Excellent Technology Co., Ltd, Suzhou, China.
Department of Bioengineering, College of Chemical and Biological Engineering, Shandong Innovation Center for Non-grain Ethanol Biorefinery Technology, Shandong University of Science and Technology, Qingdao, China.
Rapid Commun Mass Spectrom. 2022 Apr 30;36(8):e9242. doi: 10.1002/rcm.9242.
Saliva has been widely accepted as a more convenient alternative to serum or plasma in the field of clinical diagnosis. However, the detection of trace components in saliva has been a bottleneck problem. The aim of this work was to develop a highly sensitive and reliable method for simultaneously determining the trace steroid hormones including some with poor ionization efficiency in human saliva by liquid chromatography/tandem mass spectrometry (LC/MS).
Saliva was deproteinated by acetonitrile containing mixed isotope internal standards and extracted with methyl tert-butyl ether. The extraction solution was dried under a stream of nitrogen and the residue was derivatized using 50 mM O-ethylhydroxylamine hydrochloride in 80% methanol/water solution (v/v). The processed sample was determined by LC/MS in multiple reaction monitoring (MRM) mode.
The method was successfully established for the simultaneous quantification of seven steroid hormones in human saliva and showed excellent specificity and sensitivity. The limits of quantification (LOQs) of all steroid hormones were below 5 pg/mL, in particular, the LOQ of progesterone was as low as 0.15 pg/mL. The linear correlation coefficients (r) were greater than 0.9990 in the range of 2-200 pg/mL for T, DHEA, A4, P4, P5, and 17OHP4 and in the range of 5-500 pg/mL for 17OHP5. The intra-day and inter-day variability ranged from 1.86% to 7.83% and 1.95% to 10.4%, respectively. The recovery of the method ranged from 86.9% to 111.1% for all steroid hormones using three spiked concentrations.
A novel LC/MS/MS method was developed for the simultaneous quantification of seven kinds of trace steroid hormones in human saliva. The results of the methodological study showed that the method exhibited excellent sensitivity and reliability for the evaluation of free steroid hormones in the human body. It is believed that this method could provide useful information of steroid hormone metabolism for auxiliary diagnosis of some endocrine disorders.
唾液已被广泛接受为临床诊断领域中比血清或血浆更方便的替代物。然而，检测唾液中的痕量成分一直是一个瓶颈问题。本工作旨在开发一种高度敏感和可靠的方法，用于通过液相色谱/串联质谱（LC/MS）同时测定痕量甾体激素，包括一些电离效率差的甾体激素。
唾液用含混合同位素内标的乙腈沉淀蛋白，然后用甲基叔丁基醚提取。提取液在氮气流下干燥，残渣用 50 mM O-乙基羟胺盐酸盐在 80%甲醇/水溶液（v/v）中衍生化。处理后的样品通过 LC/MS 在多重反应监测（MRM）模式下进行测定。
成功建立了一种同时定量测定人唾液中七种甾体激素的方法，该方法具有良好的特异性和灵敏度。所有甾体激素的定量限（LOQ）均低于 5 pg/mL，特别是孕酮的 LOQ 低至 0.15 pg/mL。在 2-200 pg/mL 范围内，T、DHEA、A4、P4、P5 和 17OHP4 的线性相关系数（r）均大于 0.9990，在 5-500 pg/mL 范围内，17OHP5 的线性相关系数（r）大于 0.9990。日内和日间变异性分别为 1.86%-7.83%和 1.95%-10.4%。该方法在三个加标浓度下对所有甾体激素的回收率为 86.9%-111.1%。
本研究建立了一种用于同时定量测定人唾液中七种痕量甾体激素的新型 LC/MS/MS 方法。方法学研究结果表明，该方法具有良好的灵敏度和可靠性，可用于评估人体游离甾体激素。相信该方法可为某些内分泌疾病的辅助诊断提供甾体激素代谢的有用信息。
