Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2018-07-09T16:12:59Z No. of bitstreams: 2 Tese- Maysa Aparecida de Oliveira - 2013.pdf: 2303389 bytes, checksum: d6b1b0186001f327c5e50809b8d401fb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-07-10T11:10:17Z (GMT) No. of bitstreams: 2 Tese- Maysa Aparecida de Oliveira - 2013.pdf: 2303389 bytes, checksum: d6b1b0186001f327c5e50809b8d401fb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-07-10T11:10:17Z (GMT). No. of bitstreams: 2 Tese- Maysa Aparecida de Oliveira - 2013.pdf: 2303389 bytes, checksum: d6b1b0186001f327c5e50809b8d401fb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-07-16 / A simple and accurate stability-indicating high-performance liquid chromatography (HPLC-DAD) method was developed to measure nifedipine (NIF) in plasma in the presence of its degradation products. The chromatographic separation was performed on a C18 column using H3PO4 0.01%:CH3CN:CH3OH (60:20:20, v/v/v) as the mobile phase and a 1.0ml/min flow rate. The analytical validation was performed according to FDA, EMA and ANVISA (Brazilian Health Surveillance Agency) guidelines and was linear between 100.0 and 2000.0 ng/ml, precise (1.9 to 11.3%) and accurate (86.0 to 113.1%). Under the evaluated conditions, NIF in plasma samples were stable after processing and freeze-thaw cycles. The average liquid-liquid extraction recovery was 101.3 ± 5.4%. After validation, this method was applied to evaluate the influence of nitrosophenylpyridine (NO-NIF) in the pharmacokinetic of NIF in plasma of Wistar rats. This method was also validated for quality control of NO-NIF obtained after photodegradation of NIF in photostability chamber. The method showed selectivity, linearity (0.4 to 2.4mg/ml), as well as precision and accuracy. Nitrophenylpyridine (NINIF) was synthesized from the NIF. These degradation products were characterized by NMR and mass spectroscopy. In addition, a breakdown product of NO-NIF due to its contact with plasma was identified and characterized. / Um método simples, rápido e preciso por cromatografia líquida de alta eficiência (HPLC-DAD) foi desenvolvido para determinação de nifedipino (NIF) na presença de seus produtos de degradação em amostras de plasma. A separação cromatográfica foi realizada em coluna C18 empregando H3PO4 0,01%:CH3CN:CH3OH (60:20:20 v/v/v) como fase móvel e vazão de 1,0mL/min. A validação procedeu-se segundo as recomendações dos guias editados pela ANVISA, EMA e FDA e apresentou linearidade no intervalo de 100,0 a 2000,0ng/mL com precisão (1,9 a 11,3%) e exatidão 86,0 a 113,1%) adequadas. Nas condições avaliadas, as amostras de NIF mostraram-se estáveis tanto em plasma como após processamento e ciclos de congelamento e descongelamento. A eficiência do método de extração líquido-líquido demonstrou recuperação média de 101,3 ± 5,4%. Após a etapa de validação, o método foi aplicado em estudos farmacocinéticos para avaliação da influência do nitrosofenilpiridino (NO-NIF) na farmacocinética do NIF. Este método também foi validado para o controle de qualidade do NO-NIF obtido após fotodegradação do NIF em câmara de fotoestabilidade e apresentou seletividade, linearidade (0,4 a 2,4μg/mL), assim como precisão e exatidão. Além da obtenção do NO-NIF por fotodegradação, nitrofenilpiridino (NI-NIF) foi sintetizado a partir do NIF. Esses produtos de degradação foram caracterizados por RMN e espectrometria de massas. Além disso, um produto de degradação do NO-NIF decorrente do seu contato com plasma foi identificado e caracterizado.

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Measuring urinary tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide conjugate may provide the best biomarker of tobacco smoke lung carcinogen metabolism. Existence of differences in the extent of NNAL metabolism rates may be potentially related to an individuals’ lung cancer susceptibility. Low concentrations of NNAL in smokers urine (<1 ng/mL) require sensitive and selective methods for analysis. Traditionally, this involves extensive, time-consuming sample preparation that limits throughput and adds to measurement variability. Molecularly imprinted polymers (MIPs) have been developed for the analysis of urinary NNAL by offline cartridge extraction combined with LC-MS/MS. This method when reproduced demonstrated problems with matrix effects. In the first part of this work, investigation of matrix effects and related problems with sensitivity for the published offline extraction method has been conducted. In order to address the need to improve throughput and other analytical figures of merit for the original method, the second part of this work deals with development of a high-throughput online microfluidic method using capillary-columns packed with MIP beads for the analysis of urinary NNAL. The method was validated as per the FDA guidance, and enabled low volume, rapid analysis of urinary NNAL by direct injection on a microfluidic column packed with NNAL specific MIP beads. The method was used for analysis of urinary NNAL and NNAL-Gluc in smokers. Chemometric methods were used with this data to develop a potential cancer-risk-assessment tool based on pattern recognition in the concentrations of these compounds in urine. In the last part, method comparison approaches for the online and the offline sample extraction techniques were investigated. A ‘fixed’ range acceptance criterion based on combined considerations of method precision and accuracy, and the FDA bioanalytical guidance limits on precision and accuracy was proposed. Data simulations studies to evaluate the probabilities of successful transfers using the proposed criteria were performed. Various experimental designs were evaluated and a design comprised of 3 runs with 3 replicates each with an acceptance range of ±20% was found appropriate. The off-line and the on-line sample extraction methods for NNAL analysis were found comparable using the proposed fixed range acceptance criteria.