105% for monocrotaline. To determine the applicability of the LC-MS/MS method the unsaturated PA content of C. laburnifolia and C. dura were investigated. Crotalaria laburnifolia, which is regarded as non-toxic, contained low concentrations (< 20 µg.g-1) of unsaturated PAs. Crotalaria dura, on the other hand, is known to be toxic to livestock and the concentration of unsaturated PAs was significantly higher (585 µg.g-1). The toxic PA content of Senecio inaequidens was also determined after an incident of livestock poisoning. The plant material contained very high concentrations of retrorsine (11.5 mg.g-1) and senecionine (0.5 mg.g-1) which were also present in the rumen content collected post-motally. These results confirmed the suspected toxicity of S. inaequidens. The LC-MS/MS method was also used to follow variations in unsaturated PA content in C. sphaerocarpa plants during the growing season. Pyrrolizidine alkaloids were present in the roots of the growing plants as N-oxides and also found in the mature aerial parts, where it was present mainly as the basic alkaloids. The method was used to determine the concentration of unsaturated PAs, in various C. sphaerocarpa seeds from different locations, in order to calculate the allowable level of C sphaerocarpa seed in maize. Of all the seed samples analyzed, the highest unsaturated PA concentration found was 150 µg.g-1. The allowable level of seed was calculated using this result and was found to be 656 seeds per 10 kg maize, based on the Australian and New Zealand Food Authority level of 0.1 µg.kg-1.day-1. If these results are confirmed with systematic statistical samples of C. sphaerocarpa seed from different grain production areas, the allowable level could be increased substantially. This may have an economic benefit to grain producers.Thesis (PhD (Paraclinical Science))--University of Pretoria, 2006.
Paraclinical Sciences
PhD
unrestricted
45
Dubber, Mary-Jean. "Application of CE, HPLC and LC-MS-MS for the analysis and quality control of Ginkgo biloba dosage forms". Thesis, Rhodes University, 2006. http://hdl.handle.net/10962/d1003235.
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Natural products are complex mixtures of compounds with therapeutic effects which are often reported to be due to the synergistic action of multiple and sometimes unknown components. Consequently, standardization of these products is complex and a lack of effective quality control (QC) criteria in most countries has led to marketing of commercial products with questionable quality, safety and efficacy (QSE). The aim of this study was therefore to develop qualitative and quantitative analytical methods for use in the QC of Ginkgo biloba solid oral dosage forms. Initially, a micellar electrokinetic chromatography (MEKC) method was developed for the identification of the flavonol glycosides, rutin and quercitrin as well as 3 flavonol aglycones, quercetin, kaempferol and isorhamnetin in crude extracts of 4 Ginkgo biloba solid oral dosage forms using ultraviolet (UV) detection. A reversed-flow cyclodextrin-modified MEKC method was subsequently developed for the simultaneous determination of the aforementioned flavonols as well as ginkgolide A, B, C, J and bilobalide (all positive markers) in Ginkgo commercial products. A non-aqueous capillary electrophoresis (CE) method was also developed for fingerprinting the presence of ginkgolic acids (negative markers) in Ginkgo biloba leaf extracts, which are purported to be associated with toxic properties. This method was also applied to 2 Ginkgo biloba commercial products. Since the flavonols have strong UV absorbing chromophores, a reversed phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated using photo-diode-array (PDA) detection which was then successfully applied to fingerprint commercially available Ginkgo biloba solid oral dosage forms as well as quantify the relevant flavonol markers present in these extracts. Sample preparation was simple, rapid and cost efficient with minimal clean-up and the employment of a minibore column which requires low mobile phase flow rates contributed to the economy of the method. Unlike the conventional QC approach, samples were not hydrolyzed and direct determination of 2 intact flavonol glycosides, together with the usual aglycone markers was facilitated which provided maximal content information for fingerprint comparisons. On the other hand, terpene trilactones possess poor chromophores and an alternative detection