Measurement of reference intervals for urinary free adrenal steroid levels in Japanese newborn infants by using stable isotope dilution gas chromatography/mass spectrometry

Background: In newborn infants, there are no reference intervals for urinary free steroids, which are thought to reflect the bioavailable fraction of steroids in the blood. We establish a method for simultaneous measurement of urinary free adrenal steroids such as pregnenolone, progesterone, 16α-hydroxyprogesterone, 17α-hydroxyprogesterone, 21-deoxycortisone, 21-deoxycortisol, dehydroepiandrosterone, androstenedione, and 11β-hydroxyandrostenedione by using stable isotope dilution gas chromatography/mass spectrometry (SID-GC/MS) and determined the reference intervals for urinary levels of free adrenal steroids in Japanese newborn infants. Methods: Newborn pooled urine was used for validation. Spot urine samples were collected from 67 full-term Japanese newborn infants (34 male and 33 female infants) at 3-4. days of age to determine reference intervals. The extracted and purified free steroids were delivered with heptafluorobutyric anhydride and analyzed by SID-GC/MS. Results: We validated a SID-GC/MS method with good repeatability and recovery rate. The preliminary reference intervals (median [range], μmol/mol creatinine) were as follows: pregnenolone, 4.2 (0.7-31.6); progesterone, 0.5 (not detected (n.d.)-0.6); 16α-hydroxyprogesterone, 1.4 (n.d.-10.3); 17α-hydroxyprogesterone, 1.1 (n.d.-1.9); 21-deoxycortisone, n.d. (n.d.-n.d.); 21-deoxycortisol, n.d. (n.d.-n.d.); dehydroepiandrosterone, 2.2 (0.6-27.3); androstenedione, 0.7 (n.d.-5.2); and 11β-hydroxyandrostenedione, 2.9 (n.d.-26.7). Conclusions: We established a reliable SID-GC/MS method and were able to determine preliminary reference intervals for 9 urinary free adrenal steroids in newborn infants.