title: Synthesis of (2β,3α,6-²H₃cholesteryl linoleate and cholesteryl oleate as internal standards for mass spectrometry.
abstract: The accurate analysis of trace component in complex biological matrices requires the use of reliable standards. For liquid chromatography/mass spectrometry analysis, the stable isotope-labeled derivatives of the analyte molecules are the most appropriate internal standards. We report here the synthesis of (2β,3α,6-(2)H3)cholesteryl linoleate and oleate containing three non-exchangeable deuterium in the steroid ring. The principal reactions used were: (1) trans diaxial opening of 2α,3α-epoxy-6-oxo-5α-cholestane with LiAlD4 and subsequent oxidation of the resulting (2β,6α-(2)H2)-3α,6β-diol with Jones' reagent, followed by reduction of the resulting (2β-(2)H)-3,6-dione with NaBD4 leading to the (2β,3α,6α-(2)H3)-3β,6β-dihydroxy-5α-cholestane, (2) selective protection of the 3β-hydroxy group as the tert-butyldimethylsilyl ether, (3) dehydration of the 6β-hydroxy group with POCl3 and removal of tert-butyldimethylsilyloxy groups with 5M HCl in acetone, and (4) esterification of the resultant (2β,3α,6-(2)H3)cholesterol with linoleic and oleic acids using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. The isotopic purity was found to be satisfactory by mass spectrometry, and nuclear magnetic resonance properties of the new compounds were tabulated. The labeled compounds can be used as internal standards in liquid chromatography/mass spectrometry assays for clinical and biochemical studies.
authors.affiliation: Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Sapporo 060-0812, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Miura
authors.affiliation: Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Sapporo 060-0812, Japan. Electronic address: keino@hs.hokudai.ac.jp.
authors.forename: Shu-Ping
authors.initials: SP
authors.lastname: Hui
authors.affiliation: Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Sapporo 060-0812, Japan.
authors.forename: Rojeet
authors.initials: R
authors.lastname: Shrestha
authors.affiliation: Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Sapporo 060-0812, Japan.
authors.forename: Takahisa
authors.initials: T
authors.lastname: Hiruma
authors.affiliation: Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Sapporo 060-0812, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Takeda
authors.affiliation: Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Sapporo 060-0812, Japan.
authors.forename: Hirotoshi
authors.initials: H
authors.lastname: Fuda
authors.affiliation: Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Sapporo 060-0812, Japan.
authors.forename: Shigeo
authors.initials: S
authors.lastname: Ikegawa
authors.affiliation: Laboratory of Cardiovascular Disease, Novel, Non-Invasive, and Nutritional Therapeutics (CNT), Graduate School of Medicine, Osaka University, 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan; Department of Cardiovascular Medicine, Graduate School of Medicine, Osaka University, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Hirano
authors.affiliation: Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Sapporo 060-0812, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Chiba
mesh_terms: D002788:Cholesterol Esters / Q000138:chemical synthesis* / Q000737:chemistry*; D013058:Mass Spectrometry
chemical_list: D002788:Cholesterol Esters
keywords: Cholesterol; Cholesteryl ester; Liquid chromatography/mass spectrometry; Multiple deuteration; Sodium borodeuteride; Trans-diaxial opening