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Gorityala, Shashank. "TARGETED AND UNTARGETED OMICS FOR DISEASE BIOMARKERS USING LC-MS." Cleveland State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=csu1547093694357568.
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Clarin, Leona. "Development and validation of an ultrafiltration-UHPLC-MS/MS method for the quantification of unbound Beta-Lactam antibiotics cefotaxime, piperacillin, cloxacillin and flucloxacillin in plasma." Thesis, KTH, Tillämpad fysikalisk kemi, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-287570.
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Infections in critically ill patients are a problem for the healthcare system and at any one time, 70 % of all intensive care unit (ICU) patients are treated with antibiotics. Antibiotics bind toproteins in the blood, but only unbound drug can diffuse over capillary membranes and bindto the targeted receptor. Standard protein binding percentages for antibiotics have been developed from studies on healthy volunteers and dosing regimens for patients are adapted accordingly. The determination of the total concentration of antibiotics in patients’ bloodsamples is, based on the standard percentages, ordinarily representative for the pharmacological effect of the antibiotic. However, certain conditions that are common incritically ill patients can alter protein binding percentages, resulting in a larger or smaller unbound fraction. This in turn can result in toxicity or therapeutic failure. The aim of this project was to develop an analytical method for the determination of the unbound concentration of the Beta-Lactam antibiotics cefotaxime, flucloxacillin, cloxacillin and piperacillin in plasma. A method was successfully developed using ultrafiltration for the extraction of unbound analytes and ultra high performance liquid chromatography tandem mass spectrometry, UHPLC-MS/MS, for their quantification. The method was partly validated according to the European Medicines Agency’s guidelines on bioanalytical method validation.Kritiskt sjuka patienter med infektioner är en börda för sjukvården och 70 % av alla patienter på intensivvårdsavdelningar är ordinerade antibiotika. Antibiotika binder till proteiner i blodet, men enbart den icke-proteinbundna (fria) fraktionen kan diffundera över kapillära membran och binda till receptorer. Standardproteinbindningsgrad för olika antibiotika har utvecklats från studier på friska frivilliga och doseringen av läkemedlen är anpassade därefter. Den totala koncentrationen av antibiotika i patienters blod är vanligen representativ för den farmakologiska effekten. Dock kan vissa sjukdomar påverka proteinbindningsgraden vilket resulterar i en större eller mindre mängd fria antibiotika i blodcirkulationen. Det här kan i sintur resultera i toxicitet eller otillräcklig effekt av läkemedlet. Syftet med det här projektet var att utveckla en analytisk metod för att bestämma den fria koncentrationen av Beta-Lactam antibiotikan cefotaxim, flukloxacillin, kloxacillin och piperacillin i plasma. En metod utvecklades med ultrafiltrering för extraktion av den fria fraktionen och högupplösande vätskekromatografi och tandem masspektrometri, UHPLCMS/MS, för kvantifiering av analyterna. Metoden validerades delvis enligt den Europeiska Läkemedelsmyndighetens riktlinjer för bioanalytisk metodvalidering.
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Harder, Ulrike [Verfasser], and Berthold [Akademischer Betreuer] Koletzko. "Amino acid analysis in biofluids using LC-MS/MS : method development, validation and application in clinical research and dairy science / Ulrike Harder. Betreuer: Berthold Koletzko." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2014. http://d-nb.info/1048361772/34.
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Huang, Qianyang. "Development and Validation of UPLC/MS/MS Methods for Quantification of Gangliosides in the Clinical Study of Ganglioside GM3 Synthase Deficiency." Cleveland State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1472042152.
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Reinsch, Martin. "Entwicklung von Analyseverfahren zur Bestimmung von Ochratoxin A in Lebensmitteln." Doctoral thesis, [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=980847982.
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Hellmuth, Christian [Verfasser], and Berthold [Akademischer Betreuer] Koletzko. "LC-MS/MS applications in Targeted Clinical Metabolomics : method development and validation with focus on sulphur-containing amino acids and nonesterified fatty acids / Christian Hellmuth. Betreuer: Berthold Koletzko." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2014. http://d-nb.info/1049891201/34.
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Bunch, Nathan. "Oral Fluid Method Validation for Bowling Green State University." Bowling Green State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1586969951770212.
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Jakob-Rodamer, Verena [Verfasser], Fritz [Gutachter] Sörgel, Ulrike [Gutachter] Holzgrabe, and Petra [Gutachter] Högger. "Development and validation of LC-MS/MS methods to determine PK/PD parameters of anti-infectives / Verena Jakob-Rodamer. Gutachter: Fritz Sörgel ; Ulrike Holzgrabe ; Petra Högger." Würzburg : Universität Würzburg, 2015. http://d-nb.info/1112040285/34.
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Silva, Márcia Aparecida da. "Determinação de clorpropamida em plasma empregando empregando cromatografia liquida de alta eficiencia acoplada a espectrometria de massa sequencial (LC/MS/MS) e sua aplicação em um estudo de bioequivalencia." [s.n.], 2007. http://repositorio.unicamp.br/jspui/handle/REPOSIP/309495.
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Orientador: Gilberto de NucciTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
Made available in DSpace on 2018-08-11T03:36:14Z (GMT). No. of bitstreams: 1 Silva_MarciaAparecidada_D.pdf: 10125609 bytes, checksum: 145f4a9b65da8eb2521c6f76e1d715a9 (MD5) Previous issue date: 2007
Resumo: A biodisponibilidade relativa, de duas formulações de comprimido de clorpropamida 250 mg, foi avaliada em voluntários sadios de ambos os sexos. O estudo foi aberto, aleatorizado, cruzado em dois períodos com intervalo de três semanas, para o qual foram selecionados 36 voluntários. As amostras de sangue foram coletadas antes da administração de dose única de cada formulação, uma Teste (T) e outra Referência (R) e durante 72 horas após a administração. A concentração plasmática de clorpropamida foi determinada por cromatografia líquida de alta eficiência acoplada à espectrometria de massa seqüencial (LC/MS/MS), usando ionização tipo eletrospray no modo positivo com monitoramento de reação múltipla (MRM). O limite de quantificação foi de 0,1 µg/mL. O padrão interno foi a glibenclamida. A ASC0-72, que representa ASC truncada, Cmax e Tmax foram obtidas da curva de concentração plasmática em função do tempo. A ASC foi calculada empregando o método trapezoidal. Apenas ASC0-72 e Cmax de ambas as formulações foram estatisticamente comparados. A média geométrica e o intervalo de confiança (90%) da razão T/R foram respectivamente 93,99 (87,11%¿101,41%) para Cmax e 92,45 (85,96%-99,44%) para ASC0-72. Como o intervalo de confiança (90%), para ASC0-72 e Cmax, apresenta-se dentro do intervalo de confiança de 80-125% proposto pela Agência Nacional de Vigilância Sanitária (Anvisa) e pelo Food and Drug Administration (FDA-EUA), concluiu-se que ambas as formulações estudadas são bioequivalentes, no que diz respeito à magnitude e a velocidade de absorção e, portanto podem ser clinicamente intercambiáveis sem prejuízo terapêutico. Palavras-chave: Clorpropamida, bioequivalência, HPLC acoplada à espectrometria de massa seqüencial (LC/MS/MS), biodisponibilidade, farmacocinética
Abstract: The relative bioavailability between two formulations of chlorpropamide was assessed on the dosage form tablet 250 mg, in healthy volunteers of both sexes. The study was conducted using an open, randomized, two-period crossover design with the 3-week washout interval. Thirty-six subjects were selected. The blood samples were collected at the time prior to dosing and over an interval of 72 hours, of the single dose of each formulation, a Test (T) and another Reference (R). Chlorpropamide plasma concentrations were analyzed by high performance liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) with positive ion electrospray ionization using multiple reaction monitoring (MRM). The limit of quantification was 0,1 µg/mL for plasma Chlorpropamide analysis. The internal standard was the glibenclamide. The AUC 0-72, which represents AUC truncated, Cmax and Tmax were obtained from plasma concentration-time curve. The AUC was calculated using the trapezoidal rule. Only AUC0-72 and Cmax of the each other formulations were statistically compared. The geometric mean and respective 90% confidence interval (CI) of T/R ratios were 93.99 (87.11%¿101.41%) for Cmax and 92.45 (85.96%-99.44 %) for AUC0-72. Since the interval confidential (90%) for AUC0-72 and Cmax ratios were within the 80-125% interval proposed by the Agência Nacional de Vigilância Sanitária-Brazil (Brasil-Anvisa) and by the Food and Drug Administration (USA-FDA), it was concluded that both the formulations studied are bioequivalent for both the rate and extent of absorption and, therefore can be used interchangeably without impairing therapeutic effectiveness. Key words: Chlorpropamide, bioequivalence, high performance liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), bioavailability, pharmacokinetic
Doutorado
Doutor em Farmacologia
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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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Smit, Michiel Johannes. "Development and validation of selective and sensitive LC-MS/MS Methods for determination of para-aminosalicyclic acid and cycloserine/terizidone applicable to clinical studies for the treatment of tuberculosis." Master's thesis, University of Cape Town, 2018. http://hdl.handle.net/11427/29814.
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A method was validated for the quantification of para-aminosalicylic acid (PAS) in human plasma. The technique consisted of a protein precipitation extraction, followed by high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) detection. Rilmenidine was used as the internal standard (ISTD). Analyte mean extraction yields determined were ~100.3% (CV % = 3.3). The extraction procedure was followed by liquid chromatographic separation using a Phenomenex Synergi Hydro-RP (150 x 2.0 mm, 4µm) analytical column. An isocratic mobile phase containing methanol, water and formic acid (40:59.8:0.2, v/v/v) was used at a flow-rate of 300 µl per minute. The retention times for PAS and rilmenidine were, ~2.4 and ~1.6 minutes, respectively. An AB Sciex API 3000 mass spectrometer at unit resolution in the multiple reaction monitoring (MRM) mode was used to monitor the transition of the protonated precursor ions m/z 154.1 and m/z 181.2 to the product ions m/z 80.2 and m/z 95.2 for PAS and the ISTD, respectively. Electro Spray Ionisation (ESI) was used for ion production. Accuracy and precision were assessed over three consecutive, independent runs. The calibration curve fits a quadratic (weighted by 1/x concentration) regression for PAS over the range 0.391 – 100 µg/ml, based on peak area ratios. A 1:1 and 1:4 dilution of the QC Dilution sample showed that concentrations of up to 160 µg/ml of PAS in plasma could be analysed reliably when diluted into the calibration range. Endogenous matrix components were found to have an insignificant effect on the reproducibility of the method, when human plasma originating from eight different sources were analysed. PAS was found to be stable in human plasma for 21 months kept at ~-80°C, for up to 21 hours at room temperature and when subjected to 3 freeze-thaw cycles. Stock solutions of PAS in methanol were stable for 2 days when stored at ~80°C and for 24 hours when stored at room temperature, ~4°C and ~-20°C. Plasma extracts of the analyte/ISTD ratio were shown to be stable on instrument over a period of ~55 hours. Reinjection reproducibility experiments indicated that an assay batch may be re-injected within 58 hours. Quantification of PAS in plasma was not significantly affected by the presence of haemolysed blood (2%) in plasma and when Lithium Heparin was used as anti-coagulant instead of K3EDTA. The best marker for terizidone pharmacokinetics is the analysis of cycloserine, a small polar drug with limited potential for absorbing UV that makes it difficult to analyse. A method was validated for the quantification of cycloserine in human plasma, and consisted of a protein precipitation extraction and derivatization, followed by high performance liquid chromatography with MS/MS detection. No ISTD was used as no suitable match could be found. The mean extraction yield determined was ~77% (CV% = 10.7). The extraction procedure was followed by liquid chromatographic separation using a Gemini NX C18 (50 x 2.0 mm, 5µ) analytical column. An isocratic mobile phase containing acetonitrile, water and formic acid (30:69.9:0.1, v/v/v) was used at a flow-rate of 300 µl per minute. The retention time for cycloserine was ~ 1.5 minutes. An AB Sciex API 3000 mass spectrometer at unit resolution in the MRM mode was used to monitor the transition of the protonated precursor ion m/z 335.9 to the product ion m/z 157.2 for cycloserine. ESI was used for ion production. Accuracy and precision were assessed over three consecutive, independent runs. The calibration curve fits a quadratic (weighted by 1/x concentration) regression for cycloserine over the range 0.313 – 40.0 µg/ml, based on peak areas. A 1:4 dilution of the QC Dilution sample showed that concentrations of up to 64.0 µg/ml of cycloserine in plasma could be analysed reliably when diluted into the calibration range and no carry over peaks were observed. Endogenous matrix components were found to have no effect on the reproducibility of the method when human plasma originating from six different sources was analysed. Cycloserine was found to be stable in human plasma for up to 18 hours at room temperature, and when subjected to 3 freeze-thaw cycles. Stock solutions of cycloserine in water and methanol were stable for 10 days when stored at ~ -80°C and for 18 hours when stored at room temperature, ~ 4°C and ~ -20°C. Long term stability in plasma has been proven for 17 months at -80°C. Plasma extracts of the analyte were shown to be stable on instrument over a period of ~ 29 hours. Reinjection reproducibility experiments indicate that an assay batch may be re-injected within 29 hours. Cycloserine is stable in whole blood (on ice) for up to 30 minutes. Both validated methods presented performed well on clinical samples generated from a multi drug resistant TB (MDR-TB) research study in children dosed with PAS and terizidone.
