title: Immunomagnetic molecular probe with UHPLC-MS/MS: a promising way for reliable bronchial asthma diagnostics based on quantification of cysteinyl leukotrienes.
abstract: A sensitive and precise method for simultaneous quantification of cysteinyl leukotrienes (=cys LTs) - leukotriene C4 (=LTC4), leukotriene D4 (=LTD4) and leukotriene E4 (=LTE4) - essential biomarkers of bronchial asthma present in exhaled breath condensate (=EBC) was developed. An immunomagnetic molecular probe was prepared by anchoring cysteinyl leukotrienes antibody on the surface of functionalized monodispersed magnetic particles and used to selectively isolate cys LTs from biological matrices - EBC, plasma and urine. Immobilization and the immunoaffinity capture procedures were optimized to maximize the amount of separated cys LTs, which were detected "off-beads" after acidic elution by UHPLC-ESI-MS/MS operated in a multiple reaction monitoring mode. The developed method was characterized with high precision ≤13.6% (intra-day precision determined as RSD) and ≤14.5% (inter-day precision determined as RSD), acceptable accuracy ≤18.5% (determined as RE), and high recovery of immunoseparation (≥93.1%) in aforementioned biological matrices. The applicability of the method was demonstrated on EBC, plasma and urine clinical samples of patients with various subtypes of bronchial asthma (occupational, steroid-resistant, moderate with and without corticosteroids therapy) and healthy subjects where reasonable differences in cys LTs concentration levels were found. Combining extremely selective immunomagnetic separation with highly sensitive and precise detection step, the developed method was used to aid diagnosis, predict the most effective therapy, and monitor the response to treatment. The detection of elevated inflammatory mediators (cys LTs) in EBC of subjects with relatively asymptomatic asthma and normal pulmonary function tests could offer a novel way for monitoring the lung inflammation and perhaps initiating treatment in an earlier stage.
authors: Institute of Chemical Technology, Technicka 5, 166 28 Prague 6, Czech Republic.
authors: Kamila
authors: K
authors: Syslová
authors: Adéla
authors: A
authors: Böhmová
authors: Elvan
authors: E
authors: Demirbağ
authors: Kateřina
authors: K
authors: Šimková
authors: Marek
authors: M
authors: Kuzma
authors: Daniela
authors: D
authors: Pelclová
authors: Vratislav
authors: V
authors: Sedlák
authors: Petr
authors: P
authors: Čáp
authors: Pavel
authors: P
authors: Martásek
authors: Petr
authors: P
authors: Kačer
mesh_terms: D001249:Asthma / Q000175:diagnosis* / Q000188:drug therapy / Q000503:physiopathology; D001944:Breath Tests; D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005938:Glucocorticoids / Q000627:therapeutic use; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods; D017997:Leukotriene C4 / Q000032:analysis*; D017998:Leukotriene D4 / Q000032:analysis*; D017999:Leukotriene E4 / Q000032:analysis*; D008875:Middle Aged; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D005938:Glucocorticoids; D017997:Leukotriene C4; D017998:Leukotriene D4; D017999:Leukotriene E4