Determination of cysteinyl leukotrienes in exhaled breath condensate: Method combining immunoseparation with LC–ESI-MS/MS

Abstract

A rapid and precise method for the identification and quantification of cysteinyl leukotrienes (leukotriene C 4, leukotriene D 4 and leukotriene E 4), essential markers of bronchial asthma, in exhaled breath condensate was developed. The protocol consists of immunoaffinity separation and a detection step, liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS). In particular, the selected reaction monitoring mode was used for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized with a high precision (≤7.7%, determined as RSD), an acceptable accuracy (90.4–93.7%, determined as recovery), a low limit of detection (≤2 pg/ml EBC) and a low limit of quantification (≤10 pg/ml EBC). It was compared to other simple, clinically appropriate combinations of pre-treatment methods (solid phase extraction and lyophilization) with LC/MS. Finally, the method (a combination of immunoaffinity separation with LC–MS) was successfully tested in a clinical study where a significant difference was found in the concentration levels of cysteinyl leukotrienes between patients with occupational bronchial asthma and healthy subjects.