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.