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Mannem, Chandana. "IN-QUEST OF BIOMARKERS FOR ALZHEIMER’S DISEASE AND PHARMACOKINETIC PROFILE OF ANTICANCER AGENTS USING LC-MS IN HUMAN PLASMA." Cleveland State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=csu1579470084334844.
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Höfig, Carolin. "Establishment, validation and application of immunological and LC-MS/MS-based detection methods to study the role of human aromatic L-amino acid decarboxylase as an enzyme potentially involved in thyronamine biosynthesis." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16645.
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Thyronamine (TAM) sind eine neue Molekülklasse, die endokrinologische und metabolische Prozesse miteinander vereinen. Der biologisch aktive Metabolit 3-Iod-L-Thyronamin (3-T1AM) wird durch eine kombinierte Deiodierung und Decarboxylierung von Schilddrüsenhormonen (TH) gebildet. Existierende Methoden zum Nachweis und zur Quantifizierung von 3-T1AM im menschlichen Serum sind immer noch umstritten. Auch die an der Biosynthese vermutlich beteiligte TH-Decarboxylase konnte noch nicht identifiziert werden. Für die Identifizierung und Quantifizierung von TH und TAM Profilen wurde die Flüssigchromatographie-Tandem-Massenspektrometrie (LC-MS/MS) verwendet. In der bisherigen präanalytischen Aufarbeitung liefern weder Flüssig-Flüssig- noch Festphasenextraktionen reproduzierbare Ergebnisse des 3-T1AM-Gehalts im Serum. Mit der Entwicklung eines spezifischen Extraktionsverfahrens und nachfolgender Detektion mittels LC-MS/MS gelang der gleichzeitige Nachweis der häufigsten TH im humanen Serum. Parallel dazu wurden monoklonale Antikörper gegen 3-T1AM entwickelt, auf deren Basis ein quantitativer 3-T1AM Chemilumineszenz-Immunoassay entstand. Ergebnisse aus klinischen Kollektiven zeigen, dass 3-T1AM im Serum im nM Konzentrationsbereich vorkommt und dass 3-T1AM bei Patienten außerhalb der Schilddrüse produziert wird. Viele Forscher gehen davon aus, dass die aromatische L-Aminosäure Decarboxylase (AADC) die Synthese von TAM über Decarboxylierung von TH katalysiert. Diese Hypothese wurde durch Inkubation von rekombinanter humaner AADC mit TH getestet. In keinem der Experimente konnte AADC die Decarboxylierung von TH katalysieren. Zusammenfassend ist die Bestimmung von 3-T1AM im Serum mittels LC-MS/MS aufgrund der nicht reproduzierbaren präanalytischen Probenaufbereitung problematisch. In dieser Arbeit wird der erste MAb-basierte 3-T1AM assay vorgestellt, der 3-T1AM zuverlässig in humanem Serum quantifiziert. Die AADC ist wahrscheinlich nicht an der Biosynthese von TAM beteiligt.Thyronamines (TAM) are a new class of molecules linking endocrinology and metabolism. Combined deiodination and decarboxylation of thyroid hormones (TH) generates a biologically active ‘cooling’ metabolite, 3-iodo-L-thyronamine (3-T1AM).. It remains controversial, which methods are able or not to reliably detect 3-T1AM in human serum, and the presumed TH decarboxylase is still elusive. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for the simultane-ous identification and quantification of TH and TAM profiles in biological samples. Several preanalytical methods were tested for complete extraction of 3-T1AM in human serum. Thus far, neither liquid-liquid nor solid-phase extraction methods allowed reproducible extraction of 3-T1AM from human serum samples in the preanalytical sample workup. Nevertheless, a rapid and sensitive extraction procedure was developed for detection of the major TH by LC-MS/MS in a single human serum sample. In parallel, monoclonal antibodies (MAb) targeting 3-T1AM were developed and characterized, and a highly specific quantitative 3-T1AM MAb-based chemiluminescence immunoassay was developed. Studies in clinical cohorts provide evidence that 3-T1AM is present in human serum in the nM concentration range and that 3-T1AM is produced extrathyroidally. Many researchers have reasoned that the aromatic L-amino acid decarboxylase (AADC) mediates TAM synthesis via decarboxylation of TH. This hypothesis was tested by incubating recombinant human AADC with several TH. In all tested conditions, AADC failed to catalyze the decarboxylation of TH. These in vitro observations are supported by the finding that 3-T1AM is also present in plasma samples of patients with AADC deficiency. In summary, 3-T1AM detection in serum using LC-MS/MS encounters preanalytical problems. The first MAb-based 3-T1AM CLIA is presented, which reliably quantifies 3-T1AM in human serum. AADC is likely not involved in TAM biosynthesis.
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Xu, Gang. "PART I. COMPREHENSIVE STUDY OF HERBAL MEDICINE FORMULA SHUANG HUANG LIAN BY UNTARGETED PROFILING WITH UHPLC-QTOF-MS AND NETWORK PHARMACOLOGYPART II. DEVELOPMENT OF UHPLC-MS/MS-BASED ASSAY FOR CARDIOLIPIN, A BIOMARKER OF HUMAN DISEASES." Cleveland State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=csu1560269742841914.
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Wei, Ruhan. "Part I: The role of RNase L in lipid homeostasis and the development of atherosclerosisPart II: The role of RNase L in lipopolysaccharide-induced lung inflammationPart III: Development of LC-MS/MS assay for GSK3 inhibitors in plasma." Cleveland State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=csu157902853120912.
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Guedes, Jhonyson Arruda Carvalho. "Validation of analytical method employing quechers and gc-ms for multiresidue determination of pesticides in guava." Universidade Federal do CearÃ, 2014. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=12267.
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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgicoA goiaba à uma das frutas tropicais mais populares e de grande aceitaÃÃo no Brasil e no mundo. A fruta goiaba pode ser alvejada por diversas pragas, reduzindo consideravelmente o rendimento da produÃÃo ocasionando a escassez da fruta no mercado. Visando o aumento da produtividade e consequentemente atender a demanda da populaÃÃo sÃo empregadas elevadas quantidades de agrotÃxicos com o intuito de combater as possÃveis pragas que venham acarretar a deterioraÃÃo da fruta. Dessa maneira, o desenvolvimento de mÃtodos analÃticos para a determinaÃÃo multirresÃduos de agrotÃxicos em alimentos à fundamental para o monitoramento da qualidade dos produtos consumidos pela populaÃÃo, alÃm de possibilitar aos ÃrgÃos reguladores a obtenÃÃo de resultados mais rÃpidos e confiÃveis. Neste trabalho, foi adaptado e validado um mÃtodo para a determinaÃÃo de agrotÃxicos na fruta goiaba, executando-se o preparo da amostra atravÃs do mÃtodo QuEChERS modificado, substituindo-se o solvente acetonitrila apÃs a etapa de limpeza (clean up) por solvente ciclohexano / acetato de etila 1:1 e adiÃÃo de carvÃo ativo para maior limpeza do extrato. Os limites de detecÃÃo (LD) variaram entre 0,002 e 0,010 mg kg-1 e os limites de quantificaÃÃo (LQ) variaram entre 0,005 e 0,030 mg kg-1. Todas as curvas analÃticas construÃdas usando o solvente e a matriz apresentaram significÃncia estatÃstica. Quando comparadas Ãs curvas analÃticas, verificou-se forte efeito matriz para o clorobenzilato, diferentemente do hexaclorobenzeno e da ametrina onde o efeito de matriz foi pouco significativo. Nas anÃlises de amostras de goiaba comercializada na cidade de Fortaleza, foi detectado a presenÃa de agrotÃxicos em 7 amostras. Dos agrotÃxicos detectados, apenas a trifloxistrobina à permitida para a cultura da goiaba, entretanto a concentraÃÃo deste composto à inferior ao limite mÃximo de resÃduo permitido para a goiaba (LMR = 0,050 mg kg-1) segundo a ANVISA.
Guava is one of the most popular and widely accepted in Brazil and worldwide tropical fruits. The fruit can be targeted by various pests, reducing considerably the yield thus causing a slowdown or even a shortage of fruit on the market. Aiming the increase in productivity and ultimately the population's demand high amounts of pesticides are used in order to counter potential pests that may lead to a deterioration of the fruit. Thus, the development of analytical methods for multiresidue determination of pesticides in food is essential for the efficient monitoring of these compounds in the products consumed by the population, enabling regulators to obtain faster and more reliable results. In this work a method for the determination of pesticides in guava was developed and validated, running the sample preparation by modified QuEChERS method, replacing the acetonitrile solvent after cleaning step by cyclohexane / ethyl acetate solvent acetate 1:1 and addition of activated charcoal to extract greater cleanliness. The limits of detection (LOD) ranged between 0.002 and 0.010 mg kg -1 and the limits of quantification (LOQ) ranged from 0.005 to 0.030 mg kg-1 . All calibration curves constructed in solvent and in matrix showed statistical significance when compared to the analytical curves and a strong matrix effect for chlorobenzilate, unlike hexachlorobenzene and ametrina where the matrix effect was negligible. In the analyzes of samples of guava sold in the city of Fortaleza, pesticides were found in 7 samples. Pesticides detected, trifloxystrobin is only allowed for the cultivation of guava, however the concentration of this compound is below the maximum residue limit allowed for guava (MRL = 0.050 mg kg-1 ) according to ANVISA
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Dias, Jonatan Vinicius. "Determinação de resíduos de agrotóxicos em tomate para fins de acreditação." Universidade Federal de Santa Maria, 2014. http://repositorio.ufsm.br/handle/1/10557.
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Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorThe optimization and validation of an analytical method for 51 pesticides determination in tomato, using UPLC-MS/MS, was developed in order to fulfill the requirement 5.4.5 (Method Validation) of the NBR ISO/IEC 17025:2005 and following the orientation document DOQ-CGCRE-008 (Orientation of analytical methods validation). This item is very important to a laboratory to be accredited by INMETRO (Brazilian accreditation body). The method validation was carried out following all the validation parameters, such as linearity of the analytical curves, limits of detection and quantification, matrix effects, accuracy and precision of the method. The studied compounds in this work were determined by UPLC-MS/MS in the positive ESI ionization mode. The stock solutions of the pesticides were prepared in organic solvent compatible with the compound solubility. These solutions were prepared by two different analysts in order to validate the preparation by comparing the areas of the injections (n=7) of each individual solution. After evaluation, 43 from 51 studied solutions showed results between the acceptable range of ± 10% demonstrating the satisfactory quality of solutions preparation. The evaluated pesticides were extracted from tomatoes applying the mini Luke method with a mixture 1:1:1 (v/v/v) of each acetone/petroleum ether/dichloromethane solvent as extraction solvent. After extraction, the organic solvent extract was evaporate and reconstituted in appropriate solvent (acidified methanol with 0.1% acetic acid) for UPLC-MS/MS analysis. The recovery experiments were done at 10, 20 and 50 μg kg-1 spike levels, by two analysts in different days, and 7 replicates (n=7) for each spike level plus the blank matrix (without pesticides). The limits of detection and quantification for the instrument and method were estimated, as well as the linearity of the analytical curves evaluated based on determination coefficient (r²), dynamic linear range, accuracy by recovery experiments (%), precision (RSD%) and matrix effect. From the 51 studied compounds, 46 showed recoveries between the range of 81 115% for all the evaluated spike levels. About precision, 92% of the compounds showed RSD% below 18.7% in the lowest spike level. The quantification limit of the method, for 82% from all evaluated compounds, was the lowest spike level studied (10 μg kg-1). The matrix effect observed was between ± 20% for all evaluated compounds, showing that there is not considerable suppression or enhancement in the analytes signal. For all evaluated pesticides the intermediate precision was below 20% of RSD showing the good repeatability of the method.
