Rapid-Response Nitrite Probes: Intramolecular Griess Reaction for Nitrite Detection at Picogram Level

Nitrite is abundantly used not only in the food industry but also in various chemical reactions such as diazotization to furnish azo-dye compounds and the Sandmeyer reaction. When a secondary amine is in the presence of nitrite, residual levels of nitrite ions can potentially form corresponding N-nitrosamines, many of which are known to be carcinogenic. The carcinogenicity concerns with N-nitrosamines resulted in worldwide recalls of numerous marketed pharmaceutical products since 2018. Therefore, the residual nitrite assay is a critical part of N-nitrosamine risk assessment, as many components present in drug products including not only the drug substance but also excipients can be a potential source of nitrite ions. While ion chromatography serves as a primary analytical tool for the nitrite ion assay, leveraging the Griess reaction shows several benefits over ion chromatography, which includes rapid and visible responses as well as flexibility of sample preparations in organic solvent. In order to simplify the Griess reaction method and enhance reactivity toward the nitrite ion, a series of probe molecules was designed and synthesized. Upon exposure to the nitrite ion, molecular probes undergo diazotization followed by intramolecular cyclization to form benzo[c]cinnoline, which elicits dramatic absorption and emission changes as a signal readout for the nitrite assay. The reactivity toward the nitrite ion depends on nucleophilicity as well as electron-withdrawing/donating properties of substituents on probe molecules. Among a series of probe molecules, comparative kinetic studies revealed that the para-sulfonamide-substituted molecule (probe 1) has the highest reactivity with no-detectable side reaction. With probe 1, method validation was performed with two representative excipients, microcrystalline cellulose and dicalcium phosphate dihydrate, which demonstrated excellent accuracy, linearity, precision, and multilevel spike/recovery as well as very low detection limit (sub-parts-per-billion level) when coupled with a fluorescence detector. This new probe molecule offers low detection limits and wide flexibility in terms of sample preparation in a higher composition of organic solvent. Such flexibility of solvent choice enables a broader application of the method to any components in drug products including API, process intermediates, and excipients.