Free fatty acid (FFA) and acylcarnitine (AcCar) are key elements of energy metabolism. Dysregulated levels of FFA and AcCar are associated with genetic defects and other metabolic disorders. Due to differences in the physicochemical properties of these two classes of compounds, it is challenging to quantify FFA and AcCar in human plasma using a single method. In this work, we developed a chemical isotope labeling (CIL)-based liquid chromatography-multiple reaction monitoring (LC-MRM) method to simultaneously quantify FFA and AcCar. Dansylhydrazine (DnsHz) was used to label the carboxylic acid moiety on FFA and AcCar. This resulted in the formation of a permanently charged ammonium ion for facile ionization in positive ionization mode and higher hydrophobicity for enhanced retention of short-chain analogs on reversed-phase LC columns and enabled absolute quantification by using heavy labeled DnsHz analogs as internal standards. Labeling conditions including the concentration and freshness of cross-linker, reaction time, and temperature were optimized. This method can successfully quantify all short-, medium- and long-chain FFAs and AcCars with greatly enhanced sensitivity. Using this method, 25 FFAs and 13 AcCars can be absolutely quantified and validated in human plasma samples within 12 min. Simultaneous quantification of FFA and AcCar enabled by this CIL-based LC-MRM method facilitates the investigation of fatty acid metabolism and has potential in clinical applications.

中文翻译：

---

使用丹磺酰肼标记和液相色谱-三重四极杆质谱法同时定量血浆样品中的游离脂肪酸和酰基肉碱。

游离脂肪酸 (FFA) 和酰基肉碱 (AcCar) 是能量代谢的关键元素。FFA 和 AcCar 水平失调与遗传缺陷和其他代谢紊乱有关。由于这两类化合物的物理化学性质不同，使用单一方法量化人血浆中的 FFA 和 AcCar 具有挑战性。在这项工作中，我们开发了一种基于化学同位素标记 (CIL) 的液相色谱-多反应监测 (LC-MRM) 方法来同时量化 FFA 和 AcCar。丹磺酰肼 (DnsHz) 用于标记 FFA 和 AcCar 上的羧酸部分。这导致形成永久带电的铵离子，以便在正电离模式下轻松电离，并形成更高的疏水性，以增强短链类似物在反相 LC 柱上的保留，并通过使用重标记的 DnsHz 类似物作为内标实现绝对定量。对交联剂的浓度和新鲜度、反应时间和温度等标记条件进行了优化。该方法可以成功地量化所有短链、中链和长链 FFA 和 AcCar，灵敏度大大提高。使用这种方法，25 FFAs 和 13 AcCars 可以在 12 分钟内在人血浆样本中进行绝对量化和验证。