Steroids are an important class of biomolecules studied for their role in metabolism, development, nutrition, and disease. Although highly sensitive GC- and LC-MS/MS-based methods have been developed for targeted quantitation of known steroid metabolites, emerging techniques including ion mobility (IM) have shown promise in improved analysis and capacity to better identify unknowns in complex biological samples. Herein, we couple LC-IM-MS/MS with structurally selective reactions targeting hydroxyl and carbonyl functional groups to improve IM resolution and structural elucidation. We demonstrate that 1,1-carbonyldiimidazole derivatization of hydroxyl stereoisomer pairs such as testosterone/epitestosterone and androsterone/epiandrosterone results in increased IM resolution with ΔCCS > 15%. Additionally, performing this in parallel with derivatization of the carbonyl group by Girard’s Reagent P resulted in unique products based on relative differences in number of each functional group and C17 alkylation. These changes could be easily deciphered using the combination of retention time, collision cross section, accurate mass, and MS/MS fragmentation pattern. Derivatization by Girard’s Reagent P, which contains a fixed charge quaternary amine, also increased the ionization efficiency and could be explored for its potential benefit to sensitivity. Overall, the combination of these simple and easy derivatization reactions with LC-IM-MS/MS analysis provides a method for improved analysis of known target analytes while also yielding critical structural information that can be used for identification of potential unknowns.

中文翻译：

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使用衍生化方法改进类固醇的离子淌度分离和结构表征

类固醇是一类重要的生物分子，研究它们在新陈代谢、发育、营养和疾病中的作用。尽管基于 GC 和 LC-MS/MS 的高灵敏度方法已被开发用于已知类固醇代谢物的靶向定量，但包括离子淌度 (IM) 在内的新兴技术已显示出改进分析和更好识别复杂生物样品中未知物的能力的前景。在这里，我们将 LC-IM-MS/MS 与针对羟基和羰基官能团的结构选择性反应相结合，以提高 IM 分辨率和结构解析。我们证明了羟基立体异构体对（例如睾酮/表睾酮和雄酮/表雄酮）的 1,1-羰基二咪唑衍生化导致 IM 分辨率增加，ΔCCS > 15%。此外，与 Girard 试剂 P 对羰基的衍生化同时进行，基于每个官能团数量和 C17 烷基化的相对差异，产生了独特的产品。结合保留时间、碰撞截面、精确质量和 MS/MS 碎裂模式，可以轻松解读这些变化。Girard 的试剂 P 衍生化，其中包含固定电荷的季胺，也提高了电离效率，并且可以探索其对灵敏度的潜在好处。总体而言，将这些简单易行的衍生化反应与 LC-IM-MS/MS 分析相结合，为改进已知目标分析物的分析提供了一种方法，同时还产生了可用于识别潜在未知物的关键结构信息。