O desenvolvimento e validação do método analítico para determinação de 51 agrotóxicos em tomate por UPLC-MS/MS foi realizado a fim de atender ao requisito 5.4.5 (Validação de Métodos) da norma NBR ISO/IEC 17025:2005 e seguindo o documento orientativo DOQ-CGCRE-008 (Orientação sobre validação de métodos analíticos). Esse item descrito na norma é de fundamental importância para que o laboratório possa ser acreditado pelo INMETRO. A validação do método seguiu todos os parâmetros de validação, tal como a linearidade das curvas analíticas, limites de detecção e quantificação, efeito matriz, exatidão e precisão do método. Os agrotóxicos estudados neste trabalho foram determinados por UPLC- MS/MS no modo de ionização ESI positivo. As soluções estoque dos agrotóxicos foram preparadas em solvente orgânico compatível com a solubilidade de cada substância. Essas soluções foram preparadas por dois analistas distintos a fim de validar o preparo dessas a partir da comparação das áreas das injeções realizadas (n=7) de cada solução individual. Após avaliação, 43 das 51 soluções estudadas mostraram resultados dentro da faixa de ± 10% demonstrando a excelente qualidade no preparo dessas soluções. Os agrotóxicos estudados foram extraídos das amostras de tomate utilizando o método mini Luke o qual faz o uso de uma mistura 1:1: 1 (v/v/v) de acetona/éter de petróleo/diclorometano como solvente extrator. Após extração, o extrato em solvente orgânico foi evaporado e ressuspendido em solvente apropriado (metanol acidificado com 0,1% ácido acético) para análise por UPLC-MS/MS. O estudo de fortificação e recuperação dos analitos foi realizado nas concentrações de 10, 20 e 50 μg kg-1, por dois analistas, e 7 replicatas (n=7) para cada concentração, além das amostras branco (sem conter os agrotóxicos). Os limites de detecção e quantificação do instrumento e do método foram determinados, bem como a linearidade das curvas analíticas avaliadas através do coeficiente de determinação (r²), faixa linear dinâmica de trabalho, exatidão através do estudo de fortificação e recuperação (%) dos analitos, precisão (RSD%) e efeito matriz. Dos 51 agrotóxicos estudados, 46 apresentaram recuperação média na faixa de 81 a 115 % para todas as concentrações avaliadas. Com relação à precisão, 92% dos agrotóxicos demonstraram RSD% abaixo de 18,7% na menor concentração de fortificação. O limite de quantificação do método, para 82% dos agrotóxicos, foi a concentração de fortificação mais baixa estudada (10 μg kg-1). O efeito matriz observado foi inferior a ± 20% para todos os agrotóxicos avaliados, demonstrando assim que não há supressão ou aumento considerável no sinal do analito. Para todos os agrotóxicos estudados os resultados de precisão intermediária (onde foram avaliados diferentes analistas em diferentes dias) ficaram abaixo de 20% demonstrando a repetitividade satisfatória do método proposto.
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Lehto, T. (Tiina). "Evaluation of new laboratory methods for routine use." Doctoral thesis, Oulun yliopisto, 2016. http://urn.fi/urn:isbn:9789526210759.
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Abstract Laboratory medicine is under constant pressure from changes in the operating environment. Organisational changes and tendering processes have led to a trend towards shorter turn-around times and more cost-effective choices. Analysis tools that were previously only available at research laboratories, such as the mass spectrometer and polymerace chain reaction (PCR), have now made their way to university hospital laboratories and even mid-sized laboratories. Organisational changes have increased the need to monitor the pre-analytical steps. The specimen can be drawn from the patient in a satellite laboratory, which may be located several hours from the central laboratory. The increased transportation times may change the analytical properties of the specimens, which is why the stability of different analytes should be investigated thoroughly in different temperatures. It should be born in mind that doctors are treating the patients based on the results they receive from the laboratory. To avoid possible malpractice, the analytical properties should remain reliable. Traditionally, some analyses have been carried out manually, which is known to be time-consuming and carries the possibility of wide intra-observatory mistakes. For that reason, it would be reasonable to perform some manual analyses, such as body fluid analysis, in an automated manner. Automating the manual steps taken in the laboratory would release labour for other tasks and may increase the cost-effectiveness of the work. Organisational changes have redirected the needs of a clinical laboratory towards automated options instead of manual ones and finding more economically-based alternatives to replace or complement traditional methodsTiivistelmä Laboratoriolääketiede on jatkuvan muutospaineen alla. Organisaatiomuutokset ja kilpailutus ovat saaneet aikaan sen, että laboratorioiden analytiikkatarjonnan tulee olla kilpailukykyistä niin hinnan kuin tulosten vastausnopeuden suhteen. Aikaisemmin pelkästään tutkimuskäytössä olleet menetelmät, kuten PCR ja massaspektrometri, ovat jalkautuneet jo keskussairaalatasoiseen tutkimusvalikoimaan. Organisaatiomuutokset ovat saaneet aikaan myös sen, että näytteet voidaan ottaa potilaasta alueellisissa toimipisteissä ja kuljettaa päivän aikana keskuslaboratorioon analysoitavaksi. Kuljetusmatkat ja -ajat saattavat olla hyvinkin pitkiä. Tämän johdosta on erittäin tärkeää selvittää näytteiden säilyvyys niin, että tulokset pysyvät luotettavina eikä potilaan hoito kärsi. Perinteisesti osa tutkimuksista, kuten punktionesteen solut, on tehty käsin mikroskopoimalla, jonka tiedetään olevan aikaa vievää ja näin ollen myös kallista analysointia. Kyseisen tutkimuksen siirtäminen analysaattoreille tehtäväksi voi tuoda laboratoriolle taloudellisen säästön lisäksi työvoiman vapautumista manuaalisesti suoritettavalta mikroskopoinnilta. Muutospaineet laboratoriotoiminnoissa ovat saaneet aikaan tarpeen automatisaation lisääntymiselle ja taloudellisempien vaihtoehtojen löytämiselle perinteisten menetelmien rinnalle tai niiden sijaan
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Martins, FÃtima Itana Chaves CustÃdio. "Development and method validation for multiresidue determination of pesticides in mango (Mangifera indica L.) using QuEChERS and GC - MS." Universidade Federal do CearÃ, 2015. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=15591.
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Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgicoThe development of analytical methods aiming at monitoring of pesticides is essential to ensure that foods are within limits considered safe for health, and to enable the regulatory agencies to obtain faster and more reliable results. In this work, we developed and validated a method for the determination of pesticides in mango, running the sample preparation with QuEChERS method modified with the addition of graphitized carbon (CGB - Carbon Grafitized Black) to remove nonpolar pigments and to identify and quantification of the compounds to the Gas Chromatography Mass Spectrometry (GC-MS). The developed method showed selectivity for all compounds. For all compounds was identified matrix effect, so the study of quantification of the compound was made based on the curves made in the matrix extract. The limits of detection (LOD) ranged from 0,0025 to 0,01 mg.kg-1 and the limits of quantification (LOQ) ranged from 0,008 to 0,03 mg.kg-1. The compounds showed acceptable recovery levels ranging from34% to 143%. The method was applied to the determination of pesticide residues in twelve samples of mango, among which, in six samples were detected five different compounds (Chloroneb, Propachlor, α-Chlordane, Chlorpyrifos,DCPA, Chlorobenzilate and trans-Permethrin). For Chloroneb compounds, Propachlor and α-Chlordane were detected concentration above the maximum residue limit allowed under EU data
O desenvolvimento de mÃtodos analÃticos visando o monitoramento de pesticidas à fundamental para assegurar que os alimentos estejam dentro dos limites considerados seguros para a saÃde, bem como possibilitar aos ÃrgÃos fiscalizadores a obtenÃÃo de resultados mais rÃpidos e confiÃveis. Neste trabalho, foi desenvolvido e validado um mÃtodo para a determinaÃÃo de pesticidas em manga, executando-se o preparo da amostra com mÃtodo QuEChERS modificado, com a adiÃÃo de carbono grafitizado (CGB â Carbon Grafitized Black) para remover pigmentos apolares e para identificaÃÃo e quantificaÃÃo dos compostos a Cromatografia Gasosa acoplada a Espectrometria de Massas (CG-EM). O mÃtodo desenvolvido demonstrou seletividade para todos os compostos. Para todos os compostos foi identificado efeito matriz, dessa forma o estudo de quantificaÃÃo dos compostos foi feito com base nas curvas realizadas no extrato da matriz. Os limites de detecÃÃo (LD) variaram de 0,0025 a 0,01 mg.kg-1 e os limites de quantificaÃÃo (LQ) variaram de 0,008 a 0,03 mg.kg-1. Os compostos apresentaram nÃveis de recuperaÃÃo aceitÃveis compreendidos entre 34% a 143%. O mÃtodo desenvolvido foi aplicado para a determinaÃÃo de resÃduos de pesticidas em doze amostras de manga, dentre as quais, em seis amostras foram detectados cinco diferentes compostos (Cloronebe, Propacloro, α-Clordano, ClorpirifÃs, DCPA, Clorobenzilato e trans-Permetrina), com valores entre 0,004 a 0,042 mg.kg-1. Para os compostos Cloronebe, Propacloro e α-Clordano foram detectados concentraÃÃes superiores ao limite mÃximo de resÃduo permitido segundo dados da UniÃo EuropÃia
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Höfig, Carolin [Verfasser], Werner [Akademischer Betreuer] Kloas, Josef [Akademischer Betreuer] Köhrle, and Dagmar [Akademischer Betreuer] Führer-Sakel. "Establishment, validation and application of immunological and LC-MS/MS-based detection methods to study the role of human aromatic L-amino acid decarboxylase as an enzyme potentially involved in thyronamine biosynthesis / Carolin Höfig. Gutachter: Werner Kloas ; Josef Köhrle ; Dagmar Führer-Sakel." Berlin : Humboldt Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://d-nb.info/1029763844/34.
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Alamil, Helena. "Etude de profils en adduits à l'ADN comme biomarqueurs potentiels d'exposition aux polluants aériens en milieu urbain dans une approche de type adductomique." Thesis, Normandie, 2019. http://www.theses.fr/2019NORMC407/document.
