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

• Main Authors: Jonas Abdel-Khalik, Peter J. Crick, Eylan Yutuc, Yuqin Wang, William J. Griffiths
• Format: Article
• Language: English
• Published: MDPI AG 2019-02-01
• Series: Molecules
• Subjects: bile alcohol; cholestanoic acid; oxysterol; sterolomics; enzyme-assisted derivatization; electrospray ionization-mass spectrometry; Girard reagent
• Online Access: https://www.mdpi.com/1420-3049/24/3/597

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 3β-hydroxy-5-ene or 3β-hydroxy-5α-hydrogen structure, using bacterial cholesterol oxidase enzyme to convert the 3β-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 3α-hydroxysteroid dehydrogenase (3α-HSD) for cholesterol oxidase, making the method applicable to sterols with a 3α-hydroxy-5β-hydrogen structure. The 3α-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 C₂₇ acids give an intense molecular ion ([M]⁺) upon electrospray ionization and informative fragmentation spectra. The method shows promise for analysis of bile alcohols and 3α-hydroxy-5β-C₂₇-acids, enhancing the range of sterols that can be analyzed at high sensitivity in sterolomic studies.