title: Quantitative measurement of cysteinyl leukotrienes and leukotriene B₄ in human sputum using ultra high pressure liquid chromatography-tandem mass spectrometry.
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 19.5 pg/mL, respectively. The lower limit of quantitation (LLOQ) for LTE₄ and LTB₄ was 19.5 and 39.0 pg/mL, respectively. The dynamic range of the LTE₄ assay was from 9.8 to 5000 pg/mL, whereas for the LTB₄ assay was from 19.5 to 10,000 pg/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 7220 pg/mL. LTE₄ was detectable in most of the sputum samples (12.3-891 pg/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.
authors.affiliation: Clinical Development Lab, Early Stage Development, Merck & CO, Rahway, NJ, USA.
authors.forename: Gloria Paola
authors.initials: GP
authors.lastname: Chappell
authors.forename: Xiaoyao
authors.initials: X
authors.lastname: Xiao
authors.forename: Arnaldo
authors.initials: A
authors.lastname: Pica-Mendez
authors.forename: Tracey
authors.initials: T
authors.lastname: Varnell
authors.forename: Stuart
authors.initials: S
authors.lastname: Green
authors.forename: Wesley K
authors.initials: WK
authors.lastname: Tanaka
authors.forename: Omar
authors.initials: O
authors.lastname: Laterza
mesh_terms: D001249:Asthma; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015289:Leukotrienes / Q000032:analysis* / Q000737:chemistry; D016014:Linear Models; D029424:Pulmonary Disease, Chronic Obstructive; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013183:Sputum / Q000737:chemistry*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015289:Leukotrienes