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De nombreuses études dans la seconde moitié du 20ème siècle, ont mis en évidence que des génotoxiques cancérogènes réagissent avec l'ADN pour former par liaison covalente des adduits qui sont impliqués dans le processus cancérigène. Bien qu’il existe des preuves convaincantes de la présence de multiples adduits à l'ADN dans les poumons de sujets exposés au tabagisme ou en milieu professionnel à un aldéhyde donné, il est évident que c'est un domaine dans lequel des recherches supplémentaires ont été nécessaires. L’objectif de ce travail de thèse est d’établir des profils d’adduits exocycliques à l'ADN induits par le mélange d’aldéhydes, qui pourraient à terme être considérés comme un marqueur génotoxique de l’exposition aux aldéhydes, tant endogène qu’environnemental. Pour cette raison, nous avons validé une méthode en UHPLC-MS/MS rapide, sensible et précise en utilisant la dilution isotopique, pour la quantification à l’état de trace de 9 adduits exocycliques à l’ADN dérivés de 8 principaux aldéhydes exogènes et endogènes, notamment le formaldéhyde, l’acétaldéhyde, l’acroléine, le crotonaldéhyde, le malondialdéhyde, le 4-hydroxy-2-nonénal, le glyoxal et le méthylglyoxal. Ces adduits ont été synthétisés et purifiés ainsi que leurs homologues marqués au 13C10, 15N5, identifiés et quantifiés par le biais des courbes d'étalonnage allant de 0,25 à 250 ng/mL d'adduits dans l'eau et l'ADN afin de décrire les effets matrice. Des échantillons de contrôle qualité ont été préparés et analysés afin de vérifier l'exactitude et la précision de la méthode dans des situations de répétabilité et de fidélité intermédiaire. L'absence de contamination croisée a également été démontrée. La méthode est capable de différencier les 9 analytes d'intérêt et leurs étalons internes en utilisant pour chaque analyte une transition de quantification et une seconde de confirmation. Cette méthode a été validée selon les recommandations de l'Agence Européenne des Médicaments concernant les méthodes bioanalytiques. Elle répond à tous les critères essentiels pour garantir l'acceptabilité des performances et la fiabilité des résultats d'analyse. Cette méthode est la toute première validée et peut être utilisée en adductomique dans le cadre d'études sur l'exposome. En plus, nous avons simultanément mesuré par une approche in vitro les 9 adduits exocycliques dans de l’ADN de thymus de veau exposé à de différentes concentrations de chaque aldéhyde seul ou en mélanges équimolaires. Cette approche nous a permis d’établir des relations dose-dépendantes pour tous les aldéhydes à l’exception du malondialdéhyde et du méthylglyoxal. Une relation dose-réponse a également été observée avec les mélanges équimolaires d’aldéhydes. Elle a permis de définir des réactivités différentes des aldéhydes en mélange vis-à-vis de l’ADN. Les profils de ces adduits exocycliques ont été également déterminés dans l'ADN de sang de fumeurs et de non-fumeurs. La fumée de cigarette contient plusieurs aldéhydes connus de se lier par covalence aux bases de l’ADN, ainsi l’adduit à l’ADN peut être considéré comme biomarqueur d’exposition au tabac. Des différences significatives dans les niveaux d’adduits ont été obtenues entre l’ADN des fumeurs et celui des non-fumeurs à l’exception de l’adduit induit par le malondialdéhyde. Des corrélations ont été établies entre chaque adduit et les marqueurs de la consommation tabagique sans aucune corrélation significative de la totalité des adduits avec un marqueur spécifique. Par ailleurs, nous avons montré que l’exposition au formaldéhyde, au butanal et au benzaldéhyde a eu un effet sur les concentrations du MDA urinaire mesurées chez les policiers libanais stationnés au carrefour pendant 7 h par jour et après exposition de 5 jours aux émissions du trafic routier. Une augmentation du MDA plasmatique a été décrite ; les années de travail avaient une incidence sur les concentrations de ce biomarqueurMany studies in the second half of the 20th century have shown that genotoxic carcinogens, either directly or after metabolic activation, react with DNA to form covalently bonded adducts that are absolutely central in the carcinogenic process. Although there is compelling evidence of the presence of multiple DNA adducts in the lungs of subjects exposed to smoking or occupational exposure to a given aldehyde, it is clear that this is an area in which further research has been necessary. The aim of this thesis is to establish exocyclic DNA adducts profiles induced by the mixture of aldehydes, which could eventually be considered as a genotoxic marker of aldehyde exposure, both endogenous environmental. For this reason, we have validated a fast, sensitive and precise method on liquid chromatography coupled to mass spectrometry in tandem mode (UHPLC-MS/MS) using isotopic dilution, for trace quantification of 9 exocyclic DNA adducts derived from 8 major exogenous and endogenous aldehydes, including formaldehyde, acetaldehyde, acrolein, crotonaldehyde, malondialdehyde, 4-hydroxy-2-nonenal, glyoxal and methylglyoxal. These adducts were synthesized and purified as well as their labeled homologues, identified and quantified through standard curves ranging from 0.25 (LLOQ) to 250 ng/mL (ULOQ) adducts in water and in DNA to describe the matrix effects. Quality control (QC) samples were prepared and analyzed to verify the accuracy and precision of the method in repeatability and intermediate fidelity situations. The absence of cross-contamination has also been demonstrated. The method is able to differentiate the 9 analytes of interest and their internal standards using for each analyte a quantification transition and a confirmation transition. This method has been validated according to the recommendations of the European Medicines Agency (EMA) concerning bioanalytical methods. It meets all the essential criteria to guarantee the acceptability of the performances and the reliability of the analysis results. This method is the very first validated and can be used in adductomics in the context of studies on the exposome. In addition, the exocyclic adducts were simultaneously measured by an in vitro approach in calf thymus DNA exposed to different concentrations of each aldehyde apart or in equimolar mixtures. This approach allowed us to establish dose-dependent relationships for all aldehydes with the exception of malondialdehyde and methylglyoxal. A dose-response relationship was also observed with equimolar mixtures of aldehydes. It made it possible to define different reactivities of aldehydes in mixture versus DNA. The profiles of these exocyclic adducts were also determined in the blood DNA of smokers and non-smokers. Cigarette smoke contains several aldehydes known to covalently bind to DNA bases, so the DNA adduct may be considered as biomarker of tobacco exposure. Significant differences in adducts levels were obtained between smokers and non-smokers DNA with the exception of malondialdehyde-induced DNA adduct. Correlations were established between each adduct and smoking-related markers without any significant correlation of all adducts with a specific marker. Furthermore, we have shown that exposure to formaldehyde, butanal and benzaldehyde had an effect on the concentrations of urinary MDA measured in Lebanese police stationed at the intersection for 7 hours a day and after 5-day exposure to road traffic. An increase in plasma MDA has been described; years of work had an impact on the concentrations of this biomarker. These results are promising and it would be interesting to validate in population the profile of 9 exocyclic adducts as biomarkers of exposure to both exogenous and endogenous aldehydes as part of an adductomic approach to understand the carcinogenic risk in relation to aldehydes exposures in urban areas
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Meirelles, Alyne Fávero Galvão. "Uso da cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial para determinação do perfil de eicosanoides em plasma após estimulação: comparação entre pacientes com anemia falciforme e indivíduos saudáveis." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-02052016-105840/.
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Os eicosanoides, produtos do metabolismo do ácido araquidônico, apresentam papel importante na homeostasia e na patogênese de diversas doenças humanas. A biossíntese desses compostos pode ser estimulada por agentes farmacológicos como ionóforos e inibidores da Ca2+-ATPase, e também por agonistas naturais como o formil-metionil-leucil-fenialanina (fMLP). Considerando os interesses em avaliar e comparar o perfil de mediadores lipídicos, como os leucotrienos (LTs), as prostaglandinas (PGs), os ácidos epoxieicosatrienoicos (EETs), os ácidos dihidroxitetraenoicos (DiHETEs) e os ácidos hidroxieicosatetraenoicos (HETEs), na saúde e na doença, o objetivo deste trabalho foi padronizar um método analítico para determinar do perfil de eicosanoides em plasma humano após estimulação do sangue total, e assim observar diferenças entre indivíduos saudáveis e doentes. Dessa forma, um método por cromatografia líquida de alta eficiência acoplada à espectrometria de massas sequencial (HPLC-MS/MS) foi validado para quantificação de 22 eicosanoides em plasma de indivíduos saudáveis. A análise por HPLCMS/ MS foi realizada em modo negativo pelo modo de varredura por monitoramento de reações múltiplas (MRM). A linearidade do método apresentou coeficiente de correlação (r) maior que 0,98 para todos os eicosanoides analisados. A precisão e exatidão intra e inter-ensaios tiveram desvio padrão e erro relativo menores que 15%, exceto para o limite inferior de quantificação cujos valores foram menores que 20%. Para estimulação das células do sangue total, quatro estímulos (fMLP, ionomicina, A23187 e tapsigargina) foram utilizados. A análise estatística mostrou que o A23187 e a tapsigargina foram os estímulos mais potentes na indução da produção de eicosanoides. Em seguida, comparamos o perfil de eicosanoides em amostras de plasma de indivíduos saudáveis com pacientes com anemia falciforme (AF), em tratamento com hidroxiureia (HU) ou transfusão sanguínea crônica. Os resultados demonstraram que o método é preciso para determinação de diferenças entre os pacientes e indivíduos saudáveis quanto à produção dos mediadores lipídicos 5-HETE, 12-HETE, LTB4, LTE4, TXB2 e PGE2. Portanto, nosso método analítico é sensível, específico e reprodutível para identificar e quantificar diferenças no perfil de eicosanoides em amostras de sangue estimuladas in vitro, e poderá contribuir para o estabelecimento do perfil de mediadores lipídicos em diferentes doenças inflamatórias e infecciosas.Eicosanoids, products from arachidonic acid metabolism, play an important role in the homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca2+ ionophores and Ca2+-ATPase inhibitors, as well as natural agonists such as fMet-leu-Phe (fMLP) can stimulate eicosanoid biosynthesis. Considering the interests in evaluate and compare the profile of lipid mediators, as leukotriens (LTs), prostaglandins (PGs), epoxyeicosatrienoic acids (EETs), dihydroxytetraenoic acids (DiHETEs) and hydroxyeicosatetraenoic acids (HETEs), in healthy and disease, the aim of this work was to standardize a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation, and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatographytandem mass spectrometry (LC-MS/MS) method was validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient > 0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of < 15%, except for the lower limit of quantification, where these values were < 20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were used. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli to induce the production of eicosanoids. We next compared the eicosanoid profiles of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients compared to healthy subjects for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method is sensitive, specific and reproducible for indentify and quantify changes in eicosanoid profiles in whole blood stimulated in vitro, which can contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases.
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Utami, Wahyu. "Ion pairing LC-MS/MS method for analysis of intracellular phosphorylated metabolites." Thesis, University of Nottingham, 2015. http://eprints.nottingham.ac.uk/30566/.
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Nucleoside analogues have been extensively used in medication. The nucleoside analogue cordycepin is the principal bioactive compound found in the caterpillar fungi (Cordyceps and Ophiocordyceps). It has been shown to have biological activity, including anti-inflammatory, immunomodulatory and anti-proliferative activity in many kinds of malignant cells. Intracellular drug interactions at the nucleotide level can be explained by understanding the intracellular metabolism of nucleoside analogues as well as their plasma metabolism since their efficacy of therapy or toxicity does not associate with the plasma level of nucleoside. Therefore, investigation of the metabolism of nucleoside analogues is required for a full understanding of their pharmacological activity and toxicity. For that reason, here an ion pairing LC-MS/MS method has been developed for quantitative analysis of the nucleoside analogue cordycepin and the metabolites and its application to cell culture, using in vitro and in vivo studies. Several HPLC parameters and extraction techniques have been optimised, followed by optimisation of the mass spectrometry method by examining the fragmentation of nucleotides. The method was then validated and applied to study the metabolism of cordycepin in vitro and in vivo, to investigate the effect of the cordycepin treatment with or without pentostatin on the intracellular level of endogenous nucleotides, and to examine the intracellular metabolism of nucleoside analogue 4-thiouridine and the effect of its metabolite on the metabolic balance of adenine and uridine nucleotides. The study on the intracellular metabolism of cordycepin in MCF7 and HeLa cells shows that cordycepin was rapidly metabolized into the deaminated form by adenosine deaminase (ADA) in culture medium as well as in cancer cells; therefore combination with pentostatin, an ADA inhibitor, resulting in the highly accumulated phosphorylated metabolite intracellularly. In contrast, cordycepin in C. militaris extracts showed much lower degradation in non-heat-treated serum compared with pure cordycepin that indicates a strong evidence of the presence of a deaminase inhibitor in the extract of Cordyceps. Moreover, the determination of concentrations of cordycepin and the metabolites in the plasma and liver of rats dosed with cordycepin proves that the half-life of cordycepin and its metabolites are very short in the plasma; nevertheless they are accumulated in the liver with repeated administration. Treatment using cordycepin initially caused an increase in the intracellular concentrations of nucleoside triphosphate, but in the long term, the active metabolite of cordycepin likely induced a long term change in the cell resulting in a drop in nucleotide levels. Pentostatin on its own reduced nucleoside triphosphates levels in the long term and combination with cordycepin increased the effect of cordycepin on nucleotide concentrations. High levels of the accumulated cordycepin triphosphate led to a massive decline in nucleotide levels. A study on the intracellular metabolism of nucleoside analogue 4-thiouridine has shown that generally the uptake of 4-thiouridine into NIH 3T3 cells was fast and the phosphorylated metabolite rapidly was developed only after two min labelling. However, it was also shown that its phosphorylation was not very efficient, but the level of the phosphorylated metabolite increased in serum-stimulated cells likely because the enzyme was upregulated in the presence of growth factor. Moreover, the present study provides additional evidence that 4-thiouridine and its metabolite have no adverse effect on the metabolic balance of adenine and uridine nucleotides. This study confirms that pharmacological activity of nucleosides analogues and their cytotoxicity highly rely on the accumulation of their phosphorylated metabolites. Consequently, the activity and the level of the enzymes involved in their metabolism are highly influential on their pharmacological action as well as their toxicity.