method to UV was required in order to obtain suitable sensitivity. RP-HPLC with evaporative light scattering detection (ELSD) was selected for quantification of these non-volatile constituents in Ginkgo dosage forms and this method was deemed suitable for the routine QC analysis of these positive markers in commercial products. Since approximately 33 flavonoids have been identified in Ginkgo biloba leaf extracts, baseline separation using UV/PDA detection normally requires complex gradient programs and long analysis times. In addition, unequivocal identification of the flavonoids with similar UV spectra and elution times cannot be guaranteed. A liquid chromatographic tandem mass spectrometric (LC-MS-MS) method was therefore developed and validated in order to ensure accurate quantification of the selected flavonol marker compounds in Ginkgo commercial products. LC-MS-MS analysis of Ginkgo extracts revealed, in addition to rutin, the possible presence of other quercetin analogues, quercetin-3-Orhamnoside-7-O-glucoside or quercetin-3-O-glucoside-7-O-rhamnoside, previously unreported in Ginkgo biloba leaf extracts or dosage forms. In terms of evaluating the most suitable analytical method for QC, CE shows exceptional potential in the future analysis of Ginkgo biloba dosage forms while HPLC-PDA and HPLC-ELSD are currently the most affordable and practical instruments for the routine analysis of the flavonols and terpenoids, respectively. LC-MS-MS proved to be pivotal for the accurate identification and quantification of the flavonols due to interference by other flavonoid compounds with similar retention times and UV spectra to the peaks of interest. All quantitative and qualitative results revealed large discrepancies in the marker content between the products regardless of which batch was analysed and product labels disclosed little relevant information. Although currently not required by most regulatory agencies, some of the usual quality criteria applied to orthodox medicines was evaluated. In particular, dissolution analysis, disintegration, tablet hardness and weight uniformity were assessed and revealed similar inconsistencies. This thesis emphasises that implementation of effective QC criteria is long overdue and is essential to ensure consistent product QSE of commercially available Ginkgo biloba solid oral dosage forms.
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Borsò, Marco. "HPLC-MS-MS analysis of thyroid hormones and iodotyrosines in knocked-out murine and natural human DEHAL-1 deficiency". Doctoral thesis, Università di Siena, 2021. http://hdl.handle.net/11365/1128063.
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Thyroid hormones (TH), namely 3,5,3’-triiodothyronine (T3) and its precursor thyroxine (T4), are key regulators of growth processes and development, and crucially control the energy metabolism. A proper availability of iodine within the thyroid is crucial for the synthesis of TH in order to maintain the homeostasis of circulating levels. The daily dietary iodine intake is not sufficient to sustain their synthesis and, in fact, most of the intra-thyroidal iodine is recycled through the activity of the Iodotyrosine Dehalogenase-1 (DEHAL-1) enzyme. Mono-iodotyrosine (MIT) and di-iodotyrosine (DIT) precursors produced in excess during TH synthesis, are deiodinated by DEHAL-1, leading to the production of I- and Tyr that are recycled and reused within the thyroid. In the last decade, human mutations of DEHAL1 causing the impairment of its catalytic activity were reported. The clinical picture caused by these mutations recapitulates the phenotype of what is known as Iodotyrosine deiodinase deficiency (ITDD), characterized by hypothyroidism, goitre and, if not treated during childhood, by intellectual impairments. A hallmark of ITDD resides in the increase of urinary and plasmatic MIT and DIT levels.
High-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS-MS) is a powerful technique characterized by high specificity and sensitivity. Even if clinical testing of TH is still performed using immunoassays, various HPLC-MS-MS methods to assay TH at low concentrations have been proposed. HPLC-MS-MS is now emerging as a powerful technique that complements traditional testing overcoming some of their limitations. Hitherto, only one HPLC-MS-MS method have been proposed in order to assay MIT and DIT levels in urine that has been used in the discovery of one of the most recent discovered human DEHAL1 mutations.
