Characterization of oxysterols by electrospray ionization tandem mass spectrometry after one-step derivatization with dimethylglycine

We report a novel approach to derivatize the primary, secondary, and tertiary hydroxy group(s) of oxysterols with N,N-dimethylglycine (DMG) in the presence of both 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4-(N,N-dimethylamino)pyridine to yield their corresponding mono- or di-DMG esters. Eight oxysterols including 7-oxocholesterol, 5 alpha,6 alpha- and 5 beta,6 beta-epoxycholesterols, as well as 7 alpha-, 7 beta-, 24(S)-, 25-, and 27-hydroxycholesterols, were studied. Electrospray ionization tandem mass spectrometric characterization of these singly or doubly protonated derivatives demonstrates the presence of an informative fragmentation pattern for each oxysterol derivative. Potential dissociation pathways for the production of these unique fragmentation patterns are proposed and discussed. Collectively, these informative and unique fragmentation patterns allow rapid and direct discrimination of the identities of 7 alpha-, 7 beta-, 24(S)-, 25-, and 27-hydroxycholesterol isomers, as well as 5 alpha,6 alpha- and 5 beta,6 beta-epoxycholesterol isomers, thereby potentially providing a foundation for quantitative analysis of oxysterols in biological samples in combination with a chromatographic separation. Copyright (c) 2006 John Wiley & Sons, Ltd.