Simultaneous determination of 18 steroids in the hypothalamic pituitary gonadal axis based on UPLC-MS/MS with multimode ionization†

To objectively quantify changes in steroid hormones in organisms caused by adverse environmental loads, we developed a simple and sensitive UPLC-MS/MS (ultra-performance liquid chromatography triple quadrupole mass spectrometry) method for the simultaneous determination of 18 steroid hormones on the HPG axis. This analytical method was based on liquid extraction and a multimode electrospray and atmospheric pressure chemical ionization (ESCi) source, which was optimized by mass spectrometry, liquid phase and pretreatment for the quantification of cholesterol (CH), aldosterone (A), cortisone (E), hydrocortisone (F), 21-deoxycortisol (21-DF), corticosterone (B), 11-deoxycortisol (11-DF), androstenedione (A₂), estradiol (E₂), estrone (E₁), 2-methoxyestradiol (2-MeE₂), 21-hydroxyprogesterone (21-OHP), 17-α hydroxyprogesterone (17α-OHP), testosterone (T), dehydroepiandrosterone (DHEA), progesterone (P₄), dihydrotestosterone (DHT), and pregnenolone (P₅). The method exhibits linearity in the analyte-concentration range 0.03–1000 μg mL⁻¹ (r² > 0.99), the spiked recoveries for the concentration range tested are 76.22–113.66%, and the relevant parameters of precision are 7.52–1.14%. Compared to other methods, this new method not only uses a small amount of serum (only 100 μL), but also permits the analysis of the challenging steroid, cholesterol. Furthermore, the method was successfully applied to the determination of steroids in Mus musculus, Carassius auratus, Rana catesbeiana Shaw, and Rana nigromaculata serum samples from randomly selected individuals. Therefore, this method is efficient and a very useful tool for assessing changes in steroid hormones.