Olfert Matthias, Knappe Cornelius, Sievers-Engler Adrian, Masberg Benedikt, Lämmerhofer Michael
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
Anal Bioanal Chem. 2025 May;417(13):2753-2766. doi: 10.1007/s00216-024-05542-z. Epub 2024 Oct 5.
Comprehensive in-depth structural characterization of free mono-unsaturated and polyunsaturated fatty acids often requires the determination of carbon-carbon double bond positions due to their impact on physiological properties and relevance in biological samples or during impurity profiling of pharmaceuticals. In this research, we report on the evaluation of disulfides as suitable derivatization reagents for the determination of carbon-carbon double bond positions of unsaturated free fatty acids by UHPLC-ESI-QTOF-MS/MS analysis and SWATH (sequential windowed acquisition of all theoretical mass spectra) acquisition. Iodine-catalyzed derivatization of C = C double bonds with dimethyl disulfide (DMDS) enabled detection of characteristic carboxy-terminal MS2 fragments for various fatty acids in ESI negative mode. The determination of double bond positions of fatty acids with up to three double bonds, the transfer of the method to plasma samples, and its limitations have been shown. To achieve charge-switching for positive ion mode MS-detection, derivatization with 2,2'-dipyridyldisulfide (DPDS) was investigated. It enabled detection of both corresponding characteristic omega-end- and carboxy-end-fragments for fatty acids with up to two double bonds in positive ion mode. It provides a straightforward strategy for designing MRM transitions for targeted LC-MS/MS assays. Both derivatization techniques represent a simple and inexpensive way for the determination of double bond positions in fatty acids with low number of double bonds. No adaptation of MS hardware is required and the specific isotopic pattern of resulting sulfur-containing products provides additional structural confirmation. This reaction scheme opens up the avenue of structural tuning of disulfide reagents beyond DMDS and DPDS using reagents like cystine and analogs to achieve enhanced performance and sensitivity.
由于游离单不饱和脂肪酸和多不饱和脂肪酸对生理特性有影响，且在生物样品或药物杂质分析中具有相关性，因此对其进行全面深入的结构表征通常需要确定碳 - 碳双键位置。在本研究中，我们报告了对二硫化物作为合适的衍生化试剂的评估，用于通过超高效液相色谱 - 电喷雾电离 - 四极杆飞行时间串联质谱（UHPLC - ESI - QTOF - MS/MS）分析和SWATH（所有理论质谱的顺序窗口采集）采集来确定不饱和游离脂肪酸的碳 - 碳双键位置。用二甲基二硫化物（DMDS）对碳 - 碳双键进行碘催化衍生化，能够在电喷雾电离负离子模式下检测各种脂肪酸的特征性羧基末端二级质谱碎片。已展示了具有多达三个双键的脂肪酸双键位置的测定、该方法向血浆样品的转移及其局限性。为了实现正离子模式质谱检测的电荷切换，研究了用2,2'-二吡啶基二硫化物（DPDS）进行衍生化。它能够在正离子模式下检测具有多达两个双键的脂肪酸的相应特征性ω - 末端和羧基末端碎片。它为设计靶向液相色谱 - 串联质谱分析的多反应监测（MRM）转换提供了一种直接的策略。两种衍生化技术都是测定双键数量较少的脂肪酸双键位置的简单且廉价的方法。无需对质谱硬件进行调整，所得含硫产物的特定同位素模式提供了额外的结构确认。该反应方案开辟了一条超越DMDS和DPDS使用胱氨酸及其类似物等试剂对二硫化物试剂进行结构调整的途径，以实现更高的性能和灵敏度。
