In Situ Mass Spectrometric Analysis of Intermediates in Aziridination Reaction of Unsaturated Lipids

The structures of lipids determine their biological functions, and the identification of C=C double bond isomers in unsaturated lipids faces challenges. The direct nitrogen-methyl (N-Me) aziridination reaction allows for the derivatization of unsaturated lipid double bonds, which can combine with tandem mass spectrometry (MS/MS) to enable the resolving for positions of unsaturated lipid double bonds. However, current studies about the mechanism of the N-Me aziridination reaction lack direct evidence of intermediate processes. Here, a nano-electrostatic-spray ionization-mass spectrometry (nESTASI-MS) device based on a θ capillary was used to separate the reactants through the channels on both sides of the θ capillary, allowing the solutions to be mixed and react on-line during the electrospray process. This on-line mixing greatly shortened the residence time of the two reactants, providing for the successful capture and identification of short-lived intermediates during the reaction process. The device was used to study the reaction process of aziridination of unsaturated phosphatidylcholine (PC). N-Methyl-O-(2,4-dinitrophenyl)hydroxylamine (N-Me-DPH) was used as the amination reagent, PC 36:2 as a representative substrate, and Rh₂(esp)₂ as a catalyst to study the reactions of these three substances in different mixing modes. The results showed that N-Me-DPH pre-mixed with Rh₂(esp)₂ may deactivate the catalyst and the relevant intermediates and products could not be observed. In the other two mixing modes, the key rhodium-nitrene intermediate and the ternary biradical intermediate containing the first C-N bond during the reaction were successfully captured and characterized, as well as the product containing 1-methyl aziridine structure, which provided key evidence for the mechanistic study of the direct aziridination reaction of N-Me. Derivatization of N-Me aziridination could generate the 1-methyl aziridine structure at the double bond position, which was susceptible to break during mass spectrometric collision-induced dissociation (CID) to generate fragment ions providing indicative information about the position of the double bond. The dissociation behavior of the protonated peak of its aziridination product in CID mode was examined using PC 36:2 as an example. The results showed that the abundance of fragment ions breaking at the 1-methylaziridine structure was very low. Compared to the derived aziridine structure, the choline phosphate head group of PC was more likely to be lost during CID. Further collision-induced dissociation of the ion fragmentation peaks after the loss of the head group produced significant fragmentation of the aziridine structural unit, yielding fragment ions with C-N bond breaking near the acyl end and near the methyl end, respectively. The position of the double bond on the aliphatic chain could be easily determined from the mass of the fragment ions. This study provides a new in situ MS tool for the study of short-lived chemical reaction intermediates, which is expected to play an important role in the study of transient intermediate processes of chemical reactions.