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Sample Pretreatment Optimized for Steroid Analysis of Mouse Brain
Steroid hormones are small molecules, known to have a profound biological impact on target cells or organs. Steroid hormones consist of two classes, corticosteroids and sex steroids (androgens, estrogens, and progestogens). Steroids, even with quite low concentration, could influence brain development, behavior, cognition, neuroplasticity, etc. [2]. Steroids are normally extracted from the tissue homogenates by liquid-liquid extraction with organic solvents due to their hydrophobicity. However, some hydrophobic metabolites in mouse brain, such as fatty acids could interfere with steroid measurement. According to the slight difference of hydrophobicity among the lipids, solid phase extraction can be a method to isolate steroids from the biological matrices. Log P is a measured parameter of hydrophobicity. The relatively hydrophilic estriol (E3) and tetrahydrocortisol (TH-COR) have the log P values, 2.67 and 1.11, respectively, while the values of hydrophobic androsterone (An) and pregnenolone (P5) are 3.77 and 3.58 [3]. The different logP values imply the differential retention on the stationary phase.
Herein, we have pursued an optimal extraction strategy of steroids from a biological sample. Hydrophilic steroids such as E3 and TH-COR, and hydrophobic steroids like An and P5 were spiked in the mouse brain homogenate, extracted by the present method, and analyzed with the MRM mode in UPLC-MS/MS. The optimal extraction method, with respect to the high recovery of each steroid, will be discussed.