title: A liquid chromatography-tandem mass spectrometry-based method for the simultaneous determination of hydroxy sterols and bile acids.
abstract: Recently, hydroxy sterols and bile acids have gained growing interest as they are important regulators of energy homoeostasis and inflammation. The high number of different hydroxy sterols and bile acid species requires powerful analytical tools to quantify these structurally and chemically similar analytes. Here, we introduce a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for rapid quantification of 34 sterols (hydroxy sterols, primary, secondary bile acids as well as their taurine and glycine conjugates). Chromatographic baseline separation of isomeric hydroxy sterols and bile acids is obtained using a rugged amide embedded C18 (polar embedded) stationary phase. The current method features a simple extraction protocol validated for blood plasma, urine, gall bladder, liver, feces, and adipose tissue avoiding solid phase extraction as well as derivatization procedures. The total extraction recovery for representative analytes ranged between 58-86% in plasma, 85% in urine, 79-92% in liver, 76-98% in adipose tissue, 93-104% in feces and 62-79% in gall bladder. The validation procedure demonstrated that the calibration curves were linear over the selected concentration ranges for 97% of the analytes, with calculated coefficients of determination (R2) of greater than 0.99. A feeding study in wild type mice with a standard chow and a cholesterol-enriched Western type diet illustrated that the protocol described here provides a powerful tool to simultaneously quantify cholesterol derivatives and bile acids in metabolically active tissues and to follow the enterohepatic circulation.
authors.affiliation: Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.
authors.forename: Clara
authors.initials: C
authors.lastname: John
authors.affiliation: Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany.
authors.forename: Philipp
authors.initials: P
authors.lastname: Werner
authors.affiliation: Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Worthmann
authors.affiliation: Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Wegner
authors.affiliation: Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Tödter
authors.affiliation: Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany.
authors.forename: Ludger
authors.initials: L
authors.lastname: Scheja
authors.affiliation: Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany.
authors.forename: Sascha
authors.initials: S
authors.lastname: Rohn
authors.affiliation: Department of Biochemistry and Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. Electronic address: heeren@uke.de.
authors.forename: Joerg
authors.initials: J
authors.lastname: Heeren
authors.affiliation: Hamburg School of Food Science, Institute of Food Chemistry, University of Hamburg, Grindelallee 117, 20146 Hamburg, Germany. Electronic address: markus.fischer@chemie.uni-hamburg.de.
authors.forename: Markus
authors.initials: M
authors.lastname: Fischer
mesh_terms: D000818:Animals; D001647:Bile Acids and Salts / Q000032:analysis*; D001826:Body Fluids / Q000737:chemistry; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005243:Feces / Q000737:chemistry; D057230:Limit of Detection; D008099:Liver / Q000737:chemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D052616:Solid Phase Extraction; D013261:Sterols / Q000032:analysis*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001647:Bile Acids and Salts; D013261:Sterols
keywords: Bile acids; HPLC-ESI-QqQ-MS/MS; Hydroxy sterols; Polar embedded stationary phase; Sterol profiling in biological matrices lipid metabolism