title: Profiling of 3-hydroxy fatty acids as environmental markers of endotoxin using liquid chromatography coupled to tandem mass spectrometry.
abstract: 3-Hydroxy acids are constituents of the lipid A part of lipopolysaccharides and may potentially be used as chemical markers of endotoxin. While commercial enzymatic assays, such as the widely used Limulus amebocyte lysate (LAL) assay, commonly detect merely the water-soluble fraction of the bioactive endotoxin, the chemical approach aims to estimate the total amount of endotoxin present in a sample. Our objective was to develop a simple method for quantitative profiling of 3-hydroxy fatty acids in occupational and environmental samples based on detection with HPLC-MS/MS. We included eleven 3-hydroxy fatty acids (3-hydroxyoctanoic acid to 3-hydroxyoctadecanoic acid) in the HPLC-MS/MS based method, which involved base hydrolysis of filter samples using 1M sodium hydroxide and removal of the base as well as concentration of the fatty acids using solid-phase extraction on a functionalized polystyrene-divinylbenzene polymer. Recovery trials from spiked glass fiber filters, using threo-9,10-dihydroxyhexadecanoic acid as internal standard, gave an overall recovery of 54-86% for 3-hydroxy fatty acids of medium chain length (3-hydroxynonanoic to 3-hydroxypentadecanoic acid). 3-Hydroxyoctanoic acid and the longer chain fatty acids were more problematic yielding overall spike recoveries of 11-39%. While the 3-hydroxy fatty acid profile of pure lipopolysaccharides was dominated by 3-hydroxydecanoic, 3-hydroxydodecanoic and 3-hydroxytetradecanoic acid the aqueous phase from drilling mud contained in addition relatively high amounts of 3-hydroxyoctanoic and 3-hydroxynonanoic acid. Endotoxin activity as measured by the LAL assay was reasonably correlated (R(2)=0.54) to the sum of 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid and 3-hydroxytetradecanoic acid in these samples.
authors.affiliation: Department for the Chemical and Biological Work Environment, National Institute of Occupational Health, P.O. Box 8149 Dep., 0033 Oslo, Norway; Section for Chemistry and Toxicology, Norwegian Veterinary Institute, P.O. Box 750 Sentrum, 0106 Oslo, Norway. Electronic address: silvio.uhlig@stami.no.
authors.forename: Silvio
authors.initials: S
authors.lastname: Uhlig
authors.affiliation: Department for the Chemical and Biological Work Environment, National Institute of Occupational Health, P.O. Box 8149 Dep., 0033 Oslo, Norway.
authors.forename: Mariell
authors.initials: M
authors.lastname: Negård
authors.affiliation: Department for the Chemical and Biological Work Environment, National Institute of Occupational Health, P.O. Box 8149 Dep., 0033 Oslo, Norway.
authors.forename: Kari K
authors.initials: KK
authors.lastname: Heldal
authors.affiliation: Department for the Chemical and Biological Work Environment, National Institute of Occupational Health, P.O. Box 8149 Dep., 0033 Oslo, Norway.
authors.forename: Anne
authors.initials: A
authors.lastname: Straumfors
authors.affiliation: Department for the Chemical and Biological Work Environment, National Institute of Occupational Health, P.O. Box 8149 Dep., 0033 Oslo, Norway.
authors.forename: Lene
authors.initials: L
authors.lastname: Madsø
authors.affiliation: Department of Occupational Health Surveillance, National Institute of Occupational Health, P.O. Box 8149 Dep., 0033 Oslo, Norway.
authors.forename: Berit
authors.initials: B
authors.lastname: Bakke
authors.affiliation: Department for the Chemical and Biological Work Environment, National Institute of Occupational Health, P.O. Box 8149 Dep., 0033 Oslo, Norway.
authors.forename: Wijnand
authors.initials: W
authors.lastname: Eduard
mesh_terms: D015415:Biomarkers / Q000032:analysis*; D002210:Caprylates / Q000032:analysis*; D004731:Endotoxins / Q000032:analysis*; D004784:Environmental Monitoring / Q000379:methods*; D004785:Environmental Pollutants / Q000032:analysis*; D006801:Humans; D008070:Lipopolysaccharides / Q000032:analysis; D009227:Myristic Acids / Q000032:analysis*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015415:Biomarkers; D002210:Caprylates; D004731:Endotoxins; D004785:Environmental Pollutants; D008070:Lipopolysaccharides; D009227:Myristic Acids; C009893:beta-hydroxymyristic acid; C101940:3-hydroxyoctanoic acid
keywords: Endotoxin; Fatty acid; HPLC–MS/MS; Hydrolysis; LAL assay