Tert-Butyldiphenylsilyl Derivatization for Liquid Chromatography and Mass Spectrometry

Abstract

Tert−butyldiphenylsilyl (TBDPS) derivatization has been investigated for the purposes of improving the HPLC analysis of polar compounds such as fatty acids, cholesterol and bile acids with ultraviolet absorption and/or mass spectral detection. Very mild reaction conditions (room temperature, 30 min) gave quantitative conversion of carboxyls and sterically accessible hydroxyls. The sterically hindered 7α and 12α hydroxyls of bile acids were not silylated to any extent under these conditions. The TBDPS ethers and esters proved stable under HPLC conditions and could be separated on an octylsilyl (RP-8) reverse phase column using acetonitrile as the mobile phase. Detection with an ultraviolet absorption detector at 254 nm was possible but 220 nm provided maximum sensitivity. The derivatives proved to be sufficiently volatile for electron-impact mass spectrometry and the apolar nature of the mobile phase facilitated combined LC/MS with a moving belt interface. The spectra were characterized by abundant ions at [M-57]⁺ due to loss of a tert−butyl radical and m/z 199 due to Ph₂SiOH⁺.