Department of Chemistry, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada.
Department of Physical Pharmacy, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia in Katowice, 41-200, Sosnowiec, Poland.
Anal Bioanal Chem. 2020 Jul;412(17):4183-4194. doi: 10.1007/s00216-020-02657-x. Epub 2020 May 2.
Steroid hormones (SH) play a number of important physiological roles in vertebrates including fish. Changes in SH concentration significantly affect reproduction, differentiation, development, or metabolism. The objective of this study was to develop an in vitro high-throughput thin-film solid-phase microextraction (TF-SPME)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for targeted analysis of endogenous SH (cortisol, testosterone, progesterone, estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2)) in wild white sucker fish plasma where the concentrations of the analytes are substantially low. A simple TF-SPME method enabled the simultaneous determination of free and total SH concentrations. The use of biocompatible coating allowed direct extraction of these hormones from complex biological samples without prior preparation. The carryover was less than 3%, thereby ensuring reusability of the devices and reproducibility. The results showed that TF-SPME was suitable for the analysis of compounds in the polarity range between 1.28 and 4.31 such as SH at different physicochemical properties. The proposed method was validated according to bioanalytical method validation guidelines. The limit of detection (LOD) and limit of quantification(LOQ) for cortisol, testosterone, progesterone, E1, E2, and EE2 were from 0.006 to 0.150 ng/mL and from 0.020 to 0.500 ng/mL, respectively. The recovery for the method was about 85%, and the accuracy and precision of the method for cortisol, testosterone, and progesterone were ≤ 6.0% and ≤ 11.2%, respectively, whereas those for E1, E2, and EE2 were ≤ 15.0% and ≤ 10.2%, respectively. On the basis of this study, TF-SPME demonstrated several important advantages such as simplicity, sensitivity, and robustness under laboratory conditions. Graphical abstract.
甾体激素 (SH) 在包括鱼类在内的脊椎动物中发挥着许多重要的生理作用。SH 浓度的变化会显著影响生殖、分化、发育或代谢。本研究的目的是开发一种用于检测内源性 SH（皮质醇、睾酮、孕酮、雌酮 (E1)、17β-雌二醇 (E2) 和 17α-乙炔基雌二醇 (EE2)) 的体外高通量薄膜固相微萃取 (TF-SPME)-液相色谱-串联质谱 (LC-MS/MS) 方法，该方法适用于分析野生白 sucker 鱼血浆中浓度较低的 SH。一种简单的 TF-SPME 方法可同时测定游离和总 SH 浓度。使用生物相容性涂层可以直接从复杂的生物样本中提取这些激素，而无需事先进行预处理。交叉污染小于 3%，从而确保了装置的可重复使用性和重现性。结果表明，TF-SPME 适用于分析不同理化性质的 SH 等极性范围在 1.28 和 4.31 之间的化合物。该方法按照生物分析方法验证指南进行了验证。皮质醇、睾酮、孕酮、E1、E2 和 EE2 的检测限 (LOD) 和定量限 (LOQ) 分别为 0.006-0.150ng/mL 和 0.020-0.500ng/mL。该方法的回收率约为 85%，皮质醇、睾酮和孕酮的方法准确度和精密度均≤6.0%和≤11.2%，而 E1、E2 和 EE2 的方法准确度和精密度均≤15.0%和≤10.2%。基于本研究，TF-SPME 在实验室条件下具有简单、灵敏和稳健等重要优点。
