Sex steroid hormones are potential biomarkers of reproductive function in teleost fish, but their measurement continues to rely on antibody-based assays. The objective of this study was to optimize a robust and simultaneous liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for measurement of eight steroid hormones (cortisol, 11-ketotestosterone, estradiol, 17α-ethynyl estradiol, estrone, estriol, progesterone and testosterone) in fish plasma. The extraction was followed by liquid–liquid extraction with tert-butyl methyl ether and time scheduled multi-reaction monitoring (sMRM) was used for quantitation of steroids. Validation of method performance using charcoal-stripped human plasma showed extraction recoveries for eight steroids ranged from 85.5 to 108.2% with matrix effects > 80%. The limits of quantitation were 0.01 pg/μL for testosterone, 0.05 pg/μL for cortisol and progesterone, 0.1 pg/μL for 11-ketotestosterone, estradiol and estrone, 0.125 pg/μL for estriol and 0.25 pg/μL for 17α-ethynyl estradiol. The proposed method was applied to plasma samples of largemouth bass (Micropterus salmoides) collected from contaminated (Lake Apopka) and reference sites (Wildcate Lake) in Florida. Concentrations of testosterone, cortisol, estradiol and estrone were significantly different in female fish, but plasma concentration of cortisol was only statistically different in male fish between two sites (P < 0.05). This study demonstrates the application of a robust LC–MS/MS analysis for a range of sex steroid hormones representative of endocrine function in a top predator, largemouth bass.

中文翻译：

---

液相色谱-串联质谱法同时测量大口黑鲈（Micropterus salmoides）血浆中的类固醇激素

性类固醇激素是硬骨鱼生殖功能的潜在生物标志物，但其测量仍依赖于基于抗体的检测方法。这项研究的目的是优化一种可靠且同时进行的液相色谱-串联质谱（LC-MS / MS）方法，用于测量八种类固醇激素（皮质醇，11-酮睾丸激素，雌二醇，17α-乙炔基雌二醇，雌酮，雌三醇，鱼血浆中的黄体酮和睾丸激素）。萃取后，用叔酸液-液萃取丁基甲基醚和定时多反应监测（sMRM）用于甾体的定量。使用木炭剥离的人血浆对方法性能的验证表明，八种类固醇的提取回收率在85.5％至108.2％之间，基质效应> 80％。定量限为：睾丸激素为0.01 pg /μL，皮质醇和孕酮为0.05 pg /μL，11-酮睾酮，雌二醇和雌酮为0.1 pg /μL，雌三醇为0.125 pg /μL，17α-乙炔基雌二醇为0.25 pg /μL。 。该方法应用于大口鲈（Micropterus salmoides）的血浆样品。）收集自佛罗里达州受污染的（阿波普卡湖）和参考地点（野生湖）。雌性鱼类中睾丸激素，皮质醇，雌二醇和雌酮的浓度存在显着差异，但雄性鱼类中两个部位之间的血浆皮质醇浓度仅具有统计学差异（P  <0.05）。这项研究证明了强大的LC-MS / MS分析方法可用于代表顶级捕食者大嘴鲈的内分泌功能的一系列性类固醇激素。