Biochemistry and Molecular Genetics, Hospital Clínic of Barcelona, University of Barcelona, Spain.
Bioanalysis Research Group, IMIM (Institut Hospital del Mar d'Investigacions Mèdiques), Barcelona, Spain; Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain.
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Jun 1;960:8-13. doi: 10.1016/j.jchromb.2014.04.015. Epub 2014 Apr 13.
Gas chromatography-mass spectrometry (GC-MS) remains as the gold-standard technique for the study of the steroid metabolome. A main limitation is the need of performing a derivatization step since incubation with strong silylations agents for long periods of time (usually 16 h) is required for the derivatization of hindered hydroxyls present in some steroids of interest. In the present work, a rapid, simple and reproducible microwave-assisted derivatization method was developed. In the method, 36 steroids already treated with methoxyamine (2% in pyridine) were silylated with 50 μl of N-trimethylsilylimidazole by using microwave irradiation, and the formed methyloxime-trimethylsilyl derivatives were analyzed by GC-MS. Microwave power and derivatization time silylation conditions were optimized being the optimum conditions 600 W and 3 min respectively. In order to evaluate the usefulness of this technique, the urine steroid profiles for 20 healthy individuals were analyzed. The results of a comparison of microwave irradiation with the classical heating protocol showed similar derivatization yields, thus suggesting that microwave-assisted silylation is a valid tool for the rapid steroid metabolome study.
气相色谱-质谱联用（GC-MS）仍然是研究类固醇代谢组学的金标准技术。一个主要的限制是需要进行衍生化步骤，因为需要用强硅烷化试剂孵育很长时间（通常为 16 小时）才能衍生化一些感兴趣的类固醇中存在的受阻羟基。在本工作中，开发了一种快速、简单和可重复的微波辅助衍生化方法。在该方法中，已经用甲氧胺（吡啶中的 2%）处理的 36 种类固醇与 50μl N-三甲基硅咪唑进行硅烷化，并用 GC-MS 分析形成的甲氧肟-三甲基硅基衍生物。优化了微波功率和衍生化时间硅烷化条件，最佳条件分别为 600W 和 3 分钟。为了评估该技术的有用性，分析了 20 名健康个体的尿类固醇谱。微波辐射与经典加热方案比较的结果表明衍生化产率相似，因此表明微波辅助硅烷化是快速类固醇代谢组学研究的有效工具。