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Sia, Xin Rong. "Development of a rapid and in-field phenotyping tool for screening protein quality in soybeans (Glycine max) using a miniature near infrared sensor." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574800276913103.
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Lindholm, Johan. "Development and Validation of HPLC Methods for Analytical and Preparative Purposes." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, Univ.-bibl. [distributör], 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-4442.
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Miranda, e. Silva Lígia Maria 1982. "Validação de método de análise de multiresíduos de defensivos agrícolas por GC-MS/MS e LC-MS/MS." [s.n.], 2012. http://repositorio.unicamp.br/jspui/handle/REPOSIP/254815.
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Orientador: Marcelo Alexandre PradoDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: O crescente aumento populacional em escala mundial, tornou necessário um grande esforço por parte da agricultura para aumentar, a cada ano, a produção de alimentos para atender as necessidades do mercado externo e interno do Brasil. Recursos técnicos e científicos passaram então, a serem aplicados em busca da melhoria na produção dos cultivos,principalmente mediante o uso de fertilizantes e praguicidas. Com isso, a sociedade se deparou com problemas de ordem de equilíbrio ambiental e saúde pública, pois devido à contínua diversificação dos fitoparasitas, surgem, a todo momento, reduções do período de tempo entre aplicações consecutivas, e mais importante talvez, usos de doses mais altas e emprego simultâneo de diferentes pesticidas, por parte dos agricultores, objetivando complementar ações específicas ou alcançar efeitos sinérgicos para maiores rendimentos na produção. Tal situação traz como conseqüência óbvia e direta, o aumento, inaceitável, dos riscos de contaminação do meio ambiente com resíduos químicos de defensívos da área agropecuarista prejudiciais à saúde, o que leva a inúmeros problemas relativos à segurança alimentar dos produtos consumidos, e à uma preocupação de âmbito nacional evidenciada pela criação do Programa de Análise de Resíduos de Agrotóxicos em alimentos (PARA) da ANVISA. O aumento na necessidade de detecção e quantificação destes compostos, acarretou o desenvolvimento de pesquisas no setor, a fim de atingir uma melhoria na eficiência,qualidade e rapidez de resposta nas análises. A possibilidade do estudo de não apenas um de cada vez, mas de até 300 compostos sendo extraídos, detectados e quantificados simultâneamente se tornou a saída mais viável, tanto qualitativa quanto economicamente, facilitando o monitoramento contínuo do fornecimento de produtos do setor alimentício pelos chamados métodos multiresíduos. O presentre trabalho teve como princípio a validação de um método multiresíduo para análise de 14 analitos usando uma técnica de alto poder de concentração e limpeza do extrato como o GPC (Gel Permeation Chromatography) e detecção e quantificação por GC-MS/MS e LC-MS/MS. Os pesticidas investigados englobam classes como: acaricidas, inseticidas, fungicidas, nematicidas e formicidas de aplicação foliar, em sementes ou em solo, sendo que o acefato, metamidofós, acetamiprido e o thiamethoxan foram extraídos de amostras de batata e feijão e analisados por LC-MS/MS e a azoxistrobina, bifentrina, carbofuran, chlorotalonil, clorpirifós, clorfenapir, etofenprox, famoxadone,metalaxil, procimidone e o tebuconazole em amostras de batata e tomate e analisados por GCMS/MS. Os limites de detecção (LD) encontrados variaram de 0,06 a 2,89µg/L, e os coeficientes de variação (CV), de 0,036 a 2,036%. As recuperações foram determinadas em cada tipo de amostras, e os valores encontrados estavam entre 93,34% e 109,67%. Nenhuma das matrizes utilizadas apresentaram resultados insatisfatórios e o método utilizado mostrouse robusto e de fácil aplicação para todos os analitos testados
Abstract: The growing population worldwide, has required a great effort on the part of agriculture to increase each year, the production of food to meet the needs of external and internal market of Brazil. Technical and scientific resources spent then, to be applied in pursuit of improved crop production, mainly through the use of fertilizers and pesticides.With this, the company encountered problems in the balance of environmental and public health, since due to the continuous diversification of plant parasites, arise at any moment,reductions in the time period between consecutive applications, and perhaps most important,uses more doses high and simultaneous use of different pesticides by farmers, aiming to complete specific actions or to achieve synergistic effects in producing higher yields. This situation brings obvious and direct consequence, the increase unacceptable risk of environmental contamination with chemical residues from pesticides in farms are harmful to health, which leads to numerous problems relating to food safety of the products consumed, and to a concern nationwide evidenced by the creation of the Program Analysis of Pesticide Residues in Food (TO) of ANVISA. The increase in the necessity for detection and quantification of these compounds, led the development of research in the sector in order to achieve an improvement in efficiency, quality and responsiveness in the analyzes. The possibility of studying not just one at a time, but up to 300 compounds being extracted,detected and quantified simultaneously output became more viable, both qualitatively and economically, facilitating continuous monitoring of the supply of products by the food industry called methods multiresidue. The principle presentre work was the validation of a multiresidue method for analysis of 14 analytes using a technique of high power concentration and cleanup of the extract as GPC (Gel Permeation Chromatography) and detection and quantification by GC-MS/MS and LC- MS / MS. The pesticides investigated include classes such as acaricides, insecticides, fungicides, insecticides and nematicides foliar, seed or soil,and acephate, methamidophos, and Acetamiprid thiamethoxan were extracted from samples of potatoes and beans and analyzed by LC-MS / MS and azoxystrobin, bifenthrin, carbofuran,chlorothalonil, chlorpyrifos, chlorfenapyr, etofenprox, famoxadone, metalaxyl, procymidone and tebuconazole in samples of potato and tomato and analyzed by GC-MS/MS. The limits of detection (LOD) ranged from 0.06 to 2.89 mg / L, and the coefficients of variation (CV), 0.036 to 2.036%. The recoveries were determined for each type of samples, and the values were between 93.34% and 109.67%. None of the arrays used had unsatisfactory results and method proved to be robust and easy to apply for all analytes tested
Mestrado
Ciência de Alimentos
Mestra em Ciência de Alimentos
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Lundberg, Robert. "Validation of Biomarkers for the Revision of the CEN/TR 15522-2:2012 Method : A Statistical Study of Sampling, Discriminating Powers and Weathering of new Biomarkers for Comparative Analysis of Lighter Oils." Thesis, Linköpings universitet, Kemi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-156975.
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The revision of the CEN/TR 15522-2:2012 methodology contains new biomarkers to facilitate forensic fingerprinting of the variety of oil types that can be a part of different crimes and the purpose of this project is to validate the biomarkers of the new methodology. Biomarkers were validated by examining corresponding diagnostic ratios compatibility with the internationally used sampling cloth, discriminating power, correlation and simulated weathering sensibility through GC-SIM-MS analysis followed by statistical evaluation with t-tests, diagnostic power, Pearson correlation matrices and MS-PW plots respectively. Results based on most of the diagnostic ratios showed good compatibility with the internationally used sampling cloth, expected patterns of biodegradation and photo-oxidation except for observed photo-oxidation of hydro PAHs and that normative ratios and informative ratios with high diagnostic powers, but with strong correlations for some of the tested ratios, could be identified in diesel oils. Due to delimitations however such as the limited number of oils with similar origins that were analyzed the results should be regarded as guidelines that can be expanded.
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Yacoub, Kimberly. "Development of ESI-LC-MS Method for Drug Analysis." Cleveland State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=csu1524040258129489.
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Tang, Jennifer Huiqin. "DEVELOPMENT OF A LC/MS/MS ENZYME METHOD FOR N8 - ACETYLSPERMIDINE MEASUREMENTS IN ENZYME ASSAYS." Scholarly Commons, 2005. https://scholarlycommons.pacific.edu/uop_etds/607.
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This thesis describes the development of a way to study the N8 - acetylspermidine deacetylase enzyme activity. The method created in this thesis emphasizes sensitivity, accuracy and safety.
In this study, HeLa cells were cultured and extracted to yield a crude N8 - acetylspermidine deacetylase enzyme mixture. By measuring the decrease of N8 - acetylspermidine and the increase of spetmidine, N8 -acetylspermidine deacetylase enzyme activity can be determined using either a Varian 1200L LC/MS/MS or an API 3000 LC-ES (+)/MS/MS. An acetylation-derivatization method was developed because N8 -acetylspermidine and spermidine are hard to purify from a biological sample since they are not retained on a CIS solid phase extraction column or on a RP HPLC (high performance liquid chromatography) reverse phase column due to their small molecular weight and high polarity.
The quantitation of N8 -acetylspem1indine over the range 2ng/ul to 5pg/ul was fit by linear regression as y = 1.064x + 0.218 with an R-squared value of 0.9996, where y is the peak area of the fragment-ion SRM (selected reaction monitoring: m/z: 188/114) chromatograms from N8 -acetylspermindine and x is the concentration of N8 - acetylspermindine. Acetylation of spermidine (SPD) and N8-acetylspermidine (N8AcSPD) with d6-acetic anhydride produces the d9 labeled triacetylated derivative of SPD and d6 labled triacetylated spermidine derivative of N8AcSPD. These triacetylated forms are retained on a C18 column. MS/MS gives characteristic m/z fragment ions for the derivatized species: N8AcSPD (278 to 215), NlAcSPD (278 to 218) and SPD (281 to 218). The fragment-ion SRM (selected reaction monitoring) chromatograms are used for the quantitation. A plot of peak area ratios for known mixtures of N8AcSPD and total SPD versus the molar ratios of N8AcSPD and total SPD was found to fit a linear regression line withy= 0.705x + 0.035 with an R-squared value of 0.9919. Quantitation of d6- and dg-tri-acetylspermidine by LC/MS/MS is possible at the low levels of materials found in cell extracts since the separation method results in a lower limit of quantitation. This approach enables the study of N8 - acetylspermidine deacetylase enzyme activity.
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Lively, J. D. "The automation and validation of LC/MS/MS and its application to pharmacokinetic and drug metabolism studies." Thesis, Swansea University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.637929.
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The studies described in this thesis have investigated the complexities of automating Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS) systems, both in terms of sample introduction and data processing, and their subsequent testing and validation. Atmospheric pressure ionisation sources have revolutionised the use of mass spectrometry in the last 10 years. With the use of these robust and universal sources, large numbers of samples can now be processed in a single bioanalytical run. This has driven the need for high sample throughput automation. Described within are applications exploiting the versatility of the Gilson Automated Sample Processor using Extraction Columns (ASPEC) XLi Cartesian robot. In order to process the ever-increasing sample data, and to relieve this bottleneck in bioanalysis, in-house Interactive Chemical Information Software (ICIS-IIITM) scripts were written and developed. The validation of the Finnigan MAT TSQ 700 mass spectrometer and its ancillaries, including the in-house written software, to Good Laboratory Practice (GLP) standards are described in detail. In addition, and as a further demonstration of the robustness and versatility of LC/MC/MC, the analysis of a compound extracted from dog lung homogenate following a novel solid phase extraction (SPE) method is recapitulated. A strategy for the method development of LC/MS/MS bioanalytical assays is likewise included.
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Cortese, Melissa. "Développement et validation du dosage de 9 nucléotides par couplage LC-MS/MS pour la recherche dans les maladies cardiovasculaires." Doctoral thesis, Universite Libre de Bruxelles, 2019. https://dipot.ulb.ac.be/dspace/bitstream/2013/294060/4/TdM.docx.
