title: Fast LC-MS/MS analysis of free oxysterols derived from reactive oxygen species in human plasma and carotid plaque.
abstract: BACKGROUND
  A rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the quantification of reactive oxygen species (ROS) derived free oxysterols and cholesterol in human plasma and atherosclerotic plaque.
  
  
  METHODS
  In vitro autoxidation of cholesterol during sample pretreatment was avoided by applying only one protein precipitation and re-concentration step using 80 μl plasma. For preparation of 10mg atherosclerotic plaques an additional liquid-liquid extraction was included. Free 7-keto-, 7-α/ß-hydroxy-, 5,6-α-epoxy-, 5,6-β-epoxycholesterol, cholestane-3ß,5α,6ß-triol and cholesterol were separated within 7 min on a monolithic column. An API 4000 tandem mass spectrometer was applied in positive ionization mode using atmospheric pressure chemical ionization.
  
  
  RESULTS
  The detection limit was 0.1 ng/ml and the linearity ranged from 0.5 to 0.75 to 2000 ng/ml for the oxysterols and from 50 to 1000 μg/ml for cholesterol. Recovery was between 80.9 and 107.9%. Between-run imprecision ranged from 7.9 to 11.7%. Analysis of plasma samples from additional 50 middle-aged volunteers revealed a large inter-individual variability (e.g. 7-ketocholesterol 2.63-30.47 ng/ml). Oxysterol concentrations normalized to cholesterol were about 43 times higher in carotid plaque compared to plasma (n=5).
  
  
  CONCLUSIONS
  This rapid LC-MS/MS method enables reliable quantification focused on especially ROS-derived oxysterols in human plasma and atherosclerotic plaque samples under high-throughput conditions.
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Germany; LIFE - Leipzig Research Center for Civilization Diseases, University of Leipzig, Germany. Electronic address: christin.helmschrodt@medizin.uni-leipzig.de.
authors.forename: Christin
authors.initials: C
authors.lastname: Helmschrodt
authors.forename: Susen
authors.initials: S
authors.lastname: Becker
authors.forename: Jenny
authors.initials: J
authors.lastname: Schröter
authors.forename: Max
authors.initials: M
authors.lastname: Hecht
authors.forename: Gabriela
authors.initials: G
authors.lastname: Aust
authors.forename: Joachim
authors.initials: J
authors.lastname: Thiery
authors.forename: Uta
authors.initials: U
authors.lastname: Ceglarek
mesh_terms: D002138:Calibration; D002339:Carotid Arteries / Q000737:chemistry*; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000097:blood / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D006888:Hydroxycholesterols / Q000097:blood* / Q000302:isolation & purification; D007536:Isomerism; D007653:Ketocholesterols / Q000097:blood* / Q000302:isolation & purification; D057230:Limit of Detection; D059625:Liquid-Liquid Extraction; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D058226:Plaque, Atherosclerotic / Q000737:chemistry*; D017382:Reactive Oxygen Species / Q000097:blood*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: C119784:5,6-epoxycholesterol; D006888:Hydroxycholesterols; D007653:Ketocholesterols; D017382:Reactive Oxygen Species; C011724:cholest-5-en-3 beta,7 alpha-diol; D002784:Cholesterol; C003001:7-ketocholesterol
keywords: -OHC; -hydroxycholesterol; 5,6-α-EC; 5,6-α-epoxycholesterol; 5,6-β-EC; 5,6-β-epoxycholesterol; 7-KC; 7-ketocholesterol; 7-α- and 7-β-hydroxycholesterol; 7-α/β-OHC; APCI; Atherosclerotic plaque; BHT; CE; CEA; CID; CV; CXP; DP; EP; Fast chromatography; Free oxysterol; GC-MS; HCl; HPLC; HQC; LC; LC–MS/MS; LLE; LLOQ; LOD; LQC; MQC; MRM; NQC; Na(2)SO(4); NaOH; Oxysterol; PQC; QC; ROS; Reactive oxygen species; SPE; Tandem mass spectrometry; Triol; atmospheric pressure chemical ionization; butylated hydroxytoluene; carotid endarterectomy; cholestane-3β,5α,6β-triol; coefficient of variation; collision energy; collision exit potential; collision induced dissociation; declustering potential; extensions potential; gas chromatography mass spectrometry; high concentrated quality control; high-performance liquid chromatography; hydrochloric acid; limit of detection; liquid chromatography; liquid chromatography tandem mass spectrometry; liquid–liquid extraction; low concentrated quality control; lower level of quantification; medium concentrated quality control; multiple reacting monitoring; native quality control; pathological concentrated quality control; quality controls; reactive oxygen species; s/n; signal-to-noise ratio; sodium hydroxide; sodium sulfate; solid phase extraction