Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are an important family of endogenous lipids, possessing antidiabetic and anti-inflammatory functions. Therefore, analysis of FAHFAs in biological samples obtained under healthy and disease states can uncover underlying mechanisms of various relevant disorders (e.g., diabetes and autoimmune diseases). Up to now, due to their extremely low abundance, the determination of the changed levels of these species is still a huge challenge, even though great efforts have been made by utilizing liquid chromatography-tandem mass spectrometry with or without derivatization. Herein, we described a novel method for analysis of FAHFAs present in lipid extracts of biological examples after solid-phase extraction and chemical derivatization with one authentic FAHFA specie as an internal standard based on the principles of multi-dimensional mass spectrometry-based shotgun lipidomics. The approach possessed marked sensitivity, high specificity, and broad linear dynamic range of over 3 orders without obvious matrix effects. Moreover, after chemical derivatization, the molecular masses of FAHFAs shift from an overlapped region with ceramide species to a new region without overlaps, removing these contaminating signals from ceramides, and thereby reducing the false results of FAHFAs. Finally, this novel method was successfully applied for determining FAHFAs levels in varieties of representative biological samples, including plasma from lean and overweight/obese individuals of normoglycemia, and tissue samples (such as liver and white adipose tissue from diabetic (db/db) mice). We revealed significant alterations of FAHFAs in samples under patho(physio)logical conditions compared to their respective controls. Taken together, the developed method could greatly contribute to studying altered FAHFA levels under a variety of biological/biomedical conditions, and facilitate the understanding of these lipid species in the patho(physio)logical process.

中文翻译：

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通过一步衍生后的鸟枪脂质组学对生物脂质提取物中羟基脂肪酸的脂肪酸酯进行灵敏分析

羟基脂肪酸的支链脂肪酸酯 (FAHFA) 是重要的内源性脂质家族，具有抗糖尿病和抗炎功能。因此，分析在健康和疾病状态下获得的生物样品中的 FAHFA 可以揭示各种相关疾病（例如糖尿病和自身免疫性疾病）的潜在机制。到目前为止，由于它们的丰度极低，尽管已经通过使用或不衍生化的液相色谱 - 串联质谱法做出了巨大努力，但确定这些物种的变化水平仍然是一个巨大的挑战。在此处，我们根据基于多维质谱的鸟枪脂质组学原理，以一种真正的 FAHFA 物质作为内标，描述了一种分析生物实例脂质提取物中存在的 FAHFA 的新方法。该方法具有显着的灵敏度、高特异性和超过 3 个数量级的宽线性动态范围，没有明显的基质效应。此外，经过化学衍生后，FAHFA 的分子质量从与神经酰胺物质重叠的区域转移到一个没有重叠的新区域，从而消除了神经酰胺的这些污染信号，从而减少了 FAHFA 的错误结果。最后，这种新方法成功应用于测定各种代表性生物样品中的 FAHFA 水平，包括来自正常血糖的瘦和超重/肥胖个体的血浆，以及组织样本（例如来自糖尿病 (db/db) 小鼠的肝脏和白色脂肪组织）。我们揭示了在病理（生理）条件下样品中 FAHFA 与其各自对照相比的显着改变。总之，所开发的方法可以极大地有助于研究在各种生物/生物医学条件下改变的 FAHFA 水平，并促进对病理（生理）过程中这些脂质种类的理解。