title: Challenges in accurate quantitation of lysophosphatidic acids in human biofluids.
abstract: Lysophosphatidic acids (LPAs) are biologically active signaling molecules involved in the regulation of many cellular processes and have been implicated as potential mediators of fibroblast recruitment to the pulmonary airspace, pointing to possible involvement of LPA in the pathology of pulmonary fibrosis. LPAs have been measured in various biological matrices and many challenges involved with their analyses have been documented. However, little published information is available describing LPA levels in human bronchoalveolar lavage fluid (BALF). We therefore conducted detailed investigations into the effects of extensive sample handling and sample preparation conditions on LPA levels in human BALF. Further, targeted lipid profiling of human BALF and plasma identified the most abundant lysophospholipids likely to interfere with LPA measurements. We present the findings from these investigations, highlighting the importance of well-controlled sample handling for the accurate quantitation of LPA. Further, we show that chromatographic separation of individual LPA species from their corresponding lysophospholipid species is critical to avoid reporting artificially elevated levels. The optimized sample preparation and LC/MS/MS method was qualified using a stable isotope-labeled LPA as a surrogate calibrant and used to determine LPA levels in human BALF and plasma from a Phase 0 clinical study comparing idiopathic pulmonary fibrosis patients to healthy controls.
authors.affiliation: Departments of Bioanalytical and Discovery Analytical Science, Bristol-Myers Squibb Co., Princeton, NJ.
authors.forename: Joelle M
authors.initials: JM
authors.lastname: Onorato
authors.affiliation: Departments of Bioanalytical and Discovery Analytical Science, Bristol-Myers Squibb Co., Princeton, NJ.
authors.forename: Petia
authors.initials: P
authors.lastname: Shipkova
authors.affiliation: Exploratory Clinical and Translational Research, Bristol-Myers Squibb Co., Princeton, NJ.
authors.forename: Anne
authors.initials: A
authors.lastname: Minnich
authors.affiliation: Analytical and Bioanalytical Development, Bristol-Myers Squibb Co., Princeton, NJ.
authors.forename: Anne-Françoise
authors.initials: AF
authors.lastname: Aubry
authors.affiliation: Discovery Chemistry, Bristol-Myers Squibb Co., Princeton, NJ.
authors.forename: John
authors.initials: J
authors.lastname: Easter
authors.affiliation: Departments of Bioanalytical and Discovery Analytical Science, Bristol-Myers Squibb Co., Princeton, NJ.
authors.forename: Adrienne
authors.initials: A
authors.lastname: Tymiak
mesh_terms: D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods; D005260:Female; D006801:Humans; D054990:Idiopathic Pulmonary Fibrosis / Q000378:metabolism*; D008246:Lysophospholipids / Q000378:metabolism*; D008297:Male; D013058:Mass Spectrometry / Q000379:methods
chemical_list: D008246:Lysophospholipids; C032881:lysophosphatidic acid
keywords: bronchoalveolar lavage fluid; human plasma; idiopathic pulmonary fibrosis; stability