Quantitation of leukotriene B4 in human sputum as a biomarker using UPLC–MS/MS

Quantitative analysis of endogenous compounds

“…For proper assay performance, it is important to demonstrate similar matrix effect and extraction recovery of all internal standards and analytes over the entire intended concentration range in both surrogate and original matrices. Background subtraction, standard addition [53,54,56,80], or labeled internal standards or analytes methods [8,10,11] can be used to determine extraction recovery of endogenous compounds in the original matrices.…”

Effects of JNJ-40929837, a leukotriene A4 hydrolase inhibitor, in a bronchial allergen challenge model of asthma

“…After weighing, freshly expectorated sputum was poured into a 150 mm tissue culture dish, sputum fluid transferred to a 15 mL conical tube and an equal volume of Dulbecco's phosphate buffered saline added. After rocking and centrifuging at 600 Â g for 10 min at 4 C, 500 mL aliquots of supernatant were frozen at À70 C. LTB 4 quantitation was performed on thawed aliquots using a qualified UPLC-MS/MS method (lower limit of quantification, LLOQ: 0.2 ng/ ml) [12]. Selected squamous cell-free sputum plugs (0.1e0.3 g) were placed in the ACCUFILTER™ collection vial, and a dithiothreitol working solution equal to 4Â the sputum weight was added to the collection vial, and the mixture was rocked for 15 min at room temperature.…”

Leukotriene B4 levels in sputum from asthma patients

“…However, DTT interferes with immunosassays including those for LTB4 [20, 21]. Furthermore, LTB4 levels in DTT processed sputum are unstable, in contrast to non-DTT processed sputum where stable LTB4 levels are observed [22]. A two-step sputum processing procedure involving PBS processing for DTT-free supernatant collection followed by DTT processing for cell counts allows the collection of DTT-free supernatant and good quality cytospins.…”