Simultaneous Differentiation of C=C Position Isomerism in Fatty Acids through Ion Mobility and Theoretical Calculations

Fatty acids play a pivotal role in biological processes and have many isomers, particularly at the C=C position, that influence their biological function. Distinguishing between isomers is crucial to investigating their role in health and disease. However, separating the isomers poses a significant analytical challenge. In this study, we developed a simple and rapid strategy combining ion mobility spectrometry and theoretical chemical calculations to differentiate and quantify the C=C positional isomers in 2-/3-butenoic acid (BA), 2-/3-/4-pentenoic acid (PA), and 2-/3-/5-hexenoic acid (HA). C=C positional isomerism was mobility-differentiated by simple complexation with crown ethers (12C4, 15C5, and 18C6) and divalent metal ions (Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Sr2+, and Ba2+), that is, converting C=C positional isomers with small structural differences into complexes with large structural differences through the interaction with metal ions and crown ethers. Metallized isomers were formed but could not be differentiated due to their complex and overlapping extracted ion mobiliograms (EIMs). Binary crown ether-isomer complexes were not observed, indicating that C=C positional isomers could not be separated by simple mixing with crown ethers. However, significant EIM differences were obtained for the formed ternary complexes, allowing baseline separation for the isomers. Notably, all crown ethers and metal ions have a separation effect with the isomers, with a calculated separation resolution (Rp-p) of 0.07-2.44. Theoretical chemical calculations were performed to provide in-depth structural information for the complexes and explain the separation principle. Theoretical conformational space showed that the divalent metal ions act as a bridge connecting the crown ether and the isomer. Additionally, the ternary complex becomes more compact as the distance between C=C and -COOH increases. Theoretical results can reflect the features of mobility experiments, with relative errors between the experiment collision cross-section (CCS) and theoretical CCS of no more than +/- 8.06%. This method was also evaluated in terms of quantification, accuracy, and precision repeatability. Overall, this study establishes that the crown ether-metal ion pair can function as a robust unit for differentiating C=C positional isomerism.

Automated summary

In this study, a simple and rapid strategy combining ion mobility spectrometry and theoretical chemical calculations was developed to differentiate and quantify the C=C positional isomers in fatty acids. The isomers were differentiated by complexing with crown ethers and divalent metal ions, and the separation was achieved through the observation of their mobility differences. The method showed promising results in differentiating the isomers and provided structural information for the complexes.