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Les nucléotides, en tant que seconds messagers (AMPc et GMPc) et qu’agonistes des récepteurs purinergiques (ADP, ATP, UDP et UTP) jouent de nombreux rôles dans la régulation des processus physiologiques et pathologiques. Bien que les réponses physiologiques impliquant les nucléotides ne se limitent pas aux maladies cardiovasculaires, ce projet s’est focalisé sur celles-ci, en particulier sur l’athérosclérose, le dysfonctionnement endothélial et l’anévrisme cérébral étant donné que notre laboratoire s’intéresse de près aux phénomènes inflammatoires liés à ces pathologies. Il est par exemple reconnu que les variations des taux d’AMPc et de GMPc peuvent réguler la fonction de la barrière endothéliale (via les phosphodiestérases qui les hydrolysent respectivement en AMP et GMP inactives et via les cyclases qui les synthétisent à partir des nucléotides triphosphates) et donc causer le dysfonctionnement endothélial qui est une cause sous-jacente de plusieurs conditions pathologiques telle que l’athérosclérose. Mais aussi, que le relargage de l’ATP par les fibres sympathiques dans les cellules musculaires lisses peut causer une augmentation de la pression sanguine et qu’une augmentation de l’ADP extracellulaire mène à l’agrégation plaquettaire (via les récepteurs purinergiques). Ces deux phénomènes sont associés au développement de l’athérosclérose et des anévrismes. Il est également avéré que l’ATP et l’UTP libérés par les globules rouges lors de lésions de ceux-ci permettent une vasodilatation par l’activation de la NO synthase endothéliale. L’UTP libéré peut également être métabolisé en UDP, un agoniste des récepteurs P2Y6 impliqué dans l’activation des macrophages et dans la production de NO. Suite à toutes ces implications des nucléotides dans les processus inflammatoires et le fait que la littérature n’offrait aucune méthode permettant le dosage et la séparation simultanée des neufs nucléotides étudiés dans ce travail, il semblait essentiel de développer et de valider une nouvelle méthode LC-MS/MS permettant ce dosage. Dans un premier temps, deux méthodes d’extraction des nucléotides ont été mises au point, à savoir une méthode par de l’EtOH à froid suivi d’une lyophilisation puis d’une reprise par de l’H2O milliQ ;alternativement, nous avons utilisé une méthode d’extraction par du PCA 8 %. Une méthode analytique LC-MS/MS a été développée et validée pour neuf nucléotides :AMP, vi ADP, ATP, UMP, UDP, UTP, GMP, AMPc et GMPc avec l’avantage de quantifier ces neufs nucléotides simultanément en moins de 10 minutes. Dans un deuxième temps, les résultats obtenus avec la méthode LC-MS/MS pour trois des nucléotides (AMPc, ADP et ATP) ont été comparés à ceux obtenus avec des kits vendus dans le commerce (cAMP Enzyme Immunoassay kit, ADP Assay kit, ATP Bioluminescent Assay kit) pour appuyer la validation de cette méthode de quantification. Les résultats ont montré une erreur relative allant de 6,8 à 11,7 % par rapport aux valeurs obtenues avec les kits qui sont des références en la matière quant à la quantification de ces nucléotides. Dans un troisième temps, la méthode a été testée in vitro sur des cellules endothéliales EA.Hy926 stimulées par du TNF-α, des Ox-LDLs, des LDLs natives ou des Mox-LDLs en présence ou non d’inhibiteurs de phosphodiestérases, et in vivo sur du plasma et des globules rouges de donneurs sains avant et après stase veineuse et de patients atteints d’anévrisme cérébral avant et après la pose d’un stent. Ces analyses ont été possibles grâce à la validation préalable de la méthode et ont permis de nouvelles observations quant à la variation des taux de nucléotides lors de processus inflammatoires. En conclusion, ce travail nous a permis de mettre au point deux méthodes d’extraction des nucléotides et de développer une méthode LC-MS/MS précise, sensible et exacte pour la quantification de neufs nucléotides impliqués dans des phénomènes inflammatoires de pathologies cardiovasculaires. Ce projet a également permis l’analyse in vitro et in vivo de ces neufs nucléotides dans différentes matrices et dans différentes conditions physiopathologiques menant effectivement à des variations des taux de ceux-ci.Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie)
info:eu-repo/semantics/nonPublished
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Tavares, Ludmyla Santos. "Padronização e validação analítica dos métodos DPX-RAM/LC-MS e MIP-PSI-MS para análise de cocaína em fluido oral." Universidade Federal de Goiás, 2017. http://repositorio.bc.ufg.br/tede/handle/tede/7385.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
The analysis of drugs of abuse in non-conventional biological fluids is attracting a lot of interest due to recent legislation changes in Brazil and a greater police surveillance. However, complex matrix samples have a large amount of endogenous, exogenous and other interfering compounds that make direct analysis impossible in analytical systems. In this way, sample preparation has been presented as an important tool for rapid and sensitive analytical methods. The miniaturized sample preparation technique called disposable pipette extraction (DPX) has shown to be promising once it integrates the clean-up and pre-concentration of samples. Restricted access materials (RAM) are a class of materials that allow the exclusion of endogenous compounds and the extraction of analytes in a single step. In this manner, in this work a RAM extraction phase has been developed for DPX applied to the analysis of cocaine in oral fluid via liquid chromatography coupled with mass spectrometry (DPX-RAM/LC-MS). The proposed method was optimized using a 24 factorial design. According to the results obtained, the better efficiency of extraction was obtained in the following conditions: pH 9, 10 extractions cycles (draw/eject), 3 desorption cycles (draw/eject) and acetonitrile as desorption solvent. The DPX-RAM/LC-MS method showed to be linear from 10 to 100 ng.mL-1 and have R2 = 0,999. As an alternative to sample preparation and a previous separation step, it was also evaluated in this work, a direct injection system for the mass spectrometer (MS) employing an ambient ionization source called Paper Spray Ionization (PSI). For this purpose, molecularly imprinted polymers (MIP) for the analysis of cocaine were synthetized directly on the surface of a cellulose membrane and employed as paper in a PSI system. The membrane containing MIP showed to be more selective and had a greater intensity of signal when compared to the chromatographic paper normally used for PSI. MIP-PSI method showed to be linear from 1 to 100 ng.mL-1 and have R2 = 0,998.The methods proposed are precise and accurate with obtained values bellow 15%, recovery above 80% and the limits of detection and quantification are lower than the ones found in the literature. Both techniques were standardized and validated according to ANVISA’s normative.
As análises de drogas de abuso em fluidos biológicos não convencionais têm atraído muito interesse devido às recentes mudanças nas legislações nacionais e maior fiscalização policial. No entanto, amostras com matrizes complexas apresentam grande quantidade de compostos endógenos, exógenos e outros interferentes que inviabilizam a análise direta em sistemas analíticos. Neste sentido, o preparo de amostras tem se apresentado como importante ferramenta para métodos analíticos rápidos e sensíveis. A técnica miniaturizada de preparo de amostras, conhecida como extração em ponteiras descartáveis (DPX) tem se mostrado promissora uma vez que integra o clean-up e a pré-concentração das amostras. Os materiais de acesso restrito (RAM) são uma classe de materiais que permitem a exclusão dos compostos endógenos e a extração dos analitos de interesse em uma única etapa. Assim sendo, neste trabalho foi desenvolvida uma fase extratora RAM para DPX e aplicada à análises de cocaína em fluido oral por cromatografia líquida acoplada ao espectrômetro de massas (DPX-RAM/LC-MS). O método proposto foi otimizado utilizando um planejamento fatorial 24 e segundo os resultados obtidos, a maior eficiência de extração foi obtida nas seguintes condições: pH 9, 10 ciclos de extração (aspirar/dispensar), 3 ciclos de dessorção (aspirar/dispensar) e acetonitrila como solvente de dessorção. O método DPXRAM/LC-MS mostrou-se linear de 10 a 100 ng.mL-1 com R2 = 0,999. Como alternativa ao preparo de amostras e a separação prévia foi também avaliado, neste trabalho, um sistema para injeção direta no espectrômetro de massas (MS) pela técnica de ionização ambiente conhecida como Paper Spray Ionization (PSI). Para tanto, polímeros molecurlamente impressos (MIPs) para análise de cocaína foram sintetizados diretamente na superfície de uma membrana filtrante e empregados como papel em sistema PSI. A membrana contendo o MIP mostrou ser mais seletiva e apresentou maior intensidade no sinal quando comparada com o papel cromatográfico usualmente utilizado para PSI. O método PSI-MS mostrou-se linear de 1 a 100 ng.mL-1 com R2 = 0,998. Os dois métodos apresentaram valores de exatidão e precisão abaixo de 15%, recuperação acima de 80% e os limites de quantificação e detecção abaixo dos encontrados na literatura. Ambas técnicas foram padronizadas e validadas de acordo com as normativas da ANVISA.
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Hansson, Anna. "Design of an LC-MS/MS method for measuring concentrations of Cyclosporine A and Tacrolimus from dried blood spots." Thesis, Uppsala universitet, Avdelningen för analytisk farmaceutisk kemi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-261083.
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Patients that have undergone organ transplantation are life-long treated with immunosuppressant drugs and these have to be monitored regularly to get the desired effect of suppressing the immune system. To monitor the drug concentration normally a venous blood sample is collected at a clinic but the use of dried blood spots (DBS) as a matrix for drug monitoring for immunosuppressant drugs will make home sampling possible for this patient group. The aim of this study was to develop and validate a bioanalytical method for quantifying cyclosporine A and tacrolimus in dried blood spots. The method consist of punching out a 5 mm disc from a blood spot , followed by extracting the spot in a 96-well hydrophobic filter plate with 150 µL extraction solution containing internal standard (ascomycin and cyclosporine A d12) in a methanol water solution (80:20v/v%). The extract is then centrifuged through the filter plate down in a 96-deep well plate and injected on the LC-MS/MS, with an analysis time of 2.5min. The method will be validated in accordance with the guidelines set by the European Medicines Agency with additions specific to DBS. The method is not fully validated but will be in due time. The validated parameters show a robust and fast analysing method that has the prospects of being used for analysing DBS samples for patients and in the future can possibly be used by patients in home environment.
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Acheampong, Akwasi. "Development of lc-ms method to identify triacylglycerols in resinous seed oils." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA112083.
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Cette thèse a consisté à développer une nouvelle stratégie analytique en utilisant le couplage chromatographie liquide - spectrométrie de masse (LC-MSn) afin d’identifier dans les huiles et corps gras, les triglycérides (TAGs) et leur régio-spécificité. Cette méthodologie analytique générale a été appliquée à 8 huiles de graine de résineux en particulier, à l’huile de pignon de Pinus Koraiensis car ses TAGs sont constitués d’acides gras, possédant une double liaison en position Δ5, non-méthylène alternée, susceptibles de jouer un rôle anti-cholestérol chez l’homme.La thèse se décompose en quatre chapitres: les deux premiers, bibliographiques, traitent respectivement de la taxonomie des 8 résineux étudiés, de la composition en acide gras (AG) de leur graine et des techniques analytiques déjà décrites pour caractériser les TAGs. Le troisième chapitre, expérimental, est dédié au développement de nouvelles stratégies analytiques NARP-LC-MSn mises en place pour identifier les TAGs présents dans les huiles et plus particulièrement à l’huile de pignon de Pinus Koraiensis. Grâce à l’ajout post colonne de sel d’argent, il a été possible de déterminer de manière non ambigüe la structure de tous les TAGs d’un lipide, même ceux présents en faible quantité, par Ag+-NARP-LC-MS mais aussi de déterminer la structure de chaque AG constitutif d’un TAG par fragmentation de l’adduit moléculaire par Ag+-NARP-LC-MS2. Le problème de la distinction des TAGs ayant la même masse moléculaire, les mêmes longueurs de chaine mais des positions de double liaisons différentes, a été résolu en développant une méthode d’identification des TAGs à partir des lois de rétention chromatographiques qui relient linéairement le logarithme du facteur de rétention de chaque TAG soit au nombre total de carbone soit au nombre total de double liaisons. Cette étude a permis d’identifier 22 nouveaux TAGs parmi un nombre total de 58 TAGs caractérisés. Elle a amené la preuve que le résidu AG saturé à 17 atomes de carbone est ramifié et non linéaire. Elle a mis en évidence la présence de trois AGs constitutifs qui n’ont jamais été décrit: 19:1, 19:2 et 24:0 dans l’huile de pignon de Pinus Koraiensis. Le quatrième chapitre porte sur le développement de trois méthodes de détermination de la régiospécificité des TAGs, grâce à la seule SM: (1) une méthode Ag+-NARP-LC-ESI-MS2 utilisant les rapports des ions diglycériques des TAGs. Il en ressort que cette méthode n’est pas assez fiable pour déterminer la structure des TAGs. (2) la seconde méthode fait appel à des expériences MS4/ MS5 sur les adduits argent des TAGs. Elle s’avère pertinente à condition d’avoir à disposition les couples de TAGs standards. (3) enfin une troisième méthode, utilisant la MS2, s’appuie sur le principe de la méthode de dissociation compétitive d’une paire [TAGref – Li -TAG]+. En utilisant la méthode des ajouts dosés il est montré qu’elle ne nécessite que de la disponibilité d’un seul des deux TAGs stéréoisomères comme standard.Ces méthodes originales ont permis, malgré leurs limites respectives de caractériser la régiospécificité d’un certain nombre de TAGs présents dans l’huile de graine de Pinus KoraiensisThis thesis consisted of developing a new analytical strategy using liquid chromatography coupled with mass spectrometry (LC-MSn) to identify in oils and fats, triglycerides (TAGs) and their regio-specificity. The general analytical methodology was applied to 8 conifers seed oils, in particular, the seed oil of Pinus koraiensis. These conifer seed oils differ from common edible vegetable oils by having a series of unusual polyunsaturated fatty acids (UPIFA) with a polymethylene-interrupted (PMI) double bond system and a double bond at the 5 position which may have anti-cholesterol properties. This thesis is composed of four chapters: the two first chapters, literature review, are devoted respectively to TAGs of the 8 resinous seed oils studied and analytical techniques already used. The third chapter is dedicated to the development of a new analytical strategy combining HPLC with mass spectrometry method to identify TAGs in Pinus Koraiensis seed oil. Thanks to post column addition of silver salt, it was possible to determine the TAGs present by Ag+-NARP-LC-MS and also the fatty acids composition of the TAGs by Ag+-NARP-LC-MS2. Concerning the distinction between TAGs with the same mass, same chain length but differing positions of double bond on fatty acid chain, it was determined by chromatographic retention rules which link linearly the logarithm of retention factor of each TAG to the total carbon number or the total number of double bonds. This study has identified 22 new TAGs from a total of 58 TAGs characterized. It confirmed the knowledge that the saturated fatty acid with 17 carbon atoms is branched, not linear. It highlighted the presence of three constituent fatty acids that have never been described: 19:1, 19:2 and 24:0 in the seed oil of Pinus koraiensis. The last experimental part is devoted to the regiospecificity determination of TAGs. Three methodologies were developed. The first one used the ratios of diacylglycerol ions of TAGs but was not reliable enough. The second method used the LC-MS4 experiments (It is relevant if they have available couples of TAG standards). Finally, a third method, using MS2, based on the principle of the method of competitive dissociation of a pair [TAGref - Li-TAG] +. Using the method of standard additions it has been shown that it requires the availability of one of the two stereoisomers TAGs as a standard.These methods provide a significantly different approach to regioisomer characterization of TAGs and overcome most of the shortcomings of existing methodologies
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Ida, Björs. "Development of separation method for analysis of oligonucleotides using LC-UV/MS." Thesis, Uppsala universitet, Analytisk farmaceutisk kemi, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-403381.
