Darlington Daniel N
Blood and Shock Research, US Army Institute of Surgical Research, JBSA Fort Sam Houston, San Antonio, TX 78234, USA.
Department of Surgery, Trauma and Emergency Surgery, University of Texas Health, San Antonio, TX 78229, USA.
Molecules. 2025 Jan 16;30(2):341. doi: 10.3390/molecules30020341.
Conjugation of short-chain fatty acids (SDFAs) to amines containing ring structures allows for better measurement by liquid chromatography tandem mass spectroscopy (LC-MS/MS). However, collision-induced dissociation (CID) results in breaking the conjugate back to the original SCFA and amine. We therefore set out to find an amine that would remain on the SCFA after CID and create a unique daughter for selectivity of measurement. Of twenty-seven amines with ring structures, we found four that contain bicycle-type structures (two rings connected by a carbon) with nitrogen in the second ring. CID removes the second ring at the nitrogen, leaving the first ring on the daughter. Of the four amines, 4-(pyrrolidine-1-ylmethyl) benzylamine (4PyBA) showed the strongest conjugation. Conjugation of 4PyBA to SCFA (C3-C6), their isomers and their phenylated versions (and isomers) resulted in good chromatographic peaks and separation. CID resulted in unique daughters that allowed for selectivity of measurement. Using this method, standard curves were generated that show good linearity (r2 > 0.99) in the nM and μM range with lower limits of detection between 40 and 229 nM for a 10 μL sample. Finally, we used this method to measure SCFA in plasma, liver, platelets, and red blood cells, demonstrating its use in biological systems. Because SCFAs are an index of microbiome diversity in the gastrointestinal track, this method will allow us to study changes in SCFAs and the microbiome in pathologic conditions including trauma, hemorrhage, and sepsis.
短链脂肪酸（SDFAs）与含环结构的胺结合，有助于通过液相色谱串联质谱法（LC-MS/MS）进行更好的测量。然而，碰撞诱导解离（CID）会导致结合物分解回原始的短链脂肪酸和胺。因此，我们着手寻找一种在CID后仍能与短链脂肪酸结合的胺，并产生一个独特的子离子用于选择性测量。在27种含环结构的胺中，我们发现有4种含有双环型结构（两个环通过一个碳相连），且第二个环中有氮。CID会在氮处去除第二个环，使第一个环保留在子离子上。在这4种胺中，4-（吡咯烷-1-基甲基）苄胺（4PyBA）表现出最强的结合能力。4PyBA与短链脂肪酸（C3-C6）、它们的异构体及其苯基化形式（和异构体）结合后，产生了良好的色谱峰和分离效果。CID产生了独特的子离子，实现了选择性测量。使用这种方法生成的标准曲线在纳摩尔和微摩尔范围内显示出良好的线性（r2>0.99），对于10μL样品，检测下限在40至229纳摩尔之间。最后，我们使用这种方法测量了血浆、肝脏、血小板和红细胞中的短链脂肪酸，证明了其在生物系统中的应用。由于短链脂肪酸是胃肠道微生物群多样性的一个指标，这种方法将使我们能够研究在创伤、出血和脓毒症等病理状况下短链脂肪酸和微生物群的变化。
