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: Rosa Perestrelo, Catarina L Silva, José S Câmara
• Journal: Talanta (Talanta) Vol. 144 Pg. 382-9 (Nov 01 2015) ISSN: 1873-3573 [Electronic] Netherlands
• PMID: 26452837 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2015 Elsevier B.V. All rights reserved.

Chemical References

• Leukotriene B4

Topics

• Adolescent
• Asthma (urine)
• Automation
• Chromatography, High Pressure Liquid (instrumentation)
• Cost-Benefit Analysis
• Female
• Humans
• Leukotriene B4 (isolation & purification, urine)
• Limit of Detection
• Male
• Semiconductors
• Solid Phase Microextraction (economics, methods)
• Time Factors
• Urinalysis (economics, instrumentation, methods)