Developing an Enzyme-Assisted Derivatization Method for Analysis of C27 Bile Alcohols and Acids by Electrospray Ionization-Mass Spectrometry

Enzyme-assisted derivatization for sterol analysis (EADSA) is a technology designed to enhance sensitivity and specificity for sterol analysis using electrospray ionization-mass spectrometry. To date it has only been exploited on sterols with a 3 beta-hydroxy-5-ene or 3 beta-hydroxy-5 alpha-hydrogen structure, using bacterial cholesterol oxidase enzyme to convert the 3 beta-hydroxy group to a 3-oxo group for subsequent derivatization with the positively charged Girard hydrazine reagents, or on substrates with a native oxo group. Here we describe an extension of the technology by substituting 3-hydroxysteroid dehydrogenase (3 alpha-HSD) for cholesterol oxidase, making the method applicable to sterols with a 3 alpha-hydroxy-5 beta-hydrogen structure. The 3 alpha-HSD enzyme works efficiently on bile alcohols and bile acids with this stereochemistry. However, as found by others, derivatization of the resultant 3-oxo group with a hydrazine reagent does not go to completion in the absence of a conjugating double bond in the sterol structure. Nevertheless, Girard P derivatives of bile alcohols and C-27 acids give an intense molecular ion ([M](+)) upon electrospray ionization and informative fragmentation spectra. The method shows promise for analysis of bile alcohols and 3 alpha-hydroxy-5 beta-C-27-acids, enhancing the range of sterols that can be analyzed at high sensitivity in sterolomic studies.