采用质谱和离子淌度谱法对螺内酯及其代谢物用吉拉德试剂 P 衍生化后的产物进行表征。
Characterization of spironolactone and metabolites derivatized using Girard's reagent P using mass spectrometry and ion mobility spectrometry.
机构信息
Neurobiology Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.
Immunity, Inflammation, and Disease Laboratory, Division of Intramural Research, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.
出版信息
Rapid Commun Mass Spectrom. 2024 Aug 15;38(15):e9775. doi: 10.1002/rcm.9775.
RATIONALE
Spironolactone is a steroidal drug prescribed for a variety of medical conditions and is extensively metabolized quickly after administration. Measurement of spironolactone and its metabolites remains challenging using mass spectrometry (MS) due to in-source fragmentation and relatively poor ionization using electrospray ionization. Therefore, improved methods of measurements are needed, particularly in the case of small sample volumes.
METHODS
Girard's reagent P (GP) derivatization of spironolactone was employed to improve response and provide an MS-based solution to the measurement of spironolactone and its metabolites. We performed ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) and ion mobility spectrometry (IMS)-high-resolution mass spectrometry (HRMS) to fully characterize the GP derivatization products. Analytes were studied in positive ionization mode, and MS/MS was performed using nonresonance and resonance excitation collision-induced dissociation.
RESULTS
We observed the successful GP derivatization of spironolactone and its metabolites using authentic chemical standards. A signal enhancement of 1-2 orders of magnitude was observed for GP-derivatized versions of spironolactone and its metabolites. Further, GP derivatization eliminated in-source fragmentation. Finally, we performed GP derivatization and ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) in a small volume of murine serum (20 μL) from spironolactone-treated and control animals and observed multiple spironolactone metabolites only in the spironolactone-treated group.
CONCLUSIONS
GP derivatization was proven to have advantageous mass spectral performance (e.g., limiting in-source fragmentation, enhancing signals, and eliminating isobaric analytes) for spironolactone and its metabolites. This work and the detailed characterization using ultra-high-performance liquid chromatography-high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) and IMS serve as the foundation for future developments in reaction optimization and/or quantitative assay development.
原理
螺内酯是一种甾体药物，用于治疗多种医学病症，且在给药后会迅速广泛代谢。由于源内碎裂和电喷雾电离时相对较差的离子化，使用质谱（MS）测量螺内酯及其代谢物仍然具有挑战性。因此，需要改进测量方法，特别是在小样本量的情况下。
方法
采用吉拉德试剂 P（GP）对螺内酯进行衍生化，以提高响应，并为测量螺内酯及其代谢物提供基于 MS 的解决方案。我们进行了超高效液相色谱-电喷雾电离-串联质谱（UHPLC-ESI-MS/MS）和离子淌度谱-高分辨率质谱（IMS-HRMS），以充分表征 GP 衍生化产物。分析物在正离子化模式下进行研究，使用非共振和共振激发碰撞诱导解离进行 MS/MS。
结果
我们使用真实的化学标准品观察到螺内酯及其代谢物的成功 GP 衍生化。观察到 GP 衍生化的螺内酯及其代谢物的信号增强了 1-2 个数量级。此外，GP 衍生化消除了源内碎裂。最后，我们对螺内酯处理和对照动物的小体积（20 μL）鼠血清进行了 GP 衍生化和超高效液相色谱-高分辨率质谱（UHPLC-HRMS），仅在螺内酯处理组中观察到多个螺内酯代谢物。
结论
GP 衍生化已被证明对螺内酯及其代谢物具有有利的质谱性能（例如，限制源内碎裂、增强信号和消除等质量分析物）。这项工作以及使用超高效液相色谱-高分辨率串联质谱（UHPLC-HRMS/MS）和 IMS 的详细表征为反应优化和/或定量分析方法的发展奠定了基础。
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