Steroids excreted in urine by neonates with 21-hydroxylase deficiency. 2. Characterization, using GC–MS and GC–MS/MS, of pregnanes and pregnenes with an oxo- group on the A- or B-ring

Urine from neonates with 21-hydroxylase deficiency contains a large range of metabolites of 17-hydroxyprogesterone, 21-deoxycortisol and androgens but few have been previously described. We present the second part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal steroid metabolism.Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC–MS and GC–MS/MS spectra were used together to determine the structure of the A- and B-rings containing an oxo group.Fragmentations indicating presence of 3-, 6-, and 7-oxo groups and also 1β-, 2α-, 4β-, and 6β-hydroxyls are presented and discussed for the first time. Interpretation was aided by comparison with spectra of available relevant standards, of oxidation products of standards and urinary metabolites and of deuterated derivatives. Endogenous 1-enes and 2(3)-ene artifacts of non-hydrolyzed 3α-sulfates are also reported. D-ring and side chain structure was determined according to our previously published criteria. Likely metabolic relationships were also explored.We conclude that GC–MS combined with GC–MS/MS allows identification of the A- and B-ring structure of pregnane and pregnenes in the presence of an oxo group on one of these rings. Major oxygenations are 1β, 15β, 16α and 21-hydroxy and 6- and 7-oxo groups. Minor positions of hydroxylation are those at 2α, 4β and 6β. Three major metabolic streams exist in affected neonates in addition to the classical 3α-hydroxy-5β-pregnane pathway, i.e. these of the 3-oxo-4-enes as well as 3α- and 3β-hydroxy-5α-anes.

► Urinary steroid metabolites were examined in neonates with 21-hydroxylase deficiency.
► GC–MS/MS characteristics of A- and B-rings of C21 steroid metabolites are presented.
► New metabolites with 3, 6, 7-oxo and 1β, 2α, 4β, 6β-hydroxyl groups are reported.
► The metabolic pathways 3β, 5α; 3α, 5α and 3oxo-4ene are added to the known 3α, 5β.
► Major additional oxygenations are 1β, 15β, 16α, 21-hydroxyls and 6, 7-oxo groups.