Synthesis of (2β,3α,6-2H3)cholesteryl 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,6-2H3)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-2H2)-3α,6β-diol with Jones’ reagent, followed by reduction of the resulting (2β-2H)-3,6-dione with NaBD4 leading to the (2β,3α,6α-2H3)-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,6-2H3)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.