Double derivatization-enhanced LC-MS/MS for isomer-resolved identification and quantification of fatty acids

IF 6.1 2区化学 Q1 CHEMISTRY, ANALYTICAL

Analytica Chimica Acta Pub Date : 2026-03-08 Epub Date: 2026-01-20 DOI:10.1016/j.aca.2026.345133

Xingyu Shi , Xuan Liang , Shuilin Xie , Ting Zhou , Wei Xiong

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引用次数: 0

Abstract

Background

Fatty acids (FAs) are essential metabolites involved in energy storage, signaling, and inflammation regulation, and their biological functions are closely linked to the position and geometric configuration of carbon–carbon double bonds (CC). However, the isomer-resolved characterization and highly sensitive detection of FAs in complex biological matrices remain challenging due to their co-elution, similar fragmentation, and low ionization efficiency. Therefore, there remains a critical need for an efficient approach that can simultaneously enhance chromatographic separation, enable unambiguous CC characterization, and improve detection sensitivity for FA isomers in biological samples.

Results

In this study, we developed a double derivatization strategy combining magnesium monoperoxyphthalate hexahydrate (MMPP) epoxidation with N,N-diethyl-1,2-ethanediamine (DEEA) amidation, coupled with LC–MS/MS, for isomer-resolved FA analysis. The double derivatization enhanced the chromatographic resolution of positional and cis/trans isomers, enabled reliable localization of CC positions via Δ16 Da diagnostic ion pairs, and improved detection sensitivity by 16–32 fold relative to epoxidation alone. Cis-trans configurations were further supported by parallel linear relationships between retention time and CC position under identical LC conditions, providing a standards-sparing criterion for structural assignment. Applied to mouse plasma, the strategy identified 69 FAs, including 55 unsaturated species, representing an increase of 46 over the underivatized approach. Quantitative analyses and differential analyses showed most FAs were elevated in hepatitis B virus (HBV) mice, with altered isomer ratios pointing to disrupted desaturase activity and oxidative stress.

Significance

In conclusion, this double derivatization LC-MS/MS platform provided improved structural resolution, higher detection sensitivity, and more reliable configurational assignment of FAs without requiring extensive standards. Its successful application to HBV mouse plasma demonstrated its suitability for complex biological matrices and highlighted its potential for biomarker discovery and mechanistic studies of lipid-related metabolic diseases.

Abstract Image

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双衍生增强LC-MS/MS用于脂肪酸的异构体分辨鉴定和定量

脂肪酸（FAs）是参与能量储存、信号传导和炎症调节的重要代谢物，其生物学功能与碳-碳双键（CC）的位置和几何构型密切相关。然而，复杂生物基质中FAs的同分异构体分辨表征和高灵敏度检测仍然具有挑战性，因为它们的共洗脱、相似的碎片化和低电离效率。因此，仍然迫切需要一种有效的方法，可以同时增强色谱分离，使明确的CC表征，并提高生物样品中FA异构体的检测灵敏度。结果在本研究中，我们建立了一种双衍生化策略，将六水邻苯二甲酸一过氧镁（MMPP）环氧化与N，N-二乙基-1,2-乙二胺（DEEA）酰胺化结合LC-MS /MS，用于异构体分辨FA分析。双重衍生化提高了位置和顺/反式异构体的色谱分辨率，通过Δ16 Da诊断离子对实现了CC位置的可靠定位，相对于单独的环氧化反应，检测灵敏度提高了16-32倍。在相同的LC条件下，保留时间和CC位置之间的平行线性关系进一步支持顺反构型，为结构分配提供了标准保留准则。应用于小鼠血浆，该策略鉴定了69种FAs，其中包括55种不饱和脂肪酸，比未分化方法增加了46种。定量分析和差异分析显示，大多数FAs在乙型肝炎病毒（HBV）小鼠中升高，异构体比例改变，表明去饱和酶活性和氧化应激被破坏。总之，该双衍生化LC-MS/MS平台提供了更高的结构分辨率、更高的检测灵敏度和更可靠的FAs构型分配，而无需大量的标准。它在HBV小鼠血浆中的成功应用证明了它对复杂生物基质的适用性，并突出了它在脂质相关代谢疾病的生物标志物发现和机制研究方面的潜力。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Analytica Chimica Acta 化学-分析化学

CiteScore

10.40

自引率

6.50%

发文量

1081

审稿时长

38 days

期刊介绍： Analytica Chimica Acta has an open access mirror journal Analytica Chimica Acta: X, sharing the same aims and scope, editorial team, submission system and rigorous peer review. Analytica Chimica Acta provides a forum for the rapid publication of original research, and critical, comprehensive reviews dealing with all aspects of fundamental and applied modern analytical chemistry. The journal welcomes the submission of research papers which report studies concerning the development of new and significant analytical methodologies. In determining the suitability of submitted articles for publication, particular scrutiny will be placed on the degree of novelty and impact of the research and the extent to which it adds to the existing body of knowledge in analytical chemistry.