Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. Due to the labile nature of mRNA, identifying impurities that could affect product stability and efficacy is crucial to the long-term use of nucleic-acid based medicines. Herein, reversed-phase ion pair high performance liquid chromatography (RP-IP HPLC) was used to identify a class of impurity formed through lipid:mRNA reactions; such reactions are typically undetectable by traditional mRNA purity analytical techniques. The identified modifications render the mRNA untranslatable, leading to loss of protein expression. Specifically, electrophilic impurities derived from the ionizable cationic lipid component are shown to be responsible. Mechanisms implicated in the formation of reactive species include oxidation and subsequent hydrolysis of the tertiary amine. It thus remains critical to ensure robust analytical methods and stringent manufacturing control to ensure mRNA stability and high activity in LNP delivery systems.

The in vivo oxidation of sulfur and nitrogen atoms in many drugs into sulfoxide and N-oxide functionalities is a common biotransformation process. Unfortunately, the unambiguous identification of these metabolites can be challenging. In the present study, ion-molecule reactions of tris(dimethylamino)borane followed by collisionally activated dissociation (CAD) in an ion trap mass spectrometer are demonstrated to allow the identification of N-oxide and sulfoxide functionalities in protonated polyfunctional drug metabolites. Only ions with N-oxide or sulfoxide functionality formed diagnostic adducts that had lost dimethyl amine (DMA). This was demonstrated even for an analyte that contains a substantially more basic functionality than the functional group of interest. CAD of the diagnostic product ions (M) resulted mainly in type A (M - DMA) and B fragment ions (M - HO-B(N(CH3)2)2) for N-oxides, but sulfoxides also formed diagnostic C ions (M - O═BN(CH3)2), thus allowing differentiation of the functionalities. Some protonated analytes yielded abundant TDMAB adducts that had lost two DMA molecules instead of just one. This provides information on the environment of the N-oxide and sulfoxide functionalities. Quantum chemical calculations were performed to explore the mechanisms of the above-mentioned reactions. The method can be implemented on HPLC for real drug analysis.

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The reactivity of a carbon‐centered σ,σ,σ,σ‐type singlet‐ground‐state tetraradical containing two meta‐benzyne moieties was examined in the gas phase. Surprisingly, the tetraradical showed higher reactivity than its individual meta‐benzyne counterparts. The reactivity of meta‐benzynes is controlled by their (calculated) distortion energy ΔE2.3, singlet–triplet spitting ΔES–T, and electron affinity (EA2.3) of the meta‐benzyne moiety at the transition state geometry for hydrogen‐atom abstraction reactions. The addition of a second meta‐benzyne moiety to a meta‐benzyne does not significantly change EA2.3. However, ΔE2.3 is substantially decreased for both meta‐benzyne moieties in the tetraradical, and this explains their higher reactivities. The decrease in ΔE2.3 for each meta‐benzyne moiety in the tetraradical is rationalized by stabilizing spin–spin coupling between one radical site in each meta‐benzyne moiety. Therefore, spin–spin coupling between the meta‐benzyne moieties in this tetraradical increases its reactivity, whereas spin–spin coupling within each meta‐benzyne moiety decreases its reactivity.

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