Quantitative measurement of cysteinyl leukotrienes and leukotriene B4 in human sputum using ultra high pressure liquid chromatography-tandem mass spectrometry

摘要

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 B_4 (LTB_4) 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 LTE_4 and LTB_4 was 9.8 and 19.5pg/mL, respectively. The lower limit of quantitation (LLOQ) for LTE_4 and LTB_4 was 19.5 and 39.0pg/mL, respectively. The dynamic range of the LTE_4 assay was from 9.8 to 5000pg/mL, whereas for the LTB_4 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 LTE_4 and LTB_4, respectively. Spike recovery ranged from 105% to 111% for both analytes. In addition, twenty-two sputum samples were analyzed for cysLTs and LTB_4. Fourteen of these samples were purchased commercially and eight were collected during the course of a clinical trial. LTB_4 was detectable in all samples tested and it ranged from 79 to 7220pg/mL. LTE_4 was detectable in most of the sputum samples (12.3-891pg/mL), whereas LTC_4 and LTD_4 were below limit of detection for majority of sputum samples. The in vitro conversion of LTC_4 and LTD_4 into LTE_4 was observed. The measurement of LTB_4 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.