A powerful strategy based on targeted analysis for detection of asthma biomarkers: the challenge for miniaturization

• Autor(a) principal: Berenguer, Pedro Henrique Fernandes da Silva
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.13/2095
• topic: Asthma; Biomarkers; 11β-Prostaglandin F2α; Leukotriene E4; Leukotriene B4; Micro extraction by packed sorbent; Ultra high pressure liquid chromatography; Asma; 11β-Prostaglandina F2α; Leucotrieno E4; Leucotrieno B4; Micro extracção por sorvente empacotado; Cromatografia líquida de ultra alta pressão; Applied Biochemistry; .; Faculdade de Ciências Exatas e da Engenharia; Domínio/Área Científica::Ciências Médicas::Biotecnologia Médica; Domínio/Área Científica::Ciências Naturais::Ciências Químicas; Domínio/Área Científica::Ciências Médicas::Medicina Clínica

Asthma is a disease characterized by chronic airway inflammation and by its long term irreversible remodeling. The enzymatic peroxidation of the arachidonic acid leads to the formation of bioactive eicosanoids, key mediators of bronchial inflammation and response modulation in the airways contributing to the pathophysiology of this disease. The present work aimed to develop an improved ultra-high pressure liquid chromatography (UHPLC)-based strategy to characterize biomarkers of lipid peroxidation: leukotrienes E4 (LTE4) and B4 (LTB4) and 11β-prostaglandin F2α (11βPGF2α), present in urine of asthmatic patients and healthy individuals (control group). A semi-automatic eVol®-microextraction by packed sorbent (MEPS) format was used to isolate the target analytes. Several experimental parameters with influence on this procedure and on the chromatographic resolution, were evaluated and optimized. The method was fully validated with compatible values under optimal extraction (R-AX sorbent, 3 conditioning-equilibration cycles with 250 µL of ACN-H2O at 0.1% FA, 10 extract-discard cycles of 250 µL of sample at a pH of 5.1, elution with 2 times 50 µL of MeOH and concentration of the eluate until half of its volume) and chromatographic conditions (14-min analysis at a flow rate of 300 µL min-1 in an UHPLC-PDA equipped with a BEH C18 column). The developed method was successfully applied to the urine of asthmatic patients and healthy individuals. On average, the urine of asthmatic patients present significantly higher concentrations of 11βPGF2α (112.96 ng mL-1 vs 62.56 ng mL-1 in control group), LTE4 (1.27 ng mL-1 vs 0.89 ng mL-1 in control group) and LTB4 (1.39 ng mL-1 vs 0.76 ng mL-1 in control group). These results suggest the potential of the target eicosanoids on asthma diagnosis and on the follow-up of the therapeutic response, even though a larger and more extensive study will be necessary, using a bigger number of cases, to confirm the data obtained and to guarantee a greater robustness to the approach.