Conclusions:
Lithium ion adduction-based UPLC-MS/MS was applied to urinary steroid analysis and thirteen 3-OH steroids were detected. Among them, 7-OH P5 and 7-OH DHEA were identified for the first time in human urine. A total of 29 steroids and 19 free steroids were identified by combining conventional methods to detect protonated ions. The urinary steroid profile obtained in this study is useful for monitoring relatively long-term physiological conditions and diagnosing chronic diseases.
Figure 1. Biosynthesis map of steroid hormones.
Urinary steroids identified in this study are shown, of which 3-OH steroids are shown in green and newly identified steroids are shown in red.
Figure 2. MRM chromatograms of newly identified 7-OH P5 (left) and 7-OH DHEA (right) in human urine. Chromatograms were obtained for standard (2 ng for each, topmost), female-total (second), male-total (third), female-free (fourth), male-free (fifth) by the Li method (left) and the H method (right). The values above the peaks indicate retention times and the values in brackets indicate the peak-height ratio of the two peaks in each chromatogram observed at the two MRM transitions set for each compound (see Table S1). Typical fragmentation ions of lithiated 3-OH steroids are depicted in their structures.
Figure 3. Pie chart of relative amounts of major steroids in male (a) and female urine (b) and bar chart of free and total steroid levels (ng/creatinine (mg)) obtained in male (top) and female (bottom) urine (c). All the data were summarized in Table S5.