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-hydroxy-progesterone, 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 1 beta-, 2 alpha-, 4 beta-, and 6 beta-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 3 alpha-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 1 beta, 15 beta, 16 alpha and 21-hydroxy and 6- and 7-oxo groups. Minor positions of hydroxylation are those at 2 alpha, 4 beta and 6 beta. Three major metabolic streams exist in affected neonates in addition to the classical 3 beta-hydroxy-5 alpha-pregnane pathway, i.e. these of the 3-oxo-4-enes as well as 3 alpha- and 3 beta-hydroxy-5 alpha-anes. (C) 2011 Elsevier Inc. All rights reserved.