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Introduction Oligonucleotides are short nucleic acid chains, usually 19-27mer long. They bind to their corresponding chain, making a specific inhibition possible. In pharmaceuticals, this can be used to inhibit the expression of a gene or protein of interest. Oligonucleotides are usually analyzed based on separation using both hydrophobic and ion-exchange properties. In this project, the possibility to use a mixed-mode column to separate these oligonucleotides and their impurities were explored. Method Liquid chromatography is used as the separation method and the method of detection is both mass spectrometry and UV. Three different columns are evaluated; C18, DNAPac RP, and mixed-mode RP/WAX. Results and discussion Different compositions of mobile phases and gradients are evaluated based on a literature study. Triethylamine, triethylammonium acetate, ammonium formate, hexafluoroisopropanol is used along with both methanol and acetonitrile. Phosphate buffer is evaluated on LC-UV. The results from the C18 column displays a good separation of the oligonucleotides, whilst the DNAPac RP is not as sufficient using the same mobile phases. The mixed-mode column provides good separation and selectivity using phosphate buffer and UV detection. Conclusion Mixed-mode column has the potential to be used for separation of oligonucleotides and one future focus would be to make the mobile phase compatible with mass spectrometry. Phosphate buffer and UV detection seems to be the go-to mobile phase using mixed-mode column even though MS is a more powerful tool for the characterization and identification of oligonucleotides. This provides a hint about the challenge in making the mobile phase MS compatible.
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Hashimoto, Juliana Campos. "Determinação de residuos de verde de malaquita e verde de leucomalaquita em peixes por LC-ESI-MS/MS." [s.n.], 2010. http://repositorio.unicamp.br/jspui/handle/REPOSIP/322522.
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Orientador: Felix Guillermo Reyes ReyesDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Mestrado
Mestre em Ciência de Alimentos
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Elmsjö, Albert. "Selectivity in NMR and LC-MS Metabolomics : The Importance of Sample Preparation and Separation, and how to Measure Selectivity in LC-MS Metabolomics." Doctoral thesis, Uppsala universitet, Avdelningen för analytisk farmaceutisk kemi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-318296.
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Until now, most metabolomics protocols have been optimized towards high sample throughput and high metabolite coverage, parameters considered to be highly important for identifying influenced biological pathways and to generate as many potential biomarkers as possible. From an analytical point of view this can be troubling, as neither sample throughput nor the number of signals relates to actual quality of the detected signals/metabolites. However, a method’s selectivity for a specific signal/metabolite is often closely associated to the quality of that signal, yet this is a parameter often neglected in metabolomics. This thesis demonstrates the importance of considering selectivity when developing NMR and LC-MS metabolomics methods, and introduces a novel approach for measuring chromatographic and signal selectivity in LC-MS metabolomics. Selectivity for various sample preparations and HILIC stationary phases was compared. The choice of sample preparation affected the selectivity in both NMR and LC-MS. For the stationary phases, selectivity differences related primarily to retention differences of unwanted matrix components, e.g. inorganic salts or glycerophospholipids. Metabolites co-eluting with these matrix components often showed an incorrect quantitative signal, due to an influenced ionization efficiency and/or adduct formation. A novel approach for measuring selectivity in LC-MS metabolomics has been introduced. By dividing the intensity of each feature (a unique mass at a specific retention time) with the total intensity of the co-eluting features, a ratio representing the combined chromatographic (amount of co-elution) and signal (e.g. in-source fragmentation) selectivity is acquired. The calculated co-feature ratios have successfully been used to compare the selectivity of sample preparations and HILIC stationary phases. In conclusion, standard approaches in metabolomics research might be unwise, as each metabolomics investigation is often unique. The methods used should be adapted for the research question at hand, primarily based on any key metabolites, as well as the type of sample to be analyzed. Increased selectivity, through proper choice of analytical methods, may reduce the risks of matrix-associated effects and thereby reduce the false positive and false negative discovery rate of any metabolomics investigation.
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Utley, Lucas James. "Design, testing, and validation of an LC/MS compatible cell for measuring transdermal diffusion." [Gainesville, Fla.] : University of Florida, 2001. http://purl.fcla.edu/fcla/etd/anp1048.
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Thesis (Ph. D.)--University of Florida, 2001.Title from first page of PDF file. Document formatted into pages; contains xiii, 126 p.; also contains graphics. Vita. Includes bibliographical references (p. 119-125).
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Edlund, Sofie. "Optimering av vätskekromatografiska parametrar vid kvantifiering av läkemedel i serum med LC-MS/MS för klinisk diagnostik." Thesis, Malmö universitet, Institutionen för biomedicinsk vetenskap (BMV), 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:mau:diva-44344.
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Vid Klinisk kemi och farmakologi, Specialkemi, vid Skånes universitetssjukhus, Lund, utförs kvantifiering med LC-MS/MS av antipsykotiska och antidepressiva läkemedelskoncentrationer i serum med acetonitril (ACN) som mobilfas. ACN är på grund av sin höga elueringsstyrka ett av de vanligaste organiska lösningsmedlen vid reversed phase (RP) kromatografi, men uppvisar samtidigt en hög toxicitet med risk för stora leveransproblem. I syfte att reducera mängden ACN undersöktes därför möjligheten till ett mobilfasbyte till metanol (MeOH). Ordinarie metod jämfördes med tre nyutvecklade metoder med MeOH-baserad mobilfas. I en av metoderna ändrades endast mobilfas och elueringsgradient, medan två av metoderna även använde andra sorters RP-kolonner med anpassade elueringsgradienter. Samtliga analyter uppvisade godkänd separation och retention vid eluering med MeOH, men stor fluktuation från referensmetod sågs vid kvantifieringen av flera analyter, däribland olanzapin, desmetylolaznapin och mirtazapin. Liknande avvikelser med avseende på regression och kvantifieringsdifferens observerades vid eluering med andra sorters RP-kolonner. Detta indikerar att vidare optimering av andra vätskekromatografiska och masspektrometriska parametrar bör utföras innan metoderna kan valideras.The quantification of antipsychotics and antidepressants in human serum with LC-MS/MS is usually performed with acetonitrile (ACN) as mobile phase. ACN is one of the most common organic solvents in reversed phase (RP) chromatography thanks to its high elution strength but is also highly toxic and can at times suffer from major delivery problems. The present study investigated the possibility of replacing ACN with methanol (MeOH) as primary organic solvent with the purpose of reducing the total ACN-usage. Three newly developed methods for chromatographic analysis of antipsychotic and antidepressant drugs using MeOH as organic solvent as part of the mobile phase were compared to the routine method in regard to analyte separation and retention, as well as the relative quantification of substance. In one of the methods only the mobile phase and elution gradient was changed whereas different types of RP columns were applied in addition to the changed mobile phase in the other two. All substances showed acceptable separation and retention when eluted with MeOH but displayed large fluctuations in the quantification of the analyzed substances, more specifically olanzapine, desmethylolanzapine and mirtazapine, in comparison to the reference method. Similar deviations in terms of regression and quantification bias were observed when analytes were eluted with MeOH through other types of RP columns. This indicates that further optimization of other parameters relating to the chromatography and mass spectrometer should be performed before validation.
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Barclay, Victoria K. H. "Development of LC-MS/MS Methods for the Analysis of Chiral and Achiral Pharmaceuticals and Metabolites in Aqueous Environmental Matrices." Doctoral thesis, Uppsala universitet, Avdelningen för analytisk farmaceutisk kemi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-171550.
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This thesis describes the development of liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for the trace analysis of active pharmaceutical ingredients (APIs) and their metabolites in aqueous environmental matrices. The research was focused on the development of chiral LC-MS/MS methods for the analysis of fluoxetine and metoprolol, as well as their chiral metabolites in environmental water samples. A method was also developed for the achiral compounds, diazepam and nordiazepam. The LC-MS/MS methods were validated by the use of the isotope-labeled compounds. As these isotope-labeled compounds were not found in the wastewater samples, the validation could be assessed at trace level concentrations in the actual matrices in which the analytes were detected. The analytes were extracted from the water samples using solid phase extraction methods. Different types of solid phase extraction sorbents were evaluated. Fluoxetine and norfluoxetine were extracted through the use of a mixed mode polymeric based extraction sorbent. A hydrophilic and lipophilic balanced sorbent was employed for the simultaneous extraction of metoprolol and its metabolites, the base α-hydroxymetoprolol and the acidic metabolite deaminated metoprolol. Moreover, silica based C18 extraction discs were applied for the sample preparation of diazepam and nordiazepam. The chromatographic separations were conducted in reversed phase LC with MS compatible mobile phases. The enantiomers of fluoxetine and norfluoxetine were simultaneously separated using the chiral stationary phase (CSP), α1-acid glycoprotein (AGP). The Chiral AGP column was also applied for the separation of the enantiomers of deaminated metoprolol. For the simultaneous separation of the metoprolol enantiomers and the four stereoisomers of α-hydroxymetoprolol, the cellobiohydrolase (CBH) protein based CSP was used. An octadecyl silica based LC column was applied for the separation of diazepam and nordiazepam. The analytes were detected by the use of tandem quadrupole mass spectrometry operating in selective reactive monitoring mode. High resolution MS, employing a quadrupole time-of-flight (QqTOF) mass analyzer, was utilized for the identification of an unknown compound in wastewater samples. The APIs and their metabolites, as well as their respective enantiomers, were quantified in raw and treated wastewater from Uppsala, Sweden along with surface water from the River Fyris in Uppsala.
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Cai, Xiaohan. "¿¿¿¿¿¿Development of Bioanalytical Methods for Clinical Applications and Drug Screening." Cleveland State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=csu1314982525.
