title: Determination of urinary levels of leukotriene B(4) using ad highly specific and sensitive methodology based on automatic MEPS combined with UHPLC-PDA analysis.
abstract: Leukotriene B4 (LTB4) is a potent mediator of inflammation and plays a key function in the pathophysiology of chronic asthma. Detectable urinary levels of LTB4, arises from the activation of leukotriene pathways. In this study an ultra-fast, selective and sensitive analytical method based on semi-automatic microextraction by packed sorbents (MEPS) technique, using a new digitally controlled syringe (eVol®) combined with ultra-high pressure liquid chromatography (UHPLC), is proposed for the measurement of urinary LTB4 (U-LTB4) levels in a group of asthmatic patients (APs) and healthy controls (CTRL). Important parameters affecting MEPS performance, namely sorbent type, number of extraction cycles (extract-discard) and elution volume, were evaluated. The optimal experimental conditions among those investigated for the quantification of U-LTB4 in urine samples were as follows: porous graphitic carbon sorbent (PGC), 10 extractions cycle (10×250 μL of sample) and LTB4 elution with 100 μL of acetonitrile. The UHPLC optimum conditions resulted in a mobile phase consisting of 95% (v/v) of acid aqueous solution (v/v), and acetonitrile 5% (v/v); flow rate of 500 µL/min, and a column temperature of 37±0.1 °C. Under optimized conditions the proposed method exhibit good selectivity and sensitivity LOD (0.37 ng/mL) and LOQ (1.22 ng/mL). The recovery ranging from 86.4 to 101.1% for LTB4, with relative standard deviations (% RSD) no larger than 5%. In addition, the method also afforded good results in terms of linearity (r(2)>0.995) within the established concentration range, with a residual deviation for each calibration point below 6%, and intra- and inter-day repeatability in urine samples with RSD values lower than 4 and 5%, respectively. The application of the method to urine samples revealed a tendency towards the increased urinary LTB4 levels in APs (5.42±0.17 ng/mL) when compared to those of CTRL group (from ND to 1.9 ng/mL). Urinary measurement of LTB4 may be an interesting and non-invasive option to assess control of asthma.
authors.affiliation: CQM, Centro de Química da Madeira, Universidade da Madeira, 9000-390 Funchal, Portugal.
authors.forename: Rosa
authors.initials: R
authors.lastname: Perestrelo
authors.affiliation: CQM, Centro de Química da Madeira, Universidade da Madeira, 9000-390 Funchal, Portugal.
authors.forename: Catarina L
authors.initials: CL
authors.lastname: Silva
authors.affiliation: CQM, Centro de Química da Madeira, Universidade da Madeira, 9000-390 Funchal, Portugal; Centro de Ciências Exatas e de Engenharia da Universidade da Madeira, Campus Universitário da Penteada, 9000-390 Funchal, Portugal. Electronic address: jsc@uma.pt.
authors.forename: José S
authors.initials: JS
authors.lastname: Câmara
mesh_terms: D000293:Adolescent; D001249:Asthma / Q000652:urine; D001331:Automation; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation*; D003362:Cost-Benefit Analysis; D005260:Female; D006801:Humans; D007975:Leukotriene B4 / Q000302:isolation & purification* / Q000652:urine*; D057230:Limit of Detection; D008297:Male; D012666:Semiconductors; D052617:Solid Phase Microextraction / Q000191:economics / Q000379:methods*; D013997:Time Factors; D016482:Urinalysis / Q000191:economics / Q000295:instrumentation / Q000379:methods*
chemical_list: D007975:Leukotriene B4
keywords: Method validation; Microextraction by packed sorbent; Ultra-high pressure liquid chromatography; Urinary leukotriene B(4)