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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

Perestrelo, Rosa, Silva, Catarina L., Câmara, José S.
Talanta 2015 v.144 pp. 382-389
acetonitrile, aqueous solutions, asthma, graphene, inflammation, microextraction, pathophysiology, patients, sorbents, statistical analysis, temperature, ultra-performance liquid chromatography, urine
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 (r²>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.