Hydroxysteroid dehydrogenase transformations of 5β-scymnol and identification of oxoscymnol transformation products by liquid chromatography–tandem mass spectroscopy

▶ LC–MS analysis of hydroxysteroid dehydrogenase oxidations of 5β-scymnol is reported. ▶ 3-Oxoscymnol is formed in the reaction of scymnol and 3α-hydroxysteroid dehydrogenase. ▶ 7-Oxoscymnol is formed in the reaction of scymnol and 7α-hydroxysteroid dehydrogenase. ▶ 12-Oxoscymnol is formed in the reaction of scymnol and 12α-hydroxysteroid dehydrogenase. A new and sensitive high performance liquid chromatography (HPLC) separation procedure coupled with tandem mass spectroscopy (MS and MS 2) detection was developed to identify for the first time the oxidation products of 5β-scymnol [(24R)-(+)-5β-cholestan-3α,7α,12α,24,26,27-hexol] catalysed by bacterial hydroxysteroid dehydrogenase (HSD) reactions in vitro. The authentic scymnol (MW 468) standard yielded a protonated molecular ion [M+H] + at m/ z 469 Da, and higher mass adduct ions attributed to [M+NH 4] + ( m/ z 486), [M+H+CH 3OH] + ( m/ z 501) and [M+H+CH 3COOH] + ( m/ z 530). (24R)-(+)-5β-Cholestan-3-one-7α,12α,24,26,27-pentol (3-oxoscymnol, m/ z 467 Da, relative retention time (RRT) = 0.89) was identified as the principle molecular species of scymnol in the reaction with 3α-HSD pure enzyme. [S] 0.5 for the reaction of 3α-HSD with scymnol as substrate was 0.7292 mM. (24R)-(+)-5β-cholestan-7-one-3α,12α,24,26,27-pentol (7-oxoscymnol, m/ z 467 Da, RRT = 0.79) and (24R)-(+)-5β-cholestan-12-one-3α,7α,24,26,27-pentol (12-oxoscymnol, m/ z 467 Da, RRT = 0.81) were similarly identified as principle molecular species in the respective 7α-HSD and 12α-HSD reactions. Polarity of the oxoscymnol species was established as 7-oxoscymnol > 12-oxoscymnol > 3-oxoscymnol > scymnol (in order from most polar to least polar). Confirmation that 5β-scymnol is an oxidative substrate for steroid-metabolising enzymes was made possible by the use of sophisticated liquid chromatography–mass spectrometry (LC–MS) techniques that will likely provide the basis for further exploration of scymnol as a therapeutic compound.