Simultaneous determination of the 18 steroids in hypotha-lamic pituitary gonadal axis based on UPLC–MS/MS with multimode ionization

Abstract

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 based on liquid extraction and a multimode electrospray and at-mospheric pressure chemical ionization (ESCi) source, which was optimized by mass spectrometry, liquid phase and pretreatment for the quantification of choles-terol [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 range (r2 > 0.99), 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 method, this new method not only uses a small amount of serum (only 100 μL), but also permitted the analysis of the challenging steroids, cholesterol. Furthermore, the method was successfully applied to determination of steroids in Mus musculus, Carassius auratus, Rana catesbiana Shaw, and Rana nigromaculata serum samples from randomly selected individuals. This method may therefore be very useful tool as an efficient analysis method for assessing changes in steroid hormones.