Oxysterols play a key role in many (patho)physiological processes and they are potential biomarkers for oxidative stress in several diseases. Here we developed a rapid gas chromatographic-mass spectrometry-based method for the separation and quantification of 11 biologically relevant oxysterols bearing hydroxy, epoxy, and dihydroxy groups. Efficient chromatographic separation (resolution ≥ 1.9) was achieved using a medium polarity 35%-diphenyl/65%-dimethyl polysiloxane stationary phase material (30 m × 0.25 mm inner diameter and 0.25 μm film thickness). Based on thorough analysis of the fragmentation during electron ionization we developed a strategy to deduce structural information of the oxysterols. Optimized sample preparation includes (i) extraction with a mixture of n-hexane/iso-propanol, (ii) removal of cholesterol by solid phase extraction with unmodified silica, and (iii) trimethylsilylation. The method was successfully applied on the analysis of brain samples, showing consistent results with previous studies and a good intra- and interday precision of ≤20%. Finally, we used the method for the investigation of oxysterol formation during oxidative stress in HepG2 cells. Incubation with tert-butyl hydroperoxide led to a massive increase in free radical formed oxysterols (7-keto-chol > 7β-OH-chol >> 7α-OH-chol), while 24 h incubation with the glutathione peroxidase 4 inhibitor RSL3 showed no increase in oxidative stress based on the oxysterol pattern. Overall, the new method described here enables the robust analysis of a biologically meaningful pattern of oxysterols with high sensitivity and precision allowing us to gain new insights in the biological formation and role of oxysterols.

中文翻译：

---

氧甾醇的 GC-MS 分析及其在培养的肝细胞 (HepG2) 中的形成

氧甾醇在许多（病理）生理过程中起着关键作用，它们是多种疾病中氧化应激的潜在生物标志物。在这里，我们开发了一种基于快速气相色谱-质谱法的方法，用于分离和定量 11 种具有羟基、环氧基和二羟基的生物相关氧甾醇。使用中等极性 35%-二苯基/65%-二甲基聚硅氧烷固定相材料（30 m × 0.25 mm 内径和 0.25 μm 薄膜厚度）实现高效色谱分离（分辨率 ≥ 1.9）。基于对电子电离过程中碎片的全面分析，我们制定了一种策略来推断氧固醇的结构信息。优化的样品制备包括 (i) 使用正己烷/异己烷混合物进行萃取-丙醇，（ii）用未改性的二氧化硅通过固相萃取去除胆固醇，和（iii）三甲基甲硅烷基化。该方法成功应用于脑样本分析，结果与以往研究结果一致，日内日间精密度≤20%。最后，我们使用该方法研究 HepG2 细胞氧化应激过程中氧固醇的形成。孵化叔-丁基氢过氧化物导致自由基形成的氧甾醇大量增加（7-酮-胆碱 > 7β-OH-胆碱 >> 7α-OH-胆碱），而与谷胱甘肽过氧化物酶 4 抑制剂 RSL3 孵育 24 小时未显示氧化性增加基于氧固醇模式的压力。总的来说，这里描述的新方法能够以高灵敏度和精确度对具有生物学意义的氧固醇模式进行稳健分析，使我们能够对氧固醇的生物形成和作用获得新的见解。