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Yasueda, Yuuki. "A method for chemical proteomics based on the selective localization of labeling molecules in living systems." 京都大学 (Kyoto University), 2016. http://hdl.handle.net/2433/215578.
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Howard, James W. "The development of mass spectrometry-based methodologies for the high throughput quantitation of peptides in biological matrices." Thesis, Loughborough University, 2018. https://dspace.lboro.ac.uk/2134/32454.
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The aim of this research was the development of mass spectrometry-based methodologies for the high-throughput quantitation of peptides in biological matrices. Glucagon and GLP-1, which are of interest as biomarkers and in the development of therapeutics, were chosen as model peptides. Immunoassays that are traditionally used to quantify these often perform poorly; therefore, necessitating the development of alternative methodologies. Application of mass spectrometry-based methodologies to these analytes has, however, been limited, primarily due to sensitivity challenges, but also due to analytical challenges associated with their endogenous nature and instability in biological matrices. Chapter 2 describes the development and qualification of the first liquid-chromatography coupled tandem mass spectrometry (LC-MS/MS) method for the quantitation of endogenous glucagon from human plasma. A novel 2D extraction procedure was developed to ensure robustness and sensitivity, whilst a novel surrogate matrix quantitation strategy took into account the endogenous nature of the analyte. A lower limit of quantitation (LLOQ) of 25 pg/mL was qualified, which was a considerable improvement over that previously reported in the literature (250 pg/mL) for a LC-MS/MS method. Clinical samples were cross-validated against a conventional radioimmunoassay (RIA), and similar pharmacokinetic (PK) profiles resulted, demonstrating that the methods were complementary. In Chapter 2 glucagon instability in biological matrix was noted. To characterise this further, in Chapter 3 in vitro glucagon metabolites were identified using high-resolution mass spectrometry (HRMS). Metabolites observed by others (glucagon19-29, glucagon3 29 and [pGlu]3glucagon3 29) in alternative matrices were identified, alongside novel metabolites (glucagon20-29 and glucagon21-29). Cross-interference of these metabolites in immunoassays may help to explain their poor performance, whilst knowledge of metabolism may also aid the development of future stabilisation strategies. The method developed in Chapter 2 was refined in Chapter 4 to improve sensitivity, robustness and throughput, and to add GLP-1 as a secondary analyte. The sensitivity achieved (glucagon: 15 pg/mL LLOQ, GLP-1: 25 pg/mL LLOQ) is the highest reported for both peptides for an extraction avoiding immunoenrichment. Specificity of endogenous glucagon quantitation was assured using a novel approach with a supercharging mobile phase additive to access a sensitive qualifier transition. A cross-validation against established immunoassays using physiological study samples demonstrated some similarities between the methods. Differences between the immunoassay results exemplified the need to develop alternative methodologies. The resulting LC-MS/MS method is considered a viable alternative to immunoassays, for the quantitation of endogenous glucagon, dosed glucagon and/or dosed GLP-1 in human plasma.
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Strayer, Kraig Edward. "A LC-MS/MS-Based Method for the Multiplex Detection of 24 Fentanyl Analogs and Metabolites in Whole Blood at Sub ng mL-1 Concentrations." Wright State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=wright1527173022936317.
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Hasan, Haslina. "Development of an LC-MS/MS method for the analysis of triacylglycerols from meat and application in the discrimination of cooked meat products." Thesis, University of York, 2010. http://etheses.whiterose.ac.uk/1079/.
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A single stage reversed phase high performance liquid chromatography (RP HPLC) separation of animal fat triacylglycerols (TAGs) has been developed for coupling with atmospheric pressure chemical ionization tandem mass spectrometry using an ion trap mass spectrometer. The method developed offers significant improvements on existing methods for TAG analysis, giving better resolution of TAGs with similar equivalent carbon number (ECN), and good separation of TAGs with odd ECN and TAG regioisomers of animal fats. Although the analysis times for chromatographic analysis of these TAGs are long, this is compensated by better separation of highly unsaturated TAGs. Development of an ultra high performance liquid chromatography method has reduced the run time by half, while maintaining separation and resolution. The TAG profiles of fats reflect their fatty acid (FA) compositions, showing a high proportion of unsaturated FAs for chicken and pork, whereas, saturated FAs are dominant in the major TAGs detected in beef and lamb. The improved RP HPLC separation of TAGs developed in this study has been shown to give more reliable discrimination of different animal species than previous methods including analysis of FAs as the methyl esters and RP HPLC separations of intact TAGs. All animal species separated well in the principal component analysis (PCA) plot of TAG profiles, whereas in the PCA plot of FA, chicken plots very close to pork fat, particularly ham. The profiles of TAGs in animal species highlight a number of components that are important for species discrimination. The meat products of different species (beef, pork, chicken and lamb) cooked by microwave, roasting and currying are separated well in the PCA scores plot. This work shows that the discrimination of meat from different animal species is possible for both raw and cooked meat products, and reveals that the differences produced by the various cooking methods were less than the variations observed between species. The loadings values for the scores plot of TAGs for raw and cooked meat products are similar to the raw meat in different animal species and have the same important descriptors for discrimination. Hence, analysis of intact TAGs in cooked food products has considerable potential for detection of adulteration of cooked meat-based food products.
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Eriksson, Andreas. "Development of a Urinary Lipidomics Method Using LC-MS : Application in a Kidney Rejection Study." Thesis, Umeå universitet, Kemiska institutionen, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-150539.
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Alberts, P. "Development of a novel LC-MS/MS method for the detection of adulteration of South African sauvignon blanc wines with 3-alkyl-2-methoxypyrazines." Thesis, Link to the Internet, 2008. http://hdl.handle.net/10019/805.
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Oberholster, Lucas. "The Development and Validation of a Direct LC-MS/MS Assay for the Determination of Tenofovir-diphosphate in Dried Blood Spots for the Analysis of Clinical Samples." Master's thesis, Faculty of Health Sciences, 2019. http://hdl.handle.net/11427/31191.
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Tenofovir (TFV) and emtricitabine (FTC) are nucleoside reverse transcriptase inhibitors, often used in preexposure prophylaxis (PrEP) trials: where antiretroviral drugs are administered to high-risk, HIV-negative individuals to prevent HIV infection. Both drugs are safe when taken either daily or intermittently, which is ideal for PrEP regimens where adherence may not be high. The minimum number of doses estimated to confer high PrEP efficacy for a TFV/FTC regimen is four or more doses per week, resulting in a 95% lower risk of HIV acquisition. However, this is highly dependent on various host factors, of which adherence plays the largest role. The aim of the project was to develop a novel sensitive, specific, and robust direct method for the measurement of adherence, utilising tenofovir-diphosphate (TFV-DP) in dry blood spots (DBS) through LC-MS/MS analysis, to replace the current costly and laborious indirect method currently used to elucidate adherence of patients. This indirect method faces challenges, due to the polar nature of TFV and its metabolites, leading to separation and retention issues. The existing method applied a technique which separated the parent drug from the metabolite and then back-converted all metabolites to the parent drug before analysing the samples on LC-MS/MS. The developed alternative method aimed to reduce the time taken for each assay and the associated cost of consumables. TFV-DP is a highly polar compound and traditional reverse-phase chromatography has poor retention and separation capabilities when used to retain polar compounds, therefore alternative strategies were implemented. In this developed direct method, an anion exchange column was used along with a pH gradient, with the aim of improving separation and chromatography of TFV, TFV-DP, and tenofovir-monophosphate (TFV-MP). The method was optimised and validated using current U.S. Food and Drug Administration (FDA) and European Medical Agency (EMA) guidelines. The use of the anion exchange column resulted in a marked increase in retention time and allowed baseline separation of TFV, TFV-DP, and TFV-MP. Determination of TFV-DP from DBS was performed using three 3 mm DBS punches per sample, which underwent an extraction procedure followed by high-performance liquid chromatography with tandem mass spectrometry detection on an AB Sciex Qtrap 5500 mass spectrometer. The transitions of the protonated precursor ions were monitored at m/z 448.0 and 452.9 to the product ions m/z 350.0 and 354.9 for TFV-DP and the deuterated TFVDP internal standard, respectively. The method was validated over a range of 50–6400 fmol/punch for TFV-DP. The developed direct method had a lower limit of quantification (LLOQ) of 50 fmol/punch, which was higher than that of the indirect method; therefore, it had less sensitivity. The reduced sensitivity was acceptable, since the methods were meant for the measurement of adherence. The direct method had an ULOQ of 6400 fmol/punch, which was similar to that of the indirect method. The direct method also required significantly less on-bench sample processing and, therefore, was less time consuming and costly. To determine the suitability and accuracy of the direct method in comparison to the indirect method a comparative analysis was completed by analysing the same samples using both the indirect and direct method. The developed method met all the validation requirements and a strong correlation was observed between the results of the indirect and direct methods during the comparative analysis.
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Turp, Aleksandra. "Development of LC-MS method and myoblast differentiation model for studying the mechanism of DNA demethylation." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/24946.
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DNA methylation is an epigenetic modification important in many cellular processes such as maintenance of genome stability, establishment and maintenance of imprinting, transposon silencing, chromatin remodelling and control of gene expression. It is therefore important to understand how this modification is established and erased. We set out to develop a sensitive, liquid-chromatography mass-spectrometry method to measure global levels of DNA methylation (5mdC), as well as hydroxymethylation (5hmdC), a potential intermediate of DNA demethylation. With the new Agilent 6490 QQQ LC-MS we were able to detect as little as 50 amol of 5mdC and 5hmdC. We used this method to quantify levels of DNA methylation from DNA extracted from only 100 cells, allowing us to compare DNA methylation levels in early zygote development. Given that the evidence for DNA demethylation in early zygotes comes from methods using either antibody staining or bisulfite sequencing, this is the first direct demonstration that global DNA demethylation occurs in zygotes. Myoblast differentiation is a well-known model of DNA demethylation, where locus-specific DNA demethylation at the promoters and enhancer elements of myogenic regulatory factors, such as myogenin and MyoD, induce muscle specification and differentiation. It has been proposed, however, that the DNA demethylation process involves a large proportion of the genome and that it occurs in the absence of replication, indicating an active process. Therefore, in the second part of this work I set out to further investigate the global scale of epigenetic events associated with myoblast differentiation. Whilst some of the myoblast differentiation experiments showed a marked wave of DNA demethylation (up to 51%) others did not show any changes in DNA methylation level, showing that myoblast differentiation and DNA demethylation are not co-dependent. Addition of a DNA demethylating agent, 5-Aza-3'-deoxycytidine, to the growth medium of differentiating myoblast enhanced the differentiation process. On the other hand, inhibition of Poly (ADP- ribose) polymerase 1 activity, which has been shown to be mechanistically involved in DNA demethylation, inhibited the differentiation process. My work documents the kinetics of 5- v methylcytosine abundance during the myoblast differentiation together with the expression dynamics of factors previously linked to the DNA demethylation process.
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Adaway, Joanne E., Mirko Peitzsch, and Brian G. Keevil. "A novel method for the measurement of plasma metanephrines using online solid phase extraction-liquid chromatography tandem mass spectrometry." Sage, 2015. https://tud.qucosa.de/id/qucosa%3A35429.
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Background: Measurement of plasma metanephrine, normetanephrine and 3-methoxytyramine is useful in the diagnosis of phaeochromocytomas, but many assays require a large volume of plasma due to poor assay sensitivity, and often require lengthy sample preparation. Our aim was to develop a method for measurement of plasma metanephrines using a small sample volume with minimal hands-on preparation. Methods: Samples were deproteinised using 10 K spin filters prior to online solid phase extraction using a Waters Acquity UPLC Online SPE Manager (Waters, Manchester, UK) coupled to a Waters Xevo TQ-S mass spectrometer (Waters, Manchester, UK). The assay was validated and results compared to a previously published method. Results: We achieved a limit of quantification of 37.5 pmol/L for metanephrine and 3-methoxytyramine and 75 pmol/L for normetanephrine using only 150 mL of sample. The assay was linear up to 30,000 pmol/L for all analytes and in a method comparison study results showed good agreement with a previously published LC-MS/MS assay. Conclusions: We have developed a simple method for measurement of plasma metanephrine, normetanephrine and 3-methoxytyramine using only 150 mL of sample. There is minimal hands-on sample preparation required and the assay is suitable for routine use in a clinical laboratory.