A Complementary Multi-Aptamer Approach for Efficient Extraction of Steroid Hormones and Application to Their Determination in Biological Samples using Q-TOF LC-MS

Steroid hormones play a crucial role in human physiological functions, and the simultaneous quantification of multiple steroid hormones in biological matrices has considerable clinical relevance. However, the simultaneous quantification of steroid hormones in biological matrices remains analytically challenging due to their ultra-trace concentrations and substantial matrix interference. A novel complementary multi-aptamer strategy was proposed in our study. Multi-aptamer magnetic beads (MA-MBs) were synthesized by simultaneously immobilizing estrogen-specific aptamer (AptE) and corticoid-specific aptamer (AptC) on the magnetic beads. Then MA-MBs was used to extract multiple steroid hormones in biological matrices based on the combined specificity of complementary aptamers. Following extraction, the enriched steroid hormones are quantified using quadrupole time-of-flight liquid chromatography-mass spectrometry (Q-TOF LC-MS). MA-MBs demonstrated a rapid magnetic response (5 seconds), high extraction efficiency, and excellent reusability (at least 10 cycles). Under optimal extraction conditions, the proposed MA-MBs-Q-TOF LC-MS method exhibited a wide linear range (0.02-200 ng/mL), low limits of detection (0.02-1 ng/mL), and high recovery rates (92.93%-104.93%) for 10 types of steroid hormones. Finally, the validated method was applied to quantify ten clinically-relevant steroid hormones in serum samples, demonstrating its potential for advanced endocrine research applications. Furthermore, this study provides a robust platform for multiplexed targets analysis in complex biological matrices.