Center for Translational Biomedical Research, University of North Carolina at Greensboro, North Carolina Research Campus, Kannapolis, NC, 28081, USA.
Graduate Institute of Forensic Medicine, National Taiwan University, Taipei, Taiwan.
Anal Bioanal Chem. 2020 May;412(12):2841-2849. doi: 10.1007/s00216-020-02514-x. Epub 2020 Feb 20.
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.
游离脂肪酸（FFA）和酰基辅酶 A（AcCar）是能量代谢的关键元素。FFA 和 AcCar 水平的失调与遗传缺陷和其他代谢紊乱有关。由于这两类化合物在物理化学性质上存在差异，因此很难使用单一方法定量测定人血浆中的 FFA 和 AcCar。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-多重反应监测（LC-MRM）方法，可同时定量测定 FFA 和 AcCar。丹磺酰肼（DnsHz）用于标记 FFA 和 AcCar 上的羧酸部分。这导致在正离子模式下形成带永久电荷的铵离子，从而易于离子化，并且由于短链类似物在反相 LC 柱上的保留增强而具有更高的疏水性，从而可以使用重标记的 DnsHz 类似物作为内标进行绝对定量。优化了标记条件，包括交联剂的浓度和新鲜度、反应时间和温度。该方法可以成功地定量测定所有短链、中链和长链 FFA 和 AcCar，灵敏度大大提高。使用该方法，可在 12 分钟内绝对定量和验证人血浆样品中的 25 种 FFA 和 13 种 AcCar。基于 CIL 的 LC-MRM 方法可同时定量测定 FFA 和 AcCar，有助于脂肪酸代谢的研究，并具有临床应用的潜力。
