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Molecules 2019, 24(3), 597; https://doi.org/10.3390/molecules24030597

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

Swansea University Medical School, ILS1 Building, Swansea University, Singleton Park, Swansea SA2 8PP, Wales, UK
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Received: 21 December 2018 / Revised: 29 January 2019 / Accepted: 30 January 2019 / Published: 7 February 2019
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Abstract

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. View Full-Text
Keywords: bile alcohol; cholestanoic acid; oxysterol; sterolomics; enzyme-assisted derivatization; electrospray ionization-mass spectrometry; Girard reagent bile alcohol; cholestanoic acid; oxysterol; sterolomics; enzyme-assisted derivatization; electrospray ionization-mass spectrometry; Girard reagent
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Abdel-Khalik, J.; Crick, P.J.; Yutuc, E.; Wang, Y.; Griffiths, W.J. Developing an Enzyme-Assisted Derivatization Method for Analysis of C27 Bile Alcohols and Acids by Electrospray Ionization-Mass Spectrometry. Molecules 2019, 24, 597.

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