Institute for Protein Research, Osaka University, Osaka, Japan.
Rapid Commun Mass Spectrom. 2024 May 15;38(9):e9719. doi: 10.1002/rcm.9719.
As 3-OH-containing steroids are prone to dehydration by conventional electrospray ionization, reducing detection sensitivity, Li ion adduction-based ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC/MS/MS), developed to prevent dehydration and effectively detect 3-OH steroids, was applied for profiling total and free steroids in urine.
Free urinary steroids were isolated directly from urine by solid-phase extraction (SPE) with 80% acetonitrile. The total steroids were prepared by enzymatic treatment of urine with a cocktail of sulfatase and glucronidase, protein precipitation, and separation with the above SPE. In order to detect as many steroid types as possible, UHPLC/MS/MS (Li method) with Li solution added after the column was used for analysis in addition to the conventional method of detecting protonated ions (H method). The 13 3-OH steroids and the remaining 16 steroids were quantified by standard curves prepared using product ion transitions derived from [M + Li] and MH , respectively.
Two groups of human urine, male and female urine, were analyzed. 3-OH steroids could be detected with greater sensitivity using the Li method than the conventional method. The absolute amounts of each steroid were normalized based on creatinine levels. The difference between the male and female groups are clearly attributable to sex steroids.
Twenty-nine total steroids and 19 free steroids were identified in a limited volume (240 mL) of urine. Of these, 13 3-OH steroids were better detected by Li adduction-based UHPLC/MS/MS.
由于 3-OH 类固醇经传统电喷雾电离容易脱水，从而降低检测灵敏度，因此开发了基于 Li 离子加成的超高效液相色谱-串联质谱法（UHPLC/MS/MS），以防止脱水并有效检测 3-OH 类固醇，用于对尿液中的总类固醇和游离类固醇进行分析。
游离尿类固醇通过固相萃取（SPE）直接用 80%乙腈从尿液中分离。总类固醇通过用磺基水解酶和葡萄糖醛酸苷酶的混合物对尿液进行酶处理、蛋白质沉淀，并采用上述 SPE 进行分离来制备。为了尽可能多地检测到类固醇类型，除了使用常规的质子化离子检测方法（H 方法）之外，还使用在柱后添加 Li 溶液的 UHPLC/MS/MS（Li 方法）进行分析。使用源自 [M + Li] 和 MH 的产物离子转换分别制备的标准曲线来定量 13 种 3-OH 类固醇和其余 16 种类固醇。
分析了两组人类尿液，男性和女性尿液。与常规方法相比，Li 方法可以更灵敏地检测到 3-OH 类固醇。每种类固醇的绝对量均基于肌酐水平进行归一化。男性组和女性组之间的差异显然归因于性激素。
在有限的尿液体积（240 mL）中鉴定出 29 种总类固醇和 19 种游离类固醇。其中，13 种 3-OH 类固醇通过基于 Li 加成的 UHPLC/MS/MS 得到更好的检测。
