title: Gas chromatography-tandem mass spectrometry method for the simultaneous determination of oxysterols, plant sterols, and cholesterol precursors.
abstract: BACKGROUND
  Cholesterol precursors and plant sterols have considerable potential as plasma biomarkers in several disorders of sterol metabolism and intestinal sterol absorption. Oxysterols are associated with atherogenesis, neurodegeneration, and inflammation. We developed a GC-MS method for the simultaneous analysis of these species in human plasma, including 24-, 25-, 27-hydroxycholesterol; 7-ketocholesterol; lanosterol; lathosterol; 7-dehydrocholesterol; desmosterol; stigmasterol; sitosterol; and campesterol.
  
  
  METHODS
  Sterols were hydrolyzed with ethanolic potassium hydroxide solution, extracted by liquid/liquid extraction with n-hexane, and derivatized with N-methyl-N-trimethylsilyl-trifluoracetamide. Positive chemical ionization with ammonia, as reagent gas, was applied to generate high abundant precursor ions.
  
  
  RESULTS
  The definition of highly sensitive precursor/product ion transitions, especially for coeluting substances, allowed fast gas chromatography run times of under 8.5 min. Using the multiple reaction monitoring mode, detection limits in the picogram per milliliter range could be achieved for most compounds. The method was validated for precision and recovery. Intraassay and interassay CVs were mostly <15% for serum and plasma samples. The recoveries of supplemented plasma samples in different concentrations were 88%-117%. The method was applied to stratification of patients with disorders in cholesterol biosynthesis and/or cholesterol absorption in hypercholesterolemia. The method revealed associations of sterol species with thyroid dysfunction and type 2 diabetes.
  
  
  CONCLUSIONS
  This method allows high-throughput sterol profiling in various diseases.
authors.affiliation: University Hospital Regensburg, Regensburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Matysik
authors.forename: H H
authors.initials: HH
authors.lastname: Klünemann
authors.forename: G
authors.initials: G
authors.lastname: Schmitz
mesh_terms: D002784:Cholesterol / Q000097:blood; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006937:Hypercholesterolemia / Q000097:blood; D006980:Hyperthyroidism / Q000097:blood; D007037:Hypothyroidism / Q000097:blood; D052556:Niemann-Pick Disease, Type C / Q000235:genetics; D010840:Phytosterols / Q000097:blood; D010949:Plasma; D013261:Sterols / Q000097:blood*; D053719:Tandem Mass Spectrometry; D013631:Tangier Disease / Q000235:genetics; D019294:Xanthomatosis, Cerebrotendinous / Q000235:genetics
chemical_list: D010840:Phytosterols; D013261:Sterols; D002784:Cholesterol