School of Pharmacy, Shujitsu University, Nishigawara, Okayama 703-8516, Japan.
Molecules. 2022 May 18;27(10):3225. doi: 10.3390/molecules27103225.
Accurate measurement of sulfated steroid metabolite concentrations can not only enable the elucidation of the mechanisms regulating steroid metabolism, but also lead to the diagnosis of various related diseases. The present study describes a simple and sensitive method for the simultaneous determination of four sulfated steroid metabolites in saliva, pregnenolone sulfate (PREGS), dehydroepiandrosterone sulfate (DHEAS), cortisol sulfate (CRTS), and 17β-estradiol-3-sulfate (E2S), by online coupling of in-tube solid-phase microextraction (IT-SPME) and stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). These compounds were extracted and concentrated on Supel-Q PLOT capillary tubes by IT-SPME and separated and detected within 6 min by LC-MS/MS using an InertSustain swift C18 column and negative ion mode multiple reaction monitoring systems. These operations were fully automated by an online program. Calibration curves using their stable isotope-labeled internal standards showed good linearity in the range of 0.01-2 ng mL for PREGS, DHEAS, and CRTS and of 0.05-10 ng mL for E2S. The limits of detection ( = 3) of PREGS, DHEAS, CRTS, and E2S were 0.59, 0.30, 0.80, and 3.20 pg mL, respectively. Moreover, intraday and interday variations were lower than 11.1% ( = 5). The recoveries of these compounds from saliva samples were in the range of 86.6-112.9%. The developed method is highly sensitive and specific and can easily measure sulfated steroid metabolite concentrations in 50 μL saliva samples.
准确测量硫酸化甾体代谢物浓度不仅可以阐明调节甾体代谢的机制，还可以导致各种相关疾病的诊断。本研究描述了一种简单、灵敏的方法，用于通过在线耦合管内固相微萃取（IT-SPME）和稳定同位素稀释液相色谱-串联质谱（LC-MS/MS）同时测定唾液中的 4 种硫酸化甾体代谢物，即孕烯醇酮硫酸盐（PREGS）、脱氢表雄酮硫酸盐（DHEAS）、皮质醇硫酸盐（CRTS）和 17β-雌二醇-3-硫酸盐（E2S）。这些化合物通过 IT-SPME 被提取和浓缩在 Supel-Q PLOT 毛细管上，并通过 LC-MS/MS 在 6 分钟内分离和检测，使用 InertSustain swift C18 柱和负离子模式多反应监测系统。这些操作通过在线程序完全自动化。使用其稳定同位素标记的内标物的校准曲线在 PREGS、DHEAS 和 CRTS 的 0.01-2ng mL 范围内以及 E2S 的 0.05-10ng mL 范围内呈现良好的线性关系。PREGS、DHEAS、CRTS 和 E2S 的检测限（ = 3）分别为 0.59、0.30、0.80 和 3.20pg mL。此外，日内和日间变化均低于 11.1%（ = 5）。这些化合物从唾液样品中的回收率在 86.6-112.9%之间。所开发的方法具有高度的灵敏度和特异性，可轻松测量 50μL 唾液样品中的硫酸化甾体代谢物浓度。