The aim of my project was to develop a sensitive and robust HPLC-MS-MS method able to quantify MIT and DIT in urine and plasma, together with T3 and T4 in the latter case. The method was extensively validated and used to assay these molecules in urine and plasma collected from the novel Dehal1 knock-out mouse. Our method played a crucial role in the biochemical characterization of this first mammalian model of defective iodine recycling through DEHAL-1 deletion. We detected increased urinary and plasmatic levels of MIT and DIT in the knock-out mice and we demonstrated the presence of elevated concentrations of both molecules even at the first stage of life. The influence of iodine availability was tested, showing that mice were still euthyroid when levels of dietary iodine were sufficiently high. In the presence of scarce iodine availability, coupled to the impaired ability to recycle iodine through DEHAL-1, knock-out mice became hypothyroid. Our results demonstrated the importance of iodine availability in triggering hypothyroidism in the presence of ITDD.
The importance of a powerful technique able to detect MIT and DIT was demonstrated assaying these molecules in human urine collected from a consanguineous family with a diagnosed DEHAL-1 deficiency. Our HPLC-MS-MS method was able to detect elevated urinary levels of MIT and DIT in the index patient that was clinically diagnosed with ITDD showing goitre and hypothyroidism. Remarkably, we detected a massive increase of both molecules in urine collected from one of the brothers that was healthy at the time of sample collection but that developed hypothyroidism and goitre several years later.
In conclusion, our findings demonstrated the ability of our validated HPLC-MS-MS method to detect MIT, DIT together with T3 and T4 in urine and plasma samples. We showed the potential of MIT and DIT assay, especially in urine, for the early detection of hypothyroidism in DEHAL-1 deficiency. Considering the presence from the beginning of life, the detection of iodotyrosines could be potentially included in the human new-born screening for hypothyroidism.
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Wohlfarth, Michael. "Die Aufklärung der Biogenese strukturell ungewöhnlicher Alkaloide aus Triphyophyllum (Dioncophyllaceae) und Antidesma (Euphorbiaceae) und Entwicklung und Einsatz der "Triade" zur phytochemischen Online-Analytik: HPLC-MS-MS, HPLC-NMR und HPLC-CD". [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=969623372.
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Aguiar, Giovanna de Fatima Moreno. "Avaliação de métodos empregando a espectrometria de massas com plasma acoplado (ICP-MS) para determinação de impurezas elementares e especiação química de arsênio e mercúrio em fármacos e excipientes". Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/60/60134/tde-23112017-113653/.
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Com o aumento das exigências regulatórias e estabelecimento de limites mais restritos para impurezas elementares em fármacos e excipientes usados em formulações farmacêuticas, ocorreu uma busca por técnicas analíticas capazes de quantificar elementos em níveis traço e assegurar a qualidade e a segurança dos medicamentos. A espectrometria de massas com plasma acoplado indutivamente (ICP-MS) é uma técnica multielementar, que apresenta alta sensibilidade e é empregada com eficiência na análise de elementos traço em diferentes matrizes. O ICP-MS, acoplado a um sistema separador como o cromatógrafo líquido de alta eficiência (HPLC), possibilita a especiação de elementos químicos, o que é importante, pois a toxicidade pode estar diretamente ligada à forma química do elemento, como ocorre para o Hg e As. Neste sentido, os objetivos deste trabalho foram desenvolver e validar um método de análise simples, rápido e com alta sensibilidade, para determinação de 15 impurezas elementares: As, Cd, Cr, Cu, Ni, Pb, V, Ir, Pd, Pt, Rh, Ru, Hg, Os e Mo, em diferentes fármacos e excipientes, em níveis que atendam aos novos critérios estabelecidos pelos órgãos regulatórios, avaliar a utilização da cela de reação dinâmica (DRC) para eliminação de interferências espectrais na determinação de As, Cr e V, realizar a especiação de As e Hg, e aplicar esta metodologia para análise de diversas amostras comprovando sua robustez, versatilidade e vantagens para utilização em rotina. Foram avaliadas cinco estratégias de preparo de amostra: digestão em micro-ondas sistema fechado e digestão em micro-ondas - sistema aberto, digestão em banho-maria, diluição direta em meio ácido e extração utilizando ponteira de ultrassom. Através de ensaios de recuperação e materiais de referência certificados, os melhores resultados (84 a 101%) foram obtidos com a digestão das amostras em sistema fechado de micro-ondas e extração por ponteira de ultrassom. Os limites de detecção variaram