Abstract

Quantitation of endogenous steroids and their precursors is essential for diagnosis of a wide range of endocrine disorders. Usually, these analyses have been carried out using immunoassays. However, immunoassays often overestimate concentrations due to assay interference by other endogenous steroids, especially for low concentrations. Mass spectrometry based methods offer superior specificity, accuracy, and sensitivity. We therefore present a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method with automated sample preparation for determination of 17α-hydroxyprogesterone (17OHP), cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), androstenedione (A₄), testosterone (T), and estrone sulfate (E₁S). Samples were prepared using protein precipitation and 96-well filter plates, fully automated in a pipetting robot and analyzed by LC-MS/MS. Serum samples from 187 healthy children and adolescents aged 5-18 years were used to study hormone changes in relation to sex and pubertal stage. Lower limit of quantification for 17OHP was 0.7 nmol/L, for cortisol 11 nmol/L, for cortisone 2 nmol/L, for DHEAS 0.1 µmol/L, and for A₄, T, and E₁S, 0.2 nmol/L. This study showed a general increase in 17OHP, DHEAS, A₄, T and E₁S in both genders during puberty. In boys, A₄ and T increased significantly throughout pubertal development. Girls had significantly higher A₄ and E₁S concentrations, while boys had higher T concentrations. No sex- or puberty-specific differences were seen in cortisol or cortisone concentrations. To the best of our knowledge, this is the first presentation of changes in serum E₁S concentrations during pubertal development in healthy children.

摘要

内源性类固醇及其前体的定量对于诊断各种内分泌疾病至关重要。通常，这些分析是使用免疫测定法进行的。然而，由于其他内源性类固醇的测定干扰，免疫测定经常高估浓度，特别是对于低浓度。基于质谱的方法提供了卓越的特异性、准确性和灵敏度。因此，我们提出了一种液相色谱-串联质谱 (LC-MS/MS) 方法，该方法具有自动化样品制备，用于测定 17α-羟基孕酮 (17OHP)、皮质醇、可的松、硫酸脱氢表雄酮 (DHEAS)、雄烯二酮 (A ₄ )、睾酮 ( T) 和硫酸雌酮 (E ₁S)。使用蛋白质沉淀和 96 孔过滤板制备样品，在移液机器人中全自动进行，并通过 LC-MS/MS 进行分析。来自 187 名 5-18 岁健康儿童和青少年的血清样本用于研究与性别和青春期相关的激素变化。17OHP 的定量下限为 0.7 nmol/L，皮质醇为 11 nmol/L，可的松为 2 nmol/L，DHEAS 为 0.1 µmol/L，A ₄、T 和 E ₁ S 为 0.2 nmol/L。该研究表明，青春期男女的17OHP、DHEAS、A ₄、T 和 E ₁ S普遍增加。在男孩中，A ₄和 T 在整个青春期发育过程中显着增加。女孩的 A ₄和 E ₁明显更高S 浓度，而男孩的 T 浓度较高。皮质醇或可的松浓度未见性别或青春期特异性差异。据我们所知，这是健康儿童青春期发育期间血清 E ₁ S 浓度变化的首次表现。