Steroids are essential hormones that play a crucial role in homeostasis of many biological processes including sexual development, spermatogenesis, sperm physiology and fertility. Although steroids have been largely studied in many biological matrices (such as urine and plasma), there is very limited information of the steroid content and their study as potential indicators of the quality of the seminal fluid. In this study, a LC-HRMS (liquid chromatography-high resolution mass spectrometry) strategy has been developed in order to obtain the extended steroid profile of human seminal fluid. A comparison between supported liquid extraction (SLE) and solid liquid extraction (SPE) was carried out and the chosen SPE method was further optimized to evidence the largest possible number of compounds. Steroids were automatically annotated by using DynaStI, a publicly available retention time prediction tool developed in our lab, to match the experimental data (i.e. accurate mass and tR). Altogether, these resources allowed us to develop a post-targeted approach able to consistently detect 41 steroids in seminal fluid (with half of them being androgens). Such steroid pattern was found to be stable across different extraction times and injection days. In addition to accurate mass and retention time, the identity of 70% of the steroids contained in such steroid profile was confirmed by comparing their fragmentation patterns in real samples to those of pure commercial standards. Finally, the workflow was applied to compare and distinguish the steroid profile in seminal fluid from healthy volunteers (n = 7, with one of them being a vasectomized subject). In all, the developed steroidomics strategy allows to reliably monitor an extended panel of 41 steroids in human seminal fluid.

中文翻译：

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通过LC-HRMS对人精液中的类固醇进行分析。

类固醇是必不可少的激素，在许多生物过程（包括性发育，精子发生，精子生理和生育）的体内平衡中起着至关重要的作用。尽管在许多生物基质（例如尿液和血浆）中都对类固醇进行了广泛的研究，但是关于类固醇含量及其作为精液质量的潜在指标的研究却非常有限。在这项研究中，LC-HRMS（液相色谱-高分辨率质谱法）策略已得到开发，以获得人类精液的类固醇激素分布的扩展。对支持的液体萃取（SLE）和固液萃取（SPE）进行了比较，并对所选的SPE方法进行了进一步优化，以证明可能存在的最大化合物数量。使用DynaStI自动对类固醇进行注释，我们实验室开发的公开保留时间预测工具，用于匹配实验数据（即准确的质量和tR）。总而言之，这些资源使我们能够开发出一种针对性的方法，能够持续检测精液中的41种类固醇（其中一半为雄激素）。发现这种类固醇模式在不同的提取时间和注射天数内是稳定的。除了准确的质量和保留时间外，通过将真实样品中的类固醇碎片模式与纯商业标准品中的类固醇碎片模式进行比较，可以确认类固醇谱中所含类固醇的70％的身份。最后，将工作流程应用于比较和区分健康志愿者（n = 7，其中一名是输精管切除术的受试者）的精液中的类固醇特征。在所有，