Towards Improved Steroid Hormone Analysis: The Shift from Immunoassays to Mass Spectrometry

Steroid hormones, synthesized from cholesterol via cytochrome P450 enzymes, are categorized into adrenocortical hormones and sex hormones. Their monitoring is crucial in clinical and research settings for evaluating disease risk and physiological shifts, as well as for managing medical conditions. The structural similarity and concentration differences of steroid hormones in biological samples create significant challenges for analysis. Traditional immunoassays (IAs) are now less effective due to cross-reactivity and other limitations. Advances in mass spectrometry (MS), especially liquid chromatography-tandem mass spectrometry, have become preferred for steroid hormone detection, establishing a new “gold standard” in the field. The paper examines the influence of various pretreatment methods on the sensitivity and detection limits of steroid hormones, including protein precipitation, liquid–liquid extraction, solid-phase extraction, cold-induced phase separation technique, and derivatization technique, providing a crucial reference for clinical steroid detection. This paper reviews IAs and MS methods used recently (2013–2023) for detecting endogenous steroid hormones, highlighting their analytical strengths and limitations. It also discusses recent advancements and future prospects in both IA and MS methodologies.