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Abstract: The role of leukotrienes (LTs) in airway inflammatory diseases, such as asthma, has been extensively reported. The measurement of LTs in sputum supernatants, which is commonly done via enzyme immunoassays (EIAs), may prove to be useful for assessing airway inflammation. Despite the many advantages of EIA, these methods suffer from a lack of selectivity. Therefore, a selective and reliable method for the analysis of LTs in human sputum is needed. In this study we developed and validated a sensitive and specific method using ultra high pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS), to measure simultaneously cysteinyl leukotrienes (CysLTs) and leukotriene B4 (LTB4) in human sputum. Sputum supernatants obtained by ultracentrifugation were stabilized by protease inhibitors, spiked with stable isotopic internal standards, and subjected to solid phase extraction (SPE) and UHPLC separation. Multiple reaction monitoring (MRM) transitions were optimized and measured on a mass spectrometer. The limit of detection (LOD) for LTE4 and LTB4 was 9.8 and 19.5pg/mL, respectively. The lower limit of quantitation (LLOQ) for LTE4 and LTB4 was 19.5 and 39.0pg/mL, respectively. The dynamic range of the LTE4 assay was from 9.8 to 5000pg/mL, whereas for the LTB4 assay was from 19.5 to 10,000pg/mL. The intra- and inter-day % coefficient of variation (%CV) was <6.5% and <10%, for both LTE4 and LTB4, respectively. Spike recovery ranged from 105% to 111% for both analytes. In addition, twenty-two sputum samples were analyzed for cysLTs and LTB4. Fourteen of these samples were purchased commercially and eight were collected during the course of a clinical trial. LTB4 was detectable in all samples tested and it ranged from 79 to 7220pg/mL. LTE4 was detectable in most of the sputum samples (12.3–891pg/mL), whereas LTC4 and LTD4 were below limit of detection for majority of sputum samples. The in vitro conversion of LTC4 and LTD4 into LTE4 was observed. The measurement of LTB4 was sensitive to low pH and high temperature. The use of UHPLC–MS/MS method will allow a more accurate and reliable quantitation of LTs in human sputum, which in turn, may lead to a better understanding of the role of LTs in airway disease pathways and the application in associated clinical treatments. [Copyright &y& Elsevier]