title: Simultaneous determination of β-hydroxybutyrate and β-hydroxy-β-methylbutyrate in human whole blood using hydrophilic interaction liquid chromatography electrospray tandem mass spectrometry.
abstract: OBJECTIVE
  For the quantification of β-hydroxybutyrate (BHB) and β-hydroxy-β-methylbutyrate (HMB) in human whole blood, a method using hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) was developed, which does not require chemical modification of the analytes.
  
  
  METHODS
  Samples were deproteinised by a mixture of methanol and acetonitrile, and the extracts were cleaned-up using both polymeric strong cation exchange and strong anion exchange sorbents. The analytes and their structural isomers were separated using a column with a zwitterionic stationary phase. Isotope dilution of both analytes was used for quantitative analysis.
  
  
  RESULTS
  Separation of BHB from isobaric interferences was achieved through chromatography. The relative intra-laboratory reproducibility standard deviations were better than 10% for blood samples at concentration levels of 10-20μM BHB and 1μM HMB and better than 5% at concentration levels 10 times higher. The mean true extraction recoveries were close to 100%. The trueness expressed as the relative bias of test results was within ±5% at concentration levels of 10-1000μM BHB and 1-20μM HMB. The lower limits of quantification were estimated to be 3μM for BHB and 0.4μM for HMB.
  
  
  CONCLUSIONS
  A simple and highly sensitive and selective HILIC-MS/MS method was developed that is suitable for the quantification of BHB and HMB in whole blood.
authors.affiliation: Section for Forensic Chemistry, Department of Forensic Medicine, Aarhus University, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark. Electronic address: lks@forensic.au.dk.
authors.forename: Lambert K
authors.initials: LK
authors.lastname: Sørensen
authors.forename: Nikolaj F
authors.initials: NF
authors.lastname: Rittig
authors.forename: Emil F
authors.initials: EF
authors.lastname: Holmquist
authors.forename: Karl Anker
authors.initials: KA
authors.lastname: Jørgensen
authors.forename: Jens Otto Lunde
authors.initials: JO
authors.lastname: Jørgensen
authors.forename: Niels
authors.initials: N
authors.lastname: Møller
authors.forename: Mogens
authors.initials: M
authors.lastname: Johannsen
mesh_terms: D020155:3-Hydroxybutyric Acid / Q000097:blood*; D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D057230:Limit of Detection; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014631:Valerates / Q000097:blood*
chemical_list: D014631:Valerates; C004961:beta-hydroxyisovaleric acid; D020155:3-Hydroxybutyric Acid
keywords: BHB; HMB; LC–MS/MS; Whole blood; β-Hydroxy-β-methylbutyrate; β-Hydroxybutyrate