Gas chromatography-mass spectrometry (GC-MS) of urinary steroids

Steroid metabolites in 24-h urine samples were analyzed by quantitative targeted GC-MS (9, 10, 11). Briefly, free and conjugated urinary steroids were extracted by solid phase extraction and conjugates were enzymatically hydrolyzed. After recovery of hydrolyzed steroids by solid phase extraction, known amounts of internal standards (5α-androstane-3α,17α-diol, stigmasterol) were added to each extract before formation of methyloxime-trimethylsilyl ethers. GC was performed using an Optima-1 fused silica column (Macherey–Nagel, Dueren, Germany) housed in an Agilent Technologies 6890 series GC that was directly interfaced to an Agilent Technologies 5975 inert XL mass selective detector. After calibration, values for the excretion of individual steroids were determined by measuring the selected ion peak areas against the internal standard areas.

Steroid metabolites’ ratios, as described in our previous paper (11), were used to calculate the activity of the enzymes: 5α reductase (An/Et) (5αTHF/THF, 5αTHB/THB), 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) ((THF + αTHF)/THE), 3β-hydroxysteroid dehydrogenase-(3βHSD) ((THE + THF + αTHF)/P5T-17α) and 21-hydroxylase ((THE + THF + αTHF)/PT, (THE + THF + αTHF)/PO5α3α).

The study was conducted according to Helsinki declaration, and approved by the Ethics Committee of the Medical University of Silesia. Informed consent was obtained from each patient over age 16 years, a parent or a legal guardian, after full explanation of the purpose and nature of all procedures.