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.

Introduction: Severe trauma and hemorrhage in rats lead to changes in the beta diversity of the commensal bacteria found in the gut. Because short-chain fatty acids (SCFA) are produced by these bacteria, SCFA concentration may also change following trauma and hemorrhage and reflect these alterations in the microbiome. Objective: To determine whether changes in SCFA occur after trauma and hemorrhage in the feces and plasma of rodents. Materials and Methods: Polytrauma was induced in isoflurane-anesthetized Sprague-Dawley rats by damage to the small intestine, liver, right leg skeletal muscle, and femur, followed by 20% hemorrhage. Whole blood resuscitation was performed at 1 h (20%). Rats were euthanized at 2 h and feces and plasma were analyzed for short-chain fatty acids (SCFA) by liquid chromatography tandem mass spectroscopy. Results: Of 21 SCFA analyzed in the feces and plasma, 11 were measurable. In feces, five demonstrated a significant elevation after 2 h of severe trauma and hemorrhage (n-8) including propionic (37,775 ± 8,919 vs. 146,591 ± 46,734 nM/mg protein: mean ± SEM), pentanoic (10,975 ± 2,981 vs. 41,828 ± 10,645), 2-methyl propionic (2,621 ± 523 vs. 13,798 vs. 2083), 4-methyl pentanoic (1,134 ± 302 vs. 4,320 ± 1,029), and 3-phenyl propionic acid (42,194 ± 4,863 vs. 153,024 ± 38,473). The addition of whole blood resuscitation did not change these responses, but led to an additional significant elevation in butyric (68,551 ± 10,786 vs. 369,951 ± 79,515) and hexanoic acid (24,548 ± 6,791 vs. 102,002 ± 32,069). There was no change in SCFA after trauma, hemorrhage or resuscitation in the plasma (n = 6). Conclusion: Two hours of severe trauma and hemorrhage lead an elevation in many SCFAs in rat feces. No change in SCFA was found in plasma. Because SCFA are primarily derived from commensal bacteria in the gut, these data suggest that the measurement of SCFA could be used as an index of changes in the gut microbiome in pathological condition including trauma and hemorrhage.