US EPA, Great Lakes Toxicology and Ecology Division, 6201 Congdon Blvd, Duluth, MN 55804, USA.
US EPA, Great Lakes Toxicology and Ecology Division, 6201 Congdon Blvd, Duluth, MN 55804, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Jan 15;1163:122513. doi: 10.1016/j.jchromb.2020.122513. Epub 2020 Dec 24.
Exposure to endocrine-disrupting compounds (EDCs) can alter steroid hormone production in vertebrates, sometimes leading to adverse reproductive or developmental effects. Liquid chromatography mass spectrometry methods are the gold standard for analyte confirmation and quantification in biological matrices, but radioimmunoassays (RIAs) are most commonly used for measurement of select steroid hormones in aquatic toxicology studies. Existing methods for steroid quantification often employ derivatization, limiting the range of steroids that can be simultaneously measured in a single process. In the current study, a method for the simultaneous measurement of thirteen endogenous steroids in small sample volumes without derivatization using liquid chromatography atmospheric pressure photoionization tandem mass spectrometry (LC-APPI-MS/MS) was developed. Several physiologically important steroids, including 11-deoxycortisol, 11-ketotestosterone, 17α- and 17β-estradiol, 17α-hydroxyprogesterone, 17,20β-dihydroxyprogesterone, 17,20β,21-trihydroxyprogesterone, androstenedione, cortisol, estriol, estrone, progesterone, and testosterone, were selected for the analysis. The method was validated for application to small volumes of fish plasma and fish holding water. Method detection limits using only 10 µL of plasma ranged from 0.05 to 1.0 ng/mL. As a potential surrogate for plasma steroid measurements, fish holding water was analyzed to measure excreted steroids. Lower limits of quantification when using 0.25 L of water ranged from 0.05 to 1.0 ng/L. The validated method was applied to two different experiments with small fish species exposed to an EDC known to affect steroid synthesis, fadrozole. Concentrations of the 13 steroids were measured in plasma or holding water from the studies. This work demonstrates the potential application of the developed method to measure endogenous steroids for identification of EDCs in aquatic toxicology studies.
暴露于内分泌干扰化合物（EDCs）会改变脊椎动物的类固醇激素产生，有时会导致不良的生殖或发育影响。液相色谱-质谱联用方法是生物基质中分析物确证和定量的金标准，但放射免疫分析（RIA）是水生毒理学研究中测量选定类固醇激素最常用的方法。现有的类固醇定量方法通常采用衍生化，限制了在单个过程中同时测量的类固醇的范围。在当前的研究中，开发了一种无需衍生化即可使用液相色谱大气压光电离串联质谱（LC-APPI-MS/MS）同时测量小样本量中 13 种内源性类固醇的方法。选择了几种生理上重要的类固醇，包括 11-脱氧皮质醇、11-酮睾酮、17α-和 17β-雌二醇、17α-羟孕酮、17,20β-二羟孕酮、17,20β,21-三羟孕酮、雄烯二酮、皮质醇、雌三醇、雌酮、孕酮和睾酮，用于分析。该方法经过验证可应用于小体积鱼类血浆和鱼类养殖水的分析。仅使用 10 μL 血浆进行方法检测限，范围为 0.05 至 1.0 ng/mL。作为血浆类固醇测量的潜在替代物，分析鱼类养殖水以测量排泄的类固醇。使用 0.25 L 水时，定量下限范围为 0.05 至 1.0 ng/L。验证后的方法应用于两种不同的实验，研究了暴露于一种已知影响类固醇合成的 EDC 的小鱼。从研究中测量了 13 种类固醇在血浆或养殖水中的浓度。这项工作证明了所开发方法在水生毒理学研究中用于测量内源性类固醇以鉴定 EDC 的潜在应用。
