Department of Chemistry, University at Buffalo, The State University of New York (SUNY), Buffalo, New York 14260, United States.
Chem Res Toxicol. 2022 Jul 18;35(7):1277-1288. doi: 10.1021/acs.chemrestox.2c00116. Epub 2022 Jun 13.
Per- and poly-fluorinated substances (PFASs) are organic pollutants that have been linked to numerous health effects, including diabetes, cancers, and dysregulation of the endocrine system. This study aims to develop a liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay to measure changes in 17 hormones in H295R cell line (a steroid producing adrenocortical cells) upon exposure to PFASs. Due to the challenges in the analysis of steroid hormones using electrospray ionization MS, a chemical derivatization method was employed to achieve 0.07-2 μg/L detection limits in LC-MS/MS. Furthermore, a 10-fold concentration factor through solid-phase extraction (SPE) allows for consistent sub-parts per billion detections. Optimization of the derivatization conditions showed doubly-derivatized products in some hormone analytes, including progesterone, corticosterone, and cortisol, and gave improved ionization efficiency up to 20-fold higher signal than the singly-derivatized product. The use of SPE for sample cleanup to analyze hormones from cellular media using weak anion exchange sorbent yielded 80-100% recovery for the 17 targeted hormones. The method was validated by exposing H295R cells to two known endocrine disruptors, forskolin and prochloraz, which showed expected changes in hormones. An initial exposure of H295R cells with various PFAS standards and their mixtures at 1 μM showed significant increases in progestogens with some PFAS treatments, which include PFBS, PFHxA, PFOS, PFDA, and PFDS. In addition, modest changes in hormone levels were observed in cells treated with other sulfonated or carboxylated headgroup PFASs. This sensitive LC-MS/MS method for hormone analysis in H295R cells will allow for the investigations of the alterations in the hormone production caused by exposure to various environmental insults in cell-based assays and other models.
全氟和多氟化合物（PFAS）是一类有机污染物，与许多健康影响有关，包括糖尿病、癌症和内分泌系统失调。本研究旨在开发一种液相色谱-串联质谱（LC-MS/MS）测定法，以测量 H295R 细胞系（一种产生类固醇的肾上腺皮质细胞）暴露于 PFAS 后 17 种激素的变化。由于电喷雾电离 MS 分析类固醇激素存在挑战，因此采用化学衍生化方法在 LC-MS/MS 中实现了 0.07-2μg/L 的检测限。此外，固相萃取（SPE）的 10 倍浓缩因子可实现一致的亚纳克每分检测。衍生化条件的优化显示，一些激素分析物（包括孕酮、皮质酮和皮质醇）中存在双衍生化产物，其离子化效率提高了 20 倍，信号比单衍生化产物高 20 倍。采用 SPE 进行样品净化，用弱阴离子交换吸附剂分析细胞培养基中的激素，17 种靶向激素的回收率为 80-100%。该方法通过将 H295R 细胞暴露于两种已知的内分泌干扰物福司可林和丙环唑进行验证，结果显示这些激素发生了预期的变化。用 1μM 的各种 PFAS 标准品及其混合物初始暴露 H295R 细胞，一些 PFAS 处理（包括 PFBS、PFHxA、PFOS、PFDA 和 PFDS）导致孕激素显著增加。此外，用其他磺化或羧化头部 PFAS 处理的细胞中观察到激素水平的适度变化。该灵敏的 LC-MS/MS 方法可用于分析 H295R 细胞中的激素，从而可以在细胞水平测定和其他模型中研究暴露于各种环境污染物对激素产生的改变。
