Abstract：
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.
摘要：
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。这里,我们描述了一种快速和通用的液相色谱-串联质谱（LC-MS/MS）方法，旨在准确定量人血清中的内源性类固醇。样品制备包括用甲基叔丁基醚(MTBE)从180μL血清中液-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），双氢睾酮(DHT),11-氧雄激素：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.对于A4，T，定量下限（LLOQ）为0.05ng/mL，11KA4、P4和可的松，DHT为0.1ng/mL，11OHA4,11OHT,11KT,17OHP4，11OHP4，11KP4，皮质酮，醛固酮,11-脱氧皮质醇，和皮质醇,和0.5ng/mL的DHEA。准确性,精度,再现性，和回收率落在生物分析方法验证的可接受范围内。使用29名绝经前妇女在不同月经期的血清样本，我们证明了我们方法的临床实用性，它显示出足够的灵敏度，可以可靠地量化所有通常在循环中发现的目标类固醇水平，除了11OHP4和11KP4。
