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Determination of urinary levels of leukotriene B4 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 (10×250μL of sample) and LTB4 elution with 100μL 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 500µL/min, and a column temperature of 37±0.1°C. Under optimized conditions the proposed method exhibit good selectivity and sensitivity LOD (0.37ng/mL) and LOQ (1.22ng/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±0.17ng/mL) when compared to those of CTRL group (from ND to 1.9ng/mL). Urinary measurement of LTB4 may be an interesting and non-invasive option to assess control of asthma.
AuthorsRosa Perestrelo, Catarina L Silva, José S Câmara
JournalTalanta (Talanta) Vol. 144 Pg. 382-9 (Nov 1 2015) ISSN: 1873-3573 [Electronic] Netherlands
PMID26452837 (Publication Type: Journal Article)
CopyrightCopyright © 2015 Elsevier B.V. All rights reserved.

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