Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Institute of Biochemistry and Molecular Genetics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
J Steroid Biochem Mol Biol. 2024 Oct;243:106578. doi: 10.1016/j.jsbmb.2024.106578. Epub 2024 Jul 4.
Mass spectrometric-based steroidomics is a valuable analytical approach that gives a comprehensive understanding of the interlinked steroid biosynthetic pathways. Here, we describe a rapid and versatile liquid chromatography-tandem mass spectrometry (LC-MS/MS) method designed to accurately quantify endogenous steroids in human serum. Sample preparation involved liquid-liquid extraction with methyl tert-butyl ether (MTBE) from 180 µL serum. The targeted steroids for quantification included androgens: dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), 11-oxyandrogens: 11β-hydroxy-androstenedione (11OHA4), 11-keto-androstenedione (11KA4), 11β-hydroxy-testosterone (11OHT), 11-keto-testosterone (11KT), progestogens: 17α-hydroxy-progesterone (17OHP4), progesterone (P4), 11β-hydroxy-progesterone (11OHP4), 11-keto-progesterone (11KP4), mineralocorticoids: aldosterone, corticosterone, and glucocorticoids: 11-deoxycortisol, cortisol, and cortisone. The lower limits of quantification (LLOQ) were 0.05 ng/mL for A4, T, 11KA4, P4, and cortisone, 0.1 ng/mL for DHT, 11OHA4, 11OHT, 11KT, 17OHP4, 11OHP4, 11KP4, corticosterone, aldosterone, 11-deoxycortisol, and cortisol, and 0.5 ng/mL for DHEA. Accuracy, precision, reproducibility, and recovery fell within acceptable limits for bioanalytical method validation. Using serum samples from 29 premenopausal women in different menstrual phases, we demonstrated the clinical utility of our method, which showed sufficient sensitivity to reliably quantify all targeted steroids at levels typically found in circulation, except for 11OHP4 and 11KP4.
基于质谱的类固醇组学是一种有价值的分析方法，可以全面了解相互关联的类固醇生物合成途径。在这里，我们描述了一种快速而通用的液相色谱-串联质谱 (LC-MS/MS) 方法，旨在准确量化人血清中的内源性类固醇。样品制备涉及用甲基叔丁基醚 (MTBE) 从 180µL 血清中进行液-液萃取。用于定量的靶向类固醇包括雄激素：脱氢表雄酮 (DHEA)、雄烯二酮 (A4)、睾酮 (T)、二氢睾酮 (DHT)、11-氧基雄激素：11β-羟基雄烯二酮 (11OHA4)、11-酮雄烯二酮 (11KA4)、11β-羟基睾酮 (11OHT)、11-酮睾酮 (11KT)、孕激素：17α-羟基孕酮 (17OHP4)、孕酮 (P4)、11β-羟基孕酮 (11OHP4)、11-酮孕酮 (11KP4)、盐皮质激素：醛固酮、皮质酮和糖皮质激素：11-脱氧皮质醇、皮质醇和可的松。A4、T、11KA4、P4 和可的松的定量下限 (LLOQ) 为 0.05ng/mL，DHT、11OHA4、11OHT、11KT、17OHP4、11OHP4、11KP4、皮质酮、醛固酮、11-脱氧皮质醇和皮质醇的 LLOQ 为 0.1ng/mL，而 DHEA 的 LLOQ 为 0.5ng/mL。准确度、精密度、重现性和回收率均符合生物分析方法验证的可接受标准。使用来自不同月经周期的 29 名绝经前女性的血清样本，我们展示了该方法的临床实用性，该方法具有足够的灵敏度，能够可靠地定量所有靶向类固醇，除了 11OHP4 和 11KP4 之外，这些类固醇在循环中通常可以检测到。