entre 0,001 ng g-1 (103Rh) e 0,083 ng g-1 (75As). Em seguida, a metodologia analítica foi empregada para determinação dos elementos em estudo em 74 amostras de fármacos e excipientes. O elemento mais frequentemente encontrado foi o Cu, seguido por Cr, Mo, Ni, Pd e V. Já os elementos químicos Ir, Pt, Os e Ru não foram detectados. Cabe destacar que as concentrações de Pd, Rh, As, Cd, Cr, Cu, Ni, V e Mo encontraram-se acima do limite preconizado pela farmacopeia americana em algumas amostras. A análise por especiação química de As mostrou que as amostras possuíam apenas as formas inorgânicas e mais tóxicas. Em relação ao Hg, apenas uma amostra apresentou níveis detectáveis deste elemento, mas a concentração estava abaixo do limite estabelecido pela farmacopeia americana.Due to quality and safety reasons, regulatory requirements and establishment of more restricted limits for elemental impurities in drugs and excipients are increasing. These regulations promoted a search for simple and robust analytical techniques for quantification of trace elements. Inductively coupled plasma mass spectrometry (ICP-MS) is an attractive technique for this purpose, presenting multielement and high sensitivity capabilities. ICP-MS can be hyphenated to separation techniques such as high-performance liquid chromatography (HPLC), enabling chemical speciation analysis. Speciation analysis may reveal the chemical form of the element that may be more directly related to the toxicity. In this sense, the objectives of this work were to develop and validate a simple, fast and highly sensitive method for the determination of 15 elemental impurities: As, Cd, Cr, Cu, Ni, Pb, V, Ir, Pd, Rh, Ru, Hg, Os and Mo, in different drugs and excipients, at levels that meet the new criteria established by the regulatory agencies. We also evaluated the use of the dynamic reaction cell (DRC) to eliminate spectral interferences for As, Cr And V. Finally, for As and Hg, speciation methods were applied. All analysis were performed focusing on robustness, versatility and sample high-throughput. We tested five sample preparation strategies: closed microwave system digestion, microwave digestion - open system, water bath digestion, direct acid dilution and ultrasound probe extraction. Recovery studies in ordinary samples and reference certified materials were observed with the closed microwave digestion system and ultrasound probe extraction (84 and 101%). Detection limits ranged from 0.001 ng g-1 for 103Rh to 0.083 ng g-1 for 75As. The analytical methodology was then applied for the determination of the elements in study in 74 samples of drugs and excipients. The most frequently found element was Cu, followed by Cr, Mo, Ni, Pd and V. The chemical elements Ir, Pt, Os and Ru were not detected. It should be mention that the concentrations of Pd, Rh, As, Cd, Cr, Cu, Ni, V and Mo were above the limit recommended by the American pharmacopeia in some samples. Only inorganic arsenic (most toxic forms) was found in samples by applying speciation analysis. Regarding Hg, only one sample had detectable levels of this element, but the concentration was below the limit established by the American pharmacopeia
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Meiby, Elinor, Zetterberg Malin Morin, Sten Ohlson, Hernández Víctor Agmo e Katarina Edwards. "Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis". Uppsala universitet, Analytisk kemi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-197337.
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Lipodisks, also referred to as polyethylene glycol (PEG)-stabilized bilayer disks, have previously been demonstrated to hold great potential as model membranes in drug partition studies. In this study, an HPLC-MS system with stably immobilized lipodisks is presented. Functionalized lipodisks were immobilized on two different HPLC support materials either covalently by reductive amination or by streptavidin-biotin binding. An analytical HPLC column with immobilized lipodisks was evaluated by analysis of mixtures containing 15 different drug compounds. The efficiency, reproducibility, and stability of the system were found to be excellent. In situ incorporation of cyclooxygenase-1 (COX-1) in immobilized lipodisks on a column was also achieved. Specific binding of COX-1 to the immobilized lipodisks was validated by interaction studies with QCM-D. These results, taken together, open up the possibility of studying ligand interactions with membrane proteins by weak affinity chromatography.De två (2) första författarna delar förstaförfattarskapet.
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Huang, Mian. "Analysis of distributions of bacteriochlorophyll derivatives using HPLC and LC-MS". Thesis, University of York, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.534942.
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