Autor(es): Berenguer, Pedro ; Camacho, Irene C ; Câmara, José S. ; Câmara, Rita ; Oliveira, Susana
Data: 2019
Identificador Persistente: http://hdl.handle.net/10400.26/35639
Origem: Serviço de Saúde da Região Autónoma da Madeira, E.P.E.
Assunto(s): asma; biomarcadores; Região Autónoma da Madeira; Portugal; Madeira Island; eicosanóides; MEPS; UHPLC
Asthma is a heterogeneous disease characterized by chronic inflammation and long term irreversible remodeling of the airways. The enzymatic peroxidation of the arachidonic acid is part of the pathophysiology of this disease and leads to the formation of powerful inflammatory mediators, characteristic of asthma. The present work aimed to develop an easy-to-use ultra-high pressure liquid chromatography (UHPLC)-based strategy in order to characterize lipid peroxidation biomarkers: leukotrienes E4 (LTE4) and B4 (LTB4) and 11β-prostaglandin F2α (11βPGF2α), eicosanoids present in the urine of asthmatic patients and healthy individuals (control group). A semi-automatic eVol®-microextraction by packed sorbent (MEPS) format was developed in order to isolate the target analytes. The method was fully validated 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 UHPLCPDA equipped with a BEH C18 column). Our results indicated good recoveries (>95%) in addition to excellent extraction efficiency (>95%) at three concentration levels (low, mid and high) with precision (RSDs) less than 11%. The lack-of-fit, goodness-of-fit and Mandel’s fitting tests, revealed good linearity within the concentration range. Good selectivity and sensitivity were achieved with limits of detection ranging from 0.04 ng mL-1 for LTB4 to 1.12 ng mL-1 for 11βPGF2α, and limits of quantification from 0.10 ng mL- 1 for the LTB4 to 2.11 ng mL-1 for 11βPGF2α. 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 and the developed method on asthma diagnosis and on the follow-up of the therapeutic response.
