RationaleAs 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.MethodsFree 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.ResultsTwo 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.ConclusionsTwenty‐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.

中文翻译：

---

基于锂离子加合的超高效液相色谱-串联质谱法辅助分析尿类固醇

基本原理由于传统电喷雾电离很容易导致含 3-OH 类固醇脱水，从而降低检测灵敏度，基于锂离子加合的超高效液相色谱-串联质谱 (UHPLC/MS/MS) 旨在防止脱水并有效检测 3-OH 类固醇。 ‐OH 类固醇，用于分析尿液中的总类固醇和游离类固醇。方法通过使用 80% 乙腈的固相萃取 (SPE) 直接从尿液中分离出游离尿类固醇。通过用硫酸酯酶和葡糖苷酸酶的混合物对尿液进行酶处理、蛋白质沉淀并用上述SPE分离来制备总类固醇。为了检测尽可能多的类固醇类型，使用 Li 进行 UHPLC/MS/MS（Li 法）+除了检测质子化离子的常规方法（H法）外，还使用柱后添加的溶液进行分析。 13 种 3-OH 类固醇和其余 16 种类固醇通过使用 [M + Li] 衍生的产物离子跃迁制备的标准曲线进行定量+和MH+结果 分析了两组人类尿液，即男性和女性尿液。使用 Li 方法比传统方法可以更灵敏地检测 3-OH 类固醇。每种类固醇的绝对量根据肌酐水平进行标准化。男性和女性组之间的差异明显归因于性类固醇。结论在有限体积（240 mL）的尿液中鉴定出 29 种总类固醇和 19 种游离类固醇。其中，Li 可以更好地检测到 13 种 3-OH 类固醇+基于内合的 UHPLC/MS/MS。