Mass Spectrometry-Based Profiling of Deuterium-Labeled Sex Steroids Enables Non-Invasive Mapping of Steroid Dynamics in Intact Mice

Abstract Understanding the tissue-specific distribution and metabolism of sex steroids is critical for elucidating their physiological roles and pathological alterations. However, existing in vivo approaches often require surgical castration to suppress endogenous hormones, disrupting systemic endocrine balance and limiting physiological relevance. Here, we present a novel mass spectrometry method combining systemic administration of deuterium-labeled sex steroids with high-sensitivity gas chromatography-tandem mass spectrometry (GC-MS/MS) to simultaneously quantify exogenous, endogenous, and metabolized steroid species in serum and multiple tissues of non-castrated mice. This approach enables temporally resolved tracking of steroid uptake and biotransformation without perturbing endogenous hormonal status. We demonstrate the method’s high sensitivity, specificity, and capacity to reveal tissue-specific steroid metabolism previously inaccessible in physiological models. Our technique offers a versatile platform for studying steroid dynamics in vivo and has broad applications in endocrinology, neurobiology, and pharmacology. Teaser Deuterium tracing illuminates hidden steroid pathways in living animals. Competing Interest Statement The authors have declared no competing interest.