Introduction
Steroids
play important roles in various physiological functions of the organism,
including brain development, behavior, cognition, neuroplasticity, and
neuroinflammation, and are metabolized and excreted in the urine as
conjugated forms1-4. Transient concentrations of each
steroid in the blood indicate a physiological state, but these
concentrations change rapidly in response to various physiological and
even psychological changes5,6.
Urinary steroids represent the total amount of each steroid circulating
in the blood over a given time period. Profiling of urinary steroids
should be important for diagnosing relatively long-term physiological
changes such as chronic disease, breast cancer and prostate cancer1,7,8. Steroid hormones are enzymatically synthesized
from cholesterol and classified into various types of steroids,
including estrogens (female reproductive steroids), androgens (male
reproductive steroids), progestogens (pregnancy steroids), and
corticosteroids (stress steroids)2,4. After
circulating in the blood and functioning in their respective organs,
these steroids are enzymatically converted to inactive substances linked
with sulfate or glucuronic acid in several organs such as liver and
prostate, and excreted in urine4,7,9. Due to their
diverse structures, these conjugated steroids are usually converted back
to free steroids for the analysis; The advantage of urine is that it is
non-invasive, therefore it has no pain and is easy to collect in large
amounts10. LC/ESI-MS/MS has become a popular method
for steroid analysis in biological samples due to its high sensitivity,
high selectivity and the ability to run multiple analytes
simultaneously11-13. However, 26 of the 29 steroids
contain hydroxyl groups in their backbone, which are prone to
dehydration when ionized by positive-ion ESI. Such dehydration
diversifies the precursor ion, thereby reducing sensitivity and
hindering their quantification by MRM methods. To overcome this issue,
we recently reported a method to protect steroid molecules from
dehydration by using Li+ as the adduct ion to steroid (“Li
method”)14.
Application of the Li method to the analysis of human urine resulted in
the detection of 13 3-OH steroids with 1.3-20 times greater sensitivity
than conventional LC/ESI-MS/MS (“H method”). Other steroids (16
species) were identified by the H method. As a result, a total of 29
steroids were identified.