title: Bile acid preparation and comprehensive analysis by high performance liquid chromatography-high-resolution mass spectrometry.
abstract: BACKGROUND
  Accurate analytical methods for bile acid (BA) analysis are important for clinical diagnosis in newborns, adolescents, and adults. Improvements in speed, sensitivity and simplicity enable BA profiling using high performance liquid chromatography (HPLC) together with electrospray ionization (ESI) and high-resolution mass spectrometry (HR-MS).
  
  
  RESULTS
  We present a method, validated in different species and tissues, enabling a highly sensitive quantitative determination (in a range of 0.24pmol/sample to 1000pmol/sample, corresponding to 0.024-100pmol on column) of up to 36 naturally occurring BAs from as little as 10μl of plasma or serum, 1ml of urine, or 10mg of dried stool. Chromatographic separation is achieved by HPLC using a C18 reversed phase column and a water/methanol gradient. After ESI, BAs are analyzed through HR-MS using orbitrap technology in full scan mode. 30 different BAs and the corresponding internal standards are separated and analyzed in a single run. Six additional BAs are evaluated in a second run using a pentafluorophenyl (PFP) stationary phase.
  
  
  CONCLUSIONS
  This method generates detailed human and rodent BA profiles in full scan mode and accurate mass with the advantage of remarkably low required sample volume.
authors.affiliation: Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria.
authors.forename: Benno
authors.initials: B
authors.lastname: Amplatz
authors.affiliation: Department of Pediatrics and Adolescent Medicine, Medical University of Graz, Austria.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Zöhrer
authors.affiliation: Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria.
authors.forename: Christina
authors.initials: C
authors.lastname: Haas
authors.affiliation: Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria.
authors.forename: Maria
authors.initials: M
authors.lastname: Schäffer
authors.affiliation: Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria.
authors.forename: Tatjana
authors.initials: T
authors.lastname: Stojakovic
authors.affiliation: Department of Pediatrics and Adolescent Medicine, Medical University of Graz, Austria.
authors.forename: Jörg
authors.initials: J
authors.lastname: Jahnel
authors.affiliation: Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Austria. Electronic address: guenter.fauler@medunigraz.at.
authors.forename: Günter
authors.initials: G
authors.lastname: Fauler
mesh_terms: D000293:Adolescent; D000328:Adult; D053000:Analytic Sample Preparation Methods / Q000379:methods*; D001647:Bile Acids and Salts / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification / Q000652:urine; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019963:Clinical Chemistry Tests / Q000379:methods*; D006801:Humans; D007231:Infant, Newborn; D012015:Reference Standards; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D001647:Bile Acids and Salts
keywords: Bile acids; High performance liquid chromatography; High resolution-mass spectrometry; Muricholic acids; Neonates