Results and discussion
Free and total steroids were quantified for male and female urine.
Steroids were comprehensively analyzed using the Li and H methods. As
discussed in the previous paper, 3-OH steroids severely undergo
dehydration when ionized in positive ion mode14.
Furthermore, additional OH group(s) in the backbone, such as7-OH DHEA
and 7-OH P5, further promote dehydration during ionization and those are
difficult to detect by conventional LC/ESI-MS/MS. The adduction of Li
ions to steroid molecules in the ionization could protect them from
dehydration, resulting in an increase in ion intensity of the precursor
ion as a lithiated form and efficient generation of specific fragment
ions, especially, for 3-OH steroids14 (Figure 2 and
Figure S2). For 3-ketosteroids, the ”H method” could be used because
they do not contain such hydroxyl groups. Furthermore, the analytical
settings of the H-method and the Li-method are very similar, so they can
be operated in a consecutive manner with a regular LC-MS/MS, allowing
for a more comprehensive analysis of multi-class steroids in
bio-samples. Figure 2 compares MRM chromatograms of free and total 7-OH
DHEA and 7-OH P5 from female and male specimens; 7-OH P5 was only
observed in total steroids of female specimen, while 7-OH DHEA was
observed in free and total steroids prepared from female and male
samples. These two steroids were first identified by the Li method in
human urine. When these steroids were measured by the H-method, 7-OH P5
was barely observed and 7-OH DHEA was likely to be observed as a
shouldered peak, but the retention time and peak height ratio of the two
transitions differed significantly from the standard one (Figure 2,
right panel). This could be partly attributed to instability of the
protonated ion, MH+ or
(M-H2O+H)+ ions, in the ion source.
The adduction of a Li ion to steroid in the gas phase should protect it
from the dehydration or degradation during ionization. Indeed, the
sensitization ratios of 7-OH-DHEA and 7-OH-P5 were 7.1 and 11.1,
respectively (Figure 2).
19 free and 29 total steroids were identified in the urine of men and
women. The amounts of each steroid was normalized by creatinine values
obtained separately for male and female samples (Table S5). The relative
composition of free steroids was different from the total steroids, the
majority of which consists of the conjugated forms. The predominant
urinary steroids (conjugated forms) were TH-COL, TH-COR, APH, COB, THB,
An, DHEA, and 11-OH-An. Female had relatively higher contents of
7-OH-P5, 16OH-E1, E3, P5, and 7-OH-DHEA than male, while male had higher
contents of DOC, AT, TH-DOC, TE, COR, COL THB, 11-OH-An and An, a result
that clearly reflects gender differences (Figure 3). Since 7-OH-P5,
16OH-E1, E3, P5, 7-OH-DHEA, AT, TE, 11-OH-An and An are sex steroids, it
is normal for significant differences between male and female urine
samples. The stress-related corticosteroids DOC, TH-DOC, COR, COL and
THB were common in male17, however, this result needs
to be confirmed with more specimens. Note that the amounts of each
steroid were obtained for the mixture of three specimens of urine and
their levels were almost in the ranges of those reported
previously2,18-25 (Figure S5), which were obtained for
individuals’ urine or 24-hours pooled urine22, while
E2, THB were observed with higher levels than those reported previously.
It could be ascribable to inter-day or individual variation of steroid
metabolism.
The high sensitivity of the Li method also revealed the profile of free
steroids in urine (about 1.2% of total steroid levels), although their
physiological function is unclear. It is worth mentioning that since the
method allowed the reliable quantification of 7-OH DHEA, which has been
reported to be involved in “doping”, it will be useful for its
monitoring26.