APCI-Multistage Mass Spectrometry Following Liquid Chromatography for Selected 4-Desmethyl-Sterols and Their Deuterium-Labelled Analogues Unveils Characteristic Fragmentation Routes for Cholesterol and Phytosterols Identification

IF 1.8 3区化学 Q4 BIOCHEMICAL RESEARCH METHODS

Rapid Communications in Mass Spectrometry Pub Date : 2025-04-05 DOI:10.1002/rcm.10039

V. Cinquepalmi, I. Losito, A. Castellaneta, C. D. Calvano, T. R. I. Cataldi

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引用次数: 0

Abstract

Rationale

Several phytosterols (PSs), well known for their role in plant physiology and their health benefits, represent a subset of the family of 4-desmethyl-sterols. They exhibit remarkable structural variability due to differences in the number and position of C=C bonds in their tetracyclic backbone and side chain composition. When analysed using tandem mass spectrometry (MS/MS), PSs often produce complex and potentially informative spectra, as in the case of electron ionization. However, these spectra have been only partially interpreted so far. Here, a systematic interpretation of the fragmentation of PSs, specifically free sterols, was pursued through a synergic use of high- and low-resolution multistage mass spectrometry (MSⁿ, n = 2–4).

Methods

The study focused on protonated and dehydrated forms of standard 4-desmethyl-sterols ([M + H-H₂O]⁺), generated via atmospheric pressure chemical ionization (APCI) following reversed-phase liquid chromatography (RPLC). Deuterium-labelled versions of cholesterol and stigmasterol, appropriately labelled on their side chains, were examined alongside their natural counterparts and other key PS standards, including β-sitosterol, campesterol, brassicasterol, Δ⁵-avenasterol (isofucosterol) and its isomer Δ⁷-avenasterol.

Results

The use of isotopically labelled standards allowed the identification of diagnostic, low m/z, product ions associated with the side chain, demonstrating that the positive charge can localize not only at the C3 position (associated with the hydroxyl group) but also on the side chain itself (C24/C25). Furthermore, all remaining peak signals in the tandem MS spectra of PSs were successfully elucidated with the help of MS³/MS⁴ measurements, unveiling complex fragmentation pathways involving both the steroidal backbone and the side chain and indicating C17 as an additional potential site for positive charge localization.

Conclusions

The findings described in the paper offer a strong basis for identifying critical structural features of PSs, thus opening interesting perspectives for the identification of minor PSs, often isomeric with more common ones, that can be detected in vegetal matrices.

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apci -液相色谱后多级质谱分析选定的4-去甲基甾醇及其氘标记类似物揭示了胆固醇和植物甾醇鉴定的特征破碎路线

几种植物甾醇（PSs）是4-去甲基甾醇家族的一个子集，以其在植物生理和健康方面的作用而闻名。由于在其四环主链和侧链组成中C=C键的数目和位置不同，它们表现出显著的结构变异性。当使用串联质谱（MS/MS）进行分析时，ps通常会产生复杂且潜在信息丰富的光谱，如电子电离的情况。然而，到目前为止，这些光谱只得到了部分解释。在这里，通过高分辨率和低分辨率多级质谱（MSn, n = 2-4）的协同使用，对ps，特别是游离固醇的碎片化进行了系统的解释。方法采用反相液相色谱法（RPLC）对常压化学电离（APCI）生成的标准4-去甲基甾醇（[M + H-H2O]+）进行质子化和脱水研究。氘标记版本的胆固醇和豆甾醇，适当地标记在它们的侧链上，与它们的天然对应物和其他关键PS标准，包括β-谷甾醇，油菜甾醇，油菜甾醇，Δ5-avenasterol（异焦甾醇）及其异构体Δ7-avenasterol一起进行检查。结果使用同位素标记标准物可以识别诊断性的、低m/z的、与侧链相关的产物离子，表明正电荷不仅可以定位在C3位置（与羟基相关），而且可以定位在侧链本身（C24/C25）。此外，在MS3/MS4测量的帮助下，PSs串联质谱中所有剩余的峰信号都被成功地阐明，揭示了涉及甾体骨架和侧链的复杂断裂途径，并表明C17是正电荷定位的额外潜在位点。本文所描述的研究结果为鉴定PSs的关键结构特征提供了坚实的基础，从而为鉴定可以在植物基质中检测到的次要PSs（通常与更常见的PSs具有异构体）开辟了有趣的视角。

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

Rapid Communications in Mass Spectrometry 化学-分析化学

CiteScore

4.10

自引率

5.00%

发文量

219

审稿时长

2.6 months

期刊介绍： Rapid Communications in Mass Spectrometry is a journal whose aim is the rapid publication of original research results and ideas on all aspects of the science of gas-phase ions; it covers all the associated scientific disciplines. There is no formal limit on paper length ("rapid" is not synonymous with "brief"), but papers should be of a length that is commensurate with the importance and complexity of the results being reported. Contributions may be theoretical or practical in nature; they may deal with methods, techniques and applications, or with the interpretation of results; they may cover any area in science that depends directly on measurements made upon gaseous ions or that is associated with such measurements.