Göteborg Pediatric Growth Research Center, Department of Pediatrics, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Göteborg, Sweden.
Göteborg Pediatric Growth Research Center, Department of Pediatrics, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Göteborg, Sweden.
J Steroid Biochem Mol Biol. 2018 Oct;183:116-124. doi: 10.1016/j.jsbmb.2018.06.005. Epub 2018 Jun 9.
Androgen and estrogen determinations serve as important diagnostic markers in a variety of clinical conditions. However, one challenge is to enhance assay sensitivity for determination in the lowest range, such as in prepubertal children. We here present a recently developed gas chromatography-tandem mass spectrometry (GC-MS/MS) method for determination of androstenedione (A), dihydrotestosterone (DHT), testosterone (T), estrone (E), and estradiol (E) in children, which we have compared with the sensitive radioimmunoassays; E extraction-RIA and T-RIA.
Steroids were extracted in ethyl acetate n-hexane solution from serum spiked with isotopically labeled internal standard and derivatized sequentially with pentafluorobenzyl bromide, pentafluorobenzyl hydroxylamine and pentafluoropropionic acid anhydride and analyzed by GC-MS/MS using a triple quadrupole mass spectrometer operated in negative chemical ionization mode. Leftover routine samples (n = 414) were used to evaluate the concordance between GC-MS/MS and RIAs and the validity of GC-MS/MS for pediatrics; of these samples, 101 were from seemingly healthy children. Pubertal stage was recorded for reference interval evaluation.
Lower limit of detection for A, T, DHT, E, and E were 0.1 nmol/L, 0.1 nmol/L, 27 pmol/L, 9 pmol/L, and 2 pmol/L, respectively. Good agreement was found between GC-MS/MS and T-RIA (r = 0.98) as well as between GC-MS/MS and E extraction-RIA (r = 0.98, for E concentrations above 14 pmol/L). In boys, T and DHT increased significantly from prepuberty throughout pubertal development, and in girls the same increase was observed for E and E. The greatest increase in A for both genders, as well as E and E in boys and T and DHT in girls, occurred in mid to late puberty.
We report the development of a GC-MS/MS method sensitive enough to accurately determine serum levels of androgens and estrogens in children.
雄激素和雌激素的测定在各种临床情况下都是重要的诊断标志物。然而，一个挑战是提高在最低范围内的测定灵敏度，例如在青春期前的儿童中。我们在此介绍了一种最近开发的气相色谱-串联质谱（GC-MS/MS）方法，用于测定儿童的雄烯二酮（A）、二氢睾酮（DHT）、睾酮（T）、雌酮（E）和雌二醇（E），我们将其与灵敏的放射免疫分析（E 提取-RIA 和 T-RIA）进行了比较。
用乙基乙酸盐正己烷溶液从血清中提取加标内标物的类固醇，并用五氟苄基溴、五氟苄基羟胺和五氟丙酸酐依次衍生化，然后用三重四极杆质谱仪在负离子化学电离模式下进行 GC-MS/MS 分析。使用剩余的常规样本（n=414）来评估 GC-MS/MS 与 RIA 的一致性以及 GC-MS/MS 在儿科中的有效性；其中 101 份样本来自看似健康的儿童。记录青春期阶段以供参考区间评估。
A、T、DHT、E 和 E 的检测下限分别为 0.1 nmol/L、0.1 nmol/L、27 pmol/L、9 pmol/L 和 2 pmol/L。GC-MS/MS 与 T-RIA（r=0.98）以及 GC-MS/MS 与 E 提取-RIA（r=0.98，E 浓度高于 14 pmol/L 时）之间存在良好的一致性。在男孩中，T 和 DHT 从青春期前到青春期发育期间显著增加，而在女孩中，E 和 E 也观察到相同的增加。男女两性的 A 增加最大，男孩的 E 和 E 以及女孩的 T 和 DHT 也增加最大，发生在从中期到晚期青春期。
我们报告了一种 GC-MS/MS 方法的开发，该方法足够灵敏，可以准确测定儿童血清中的雄激素和雌激素水平。
