Development of an LC-MS/MS method for 32 steroid hormones and exploration of their gestational variations

ZHOU Qiqi; CHEN Junhe; NIE Chengtao; WANG Qian; XIA Fangbo; ZHOU Hongwei

doi:10.12122/j.issn.1673-4254.2026.02.08

Abstract：Objective To establish a highly sensitive and specific liquid chromatography-tandem mass spectrometry(LC-MS/MS)-based analytical method for detecting steroid hormones in human serum.Methods Solid-phase extraction(SPE)was used for sample pretreatment.Chromatographic conditions were optimized to achieve baseline separation,and mass spectrometry parameters were adjusted to ensure accurate detection of trace amounts of hormones.After validation of the linear range,accuracy,and precision,the method was applied in detection of serum samples from normal pregnant women for analyzing dynamic variation characteristics of 32 steroid hormones during the first,second,and third trimesters of pregnancy.Results The linear range of the method was 0.001-7500 ng/mL,with a limit of detection(LOD)of 0.0007 ng/mL.Both intra-day and inter-day coefficients of variation(CV)of the method were less than 10%,the spiked recovery ranged from 86.21%to 112.67%,the extraction efficiency was 85.05%-106.3%,and the matrix effects were 85.60%-113.22%.The results of detection using this method for serum steroid hormones during pregnancy revealed significant elevation of most of the hormones(such as 11-deoxycortisol,11β-hydroxyprogesterone,dehydroepiandrosterone sulfate,and estriol)with the progression of pregnancy,indicating a close correlation with the development of fetal-placental function.Some hormones increased in the third trimester,possibly due to maternal adaptive regulation and maturation of fetal adrenal function.Conclusion The detection results using the method established in this study reveal dynamic variations of steroid hormones during pregnancy,which provides clues for further physiological and pathological studies of these hormones.